Breast and ovarian cancer vaccine

Abstract

The compositions described herein comprise epitopes of peptides that can elicit an immune response in a subject upon administration. The compositions can comprise nucleic acids. The compositions can comprise peptides. The methods described herein include administering to a subject in need thereof a composition comprising an epitope of a peptide.

Description

[0001] Cross Reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 972,176, filed March 28, 2014, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made entirely with U.S. government support under Grant No. W81XWH-11-1-0760 from the Department of Defense, Grant No. P50CA083636 from the National Cancer Institute, and Grant No. R01CA098761 from the National Cancer Institute. background

[0005] Cancer treatment is traditionally completed by surgery to reduce tumor weight and subsequent chemotherapy and / or radiotherapy. This strategy can reduce tumors and, in earlier stages, usually results in complete remission. Unfortunately, the prognosis for more advanced tumors has remained virtually unchanged over the past 50 years, and a large number of cancer-related deaths have occurred due to subsequent metastasis. New preventive and therapeutic treatments are needed to combat the increasing number of cancers.

[0006] Every year the whole world has more than one million people to diagnose breast cancer and every year more than 400000 people have to die from breast cancer.Estimation has 1 in every 8 women to diagnose breast cancer at some time point in their life span.Preventing breast cancer from developing can have significant health and economic benefits to all individuals.If people no longer need to receive expensive cancer-related monitoring and therapeutic intervention, then billions of dollars will be saved.Need new methods for preventing and treating breast cancer. SUMMARY OF THE INVENTION

[0007] In some aspects, the compositions described herein include a composition comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen expressed by cells associated with breast cancer; and a second nucleotide sequence encoding a second epitope of a second antigen expressed by cells associated with breast cancer, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0008] In another aspect, the present invention provides a composition comprising: a first plasmid comprising a first nucleotide sequence, the first nucleotide sequence encoding a first epitope of a first antigen, the first epitope being part of a HIF-1α peptide, wherein the first nucleotide sequence is located in a plasmid. In another aspect, the present invention includes a composition comprising: a first plasmid comprising a first nucleotide sequence, the first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence, the second nucleotide sequence encoding a second epitope of a second antigen, wherein the first and second epitopes are part of a HIF-1α peptide, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0009] In some aspects, the compositions described herein include, comprising the following compositions: comprising a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from CD105, Yb-1, SOX-2, CDH3 or MDM2, wherein the first nucleotide sequence is located in a plasmid. In other aspects, the present invention includes a composition comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen; and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second epitope of a second antigen, wherein the first and second epitopes are independently selected from CD105, Yb-1, SOX-2, CDH3 or MDM2, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0010] In some aspects, the compositions described herein include a composition comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from IGFBP-2, HER-2, and IGF-1R, wherein the first nucleotide sequence is located in a plasmid. In other aspects, the present invention includes a composition comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen; and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second epitope of a second antigen, wherein the first and second epitopes are independently selected from IGFBP-2, HER-2, or IGF-1R, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0011] In some aspects, the compositions described herein comprise a composition comprising: a first epitope of a first antigen expressed by cells associated with breast cancer; and a second epitope of a second antigen expressed by cells associated with breast cancer.

[0012] In another aspect, the present invention includes a composition comprising: at least a first epitope of a first antigen, wherein the first epitope is a portion of a peptide from HIF-1α. In other aspects, the present invention includes a composition comprising: at least a first epitope of a first antigen, and at least a second epitope of a second antigen, wherein the first and second epitopes are from HIF-1α.

[0013] In other aspects, the present invention includes a composition comprising: at least a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from CD105, Yb-1, SOX-2, CDH3, or MDM2. In other aspects, the present invention includes a composition comprising: at least a first epitope of a first antigen, at least a second epitope of a second antigen, wherein the first and second epitopes are independently selected from CD105, Yb-1, SOX-2, CDH3, or MDM2.

[0014] In some cases, the present invention includes a composition comprising an isolated and purified plasmid comprising a nucleotide sequence encoding a polypeptide and an excipient, wherein the polypeptide comprises a plurality of epitopes. Sometimes, the plurality of epitopes comprises one or more epitopes having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, 32-34, 46-56, 60-62, 66-75, 82-85, and 87.

[0015] Sometimes, the isolated and purified plasmid may further comprise a first nucleotide sequence encoding a first epitope of a first antigen expressed by cells associated with breast cancer. In some cases, the composition further comprises a second nucleotide sequence encoding a second epitope of a second antigen expressed by cells associated with breast cancer. The first nucleotide sequence and the second nucleotide sequence may be located in one or more isolated and purified plasmids. The first epitope and the second epitope may be independently selected from a portion of a HIF-1α peptide that has at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 82-84. The first and second epitopes may be independently selected from CD105, Yb-1, SOX-2, CDH3, or MDM2, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more isolated and purified plasmids. The first and second epitopes may be independently selected from IGFBP-2, HER-2, or IGF-1R, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more isolated and purified plasmids. The first and second nucleic acid sequences may be located on a first isolated and purified plasmid. The second nucleic acid sequence may be located on a second isolated and purified plasmid.

[0016] In some cases, the present invention includes a composition comprising a first epitope of a first antigen expressed by cells associated with breast cancer or ovarian cancer; and a second epitope of a second antigen expressed by cells associated with breast cancer or ovarian cancer; wherein the first and second epitopes independently have at least 90% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 6, 8-10, 14-16, 20, 25-28, 32-34, 46-56, 60-62, 66-75, 82-85, and 87.

[0017] In some cases, the present invention includes a composition comprising a plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 70% sequence identity to an epitope sequence selected from SEQ ID NOs: 54, 73, 85, and 87, and an excipient.

[0018] In some cases, the present invention includes a composition comprising a plasmid and an excipient, wherein the plasmid comprises four nucleotide sequences, wherein each of the four nucleotide sequences independently encodes a polypeptide having at least 70% sequence identity to an epitope sequence selected from SEQ ID NOs: 54, 73, 85, and 87.

[0019] Sometimes, the invention includes a composition comprising a plasmid comprising a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to SEQ ID NO: 89 and an excipient.

[0020] Sometimes, the invention includes a composition comprising a polypeptide having at least 80% sequence identity to SEQ ID NO:89.

[0021] Disclosed herein, in some cases, are methods of administering to a subject one or more of the compositions described herein. Sometimes, a subject may be in need of one or more compositions.

[0022] Sometimes, described herein are methods of preventing breast cancer or ovarian cancer in a subject, wherein the method comprises administering to the subject a composition described herein. Sometimes, the cancer can be ovarian cancer. The cancer can be breast cancer. Described herein are methods of preventing breast cancer in a subject, wherein the method comprises administering to the subject a composition described herein.

[0023] Sometimes, described herein are methods of treating breast cancer or ovarian cancer in a subject, wherein the method comprises administering to the subject a composition described herein. Sometimes, the cancer can be ovarian cancer. The cancer can be breast cancer. Described herein are methods of treating breast cancer in a subject, wherein the method comprises administering to the subject a composition described herein.

[0024] Sometimes, administering further comprises delivering at least one dose of a composition described herein to a subject. Sometimes, administering further comprises delivering a composition described herein to a subject by subcutaneous injection, intradermal injection, intramuscular injection, intravascular injection, topical application, or inhalation.

[0025] In some cases, described herein are methods of generating an immune response in a subject having breast or ovarian cancer, comprising administering to the subject a composition described herein.

[0026] The present invention also includes an isolated and purified plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 90% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 82-84. The isolated and purified plasmid may comprise a set of two or more nucleotide sequences, wherein the two or more nucleotide sequences each independently encode a polypeptide having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 82-84. The isolated and purified plasmid may comprise two or more nucleotide sequences, wherein the two or more nucleotide sequences each encode a polypeptide having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 82-84; and each nucleotide within the set of two or more nucleotide sequences is different.

[0027] The present invention may also include an isolated and purified plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 90% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, and 32-34. The isolated and purified plasmid may comprise a group of two or more nucleotide sequences, wherein the two or more nucleotide sequences each independently encode a polypeptide having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, and 32-34. The isolated and purified plasmid may comprise a group of two or more nucleotide sequences, wherein the two or more nucleotide sequences each encode a polypeptide having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, and 32-34; and each nucleotide within the group of two or more nucleotide sequences is different.

[0028] The present invention may include an isolated and purified plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 90% sequence identity to an epitope sequence selected from SEQ ID NOs: 46-56, 60-62, or 66-75. The isolated and purified plasmid may comprise a set of two or more nucleotide sequences, wherein the two or more nucleotide sequences each independently encode a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NOs: 46-56, 60-62, or 66-75. The isolated and purified plasmid may comprise a set of two or more nucleotide sequences, wherein the two or more nucleotide sequences each encode a polypeptide having at least 90% sequence identity to a sequence selected from SEQ ID NOs: 46-56, 60-62, or 66-75; and each nucleotide within the set of two or more nucleotide sequences is different.

[0029] Sometimes, the present invention may also include an isolated and purified plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 70% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87.

[0030] Incorporation by Reference

[0031] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The novel features of the present invention are described with particularity in the appended claims. The features and advantages of the present invention may be better understood with reference to the following description and accompanying drawings, which set forth illustrative embodiments utilizing the principles of the present invention:

[0034] Figure 1 Demonstrated that Th1 and Th2 epitopes differ in functional avidity.

[0035] Figure 2 Th2 eliminates the anti-tumor efficacy of Th1.

[0036] Figure 3 Antigen-specific IgG immunity is shown.

[0037] Figure 4 Population-based epitope screening is shown.

[0038] Figure 5 Characterization of breast cancer subjects was demonstrated.

[0039] Figure 6 Antigen-specific IFNγ responses to stem cell / EMT proteins are shown.

[0040] Figure 7 Antigen-specific IL-10 responses to stem cell / EMT proteins are shown.

[0041] Figure 8 The epitope showing the extended sequence was verified to be the native epitope with the CD105 extended epitope (52 aa) QNGTWPREVLLVLSVNSSVFLHL QALGI PLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: 1).

[0042] Figure 9 The extended epitope of Yb-1 based on the IFNγ / IL-10 activity ratio is shown.

[0043] Figure 10 The magnitude and incidence of IFNγ dominance and the ratio of IFNγ / IL-10 activity for the CDH3 antigen are shown.

[0044] Figure 11 The magnitude and incidence of IFNγ-dominant IFNγ / IL-10 activity ratios for HIF1α antigens are shown.

[0045] Figure 12 Graphs showing simultaneous in vivo evaluations in mice are shown.

[0046] Figure 13 The immunogenicity and efficacy of an exemplary Yb-1 plasmid-based vaccine in mice are shown.

[0047] Figure 14 Figures, immunogenicity, and exemplary sequences of the compositions described herein are shown. SEQ ID NO: 39 Figure 14 shown.

[0048] Figure 15A Exemplary validation of peptide-specific T-cells as native epitopes is shown.

[0049] Figure 15B A timeline of clinical trials using the compositions described herein is shown.

[0050] Figure 15C The study protocol for a Phase I clinical trial using the composition described herein is shown.

[0051] Figure 16 Shown are Western blot analyses of HIF1α expression in a single plasmid, pHIF1α, and the plasmid BCMA5 encoding five antigens.

[0052] Figure 17 The magnitude and incidence of IFNγ dominance and the ratio of IFNγ / IL-10 activity for the CD105 antigen are shown.

[0053] Figure 18 The magnitude and incidence of IFNγ dominance are shown. The ratio of IFNγ / IL-10 activity for the MDM-2 antigen is shown.

[0054] Figure 19 The magnitude and incidence of IFNγ dominance are shown. The IFNγ / IL-10 activity ratio for the SOX-2 antigen is shown.

[0055] Figure 20 The magnitude and incidence of IFNγ dominance are shown. The IFNγ / IL-10 activity ratio of the Yb-1 antigen is shown.

[0056] Figure 21 The immunogenicity and efficacy of HIF1α peptide and plasmid vaccines in mice were demonstrated.

[0057] Figure 22 The immunogenicity and efficacy of CD105 peptide and plasmid vaccines in mice were demonstrated.

[0058] Figure 23 The immunogenicity and efficacy of CDH3 peptide and plasmid vaccines in mice were demonstrated.

[0059] Figure 24 The immunogenicity and efficacy of SOX2 peptide and plasmid vaccines in mice were demonstrated.

[0060] Figure 25 The immunogenicity and efficacy of MDM2 peptide and plasmid vaccines in mice were demonstrated.

[0061] Figure 26 Shown are the mouse masses 3 months after the last vaccination.

[0062] Figure 27 Shown are the mouse masses 10 days after the last vaccination.

[0063] Figure 28 It was demonstrated that the IGFBP-2 C-terminus is enriched for epitopes that induce IL-10-secreting T-cells compared to the N-terminus.

[0064] Figure 29 It was shown that N-terminal, but not C-terminal, IGFBP-2 vaccines simultaneously stimulated type I immunity and inhibited tumor growth.

[0065] Figure 30 It was shown that IGFBP-2 vaccine-induced Th2 abolished the anti-tumor effect of IGFBP-2-specific Th1.

[0066] Figure 31 demonstrated that a vaccine based on a HER2 Th1 epitope, which is associated with disseminated epitopes, increased survival in patients with advanced HER2+ breast cancer.

[0067] Figure 32 Extended Th plasmid-based vaccines proved to be more effective than peptide-based vaccines in generating tumor antigen-specific Th1 immunity.

[0068] Figure 33 demonstrated sustained HER2 ICD-specific immunity for more than one year after completion of plasmid DNA-based immunization.

[0069] Figure 34 IGF-1R epitopes screened for IFNγ and IL-10 T-cell secretion by ELISPOT are shown.

[0070] Figure 35 Demonstrated that a multi-epitope IGF-1R vaccine inhibits the growth of implanted breast cancer.

[0071] Figure 36 A multi-antigen, multi-epitope vaccine was shown to prevent breast cancer development in mice.

[0072] Figure 37 Exemplary cytokine secretion patterns induced by HER2 immunization are shown.

[0073] Figure 38 ROC analysis of stem cell / EMT antigens is shown.

[0074] Figure 39 Candidate proteins overexpressed in stem cells and / or EMT are shown.

[0075] Figure 40 Cartoons of the constructs described herein are shown.

[0076] Figure 41 Western blot images of IGFBP-2, survivin, HIF-1A, and IGF-IR expression are shown.

[0077] Figures 42A-42D The antitumor effect of the multi-antigen vaccine in the ID8 ovarian cancer xenograft model was shown. Mice were imaged on an IVIS bioluminescence imager three weeks after implantation of ID8-Luc. Figure 42A ) and metastatic sites ( Figure 42B ) were measured in total flux (photons / second). The corresponding animals are shown as adjuvant only ( Figure 42C ), and triple antigen immunization ( Figure 42D ).

[0078] Figure 43A and Figure 43BThe TH1 response is shown as a function of protein sequence. Selective TH1-inducing sequences were identified in the C-terminus of survivin and the N-terminus of HIF1a. The average cSPW x incidence of each peptide is shown by donor type. The positive y-axis shows the IFN-g cSPW x incidence of volunteer donors (n=20) (white) and cancer donors (n=20) (grey). The negative y-axis shows the IL-10 cSPW x incidence of volunteer donors (solid black) and cancer donors (dashed black). Figure 43A TH1 responses to HIF-1A peptides are shown. Figure 43B TH1 responses to survivin peptides are shown. Vertical lines show selected sequences.

[0079] Figures 44A-44D Comparison of IgG antibody expression levels in ovarian cancer patients and volunteers is shown. IgG antibodies specific for the candidate antigen were significantly elevated in ovarian cancer patients compared to supporter controls. IGF-IR ( Figure 44A )、IGFBP-2( Figure 44B )、HIF-1A( Figure 44C ), and survivin ( Figure 44D ) of IgG (in ug / ml) (y-axis) and experimental group (x-axis). Mean and 2 standard deviations of volunteer control (dashed line), *p<0.05; **p<0.01; ***p<0.001. Detailed Description of the Invention

[0080] The present invention provides compositions of breast cancer vaccines and ovarian cancer vaccines, generally for use in preventing or treating breast cancer or ovarian cancer. The present invention also provides methods for administering the breast cancer vaccine or ovarian cancer vaccine to a subject. The compositions described herein can be used in conjunction with the methods described herein to prevent or treat breast cancer or ovarian cancer.

[0081] In some cases, the composition may include: a nucleic acid sequence encoding an epitope of a breast cancer or ovarian cancer antigen that can elicit an immune response in a subject, a plasmid containing a sequence described herein, an adjuvant, a drug carrier, and an inert chemical suitable for use in a pharmaceutical composition. A breast cancer or ovarian cancer antigen can be at least one of any antigens expressed in a subject that may suffer from or develop breast cancer or ovarian cancer. Typically, breast cancer or ovarian cancer antigens are expressed by breast cancer cells, ovarian cancer cells, and / or tissues such as breast cancer or ovarian cancer stem cells (CSCs). CSCs may exhibit the ability to self-renew, unregulated growth, and drug resistance. In some cases, CSCs may express proteins (e.g., antigens), and for example, the level of proteins (e.g., antigens) expressed by CSCs may be upregulated (e.g., expression increased relative to a given amount) or downregulated (e.g., expression decreased relative to a given amount). In some cases, proteins upregulated by CSCs compared to normal tissues or cells may be involved in the development and / or progression of breast cancer or ovarian cancer. For example, the compositions and methods described herein can be used to identify proteins and target antigenic epitopes.

[0082] In some cases, a breast cancer or ovarian cancer antigen epitope can be used in the composition. In other cases, more than one breast cancer or ovarian cancer antigen epitope can be used in the composition. In other cases, more than 2 antigens, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 15, more than 20, more than 25 or more than 30 breast cancer or ovarian cancer antigens can be used in the composition. In some cases, the antigens can be the same. In other cases, the antigens can be different. The composition of the breast cancer or ovarian cancer vaccine described herein can be formulated for preventing breast cancer or ovarian cancer. For example, the preventive composition can eliminate cells (e.g., CSC, such as breast CSC or ovarian CSC) with abnormal (e.g., up-regulated) expressed proteins to prevent breast cancer or ovarian cancer.

[0083] In some cases, one and / or more epitopes can be on the same breast or ovarian cancer antigen, or one and / or more epitopes can be on different breast or ovarian cancer antigens. In some cases, an epitope on one breast or ovarian cancer antigen can be used in the composition. In other cases, epitopes on more than one breast or ovarian cancer antigen, more than two antigens, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than ten, more than 15, more than 20, more than 25, or more than 30 breast or ovarian cancer antigens can be used in the composition.

[0084] The compositions and methods described herein can elicit an immune response in a subject. The immune response can be an immune response to an epitope of an antigen in a composition (e.g., a vaccine). The vaccine equips the immune system of the subject so that the immune system can detect and destroy those substances containing the antigen of the vaccine in the subject. The compositions and methods described herein can elicit a type 1 (Th1) immune response in a subject. The Th1 immune response can include secretion of inflammatory cytokines (e.g., IFNγ, TNFα) by a subgroup of immune cells (e.g., antigen-specific T cells). In some cases, inflammatory cytokines activate another subtype of immune cells (e.g., cytotoxic T cells), which can destroy those substances containing antigens in the subject.

[0085] Using the screening methods described herein to identify epitopes and in combination with peptides from tumor antigens, epitopes from a variety of antigens can be screened for inducing a Th1 immune response. For example, the screening methods can identify epitopes from at least one tumor antigen that elicit a Th1 response (e.g., preferably resulting in Th1 cytokine secretion) against breast cancer or ovarian cancer antigens, including CSC antigens (e.g., breast CSCs or ovarian CSCs), as described herein.

[0086] In some cases, the epitopes and / or antigens used in the compositions and methods described herein can be recognized by the subject's immune system to trigger a Th1 immune response and release type I cytokines. A Th1 response can be triggered by an interaction between the epitope and a T cell, more specifically, a major histocompatibility complex (MHC) expressed by the T cell. For example, high-affinity binding of the epitope to an MHC receptor can stimulate a Th1 response. The MHC receptor can be at least one of a variety of types. The MHC receptors on T cells can vary between individuals in a population.

[0087] In addition to nucleic acids encoding antigenic epitopes, the compositions described herein may include other components. In some cases, the composition may include at least one adjuvant. In some cases, the composition may include at least one pharmaceutical carrier. In some cases, the composition may include at least one inert chemical suitable for pharmaceutical compositions. In some cases, the composition may include at least one adjuvant and at least one pharmaceutical carrier. In some cases, the composition may include at least one adjuvant and at least one inert chemical suitable for pharmaceutical compositions. In some cases, the composition may include at least one inert chemical suitable for pharmaceutical compositions and a pharmaceutical carrier. In some cases, the composition may contain multiple adjuvants, multiple pharmaceutical carriers, and multiple inert chemicals suitable for pharmaceutical compositions.

[0088] In some cases, one adjuvant may be used in the composition. In other cases, more than one adjuvant, more than two adjuvants, more than three adjuvants, more than four adjuvants, more than five adjuvants, more than six adjuvants, more than seven adjuvants, more than eight adjuvants, more than nine adjuvants, or more than ten adjuvants may be used in the composition. In some cases, one pharmaceutical carrier may be used in the composition. In other cases, more than one pharmaceutical carrier, more than two pharmaceutical carriers, more than three pharmaceutical carriers, more than four pharmaceutical carriers, more than five pharmaceutical carriers, more than six pharmaceutical carriers, more than seven pharmaceutical carriers, more than eight pharmaceutical carriers, more than nine pharmaceutical carriers, or more than ten pharmaceutical carriers may be used in the composition. In some cases, one chemical may be used in the composition. In other cases, more than 1 chemical, more than 2 chemicals, more than 3 chemicals, more than 4 chemicals, more than 5 chemicals, more than 6 chemicals, more than 7 chemicals, more than 8 chemicals, more than 9 chemicals, or more than 10 chemicals may be used in the composition.

[0089] The present invention also describes a method for administering a breast cancer vaccine or an ovarian cancer vaccine to an object. In some cases, the method may include constructing a plasmid-based vaccine targeting these antigens and determining whether administering the vaccine is safe, immunogenic, and effectively prevents breast cancer development. For example, the composition may be a vaccine based on a multi-antigen Th1 multi-epitope plasmid. In some cases, the method may include conducting at least one clinical trial to determine the safety and immunogenicity of a plasmid-based vaccine in an object suffering from breast cancer or ovarian cancer. For example, an antigen may be expressed by or associated with CSC (e.g., breast CSC or ovarian CSC), and / or cells may be converted from epithelial cells into mesenchymal cells (EMT). In some cases, the epitope of the composition may be derived from an antigen, wherein the epitope may elicit a Th1 immune response in the object. For example, a Th1 immune response may include immune cells, typically CD4+ T cells. In some cases, the composition may be a nucleic acid (e.g., a plasmid-based vaccine), which may include encoding a nucleic acid encoding more than one antigen or more than one (individual) epitope of an antigen. In some cases, the method can be used to determine whether the compositions described herein prevent breast cancer or ovarian cancer development in a variety of organisms, for example, in a cancer (e.g., breast cancer or ovarian cancer) model using genetically similar rodents (e.g., mice), using genetically diverse rodents (e.g., mice), and in subjects who may or may not have breast cancer or ovarian cancer. In some cases, the cancer may be breast cancer. In some cases, the breast cancer may be triple-negative breast cancer (TNBC).

[0090] Antigen identification

[0091] The compositions and methods described herein include identifying and engineering breast cancer or ovarian cancer antigens in pharmaceutical compositions (e.g., vaccines). Although any technology known to those of ordinary skill in the art can be used to identify antigens expressed by objects suffering from breast cancer or ovarian cancer, in exemplary cases, the methods described herein can be used to identify suitable antigens. In some cases, the method may include screening serum from the object. In some cases, the screening may be an antibody screening. For example, the antibody screened may be an IgG antibody. In some cases, serum may be from an object suffering from breast cancer or ovarian cancer. In other cases, serum may be from an object not suffering from breast cancer or ovarian cancer.

[0092] Cancer antigens, for example, breast cancer antigens or ovarian cancer antigens, can be portions of proteins, peptides, or polyamino acids. In some cases, the portion can be a percentage of the protein, peptide, or polyamino acid. In some cases, the percentage can be less than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the protein, peptide, or polyamino acid. In some cases, the portion can be located at the C-terminus of the protein, peptide, or polyamino acid. In other cases, the portion can be located near the C-terminus of the protein, peptide, or polyamino acid. For example, the portion near the C-terminus can be within 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the midpoint of the total protein, peptide, or polyamino acid length. In some cases, the portion may be located at the N-terminus of a protein, peptide, or polyamino acid. In other cases, the portion may be located near the N-terminus of the protein, peptide, or polyamino acid. For example, near the N-terminus may be within 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the midpoint of the total protein, peptide, or polyamino acid length. In some cases, the portion may be located near the middle of a protein, peptide, or polyamino acid. In other cases, the portion may be located near the middle of a protein, peptide, or polyamino acid. For example, near the middle may be within 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the midpoint of the total protein, peptide, or polyamino acid length.

[0093] At least one antigen can be identified and screened for suitability as an antigen in a composition (e.g., a vaccine) described herein. In some cases, one antigen can be identified and screened. In other cases, more than 1 antigen can be identified and screened, more than 2 antigens can be identified and screened, more than 3 antigens can be identified and screened, more than 4 antigens can be identified and screened, more than 5 antigens can be identified and screened, more than 6 antigens can be identified and screened, more than 7 antigens can be identified and screened, more than 8 antigens can be identified and screened, more than 9 antigens can be identified and screened, more than 10 antigens can be identified and screened, more than 11 antigens can be identified and screened, more than 12 antigens can be identified and screened, more than 13 antigens can be identified and screened, more than 14 antigens can be identified and screened, more than 15 antigens can be identified and screened, more than 20 antigens can be identified and screened, more than 25 antigens can be identified and screened, more than 30 antigens can be identified and screened, more than 35 antigens can be identified and screened, more than 40 antigens can be identified and screened, more than 45 antigens can be identified and screened, and more than 50 antigens can be identified and screened for vaccine suitability. In an exemplary case, 5 antigens are identified and screened for vaccine suitability.

[0094] Using screening techniques known to those of ordinary skill in the art, the antigens for screening vaccine suitability can be derived from any protein detected in the serum of a subject suffering from breast cancer or ovarian cancer. In some cases, screening can generally be antibody screening. Although the protein can be any protein detected in the serum of a subject suffering from breast cancer or ovarian cancer, in exemplary cases, the protein derived from the antigen can be classified as stem cell protein and / or EMT protein. For example, in breast cancer, stem cell / EMT proteins may include SOX2, YB1, CD105, MDM2, CDH3+ / - and HIF1α. Typically, the antigen may have immunogenicity in a breast cancer subject and in a subject without breast cancer.

[0095] Epitope mapping

[0096] The compositions and methods provided herein include mapping at least one epitope(s) within an antigen so that the epitope(s) produce a Th1 immune response when administered to a subject. In some cases, the epitope(s) can be administered as a breast cancer vaccine or an ovarian cancer vaccine. While any technique known to those of ordinary skill in the art can be used to identify epitopes that can elicit a Th1 immune response produced by a subject, it is still preferred to use the methods described herein. In some cases, the epitope(s) can be a portion of an antigen (e.g., identified above). For example, the epitope(s) can be a peptide of an antigenic protein and / or a portion of an antigenic protein.

[0097] In some cases, the epitope can be a human leukocyte antigen (HLA) class I epitope derived from a breast cancer or ovarian cancer antigen. For example, an HLA class I epitope can include epitopes that bind to HLA-A, -B, and -C molecules. In some cases, the epitope can be a class II epitope derived from a breast cancer or ovarian cancer antigen used in the development of a cancer vaccine (e.g., breast cancer or ovarian cancer). For example, an HLA class II epitope can include epitopes that bind to HLA-DP, -DM, -DOA, -DOB, -DQ, and -DR molecules. In some cases, in addition to the methods described herein, the following steps can be used to map the epitope: (1) determine whether the epitope binds to MHC (e.g., with high affinity) via at least one HLA allele (e.g., HLA-DR, i.e., a universal epitope), (2) determine whether the epitope stimulates IFNγ but not IL-10 secretion (e.g., from antigen-specific T-cells), and (3) determine whether T-cells can recognize the peptide (e.g., epitope) processed by antigen presenting cells (APCs), i.e., it is a native epitope. In some cases, a T-cell line can be used. For example, the T-cell line can be an epitope-derived T-cell line. In some cases, the T-cell can be an exogenous T-cell engineered to express a chimeric antigen receptor construct that binds the epitope with high selectivity and affinity. In some cases, the epitope can be derived from a protein (e.g., a recombinant protein). In other cases, the protein can be a native protein. In some cases, the protein can be endogenously processed. In other cases, the protein can be exogenously processed. In some cases, the protein may be processed endogenously by autologous APCs. In other cases, the protein may be processed exogenously by autologous APCs.

[0098] In all cases, peptides are epitopes mapped from antigens, and can be identified for selecting peptide epitopes using the methods described herein. In some cases, epitopes can be derived from human proteins that can be used directly in vaccines based on peptides. In other cases, epitopes can be derived from human proteins and the encoding nucleic acid sequence can be integrated into a nucleic acid construct designed to induce epitope expression in an object after administration. For example, a nucleic acid construct can allow for an immune response to at least one (individual) epitope to be produced, amplified, weakened, suppressed or eliminated from a specific group of proteins. In some cases, peptides or nucleic acid constructs can be optimized to induce, amplify or produce a Th1 immune response based on protein or plasmid immunity. In some cases, the epitope can be an extended Th1 epitope. In other cases, peptides or nucleic acid constructs can be optimized to suppress, weaken or eliminate pathogenicity responses in an object (e.g., human or animal) in need thereof based on protein or plasmid immunity.

[0099] In some cases, the peptide is located within a portion of a protein, peptide, or polyamino acid, such that the protein, peptide, or polyamino acid stimulates IFNγ secretion. In some cases, the peptide is located within a portion of a protein, peptide, or polyamino acid, such that the protein, peptide, or polyamino acid inhibits IFNγ secretion. In some cases, the peptide is located within a portion of a protein, peptide, or polyamino acid, such that the protein, peptide, or polyamino acid stimulates IL-10 secretion. In some cases, the peptide is located within a portion of a protein, peptide, or polyamino acid, such that the protein, peptide, or polyamino acid inhibits IL-10 secretion. In some cases, the peptide can stimulate IFNγ secretion and inhibit IL-10 secretion. In other cases, the peptide can stimulate IL-10 secretion and inhibit IFNγ secretion. In some cases, the peptide can stimulate IFNγ secretion and stimulate IL-10 secretion. In other cases, the peptide can inhibit IL-10 secretion and inhibit IFNγ secretion.

[0100] In some cases, the amino acids comprising the peptide can be adjusted so that the desired effect of the peptide on IFNγ secretion and / or the desired effect of the peptide on IL-10 secretion can be achieved. For example, a peptide that stimulates both IFNγ and IL-10 secretion can be adjusted so that the length of the peptide is shortened to eliminate amino acids that stimulate IL-10 secretion, so that the peptide only stimulates IFNγ secretion.

[0101] In some cases, the identified epitope can be included in the vaccine composition of an extended peptide vaccine. In some cases, the extended epitope can be a 40-80 polymer peptide. In an exemplary case, a nucleic acid sequence or a peptide sequence is used in parallel to construct an extended peptide sequence. The juxtaposition of peptides selected within the parent protein (for example, within 10 amino acids of each other) allows the construction of a serially extended epitope, which may contain tolerance and / or inhibitory epitopes. For example, a serially extended epitope may contain a short intervening sequence of <10 amino acids. Any of these peptides and / or extended epitopes alone (embodied as the peptide itself, or the corresponding nucleic acid construct) or any combination can be optimized into protein- or plasmid-based immunization, which will specifically induce, amplify or produce a protective immune response in an object (animal or human) in need thereof, or will suppress, weaken or eliminate the pathogenic response.

[0102] In some cases, an epitope can be a certain length of amino acids. In some cases, an epitope can be less than 5 amino acids, less than 10 amino acids, less than 15 amino acids, less than 20 amino acids, less than 25 amino acids, less than 30 amino acids, less than 35 amino acids, less than 40 amino acids, less than 45 amino acids, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 70 amino acids, less than 75 amino acids, less than 80 amino acids, less than 85 amino acids, less than 90 amino acids, less than 95 amino acids, less than 100 amino acids, less than 110 amino acids, less than 120 amino acids, less than 130 amino acids. , less than 140 amino acids, less than 150 amino acids, less than 160 amino acids, less than 170 amino acids, less than 180 amino acids, less than 190 amino acids, less than 200 amino acids, less than 210 amino acids, less than 220 amino acids, less than 230 amino acids, less than 240 amino acids, less than 250 amino acids, less than 260 amino acids, less than 270 amino acids, less than 280 amino acids, less than 290 amino acids, less than 300 amino acids, less than 350 amino acids, less than 400 amino acids, less than 450 amino acids, or less than 500 amino acids.

[0103] In some cases, the present invention provides a composition comprising an isolated and purified plasmid and an excipient, wherein the plasmid comprises a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises a plurality of epitopes. In some cases, the plurality of epitopes comprises one or more epitopes having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, 32-34, 46-56, 60-62, 66-75, 82-85, and 87. In some cases, the plurality of epitopes comprises one or more epitopes having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 82-84. In some cases, the plurality of epitopes comprises one or more epitopes having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, or 32-34. In some cases, the plurality of epitopes comprises one or more epitopes having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 46-56, 60-62, or 66-75. In some cases, the plurality of epitopes comprises one or more epitopes having at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 54, 73, 85, and 87. In some cases, the plurality of epitopes comprises one or more epitopes selected from SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, 32-34, 46-56, 60-62, 66-75, 82-85, and 87.

[0104] In some cases, the multiple (individual) epitopes are multiple adjacent epitopes. In some cases, the adjacent epitopes also include a linker between one or more epitope sequences. In some cases, the amino acid sequences of the first and second epitopes are separated by a linker amino acid sequence. In some cases, the amino acid sequence of the first epitope is adjacent to the amino acid sequence of the second epitope.

[0105] In some cases, the composition further comprises additional isolated and purified plasmids comprising additional nucleotide sequences encoding additional polypeptides, wherein the additional polypeptides comprise a plurality of epitopes, including one or more epitopes having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, 32-34, 46-56, 60-62, 66-75, 82-85, and 87. Sometimes, the composition further comprises additional isolated and purified plasmids comprising additional nucleotide sequences encoding additional polypeptides, wherein the additional polypeptides comprise a plurality of epitopes selected from the group consisting of SEQ ID NOs: 1, 6, 8-10, 14-16, 20, 25-28, 32-34, 46-56, 60-62, 66-75, 82-85, and 87. In some cases, the sequences of the polypeptides and the additional polypeptides are different.

[0106] In some cases, the immune response is a Type 1 immune response. In some cases, the immune response is characterized by a ratio of Type I cytokine production to Type II cytokine production that is greater than 1. In some cases, the immune response is characterized by a ratio of Type I cytokine production to Type II cytokine production that is less than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production that is greater than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production that is less than 1.

[0107] In some cases, the composition is administered to a subject. In some cases, the subject is in need of administration of the composition. In some cases, the composition effectively elicits an immune response in the subject. In some cases, the composition effectively eliminates a variety of cells associated with breast cancer or ovarian cancer in the subject. In some cases, the composition can be used to prevent the growth of cells associated with breast cancer or ovarian cancer in the subject.

[0108] In some cases, the cancer is breast cancer. In some cases, the breast cancer is recurrent or refractory or metastatic breast cancer. In some cases, the cancer is ovarian cancer. In some cases, the ovarian cancer is recurrent or refractory or metastatic ovarian cancer.

[0109] In some cases, at least the first epitope is contained in a pharmaceutical composition. In some cases, at least the first epitope is contained in a pharmaceutical composition that also includes a pharmaceutical carrier. In some cases, at least the first epitope is contained in a pharmaceutical composition that also includes a pharmaceutical carrier and an adjuvant. In some cases, at least the first epitope is contained in a pharmaceutical composition that also includes an adjuvant. In some cases, the composition further includes an adjuvant and a pharmaceutical carrier. In some cases, the adjuvant is GM-CSF.

[0110] The present invention also provides kits for preparing the compositions described herein, the kits comprising instructions for preparing the compositions. The present invention also provides kits for administering the compositions described herein, the kits comprising instructions for administering the compositions.

[0111] Compositions comprising epitopes for breast cancer vaccines

[0112] The compositions described herein include compositions comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen expressed by cells associated with breast cancer; and a second nucleotide sequence encoding a second epitope of a second antigen expressed by cells associated with breast cancer, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids. In some cases, the composition may include nucleic acids encoding epitopes from the following proteins: CD105, HIF1α, MDM2, Yb1, SOX-2, HER-2, IGFBP2, IGF-1R, CDH3, and survivin

[0113] In some cases, the composition may include a composition comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from CD105, Yb-1, SOX-2, CDH3 or MDM2, wherein the first nucleotide sequence is located in a plasmid. In other cases, the composition may include a composition comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen; and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second epitope of a second antigen, wherein the first and second epitopes are independently selected from CD105, Yb-1, SOX-2, CDH3 or MDM2, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0114] In some cases, the composition may include an epitope and nucleic acid encoding an epitope from the following proteins: CD105, MDM2, Yb-1, SOX-2, and CDH3. In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide CD105 selected from the group consisting of: ... NO: 2) a nucleotide sequence having at least 90% sequence identity; and a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence ACCGTGTTCATGCGCCTGAACATCATCTCCCCCGACCTGTCCGGCTGCACCTCCAAGGGCCTGGTGCTGCCCGCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCTCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 3) a nucleotide sequence having at least 90% sequence identity; a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGGCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 4);a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 5); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO: 6); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence QNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: NO:7) having at least 90% sequence identity; a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence TVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO:8); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence;

[0115] TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 9); or a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence

[0116] TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 10) has at least 90% sequence identity. In some cases, the composition may include a nucleic acid sequence encoding an epitope of peptide Yb-1 selected from the group consisting of a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACCGCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAAC (SEQ ID NO: 11); a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 12); a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 13); a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence EDVFVHQTAIKKNNPRK (SEQ ID NO: 14); a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence YRRNFNYRRRRPEN (SEQ ID NO: 15);Or a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence GVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN (SEQ ID NO: 16). In some cases, the composition can include a nucleic acid sequence encoding an epitope of peptide SOX-2 selected from the group consisting of a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTG (SEQ ID NO: 17); a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 18); NO: 18) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 19);or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSV (SEQ ID NO: 20). In some cases, the composition can include a nucleic acid sequence encoding an epitope of the peptide CDH3 selected from the group consisting of a nucleic acid sequence encoding an epitope of the peptide CDH3 having at least 90% sequence identity to the amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSV (SEQ ID NO: 20). In some cases, the composition can include a nucleic acid sequence encoding an epitope of the peptide CDH3 selected from the group consisting of a nucleic acid sequence encoding an epitope of the peptide CDH3 having at least 90% sequence identity to the amino acid sequence AGGTCACTGAAGGAAAGGAATCCATTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 21) has at least 90% sequence identity with a nucleotide sequence; and a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence TTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 22) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GCCATGCACTCCCCCCCCACCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTTCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGATCTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGTCC (SEQ ID NO: 23);a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence GTGATGAACTCCCCCCCCTCCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTCCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGCTGTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGACC (SEQ ID NO: 24); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: NO: 25); a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence LKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 26); a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence AMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKES (SEQ ID NO: 27);or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence VMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKET (SEQ ID NO: 28). In some cases, the composition can include a nucleic acid sequence encoding an epitope of the peptide MDM2 selected from the group consisting of ... NO: 29) has at least 90% sequence identity with a nucleotide sequence; and a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence ACCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGCCGTGTCCCAGCAGGACTCCGGCACCTCCCTGTCC (SEQ ID a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence ATCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGTGGTGTCCCAGCAGGACTCCGGCACCTCCCCCTCC (SEQ ID NO: 31); a nucleotide sequence encoding an amino acid sequence, the amino acid sequence being identical to the amino acid sequence;

[0117] TYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSV (SEQ ID NO: 32); a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence TYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLS (SEQ ID NO: 33); or a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence IYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPS (SEQ ID NO: 34).

[0118]

[0119] PEGVFTIEKETRSAGE

[0120] IYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPSRS (SEQ ID NO: 42) has at least 90% sequence identity.

[0121]

[0122] In some cases, the composition may include a first and a second epitope independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may include a third epitope, wherein the first, second, and third epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may include a third and a fourth epitope, wherein the first, second, third, and fourth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may include a third, fourth, and fifth epitope, wherein the first, second, third, fourth, and fifth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2.

[0123] In some cases, the composition can include a first and a second epitope independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R. In some cases, the composition can include a third epitope, the first, second, and third epitopes independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R.

[0124] In some cases, the composition can be administered to a subject. In some cases, the subject needs to be administered the composition. In some cases, the composition effectively elicits an immune response in the subject. In some cases, the composition effectively eliminates a variety of cells associated with breast cancer in the subject. In some cases, the composition can be used to prevent the growth of cells associated with breast cancer in the subject.

[0125] In some cases, the first and second nucleic acid sequences are located on a first plasmid. In some cases, the second nucleic acid sequence is located on a second plasmid.

[0126] In some cases, the cell associated with breast cancer is selected from the group consisting of a breast cell expressing an aberrant signature, a pre-neoplastic breast cell, a breast cancer cell, a pre-invasive breast cancer cell, a breast cancer stem cell, an epithelial cell, a mesenchymal cell, a somatic cell, or a combination thereof.

[0127] In some cases, the first and second nucleic acid sequences are purified to at least 70% purity. In some cases, the first and second nucleic acid sequences are located on a first plasmid and are separated by a linker nucleic acid sequence. In some cases, the first nucleic acid sequence is adjacent to the second nucleic acid sequence on the first plasmid.

[0128] In some cases, at least the first plasmid is contained in a pharmaceutical composition. In some cases, at least the first plasmid is contained in a pharmaceutical composition that also includes a pharmaceutical carrier. In some cases, at least the first plasmid is contained in a pharmaceutical composition that also includes a pharmaceutical carrier and an adjuvant. In some cases, at least the first plasmid is contained in a pharmaceutical composition that also includes an adjuvant. In some cases, the composition further includes an adjuvant and a pharmaceutically acceptable carrier. In some cases, the adjuvant is GM-CSF.

[0129] In some cases, the subject is selected from: a human with breast cancer, a mouse with breast cancer, or a rat with breast cancer. In some cases, the subject is selected from: a human without breast cancer, a mouse without breast cancer, or a rat without breast cancer.

[0130] In some cases, the immune response is a Type 1 immune response. In some cases, the first nucleic acid sequence is a species selected from humans, mice, or rats. In some cases, the second nucleic acid sequence is a species selected from humans, mice, or rats. In some cases, the immune response is characterized by a ratio of type I cytokine production to type II cytokine production greater than 1. In some cases, the immune response is characterized by a ratio of type I cytokine production to type II cytokine production less than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production greater than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production less than 1.

[0131] In some cases, the composition includes a composition comprising: a first plasmid comprising a first nucleotide sequence, the first nucleotide sequence encoding a first epitope of a first antigen, the first epitope being part of a HIF-1α peptide, wherein the first nucleotide sequence is located in a plasmid. In other cases, the composition includes a composition comprising: a first plasmid comprising a first nucleotide sequence, the first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence, the second nucleotide sequence encoding a second epitope of a second antigen, wherein the first and second epitopes are part of a HIF-1α peptide, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0132] The nucleic acid sequence encoding the epitope of the following proteins is different from those listed herein: CD105, HIF1α, MDM2, Yb1, SOX-2, HER-2, IGFBP2, IGF-1R and CDH3. In some cases, nucleic acid sequences that are more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or more than 50% homologous to those described herein can be used in the compositions described herein.

[0133] In some cases, the compositions described herein may include a composition comprising: a first epitope of a first antigen expressed by cells associated with breast cancer; and a second epitope of a second antigen expressed by cells associated with breast cancer.

[0134] In some cases, the composition may comprise: at least a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may comprise: at least a first epitope of a first antigen, and at least a second epitope of a second antigen, wherein the first and second epitopes are independently selected from CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, at least the first epitope of peptide CD105 is selected from the group consisting of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO: 6); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence QNGTWPREVLLVLSVNS SVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: 1); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence TVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 8); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: NO:9); or an amino acid sequence that has at least 90% sequence identity to the amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSK REPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO:10). In some cases, at least the first epitope of peptide Yb-1 is selected from the group consisting of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence EDVFVHQTAIKKNNPRK (SEQ ID NO:14); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence YRRNFNYRRRRPEN (SEQ ID NO:15); or an amino acid sequence that has at least 90% sequence identity to the amino acid sequence GVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN (SEQ ID NO:16).In some cases, at least the first epitope of peptide SOX-2 is selected from the group consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSV (SEQ ID NO: 20). In some cases, at least the first epitope of the peptide CDH3 is selected from the group consisting of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 25); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of LKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 26); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of AMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKES (SEQ ID NO: NO: 27) has at least 90% sequence identity; or an amino acid sequence that has at least 90% sequence identity to the amino acid sequence VMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKET (SEQ ID NO: 28).In some cases, at least the first epitope of peptide MDM-2 is selected from the group consisting of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of TYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSV (SEQ ID NO: 32); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of TYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLS (SEQ ID NO: 33); or an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of IYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPS (SEQ ID NO: 34).

[0135] In some cases, the compositions described herein include an amino acid sequence of a fusion peptide of five (or more) epitopes selected from the group consisting of an amino acid sequence that is the same as the amino acid sequence MAVPMQLSCSRQNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTELRSTGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLRSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID an amino acid sequence having at least 90% sequence identity to the amino acid sequence of MAVPMTVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO:40);The amino acid sequence is consistent with the amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGS PTYSMSYSQQGTPGMALGSMGSVRSQLAMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKESRSAGETYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLSRS(SEQ ID NO: 41) having at least 90% sequence identity; or an amino acid sequence having at least 90% sequence identity to the amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLVMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKETRSAGEIYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPSRS (SEQ ID NO: 42);

[0136] The compositions described herein also include compositions comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from IGFBP-2, HER-2, or IGF-1R, wherein the first nucleotide sequence is located in a plasmid. In some cases, the composition may comprise: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen; and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second epitope of a second antigen, wherein the first and second epitopes are independently selected from IGFBP-2, HER-2, or IGF-1R, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids. In some cases, at least the first epitope of the peptide IGFBP-2 is selected from the group consisting of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of NHVDSTMNMLGGGGS (SEQ ID NO: 46); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of ELAVFREKVTEQHRQ (SEQ ID NO: 47); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of LGLEEPKKLRPPPAR (SEQ ID NO: 48); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of DQVLERISTMRLPDE (SEQ ID NO: 49); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of GPLEHLYSLHIPNCD (SEQ ID NO: 50); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of KHGLYNLKQCKMSLN (SEQ ID NO: an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of MLPRVGCPALPLPPPPLLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVE (SEQ ID NO: 54);an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVE (SEQ ID NO: 55); or an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACG PPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELP LKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVE (SEQ ID NO: 56). In some cases, at least the first epitope of the peptide HER-2 is selected from the group consisting of a nucleotide sequence encoding an amino acid sequence that is identical to the amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVY KGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESIL RRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL(SEQ ID NO: 60) has at least 90% sequence identity;The nucleotide sequence encoding the amino acid sequence is the same as the amino acid sequence TMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 61) has at least 90% sequence identity; or a nucleotide sequence encoding an amino acid sequence, the amino acid sequence being the same as the amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAA In some cases, the nucleic acid sequence encoding the epitope of the peptide IGF-1R is selected from the group consisting of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence DYRSYRFPKLTVITE (SEQ ID NO: 66); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence IRGWKLFYNYALVIF (SEQ ID NO: 67);an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of VVTGYVKIRHSHALV (SEQ ID NO: 68); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of FFYVQAKTGYENFIH (SEQ ID NO: 69); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of LIIALPVAVLLIVGG (SEQ ID NO: 70); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of LVIMLYVFHRKRNNS (SEQ ID NO: 71); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of NCHHVVRLLGVVSQG (SEQ ID NO: 72); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence of WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELM RMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKN ERALP (SEQ ID NO: 73); an amino acid sequence that has at least 90% sequence identity to the amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 74); or an amino acid sequence that has at least 90% sequence identity to the amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 75).

[0137] The composition described herein may further comprise a nucleic acid sequence encoding a fusion protein of three (or more) epitopes selected from the group consisting of: an amino acid sequence having at least 90% sequence identity to the amino acid sequence (SEQ ID NO: 79);An amino acid sequence that has at least 90% sequence identity with the amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 80);or an amino acid sequence that has at least 90% sequence identity with the amino acid sequence (SEQ ID NO: 81). ;

[0138] In some cases, the composition comprises a first and a second epitope independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition further comprises a third epitope, wherein the first, second, and third epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition further comprises a third and a fourth epitope, wherein the first, second, third, and fourth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition further comprises a third, fourth, and fifth epitope, wherein the first, second, third, fourth, and fifth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2.

[0139] In some cases, the composition comprises a first and a second epitope independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R. In some cases, the composition further comprises a third epitope, the first, second, and third epitopes independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R.

[0140] In some cases, the composition may comprise: at least a first epitope of a first antigen, the first epitope being part of a peptide derived from HIF-1α. In some cases, the composition may comprise: at least a first epitope of a first antigen, at least a second epitope of a second antigen, the first and second epitopes being derived from HIF-1α.

[0141] In some cases, the composition can include a nucleotide sequence encoding an epitope of the peptide HIF-1α selected from the group consisting of: a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence DSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTK THHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYN (SEQ ID NO: 82); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence SDNVNKYMGLTQFELTGHSVFDFTHP (SEQ ID NO: 83); and a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence GGYVWVETQATVIYNTKNSQ (SEQ ID NO: 84).

[0142] In some cases, the composition can include at least a first epitope of a peptide HIF-1α selected from the group consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence DSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTK THHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYN (SEQ ID NO: 82); an amino acid sequence having at least 90% sequence identity to the amino acid sequence SDNVNKYMGLTQFELTGHSVFDFTHP (SEQ ID NO: 83); and an amino acid sequence having at least 90% sequence identity to the amino acid sequence GGYVWVETQATVIYNTKNSQ (SEQ ID NO: 84).

[0143] The amino acid sequence encoding an epitope selected from the group consisting of CD105, HIF1α, MDM2, Yb1, SOX-2, HER-2, IGFBP2, IGF-1R, and CDH3 is different from those listed herein. In some cases, amino acid sequences that are more than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or more than 50% homologous to those described herein can be used in the compositions described herein.

[0144] In some cases, the first amino acid sequence is selected from the group consisting of: human, mouse, and rat. In some cases, the second amino acid sequence is selected from the group consisting of: human, mouse, and rat.

[0145] In some cases, the first and second nucleic acid sequences are located on a first plasmid. In some cases, the second nucleic acid sequence is located on a second plasmid. In some cases, the amino acid sequences of the first and second epi-positions are separated by a linker amino acid sequence. In some cases, the amino acid sequence of the first epi-position is adjacent to the amino acid sequence of the second epi-position.

[0146] In some cases, the immune response is a Type 1 immune response. In some cases, the immune response is characterized by a ratio of Type I cytokine production to Type II cytokine production that is greater than 1. In some cases, the immune response is characterized by a ratio of Type I cytokine production to Type II cytokine production that is less than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production that is greater than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production that is less than 1.

[0147] In some cases, the composition is administered to a subject. In some cases, the subject is in need of administration of the composition. In some cases, the composition effectively elicits an immune response in the subject. In some cases, the composition effectively eliminates a variety of cells associated with breast cancer in the subject. In some cases, the composition can be used to prevent the growth of cells associated with breast cancer in the subject.

[0148] In some cases, the subject is selected from the group consisting of a human with breast cancer, a mouse with breast cancer, and a rat with breast cancer. In some cases, the subject is selected from the group consisting of a human without breast cancer, a mouse without breast cancer, and a rat without breast cancer.

[0149] In some cases, the cell associated with breast cancer is selected from the group consisting of a breast cell expressing an aberrant signature, a pre-neoplastic breast cell, a breast cancer cell, a pre-invasive breast cancer cell, a breast cancer stem cell, an epithelial cell, a mesenchymal cell, a somatic cell, or a combination thereof.

[0150] In some cases, at least the first epitope is contained in a pharmaceutical composition. In some cases, at least the first epitope is contained in a pharmaceutical composition that also includes a pharmaceutical carrier. In some cases, at least the first epitope is contained in a pharmaceutical composition that also includes a pharmaceutical carrier and an adjuvant. In some cases, at least the first epitope is contained in a pharmaceutical composition that also includes an adjuvant. In some cases, the composition further includes an adjuvant and a pharmaceutical carrier. In some cases, the adjuvant is GM-CSF.

[0151] In some cases, the composition may be administered to the subject. In some cases, the subject has such a need. In some cases, provided herein is a method for preventing breast cancer in a subject, such that the method comprises administering a composition as described herein to the subject. In some cases, provided herein is a method for treating breast cancer in a subject, such that the method comprises administering a composition as described herein to the subject. In some cases, administration further comprises delivering at least one dose of the composition as described herein to the subject. In some cases, administration further comprises delivering the composition as described herein to the subject by subcutaneous injection, intradermal injection, intramuscular injection, intravascular injection, topical application, or inhalation. In some cases, the subject is selected from the group consisting of: a human with breast cancer, a mouse with breast cancer, and a rat with breast cancer. In some cases, the subject is selected from the group consisting of: a human without breast cancer, a mouse without breast cancer, and a rat without breast cancer.

[0152] The present invention also provides kits for preparing the compositions described herein, the kits comprising instructions for preparing the compositions. The present invention also provides kits for administering the compositions described herein, the kits comprising instructions for administering the compositions.

[0153] Compositions comprising an epitope selected from survivin, HIF-1α, IGFBP-2, and IGF-1R

[0154] The compositions described herein comprise a plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 70% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87, and an excipient. The composition may comprise a plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 80% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The composition may comprise a plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 90% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The composition may comprise a plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 95% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The composition may comprise a plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 99% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The composition may comprise a plasmid comprising at least one nucleotide sequence encoding a polypeptide having 100% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The composition may comprise a plasmid comprising at least one nucleotide sequence encoding a polypeptide having 100% sequence identity to the entire length of an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87.

[0155] Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 70% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 80% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 90% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 95% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 99% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 100% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85.

[0156] Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 70% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 80% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 90% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 95% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 99% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. Sometimes, at least one nucleotide sequence encodes a polypeptide having at least 100% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87.

[0157] In some cases, the composition comprises an isolated plasmid comprising at least four nucleotide sequences. Sometimes, each of the at least four nucleotide sequences independently encodes a polypeptide having at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to an epitope sequence selected from SEQ ID NOs: 54, 73, 85, and 87. Sometimes, the isolated plasmid may comprise four nucleotide sequences. Sometimes, each of the four nucleotide sequences independently encodes a polypeptide having at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to an epitope sequence selected from SEQ ID NOs: 54, 73, 85, and 87. Additionally, each of the four nucleotide sequences may encode a different polypeptide. Sometimes, the different polypeptides can each have at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to an epitope sequence selected from SEQ ID NOs: 54, 73, 85, and 87.

[0158] Sometimes, one of the four nucleotide sequences may encode a polypeptide having at least 70% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. One of the four nucleotide sequences may encode a polypeptide having at least 80% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. One of the four nucleotide sequences may encode a polypeptide having at least 90% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. One of the four nucleotide sequences may encode a polypeptide having at least 95% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85. One of the four nucleotide sequences may encode a polypeptide having at least 100% sequence identity to at least 20 contiguous amino acids of SEQ ID NO:85.

[0159] Sometimes, one of the four nucleotide sequences may encode a polypeptide having at least 70% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. One of the four nucleotide sequences may encode a polypeptide having at least 80% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. One of the four nucleotide sequences may encode a polypeptide having at least 90% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. One of the four nucleotide sequences may encode a polypeptide having at least 95% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87. One of the four nucleotide sequences may encode a polypeptide having at least 100% sequence identity to at least 60 contiguous amino acids of SEQ ID NO:87.

[0160] In some cases, the four nucleotide sequences are arranged in tandem within the plasmid. The four nucleotide sequences can be separated by a linker nucleic acid sequence. The length of the linker nucleic acid sequence can be from about 1 to about 150, from about 5 to about 100, or from about 10 to about 50 nucleic acids. In some cases, the nucleic acid can encode one or more amino acid residues. Sometimes, the amino acid sequence length of the linker can be from about 1 to about 50, or from about 5 to about 25 amino acid residues. Sometimes, the linker can include, for example, Figure 14 The linker is underlined (SEQ ID NO: 14).

[0161] Sometimes, a composition can also include at least one additional isolated plasmid. Sometimes, a composition can also include at least 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 or more additional isolated plasmids.

[0162] In some cases, at least one other isolated plasmid comprises a nucleotide sequence encoding a polypeptide having at least 70% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. At least one other isolated plasmid may comprise a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. At least one other isolated plasmid may comprise a nucleotide sequence encoding a polypeptide having at least 90% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. At least one other isolated plasmid may comprise a nucleotide sequence encoding a polypeptide having at least 95% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. At least one other isolated plasmid may comprise a nucleotide sequence encoding a polypeptide having at least 99% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. At least one other isolated plasmid can comprise a nucleotide sequence encoding a polypeptide having 100% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87.

[0163] Sometimes, the compositions described herein may include a composition comprising a plasmid comprising a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to SEQ ID NO: 89 and an excipient. The composition may include a plasmid comprising a nucleotide sequence encoding a polypeptide having at least 90% sequence identity to SEQ ID NO: 89. The composition may include a plasmid comprising a nucleotide sequence encoding a polypeptide having at least 95% sequence identity to SEQ ID NO: 89. The composition may include a plasmid comprising a nucleotide sequence encoding a polypeptide having 100% sequence identity to SEQ ID NO: 89. The composition may include a plasmid comprising a nucleotide sequence encoding a polypeptide having 100% sequence identity to SEQ ID NO: 89.

[0164] The compositions described herein may include a composition comprising a polypeptide having at least 80% sequence identity to SEQ ID NO: 89. The composition may include a polypeptide having at least 90% sequence identity to SEQ ID NO: 89. The composition may include a polypeptide having at least 95% sequence identity to SEQ ID NO: 89. The composition may include a polypeptide having 100% sequence identity to SEQ ID NO: 89. The composition may include a polypeptide having 100% sequence identity to SEQ ID NO: 89.

[0165] The composition can be formulated for treating breast cancer or ovarian cancer in a subject. The breast cancer can be recurrent or refractory breast cancer. The ovarian cancer can be recurrent or refractory ovarian cancer. The breast cancer can be metastatic breast cancer. The ovarian cancer can be metastatic ovarian cancer.

[0166] The composition can elicit an immune response. The immune response can be characterized by a ratio of type I cytokine production to type II cytokine production that is greater than 1. The immune response can be characterized by a ratio of type I cytokine production to type II cytokine production that is less than 1. The immune response can be characterized by a ratio of IFN-γ production to IL-10 production that is greater than 1. The immune response can be characterized by a ratio of IFN-γ production to IL-10 production that is less than 1.

[0167] Sometimes, the composition can also include an adjuvant. Sometimes, the adjuvant is GM-CSF.

[0168] The composition may further comprise an excipient. The excipient may be a pharmaceutically acceptable carrier.

[0169] Typically, the compositions will be formulated for subcutaneous, intramuscular, or intradermal administration.

[0170] The present invention also provides kits for preparing the compositions described herein, the kits comprising instructions for preparing the compositions. The present invention also provides kits for administering the compositions described herein, the kits comprising instructions for administering the compositions.

[0171] Plasmids for pharmaceutical compositions

[0172] In some cases, epitopes can be derived from human proteins that can be used directly in peptide-based vaccines. In other cases, epitopes can be derived from human proteins and the encoding nucleic acid sequence encoding the epitope can be integrated into a nucleic acid construct designed to induce epitope expression in a subject after administration. For example, an epitope encoded from a nucleic acid construct can allow an immune response to at least one (individual) epitope to be produced, amplified, weakened, suppressed or eliminated for a specific group of proteins (e.g., from proteins). In some cases, peptides or nucleic acid constructs can be optimized to induce, amplify or produce a Th1 immune response based on protein or plasmid immunity. In some cases, the epitope can be an extended Th1 epitope. In other cases, peptides or nucleic acid constructs can be optimized to suppress, weaken or eliminate pathogenicity responses in a subject (e.g., human or animal) in need thereof based on protein or plasmid immunity.

[0173] The compositions described herein may include plasmids containing nucleic acid sequences to express at least one epitope(s) in a subject after administration of the composition (e.g., a vaccine). Any plasmid backbone (e.g., a vector) known to those of ordinary skill in the art for pharmaceutical applications of expressing nucleic acids can be used in the compositions described herein. In some cases, a commercially available plasmid backbone can be used. For example, plasmid pUMVC3 can be used. In some cases, a commercially available plasmid backbone can be modified, mutated, engineered, or cloned prior to use. In other cases, a non-commercially available plasmid backbone can be used.

[0174] Prior to insertion of the nucleic acid sequence for at least one epitope(s), the length of the plasmid backbone may be less than about 500 bp, about 1.0 kB, about 1.2 kB, about 1.4 kB, about 1.6 kB, about 1.8 kB, about 2.0 kB, about 2.2 kB, about 2.4 kB, about 2.6 kB, about 2.8 kB, about 3.0 kB, about 3.2 kB, about 3.4 kB, about 3.6 kB, about 3.8 kB, about 4.0 kB, about 4.2 kB, about 4.4 kB, about 4.6 kB, about 4.8 kB, about 5. 0kB, about 5.2kB, about 5.4kB, about 5.6kB, about 5.8kB, about 6.0kB, about 6.2kB, about 6.4kB, about 6.6kB, about 6.8kB, about 7.0kB, about 7.2kB, about 7.4kB, about 7.6kB, about 7.8kB, about 8.0kB, about 8.2kB, about 8.4kB, about 8.6kB, about 8.8kB, about 9.0kB, about 9.2kB, about 9.4kB, about 9.6kB, about 9.8kB, about 10.0kB, about 10.2kB, about 10.4kB, about 10.6kB, about 10.8kB, about 11.0kB, about 11.2kB, about 11.4kB, about 11.6kB, about 11.8kB, about 12.0kB, about 12.2kB, about 12.4kB, about 12.6kB, about 12.8kB, about 13.0kB, about 13.2kB, about 13.4kB, about 13.6kB, about 13.8kB, about 14kB, about 14.5kB, about 15kB, about 15.5kB, kB, about 16 kB, about 16.5 kB, about 17 kB, about 17.5 kB, about 18 kB, about 18.5 kB, about 19 kB, about 19.5 kB, about 20 kB, about 30 kB, about 40 kB, about 50 kB, about 60 kB, about 70 kB, about 80 kB, about 90 kB, about 100 kB, about 110 kB, about 120 kB, about 130 kB, about 140 kB, about 150 kB, about 160 kB, about 170 kB, about 180 kB, about 190 kB or about 200 kB. In an exemplary case, the plasmid is about 4 kB in length before the nucleic acid sequence encoding at least one epitope(s) is added.

[0175] In some cases, the compositions described herein may include one plasmid. In other cases, the compositions described herein may include more than one plasmid. For example, the compositions described herein may include 2 plasmids, 3 plasmids, 4 plasmids, 5 plasmids, 6 plasmids, 7 plasmids, 8 plasmids, 9 plasmids, 10 plasmids, 11 plasmids, 12 plasmids, 13 plasmids, 14 plasmids, 15 plasmids, 16 plasmids, 17 plasmids, 18 plasmids, 19 plasmids, 20 plasmids, or more than 20 plasmids.

[0176] In some cases, the nucleic acid encoding at least one epitope(s) of the plasmid can be a deoxyribonucleic acid. For example, the deoxyribonucleic acid can be single-stranded, double-stranded, or complementary. The deoxyribonucleic acid can be from genomic, mitochondrial, or plasmid deoxyribonucleic acid. In other cases, the nucleic acid of the plasmid can be an ribonucleic acid. For example, the ribonucleic acid can be single-stranded or double-stranded. In some cases, the ribonucleic acid can be a micro, antisense, short hairpin, small interfering, messenger, transfer, ribosomal RNA, etc. In some cases, the nucleic acid of the plasmid can be a portion of a deoxyribonucleic acid or a portion of an ribonucleic acid.

[0177] The nucleic acid encoding at least one epitope(s) of the plasmid can be derived from any species such that the epitope expressed from the nucleic acid generates an immune response in a subject. In some cases, the subject can be a rodent, a non-human primate, or a human. The nucleic acid encoding the epitope of the plasmid can be isolated from any nucleic acid source using methods and techniques known to those skilled in the art. The nucleic acid encoding the epitope of the plasmid can be cloned into the plasmid backbone using methods and techniques known to those skilled in the art.

[0178] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence of human CD105 can be used to express CD105 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence of non-human CD105 can be used to express CD105 in a human.

[0179] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the natural nucleic acid sequence of CD105 in the genome of a species can be used to express CD105 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the natural nucleic acid sequence of CD105 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and can be used to express CD105 in a subject.

[0180] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence of human CD105 can be used to express CD105 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence of non-human CD105 can be used to express CD105 in a human.

[0181] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the natural nucleic acid sequence of CD105 in the genome of a species can be used to express CD105 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the natural nucleic acid sequence of CD105 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and can be used to express CD105 in a subject.

[0182] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human HIF-1A can be used to express HIF-1A in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human HIF-1A can be used to express HIF-1A in a human.

[0183] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of HIF-1A in the genome of a species can be used to express HIF-1A in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of HIF-1A in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express HIF-1A in a subject.

[0184] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human HIF-1A can be used to express HIF-1A in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human HIF-1A can be used to express HIF-1A in a human.

[0185] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of HIF-1A in the genome of a species can be used to express HIF-1A in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of HIF-1A in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express HIF-1A in a subject.

[0186] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human MDM2 can be used to express MDM2 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human MDM2 can be used to express MDM2 in a human.

[0187] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of MDM2 in the genome of a species can be used to express MDM2 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of MDM2 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express MDM2 in a subject.

[0188] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human MDM2 can be used to express MDM2 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human MDM2 can be used to express MDM2 in a human.

[0189] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of MDM2 in the genome of a species can be used to express MDM2 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of MDM2 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express MDM2 in a subject.

[0190] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human Yb-1 can be used to express Yb-1 in humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human Yb-1 can be used to express Yb-1 in humans.

[0191] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of Yb-1 in the genome of a species can be used to express Yb-1 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of Yb-1 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express Yb-1 in the subject.

[0192] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human Yb-1 can be used to express Yb-1 in humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human Yb-1 can be used to express Yb-1 in humans.

[0193] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of Yb-1 in the genome of a species can be used to express Yb-1 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of Yb-1 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express Yb-1 in the subject.

[0194] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human SOX-2 can be used to express SOX-2 in humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human SOX-2 can be used to express SOX-2 in humans.

[0195] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of SOX-2 in the genome of a species can be used to express SOX-2 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of SOX-2 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express SOX-2 in a subject.

[0196] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human SOX-2 can be used to express SOX-2 in humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human SOX-2 can be used to express SOX-2 in humans.

[0197] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of SOX-2 in the genome of a species can be used to express SOX-2 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of SOX-2 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express SOX-2 in a subject.

[0198] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence from a human HER-2 can be used to express HER-2 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence from a non-human HER-2 can be used to express HER-2 in a human.

[0199] In some cases, the nucleic acid sequence for expressing the antigenic epitope can be a wild-type nucleic acid sequence. For example, the natural nucleic acid sequence of HER-2 in the species genome can be used to express HER-2 in a subject. In other cases, the nucleic acid sequence for expressing the epitope can be a synthetic nucleic acid sequence. For example, the natural nucleic acid sequence of HER-2 in the subject genome can be modified using molecular techniques known to those of ordinary skill in the art and can be used to express HER-2 in a subject.

[0200] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence from a human HER-2 can be used to express HER-2 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence for the subject. For example, a nucleic acid sequence from a non-human HER-2 can be used to express HER-2 in a human.

[0201] In some cases, the nucleic acid sequence for expressing the antigenic epitope can be a wild-type nucleic acid sequence. For example, the natural nucleic acid sequence of HER-2 in the species genome can be used to express HER-2 in a subject. In other cases, the nucleic acid sequence for expressing the epitope can be a synthetic nucleic acid sequence. For example, the natural nucleic acid sequence of HER-2 in the subject genome can be modified using molecular techniques known to those of ordinary skill in the art and can be used to express HER-2 in a subject.

[0202] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human IGFBP2 can be used to express IGFBP2 in humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human IGFBP2 can be used to express IGFBP2 in humans.

[0203] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGFBP2 in the genome of a species can be used to express IGFBP2 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of IGFBP2 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express IGFBP2 in a subject.

[0204] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human IGFBP2 can be used to express IGFBP2 in humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human IGFBP2 can be used to express IGFBP2 in humans.

[0205] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGFBP2 in the genome of a species can be used to express IGFBP2 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of IGFBP2 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express IGFBP2 in a subject.

[0206] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from a human IGF-1R can be used to express IGF-1R in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from a non-human IGF-1R can be used to express IGF-1R in a human.

[0207] In some cases, the nucleic acid sequence used to express an antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a species can be used to express IGF-1R in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express IGF-1R in a subject.

[0208] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from a human IGF-1R can be used to express IGF-1R in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from a non-human IGF-1R can be used to express IGF-1R in a human.

[0209] In some cases, the nucleic acid sequence used to express an antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a species can be used to express IGF-1R in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express IGF-1R in a subject.

[0210] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human CDH3 can be used to express CDH3 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human CDH3 can be used to express CDH3 in a human.

[0211] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of CDH3 in the genome of a species can be used to express CDH3 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of CDH3 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express CDH3 in a subject.

[0212] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human CDH3 can be used to express CDH3 in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human CDH3 can be used to express CDH3 in a human.

[0213] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of CDH3 in the genome of a species can be used to express CDH3 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of CDH3 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express CDH3 in a subject.

[0214] The compositions described herein include compositions comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen expressed by cells associated with breast cancer; and a second nucleotide sequence encoding a second epitope of a second antigen expressed by cells associated with breast cancer, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids. In some cases, the composition may include nucleic acids encoding epitopes from the following proteins: CD105, HIF1α, MDM2, Yb-1, SOX-2, HER-2, IGFBP2, IGF-1R, and CDH3.

[0215] In some cases, the composition may include a composition comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from CD105, Yb-1, SOX-2, CDH3 or MDM2, wherein the first nucleotide sequence is located in a plasmid. In other cases, the composition may include a composition comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen; and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second epitope of a second antigen, wherein the first and second epitopes are independently selected from CD105, Yb-1, SOX-2, CDH3 or MDM2, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0216] In some cases, the composition may include an epitope and nucleic acid encoding an epitope from the following proteins: CD105, MDM2, Yb-1, SOX-2, and CDH3. In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide CD105 selected from the group consisting of: ... NO: 2) a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence ACCGTGTTCATGCGCCTGAACATCATCTCCCCCGACCTGTCCGGCTGCACCTCCAAGGGCCTGGTGCTGCCCGCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCTCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO:3) nucleotide sequences that have at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;With the nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATC TACTCCCACACCCGCGGCCTCCAAGCGCGAGCCCGTGGTGCCGTGGCCGCCCCCGCCTCCTCCGAGTCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC(SEQ ID NO: 4) a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO:5) a nucleotide sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO:6); a nucleotide sequence that encodes an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO:6);A nucleotide sequence encoding an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence QNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNS SLVTFQEPPGVNTTEL (SEQ ID NO: 1); a nucleotide sequence encoding an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence TVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 2) NO: 8) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; a nucleotide sequence encoding an amino acid sequence that is identical to the amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSK REPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 8); NO:9) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO:10). In some cases, the composition can include a nucleic acid sequence encoding an epitope of peptide Yb-1 selected from the group consisting of a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence GGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACC GCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAAC (SEQ ID NO: 11);Nucleotide sequences having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 12); nucleotide sequences having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 12); NO: 12) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence EDVFVHQTAIKKNNPRK (SEQ ID NO: 14); a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence YRRNFNYRRRRPEN (SEQ ID NO: 15) or a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence YRRNFNYRRRRPEN (SEQ ID NO: 16) or a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, ID NO: 15) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;or a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence GVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN (SEQ ID NO: 16). In some cases, the composition can include a nucleic acid sequence encoding an epitope of the peptide SOX-2 selected from the group consisting of ... NO: 17) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 18) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 18);or a nucleotide sequence encoding an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSV (SEQ ID NO: 20). In some cases, the composition may include a nucleic acid sequence encoding a peptide CDH3 selected from the group consisting of: and the nucleotide sequence AGGTCACTGAAGGAAAGGAATCCATTGAAAATCTTCCCATCCAAACGTATCTTACGA AGACACA AGAGAGATTGGGTGGTTGCTCCA ATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 21) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence TTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 22) nucleotide sequences having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;With the nucleotide sequence GCCATGCACTCCCCCCCCCACCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCCATCTTCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGATCTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCGAGGGGCGTGTTCACCATCGAGAAGGAGTCC(SEQ ID NO: 23) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence GTGATGAACTCCCCCCCCTCCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTCCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGCTGTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGACC (SEQ ID ... NO: 24) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; a nucleotide sequence encoding an amino acid sequence that is identical to the amino acid sequence RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 24); a nucleotide sequence encoding an amino acid sequence that is identical to the amino acid sequence RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: NO:25) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence LKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO:26);A nucleotide sequence encoding an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of AMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKES (SEQ ID NO: 27); or a nucleotide sequence encoding an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of VMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKET (SEQ ID NO: NO: 28) has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the composition can include a nucleic acid sequence encoding an epitope of peptide MDM2 selected from the group consisting of a nucleotide sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence ACCTACACCATGAAGGAGGTGCTGTTCTACCTGGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACCTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAaATCTACACCATGATCTACCGCAACCTGGTGGTGGTGAACCAGCAGGAGTCCTCCGACTCCGGCACCTCCGTGTCC (SEQ ID NO: 29);With the nucleotide sequence ACCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGCCGTGTCCCAGCAGGACTCCGGCACCTCCCTGTCC (SEQ ID NO: 30) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence ATCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGTGGTGTCCCAGCAGGACTCCGGCACCTCCCCCTCC (SEQ ID NO: 31) a nucleotide sequence encoding an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of TYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSV (SEQ ID NO:32);A nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence TYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLS (SEQ ID NO: 33); or a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence TYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVAVSQQDSGTSPS (SEQ ID NO: 34). NO: 34) has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.;

[0217]

[0218]

[0219] In some cases, the composition may include a first and a second epitope independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may include a third epitope, wherein the first, second, and third epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may include a third and a fourth epitope, wherein the first, second, third, and fourth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may include a third, fourth, and fifth epitope, wherein the first, second, third, fourth, and fifth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2.

[0220] In some cases, the composition can include a first and a second epitope independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R. In some cases, the composition can include a third epitope, the first, second, and third epitopes independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R.

[0221] In some cases, the composition can be administered to a subject. In some cases, the subject needs to be administered the composition. In some cases, the composition effectively elicits an immune response in the subject. In some cases, the composition effectively eliminates a variety of cells associated with breast cancer in the subject. In some cases, the composition can be used to prevent the growth of cells associated with breast cancer in the subject.

[0222] In some cases, the first and second nucleic acid sequences are located on a first plasmid. In some cases, the second nucleic acid sequence is located on a second plasmid.

[0223] In some cases, the cell associated with breast cancer is selected from the group consisting of a breast cell expressing an aberrant signature, a pre-neoplastic breast cell, a breast cancer cell, a pre-invasive breast cancer cell, a breast cancer stem cell, an epithelial cell, a mesenchymal cell, a somatic cell, or a combination thereof.

[0224] In some cases, the first and second nucleic acid sequences are purified to at least 70% purity. In some cases, the first and second nucleic acid sequences are located on a first plasmid and are separated by a linker nucleic acid sequence. In some cases, the first nucleic acid sequence is adjacent to the second nucleic acid sequence on the first plasmid.

[0225] In some cases, at least the first plasmid is contained in a pharmaceutical composition. In some cases, at least the first plasmid is contained in a pharmaceutical composition that also includes a pharmaceutical carrier. In some cases, at least the first plasmid is contained in a pharmaceutical composition that also includes a pharmaceutical carrier and an adjuvant. In some cases, at least the first plasmid is contained in a pharmaceutical composition that also includes an adjuvant. In some cases, the composition further includes an adjuvant and a pharmaceutically acceptable carrier. In some cases, the adjuvant is GM-CSF.

[0226] In some cases, the subject is selected from: a human with breast cancer, a mouse with breast cancer, or a rat with breast cancer. In some cases, the subject is selected from: a human without breast cancer, a mouse without breast cancer, or a rat without breast cancer.

[0227] In some cases, the immune response is a Type 1 immune response. In some cases, the first nucleic acid sequence is a species selected from humans, mice, or rats. In some cases, the second nucleic acid sequence is a species selected from humans, mice, or rats. In some cases, the immune response is characterized by a ratio of type I cytokine production to type II cytokine production greater than 1. In some cases, the immune response is characterized by a ratio of type I cytokine production to type II cytokine production less than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production greater than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production less than 1.

[0228] In some cases, the composition includes a composition comprising: a first plasmid comprising a first nucleotide sequence, the first nucleotide sequence encoding a first epitope of a first antigen, the first epitope being part of a HIF-1α peptide, wherein the first nucleotide sequence is located in a plasmid. In other cases, the composition includes a composition comprising: a first plasmid comprising a first nucleotide sequence, the first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence, the second nucleotide sequence encoding a second epitope of a second antigen, wherein the first and second epitopes are part of a HIF-1α peptide, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

[0229] The nucleic acid sequence encoding the epitope of the following protein is different from those listed herein: CD105, HIF1α, MDM2, Yb-1, SOX-2, HER-2, IGFBP2, IGF-1R and CDH3. In some cases, nucleic acid sequences more than 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or more than 50% homologous to those described herein can be used in the compositions described herein.

[0230] In some cases, the compositions described herein may include a composition comprising: a first epitope of a first antigen expressed by cells associated with breast cancer; and a second epitope of a second antigen expressed by cells associated with breast cancer.

[0231] In some cases, the composition may comprise: at least a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition may comprise: at least a first epitope of a first antigen, and at least a second epitope of a second antigen, wherein the first and second epitopes are independently selected from CD105, Yb-1, SOX-2, CDH3, or MDM2.In some cases, at least the first epitope of the peptide CD105 is selected from the group consisting of an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO: 6); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence QNGTWPREVLLVLSVNS SVFLHLQALGIPLHLAYNS SLVTFQEPPGVNTTEL (SEQ ID NO: NO: 1) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of TVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 8); an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSK REPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO:9) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSK REPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO:10).In some cases, at least the first epitope of peptide Yb-1 is selected from the group consisting of an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of EDVFVHQTAIKKNNPRK (SEQ ID NO: 14); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of YRRNFNYRRRRPEN (SEQ ID NO: 15); or an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of GVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN (SEQ ID NO: In some cases, at least the first epitope of the peptide SOX-2 is selected from the group consisting of an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSV (SEQ ID NO: 20).In some cases, at least the first epitope of the peptide CDH3 is selected from the group consisting of an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 25); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of LKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: NO:26) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence AMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKES (SEQ ID NO:27); or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence AMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKES (SEQ ID NO:28) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence VMNSPP SRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKET (SEQ ID NO: 28) has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.In some cases, at least the first epitope of peptide MDM-2 is selected from the group consisting of an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence TYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSV (SEQ ID NO: 32); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence TYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSV (SEQ ID NO: 33); NO: 33) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence IYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPS (SEQ ID NO: 34).

[0232] In some cases, the compositions described herein include an amino acid sequence of a fusion peptide of five (or more) epitopes selected from the group consisting of an amino acid sequence that is the same as the amino acid sequence MAVPMQLSCSRQNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTELRSTGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLRSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 39) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence that is identical to the amino acid sequence MAVPMTVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN TRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDT KIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVRS(SEQ ID NO: 40) has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;The amino acid sequence is consistent with the amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGS PTYSMSYSQQGTPGMALGSMGSVRSQLAMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKESRSAGETYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLSRS(SEQ ID NO:41) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or an amino acid sequence that is identical to the amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQ NTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLVMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDR GTKLFYSITGPGADSPPEGVFTIEKETRSAGEIYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPSRS(SEQ ID NO: 42) have at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. ;

[0233] The compositions described herein also include compositions comprising: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen, wherein the first epitope is part of a peptide selected from IGFBP-2, HER-2, or IGF-1R, wherein the first nucleotide sequence is located in a plasmid. In some cases, the composition may comprise: a first plasmid comprising a first nucleotide sequence, wherein the first nucleotide sequence encodes a first epitope of a first antigen; and a second nucleotide sequence, wherein the second nucleotide sequence encodes a second epitope of a second antigen, wherein the first and second epitopes are independently selected from IGFBP-2, HER-2, or IGF-1R, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids. In some cases, at least the first epitope of the peptide IGFBP-2 is selected from the group consisting of an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of NHVDSTMNMLGGGGS (SEQ ID NO: 46); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ELAVFREKVTEQHRQ (SEQ ID NO: 47); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of LGLEEPKKLRPPPAR (SEQ ID NO: NO:48) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence DQVLERISTMRLPDE (SEQ ID NO:49); an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence GPLEHLYSLHIPNCD (SEQ ID NO: NO:50) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence KHGLYNLKQCKMSLN (SEQ ID NO:51);an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of PECHLFYNEQQEARG (SEQ ID NO: 53); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVE (SEQ ID NO: ID NO:54) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVE (SEQ ID NO:55);or an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVE (SEQ ID NO: 56). In some cases, at least the first epitope of the peptide HER-2 is selected from the group consisting of a nucleotide sequence encoding an amino acid sequence that is identical to the amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRD LAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTID VYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL(SEQ IDNO: 60) has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;The nucleotide sequence encoding the amino acid sequence is the same as the amino acid sequence TMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 61) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;or a nucleotide sequence encoding an amino acid sequence, the amino acid sequence being the same as the amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO:62) has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the composition can include a nucleic acid sequence encoding an epitope of the peptide IGF-1R selected from the group consisting of an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence DYRSYRFPKLTVITE (SEQ ID NO: 66); an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence IRGWKLFYNYALVIF (SEQ ID NO: 67); an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence VVTGYVKIRHSHALV (SEQ ID NO: NO:68) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence FFYVQAKTGYENFIH (SEQ ID NO:69);an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of LIIALPVAVLLIVGG (SEQ ID NO: 70); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of LVIMLYVFHRKRNNS (SEQ ID NO: 71); an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of NCHHVVRLLGVVSQG (SEQ ID NO: NO: 72) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALP (SEQ ID NO: 73) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO:74) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANER ALP (SEQ ID NO:75).

[0234] The compositions described herein may further comprise a nucleic acid sequence encoding a fusion protein of three (or more) epitopes selected from the group consisting of an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence (SEQ ID NO: 79);An amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 80);or an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 81);

[0235] In some cases, the composition comprises a first and a second epitope independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition further comprises a third epitope, wherein the first, second, and third epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition further comprises a third and a fourth epitope, wherein the first, second, third, and fourth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2. In some cases, the composition further comprises a third, fourth, and fifth epitope, wherein the first, second, third, fourth, and fifth epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, or MDM2.

[0236] In some cases, the composition comprises a first and a second epitope independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R. In some cases, the composition further comprises a third epitope, the first, second, and third epitopes independently selected from the group consisting of IGFBP2, HER-2, or IGF-1R.

[0237] In some cases, the composition may comprise: at least a first epitope of a first antigen, the first epitope being derived from a peptide portion of HIF-1α. In some cases, the composition may comprise: at least a first epitope of a first antigen, at least a second epitope of a second antigen, the first and second epitopes being derived from HIF-1α.

[0238] In some cases, the composition can include a nucleotide sequence encoding an epitope of the peptide HIF-1α selected from the group consisting of a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence DSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYN (SEQ ID NO: 82); a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence SDNVNKYMGLTQFELTGHSVFDFTHP (SEQ ID NO: NO:83) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and a nucleotide sequence encoding an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence GGYVWVETQATVIYNTKNSQ (SEQ ID NO:84).

[0239] In some cases, the composition can include at least a first epitope of a peptide HIF-1α selected from the group consisting of an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence DSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYN (SEQ ID NO: 82); an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence SDNVNKYMGLTQFELTGHSVFDFTHP (SEQ ID NO: NO:83) having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence GGYVWVETQATVIYNTKNSQ (SEQ ID NO:84).

[0240] The compositions described herein may contain short epitopes encoded on a single plasmid backbone. In some cases, the plasmid backbone may encode one short epitope. In other cases, the plasmid described herein may encode more than one short epitope. For example, the compositions described herein may encode 2 short epitopes, 3 short epitopes, 4 short epitopes, 5 short epitopes, 6 short epitopes, 7 short epitopes, 8 short epitopes, 9 short epitopes, 10 short epitopes, 11 short epitopes, 12 short epitopes, 13 short epitopes, 14 short epitopes, 15 short epitopes, 16 short epitopes, 17 short epitopes, 18 short epitopes, 19 short epitopes, 20 short epitopes or more than 20 short epitopes. In an exemplary case, the plasmid encodes no more than 6 short epitopes.

[0241] The compositions described herein may contain extended epitopes encoded on a single plasmid backbone. In some cases, the plasmid may encode one extended epitope. In other cases, the composition may encode more than one extended epitope. For example, the plasmid may encode 2 extended epitopes, 3 extended epitopes, 4 extended epitopes, 5 extended epitopes, 6 extended epitopes, 7 extended epitopes, 8 extended epitopes, 9 extended epitopes, 10 extended epitopes, 11 extended epitopes, 12 extended epitopes, 13 extended epitopes, 14 extended epitopes, 15 extended epitopes, 16 extended epitopes, 17 extended epitopes, 18 extended epitopes, 19 extended epitopes, 20 extended epitopes or more than 20 extended epitopes. In an exemplary case, the plasmid encodes no more than 4 extended epitopes.

[0242] The composition of the breast cancer vaccine described herein may contain short epitopes and extended epitopes on a single plasmid backbone. In some cases, the plasmid may comprise a short epitope. In other cases, the composition of the plasmid described herein may comprise more than one short epitope. For example, the composition of the plasmid described herein may comprise 2 short epitopes, 3 short epitopes, 4 short epitopes, 5 short epitopes, 6 short epitopes, 7 short epitopes, 8 short epitopes, 9 short epitopes, 10 short epitopes, 11 short epitopes, 12 short epitopes, 13 short epitopes, 14 short epitopes, 15 short epitopes, 16 short epitopes, 17 short epitopes, 18 short epitopes, 19 short epitopes, 20 short epitopes or more than 20 short epitopes.

[0243] In some cases, the plasmid can encode one extended epitope. In other cases, the compositions described herein can encode more than one extended epitope. For example, the compositions described herein can encode 2 extended epitopes, 3 extended epitopes, 4 extended epitopes, 5 extended epitopes, 6 extended epitopes, 7 extended epitopes, 8 extended epitopes, 9 extended epitopes, 10 extended epitopes, 11 extended epitopes, 12 extended epitopes, 13 extended epitopes, 14 extended epitopes, 15 extended epitopes, 16 extended epitopes, 17 extended epitopes, 18 extended epitopes, 19 extended epitopes, 20 extended epitopes, or more than 20 extended epitopes.

[0244] Plasmids used for compositions containing more than one sequence encoding an epitope may contain spacers between each epitope sequence. In some cases, sequences encoding a short epitope may be concatenated without a spacer. In some cases, sequences encoding an extended epitope may be concatenated without a spacer. In some cases, sequences encoding a short epitope may be concatenated with a spacer. In some cases, sequences encoding an extended epitope may be concatenated with a spacer.

[0245] In an exemplary embodiment, the composition can be a plasmid-based vaccine containing short and extended antigenic epitopes. A 4kB plasmid backbone (e.g., pUMVC3 or pNGVL3) can be used to construct one or more plasmids of the vaccine. Typically, the plasmid can contain an antibiotic resistance gene. For example, pUMVC3 contains a kanamycin resistance gene in addition to a replication origin for selection and proliferation in bacteria. In some cases, the multiple cloning site in pUMVC3 can be flanked by eukaryotic transcription control elements to promote expression of inserted sequences (e.g., gene cassettes) in eukaryotic cells. For example, the inserted sequence can be an epitope.

[0246] In an exemplary case, the nucleic acid coding sequence of the antigenic epitope peptide can be assembled using the Kozak consensus translation initiation sequence, stop codon, and cloning site in the plasmid backbone. Standard molecular techniques known to those of ordinary skill in the art include synthetic oligonucleotides, polymerase chain reaction amplification, restriction endonucleases, and nucleic acid ligases (e.g., DNA ligase) to generate nucleic acids (e.g., DNA fragments) and insert the nucleic acid fragments into the plasmid vector backbone.

[0247] In some cases, the plasmid may contain a nucleic acid sequence encoding at least one tag. In some cases, the tag can be translated into a peptide. Any nucleic acid sequence of a tag known to those skilled in the art can be used in conjunction with the plasmids described herein. For example, the tag can be a histidine tag with 3 histidine residues, a histidine tag with 4 histidine residues, a histidine tag with 5 histidine residues, or a histidine tag with 6 histidine residues, etc. Any suitable technique known to those skilled in the art can be used to determine the expression of the tag in a subject.

[0248] In some cases, the plasmid can be sequenced using any sequencing technology known to those of ordinary skill in the art, such that the sequencing results provide a nucleotide-level analysis of the entire plasmid.

[0249] In some aspects, the composition can be a multi-antigen breast cancer vaccine. For example, a multi-antigen breast cancer vaccine can contain multiple antigens. In some cases, the expression of one antigen can affect the expression of different antigens. In some cases, the expression of more than one antigen can affect the expression of different antigens. In some cases, the expression of one antigen can affect the expression of more than one different antigen. In some cases, the expression of one antigen may not affect the expression of different antigens. In some cases, the expression of more than one antigen may not affect the expression of different antigens. In some cases, the expression of one antigen may not affect the expression of more than one different antigen. For example, the antigenic composition can limit the immunogenicity of the multi-antigen vaccine. Any technique known to those of ordinary skill in the art can be used to determine whether the immune response elicited after administration of the multi-antigen vaccine is of a magnitude comparable to that of each antigen of a single antigen vaccine. For example, ELISPOT (e.g., for secretion of IFNγ) can determine the magnitude of the immune response. In some cases, ELISPOT can detect rodents, non-human primates, or human peptides.

[0250] Plasmids - Survivin, HIF-1A, IGF-1R, and / or IGFBP2

[0251] The compositions described herein may include nucleic acid-based vaccines comprising a plasmid encoding one or more epitopes selected from survivin, HIF-1A, IGF-1R, or IGFBP2. Sometimes, the epitope can be derived from a human protein and the nucleic acid sequence encoding the epitope can be incorporated into a nucleic acid construct designed to induce expression of the epitope in a subject after administration. For example, the epitope encoded from the nucleic acid construct can allow an immune response to at least one epitope to be generated, amplified, attenuated, inhibited, or eliminated against a specific set of proteins (e.g., from a protein).

[0252] In some cases, the vaccines described herein are peptide-based vaccines. Peptide-based vaccines may comprise a plasmid encoding one or more epitopes selected from survivin, HIF-1A, IGF-1R, or IGFBP2. The epitopes may be derived from human proteins that can be used directly in peptide-based vaccines.

[0253] In some cases, the peptide or nucleic acid construct can be optimized to induce, amplify or generate a TH1 immune response based on protein or plasmid. In some cases, the epitope can be an extended TH1 epitope. In other cases, the peptide or nucleic acid construct can be optimized to inhibit, attenuate or eliminate a pathogenic response in a subject (e.g., a human or animal) based on protein or plasmid immunity.

[0254] The compositions described herein can include a plasmid containing a nucleic acid sequence to express at least one epitope(s) in a subject following administration of the composition (eg, vaccine).

[0255] Any plasmid backbone (eg, vector) known to those of ordinary skill in the art to be suitable for pharmaceutical applications of expressing nucleic acids can be used in the compositions described herein.

[0256] The vector can be a circular plasmid or a linear nucleic acid. A circular plasmid or a linear nucleic acid can guide the expression of a specific nucleotide sequence in a suitable target cell. The vector can have a promoter operably linked to the nucleotide sequence encoding the polypeptide, which is operably linked to a termination signal. The vector can also contain sequences required for the appropriate translation of the nucleotide sequence. The vector containing the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous relative to at least one of the other components. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, which can trigger transcription only when the host cell is exposed to some specific external stimulus.

[0257] The vector may be a plasmid. The plasmid can be used to transfect cells with a nucleic acid encoding a polypeptide, wherein the transfected host cells can be cultured and maintained under conditions where expression of the polypeptide occurs.

[0258] Plasmids may contain nucleic acid sequences encoding one or more of the various polypeptides described herein. A single plasmid may contain the coding sequence for a single polypeptide, or for more than one polypeptide. Sometimes, a plasmid may also contain a coding sequence encoding an adjuvant, such as an immunostimulatory molecule, such as a cytokine.

[0259] The plasmid can also comprise an initiation codon, which can be upstream of the coding sequence, and a termination codon, which can be downstream of the coding sequence. The initiation codon and termination codon can be in the frame of the coding sequence. The plasmid can also comprise a promoter operably linked to the coding sequence, and an enhancer upstream of the coding sequence. The enhancer can be human actin, human myosin, human hemoglobin, human muscle sarcosine or a viral enhancer such as an enhancer from CMV, FMDV, RSV or EBV. Polynucleotide functional enhancers are described in U.S. Patent numbers 5,593,972, 5,962,428 and WO94 / 016737.

[0260] The plasmid may also contain a mammalian origin of replication to allow the plasmid to be maintained extrachromosomally and to generate multiple copies of the plasmid in the cell. Examples may be pVAXI, pCEP4, or pREP4 from Invitrogen (San Diego, CA).

[0261] The plasmid may also contain regulatory sequences, which are also suitable for gene expression in the cell to which the plasmid is administered.The coding sequence may contain codons, which may allow for more efficient transcription of the coding sequence in the host cell.

[0262] In some cases, a commercially available plasmid backbone can be used. For example, plasmid pUMVC3 can be used. In some cases, a commercially available plasmid backbone can be modified, mutated, engineered, or cloned prior to use. In other cases, a non-commercially available plasmid backbone can be used.

[0263] Other plasmids may include pSE420 (Invitrogen, San Diego, California), which can be used to produce proteins in Escherichia coli (E. coli). The plasmid may also be pYES2 (Invitrogen, San Diego, California), which can be used to produce proteins in yeast strains of Saccharomyces cerevisiae. The plasmid may also be the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, California), which can be used to produce proteins in insect cells. The plasmid may also be pcDNAI or pcDNA3 (Invitrogen, San Diego, California), which can be used to produce proteins in mammalian cells such as Chinese Hamster Ovary (CHO) cells.

[0264] The vector can be a circular plasmid that can transform target cells by being integrated into the cell genome or being present outside the chromosome (e.g., a self-replicating plasmid with a replication origin). Exemplary vectors include pVAX, pcDNA3.0, or provax or any other expression vector that can express the DNA encoding the antigen and enable the cell to translate the sequence into an antigen recognized by the immune system.

[0265] Nucleic acid-based vaccines can be linear nucleic acid vaccines, or linear expression cassettes ("LECs"), which can be efficiently delivered to a subject by electroporation and express one or more polypeptides described herein. The LEC can be any linear DNA with the phosphate backbone removed. The DNA can encode one or more polypeptides described herein. The LEC can contain a promoter, introns, a stop codon, and / or a polyadenylation signal. Expression of the polypeptide can be controlled by the promoter. The LEC may not contain any antibiotic resistance genes and / or a phosphate backbone. The LEC may not contain other nucleic acid sequences not related to polypeptide expression.

[0266] LECs can be derived from any plasmid that can be linearized. Plasmids can express polypeptides. Exemplary plasmids include pNP (Puerto Rico / 34), pM2 (New Caledonia / 99), WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that can express DNA encoding an antigen and enable cells to translate the sequence into an antigen recognized by the immune system.

[0267] Prior to insertion of the nucleic acid sequence for at least one epitope(s), the length of the plasmid backbone may be less than about 500 bp, about 1.0 kB, about 1.2 kB, about 1.4 kB, about 1.6 kB, about 1.8 kB, about 2.0 kB, about 2.2 kB, about 2.4 kB, about 2.6 kB, about 2.8 kB, about 3.0 kB, about 3.2 kB, about 3.4 kB, about 3.6 kB, about 3.8 kB, about 4.0 kB, about 4.2 kB, about 4.4 kB, about 4.6 kB, about 4.8 kB, about 5. 0kB, about 5.2kB, about 5.4kB, about 5.6kB, about 5.8kB, about 6.0kB, about 6.2kB, about 6.4kB, about 6.6kB, about 6.8kB, about 7.0kB, about 7.2kB, about 7.4kB, about 7.6kB, about 7.8kB, about 8.0kB, about 8.2kB, about 8.4kB, about 8.6kB, about 8.8kB, about 9.0kB, about 9.2kB, about 9.4kB, about 9.6kB, about 9.8kB, about 10.0kB, about 10.2kB, about 10.4kB, about 10.6kB, about 10.8kB, about 11.0kB, about 11.2kB, about 11.4kB, about 11.6kB, about 11.8kB, about 12.0kB, about 12.2kB, about 12.4kB, about 12.6kB, about 12.8kB, about 13.0kB, about 13.2kB, about 13.4kB, about 13.6kB, about 13.8kB, about 14kB, about 14.5kB, about 15kB, about 15.5kB, kB, about 16 kB, about 16.5 kB, about 17 kB, about 17.5 kB, about 18 kB, about 18.5 kB, about 19 kB, about 19.5 kB, about 20 kB, about 30 kB, about 40 kB, about 50 kB, about 60 kB, about 70 kB, about 80 kB, about 90 kB, about 100 kB, about 110 kB, about 120 kB, about 130 kB, about 140 kB, about 150 kB, about 160 kB, about 170 kB, about 180 kB, about 190 kB or about 200 kB. In an exemplary case, the plasmid is about 4 kB in length before the nucleic acid sequence encoding at least one epitope(s) is added.

[0268] In some cases, the compositions described herein may include one plasmid. In other cases, the compositions described herein may include more than one plasmid. For example, the compositions described herein may include 2 plasmids, 3 plasmids, 4 plasmids, 5 plasmids, 6 plasmids, 7 plasmids, 8 plasmids, 9 plasmids, 10 plasmids, 11 plasmids, 12 plasmids, 13 plasmids, 14 plasmids, 15 plasmids, 16 plasmids, 17 plasmids, 18 plasmids, 19 plasmids, 20 plasmids, or more than 20 plasmids.

[0269] The nucleic acid encoding at least one epitope(s) of the plasmid can be derived from any species such that the epitope expressed from the nucleic acid generates an immune response in a subject. In some cases, the subject can be a rodent, a non-human primate, or a human. The nucleic acid encoding the epitope of the plasmid can be isolated from any nucleic acid source using methods and techniques known to those skilled in the art. The nucleic acid encoding the epitope of the plasmid can be cloned into the plasmid backbone using methods and techniques known to those skilled in the art.

[0270] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence of survivin from a human can be used to express survivin in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence of survivin from a non-human source can be used to express survivin in a human.

[0271] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of survivin in the genome of a species can be used to express survivin in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of survivin in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express survivin in a subject.

[0272] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human HIF-1A can be used to express HIF-1A in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human HIF-1A can be used to express HIF-1A in a human.

[0273] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of HIF-1A in the genome of a species can be used to express HIF-1A in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of HIF-1A in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express HIF-1A in a subject.

[0274] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from human IGFBP2 can be used to express IGFBP2 in humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from non-human IGFBP2 can be used to express IGFBP2 in humans.

[0275] In some cases, the nucleic acid sequence used to express the antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGFBP2 in the genome of a species can be used to express IGFBP2 in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of IGFBP2 in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express IGFBP2 in a subject.

[0276] In some cases, the nucleic acid sequence encoding the epitope can be an endogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from a human IGF-1R can be used to express IGF-1R in a human. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, a nucleic acid sequence from a non-human IGF-1R can be used to express IGF-1R in a human.

[0277] In some cases, the nucleic acid sequence used to express an antigenic epitope can be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a species can be used to express IGF-1R in a subject. In other cases, the nucleic acid sequence expressing the epitope can be a synthetic nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a subject can be modified using molecular techniques known to those of ordinary skill in the art and used to express IGF-1R in a subject.

[0278] In some cases, the composition may include nucleic acids encoding one or more epitopes from the following proteins: survivin, HIF-1A, IGFBP2, and IGF-1R. In some cases, the plasmid may include a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may include a nucleic acid sequence encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may include a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may include a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to IGFBP-2 (SEQ ID NO: 54). Sometimes, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to IGFBP-2 (SEQ ID NO: 54). Sometimes, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to IGFBP-2 (SEQ ID NO: 54). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to IGFBP-2 (SEQ ID NO: 54). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to IGFBP-2 (SEQ ID NO: 54).

[0279] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 100 to at least 163 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 105 to at least 160, at least 110 to at least 155, or at least 120 to at least 145 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54).

[0280] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 100 to at least 163 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 105 to at least 160, at least 110 to at least 155, or at least 120 to at least 145 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54).

[0281] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 100 to at least 163 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 105 to at least 160, at least 110 to at least 155, or at least 120 to at least 145 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54).

[0282] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 100 to at least 163 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 105 to at least 160, at least 110 to at least 155, or at least 120 to at least 145 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54).

[0283] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 100 to at least 163 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 105 to at least 160, at least 110 to at least 155, or at least 120 to at least 145 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54).

[0284] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that is 100% identical to at least 100 to at least 163 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that is 100% identical to at least 105 to at least 160, at least 110 to at least 155, or at least 120 to at least 145 consecutive amino acids of IGFBP-2 (SEQ ID NO: 54).

[0285] In some cases, the plasmid may comprise a nucleic acid sequence that has at least 50% sequence identity to IGFBP-2 (SEQ ID NO: 43). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IGFBP-2 (SEQ ID NO: 43). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 70% sequence identity to IGFBP-2 (SEQ ID NO: 43). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 80% sequence identity to IGFBP-2 (SEQ ID NO: 43). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 90% sequence identity to IGFBP-2 (SEQ ID NO: 43). Sometimes, a plasmid may comprise a nucleic acid sequence that has at least 95% sequence identity to IGFBP-2 (SEQ ID NO: 43). Sometimes, a plasmid may comprise a nucleic acid sequence that has at least 99% sequence identity to IGFBP-2 (SEQ ID NO: 43). In some cases, a plasmid may comprise a nucleic acid sequence that has 100% sequence identity to IGFBP-2 (SEQ ID NO: 43). In some cases, a plasmid may comprise a nucleic acid sequence that has 100% sequence identity to IGFBP-2 (SEQ ID NO: 43).

[0286] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to survivin (SEQ ID NO: 85). Sometimes, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to survivin (SEQ ID NO: 85). Sometimes, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to survivin (SEQ ID NO: 85). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to survivin (SEQ ID NO: 85). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to survivin (SEQ ID NO: 85).

[0287] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 10 to at least 38 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 12 to at least 35, at least 15 to at least 30, or at least 20 to at least 25 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, or 36 consecutive amino acids of survivin (SEQ ID NO: 85).

[0288] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 10 to at least 38 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 12 to at least 35, at least 15 to at least 30, or at least 20 to at least 25 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, or 36 consecutive amino acids of survivin (SEQ ID NO: 85).

[0289] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 10 to at least 38 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 12 to at least 35, at least 15 to at least 30, or at least 20 to at least 25 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, or 36 consecutive amino acids of survivin (SEQ ID NO: 85).

[0290] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 10 to at least 38 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 12 to at least 35, at least 15 to at least 30, or at least 20 to at least 25 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, or 36 consecutive amino acids of survivin (SEQ ID NO: 85).

[0291] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 10 to at least 38 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 12 to at least 35, at least 15 to at least 30, or at least 20 to at least 25 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, or 36 consecutive amino acids of survivin (SEQ ID NO: 85).

[0292] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 100% sequence identity to at least 10 to at least 38 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 100% sequence identity to at least 12 to at least 35, at least 15 to at least 30, or at least 20 to at least 25 consecutive amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 100% sequence identity to at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, or 36 consecutive amino acids of survivin (SEQ ID NO: 85).

[0293] In some cases, the plasmid may comprise a nucleic acid sequence that has at least 50% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 70% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 80% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 90% sequence identity to survivin (SEQ ID NO: 86). Sometimes, the plasmid may comprise a nucleic acid sequence that has at least 95% sequence identity to survivin (SEQ ID NO: 86). Sometimes, the plasmid may comprise a nucleic acid sequence that has at least 99% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence that has 100% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence that has 100% sequence identity to survivin (SEQ ID NO: 86).

[0294] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to HIF-1A (SEQ ID NO: 87). Sometimes, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to HIF-1A (SEQ ID NO: 87). Sometimes, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to HIF-1A (SEQ ID NO: 87).

[0295] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 40 to at least 89 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 40 to at least 85, at least 50 to at least 80, or at least 55 to at least 75, or at least 60 to at least 70 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 consecutive amino acids of HIF-1A (SEQ ID NO: 87).

[0296] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 40 to at least 89 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 40 to at least 85, at least 50 to at least 80, or at least 55 to at least 75, or at least 60 to at least 70 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 consecutive amino acids of HIF-1A (SEQ ID NO: 87).

[0297] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 40 to at least 89 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 40 to at least 85, at least 50 to at least 80, or at least 55 to at least 75, or at least 60 to at least 70 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 consecutive amino acids of HIF-1A (SEQ ID NO: 87).

[0298] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 40 to at least 89 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 40 to at least 85, at least 50 to at least 80, or at least 55 to at least 75, or at least 60 to at least 70 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 consecutive amino acids of HIF-1A (SEQ ID NO: 87).

[0299] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 40 to at least 89 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 40 to at least 85, at least 50 to at least 80, or at least 55 to at least 75, or at least 60 to at least 70 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 consecutive amino acids of HIF-1A (SEQ ID NO: 87).

[0300] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 100% sequence identity to at least 40 to at least 89 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 100% sequence identity to at least 40 to at least 85, at least 50 to at least 80, or at least 55 to at least 75, or at least 60 to at least 70 consecutive amino acids of HIF-1A (SEQ ID NO: 87). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide that is 100% identical to at least 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88 consecutive amino acids of HIF-1A (SEQ ID NO: 87).

[0301] In some cases, the plasmid may comprise a nucleic acid sequence that has at least 50% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 70% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 80% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 90% sequence identity to HIF-1A (SEQ ID NO: 88). Sometimes, a plasmid may comprise a nucleic acid sequence that has at least 95% sequence identity to HIF-1A (SEQ ID NO: 88). Sometimes, a plasmid may comprise a nucleic acid sequence that has at least 99% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid may comprise a nucleic acid sequence that has 100% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid may comprise a nucleic acid sequence that has 100% sequence identity to HIF-1A (SEQ ID NO: 88).

[0302] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to IGF-IR (SEQ ID NO: 73). Sometimes, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to IGF-IR (SEQ ID NO: 73). Sometimes, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to IGF-IR (SEQ ID NO: 73). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to IGF-IR (SEQ ID NO: 73). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to IGF-IR (SEQ ID NO: 73).

[0303] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 50 to at least 104 consecutive amino acids of IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to at least 55 to at least 100, at least 60 to at least 90, or at least 70 to at least 80 consecutive amino acids of IGF-IR (SEQ ID NO: 73).

[0304] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 50 to at least 104 consecutive amino acids of IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 55 to at least 100, at least 60 to at least 90, or at least 70 to at least 80 consecutive amino acids of IGF-IR (SEQ ID NO: 73).

[0305] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 50 to at least 104 consecutive amino acids of IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to at least 55 to at least 100, at least 60 to at least 90, or at least 70 to at least 80 consecutive amino acids of IGF-IR (SEQ ID NO: 73).

[0306] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 50 to at least 104 consecutive amino acids of IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 55 to at least 100, at least 60 to at least 90, or at least 70 to at least 80 consecutive amino acids of IGF-IR (SEQ ID NO: 73).

[0307] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 50 to at least 104 consecutive amino acids of IGF-1R (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 55 to at least 100, at least 60 to at least 90, or at least 70 to at least 80 consecutive amino acids of IGF-1R (SEQ ID NO: 73).

[0308] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 100% sequence identity to at least 50 to at least 104 consecutive amino acids of IGF-1R (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 100% sequence identity to at least 55 to at least 100, at least 60 to at least 90, or at least 70 to at least 80 consecutive amino acids of IGF-1R (SEQ ID NO: 73).

[0309] In some cases, the plasmid may comprise a nucleic acid sequence that has at least 50% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 70% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 80% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 90% sequence identity to IGF-IR (SEQ ID NO: 63). Sometimes, a plasmid may comprise a nucleic acid sequence that has at least 95% sequence identity to IGF-IR (SEQ ID NO: 63). Sometimes, a plasmid may comprise a nucleic acid sequence that has at least 99% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, a plasmid may comprise a nucleic acid sequence that has 100% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, a plasmid may comprise a nucleic acid sequence that has 100% sequence identity to IGF-IR (SEQ ID NO: 63).

[0310] Sometimes, the isolated and purified plasmid may comprise at least one nucleotide sequence encoding a polypeptide having at least 70% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least one nucleotide sequence encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least one nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to an epitope selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least one nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least one nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least one nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least one nucleic acid sequence encoding a polypeptide having 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least one nucleic acid sequence encoding a polypeptide having 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87.

[0311] Sometimes, the isolated and purified plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 70% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 80% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 90% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 95% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 99% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise at least four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. In some cases, the at least four nucleic acid sequences independently encode polypeptides directed against an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. In other cases, the at least four nucleic acid sequences encode different polypeptides directed against an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87.

[0312] Sometimes, a plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 70% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. A plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. A plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 80% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 90% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 95% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having at least 99% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. The plasmid may comprise four nucleic acid sequences, wherein each of the four nucleic acid sequences encodes a polypeptide having 100% sequence identity to an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. In some cases, the four nucleic acid sequences independently encode polypeptides directed against an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87. In other cases, the four nucleic acid sequences encode different polypeptides directed against an epitope sequence selected from the group consisting of SEQ ID NOs: 54, 73, 85, and 87.

[0313] In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to SEQ ID NO:89. In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:89. The plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to SEQ ID NO:89. The plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to SEQ ID NO:89. The plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to SEQ ID NO:89. The plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 99% sequence identity to SEQ ID NO:89. The plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to SEQ ID NO:89. The plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to SEQ ID NO:89.

[0314] In some cases, the plasmid may comprise a nucleic acid sequence that has at least 70% sequence identity to SEQ ID NO:90. In some cases, the plasmid may comprise a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90. The plasmid may comprise a nucleic acid sequence that has at least 80% sequence identity to SEQ ID NO:90. The plasmid may comprise a nucleic acid sequence that has at least 90% sequence identity to SEQ ID NO:90. The plasmid may comprise a nucleic acid sequence that has at least 95% sequence identity to SEQ ID NO:90. The plasmid may comprise a nucleic acid sequence that has at least 99% sequence identity to SEQ ID NO:90. The plasmid may comprise a nucleic acid sequence that has 100% sequence identity to SEQ ID NO:90. The plasmid may comprise a nucleic acid sequence that is 100% identical to SEQ ID NO:90.

[0315] In some cases, the plasmid may comprise a nucleic acid sequence that has at least 70% sequence identity to SEQ ID NO: 91. In some cases, the plasmid may comprise a nucleic acid sequence that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 91. The plasmid may comprise a nucleic acid sequence that has at least 80% sequence identity to SEQ ID NO: 91. The plasmid may comprise a nucleic acid sequence that has at least 90% sequence identity to SEQ ID NO: 91. The plasmid may comprise a nucleic acid sequence that has at least 95% sequence identity to SEQ ID NO: 91. The plasmid may comprise a nucleic acid sequence that has at least 99% sequence identity to SEQ ID NO: 91. The plasmid may comprise a nucleic acid sequence that has 100% sequence identity to SEQ ID NO: 91. The plasmid may comprise a nucleic acid sequence that is 100% identical to SEQ ID NO:91.

[0316] Sometimes, the plasmid comprising more than one (individual) epitope sequence can include a spacer between each epitope sequence. In some cases, the epitope sequence can be encoded in series without a spacer. In some cases, a spacer can be used to encode the epitope sequence in series. In some cases, the spacer can include a sequence encoding about 1 to about 50, about 3 to about 40, about 5 to about 35, or about 10 to about 30 amino acid residues. In some cases, the spacer can include a sequence encoding about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 amino acid residues.

[0317] In some cases, the plasmid may contain a nucleic acid sequence encoding at least one tag. In some cases, the tag can be translated into a peptide. Any nucleic acid sequence of a tag known to those skilled in the art can be used in conjunction with the plasmids described herein. For example, the tag can be a histidine tag with 3 histidine residues, a histidine tag with 4 histidine residues, a histidine tag with 5 histidine residues, or a histidine tag with 6 histidine residues, etc. Any suitable technique known to those skilled in the art can be used to determine the expression of the tag in a subject.

[0318] In some cases, the plasmid can be sequenced using any sequencing technology known to those of ordinary skill in the art, such that the sequencing results provide a nucleotide-level analysis of the entire plasmid.

[0319] In some aspects, the composition can be a multi-antigen breast cancer vaccine or a multi-antigen ovarian cancer vaccine. For example, a multi-antigen breast cancer vaccine or a multi-antigen ovarian cancer vaccine may contain multiple antigens. In some cases, the expression of one antigen may affect the expression of different antigens. In some cases, the expression of more than one antigen may affect the expression of different antigens. In some cases, the expression of one antigen may affect the expression of more than one different antigen. In some cases, the expression of one antigen may not affect the expression of different antigens. In some cases, the expression of more than one antigen may not affect the expression of different antigens. In some cases, the expression of one antigen may not affect the expression of more than one different antigen. For example, the antigenic composition can limit the immunogenicity of the multi-antigen vaccine. Any technique known to those of ordinary skill in the art can be used to determine whether the immune response elicited after administration of the multi-antigen vaccine is of a magnitude comparable to that of the individual antigens of a single antigen vaccine. For example, ELISPOT (e.g., for secretion of IFNγ) can determine the magnitude of the immune response. In some cases, ELISPOT can detect rodent, non-human primate, or human peptides. In some cases, a multi-antigen breast or ovarian cancer vaccine may comprise multiple epitopes derived from multiple antigens selected from survivin, HIF-1α, IGF-1R, and / or IGFBP-2.

[0320] Nucleic Acids

[0321] An isolated nucleic acid molecule is one that has been removed (i.e., artificially manipulated) from its natural environment, which is the genome or chromosome in which it is naturally found. Thus, "isolated" does not necessarily reflect the degree of purification of the nucleic acid molecule, but rather indicates that the molecule does not contain the entire genome or chromosome in which it is naturally found. An isolated nucleic acid molecule may contain a gene. An isolated nucleic acid molecule that contains a gene is not a chromosomal fragment containing the gene, but rather contains the coding and regulatory regions associated with the gene, but is free of other genes naturally found on the same chromosome. An isolated nucleic acid molecule may also contain a specific nucleic acid sequence that is flanked (i.e., at the 5' and / or 3' ends of the sequence) by other nucleic acids that are not normally flanked by the specific nucleic acid sequence in nature (i.e., heterologous sequences).

[0322] The isolated nucleic acid molecule can be DNA, including genomic and cDNA, RNA, or a hybrid, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and a combination 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. Although the term "nucleic acid molecule" primarily refers to a physical nucleic acid molecule and the term "nucleic acid sequence" primarily refers to a sequence of nucleotides on a nucleic acid molecule, the two terms are used interchangeably, particularly with respect to nucleic acid molecules, or nucleic acid sequences, that can encode a protein or protein domain.

[0323] A nucleic acid molecule may refer to at least two nucleotides covalently linked together. Although in many cases described below (e.g., in the construction of primers and probes such as labeled probes), the nucleic acids described herein may contain phosphodiester bonds, nucleic acid analogs having alternating backbones may be included, including, for example, phosphoramidites (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 (1986)), phosphorothioates (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioates (Briu et al., J. Am. Chem. Soc. 111:2321 (1989)), O-methylphosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach Approach, Oxford University Press), and peptide nucleic acid (also referred to herein as "PNA") backbones and linkages (see Egholm, J. Am. Chem. Soc. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31: 1008 (1992); Nielsen, Nature, 365: 566 (1993); Carlsson et al., Nature 380: 207 (1996), all of which are incorporated herein by reference).Other similar nucleic acids include those with a double ring structure, including locked nucleic acids (also referred to herein as "LNA"), Koshkin et al., J. Am. Chem. Soc. 120:132523 (1998); positively charged backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995)); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", edited by YS Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", edited by YS Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995), pp. 169-176. Several nucleic acid analogs are described in Rawls, C&E News, June 2, 1997, p. 35. "Locked nucleic acids" are also included in the definition of nucleic acid analogs. LNA is a class of nucleic acid analogs in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom to the 4'-C atom. All of these references are incorporated herein by reference. These modifications of the ribose-phosphate backbone can be performed to increase the stability and half-life of such molecules in physiological environments.For example, PNA:DNA and LNA-DNA hybrids may exhibit increased stability and therefore may be used in some embodiments. The target nucleic acid may be single-stranded or double-stranded, as specifically described herein, or contain portions of both double-stranded and single-stranded sequences. Depending on the application, the nucleic acid may be DNA (including, for example, genomic DNA, mitochondrial DNA, and cDNA), RNA (including, for example, mRNA and rRNA), or a hybrid, wherein the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, hypoxanthine, isocytosine, and isoguanine, among others.

[0324] A recombinant nucleic acid molecule is a molecule that includes at least one of any nucleic acid sequences encoding any one or more of the proteins described herein, operably linked to at least one of any transcriptional control sequences effective to regulate expression of the nucleic acid molecule in the cell to be transfected. While the term "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the term "nucleic acid sequence" primarily refers to the sequence of nucleotides on a nucleic acid molecule, the two terms are used interchangeably, particularly with respect to nucleic acid molecules, or nucleic acid sequences, that are capable of encoding a protein. Additionally, the term "recombinant molecule" primarily refers to a nucleic acid molecule operably linked to a transcriptional control sequence, but is used interchangeably with the term "nucleic acid molecule" for administration to an animal.

[0325] Recombinant nucleic acid molecule comprises recombinant vector, and it is any nucleotide sequence, is generally a heterologous sequence, it is operably connected to the isolated nucleic acid molecule of encoding fusion rotein of the present invention, it can make fusion rotein recombinant production, and it can deliver nucleic acid molecule to host cell according to the present invention.This carrier can contain the nucleotide sequence that is not found adjacent to the isolated nucleic acid molecule to be inserted into carrier in nature.This carrier can be eukaryotic or prokaryotic RNA or DNA, and is preferably a virus or plasmid in the present invention.Recombinant vector can be used for cloning, order-checking and / or operation of nucleic acid molecule, and can be used for delivering this class molecule (for example, in DNA composition or in composition based on viral vector).Recombinant vector is preferably used for the expression of nucleic acid molecule, and also can be referred to as expression vector.Preferred recombinant vector can be expressed in the host cell of transfection.

[0326] In the recombinant molecules of the present invention, nucleic acid molecules are operably linked to expression vectors containing regulatory sequences such as transcriptional control sequences, translational control sequences, replication origins, and other regulatory sequences that are compatible with the host cell and control the expression of the nucleic acid molecules of the present invention. Specifically, the recombinant molecules of the present invention include nucleic acid molecules that are operably linked to one or more expression control sequences. The term "operably linked" refers to a nucleic acid molecule that is linked to an expression control sequence in such a way that the molecule is expressed when transfected (i.e., transformed, transduced, or transfected) into a host cell.

[0327] Pharmaceutical composition

[0328] The immunogenic compositions of the present invention are preferably formulated as vaccines for in vivo administration to subjects such that they confer antibody titers to each antigenic component that are superior to the seroprotection standard for acceptable percentages of subjects. Antibody titers associated with an antigen, above which a subject is considered seroconverted to that antigen, are well known and are published by organizations such as the World Health Organization. Preferably, greater than 80% of a statistically significant sample of subjects seroconverts, more preferably greater than 90%, even more preferably greater than 93%, and most preferably 96-100%.

[0329] adjuvant

[0330] The immunogenic compositions of the present invention are preferably adjuvanted. Adjuvants can be used to enhance the immune response (humoral and / or cellular) elicited in a patient receiving the vaccine. In some cases, adjuvants can elicit a TH1-type response. In other cases, adjuvants can elicit a TH2-type response. A TH1-type response can be characterized by the production of cytokines such as IFN-γ, in contrast to a TH2-type response, which can be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

[0331] Adjuvants may include stimulatory molecules such as cytokines. Non-limiting examples of cytokines include: CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFa, TNFp, granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), skin T cell-attracting chemokine (CTACK), epidermal thymus-expressed chemokine (TECK), mucosa-associated epidermal chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor, IL-7, nerve growth factor, vascular epidermal growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP In some cases, the adjuvant is GM-CSF.

[0332] Other adjuvants include: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular epidermal growth factor, Fas, TNF receptor, Fit , Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactivated NIK, SAPK, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

[0333] In some aspects, the adjuvant can be a modulator of a toll-like receptor. Examples of modulators of toll-like receptors include TLR-9 antagonists and are not limited to small molecule modulators, such as toll-like receptors such as imiquimod. Other examples of adjuvants that can be used in combination with the vaccines described herein include, but are not limited to, saponins, CpG ODN, and the like.

[0334] Sometimes, the adjuvant may include an aluminum salt, such as aluminum hydroxide gel (a1um), aluminum phosphate, calcium, iron or zinc salts, or may be an insoluble suspension of acylated tyrosine, or an acylated sugar, a cationically or anionically derivatized polysaccharide, or a polyphosphazene.

[0335] Sometimes, suitable adjuvant systems that promote a predominantly Th1 response include monophosphoryl lipid A or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A, and combinations of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL), with an aluminum salt. Boosting systems include combinations of monophosphoryl lipid A and saponin derivatives, particularly QS21 and 3D-MPL, as described in WO 94 / 00153, or less reactive compositions in which QS21 is quenched with cholesterol, as described in WO 96 / 33739. A particularly potent adjuvant formulation comprising QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95 / 17210. The vaccine may also comprise a saponin, more preferably QS21. The formulation may also comprise an oil-in-water emulsion and tocopherol (WO 95 / 17210). Oligonucleotides containing unmethylated CpG (WO 96 / 02555) are also potential inducers of Th1 responses and are suitable for use in the present invention.

[0336] In some cases, aluminum salts are used. Sometimes, in order to minimize the level of adjuvant in the compositions of the invention, the polysaccharide conjugate may be non-adjuvanted.

[0337] Sometimes, suitable adjuvant systems may include adjuvants or immunostimulants, such as, but not limited to, detoxified lipid A and non-toxic derivatives of lipid A from any source, saponins, and other agents capable of stimulating a TH1 type response. Enterobacterial lipopolysaccharide (LPS) is known to be a potent stimulator of the immune system, although its use in adjuvants has been limited by its toxic effects. Ribi et al. (1986, Immunology and hnmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, pp. 407-419) have described a non-toxic derivative of LPS, monophosphoryl lipid A (MPL), which is produced by removing the core sugar group and phosphate from a terminally shortened glucosamine.

[0338] A further detoxified form of MPL is produced by removing the acyl chain from the 3-position of the disaccharide backbone and is termed 3-O-deacylated monophosphoryl lipid A (3D-MPL). It has been purified and prepared by the methods described in GB 2122204B, which also discloses the preparation of diphosphoryl lipid A and its 3-O-deacylated variants.

[0339] In some cases, 3D-MPL is in the form of an emulsion with a small particle size of less than 0.2 μm in diameter, and methods for its preparation are described in WO 94 / 21292. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO9843670A2. Bacterial lipopolysaccharide-derived adjuvants to be formulated in the compositions of the present invention may be purified and processed from bacterial sources, or they may be synthetic. For example, purified monophosphoryl lipid A is described in Ribi et al., 1986 (supra), and diphosphoryl lipid A or 3-O-deacylated monophosphoryl lipid A derived from Salmonella are described in GB 2220211 and US4912094. Other purified and synthetic lipopolysaccharides have been described (Hilgers et al., 1986, Int. Arch Allergy. Immunol, 79(4):392-6; Hilgers et al., 1987, Immunology, 60(1):141-6; and EP 0 549 074 B1). A particularly preferred bacterial lipopolysaccharide adjuvant is 3D-MPL.

[0340] Thus, LPS derivatives useful in the present invention are those immunostimulants that are structurally similar to LPS or MPL or 3D-MPL. In another aspect of the present invention, the LPS derivative may be an acylated monosaccharide that is a sub-region of the above structure of MPL.

[0341] Saponins are described in Lacaille-Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine, Vol. 2, pp. 363-386). Saponins are steroid or triterpenoid saponins that are widely distributed in the plant and marine animal kingdoms. Saponins are used to form colloidal solutions in water that form foam when shaken and are used to precipitate cholesterol. When saponins approach cell membranes, they create pore-like structures in the membranes that cause membrane rupture. Hemolysis of red blood cells is an example of this phenomenon, which is a property of some, but not all, saponins.

[0342] Saponins are known as adjuvants in systemic vaccines. The adjuvant and hemolytic activity of individual saponins have been extensively studied in the art (Lacaille-Dubois and Wagner, supra). For example, Quil A (derived from the bark of the South American soapberry tree) and portions thereof are described in US 5,057,540 and "Saponins as vaccine adjuvants", Kensil, CR, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0362279B1.

[0343] Particulate structures containing portions of Quil A, called immunostimulatory complexes (ISCOMS), are hemolytic and have been used to prepare vaccines (Morein, B., EP 0 109 942 B1; WO 96 / 11711; WO 96 / 33739). The hemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and their production methods are described in U.S. Pat. No. 5,057,540 and EP 0 362 279 B1. Other saponins that have been used in systemic immunization studies include those derived from other plant species such as Dianthus and Saponaria officinalis (Bomford et al., Vaccine, 10(9): 572-577, 1992).

[0344] Enhanced systems include combinations of non-toxic lipid A derivatives and saponin derivatives, particularly QS21 and 3D-MPL, as described in WO 94 / 00153, or less reactogenic compositions in which QS21 is quenched with cholesterol, as described in WO 96 / 33739.

[0345] Sometimes, the adjuvant is selected from a bacterial toxoid, a polyoxypropylene-polyoxyethylene block polymer, an aluminum salt, a liposome, a CpG polymer, an oil-in-water emulsion, or a combination thereof.

[0346] Sometimes, the adjuvant is an oil-in-water emulsion. The oil-in-water emulsion may comprise at least one oil and at least one surfactant, wherein the oil and surfactant are biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion are typically less than 5 μm in diameter and may even have a submicron diameter, which is achieved by microfluidization to provide a stable emulsion. Droplets less than 220 nm in size are preferred because they can be filter sterilized.

[0347] The oil used may include oils of animal (e.g., fish) or plant origin. The sources of vegetable oils may include nuts, seeds, and cereals. Examples of the most common nut oils include peanut oil, soybean oil, coconut oil, and olive oil. Jojoba oil, for example, obtained from jojoba beans, may be used. Seed oils include safflower oil, cottonseed oil, sunflower oil, sesame seed oil, and the like. Cereal groups may include: corn oil and oils from other cereals such as wheat, oats, rye, rice, teff, triticale, and the like. Although 6-10 carbon fatty acid esters of glycerol and 1,2-propylene glycol do not naturally occur in seed oils, they can be prepared from nuts and seed oils by hydrolysis, separation, and esterification of suitable substances. Fats and oils from mammalian emulsions may be metabolizable and therefore may be used in vaccines as described herein. The processes of separation, purification, saponification, and other methods necessary to obtain pure oils of animal origin are well known in the art. Fish may contain easily recyclable metabolizable oils. For example, several examples of fish oils that can be used herein include cod liver oil, shark liver oil, and whale oil (such as spermaceti). Many branched oils, collectively referred to as terpenes, can be synthesized biochemically from 5-carbon isoprene units. Shark liver oil contains a branched unsaturated terpenoid compound called squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Squalane, a saturated analog of squalene, can also be used. Fish oils including squalene and squalane are readily available from commercial sources or can be obtained by methods known in the art.

[0348] Other available oils include tocopherol, which may be included in vaccines for elderly patients (as patients over 60 years old), because it is reported that vitamin E has a positive effect on immune response in this patient population. In addition, tocopherol has antioxidant properties, and this characteristic helps to stabilize this emulsion. There are various tocopherols (α, β, γ, δ, ε or ξ), but α-tocopherol is used conventionally. The example of α-tocopherol is DL-α-tocopherol. Succinic acid α-tocopherol is compatible with cancer vaccines and is a useful preservative that replaces mercury-containing compounds.

[0349] Oil mixtures may be used, for example, squalene and alpha-tocopherol. Oil levels of 2-20% by volume may be used.

[0350] Surfactants can be classified by their "HLB" (hydrophile / lipophile balance). In some cases, the HLB of the surfactant is at least 10, at least 15, and / or at least 16. Surfactants can include, but are not limited to: polyoxyethylene sorbitan ester surfactants (commonly known as Tweens), particularly polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), such as linear EO / PO block copolymers, sold under the trade name DOWFAX™; octoxynol with varying numbers of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol 9 (Triton X-100 or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids, such as phosphatidylcholine (lecithin); nonylphenol ethanol esters, such as Tergito1™ NP series; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol (called Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as Span (SPAN)), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Nonionic surfactants can be used herein.

[0351] Mixtures of surfactants can be used, such as a Tween 80 / Span 85 mixture. Combinations of polyoxyethylene sorbitan esters and octoxynol are also suitable. Another combination includes laureth-9 plus polyoxyethylene sorbitan esters and / or octoxynol.

[0352] The amount (weight %) of surfactant can be: polyoxyethylene sorbitan ester (such as Tween 80) 0.01-1%, in particular about 0.1%; octyl- or nonyl-phenoxy polyoxyethanol (such as Triton X100 or other detergents of the Triton series) 0.001-0.1%, in particular 0.005-0.02%; polyoxyethylene ether (such as laureth 9) 0.1-20%, preferably 0.1-10%, in particular 0.1-1% or about 0.5%.

[0353] Specific oil-in-water emulsion adjuvants include, but are not limited to:

[0354] A submicron emulsion of squalene, polysorbate 80, and sorbitan trioleate. The emulsion may have a volume composition of about 5% squalene, about 0.5% polysorbate 80, and about 0.5% Span 85. By weight, these proportions are 4.3% squalene, 0.5% polysorbate 80, and 0.48% Span 85. This adjuvant is referred to as "MF59." The MF59 emulsion preferably contains citrate ions, such as in a 10 mM sodium citrate buffer.

[0355] Submicron emulsions of squalene, tocopherol, and polysorbate 80. These emulsions may contain 2-10% squalene, 2-10% tocopherol, and 0.3-3% polysorbate 80, with the weight ratio of squalene:tocopherol preferably being ≤1 (e.g., 0.90) as this provides a more stable emulsion. The volume ratio of squalene to polysorbate 80 may be approximately 5:2, or the weight ratio may be approximately 11:5. One such emulsion can be prepared by dissolving Tween 80 in PBS to obtain a 2% solution, mixing 90 ml of this solution with a mixture of 5 g DL-α-tocopherol and 5 ml squalene, and then microfluidizing the mixture. The resulting emulsion contains submicron oil droplets having an average diameter of, for example, 100-250 nm, preferably about 180 nm. The emulsion may also contain 3-de-O-acylated monophosphoryl lipid A (3d-MPL). Another useful emulsion of this type may contain (per human dose) 0.5-10 mg squalene, 0.5-11 mg tocopherol and 0.1-4 mg polysorbate 80.

[0356] An emulsion of squalene, tocopherol and a triton detergent (such as triton X-100). The emulsion may also contain 3d-MPL (see below). The emulsion may contain a phosphate buffer.

[0357] An emulsion containing a polysorbate (e.g., polysorbate 80), a triton detergent (e.g., triton X-100), and a tocopherol (e.g., α-tocopheryl succinate). The emulsion may contain these three components in a mass ratio of approximately 75:11:10 (e.g., 750 μg / ml polysorbate 80, 110 μg / ml triton X-100, and 100 μg / ml α-tocopheryl succinate), with these concentrations accounting for the contribution of these components to the antigen. The emulsion may also contain squalene. The emulsion may also contain 3d-MPL. The aqueous phase may contain phosphate buffer.

[0358] Squalane, polysorbate 80 and poloxamer 401 (“Pluronic TM L121"). The emulsion can be formulated with phosphate buffered saline at pH 7.4. The emulsion is a useful muramyl dipeptide delivery vehicle and can be used with a "SAF-1" adjuvant (0.05-1% Thr-MDP, 5% squalene, 2.5% Pluronic L121, and 0.2% polysorbate 80) containing threonyl-MDP. It can also be used without Thr-MDP, for example with an "AF" adjuvant (5% squalene, 1.25% Pluronic L121, and 0.2% polysorbate 80).

[0359] An emulsion containing squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (such as polyoxyethylene (12) ceteareth-24 ether) and a hydrophobic nonionic surfactant (such as a sorbitan ester or a mannide ester, such as sorbitan monooleate or "Span 80"). The emulsion may be thermoreversible and / or wherein at least 90% of the oil droplets (by volume) are less than 200 nm in size. The emulsion may also contain one or more of the following: a sugar alcohol; a cryoprotectant (e.g., a sugar, such as dodecyl maltoside and / or sucrose); and / or an alkyl polyglycoside. The emulsion may contain a TLR4 agonist. Such an emulsion may be lyophilized.

[0360] Emulsion of Squalene, Poloxamer-105, and Abil-Care. The final concentration (by weight) of these components in the adjuvanted vaccine can be 5% squalene, 4% Poloxamer-105 (Pluronic Polyol), and 2% Abil-Care 85 (Bis-PEG / PPG-16 / 16PEG / PPG-16 / 16 Dimethicone; Caprylic / Capric Triglyceride).

[0361] An emulsion containing 0.5-50% oil, 0.1-10% phospholipids, and 0.05-5% nonionic surfactant. The phospholipid component may include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin, and cardiolipin. Submicron droplet size is preferred.

[0362] A submicron oil-in-water emulsion of a non-metabolizable oil (e.g., light mineral oil) and at least one surfactant (e.g., lecithin, Tween 80, or Span 80). Additives may include, for example, Quil A saponin, cholesterol, a saponin-lipophile conjugate (e.g., GPI-0100, produced by adding an aliphatic amine to a deacylated saponin via the carboxyl group of glucuronic acid), dimethyldioctadecyl ammonium bromide, and / or N,N-dioctadecyl-N,N-bis(2-hydroxyethyl)propylenediamine.

[0363] Carriers and excipients

[0364] In some cases, the compositions described herein may further comprise carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, antibacterial agents, chelating agents, suspending agents, thickeners and / or preservatives), water, oils, including those from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., saline solutions, aqueous dextrose and glycerol solutions, flavors, colorants, anti-adherents and other acceptable additives, adjuvants, or binders, other pharmaceutically acceptable auxiliary substances required to simulate physiological conditions, such as pH buffers, tonicity adjusters, emulsifiers, wetting agents, etc. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. In other cases, the pharmaceutical preparation is preferably free of preservatives. In other cases, the pharmaceutical preparation may contain at least one preservative. General methods for pharmaceutical dosage forms are found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999)). It will be appreciated that while any suitable carrier known to those of ordinary skill in the art can be used to administer the pharmaceutical compositions described herein, the type of carrier will vary depending on the mode of administration.

[0365] In some cases, the composition may include a surfactant. Exemplary surfactants may include octylphenoxypolyoxyethanol and polyoxyethylene sorbitan esters, as described in Surfactant Systems (1983, Chapman and Hall, eds., Attwood and Florence). Octylphenoxypolyoxyethanol (octoxynol) includes tert-octylphenoxypolyethoxyethanol (Triton X-100™), also described in Merck Index Entry 6858 (page 1162, 12th edition, Merck & Co. Inc., Whitehouse Station, NJ, USA; ISBN 0911910-12-3). Polyoxyethylene sorbitan esters include polyoxyethylene sorbitan monoesters (Tween 80™), also described in Merck Index Entry 7742 (page 1308, 12th ed., Merck & Co. Inc., Whitehouse Station, NJ, USA; ISBN 0911910-12-3). Both can be prepared using the methods described herein or purchased from commercial sources such as Sigma Inc.

[0366] Exemplary nonionic surfactants can include Triton X-45, tert-octylphenoxy-polyethoxyethanol (Triton X-100), Triton X-102, Triton X-114, Triton X-165, Triton X-205, Triton X-305, Triton -57, Triton -101, Triton -128, Breij 35, polyoxyethylene -9-lauryl ether (laureth9) and polyoxyethylene -9-stearyl ether (steareth9). Polyoxyethylene ethers can include polyoxyethylene -8-stearyl alcohol ether, polyoxyethylene -4-lauryl ether, polyoxyethylene -35-lauryl ether, and polyoxyethylene -23-lauryl ether.

[0367] Other terms or names for polyoxyethylene lauryl ethers are described in the CAS registry. The CAS registry number for polyoxyethylene-9-lauryl ether is 9002-92-0. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck Index (12th edition: entry 7717, Merck & Co. Inc., Whitehouse Station, NJ, USA; ISBN 0911910-12-3). Laureth 9 is formed by reacting ethylene oxide with dodecanol and has an average of 9 ethylene oxide units.

[0368] The ratio of the length of the polyoxyethylene portion to the length of the alkyl chain in the surfactant (i.e., the ratio of n:alkyl chain length) affects the solubility of this type of surfactant in aqueous media. Therefore, the surfactants of the present invention can be in solution or can form particulate structures, such as micelles or vesicles. As solutions, the surfactants of the present invention are safe, easy to sterilize, and simple to administer, and can be prepared in a simple manner without the GMP and QC issues associated with the formation of uniform particulate structures. Some polyoxyethylene ethers, such as laureth 9, can form non-vesicular solutions. However, polyoxyethylene-8 palmitoyl ether (C18E8) can form vesicles. Therefore, vesicles of polyoxyethylene-8 palmitoyl ether in combination with at least one other non-ionic surfactant can be used in the formulations of the present invention.

[0369] Within the inherent experimental error of this biological assay, about 0.5-0.0001%, more preferably 0.05-0.0001%, more preferably 0.005-0.0001%, and most preferably 0.003-0.0004% of the polyoxyethylene ether or surfactant of general formula (I) of the present invention preferably has hemolytic activity. Ideally, the polyoxyethylene ether or ester should have similar hemolytic activity to polyoxyethylene-9 lauryl ether or polyoxyethylene-8 stearyl ether (i.e., within a 10-fold difference).

[0370] Two or more nonionic surfactants from different groups of surfactants may be present in the vaccine formulations described herein. In particular, polyoxyethylene sorbitan esters such as polyoxyethylene sorbitan monooleate (Tween 80 TM Another particularly preferred combination of nonionic surfactants comprises laureth 9 plus polyoxyethylene sorbitan esters or octoxynol or both.

[0371] Preferably, each nonionic surfactant is present in the final vaccine formulation at a concentration of 0.001 to 20%, more preferably 0.01 to 10%, and most preferably up to 2% (w / v). Where one or two surfactants are present, they are typically present in the final formulation at a concentration of up to about 2%, typically up to about 0.6%. One or more other surfactants may be present, typically at a concentration of up to about 1%, and typically up to about 0.2% or 0.1%. Any mixture of surfactants may be present in the vaccine formulation of the present invention. Nonionic surfactants such as those described above have the following preferred concentrations in the final vaccine composition: polyoxyethylene sorbitan esters such as Tween 80™: 0.01 to 1%, most preferably about 0.1% (w / v); octyl- or nonylphenoxypolyoxyethanols such as Triton X-100™ or other detergents of the Triton series: 0.001 to 0.1%, most preferably 0.005 to 0.02% (w / v); polyoxyethylene ethers of general formula (I) such as laureth 9: 0.1 to 20%, preferably 0.1 to 10% and most preferably 0.1 to 1% or about 0.5% (w / v).

[0372] The composition can also be encapsulated in liposomes using well-known techniques. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344, and 5,942,252.

[0373] The composition can be administered into liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to patients are well known to those skilled in the art. U.S. Patent No. 4,789,734 describes a method for encapsulating biomaterials into liposomes, the contents of which are incorporated herein by reference. Specifically, the material is dissolved in an aqueous solution, suitable phospholipids and lipids and the required surfactants are added, and the material is dialyzed or sonicated as needed. G. Gregoriadis, Chapter 14, "Liposomes," Drug Carriers in Biology and Medicine, 2 sup. 87-341 (Academic Press, 1979) provides a review of known methods.

[0374] Microspheres formed from polymers or proteins are well known to those skilled in the art and can be adapted for direct entry into the bloodstream via the gastrointestinal tract. Alternatively, a compound can be incorporated and the microspheres or microsphere complexes can be implanted for sustained release over a period of days to months. See, for example, U.S. Patents 4,906,474, 4,925,673, and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contents of which are incorporated herein by reference.

[0375] The composition may contain a preservative, such as thimerosal or 2-phenoxyethanol. In some cases, the vaccine is substantially free (eg, <10 μg / ml) of mercury-containing substances, such as free of thimerosal. Alpha-tocopheryl succinate may be used as a substitute for mercury-containing compounds.

[0376] To control tonicity, the vaccine may include physiological salts such as sodium salts. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and / or magnesium chloride.

[0377] The composition may have an osmolarity within the range of 200 mOsm / kg to 400 mOsm / kg, 240 to 360 mOsm / kg, or 290 to 310 mOsm / kg.

[0378] The composition may include one or more buffers, such as Tris buffer, borate buffer, succinate buffer, histidine buffer (particularly when aluminum hydroxide adjuvant is present), or citrate buffer. In some cases, the buffer is included at a concentration generally in the range of 5-20 mM.

[0379] The pH of the composition can be from about 5.0 to about 8.5, from about 6.0 to about 8.0, from about 6.5 to about 7.5, or from about 7.0 to about 7.8.

[0380] The composition may be sterile. The vaccine may be pyrogen-free, such as less than 1 EU / dose (endotoxin unit, a standard measure), and may be less than 0.1 EU / dose. The composition may be gluten-free.

[0381] The composition may include detergents such as polyoxyethylene sorbitan ester surfactants (known as "Tweens"), octoxynol (e.g., octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB"), or sodium deoxycholate, particularly for split or surface antigen vaccines. Detergents may be present only in trace amounts. Thus, the vaccine may include octoxynol-10 and polysorbate 80 at levels of less than 1 mg / ml each. Other trace residual components may be antibiotics (e.g., neomycin, kanamycin, polymyxin B).

[0382] The composition can be formulated as a sterile solution or suspension, with a suitable carrier, as is well known in the art. The pharmaceutical composition can be sterilized by conventional sterilization techniques known in the art, or can be sterile filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized preparation can be combined with a sterile solution prior to administration. Suitable formulations and other carriers are described in Remington: The Science and Practice of Pharmacy (20th ed., Lippincott Williams & Wilkins, Baltimore Md.), which is incorporated herein by reference in its entirety.

[0383] The compositions can be formulated with one or more pharmaceutically acceptable salts. Pharmaceutically acceptable salts can include those of inorganic ions, for example, sodium, potassium, calcium, magnesium ions, etc. Such salts can include inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, mandelic acid, malic acid, citric acid, tartaric acid or maleic acid salts. In addition, if the reagent contains a carboxyl group or other acidic groups, it can be converted into a pharmaceutically acceptable addition salt with an inorganic or organic base. Examples of suitable alkalis include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl, ethanolamine, diethanolamine, triethanolamine, etc.

[0384] The addition salt may comprise bile acid or its derivative. These include bile acid derivatives and salts thereof, in particular sodium salts of bile acid or bile acid derivatives. Examples of bile acids and their derivatives include cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid and derivatives such as sugars of the aforementioned bile acids -, taurine -, aminopropyl-1-propanesulfonic acid -, aminopropyl-2-hydroxy-1-propanesulfonic acid derivatives, or N, N-bis (3D glucosamidopropyl) deoxycholamin. A particularly preferred example is sodium deoxycholate (NaDOC), which may be present in the final vaccine formulation.

[0385] The compositions described herein comprising active adjuvants such as peptides or nucleic acids and one or more adjuvants can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients, diluents and / or adjuvants, e.g., which facilitate processing of the active agent into a formulation that can be administered. Suitable agents may depend, at least in part, on the selected route of administration. A variety of routes or modes of administration can be used to deliver the agents described herein, including oral, buccal, topical, rectal, transdermal, mucosal, subcutaneous, intravenous, and intramuscular application, as well as by inhalation.

[0386] The active agent can also be formulated for parenteral administration (e.g., injection, such as bolus injection or continuous infusion) and can be prepared in unit dosage form in ampoules, prefilled syringes, small volume infusion bottles, or multi-dose containers containing preservatives. The composition can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example, solutions in aqueous polyethylene glycol.

[0387] For injection preparations, carrier can be selected from suitable ones known in the art, including aqueous solutions or oil suspensions, or emulsions, and sesame oil, corn oil, cottonseed oil or peanut oil, and elixirs, mannitol, dextrose or sterile aqueous solutions, and similar pharmaceutical carriers. Preparations can also include polymer compositions, which are biocompatible or biodegradable, such as poly (lactic acid-co-glycolic acid). These materials can be made into microspheres or nanospheres, loaded with drugs and further coated or derivatized to produce excellent sustained release properties. Carriers suitable for periocular or intraocular injections include, for example, therapeutic agent suspensions in injection-grade water, liposomes and carriers suitable for lipophilic substances. Other carriers for periocular or intraocular injections are well known in the art.

[0388] In some cases, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional methods. Compositions for intravenous administration are typically solutions dissolved in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic (such as lidocaine) to alleviate pain at the injection site. Typically, the components are provided individually or mixed together in a unit dosage form, for example as a lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the active substance content. When the composition is administered by infusion, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided to mix with the pharmaceutical ingredients before administration.

[0389] When administered by injection, the active agent can be formulated in an aqueous solution, specifically in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution or physiological saline buffer. The solution may contain formulating agents, such as suspending agents, stabilizers and / or dispersants. Alternatively, the active compound may be in the form of a powder that is reconstituted with a suitable vehicle, such as sterile, pyrogen-free water, before use. In another embodiment, the pharmaceutical composition does not contain an adjuvant or any other substance that is added to enhance the immune response stimulated by the peptide. In another embodiment, the pharmaceutical composition contains a substance that suppresses the immune response to the peptide. Methods of formulation are known in the art, for example, as described in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton P.

[0390] In addition to the above-mentioned preparations, active agents can also be formulated into long-acting preparations. Such long-acting preparations can be given by implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection or use of a transdermal patch. Thus, for example, agents can also be formulated with suitable polymeric materials or hydrophobic materials (e.g., formulated into emulsions with acceptable oils) or ion exchange resins, or formulated into slightly soluble derivatives, e.g., slightly soluble salts.

[0391] In some cases, compositions comprising one or more agents exhibit local and regional effects when administered topically or injected at or near a specific injection site. Direct topical application of viscous liquids, solutions, suspensions, dimethyl sulfoxide (DMSO)-based solutions, liposomal formulations, gels, jelly, creams, emulsions, ointments, suppositories, foams, or aerosol sprays can be used for topical administration to produce, for example, local and / or regional effects. Pharmaceutically suitable carriers for such formulations include, for example, lower fatty alcohols, polyols (e.g., glycerol, or polyethylene glycol), esters of fatty acids, oils, fats, silicones, etc. Such preparations may also include preservatives (e.g., parabens) and / or antioxidants (e.g., ascorbic acid and tocopherol). See also Dermatological Formulations: Percutaneous Absorption, Barry (ed.), Marcel Dekker Incl, 1983. In another embodiment, topical formulations comprising transport agents, carriers, or ion channel inhibitors can be used to treat epidermal or mucosal viral infections.

[0392] The composition may contain a cosmetic or skin-acceptable carrier. Such carriers are compatible with skin, nails, mucous membranes, tissues and / or hair and may include any conventional cosmetic or skin carrier that meets these requirements. One of ordinary skill in the art can easily select such carriers. In formulating skin ointments, the agent or combination of agents may be formulated in an oily hydrocarbon base, an aqueous absorption base, an oil-in-water absorption base, an oil-in-water removable base and / or a water-soluble base. Examples of such carriers and excipients include, but are not limited to, humectants (e.g., urea), glycols (e.g., polyethylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleic acid), surfactants (e.g., isopropyl myristate and sodium lauryl sulfate), pyrrolidone, glyceryl monolaurate, sulfoxides, terpenes (e.g., menthol), amines, amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

[0393] Available aqueous or oily base, add suitable thickener and / or gel to prepare ointment and cream.Lotion available aqueous or oily base preparation, and usually also contain one or more emulsifiers, stabilizer, dispersant, suspending agent, thickener or coloring agent.Be well known in the art for the construction and use of transdermal patch of delivering pharmaceutical agent.Referring to U.S. Patent Nos. 5,023,252,4,992,445 and 5,001,139.Can construct this type of patch for continuous, pulse or as needed delivery pharmaceutical agent.

[0394] Lubricants that can be used to form pharmaceutical compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerol, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, or mixtures thereof. Other lubricants include, for example, syloid silica gel, a condensed aerosol of synthetic silica, or mixtures thereof. Lubricants can optionally be added in an amount less than about 1% by weight of the pharmaceutical composition.

[0395] The compositions may be in any form suitable for topical application, including aqueous, aqueous-alcoholic or oily solutions, emulsions or serum dispersions, aqueous, anhydrous or oily gels, emulsions obtained by dispersing a fatty phase in an aqueous phase (O / W or oil-in-water), or vice versa (W / O or water-in-oil), microemulsions or microcapsules, microparticles or dispersions of ionic and / or nonionic lipid carriers. These compositions may be prepared according to conventional methods. In addition to the agents of the present invention, the amounts of the various components of the compositions of the present invention are selected according to conventional techniques in the art. These compositions may be specifically constituted as protective, therapeutic or care ointments, milks, lotions or foams for use on the face, hands, body and / or mucous membranes, or for cleansing the skin. The compositions may also consist of solid preparations constituting soaps or cleansing bars.

[0396] The composition may contain adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, sunscreens, deodorants and dyes. The amount of these adjuvants is conventional for use in the field under consideration, for example, from about 0.01% to about 20% of the total weight of the composition. Depending on their nature, these adjuvants can be introduced into the fatty phase, into the aqueous phase and / or into the lipid vesicles.

[0397] For oral administration, the active agent can be readily formulated by combining the active agent with a pharmaceutically acceptable carrier well known in the art. The agents of the present invention can be formulated, for example, into tablets (including chewable tablets), pills, dragees, capsules, lozenges, hard candies, liquids, gels, syrups, slurries, powders, suspensions, elixirs, wafers, and the like, which are ingested orally by the patient to be treated. Such formulations may contain pharmaceutically acceptable carriers, including solid diluents or fillers, sterile aqueous media, and various non-toxic organic solvents. The solid carrier may be one or more substances that can also serve as a diluent, flavoring agent, stabilizer, lubricant, suspending agent, binder, preservative, tablet disintegrant, or encapsulating material. In powders, the carrier is generally a finely divided solid that is mixed with the finely divided active ingredient. In tablets, the active ingredient is generally mixed with a carrier having the desired binding capacity in an appropriate ratio and compressed into the desired shape and size. Powders and tablets contain from about 1% to about 70% of the active compound. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting waxes, cocoa butter, etc. Generally, an amount sufficient to provide the desired unit dosage is included in an amount of about 0.5%, about 5%, about 10%, about 20%, or about 30% to about 50%, about 60%, about 70%, about 80%, or about 90% of the active agent by weight of the total composition for oral dosage forms.

[0398] Aqueous suspensions for oral use may contain the active agent and pharmaceutically acceptable excipients such as suspending agents (e.g., methylcellulose), wetting agents (e.g., lecithin, lysolecithin and / or long-chain fatty alcohols), as well as colorants, preservatives, flavorings, and the like.

[0399] An oil or non-aqueous solvent may be required to bring the active agent into solution due to, for example, the presence of a large lipophilic moiety. Alternatively, an emulsion, suspension, or other preparation, for example, a liposomal preparation, may be used. For liposomal preparations, any known method for preparing liposomes for treating a condition may be used. See, for example, Bangham et al., J. Mol. Biol. 23:238-252 (1965) and Szoka et al., Proc. Natl. Acad. Sci. USA 75:4194-4198 (1978), incorporated herein by reference. Ligands may also be attached to liposomes to direct these compositions to specific sites of action.

[0400] Pharmaceutical preparations for oral use can be obtained by the following method: with solid excipients, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable adjuvants (if necessary) to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; flavor elements, cellulose products such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate can be added. The agent can also be formulated into a sustained-release preparation.

[0401] The dragee core can be provided with a suitable coating. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments can be added to the tablets or dragee coatings for identification or characterization of different combinations of active agents.

[0402] Orally available pharmaceutical formulations include push-fit capsules made of gelatin and sealed soft capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). Push-fit capsules can contain the active ingredient, which can be mixed with a filler such as lactose, a binder such as starch and / or a lubricant such as talc or magnesium stearate and an optional stabilizer. In the soft capsule, the active agent can be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin or liquid polyethylene glycol. In addition, a stabilizer can be added. The dosage of all orally administered formulations should be suitable for the mode of administration.

[0403] Other forms suitable for oral administration include liquid form preparations, including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations, which can be converted to liquid form preparations shortly before use. Emulsions can be prepared as solutions, for example, aqueous polyethylene glycol solutions or can contain emulsifiers, for example, lecithin, sorbitan monooleate, or gum arabic. Aqueous solutions can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavorings, stabilizers, and thickeners. Aqueous suspensions can be prepared by dispersing the finely divided active ingredient in water with a viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. Suitable fillers or vehicles that can be administered with the composition include sugars, alcohols, fats, lactose, starch, cellulose derivatives, polysaccharides, polyvinyl pyrrolidone, silicon oxide, sterile saline, etc., or mixtures thereof, used in appropriate amounts. Solid form preparations include solutions, suspensions, and emulsions and may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0404] Syrups or suspensions can be prepared by adding the active compound to a concentrated aqueous solution of a sugar, for example sucrose, to which any auxiliary agents may be added. Such auxiliary agents may include flavorings, agents to prevent crystallization of the sugar or agents to increase the solubility of any other ingredients, for example, polyols such as glycerol or sorbitol.

[0405] When formulating a compound for oral administration, it may be desirable to employ a gastroretentive formulation to enhance absorption from the gastrointestinal (GI) tract. Formulations that remain in the stomach for several hours can slowly release the compound of the invention and provide sustained release for use herein. Such gastroretentive formulations are described in Klausner, E.A.; Lavy, E.; Barta, M.; Cserepes, E.; Friedman, M.; Hoffman, A. 2003 "Novel gastroretentive dosage forms: evaluation of gastroretentivity and its effect on levodopa in humans." Pharm. Res. 20, 1466-73. Hoffman, A.; Stepensky, D.; Lavy, E.; Eyal, S. Klausner, E.; Friedman, M. 2004 "Pharmacokinetic and pharmacodynamic aspects of gastroretentive dosage forms." aspects of gastroretentive dosage forms" Int. J. Pharm. 11, 141-53, Streubel, A.; Siepmann, J; Bodmeier, R.; 2006 "Gastroretentive drug delivery systems" Expert Opin. Drug Deliver. 3, 217-3, and Chavanpatil, MD; Jain, P.; Chaudhari, S.; Shear, R.; Vavia, PR "Novel sustained release, swellable and bioadhesive gastroretentive drug delivery system for olfoxacin" Int. J. Pharm. 2006 epub Mar 24. Swellable floating bioadhesive technology can be used to maximize the absorption of the compounds of the present invention.

[0406] The solubility of the composition components can be enhanced by surfactants or other suitable cosolvents in the composition. Such cosolvents include polysorbates 20, 60, and 80, Pluronic F68, F-84, and P-103, cyclodextrins, or other agents known to those skilled in the art.

[0407] These cosolvents are typically used at levels of about 0.01 to 2 weight percent.

[0408] The compositions can be packaged in multiple doses. A preservative is preferably used to prevent microbial contamination during use. Suitable agents include benzalkonium chloride, thimerosal, chlorobutanol, methylparaben, propylparaben, phenylethyl alcohol, disodium edetate, sorbic acid, Onamer M, or other agents known to those skilled in the art. In prior art ophthalmic products, such preservatives may be employed at levels of 0.004% to 0.02%. In the compositions of the present invention, the preservative, preferably benzalkonium chloride, may be employed at levels of 0.001% to less than 0.01% by weight, for example, 0.001% to 0.008% by weight, and preferably about 0.005% by weight. A benzalkonium chloride concentration of 0.005% has been found to be sufficient to protect the compositions of the present invention from microbial attack.

[0409] In cases associated with topical administration, the composition may include one or more penetration enhancers. For example, the formulation may include a suitable solid or gel phase carrier or excipient that increases penetration and aids in the delivery of the agent or combination of agents of the invention across a permeability barrier, e.g., the skin. Many such penetration enhancing compounds are known to those skilled in the art of topical formulations and include, for example, water, alcohols (e.g., terpenes such as methanol, ethanol, 2-propanol), sulfoxides (e.g., dimethyl sulfoxide, decyl methyl sulfoxide, tetradecyl methyl sulfoxide), pyrrolidones (e.g., 2-pyrrolidone, N-methyl-2-pyrrolidone, N-(2-hydroxyethyl)pyrrolidone), laurocapram, acetone, dimethylacetamide, dimethylformamide, tetrahydrofurfuryl alcohol, L-α-amino acids, anionic, cationic, amphoteric or nonionic surfactants (e.g., isopropyl myristate and sodium lauryl sulfate), fatty acids, fatty alcohols (e.g., oleic acid), amines, amides, clofibric acid amide, hexamethylene lauramide, proteolytic enzymes, α-bisabolol, d-limonene, urea and N,N-diethyl-m-toluamide, among others. Other examples include humectants (e.g., urea), glycols (e.g., propylene glycol and polyethylene glycol), glyceryl monolaurate, alkanes, alkanols, water, organase, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and / or other polymers. In another embodiment, the composition may include one or more such penetration enhancers.

[0410] Compositions for topical application may contain one or more antimicrobial preservatives such as quaternary ammonium compounds, organomercuries, parabens, aromatic alcohols, chlorobutanol, and the like.

[0411] The composition can be formulated into an aerosol solution, suspension or dry powder. The aerosol can be administered through the respiratory system or nasal passages. For example, those skilled in the art will recognize that the composition of the present invention can be suspended or dissolved in a suitable carrier, for example, a pharmaceutically acceptable propellant, and directly administered to the lungs using a nasal spray or inhaler. For example, an aerosol formulation comprising a transporter, a carrier, or an ion channel inhibitor can be dissolved, suspended or emulsified in a propellant or a mixture of a solvent and a propellant, for example, for administration with a nasal spray or inhaler. The aerosol formulation can contain any acceptable propellant under pressure, such as a makeup or skin or pharmaceutically acceptable propellant, as conventionally used in the art.

[0412] Aerosol formulations for nasal administration are generally aqueous solutions designed to be administered to the nasal passages as drops or sprays. Nasal solutions can be similar to nasal secretions, i.e., they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values ​​outside this range can additionally be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0413] Aerosol preparations or inhalants that can be designed for suction are so that reagent or reagent combination are carried in the respiratory tract of object when giving by nose or oral respiratory route.Can for example give suction solution by nebulizer.Comprise the suction of fine powder or liquid medicine or be blown into the pharmaceutical aerosol of the solution or suspension of reagent or reagent combination in the propellant and be delivered to respiratory system, for example, auxiliary compensation.Propellant can be liquefied gas, comprises halogenated hydrocarbon, for example, and fluorocarbon is as fluorinated chlorinated hydrocarbon, hydrochlorofluorocarbon and hydrochlorocarbon and hydrocarbon and hydrocarbon ether.

[0414] Halogenated hydrocarbon propellants may include fluorocarbon propellants (in which all hydrogens are replaced by fluorine), chlorofluorocarbon propellants (in which all hydrogens are replaced by chlorine and at least one fluorine), hydrofluorocarbon propellants, and hydrochlorofluorocarbon propellants. Halogenated hydrocarbon propellants are described in U.S. Patent No. 5,376,359, issued December 27, 1994 to Johnson; U.S. Patent No. 5,190,029, issued March 2, 1993 to Byron et al.; and U.S. Patent No. 5,776,434, issued July 7, 1998 to Purewal et al. Hydrocarbon propellants useful in the present invention include, for example, propane, isobutane, n-butane, pentane, isopentane, and neopentane. Blends of hydrocarbons may also be used as propellants. Ether propellants include, for example, dimethyl ether and ether. The aerosol formulations of the present invention may also contain more than one propellant. For example, aerosol formulations can comprise more than one propellant of the same class, such as two or more fluorocarbons; or more than one, more than two, or more than three propellants from different classes, such as fluorocarbons and hydrocarbons. Pharmaceutical compositions of the present invention can also be dispersed with compressed gases, such as inert gases such as carbon dioxide, nitric oxide, or nitrogen.

[0415] Aerosol formulations may also include other components, for example, ethanol, isopropyl alcohol, propylene glycol, and surfactants or other components such as oils and detergents. These components may be used to stabilize the formulation and / or lubricating components.

[0416] Aerosol formulations can be packaged under pressure and can be formulated as aerosols for use solutions, suspensions, emulsions, powders and semisolid preparations. For example, solution aerosol formulations can be included in a reagent of the present invention in a (substantially) pure propellant, such as a solution of a transporter, carrier or ion channel inhibitor, or a mixture of a propellant and a solvent. Solvents can be used for dissolving reagents and / or retarding propellant evaporation. Solvents can include, for example, water, ethanol and glycol. Any combination of suitable solvents can optionally be used in conjunction with preservatives, antioxidants, and / or other aerosol components.

[0417] Aerosol formulations can be dispersions or suspensions. Suspension aerosol formulations can comprise a suspension of an agent or combination of agents of the present invention, e.g., a transport agent, a carrier, or an ion channel inhibitor, and a dispersant. Dispersants can include, for example, sorbitan trioleate, oleoyl alcohol, oleic acid, lecithin, and corn oil. Suspension aerosol formulations can also include lubricants, preservatives, antioxidants, and / or other aerosol components.

[0418] Aerosol formulations can be formulated similarly to emulsions. Emulsion aerosol formulations can include, for example, an alcohol such as ethanol, a surfactant, water, and a propellant, as well as an agent or combination of agents of the present invention, such as a transport agent, a carrier, or an ion channel. The surfactant used can be nonionic, anionic, or cationic. An example of an emulsion aerosol formulation includes, for example, ethanol, a surfactant, water, and a propellant. Another example of an emulsion aerosol formulation includes, for example, a vegetable oil, glyceryl monostearate, and propane.

[0419] The compound can also be formulated for administration as a suppository. A low melting wax, such as a mixture of triglycerides, fatty acid glycerides, Witepsol S55 (a trademark of Dynamite Nobel Chemical, Germany), or cocoa butter is first melted and the active ingredient is evenly dispersed, for example, by stirring. The homogeneous melted mixture is then poured into convenient sized molds, allowed to cool, and thereby solidify.

[0420] The compounds may be formulated for vaginal administration. Pessaries, tampons, creams, gels, patches, foams or sprays known in the art may also be suitable.

[0421] The compound can be releasably attached to a biocompatible polymer for a sustained release formulation, or attached to an inert object for topical, intraocular, periocular, or systemic administration. Controlled release from biocompatible polymers can also be used with water-soluble polymers to form perfusable formulations. Controlled release from biocompatible polymers such as PLGA microspheres or nanospheres can be used for formulations that are implanted in the eye or injected for sustained release administration. Any suitable biodegradable and biocompatible polymer can be used.

[0422] Dosage, route of administration, and treatment regimen

[0423] The compositions and methods described herein can elicit an immune response in a subject against an epitope of an antigenic peptide. In some cases, the composition can be a breast cancer vaccine or an ovarian cancer vaccine. In some cases, the breast cancer vaccine can be a multi-antigen breast cancer vaccine. In some cases, the ovarian cancer vaccine can be a multi-antigen ovarian cancer vaccine.

[0424] In some cases, the subject may have a tumor before the vaccine is administered. In other cases, the subject may not have a tumor before the vaccine is administered. In other cases, the subject may not have a tumor before the vaccine is administered, but may have a tumor after the vaccine is administered. In other cases, the subject may not have a tumor before the vaccine is administered and may not have a tumor after the vaccine is administered. In some cases, the tumor may be a breast cancer tumor. In some cases, the breast cancer tumor in rodents is a DMBA-induced tumor. For example, the breast cancer tumor in rodents may be derived from M6 or MMC cells. Typically, the breast cancer tumor in humans is a triple-negative tumor in humans.

[0425] The compositions described herein can be administered as vaccines to subjects in need thereof. In some cases, a multi-antigen breast cancer vaccine or a multi-antigen ovarian cancer vaccine can be used to immunize a subject. For example, the vaccine can be a breast cancer vaccine (e.g., a multi-antigen vaccine) or an ovarian cancer vaccine (e.g., a multi-antigen vaccine).

[0426] Vaccine as herein described can be delivered by various approaches. Delivery route can include oral (including buccal and sublingual), rectal, nasal, local, transdermal patch, through lung, vagina, suppository or parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) administration or be suitable for by aerosolization, suction or insufflation administration form. The general content of drug delivery system is found in Ansel etc., " pharmaceutical dosage form and drug delivery system " (Pharmaceutical Dosage FormsandDrug Delivery Systems) (Lippencott Williams&Wilkins, Baltimore Md.(1999)). Vaccine as herein described can give muscle, or can be given by intradermal or subcutaneous injection, or transdermal, such as by iontophoresis. The epidermis of vaccine can be used to give.

[0427] In some cases, vaccines can also be formulated for administration through the nasal passages. Formulations suitable for nasal administration (where the carrier is a solid) include coarse powders having a particle size range of about 10 to about 500 microns, which are administered in the manner of snuff, i.e., rapidly inhaled through the nasal passages from a powder container held near the nose. The formulation can be administered as a nasal spray, nasal drops, or by aerosol administration via a nebulizer. The formulation can comprise an aqueous or oily solution of the vaccine.

[0428] The vaccine may be a liquid preparation such as a suspension, syrup or elixir. The vaccine may also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (eg, administration by injection), such as a sterile suspension or emulsion.

[0429] The vaccine may contain material for a single immunization, or it may contain material for multiple immunizations (i.e., a "multi-dose" kit). Multi-dose formulations preferably contain a preservative. As an alternative (or in addition) to including a preservative in a multi-dose composition, the composition may be contained in a container equipped with a sterile adapter for removal of the material.

[0430] The vaccine can be administered in a dose volume of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mL. Sometimes, the vaccine can be administered in a higher dose, for example, greater than 1 ml.

[0431] In some cases, a single dose of a vaccine may be used to immunize a subject. In other cases, more than one dose of a vaccine may be used to immunize a subject. For example, a subject may be immunized with more than one dose, more than two doses, more than three doses, more than four doses, more than five doses, more than six doses, more than seven doses, more than eight doses, more than nine doses, more than ten doses, more than eleven doses, more than twelve doses, more than thirteen doses, more than fourteen doses, more than fifteen doses, more than sixteen doses, more than seventeen doses, more than eighteen doses, more than nineteen doses, or more than twenty doses of a vaccine. In an exemplary case, a subject is immunized with three doses of a vaccine.

[0432] In the case where the object receives more than 1 dose of vaccine, time can pass between the first dose of vaccine and each subsequent dose. In some cases, the time that passes between the first dose of vaccine and each subsequent dose can be seconds, minutes, hours, days, weeks, months or years. For example, the object can be given more than 1 dose at intervals. In some cases, the interval can be seconds, minutes, hours, days, weeks, months or years. In some cases, the object can accept a booster. For example, a vaccine that exceeds 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses, 13 doses, 14 doses, 15 doses, 16 doses, 17 doses, 18 doses, 19 doses or 20 doses of boosters can be used to give a booster to the object. In an exemplary case, the object can accept up to 3 doses of booster vaccines.

[0433] In some cases, the interval may be the same between doses of the vaccine. In some cases, the interval may be the same between booster doses of the vaccine. In some cases, the interval may vary between doses of the vaccine. In some cases, the interval may vary between booster doses of the vaccine.

[0434] In exemplary cases, more than one dose is administered to a subject at intervals of at least 1 day. In some cases, the intervals can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days apart. In other cases, the interval can be a range of days, for example, the range of days can be 1-5 days, 1-7 days, 1-10 days, 3-15 days, 5-10 days, 5-15 days, 5-20 days, 7-10 days, 7-15 days, 7-20 days, 7-25 days, 10-15 days, 10-20 days, 10-25 days, 15-20 days, 15-25 days, 15-30 days, 20-30 days, 20-35 days, 20-40 days, 20-50 days, 25-50 days, 30-50 days, 35-50 days, or 40-50 days.

[0435] Can evaluate object after giving vaccine.In some cases, can evaluate object in 1 month (for example, short-term) at last giving vaccine.For example, short-term can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or 31 days after last giving vaccine.In some cases, can evaluate object in 4 months (for example, regular period) at last giving vaccine. For example, a short term can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, or 31 weeks after the last administration of the vaccine.

[0436] In some cases, a subject may receive at least one vaccine booster dose after the last vaccine dose is administered. For example, at least one booster dose may be administered to a subject 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, or 31 weeks after the last vaccine dose is administered. In some cases, a subject may receive 1 booster, 2 boosters, 3 boosters, 4 boosters, 5 boosters, 6 boosters, 7 boosters, 8 boosters, 9 boosters, 10 boosters, 11 boosters, 12 boosters, 13 boosters, 14 boosters, 15 boosters, 16 boosters, 17 boosters, 18 boosters, 19 boosters, 20 boosters, 21 boosters, 22 boosters, 23 boosters, 24 boosters, 25 boosters, 26 boosters, 27 boosters, 28 boosters, 29 boosters, or 30 boosters.

[0437] In another aspect, the present invention provides a kit comprising an intradermal drug delivery device and a vaccine formulation as described herein. The device is preferably filled with the vaccine. Preferably, the vaccine is in a liquid volume smaller than that of conventional intramuscular vaccines, as described herein, specifically a volume of about 0.05 ml to 0.2 ml. Preferably, the device is a short-needle delivery device for administering the vaccine to the dermis.

[0438] Suitable devices for the intradermal vaccines described herein include short needle devices such as those described in US 4,886,499, US 5.190,521, US 5,328,483, US 5,527,288, US 4,270,537, US 5,015,235, US 5,141,496, US 5,417,662. Intradermal vaccines may also be administered by devices that limit the effective penetration length of the needle in the skin and their functional equivalents, such as those described in WO 99 / 34850, which is incorporated herein by reference. Jet injection devices are also suitable, which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle that penetrates the stratum corneum and produces a jet that reaches the dermis. Jet injection devices are described, for example, in US 5,480,381, US 5,599,302, US 5,334,144, US 5,993,412, US 5,649,912, US 5,569,189, US 5,704,911, US 5,383,851, US 5,893,397, US 5,466,220, US 5,339,163, US 5,312,335, US 5,503,627, US 5,064,413, US 5,520,639, US 4,596,556, US 4,790,824, US 4,941,880, US 4,940,460, WO 97 / 37705 and WO 97 / 13537. Ballistic powder / particle delivery systems are also suitable, which use compressed gas to accelerate the vaccine in powder form through the outer layer of the skin to the dermis. In addition, conventional syringes can be used in the classic Mantoux test for intradermal administration. However, the use of conventional syringes requires a highly skilled operator, and a device that can accurately deliver without requiring a highly skilled user is preferred.

[0439] Another aspect of the present invention relates to a method for immunizing a subject or group of subjects against a disease to prevent the disease and / or reduce the severity of the disease in the subject or group of subjects. The method comprises the step of administering a composition of the present invention to a subject or group of subjects that is not infected with the disease (or is believed to be not infected with the disease).

[0440] The composition of one embodiment of the present invention can be administered using techniques well known to those skilled in the art. Preferably, the compound can be formulated and administered by genetic immunization. The technology of formulation and administration can be found in Remington's Pharmaceutical Sciences, 18th edition, 1990, Mark Publishing Company of Easton, Pennsylvania. Suitable routes may include parenteral delivery, such as intramuscular, intradermal, subcutaneous, intramedullary, and intrathecal, direct intraventricular, intravenous, intraperitoneal, or intraocular injection, etc. Other routes include oral or transdermal delivery. With regard to injection, the composition of one embodiment of the present ...

Claims

1. An isolated and purified plasmid comprising at least one nucleotide sequence encoding a polypeptide having at least 95% sequence identity with SEQ ID NO:89, and wherein said polypeptide has 100% sequence identity with each of SEQ ID NO:54, 73, 85, and 87.

2. The plasmid according to claim 1, wherein The at least one nucleotide sequence encodes a polypeptide having 100% sequence identity with SEQ ID NO:

89.

3. The plasmid according to any one of claims 1-2, characterized in that The plasmid is 50%, 60%, 70%, 80%, 90%, or 100% pure.

4. The plasmid according to any one of claims 1-2, characterized in that, The plasmid is an expression vector.

5. The plasmid according to claim 4, characterized in that, The expression vector includes pUMVC3.

6. A composition comprising: a) A plasmid comprising a nucleotide sequence encoding a polypeptide having at least 95% sequence identity with SEQ ID NO:89, and wherein said polypeptide has 100% sequence identity with each of SEQ ID NO:54, 73, 85, and 87; and b) An excipient.

7. The composition according to claim 6, wherein The plasmid comprises a nucleotide sequence encoding a polypeptide having 100% sequence identity with SEQ ID NO:

89.

8. A composition comprising a polypeptide having at least 95% sequence identity with SEQ ID NO:89, and wherein said polypeptide has 100% sequence identity with each of SEQ ID NO:54, 73, 85, and 87.

9. The composition according to claim 8, wherein The polypeptide has 100% sequence identity with SEQ ID NO:

89.

10. The composition according to claim 6 or 8, further comprising an adjuvant.

11. The composition according to claim 10, wherein, The adjuvant includes GM-CSF.

12. The composition according to any one of claims 6-9, characterized in that, The composition is administered by subcutaneous injection, intradermal injection, intramuscular injection, intravascular injection, topical application, or inhalation.

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