1. An isolated and purified plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, or 87, having at least 95% sequence identity to the epitope sequence of the polypeptide.

2. The plasmid of claim 1 wherein said at least one nucleotide sequence encodes a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, or 87, having at least 100% sequence identity.

3. The plasmid of claim 1 wherein said at least one nucleotide sequence encodes a polypeptide that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 54. 73, 85, or 87, and 100% sequence identity over the entire length of the epitope sequence.

4. The plasmid of any one of claims 1-3, wherein said at least one nucleotide sequence encodes a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 95% sequence identity to at least 20 contiguous amino acids.

5. The plasmid of any one of claims 1-4, wherein said at least one nucleotide sequence encodes a nucleotide sequence identical to SEQ ID NO: 87 having at least 95% sequence identity to at least 60 contiguous amino acids.

6. The plasmid of any one of claims 1-5, wherein the isolated plasmid comprises at least 4 nucleotide sequences.

7. The plasmid of claim 6 wherein each of said at least 4 nucleotide sequences independently encodes a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 54. 73, 85, or 87, having at least 95% sequence identity.

8. The plasmid of any one of claims 1-7, wherein the isolated plasmid comprises 4 nucleotide sequences.

9. The plasmid of claim 8 wherein each of said 4 nucleotide sequences independently encodes a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 54. 73, 85, or 87, having at least 95% sequence identity.

10. The plasmid of claim 8 wherein each of said 4 nucleotide sequences encodes a different polypeptide.

11. The plasmid of claim 10 wherein each of said different polypeptides is identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, or 87, has at least 95% sequence identity.

12. The plasmid of any one of claims 10-11 wherein said 4 nucleotide sequences are joined in tandem in said plasmid.

13. The plasmid of any one of claims 10 to 12 wherein the 4 nucleotide sequences are separated by a linker nucleic acid sequence.

14. The plasmid of any one of claims 1-13, wherein the plasmid is about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% pure.

15. The plasmid of any one of claims 1-14, wherein the plasmid is an expression vector.

16. The plasmid of claim 15 wherein said expression vector comprises pUMCC 3.

17. A composition, comprising:

a) a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to SEQ ID NO: 89 nucleotide sequences of polypeptides having at least 95% sequence identity; and

b) and (3) an excipient.

18. The composition of claim 17, wherein the plasmid comprises a nucleotide sequence encoding a polypeptide substantially identical to SEQ ID NO: 89 and a polypeptide having 100% sequence identity thereto.

19. The composition of claim 17, wherein the plasmid comprises a nucleotide sequence encoding a polypeptide that hybridizes to SEQ ID NO: 89, is 100%.

20. A composition comprising a peptide that is identical to SEQ ID NO: 89 polypeptide having at least 95% sequence identity.

21. The composition of claim 20, wherein the polypeptide hybridizes to SEQ ID NO: 89 has 100% sequence identity.

22. The composition of claim 20, wherein the polypeptide hybridizes to SEQ ID NO: 89, is 100%.

Background

Cancer treatment has traditionally been accomplished by surgical reduction of tumor weight followed by chemotherapy and/or radiation therapy. This strategy may reduce the tumor and, at an earlier stage, often results in complete remission. Unfortunately, the prognosis for more advanced tumors has changed little over the past 50 years and a large number of cancer-related deaths have resulted from subsequent metastases. New prophylactic and therapeutic treatments are needed to combat the growing number of cancers.

More than one million people worldwide are diagnosed with breast cancer each year and more than 400000 people die of breast cancer each year. It is estimated that 1 out of 8 women will be diagnosed with breast cancer at some point in their life. Preventing the development of breast cancer would have significant health and economic benefits for all individuals. If one no longer needs to receive expensive cancer-related monitoring and therapeutic intervention, one would save billions of dollars. New methods for preventing and treating breast cancer are needed.

Summary of The Invention

In some aspects, 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 a cell associated with breast cancer; and a second nucleotide sequence encoding a second epitope of a second antigen expressed by a cell associated with breast cancer, wherein the first and second nucleotide sequences are located in one or more plasmids.

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

In some aspects, the compositions described herein comprise a composition comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen, said first epitope being part of a peptide selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, and MDM2, wherein said first nucleotide sequence is located in the plasmid. In other aspects, the invention includes a composition comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence encoding a second epitope of a second antigen, wherein the first and second epitopes are independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3, and MDM2, wherein the first nucleotide sequence and the second nucleotide sequence are located in one or more plasmids.

In some aspects, the compositions described herein comprise a composition comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen, said first epitope being part of a peptide selected from the group consisting of IGFBP-2, HER-2, IGF-1R, wherein said first nucleotide sequence is located in the plasmid. In other aspects, the invention includes a composition comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence encoding 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 and second nucleotide sequences are located in one or more plasmids.

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

In another aspect, the invention includes a composition comprising: at least a first epitope of a first antigen, said first epitope being part of a peptide from HIF-1 α. In other aspects, the invention includes a composition comprising: at least a first epitope of a first antigen, at least a second epitope of a second antigen, said first and second epitopes being from HIF-1 α.

In other aspects, the invention includes a composition comprising: at least a first epitope of a first antigen, said first epitope being part of a peptide selected from the group consisting of CD105, Yb-1, SOX-2, CDH3 or MDM 2. In other aspects, the invention includes a composition comprising: at least a first epitope of a first antigen, at least a second epitope of a second antigen, said first and second epitopes being independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3 or MDM 2.

In some cases, the invention includes a composition comprising an isolated and purified plasmid comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises a plurality of epitope(s), and an excipient. Sometimes, the plurality(s) of epitopes includes at least one epitope that is identical to a sequence 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, comprise one or more epitopes having at least 90% sequence identity.

In some cases, the isolated and purified plasmid may further comprise a first nucleotide sequence encoding a first epitope of a first antigen expressed by a cell 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 a cell 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 the group consisting of SEQ ID NOs: 82-84, or a fragment thereof, having at least 90% sequence identity to the amino acid sequence of HIF-1 alpha peptide. 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 on 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.

In some cases, the invention includes a composition comprising a first epitope of a first antigen expressed by a cell associated with breast or ovarian cancer; and a second epitope of a second antigen expressed by a cell associated with breast or ovarian cancer; wherein the first and second epitopes are independently identical to a sequence 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, have at least 90% sequence identity.

In some cases, the present invention includes a composition comprising a plasmid comprising at least one nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 70% sequence identity to the epitope sequences of said polypeptides.

In some cases, the present invention includes a composition comprising a plasmid comprising 4 nucleotide sequences, wherein each of the 4 nucleotide sequences independently encodes a polypeptide having an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 70% sequence identity to the epitope sequence.

In some cases, the invention includes a composition comprising a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to SEQ ID NO: 89, or a polypeptide having at least 80% sequence identity thereto.

In some cases, the invention includes compositions comprising a polypeptide that hybridizes to SEQ ID NO: 89 has at least 80% sequence identity.

In some cases, disclosed herein are methods of administering one or more of the compositions described herein to a subject. At times, the subject may require one or more compositions.

Sometimes, described herein is a method 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 may be ovarian cancer. The cancer may 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.

Sometimes, described herein is a method 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 may be ovarian cancer. The cancer may 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.

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

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.

The present invention also includes an isolated and purified plasmid comprising at least one nucleic acid sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 82-84, and a polypeptide having at least 90% sequence identity to the epitope sequence. The isolated and purified plasmid may comprise a set of 2 or more nucleotide sequences, wherein each of the 2 or more nucleotide sequences independently encodes a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 82-84, or a polypeptide having at least 90% sequence identity thereto. The isolated and purified plasmid may comprise 2 or more nucleotide sequences, wherein each of the 2 or more nucleotide sequences encode a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 82-84, having at least 90% sequence identity; and each nucleotide within the group of 2 or more nucleotide sequences is not identical.

The present invention may also include an isolated and purified plasmid comprising at least one nucleic acid sequence encoding a polypeptide substantially identical 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 a polypeptide having at least 90% sequence identity to the epitope sequence. The isolated and purified plasmid may comprise a set of 2 or more nucleotide sequences, wherein each of the 2 or more nucleotide sequences independently encodes a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1. 6, 8-10, 14-16, 20, 25-28, and 32-34, and at least 90% sequence identity. The isolated and purified plasmid may comprise a set of 2 or more nucleotide sequences, wherein each of the 2 or more nucleotide sequences encodes a nucleotide sequence identical 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 at least 90% sequence identity; and each nucleotide within the group of 2 or more nucleotide sequences is not identical.

The present invention may include an isolated and purified plasmid comprising at least one nucleic acid sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 46-56, 60-62, or 66-75, or a polypeptide having at least 90% sequence identity to the epitope sequence. The isolated and purified plasmid may comprise a set of 2 or more nucleotide sequences, wherein each of the 2 or more nucleotide sequences independently encodes a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 46-56, 60-62, or 66-75, or at least 90% sequence identity. The isolated and purified plasmid may comprise a set of 2 or more nucleotide sequences, wherein each of the 2 or more nucleotide sequences encodes a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 46-56, 60-62, or 66-75, or a polypeptide having at least 90% sequence identity; and each nucleotide within the group of 2 or more nucleotide sequences is not identical.

In some cases, the invention may also include an isolated and purified plasmid comprising at least one nucleic acid sequence encoding a polypeptide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 70% sequence identity to the epitope sequences of said polypeptides.

Is incorporated by reference

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.

Brief description of the drawings

The novel features believed characteristic of the invention are set forth 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 in which the principles of the invention may be utilized:

fig. 1 demonstrates that the Th1 and Th2 epitopes differ in functional affinity.

Fig. 2 shows the antitumor efficacy of Th2 in eliminating Th 1.

Figure 3 shows antigen-specific IgG immunization.

Figure 4 shows population-based epitope screening.

Fig. 5 demonstrates the characteristics of breast cancer subjects.

Figure 6 shows antigen specific IFN γ responses against stem cell/EMT proteins.

FIG. 7 shows antigen specific IL-10 responses to stem cell/EMT proteins.

FIG. 8 shows epitope verification of the extended sequence as a native epitope with the CD105 extended epitope (52aa) QNGTWPREVLLVLSVNS SVFLHL QALGI PLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: 1).

FIG. 9 shows an extended epitope of Yb-1 based on the IFN γ/IL-10 activity ratio.

Figure 10 shows the magnitude and incidence of IFN γ predomination. CDH3 antigen IFN gamma/IL-10 activity ratio.

Figure 11 shows the magnitude and incidence of IFN γ predomination. The ratio of IFN γ/IL-10 activity of HIF1 α antigen.

Figure 12 shows a graph of simultaneous in vivo evaluation in mice.

FIG. 13 shows the immunogenicity and efficacy of an exemplary Yb-1 plasmid-based vaccine in mice.

Figure 14 shows a map, immunogenicity, and exemplary sequences of the compositions described herein. SEQ ID NO: 39 as shown in figure 14.

FIG. 15A shows exemplary validation of peptide-specific T-cells as native epitopes.

Figure 15B shows a timeline of a clinical trial using the compositions described herein.

Figure 15C shows a study protocol for a phase I clinical trial using the compositions described herein.

FIG. 16 shows a Western blot analysis of HIF1 α expression in single particles, pHIF1 α, and plasmid BCMA5 encoding 5 antigens.

Figure 17 shows the magnitude and incidence of IFN γ predomination. Ratio of IFN γ/IL-10 activity of CD105 antigen.

Figure 18 shows the magnitude and incidence of IFN γ predomination. The ratio of IFN γ/IL-10 activity of MDM-2 antigen.

Figure 19 shows the magnitude and incidence of IFN γ predomination. Ratio of IFN γ/IL-10 activity of SOX-2 antigen.

Figure 20 shows the magnitude and incidence of IFN γ predomination. IFN gamma/IL-10 activity ratio of Yb-1 antigen.

Figure 21 shows the immunogenicity and efficacy of HIF1 a peptide and plasmid vaccines in mice.

Figure 22 shows the immunogenicity and efficacy of CD105 peptide and plasmid vaccines in mice.

Figure 23 shows the immunogenicity and efficacy of CDH3 peptide and plasmid vaccine in mice.

Figure 24 shows the immunogenicity and efficacy of SOX2 peptide and plasmid vaccines in mice.

Figure 25 shows the immunogenicity and efficacy of MDM2 peptide and plasmid vaccines in mice.

Figure 26 shows the mouse mass 3 months after the last vaccine.

Figure 27 shows the mouse mass 10 days after the last vaccine.

FIG. 28 demonstrates that IGFBP-2C-terminal versus N-terminal induces epitope enrichment of IL-10-secreting T-cells.

FIG. 29 shows that N-terminal, but not C-terminal IGFBP-2 vaccines simultaneously stimulate type I immunity and inhibit tumor growth.

FIG. 30 shows the anti-tumor effect of IGFBP-2 vaccine-induced Th2 on the elimination of IGFBP-2-specific Th 1.

Figure 31 demonstrates that a vaccine based on the HER2 Th1 epitope can increase survival rates, which correlates with an epitope spreading in advanced HER2+ breast cancer patients.

Figure 32 demonstrates that extended Th plasmid based vaccines are more effective than peptide based vaccines in generating tumor antigen specific Th1 immunity.

Figure 33 shows persistent HER2 ICD specific immunity for more than one year after plasmid DNA-based immunization was concluded.

FIG. 34 shows IGF-1R epitopes screened for IFN γ and IL-10T-cell secretion by ELISPOT.

FIG. 35 demonstrates that the polyepitope IGF-1R vaccine inhibits growth of implanted breast cancer.

Figure 36 shows that the multiple antigen multiple epitope vaccine prevents breast cancer development in mice.

Figure 37 shows an exemplary cytokine secretion pattern induced by HER2 immunization.

Figure 38 shows ROC analysis of stem cells/EMT antigens.

Figure 39 shows candidate proteins that are overexpressed in stem cells and/or EMTs.

FIG. 40 shows an animated representation of the constructs described herein.

FIG. 41 shows a Western blot image of IGFBP-2, survivin, HIF-1A, and IGF-IR expression.

Fig. 42A-fig. 42D show the anti-tumor effect of the multiple antigen vaccine in the ID8 ovarian cancer transplantation model. Three weeks after implantation of ID8-Luc, mice were imaged on an IVIS bioluminescence imager. Total flow (photons/sec) was measured in the primary implant (fig. 42A) and the metastatic site (fig. 42B). Corresponding animals were shown adjuvant only (fig. 42C), and three antigen immunizations (fig. 42D).

Fig. 43A and 43B show TH1 response as a function of protein sequence. Selective TH1 inducible sequences were identified in the C-terminus of survivin and the N-terminus of HIF1 a. The average cSPW x incidence for each peptide is shown by donor type. The incidence of IFN-g cSPW x is shown on the positive y-axis for volunteer donors (n ═ 20) (white) and cancer donors (n ═ 20) (grey). The incidence of IL-10cSPW x is shown on the negative y-axis for volunteer donors (solid black) and cancer donors (dashed black). FIG. 43A shows TH1 responses for HIF-1A peptide. Fig. 43B shows TH1 response for survivin peptide. The vertical lines show the selected sequence.

FIGS. 44A-44D show a comparison of IgG antibody expression levels in ovarian cancer patients and volunteers. IgG antibodies specific for the candidate antigen were significantly elevated in ovarian cancer patients compared to the helper controls. IgG (in ug/ml) (y-axis) and experimental populations (X-axis) are shown for IGF-IR (FIG. 44A), IGFBP-2 (FIG. 44B), HIF-1A (FIG. 44C), and survivin (FIG. 44D). Mean and 2 standard deviations of volunteer controls (dashed line), p < 0.05; p < 0.01; p < 0.001.

Detailed Description

The present invention provides compositions of breast cancer vaccines and ovarian cancer vaccines, typically for the prevention or treatment of breast cancer or ovarian cancer. The invention also provides methods of administering a breast cancer vaccine or an ovarian cancer vaccine to a subject. The compositions set forth herein may be used in conjunction with the methods set forth herein to prevent or treat breast or ovarian cancer.

In some cases, the composition may include: a nucleic acid sequence encoding an epitope of a breast or ovarian cancer antigen that elicits an immune response in a subject, a plasmid comprising the sequence described herein, an adjuvant, a pharmaceutical carrier, and an inert chemical suitable for use in a pharmaceutical composition. The breast or ovarian cancer antigen can be at least one of any antigen expressed in a subject who may have or may develop breast or ovarian cancer. Typically, breast or ovarian cancer antigens are expressed by breast cancer cells, ovarian cancer cells, and/or tissues such as breast or ovarian Cancer Stem Cells (CSCs). CSCs may exhibit the ability to self-renew, unregulated growth, and drug resistance. In some cases, a CSC may express a protein (e.g., an antigen), and for example, the level of protein (e.g., an antigen) expressed by a CSC may be up-regulated (e.g., increased expression relative to a given amount) or down-regulated (e.g., decreased expression relative to a given amount). In some cases, proteins upregulated by CSCs as compared to normal tissues or cells may be involved in the development and/or progression of breast or ovarian cancer. For example, proteins can be identified and epitopes targeted using the compositions and methods described herein.

In some cases, one breast or ovarian cancer epitope may be used in the composition. In other cases, more than one epitope of breast or ovarian cancer may 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 or ovarian cancer antigens may be used in the composition. In some cases, the antigens may be the same. In other cases, the antigens may be different. The compositions of the breast cancer or ovarian cancer vaccines described herein can be formulated for the prevention of breast cancer or ovarian cancer. For example, a prophylactic composition can eliminate cells (e.g., CSCs, such as breast CSCs or ovarian CSCs) having an aberrantly (e.g., upregulated) expressed protein to prevent breast cancer or ovarian cancer.

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 a breast or ovarian cancer antigen may be used in the composition. In other cases, more than one epitope on a breast or ovarian cancer antigen, 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 epitopes on a breast or ovarian cancer antigen may be used in the composition.

The compositions and methods described herein can elicit an immune response in a subject. The immune response can be an immune response against an epitope of an antigen in a composition (e.g., a vaccine). The vaccine arms the subject's immune system so that it can detect and destroy those substances in the subject that contain the antigen of the vaccine. The compositions and methods described herein can elicit a type 1 (Th1) immune response in a subject. The Th1 immune response may include secretion of inflammatory cytokines (e.g., IFN γ, TNF α) by a subset of immune cells (e.g., antigen-specific T cells). In some cases, the inflammatory cytokine activates another subset of immune cells (e.g., cytotoxic T cells), which can destroy those substances in the subject that contain the antigen.

Epitopes of various antigens can be screened for induction of a Th1 immune response using the screening methods described herein to identify epitopes and bind peptides from tumor antigens. For example, the screening method can identify epitopes (e.g., preferably resulting in secretion of Th1 cytokines) from at least one tumor antigen that elicits a Th1 response against breast or ovarian cancer antigens, including CSC antigens (e.g., breast CSCs or ovarian CSCs), as described herein.

In some cases, the epitopes and/or antigens for use in the compositions and methods described herein may be recognized by the subject's immune system to elicit a Th1 immune response and release a type I cytokine. Th1 responses can be elicited by interactions between epitopes and T cells, more specifically the Major Histocompatibility Complex (MHC) expressed by T cells. For example, high affinity binding of epitopes to MHC receptors can stimulate Th1 responses. The MHC receptor can be at least one of a plurality of types of MHC receptors. MHC receptors on T cells can vary between individuals in a population.

In addition to the nucleic acid encoding the epitope, the compositions described herein can 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 use in a pharmaceutical composition. 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 use in pharmaceutical compositions. In some cases, the composition may include at least one inert chemical suitable for use in a pharmaceutical composition and a pharmaceutical carrier. In some cases, the composition may contain various adjuvants, various pharmaceutical carriers, and various inert chemicals suitable for use in pharmaceutical compositions.

In some cases, an adjuvant may be used in the composition. In other cases, more than 1 adjuvant, more than 2 adjuvants, more than 3 adjuvants, more than 4 adjuvants, more than 5 adjuvants, more than 6 adjuvants, more than 7 adjuvants, more than 8 adjuvants, more than 9 adjuvants, or more than 10 adjuvants may be used in the composition. In some cases, a pharmaceutical carrier may be used in the composition. In other cases, more than 1 pharmaceutical carrier, more than 2 pharmaceutical carriers, more than 3 pharmaceutical carriers, more than 4 pharmaceutical carriers, more than 5 pharmaceutical carriers, more than 6 pharmaceutical carriers, more than 7 pharmaceutical carriers, more than 8 pharmaceutical carriers, more than 9 pharmaceutical carriers, or more than 10 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.

The invention also describes methods of administering a breast cancer vaccine or an ovarian cancer vaccine to a subject. In some cases, the method can include constructing a plasmid-based vaccine that targets these antigens and determining whether administration of the vaccine is safe, immunogenic, and effective in preventing breast cancer development. For example, the composition may be a vaccine based on a multi-antigenic Th1 multi-epitope plasmid. In some cases, the method can include performing at least one clinical trial to determine the safety and immunogenicity of the plasmid-based vaccine in a subject having breast or ovarian cancer. For example, an antigen may be expressed by or associated with a CSC (e.g., a breast CSC or an ovarian CSC), and/or convert a cell from an epithelial cell to a mesenchymal cell (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 a subject. For example, a Th1 immune response may include immune cells, typically CD4+ T cells. In some cases, a composition can be a nucleic acid (e.g., a plasmid-based vaccine) that can include a nucleic acid encoding more than one antigen or more than one epitope(s) of an antigen. In some cases, the methods can be used to determine whether a composition described herein prevents breast or ovarian cancer development in a variety of organisms, for example, in cancer (e.g., breast or ovarian cancer) models using genetically similar rodents (e.g., mice), using genetically diverse rodents (e.g., mice), and in subjects that may or may not have breast 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).

Antigen identification

The compositions and methods described herein include identifying and engineering breast or ovarian cancer antigens in a pharmaceutical composition (e.g., a vaccine). Although any technique known to one of ordinary skill in the art can be used to identify antigens expressed by a subject having breast or ovarian cancer, in exemplary cases, the methods described herein can be used to identify suitable antigens. In some cases, the method may comprise screening serum from the subject. In some cases, the screen may be an antibody screen. For example, the antibody screened may be an IgG antibody. In some cases, the serum may be from a subject with breast or ovarian cancer. In other cases, the serum may be from a subject that does not have breast or ovarian cancer.

A cancer antigen, for example, a breast cancer antigen or an ovarian cancer antigen, may be a portion of a protein, a portion of a peptide, or a portion of a polyamino acid. In some cases, the fraction may be a percentage of protein, a percentage of peptide, or a percentage of polyamino acids. In some cases, the percentage may 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 moiety may be located at the C-terminus of the protein, peptide, or polyamino acid. In other cases, the moiety may be located near the C-terminus of the protein, peptide or polyamino acid. For example, the vicinity of the C-terminus may be within 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the length of the total protein, peptide or polyamino acid from the midpoint. In some cases, the moiety may be located at the N-terminus of the protein, peptide, or polyamino acid. In other cases, the moiety may be located near the N-terminus of the protein, peptide or polyamino acid. For example, the vicinity of the N-terminus may be within 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the length of the total protein, peptide or polyamino acid from the midpoint. In some cases, the moiety may be located near the middle of a protein, peptide, or polyamino acid. In other cases, the moiety may be located near the middle of a protein, peptide, or polyamino acid. For example, the immediate vicinity may be within 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the total protein, peptide or polyamino acid length from the terminus.

At least one antigen can be identified and screened for suitability as an antigen in a composition (e.g., vaccine) described herein. In some cases, an antigen may be identified and screened. In other instances, 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, a method for screening a protein-related proteins, 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 were identified and screened for vaccine suitability.

The antigen for which the vaccine suitability is screened may be derived from any protein detected in serum from a subject having breast or ovarian cancer using screening techniques known to those of ordinary skill in the art. In some cases, the screen may generally be an antibody screen. While the protein may be any protein detected in serum from a subject having breast or ovarian cancer, in an exemplary case, the antigen-derived protein may be classified as a stem cell protein and/or an EMT protein. For example, in breast cancer, stem cell/EMT proteins may include SOX2, YB1, CD105, MDM2, CDH3 +/-and HIF1 alpha. In general, the antigen may be immunogenic both in a subject with breast cancer and in a subject without breast cancer.

Epitope mapping

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

In some cases, the epitopes may be Human Leukocyte Antigen (HLA) class I epitopes derived from breast or ovarian cancer antigens. For example, HLA class I epitopes can include epitopes that bind to HLA-A, -B, and-C molecules. In some cases, the epitope may be a class II epitope derived from a breast or ovarian cancer antigen for use in the development of a cancer vaccine (e.g., breast or ovarian cancer). For example, HLA class II epitopes 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 epitopes can be mapped using (1) determining whether the epitope binds to MHC (e.g., with high affinity) by at least one HLA allele (e.g., HLA-DR, a universal epitope), (2) determining whether the epitope stimulates IFN γ secretion other than IL-10 (e.g., from antigen-specific T-cells), and (3) determining whether the T-cells can recognize a peptide (e.g., an epitope) that is treated by an Antigen Presenting Cell (APC), i.e., a native epitope. In some cases, T-cell lines may be used. For example, the T-cell line may 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 to an epitope with high selectivity and avidity. In some cases, the epitope can be derived from a protein (e.g., a recombinant protein). In other cases, the protein may be a native protein. In some cases, the protein may be processed endogenously. In other cases, the protein may be processed exogenously. In some cases, the protein may be processed endogenously by autologous APCs. In other cases, the protein may be processed exogenously from the autologous APC.

In all cases, the peptide is an epitope mapped from an antigen, and can be identified for selection of peptide epitopes using the methods described herein. In some cases, the epitopes may be derived from human proteins that can be used directly in peptide-based vaccines. In other cases, the epitope may be derived from a human protein and the encoding nucleic acid sequence may be incorporated into a nucleic acid construct designed to induce expression of the epitope in a subject upon administration. For example, the nucleic acid construct may allow an immune response against at least one epitope(s) to be generated, amplified, attenuated, suppressed or eliminated from a specific set of proteins. In some cases, the peptide or nucleic acid construct may be optimized to induce, amplify, or generate a Th1 immune response based on protein or plasmid immunization. In some cases, the epitope may be an extended Th1 epitope. In other cases, the peptide or nucleic acid construct may be optimized for protein or plasmid-based immunization to suppress, attenuate, or eliminate a pathogenic response in a subject (e.g., a human or animal) in need thereof.

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

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

In some cases, the identified epitopes can be included in a vaccine composition of an extended peptide vaccine. In some cases, the extended epitope may be a 40-80 mer peptide. In one exemplary case, the nucleic acid sequence or peptide sequence is used in parallel to construct an extended peptide sequence. The juxtaposition of selected peptides within the parent protein (e.g., within 10 amino acids of each other) allows for the construction of tandem extended epitopes, which may contain tolerance and/or inhibition epitopes. For example, a tandem extended epitope may contain a short intervening < 10 amino acid sequence. Any of these peptides and/or extended epitopes alone (embodied as the peptides themselves, or the corresponding nucleic acid constructs) or any combination thereof may be optimized into protein or plasmid based immunity, which will specifically induce, amplify or generate a protective immune response in a subject (animal or human) in need thereof, or which will suppress, attenuate or eliminate a pathogenic response.

In some cases, an epitope may be a 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 20 amino acids, less than 25 amino acids, less than 30 amino acids, less than 80 amino acids, less than 100 amino acids, less than 110 amino acids, less than 60 amino acids, or less than 60 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.

In some cases, the present invention provides compositions comprising an isolated and purified plasmid comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises a plurality of epitope(s), and an excipient. In some cases, the plurality of epitope(s) includes a sequence identical to a sequence 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, comprise one or more epitopes having at least 90% sequence identity. In some cases, the plurality of epitope(s) includes a sequence identical to a sequence selected from SEQ ID NOs: the amino acid sequence of 82-84 contains one or more epitopes with at least 90% sequence identity. In some cases, the plurality of epitope(s) includes a sequence identical to a sequence selected from SEQ ID NOs: 1. 6, 8-10, 14-16, 20, 25-28, or 32-34, comprises one or more epitopes having at least 90% sequence identity. In some cases, the plurality of epitope(s) includes a sequence identical to a sequence selected from SEQ ID NOs: 46-56, 60-62, or 66-75, comprises one or more epitopes having at least 90% sequence identity. In some cases, the plurality of epitope(s) includes a sequence identical to a sequence selected from SEQ ID NOs: 54. the amino acid sequences of 73, 85, and 87 contain one or more epitopes that are at least 90% sequence identical. In some cases, the plurality of epitope(s) includes a sequence 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.

In some cases, the plurality of epitope(s) is a plurality of adjacent epitopes. In some cases, 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.

In some cases, the compositions also include other isolated and purified plasmids comprising other nucleotide sequences encoding additional polypeptides, wherein the other polypeptides comprise multiple epitope(s), including sequences that hybridize to a sequence selected from the group consisting of SEQ ID NOs: 1. amino acid sequences of 6, 8-10, 14-16, 20, 25-28, 32-34, 46-56, 60-62, 66-75, 82-85, and 87 contain one (more) or more (more) epitope(s) of at least 90% sequence identity. In some cases, the composition further comprises an additional isolated and purified plasmid comprising an additional nucleotide sequence encoding an additional polypeptide, wherein the additional polypeptide comprises 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 sequences of the polypeptide and the additional polypeptide are different.

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 IFN γ production to IL-10 production that is greater than 1. In some cases, the immune response is characterized by a ratio of IFN γ production to IL-10 production of less than 1.

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 is effective to elicit an immune response in a subject. In some cases, the composition is effective to eliminate a plurality of cells associated with breast cancer or ovarian cancer in the subject. In some cases, the compositions can be used to prevent the growth of cells associated with breast cancer or ovarian cancer in a subject.

In some cases, the cancer is breast cancer. In some cases, the breast cancer is a relapsed or refractory or metastatic breast cancer. In some cases, the cancer is ovarian cancer. In some cases, the ovarian cancer is a recurrent or refractory or metastatic ovarian cancer.

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

The invention also provides a kit for preparing a composition as described herein, the kit comprising instructions for preparing the composition. The invention also provides a kit for administering the compositions described herein, the kit comprising instructions for administering the compositions.

Compositions comprising epitopes of breast cancer vaccines

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 a cell associated with breast cancer; and a second nucleotide sequence encoding a second epitope of a second antigen expressed by a cell associated with breast cancer, wherein the first and second nucleotide sequences are located in one or more plasmids. In some cases, a composition may include a nucleic acid encoding an epitope from a protein: CD105, HIF1 alpha, MDM2, Yb1, SOX-2, HER-2, IGFBP2, IGF-1R, CDH3 and survivin

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

In some cases, the composition may include epitopes and nucleic acids encoding proteins from: CD105, MDM2, Yb-1, SOX-2, and CDH 3. In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide CD105 selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence CAGAACGGCACCTGGCCCCGCGAGGTGCTGCTGGTGCTGTCCGTGAACTCCTCCGTGTTCCTGCACCTACAGGCCCTGGGCATCCCCCTGCACCTGGCCTACAACTCCTCCCTGGTGACCTTCCAGGAGCCCCCCGGCGTGAACACCACCGAGCTG (SEQ ID NO: 2); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACCGTGTTCATGCGCCTGAACATCATCTCCCCCGACCTGTCCGGCTGCACCTCCAAGGGCCTGGTGCTGCCCGCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCTCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 3); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGGCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 4); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 5); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO: 6); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence QNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: 7); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 8); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 9); or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 10). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide Yb-1 selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACCGCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAAC (SEQ ID NO: 11); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 12); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 13); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence EDVFVHQTAIKKNNPRK (SEQ ID NO: 14); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence YRRNFNYRRRRPEN (SEQ ID NO: 15); or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence GVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN (SEQ ID NO: 16). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide SOX-2 selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTG (SEQ ID NO: 17); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 18); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 19); or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSV (SEQ ID NO: 20). In some cases, the composition may include a nucleic acid sequence encoding an epitope of peptide CDH3 selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence AGGTCACTGAAGGAAAGGAATCCATTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 21); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence TTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 22); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GCCATGCACTCCCCCCCCACCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTTCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGATCTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGTCC (SEQ ID NO: 23); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence GTGATGAACTCCCCCCCCTCCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTCCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGCTGTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGACC (SEQ ID NO: 24); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 25); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence LKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 26); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence AMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKES (SEQ ID NO: 27); or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence VMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKET (SEQ ID NO: 28). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide MDM2 selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACCTACACCATGAAGGAGGTGCTGTTCTACCTGGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACCTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAaATCTACACCATGATCTACCGCAACCTGGTGGTGGTGAACCAGCAGGAGTCCTCCGACTCCGGCACCTCCGTGTCC (SEQ ID NO: 29); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGCCGTGTCCCAGCAGGACTCCGGCACCTCCCTGTCC (SEQ ID NO: 30); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGTGGTGTCCCAGCAGGACTCCGGCACCTCCCCCTCC (SEQ ID NO: 31); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSV (SEQ ID NO: 32); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLS (SEQ ID NO: 33); or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence IYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPS (SEQ ID NO: 34).

In exemplary cases, the composition may include a nucleic acid sequence encoding a fusion peptide of 5 (one) epitopes selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGGCGGTACCCATGCAACTGTCCTGCTCTAGACAGAACGGCACCTGGCCCCGCGAGGTGCTGCTGGTGCTGTCCGTGAACTCCTCCGTGTTCCTGCACCTACAGGCCCTGGGCATCCCCCTGCACCTGGCCTACAACTCCTCCCTGGTGACCTTCCAGGAGCCCCCCGGCGTGAACACCACCGAGCTGAGATCCACCGGTGGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACCGCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAACACGCGTGGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGAGATCCCAATTGAGGTCACTGAAGGAAAGGAATCCATTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACAAGATCCGCCGGCGAAACCTACACCATGAAGGAGGTGCTGTTCTACCTGGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACCTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAAATCTACACCATGATCTACCGCAACCTGGTGGTGGTGAACCAGCAGGAGTCCTCCGACTCCGGCACCTCCGTGTCCAGATCTTAG (SEQ ID NO: 35); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGGCGGTACCCATGACCGTGTTCATGCGCCTGAACATCATCTCCCCCGACCTGTCCGGCTGCACCTCCAAGGGCCTGGTGCTGCCCGCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCTCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCCACCGGTGGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACCGCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAACACGCGTGGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGAGATCCCAATTGTTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACAAGATCCGCCGGCGAAACCTACACCATGAAGGAGGTGCTGTTCTACCTGGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACCTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAAATCTACACCATGATCTACCGCAACCTGGTGGTGGTGAACCAGCAGGAGTCCTCCGACTCCGGCACCTCCGTGTCCAGATCTTAG (SEQ ID NO: 36); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGGCGGTACCCATGACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGGCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCCACCGGTGGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAACACGCGTGGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTGAGATCCCAATTGGCCATGCACTCCCCCCCCACCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTTCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGATCTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGTCCAGATCCGCCGGCGAAACCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGCCGTGTCCCAGCAGGACTCCGGCACCTCCCTGTCCAGATCTTAG (SEQ ID NO: 37); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGGCGGTACCCATGACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCCACCGGTGGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAACACGCGTGGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTGAGATCCCAATTGGTGATGAACTCCCCCCCCTCCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTCCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGCTGTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGACCAGATCCGCCGGCGAAATCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGTGGTGTCCCAGCAGGACTCCGGCACCTCCCCCTCCAGATCTTAG (SEQ ID NO: 38); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMQLSCSRQNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTELRSTGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLRSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 39); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMTVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 40); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLAMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKESRSAGETYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLSRS (SEQ ID NO: 41); or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLVMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKETRSAGEIYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPSRS (SEQ ID NO: 42).

In some cases, the composition may include epitopes and nucleic acids encoding proteins from: HER-2, IGFBP2, and IGF-1R. In some cases, a composition may comprise a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen that is part of a peptide selected from the group consisting of IGFBP-2, HER-2, IGF-1R, wherein the first nucleotide sequence is located on the plasmid. In some cases, the composition may comprise: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence encoding 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 and second nucleotide sequences are located in one or more plasmids. In some cases, a composition may include a nucleic acid sequence encoding an epitope of peptide IGFBP-2 selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGCTGCCGAGAGTGGGCTGCCCCGCGCTGCCGCTGCCGCCGCCGCCGCTGCTGCCGCTGCTGCCGCTGCTGCTGCTGCTACTGGGCGCGAGTGGCGGCGGCGGCGGGGCGCGCGCGGAGGTGCTGTTCCGCTGCCCGCCCTGCACACCCGAGCGCCTGGCCGCCTGCGGGCCCCCGCCGGTTGCGCCGCCCGCCGCGGTGGCCGCAGTGGCCGGAGGCGCCCGCATGCCATGCGCGGAGCTCGTCCGGGAGCCGGGCTGCGGCTGCTGCTCGGTGTGCGCCCGGCTGGAGGGCGAGGCGTGCGGCGTCTACACCCCGCGCTGCGGCCAGGGGCTGCGCTGCTATCCCCACCCGGGCTCCGAGCTGCCCCTGCAGGCGCTGGTCATGGGCGAGGGCACTTGTGAGAAGCGCCGGGACGCCGAGTATGGCGCCAGCCCGGAGCAGGTTGCAGACAATGGCGATGACCACTCAGAAGGAGGCCTGGTGGAG (SEQ ID NO: 43); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCACCACCCCCCAGCAGGTGGCCGACTCCGACGACGACCACTCCGAGGGCGGCCTGGTGGAG (SEQ ID NO: 44); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCACCACCCCCCAGCAGGTGGCCGACTCCGAGGACGACCACTCCGAGGGCGGCCTGGTGGAG (SEQ ID NO: 45); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence NHVDSTMNMLGGGGS (SEQ ID NO: 46); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence ELAVFREKVTEQHRQ (SEQ ID NO: 47); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence LGLEEPKKLRPPPAR (SEQ ID NO: 48); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence DQVLERISTMRLPDE (SEQ ID NO: 49); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence GPLEHLYSLHIPNCD (SEQ ID NO: 50); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence KHGLYNLKQCKMSLN (SEQ ID NO: 51); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence PNTGKLIQGAPTIRG (SEQ ID NO: 52); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence PECHLFYNEQQEARG (SEQ ID NO: 53); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVE (SEQ ID NO: 54); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVE (SEQ ID NO: 55); and a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVE (SEQ ID NO: 56). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide HER-2 selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGCTCCCGGAGCTGGCGGCATGGTGCACCACAGGCACCGCAGCTCATCTCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGCCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTG (SEQ ID NO: 57); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCGTGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGCTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGAGGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGGCCCTGGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCCCCCCCCCATCCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTG (SEQ ID NO: 58); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCATGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGGTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGACGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGACCCCCGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCTGCCCCCCGTGCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTG (SEQ ID NO: 59); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 60); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 61); and a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 62). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide IGF-1R selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence TGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGAGGCCGCAAGAACGAGCGCGCCCTGCCC (SEQ ID NO: 63); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence TGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCC (SEQ ID NO: 64); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence TGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCC (SEQ ID NO: 65); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence DYRSYRFPKLTVITE (SEQ ID NO: 66); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence IRGWKLFYNYALVIF (SEQ ID NO: 67); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence VVTGYVKIRHSHALV (SEQ ID NO: 68); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence FFYVQAKTGYENFIH (SEQ ID NO: 69); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence LIIALPVAVLLIVGG (SEQ ID NO: 70); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence LVIMLYVFHRKRNNS (SEQ ID NO: 71); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence NCHHVVRLLGVVSQG (SEQ ID NO: 72); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALP (SEQ ID NO: 73); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 74); and a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 75). In some cases, the composition may include a nucleic acid sequence encoding a fusion protein of three peptides selected from the group consisting of: a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGGCGGTACCAATGCTGCCGAGAGTGGGCTGCCCCGCGCTGCCGCTGCCGCCGCCGCCGCTGCTGCCGCTGCTGCCGCTGCTGCTGCTGCTACTGGGCGCGAGTGGCGGCGGCGGCGGGGCGCGCGCGGAGGTGCTGTTCCGCTGCCCGCCCTGCACACCCGAGCGCCTGGCCGCCTGCGGGCCCCCGCCGGTTGCGCCGCCCGCCGCGGTGGCCGCAGTGGCCGGAGGCGCCCGCATGCCATGCGCGGAGCTCGTCCGGGAGCCGGGCTGCGGCTGCTGCTCGGTGTGCGCCCGGCTGGAGGGCGAGGCGTGCGGCGTCTACACCCCGCGCTGCGGCCAGGGGCTGCGCTGCTATCCCCACCCGGGCTCCGAGCTGCCCCTGCAGGCGCTGGTCATGGGCGAGGGCACTTGTGAGAAGCGCCGGGACGCCGAGTATGGCGCCAGCCCGGAGCAGGTTGCAGACAATGGCGATGACCACTCAGAAGGAGGCCTGGTGGAGCAATTGACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGCTCCCGGAGCTGGCGGCATGGTGCACCACAGGCACCGCAGCTCATCTCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGCCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTGGGCCGGCCGGTACCTTGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGAGGCCGCAAGAACGAGCGCGCCCTGCCCGCGGCCGCATAG (SEQ ID NO: 76); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGGCGGTACCAATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCACCACCCCCCAGCAGGTGGCCGACTCCGACGACGACCACTCCGAGGGCGGCCTGGTGGAGCAATTGACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCGTGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGCTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGAGGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGGCCCTGGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCCCCCCCCCATCCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTGGGCCGGCCGGTACCTTGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCCGCGGCCGCATAG (SEQ ID NO: 77); a nucleotide sequence having at least 90% sequence identity to nucleotide sequence ATGGCGGTACCAATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCGCCACCCCCCAGCAGGTGGCCGACTCCGAGGACGACCACTCCGAGGGCGGCCTGGTGGAGCAATTGACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCATGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGGTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGACGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGACCCCCGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCTGCCCCCCGTGCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTGGGCCGGCCGGTACCTTGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCCGCGGCCGCATAG (SEQ ID NO: 78); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPAAA (SEQ ID NO: 79); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 80); and a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 81).

In some cases, the composition may include first and second epitopes independently selected from: CD105, Yb-1, SOX-2, CDH3 or MDM 2. In some cases, the composition can include a third epitope, the first, second, and third epitopes being independently selected from: CD105, Yb-1, SOX-2, CDH3 or MDM 2. In some cases, the composition can include a third and a fourth epitope, the first, second, third, and fourth epitopes independently selected from the group consisting of: CD105, Yb-1, SOX-2, CDH3 or MDM 2. In some cases, the composition can include a third, fourth, and fifth epitope, the first, second, third, fourth, and fifth epitope independently selected from the group consisting of: CD105, Yb-1, SOX-2, CDH3 or MDM 2.

In some cases, the composition may include first and second epitopes independently selected from: IGFBP2, HER-2, or IGF-1R. In some cases, the composition can include a third epitope, the first, second, and third epitopes being independently selected from: IGFBP2, HER-2, or IGF-1R.

In some cases, the composition can be administered to a subject. In some cases, the subject is in need of administration of the composition. In some cases, the composition is effective to elicit an immune response in a subject. In some cases, the composition is effective to eliminate a plurality 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 a subject.

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 cells associated with breast cancer are selected from: breast cells, pre-tumor breast cells, breast cancer cells, pre-invasive breast cancer cells, breast cancer stem cells, epithelial cells, mesenchymal cells, somatic cells, or combinations thereof expressing abnormal characteristics.

In some cases, the first and second nucleic acid sequences are purified to at least 70% pure. 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 contiguous with the second nucleic acid sequence on the first plasmid.

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

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 not having breast cancer, a mouse not having breast cancer, or a rat not having breast cancer.

In some cases, the immune response is a type 1 immune response. In some cases, the first nucleic acid sequence is of a species selected from human, mouse, or rat. In some cases, the second nucleic acid sequence is of a species selected from human, mouse, or rat. 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 IFN γ production to IL-10 production that is greater than 1. In some cases, the immune response is characterized by a ratio of IFN γ production to IL-10 production of less than 1.

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

The nucleic acid sequence encoding an epitope of a protein selected from the group consisting of: CD105, HIF1 α, MDM2, Yb1, SOX-2, HER-2, IGFBP2, IGF-1R, and CDH 3. 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.

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

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

In some cases, the compositions described herein include the amino acid sequence of a fusion peptide of 5 (one) epitopes selected from the group consisting of: an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMQLSCSRQNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTELRSTGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLRSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 39); an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMTVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 40); an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLAMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKESRSAGETYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLSRS (SEQ ID NO: 41); or an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLVMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKETRSAGEIYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPSRS (SEQ ID NO: 42).

The compositions described herein also include compositions comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen, said first epitope being part of a peptide selected from the group consisting of IGFBP-2, HER-2, and IGF-1R, wherein said first nucleotide sequence is located in the plasmid. In some cases, the composition may comprise: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence encoding 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 and second nucleotide sequences are located in one or more plasmids. In some cases, at least a first epitope of peptide IGFBP-2 is selected from the group consisting of: an amino acid sequence having at least 90% sequence identity to amino acid sequence NHVDSTMNMLGGGGS (SEQ ID NO: 46); an amino acid sequence having at least 90% sequence identity to amino acid sequence ELAVFREKVTEQHRQ (SEQ ID NO: 47); an amino acid sequence having at least 90% sequence identity to amino acid sequence LGLEEPKKLRPPPAR (SEQ ID NO: 48); an amino acid sequence having at least 90% sequence identity to amino acid sequence DQVLERISTMRLPDE (SEQ ID NO: 49); an amino acid sequence having at least 90% sequence identity to amino acid sequence GPLEHLYSLHIPNCD (SEQ ID NO: 50); an amino acid sequence having at least 90% sequence identity to amino acid sequence KHGLYNLKQCKMSLN (SEQ ID NO: 51); an amino acid sequence having at least 90% sequence identity to amino acid sequence PECHLFYNEQQEARG (SEQ ID NO: 53); an amino acid sequence having at least 90% sequence identity to amino acid sequence MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVE (SEQ ID NO: 54); an amino acid sequence having at least 90% sequence identity to amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVE (SEQ ID NO: 55); or an amino acid sequence having at least 90% sequence identity to amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVE (SEQ ID NO: 56). In some cases, at least a first epitope of the peptide HER-2 is selected from the group consisting of: a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 60); a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 61); or a nucleotide sequence encoding an amino acid sequence having at least 90% sequence identity to amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 62). In some cases, the nucleic acid sequence encoding an epitope of the peptide IGF-1R is selected from the group consisting of: an amino acid sequence having at least 90% sequence identity to amino acid sequence DYRSYRFPKLTVITE (SEQ ID NO: 66); an amino acid sequence having at least 90% sequence identity to amino acid sequence IRGWKLFYNYALVIF (SEQ ID NO: 67); an amino acid sequence having at least 90% sequence identity to amino acid sequence VVTGYVKIRHSHALV (SEQ ID NO: 68); an amino acid sequence having at least 90% sequence identity to amino acid sequence FFYVQAKTGYENFIH (SEQ ID NO: 69); an amino acid sequence having at least 90% sequence identity to amino acid sequence LIIALPVAVLLIVGG (SEQ ID NO: 70); an amino acid sequence having at least 90% sequence identity to amino acid sequence LVIMLYVFHRKRNNS (SEQ ID NO: 71); an amino acid sequence having at least 90% sequence identity to amino acid sequence NCHHVVRLLGVVSQG (SEQ ID NO: 72); an amino acid sequence having at least 90% sequence identity to amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALP (SEQ ID NO: 73); an amino acid sequence having at least 90% sequence identity to amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 74); or an amino acid sequence having at least 90% sequence identity to amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 75).

The compositions described herein may further comprise a nucleic acid sequence encoding a fusion protein of three (one) epitopes selected from the group consisting of: an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPAAA (SEQ ID NO: 79); an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 80); or an amino acid sequence having at least 90% sequence identity to amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 81).

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

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

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

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

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

The amino acid sequence encoding an epitope of a protein selected from the group consisting of: CD105, HIF1 α, MDM2, Yb1, SOX-2, HER-2, IGFBP2, IGF-1R, and CDH 3. 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.

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.

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 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.

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 IFN γ production to IL-10 production that is greater than 1. In some cases, the immune response is characterized by a ratio of IFN γ production to IL-10 production of less than 1.

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 is effective to elicit an immune response in a subject. In some cases, the composition is effective to eliminate a plurality 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 a subject.

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: humans not having breast cancer, mice not having breast cancer, and rats not having breast cancer.

In some cases, the cells associated with breast cancer are selected from: breast cells, pre-tumor breast cells, breast cancer cells, pre-invasive breast cancer cells, breast cancer stem cells, epithelial cells, mesenchymal cells, somatic cells, or combinations thereof expressing abnormal characteristics.

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

In some cases, the composition may be administered to a subject. In some cases, the subject has this need. In some cases, provided herein is a method for preventing breast cancer in a subject, such that the method comprises administering to the subject a composition described herein. In some cases, provided herein is a method for treating breast cancer in a subject, such that the method comprises administering to the subject a composition described herein. In some cases, administering further comprises delivering at least one dose of a composition described herein to the subject. In some cases, administering further comprises delivering a composition described herein to a 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: humans not having breast cancer, mice not having breast cancer, and rats not having breast cancer.

The invention also provides a kit for preparing a composition as described herein, the kit comprising instructions for preparing the composition. The invention also provides a kit for administering the compositions described herein, the kit comprising instructions for administering the compositions.

Compositions comprising an epitope selected from survivin, HIF-1 alpha, IGFBP-2, and IGF-1R

The compositions described herein comprise a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 70% sequence identity to an epitope of said polypeptide. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 80% sequence identity to an epitope of said polypeptide. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 90% sequence identity to an epitope of said polypeptide. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 95% sequence identity to an epitope of said polypeptide. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 99% sequence identity to an epitope of said polypeptide. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, and at least one nucleotide sequence of a polypeptide having 100% sequence identity to an epitope of said polypeptide. The composition can comprise a plasmid comprising a nucleic acid sequence encoding a polypeptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, and at least one nucleotide sequence of a polypeptide having 100% sequence identity over the entire length of the epitope.

In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 70% sequence identity to at least 20 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 80% sequence identity to at least 20 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 90% sequence identity to at least 20 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 95% sequence identity to at least 20 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 99% sequence identity to at least 20 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 100% sequence identity to at least 20 contiguous amino acids.

In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 70% sequence identity to at least 60 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 80% sequence identity to at least 60 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 90% sequence identity to at least 60 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 95% sequence identity to at least 60 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 99% sequence identity to at least 60 contiguous amino acids. In some cases, at least one nucleotide sequence may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 100% sequence identity to at least 60 contiguous amino acids.

In some cases, the composition comprises an isolated plasmid comprising at least 4 nucleotide sequences. Sometimes, each of the at least 4 nucleotide sequences independently encodes a nucleotide sequence identical to a nucleotide sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity. Sometimes, an isolated plasmid may comprise a 4 nucleotide sequence. Sometimes, each of the 4 nucleotide sequences may independently encode a nucleotide sequence identical to a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity. In addition, each of the 4 nucleotide sequences may encode a different polypeptide. Sometimes, each of the different polypeptides may be identical to a polypeptide selected from SEQ ID NOs: 54. 73, 85, and 87 have at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity.

Sometimes, one of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 70% sequence identity to at least 20 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 80% sequence identity to at least 20 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 90% sequence identity to at least 20 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 95% sequence identity to at least 20 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 85, or a polypeptide having at least 100% sequence identity to at least 20 contiguous amino acids.

Sometimes, one of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 70% sequence identity to at least 60 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 80% sequence identity to at least 60 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 90% sequence identity to at least 60 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 95% sequence identity to at least 60 contiguous amino acids. One of the 4 nucleotide sequences may encode a nucleotide sequence identical to SEQ ID NO: 87 having at least 100% sequence identity to at least 60 contiguous amino acids.

In some cases, the 4 nucleotide sequences are arranged in tandem within the plasmid. The 4 nucleotide sequences may be separated by a linker nucleic acid sequence. The linker nucleic acid sequence may be about 1 to about 150, about 5 to about 100, or about 10 to about 50 nucleic acids in length. In some cases, the nucleic acid may encode one or more amino acid residues. Sometimes, the amino acid sequence length of the linker may be from about 1 to about 50, or from about 5 to about 25 amino acid residues. Sometimes, the linker may include a linker as underlined in FIG. 14 (SEQ ID NO: 14).

In some cases, the composition may further comprise at least one additional isolated plasmid. In some cases, the composition can further comprise 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.

In some cases, at least one other isolated plasmid comprises a nucleic acid sequence encoding a polypeptide sequence identical to a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 70% sequence identity to the epitope of said polypeptide. The at least one additional isolated plasmid may comprise a nucleic acid sequence encoding a polypeptide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 80% sequence identity to the epitope of said polypeptide. The at least one additional isolated plasmid may comprise a nucleic acid sequence encoding a polypeptide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 90% sequence identity to the epitope of said polypeptide. The at least one additional isolated plasmid may comprise a nucleic acid sequence encoding a polypeptide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 95% sequence identity to the epitope of said polypeptide. The at least one additional isolated plasmid may comprise a nucleic acid sequence encoding a polypeptide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 99% sequence identity to the epitope of said polypeptide. The at least one additional isolated plasmid may comprise a nucleic acid sequence encoding a polypeptide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, and a nucleotide sequence of a polypeptide having 100% sequence identity to the epitope.

In some cases, the compositions described herein can include a composition comprising a plasmid comprising a nucleotide sequence encoding a polypeptide substantially identical to SEQ ID NO: 89, or a polypeptide having at least 80% sequence identity thereto. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 89 and a polypeptide having at least 90% sequence identity thereto. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 89 and a polypeptide having at least 95% sequence identity thereto. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 89 and a polypeptide having 100% sequence identity thereto. The composition can comprise a plasmid comprising a nucleotide sequence encoding a polypeptide corresponding to SEQ ID NO: 89 is 100% of the nucleotide sequence of the polypeptide.

The compositions described herein may include compositions comprising a polypeptide that hybridizes to SEQ ID NO: 89 has at least 80% sequence identity. The composition can include a polypeptide that is substantially identical to SEQ ID NO: 89 have at least 90% sequence identity. The composition can include a polypeptide that is substantially identical to SEQ ID NO: 89 have at least 95% sequence identity. The composition can include a polypeptide that is substantially identical to SEQ ID NO: 89 has 100% sequence identity. The composition can include a polypeptide that is substantially identical to SEQ ID NO: 89, is 100%.

The composition can be formulated for treating breast cancer or ovarian cancer in a subject. The breast cancer may be relapsed or refractory breast cancer. The ovarian cancer may be recurrent or refractory ovarian cancer. The breast cancer may be metastatic breast cancer. The ovarian cancer may be metastatic ovarian cancer.

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 greater than 1. The immune response can be characterized by a ratio of type I cytokine production to type II cytokine production of less than 1. The immune response can be characterized by a ratio of IFN- γ production to IL-10 production greater than 1. The immune response can be characterized by a ratio of IFN- γ production to IL-10 production of less than 1.

Sometimes, the composition may also comprise an adjuvant. Sometimes, the adjuvant is GM-CSF.

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

Generally, the compositions may be formulated for subcutaneous, intramuscular, or intradermal administration.

The invention also provides a kit for preparing a composition as described herein, the kit comprising instructions for preparing the composition. The invention also provides a kit for administering the compositions described herein, the kit comprising instructions for administering the compositions.

Plasmids for pharmaceutical compositions

In some cases, the epitopes may be derived from human proteins that can be used directly in peptide-based vaccines. In other cases, the epitope may be derived from a human protein and the encoding nucleic acid sequence encoding the epitope may be incorporated into a nucleic acid construct designed to induce expression of the epitope in a subject upon administration. For example, an epitope encoded from a nucleic acid construct may allow an immune response against a particular set of at least one epitope(s) of a protein (e.g., from a protein) to be generated, amplified, attenuated, suppressed, or eliminated. In some cases, the peptide or nucleic acid construct may be optimized to induce, amplify, or generate a Th1 immune response based on protein or plasmid immunization. In some cases, the epitope may be an extended Th1 epitope. In other cases, the peptide or nucleic acid construct may be optimized for protein or plasmid-based immunization to suppress, attenuate, or eliminate a pathogenic response in a subject (e.g., a human or animal) in need thereof.

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 (e.g., a vaccine). Any plasmid backbone (e.g., vector) known to one of ordinary skill in the art to be suitable for pharmaceutical applications for 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 pUMCV 3 can be used. In some cases, the commercially available plasmid backbone can be modified, mutated, engineered, or cloned prior to use. In other cases, non-commercially available plasmid backbones can be used.

The length of the plasmid backbone prior to insertion of the nucleic acid sequence of the at least one epitope(s) may be less than about 500bp, about 1.0kB, about 1.2kB, about 1.4kB, about 1.6kB, about 1.8kB, about 2.0kB, about 2.2kB, about 2.4kB, about 2.6kB, about 2.8kB, about 3.0kB, about 3.2kB, about 3.4kB, about 3.6kB, about 3.8kB, about 4.0kB, about 4.2kB, about 4.4kB, about 4.6kB, about 4.8kB, about 5.0kB, about 5.2kB, about 5.4kB, about 5.6kB, about 5.8kB, about 6.0kB, about 6kB, about 6.7.8 kB, about 8kB, about 10kB, about 8kB, about 8.8kB, about 6kB, about 8kB, about 10.7.8 kB, about 8kB, about 10kB, about 8kB, about 10.7.7.7.7.7.7.7.7 kB, about 10kB, about 8kB, about 10kB, about 8kB, about 6kB, about 8kB, about 8.7.7.7.7.7.7.7.7.7.7.7.7.7.0 kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 2.0kB, about 10.0kB, about 8kB, about 2kB, about 10.0kB, about 8kB, about 2kB, about 8kB, about 2kB, 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, about 16kB, about 16.5kB, about 17kB, about 17.5kB, about 18kB, about 18.5kB, about 19kB, about 19.5kB, about 20kB, about 30kB, about 40kB, about 50kB, about 60kB, about 70kB, about 80kB, about 90kB, about 100kB, about 110kB, about 120kB, about 130kB, about 140kB, about 150kB, about 160kB, about 170kB, about 180kB, about 190kB or about 200 kB. In an exemplary case, the plasmid is about 4kB in length prior to the addition of the nucleic acid sequence encoding the at least one epitope(s).

In some cases, a composition described herein can include a plasmid. In other instances, a composition described herein can include more than one plasmid. For example, a composition described herein can 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.

In some cases, the nucleic acid encoding at least one epitope(s) of the plasmid can be a deoxyribonucleic acid. For example, deoxyribonucleic acids 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 may be ribonucleic acid. For example, ribonucleic acids may be single-stranded or double-stranded. In some cases, the ribonucleic acid can be a micro, antisense, short hairpin, small interference, messenger, transfer, ribosomal ribonucleic acid, and the like. In some cases, the nucleic acid of the plasmid may be part of a deoxyribonucleic acid or part of a ribonucleic acid.

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

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, a nucleic acid sequence from human CD105 can be used to express CD105 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 a non-human CD105 can be used to express CD105 in humans.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, CD105 can be expressed in a subject using the native nucleic acid sequence of CD105 in the genome of the species. 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 CD105 in a subject's genome 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.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, a nucleic acid sequence from human CD105 can be used to express CD105 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 a non-human CD105 can be used to express CD105 in humans.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, CD105 can be expressed in a subject using the native nucleic acid sequence of CD105 in the genome of the species. 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 CD105 in a subject's genome 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.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, the nucleic acid sequence of HIF-1A from humans can be used to express HIF-1A 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 derived from non-human HIF-1A can be used to express HIF-1A in humans.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, a nucleic acid sequence native to 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 can be used to express HIF-1A in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, the nucleic acid sequence of HIF-1A from humans can be used to express HIF-1A 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 derived from non-human HIF-1A can be used to express HIF-1A in humans.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, a nucleic acid sequence native to 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 can be used to express HIF-1A in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, MDM2 may be expressed in humans using the nucleic acid sequence of MDM2 from humans. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, MDM2 may be expressed in humans using nucleic acid sequences from non-human MDM 2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, MDM2 may be expressed in a subject using the native nucleic acid sequence of MDM2 in the genome of the species. 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 can be used to express MDM2 in a subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, the nucleic acid sequence of MDM2 from humans can be used to express MDM2 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, MDM2 may be expressed in humans using nucleic acid sequences from non-human MDM 2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, MDM2 may be expressed in a subject using the native nucleic acid sequence of MDM2 in the genome of the species. 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 can be used to express MDM2 in a subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, a nucleic acid sequence of Yb-1 from human may be used to express Yb-1 in 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 Yb-1 can be used to express Yb-1 in humans.

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

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, a nucleic acid sequence of Yb-1 from human may be used to express Yb-1 in 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 Yb-1 can be used to express Yb-1 in humans.

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

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, a nucleic acid sequence for SOX-2 from a human may be used to express SOX-2 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, SOX-2 can be expressed in humans using nucleic acid sequences from non-human SOX-2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, SOX-2 can be expressed in a subject using the native nucleic acid sequence of SOX-2 in the genome of the species. 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 can be used to express SOX-2 in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, a nucleic acid sequence for SOX-2 from a human may be used to express SOX-2 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, SOX-2 can be expressed in humans using nucleic acid sequences from non-human SOX-2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, SOX-2 can be expressed in a subject using the native nucleic acid sequence of SOX-2 in the genome of the species. 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 can be used to express SOX-2 in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, HER-2 can be expressed in humans using nucleic acid sequences from human HER-2. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, HER-2 can be expressed in humans using nucleic acid sequences derived from non-human HER-2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, HER-2 can be expressed in a subject using the native nucleic acid sequence of HER-2 in the genome of the species. 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 HER-2 in a subject's 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 the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, HER-2 can be expressed in humans using nucleic acid sequences from human HER-2. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, HER-2 can be expressed in humans using nucleic acid sequences derived from non-human HER-2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, HER-2 can be expressed in a subject using the native nucleic acid sequence of HER-2 in the genome of the species. 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 HER-2 in a subject's 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 the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, IGFBP2 may be expressed in humans using nucleic acid sequences from human IGFBP 2. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, IGFBP2 may be expressed in humans using nucleic acid sequences from non-human IGFBP 2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, IGFBP2 can be expressed in a subject using the native nucleic acid sequence of IGFBP2 in the genome of the species. 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 can be used to express IGFBP2 in a subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, IGFBP2 may be expressed in humans using nucleic acid sequences from human IGFBP 2. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, IGFBP2 may be expressed in humans using nucleic acid sequences from non-human IGFBP 2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, IGFBP2 can be expressed in a subject using the native nucleic acid sequence of IGFBP2 in the genome of the species. 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 can be used to express IGFBP2 in a subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, IGF-1R may be expressed in humans using nucleic acid sequences from human IGF-1R. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, IGF-1R may be expressed in humans using nucleic acid sequences from non-human IGF-1R.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a species may 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 a subject's genome may be modified using molecular techniques known to those of ordinary skill in the art and may be used to express IGF-1R in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, IGF-1R may be expressed in humans using nucleic acid sequences from human IGF-1R. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, IGF-1R may be expressed in humans using nucleic acid sequences from non-human IGF-1R.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a species may 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 a subject's genome may be modified using molecular techniques known to those of ordinary skill in the art and may be used to express IGF-1R in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, the nucleic acid sequence of CDH3 from humans can be used to express CDH3 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, CDH3 can be expressed in humans using nucleic acid sequences from non-human CDH 3.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, CDH3 can be expressed in a subject using the native nucleic acid sequence of CDH3 in the genome of the species. 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 can be used to express CDH3 in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, the nucleic acid sequence of CDH3 from humans can be used to express CDH3 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, CDH3 can be expressed in humans using nucleic acid sequences from non-human CDH 3.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, CDH3 can be expressed in a subject using the native nucleic acid sequence of CDH3 in the genome of the species. 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 can be used to express CDH3 in the subject.

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 a cell associated with breast cancer; and a second nucleotide sequence encoding a second epitope of a second antigen expressed by a cell associated with breast cancer, wherein the first and second nucleotide sequences are located in one or more plasmids. In some cases, a composition may include a nucleic acid encoding an epitope from the following proteins, CD105, HIF1 α, MDM2, Yb-1, SOX-2, HER-2, IGFBP2, IGF-1R, and CDH 3.

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

In some cases, the composition may include epitopes and nucleic acids encoding proteins from: CD105, MDM2, Yb-1, SOX-2, and CDH 3. In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide CD105 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 nucleotide sequence CAGAACGGCACCTGGCCCCGCGAGGTGCTGCTGGTGCTGTCCGTGAACTCCTCCGTGTTCCTGCACCTACAGGCCCTGGGCATCCCCCTGCACCTGGCCTACAACTCCTCCCTGGTGACCTTCCAGGAGCCCCCCGGCGTGAACACCACCGAGCTG (SEQ ID 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 to nucleotide sequence ACCGTGTTCATGCGCCTGAACATCATCTCCCCCGACCTGTCCGGCTGCACCTCCAAGGGCCTGGTGCTGCCCGCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCTCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 3); 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 nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGGCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (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 to nucleotide sequence ACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCC (SEQ ID NO: 5); 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 amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO: 6); 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 amino acid sequence QNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: 1); 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 amino acid sequence TVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 8); 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 amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 9); 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 amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 10). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the 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 nucleotide sequence GGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACCGCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAAC (SEQ ID NO: 11); 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 nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 12); 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 nucleotide sequence GGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAAC (SEQ ID NO: 12); 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 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 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 amino acid sequence GVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN (SEQ ID NO: 16). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide SOX-2 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 nucleotide sequence GGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTG (SEQ ID NO: 17); 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 nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 18); 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 nucleotide sequence GGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTG (SEQ ID NO: 18); 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 amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTP GMALGSMGSV (SEQ ID NO: 20). . In some cases, the composition can include a nucleic acid sequence encoding the peptide CDH3 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 nucleotide sequence AGGTCACTGAAGGAAAGGAATCCATTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 21); 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 nucleotide sequence TTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACA (SEQ ID NO: 22); 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 nucleotide sequence GCCATGCACTCCCCCCCCACCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTTCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGATCTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGTCC (SEQ ID NO: 23); 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 nucleotide sequence GTGATGAACTCCCCCCCCTCCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTCCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGCTGTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGACC (SEQ ID NO: 24); 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 amino acid sequence RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 25); 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 amino acid sequence LKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 26); 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 amino acid sequence AMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKES (SEQ ID NO: 27); 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 amino acid sequence VMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKET (SEQ ID NO: 28). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the 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 nucleotide sequence ACCTACACCATGAAGGAGGTGCTGTTCTACCTGGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACCTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAaATCTACACCATGATCTACCGCAACCTGGTGGTGGTGAACCAGCAGGAGTCCTCCGACTCCGGCACCTCCGTGTCC (SEQ ID NO: 29); 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 nucleotide sequence ACCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGCCGTGTCCCAGCAGGACTCCGGCACCTCCCTGTCC (SEQ ID NO: 30); 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 nucleotide sequence ATCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGTGGTGTCCCAGCAGGACTCCGGCACCTCCCCCTCC (SEQ ID NO: 31); 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 amino acid sequence 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 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 amino acid sequence IYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPS (SEQ ID NO: 34).

In one exemplary case, the composition can include a nucleic acid sequence encoding a fusion peptide of 5 (one) epitopes 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 nucleotide sequence ATGGCGGTACCCATGCAACTGTCCTGCTCTAGACAGAACGGCACCTGGCCCCGCGAGGTGCTGCTGGTGCTGTCCGTGAACTCCTCCGTGTTCCTGCACCTACAGGCCCTGGGCATCCCCCTGCACCTGGCCTACAACTCCTCCCTGGTGACCTTCCAGGAGCCCCCCGGCGTGAACACCACCGAGCTGAGATCCACCGGTGGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACCGCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAACACGCGTGGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGAGATCCCAATTGAGGTCACTGAAGGAAAGGAATCCATTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACAAGATCCGCCGGCGAAACCTACACCATGAAGGAGGTGCTGTTCTACCTGGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACCTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAAATCTACACCATGATCTACCGCAACCTGGTGGTGGTGAACCAGCAGGAGTCCTCCGACTCCGGCACCTCCGTGTCCAGATCTTAG (SEQ ID NO: 35); 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 nucleotide sequence ATGGCGGTACCCATGACCGTGTTCATGCGCCTGAACATCATCTCCCCCGACCTGTCCGGCTGCACCTCCAAGGGCCTGGTGCTGCCCGCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCTCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCCACCGGTGGAGTGCCAGTGCAGGGCTCCAAGTACGCTGCCGACCGCAACCACTACCGCCGCTACCCACGCCGTCGCGGCCCACCCCGCAACTACCAGCAGAACACGCGTGGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGAGATCCCAATTGTTGAAAATCTTCCCATCCAAACGTATCTTACGAAGACACAAGAGAGATTGGGTGGTTGCTCCAATATCTGTCCCTGAAAATGGCAAGGGTCCCTTCCCACAGAGACTGAATCAGCTCAAGTCTAATAAAGATAGAGACACCAAGATTTTCTACAGCATCACGGGGCCGGGTGCAGACAGCCCACCTGAGGGTGTCTTCGCTGTAGAGAAGGAGACAAGATCCGCCGGCGAAACCTACACCATGAAGGAGGTGCTGTTCTACCTGGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACCTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAAATCTACACCATGATCTACCGCAACCTGGTGGTGGTGAACCAGCAGGAGTCCTCCGACTCCGGCACCTCCGTGTCCAGATCTTAG (SEQ ID NO: 36); 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 nucleotide sequence ATGGCGGTACCCATGACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGGCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCCACCGGTGGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAACACGCGTGGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTGAGATCCCAATTGGCCATGCACTCCCCCCCCACCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTTCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGATCTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGTCCAGATCCGCCGGCGAAACCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGCCGTGTCCCAGCAGGACTCCGGCACCTCCCTGTCCAGATCTTAG (SEQ ID NO: 37); 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 nucleotide sequence ATGGCGGTACCCATGACCGTGTCCATGCGCCTGAACATCGTGTCCCCCGACCTGTCCGGCAAGGGCCTGGTGCTGCCCTCCGTGCTGGGCATCACCTTCGGCGCCTTCCTGATCGGCGCCCTGCTGACCGCCGCCCTGTGGTACATCTACTCCCACACCCGCGCCCCCTCCAAGCGCGAGCCCGTGGTGGCCGTGGCCGCCCCCGCCTCCTCCGAGTCCTCCTCCACCAACCACTCCATCGGCTCCACCCAGTCCACCCCCTGCTCCACCTCCTCCATGGCCACCGGTGGCGTGCCCGTGCAGGGCTCCAAGTACGCCGCCGACCGCAACCACTACCGCCGCTACCCCCGCCGCCGCGGCCCCCCCCGCAACTACCAGCAGAACACGCGTGGCCTGAACGCCCACGGCGCCGCCCAGATGCAGCCCATGCACCGCTACGACGTGTCCGCCCTGCAGTACAACTCCATGACCTCCTCCCAGACCTACATGAACGGCTCCCCCACCTACTCCATGTCCTACTCCCAGCAGGGCACCCCCGGCATGGCCCTGGGCTCCATGGGCTCCGTGAGATCCCAATTGGTGATGAACTCCCCCCCCTCCCGCATCCTGCGCCGCCGCAAGCGCGAGTGGGTGATGCCCCCCATCTCCGTGCCCGAGAACGGCAAGGGCCCCTTCCCCCAGCGCCTGAACCAGCTGAAGTCCAACAAGGACCGCGGCACCAAGCTGTTCTACTCCATCACCGGCCCCGGCGCCGACTCCCCCCCCGAGGGCGTGTTCACCATCGAGAAGGAGACCAGATCCGCCGGCGAAATCTACACCATGAAGGAGATCATCTTCTACATCGGCCAGTACATCATGACCAAGCGCCTGTACGACGAGAAGCAGCAGCACATCGTGTACTGCTCCAACGACCTGCTGGGCGACGTGTTCGGCGTGCCCTCCTTCTCCGTGAAGGAGCACCGCAAGATCTACGCCATGATCTACCGCAACCTGGTGGTGGTGTCCCAGCAGGACTCCGGCACCTCCCCCTCCAGATCTTAG (SEQ ID NO: 38); 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 amino acid sequence MAVPMQLSCSRQNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTELRSTGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLRSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 39); 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 amino acid sequence MAVPMTVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 40); 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 amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLAMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKESRSAGETYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLSRS (SEQ ID NO: 41); 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 amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLVMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKETRSAGEIYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPSRS (SEQ ID NO: 42).

In some cases, the composition may include epitopes and nucleic acids encoding proteins from: HER-2, IGFBP2, and IGF-1R. In some cases, a composition may comprise a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen that is part of a peptide selected from the group consisting of IGFBP-2, HER-2, IGF-1R, wherein the first nucleotide sequence is located on the plasmid. In some cases, the composition may comprise: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence encoding 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 and second nucleotide sequences are located in one or more plasmids. In one exemplary case, the composition can include a nucleic acid sequence encoding an epitope of peptide IGFBP-2 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 nucleotide sequence ATGCTGCCGAGAGTGGGCTGCCCCGCGCTGCCGCTGCCGCCGCCGCCGCTGCTGCCGCTGCTGCCGCTGCTGCTGCTGCTACTGGGCGCGAGTGGCGGCGGCGGCGGGGCGCGCGCGGAGGTGCTGTTCCGCTGCCCGCCCTGCACACCCGAGCGCCTGGCCGCCTGCGGGCCCCCGCCGGTTGCGCCGCCCGCCGCGGTGGCCGCAGTGGCCGGAGGCGCCCGCATGCCATGCGCGGAGCTCGTCCGGGAGCCGGGCTGCGGCTGCTGCTCGGTGTGCGCCCGGCTGGAGGGCGAGGCGTGCGGCGTCTACACCCCGCGCTGCGGCCAGGGGCTGCGCTGCTATCCCCACCCGGGCTCCGAGCTGCCCCTGCAGGCGCTGGTCATGGGCGAGGGCACTTGTGAGAAGCGCCGGGACGCCGAGTATGGCGCCAGCCCGGAGCAGGTTGCAGACAATGGCGATGACCACTCAGAAGGAGGCCTGGTGGAG (SEQ ID NO: 43); 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 nucleotide sequence ATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCACCACCCCCCAGCAGGTGGCCGACTCCGACGACGACCACTCCGAGGGCGGCCTGGTGGAG (SEQ ID NO: 44); 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 nucleotide sequence ATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCACCACCCCCCAGCAGGTGGCCGACTCCGAGGACGACCACTCCGAGGGCGGCCTGGTGGAG (SEQ ID NO: 45); 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 amino acid sequence NHVDSTMNMLGGGGS (SEQ ID NO: 46); 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 amino acid sequence ELAVFREKVTEQHRQ (SEQ ID NO: 47); 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 amino acid sequence LGLEEPKKLRPPPAR (SEQ ID NO: 48); 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 amino acid sequence DQVLERISTMRLPDE (SEQ ID NO: 49); 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 amino acid sequence GPLEHLYSLHIPNCD (SEQ ID NO: 50); 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 amino acid sequence KHGLYNLKQCKMSLN (SEQ ID NO: 51); 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 amino acid sequence PNTGKLIQGAPTIRG (SEQ ID NO: 52); 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 amino acid sequence PECHLFYNEQQEARG (SEQ ID NO: 53); 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 amino acid sequence MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVE (SEQ ID NO: 54); 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 amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVE (SEQ ID NO: 55); 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 amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVE (SEQ ID NO: 56). In some cases, the composition may include a nucleic acid sequence encoding an epitope of the peptide HER-2 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 nucleotide sequence ACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGCTCCCGGAGCTGGCGGCATGGTGCACCACAGGCACCGCAGCTCATCTCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGCCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTG (SEQ ID NO: 57); 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 nucleotide sequence ACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCGTGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGCTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGAGGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGGCCCTGGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCCCCCCCCCATCCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTG (SEQ ID NO: 58); 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 nucleotide sequence ACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCATGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGGTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGACGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGACCCCCGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCTGCCCCCCGTGCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTG (SEQ ID NO: 59); 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 amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 60); 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 amino acid sequence TMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 61); 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 amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 62). In one exemplary case, the composition can include a nucleic acid sequence encoding an epitope of the peptide IGF-1R 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 nucleotide sequence TGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGAGGCCGCAAGAACGAGCGCGCCCTGCCC (SEQ ID NO: 63); 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 nucleotide sequence TGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCC (SEQ ID NO: 64); 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 nucleotide sequence TGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCC (SEQ ID NO: 65); 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 amino acid sequence DYRSYRFPKLTVITE (SEQ ID NO: 66); 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 amino acid sequence IRGWKLFYNYALVIF (SEQ ID NO: 67); 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 amino acid sequence VVTGYVKIRHSHALV (SEQ ID NO: 68); 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 amino acid sequence FFYVQAKTGYENFIH (SEQ ID NO: 69); 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 amino acid sequence LIIALPVAVLLIVGG (SEQ ID NO: 70); 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 amino acid sequence LVIMLYVFHRKRNNS (SEQ ID NO: 71); 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 amino acid sequence NCHHVVRLLGVVSQG (SEQ ID NO: 72); 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 amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALP (SEQ ID NO: 73); 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 amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 74); 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 amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 75). In some cases, the composition may include a nucleic acid sequence encoding a fusion peptide of 3 (n) epitopes 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 nucleotide sequence ATGGCGGTACCAATGCTGCCGAGAGTGGGCTGCCCCGCGCTGCCGCTGCCGCCGCCGCCGCTGCTGCCGCTGCTGCCGCTGCTGCTGCTGCTACTGGGCGCGAGTGGCGGCGGCGGCGGGGCGCGCGCGGAGGTGCTGTTCCGCTGCCCGCCCTGCACACCCGAGCGCCTGGCCGCCTGCGGGCCCCCGCCGGTTGCGCCGCCCGCCGCGGTGGCCGCAGTGGCCGGAGGCGCCCGCATGCCATGCGCGGAGCTCGTCCGGGAGCCGGGCTGCGGCTGCTGCTCGGTGTGCGCCCGGCTGGAGGGCGAGGCGTGCGGCGTCTACACCCCGCGCTGCGGCCAGGGGCTGCGCTGCTATCCCCACCCGGGCTCCGAGCTGCCCCTGCAGGCGCTGGTCATGGGCGAGGGCACTTGTGAGAAGCGCCGGGACGCCGAGTATGGCGCCAGCCCGGAGCAGGTTGCAGACAATGGCGATGACCACTCAGAAGGAGGCCTGGTGGAGCAATTGACGATGCGGAGACTGCTGCAGGAAACGGAGCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGGTGCCCATCAAGTGGATGGCGCTGGAGTCCATTCTCCGCCGGCGGTTCACCCACCAGAGTGATGTGTGGAGTTATGGTGTGACTGTGTGGGAGCTGATGACTTTTGGGGCCAAACCTTACGATGGGATCCCAGCCCGGGAGATCCCTGACCTGCTGGAAAAGGGGGAGCGGCTGCCCCAGCCCCCCATCTGCACCATTGATGTCTACATGATCATGGTCAAATGTTGGATGATTGACTCTGAATGTCGGCCAAGATTCCGGGAGTTGGTGTCTGAATTCTCCCGCATGGCCAGGGACCCCCAGCGCTTTGTGGTCATCCAGAATGAGGACTTGGCTCCCGGAGCTGGCGGCATGGTGCACCACAGGCACCGCAGCTCATCTCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGCCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTGGGCCGGCCGGTACCTTGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGAGGCCGCAAGAACGAGCGCGCCCTGCCCGCGGCCGCATAG (SEQ ID NO: 76); 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 nucleotide sequence ATGGCGGTACCAATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCACCACCCCCCAGCAGGTGGCCGACTCCGACGACGACCACTCCGAGGGCGGCCTGGTGGAGCAATTGACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCGTGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGCTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGAGGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGGCCCTGGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCCCCCCCCCATCCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTGGGCCGGCCGGTACCTTGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCCGCGGCCGCATAG (SEQ ID NO: 77); 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 nucleotide sequence ATGGCGGTACCAATGCTGCCCCGCCTGGGCGGCCCCGCCCTGCCCCTGCTGCTGCCCTCCCTGCTGCTGCTGCTGCTGCTGGGCGCCGGCGGCTGCGGCCCCGGCGTGCGCGCCGAGGTGCTGTTCCGCTGCCCCCCCTGCACCCCCGAGCGCCTGGCCGCCTGCGGCCCCCCCCCCGACGCCCCCTGCGCCGAGCTGGTGCGCGAGCCCGGCTGCGGCTGCTGCTCCGTGTGCGCCCGCCAGGAGGGCGAGGCCTGCGGCGTGTACATCCCCCGCTGCGCCCAGACCCTGCGCTGCTACCCCAACCCCGGCTCCGAGCTGCCCCTGAAGGCCCTGGTGACCGGCGCCGGCACCTGCGAGAAGCGCCGCGTGGGCGCCACCCCCCAGCAGGTGGCCGACTCCGAGGACGACCACTCCGAGGGCGGCCTGGTGGAGCAATTGACCATGCGCCGCCTGCTGCAGGAGACCGAGCTGGTGGAGCCCCTGACCCCCTCCGGCGCCATGCCCAACCAGGCCCAGATGCGCATCCTGAAGGAGACCGAGCTGCGCAAGGTGAAGGTGCTGGGCTCCGGCGCCTTCGGCACCGTGTACAAGGGCATCTGGATCCCCGACGGCGAGAACGTGAAGATCCCCGTGGCCATCAAGGTGCTGCGCGAGAACACCTCCCCCAAGGCCAACAAGGAGATCCTGGACGAGGCCTACGTGATGGCCGGCGTGGGCTCCCCCTACGTGTCCCGCCTGCTGGGCATCTGCCTGACCTCCACCGTGCAGCTGGTGACCCAGCTGATGCCCTACGGCTGCCTGCTGGACCACGTGCGCGAGCACCGCGGCCGCCTGGGCTCCCAGGACCTGCTGAACTGGTGCGTGCAGATCGCCAAGGGCATGTCCTACCTGGAGGACGTGCGCCTGGTGCACCGCGACCTGGCCGCCCGCAACGTGCTGGTGAAGTCCCCCAACCACGTGAAGATCACCGACTTCGGCCTGGCCCGCCTGCTGGACATCGACGAGACCGAGTACCACGCCGACGGCGGCAAGGTGCCCATCAAGTGGATGGCCCTGGAGTCCATCCTGCGCCGCCGCTTCACCCACCAGTCCGACGTGTGGTCCTACGGCGTGACCGTGTGGGAGCTGATGACCTTCGGCGCCAAGCCCTACGACGGCATCCCCGCCCGCGAGATCCCCGACCTGCTGGAGAAGGGCGAGCGCCTGCCCCAGCCCCCCATCTGCACCATCGACGTGTACATGATCATGGTGAAGTGCTGGATGATCGACTCCGAGTGCCGCCCCCGCTTCCGCGAGCTGGTGTCCGAGTTCTCCCGCATGGCCCGCGACCCCCAGCGCTTCGTGGTGATCCAGAACGAGGACCTGACCCCCGGCACCGGCTCCACCGCCCACCGCCGCCACCGCTCCTCCTCCCCCCTGCCCCCCGTGCGCCCCGCCGGCGCCACCCTGGAGCGCCCCAAGACCCTGTCCCCCGGCAAGAACGGCGTGGTGAAGGACGTGTTCGCCTTCGGCGGCGCCGTGGAGAACCCCGAGTACCTGGGCCGGCCGGTACCTTGGTCCTTCGGCGTGGTGCTGTGGGAGATCGCCACCCTGGCCGAGCAGCCCTACCAGGGCCTGTCCAACGAGCAGGTGCTGCGCTTCGTGATGGAGGGCGGCCTGCTGGACAAGCCCGACAACTGCCCCGACATGCTGTTCGAGCTGATGCGCATGTGCTGGCAGTACAACCCCAAGATGCGCCCCTCCTTCCTGGAGCACAAGGCCGAGAACGGCCCCGGCGTGCTGGTGCTGCGCGCCTCCTTCGACGAGCGCCAGCCCTACGCCCACATGAACGGCGGCCGCGCCAACGAGCGCGCCCTGCCCGCGGCCGCATAG (SEQ ID NO: 78); 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 amino acid sequence MAVPMLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPAAA (SEQ ID NO: 79); 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 amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 80); 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 amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 81).

In some cases, the composition may include first and second epitopes independently selected from: CD105, Yb-1, SOX-2, CDH3 or MDM 2. In some cases, the composition can include a third epitope, the first, second, and third epitopes being independently selected from: CD105, Yb-1, SOX-2, CDH3 or MDM 2. In some cases, the composition can include a third and a fourth epitope, the first, second, third, and fourth epitopes independently selected from the group consisting of: CD105, Yb-1, SOX-2, CDH3 or MDM 2. In some cases, the composition can include a third, fourth, and fifth epitope, the first, second, third, fourth, and fifth epitope independently selected from the group consisting of: CD105, Yb-1, SOX-2, CDH3 or MDM 2.

In some cases, the composition may include first and second epitopes independently selected from: IGFBP2, HER-2, or IGF-1R. In some cases, the composition can include a third epitope, the first, second, and third epitopes being independently selected from: IGFBP2, HER-2, or IGF-1R.

In some cases, the composition can be administered to a subject. In some cases, the subject is in need of administration of the composition. In some cases, the composition is effective to elicit an immune response in a subject. In some cases, the composition is effective to eliminate a plurality 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 a subject.

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 cells associated with breast cancer are selected from: breast cells, pre-tumor breast cells, breast cancer cells, pre-invasive breast cancer cells, breast cancer stem cells, epithelial cells, mesenchymal cells, somatic cells, or combinations thereof expressing abnormal characteristics.

In some cases, the first and second nucleic acid sequences are purified to at least 70% pure. 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 contiguous with the second nucleic acid sequence on the first plasmid.

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

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 not having breast cancer, a mouse not having breast cancer, or a rat not having breast cancer.

In some cases, the immune response is a type 1 immune response. In some cases, the first nucleic acid sequence is of a species selected from human, mouse, or rat. In some cases, the second nucleic acid sequence is of a species selected from human, mouse, or rat. 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 IFN γ production to IL-10 production that is greater than 1. In some cases, the immune response is characterized by a ratio of IFN γ production to IL-10 production of less than 1.

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

The nucleic acid sequence encoding an epitope of a protein selected from the group consisting of: CD105, HIF1 α, MDM2, Yb-1, SOX-2, HER-2, IGFBP2, IGF-1R, and CDH 3. In some cases, nucleic acid sequences that are 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.

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

In some cases, the composition may comprise: at least a first epitope of a first antigen, said first epitope being part of a peptide selected from the group consisting of CD105, Yb-1, SOX-2, CDH3 or MDM 2. 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, said first and second epitopes being independently selected from the group consisting of CD105, Yb-1, SOX-2, CDH3 or MDM 2. In some cases, at least the first epitope of peptide CD105 is 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 amino acid sequence EARMLNASIVASFVELPL (SEQ ID NO: 6); 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 amino acid sequence QNGTWPREVLLVLSVNS SVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: 1); 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 amino acid sequence 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 amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 9); 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 amino acid sequence TVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMA (SEQ ID NO: 10). In some cases, at least a first epitope of the peptide Yb-1 is 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 amino acid sequence EDVFVHQTAIKKNNPRK (SEQ ID NO: 14); 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 amino acid sequence YRRNFNYRRRRPEN (SEQ ID NO: 15); 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 amino acid sequence GVPVQGSKYAADRNHYRRYPRRRGPPRNYQQN (SEQ ID NO: 16). In some cases, at least a first epitope of peptide SOX-2 is 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 amino acid sequence GLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSV (SEQ ID NO: 20). In some cases, at least a first epitope of peptide CDH3 is 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 amino acid sequence RSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 25); 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 amino acid sequence LKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKET (SEQ ID NO: 26); 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 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 amino acid sequence VMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKET (SEQ ID NO: 28). In some cases, at least the first epitope of the peptide MDM-2 is 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 amino acid sequence TYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSV (SEQ ID NO: 32); 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 amino acid sequence TYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLS (SEQ ID NO: 33); 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 amino acid sequence IYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPS (SEQ ID NO: 34).

In some cases, the compositions described herein include the amino acid sequence of a fusion peptide of 5 (one) 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 amino acid sequence MAVPMQLSCSRQNGTWPREVLLVLSVNSSVFLHLQALGIPLHLAYNSSLVTFQEPPGVNTTELRSTGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLRSLKERNPLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 39); 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 amino acid sequence MAVPMTVFMRLNIISPDLSGCTSKGLVLPAVLGITFGAFLIGALLTAALWYIYSHTRSPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLLKIFPSKRILRRHKRDWVVAPISVPENGKGPFPQRLNQLKSNKDRDTKIFYSITGPGADSPPEGVFAVEKETRSAGETYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSRS (SEQ ID NO: 40); 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 amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRGPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLAMHSPPTRILRRRKREWVMPPIFVPENGKGPFPQRLNQLKSNKDRGTKIFYSITGPGADSPPEGVFTIEKESRSAGETYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVAVSQQDSGTSLSRS (SEQ ID NO: 41); 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 amino acid sequence MAVPMTVSMRLNIVSPDLSGKGLVLPSVLGITFGAFLIGALLTAALWYIYSHTRAPSKREPVVAVAAPASSESSSTNHSIGSTQSTPCSTSSMATGGVPVQGSKYAADRNHYRRYPRRRGPPRNYQQNTRGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVRSQLVMNSPPSRILRRRKREWVMPPISVPENGKGPFPQRLNQLKSNKDRGTKLFYSITGPGADSPPEGVFTIEKETRSAGEIYTMKEIIFYIGQYIMTKRLYDEKQQHIVYCSNDLLGDVFGVPSFSVKEHRKIYAMIYRNLVVVSQQDSGTSPSRS (SEQ ID NO: 42).

The compositions described herein also include compositions comprising: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen, said first epitope being part of a peptide selected from the group consisting of IGFBP-2, HER-2, and IGF-1R, wherein said first nucleotide sequence is located in the plasmid. In some cases, the composition may comprise: a first plasmid comprising a first nucleotide sequence encoding a first epitope of a first antigen; and a second nucleotide sequence encoding 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 and second nucleotide sequences are located in one or more plasmids. In some cases, at least a first epitope of peptide IGFBP-2 is 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 amino acid sequence NHVDSTMNMLGGGGS (SEQ ID NO: 46); 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 amino acid sequence ELAVFREKVTEQHRQ (SEQ ID NO: 47); 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 amino acid sequence LGLEEPKKLRPPPAR (SEQ ID NO: 48); 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 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 amino acid sequence GPLEHLYSLHIPNCD (SEQ ID NO: 50); 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 amino acid sequence KHGLYNLKQCKMSLN (SEQ ID NO: 51); 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 amino acid sequence PECHLFYNEQQEARG (SEQ ID NO: 53); 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 amino acid sequence MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVE (SEQ ID NO: 54); 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 amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVE (SEQ ID NO: 55); 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 amino acid sequence MLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVE (SEQ ID NO: 56). In some cases, at least a first epitope of the peptide HER-2 is 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 amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 60); 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 amino acid sequence TMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 61); 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 amino acid sequence TMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYL (SEQ ID NO: 62). In some cases, the composition may 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 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 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 amino acid sequence VVTGYVKIRHSHALV (SEQ ID NO: 68); 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 amino acid sequence FFYVQAKTGYENFIH (SEQ ID NO: 69); 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 amino acid sequence LIIALPVAVLLIVGG (SEQ ID NO: 70); 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 amino acid sequence LVIMLYVFHRKRNNS (SEQ ID NO: 71); 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 amino acid sequence NCHHVVRLLGVVSQG (SEQ ID NO: 72); 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 amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALP (SEQ ID NO: 73); 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 amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 74); 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 amino acid sequence WSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALP (SEQ ID NO: 75). .

The compositions described herein may further comprise a nucleic acid sequence encoding a fusion protein of three (one) 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 amino acid sequence MAVPMLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLAPGAGGMVHHRHRSSSPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRKNERALPAAA (SEQ ID NO: 79); 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 amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGTTPQQVADSDDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAVPNQAQMRILKETELRKLKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEEVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLALGTGSTAHRRHRSSSPPPPIRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 80); 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 amino acid sequence MAVPMLPRLGGPALPLLLPSLLLLLLLGAGGCGPGVRAEVLFRCPPCTPERLAACGPPPDAPCAELVREPGCGCCSVCARQEGEACGVYIPRCAQTLRCYPNPGSELPLKALVTGAGTCEKRRVGATPQQVADSEDDHSEGGLVEQLTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVREHRGRLGSQDLLNWCVQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLTPGTGSTAHRRHRSSSPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLGRPVPWSFGVVLWEIATLAEQPYQGLSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNPKMRPSFLEHKAENGPGVLVLRASFDERQPYAHMNGGRANERALPAAA (SEQ ID NO: 81).

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

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

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

In some cases, a composition can include a nucleotide sequence encoding an epitope of the peptide HIF-1 α 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 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 amino acid sequence SDNVNKYMGLTQFELTGHSVFDFTHP (SEQ ID NO: 83); 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 amino acid sequence GGYVWVETQATVIYNTKNSQ (SEQ ID NO: 84).

In some cases, a composition can include at least a first epitope of the peptide HIF-1 α 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 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 amino acid sequence SDNVNKYMGLTQFELTGHSVFDFTHP (SEQ ID NO: 83); 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 amino acid sequence GGYVWVETQATVIYNTKNSQ (SEQ ID NO: 84).

The compositions described herein may contain short epitopes encoded on a single particle backbone. In some cases, the plasmid backbone can encode a short epitope. In other cases, the plasmids described herein can encode more than one short epitope. For example, a composition described herein can 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.

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

The compositions of the breast cancer vaccines described herein may contain short and extended epitopes on a single particulate backbone. In some cases, the plasmid may comprise a short epitope. In other cases, a composition of plasmids described herein can comprise more than one short epitope. For example, a composition of plasmids described herein can 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.

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

Plasmids for compositions containing more than one sequence encoding an epitope may contain spacers between the epitope sequences. In some cases, sequences encoding short epitopes can be concatenated without the use of spacers. In some cases, sequences encoding extended epitopes can be concatenated without the use of spacers. In some cases, sequences encoding short epitopes can be used that are linked by spacers. In some cases, a spacer may be used to tandem encode a sequence extending an epitope.

In one exemplary case, the composition can be a plasmid-based vaccine that contains short and extended antigenic epitopes. The plasmid(s) of the vaccine may be constructed using a 4kB plasmid backbone (e.g., pomvc 3 or pNGVL 3). Typically, the plasmid may contain an antibiotic resistance gene. For example, in addition to the origin of replication for selection and propagation in bacteria, pUMVC3 contains the kanamycin resistance gene. In some cases, the multiple cloning site in pomvc 3 may be flanked by eukaryotic transcriptional control elements to facilitate expression of the inserted sequences (e.g., gene cassettes) in eukaryotic cells. For example, the inserted sequence may be an epitope.

In an exemplary case, the nucleic acid coding sequence for the antigenic epitope peptide can be assembled using a Kozak consensus translational start sequence, stop codon, and cloning site in the plasmid backbone. Standard molecular techniques known to those of ordinary skill in the art including oligonucleotide synthesis, polymerase chain reaction amplification, restriction endonucleases, and nucleic acid ligases (e.g., DNA ligases) can be used to generate nucleic acids (e.g., DNA fragments) and insert the nucleic acid fragments into the plasmid vector backbone.

In some cases, the plasmid may contain a nucleic acid sequence encoding at least one tag. In some cases, the tag may 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 may be a histidine tag having 3 histidine residues, a histidine tag having 4 histidine residues, a histidine tag having 5 histidine residues, a histidine tag having 6 histidine residues, or the like. Any suitable technique known to those skilled in the art may be used to determine the expression of a tag in a subject.

In some cases, the plasmid may be sequenced using any sequencing technique known to those of ordinary skill in the art such that the results of the sequencing provide nucleotide level resolution of the entire plasmid.

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

Plasmid-survivin, HIF-1A, IGF-1R, and/or IGFBP2

The compositions described herein may include nucleic acid-based vaccines comprising a plasmid encoding one or more epitope(s) selected from survivin, HIF-1A, IGF-1R, or IGFBP 2. In some cases, the epitope may be derived from a human protein and the encoding nucleic acid sequence encoding the epitope may be incorporated into a nucleic acid construct designed to induce expression of the epitope in a subject upon administration. For example, an epitope encoded from a nucleic acid construct may allow an immune response against a particular set of at least one epitope(s) of a protein (e.g., from a protein) to be generated, amplified, attenuated, suppressed, or eliminated.

In some cases, the vaccines described herein are peptide-based vaccines. Peptide-based vaccines can comprise a plasmid encoding one or more epitope(s) selected from survivin, HIF-1A, IGF-1R, or IGFBP 2. Epitopes may be derived from human proteins that can be used directly in peptide-based vaccines.

In some cases, the peptide or nucleic acid construct may be optimized to induce, amplify, or generate a TH1 immune response based on immunization by a protein or plasmid. In some cases, the epitope may be an extended TH1 epitope. In other cases, the peptide or nucleic acid construct may be optimized for protein or plasmid-based immunization to suppress, attenuate, or eliminate a pathogenic response in a subject (e.g., a human or animal) in need thereof.

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 (e.g., a vaccine).

Any plasmid backbone (e.g., vector) known to one of ordinary skill in the art to be suitable for pharmaceutical applications for expressing nucleic acids can be used in the compositions described herein.

The vector may be a circular plasmid or a linear nucleic acid. Circular plasmids or linear nucleic acids are capable of directing the expression of a particular nucleotide sequence in a suitable subject cell. The vector may have a promoter operably linked to a nucleotide sequence encoding a polypeptide, which is operably linked to a termination signal. The vector may also contain sequences required for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, which may initiate transcription only when the host cell is exposed to some specific external stimulus.

The vector may be a plasmid. The plasmid can be used to transfect a cell with a nucleic acid encoding a polypeptide, where the transfected host cell can be cultured and maintained under conditions in which expression of the polypeptide occurs.

The plasmid may comprise a nucleic acid sequence encoding one(s) or more(s) of the plurality of polypeptides described herein. A single plasmid may contain the coding sequence for a single polypeptide, or the coding sequence for more than one polypeptide. Sometimes, the plasmid may also contain a coding sequence encoding an adjuvant, such as an immunostimulatory molecule, such as a cytokine.

The plasmid may further comprise a start codon, which may be upstream of the coding sequence, and a stop codon, which may be downstream of the coding sequence. The start codon and stop codon can be in the frame of the coding sequence. The plasmid may also comprise a promoter operably linked to the coding sequence, and an enhancer upstream of the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle sarcosine or a viral enhancer such as from CMV, FMDV, RSV or EBV. Polynucleotide functional enhancers are described in U.S. Pat. Nos. 5,593,972, 5,962,428 and W094/016737.

The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and to produce multiple copies of the plasmid in the cell. Examples may be pVAXI, pCEP4 or pREP ep4 from invitrogen (san diego, california).

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

In some cases, a commercially available plasmid backbone can be used. For example, plasmid pUMCV 3 can be used. In some cases, the commercially available plasmid backbone can be modified, mutated, engineered, or cloned prior to use. In other cases, non-commercially available plasmid backbones can be used.

Other plasmids may include pSE420 (invitrogen, san diego, california), which can be used to produce proteins in e. The plasmid may also be pYES2 (Invitrogen, san Diego, Calif.) which can be used to produce proteins in a Saccharomyces cerevisiae strain of yeast. The plasmid may also be MAXBAC^TMA complete baculovirus expression system (invitrogen, san diego, california) which can be used to produce proteins in insect cells. The plasmid may also be pcDNA I or pcDNA3 (Invitrogen, san Diego, Calif.) which can be used to produce proteins in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

The vector may be a circular plasmid that can transform a target cell by integration into the genome of the cell or by being present extrachromosomally (e.g., a self-replicating plasmid with an origin of replication). Exemplary vectors include pVAX, pcdna3.0, or provax or any other expression vector capable of expressing DNA encoding an antigen and enabling a cell to translate the sequence into an antigen recognized by the immune system.

The nucleic acid-based vaccine can be a linear nucleic acid vaccine, or a linear expression cassette ("LEC") that can be efficiently delivered into a subject by electroporation and express one or more of the polypeptides described herein. LECs can be any linear DNA with the phosphate backbone removed. The DNA may encode one or more of the polypeptides described herein. LECs may contain promoters, introns, stop codons and/or polyadenylation signals. Expression of the polypeptide may be under the control of a promoter. LECs may not contain any antibiotic resistance genes and/or phosphate backbones. LECs may not contain other nucleic acid sequences unrelated to polypeptide expression.

LECs can be derived from any plasmid that can be linearized. The plasmid may express the polypeptide. Exemplary plasmids include pNP (Puerto Rico/34), pM2(New Caledonia/99), WLV009, pVAX, pcDNA3.0, or provax or any other expression vector capable of expressing DNA encoding an antigen and enabling a cell to translate the sequence into an antigen recognized by the immune system.

The length of the plasmid backbone prior to insertion of the nucleic acid sequence of the at least one epitope(s) may be less than about 500bp, about 1.0kB, about 1.2kB, about 1.4kB, about 1.6kB, about 1.8kB, about 2.0kB, about 2.2kB, about 2.4kB, about 2.6kB, about 2.8kB, about 3.0kB, about 3.2kB, about 3.4kB, about 3.6kB, about 3.8kB, about 4.0kB, about 4.2kB, about 4.4kB, about 4.6kB, about 4.8kB, about 5.0kB, about 5.2kB, about 5.4kB, about 5.6kB, about 5.8kB, about 6.0kB, about 6kB, about 6.7.8 kB, about 8kB, about 10kB, about 8kB, about 8.8kB, about 6kB, about 8kB, about 10.7.8 kB, about 8kB, about 10kB, about 8kB, about 10.7.7.7.7.7.7.7.7 kB, about 10kB, about 8kB, about 10kB, about 8kB, about 6kB, about 8kB, about 8.7.7.7.7.7.7.7.7.7.7.7.7.7.0 kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 10kB, about 8kB, about 2.0kB, about 10.0kB, about 8kB, about 2kB, about 10.0kB, about 8kB, about 2kB, about 8kB, about 2kB, 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, about 16kB, about 16.5kB, about 17kB, about 17.5kB, about 18kB, about 18.5kB, about 19kB, about 19.5kB, about 20kB, about 30kB, about 40kB, about 50kB, about 60kB, about 70kB, about 80kB, about 90kB, about 100kB, about 110kB, about 120kB, about 130kB, about 140kB, about 150kB, about 160kB, about 170kB, about 180kB, about 190kB or about 200 kB. In an exemplary case, the plasmid is about 4kB in length prior to the addition of the nucleic acid sequence encoding the at least one epitope(s).

In some cases, a composition described herein can include a plasmid. In other instances, a composition described herein can include more than one plasmid. For example, a composition described herein can 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.

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

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, a nucleic acid sequence for survivin from a human may be used to express survivin 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 a non-human survivin may be used to express survivin in humans.

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

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, the nucleic acid sequence of HIF-1A from humans can be used to express HIF-1A 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 derived from non-human HIF-1A can be used to express HIF-1A in humans.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, a nucleic acid sequence native to 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 can be used to express HIF-1A in the subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, IGFBP2 may be expressed in humans using nucleic acid sequences from human IGFBP 2. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, IGFBP2 may be expressed in humans using nucleic acid sequences from non-human IGFBP 2.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, IGFBP2 can be expressed in a subject using the native nucleic acid sequence of IGFBP2 in the genome of the species. 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 can be used to express IGFBP2 in a subject.

In some cases, the nucleic acid sequence encoding the epitope can be a nucleic acid sequence that is endogenous to the subject. For example, IGF-1R may be expressed in humans using nucleic acid sequences from human IGF-1R. In other cases, the nucleic acid sequence encoding the antigenic epitope can be an exogenous nucleic acid sequence to the subject. For example, IGF-1R may be expressed in humans using nucleic acid sequences from non-human IGF-1R.

In some cases, the nucleic acid sequence used to express the antigenic epitope may be a wild-type nucleic acid sequence. For example, the native nucleic acid sequence of IGF-1R in the genome of a species may 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 a subject's genome may be modified using molecular techniques known to those of ordinary skill in the art and may be used to express IGF-1R in the subject.

In some cases, a composition may include a nucleic acid encoding one or more epitopes from a protein: survivin, HIF-1A, IGFBP2 and IGF-1R. In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to IGFBP-2(SEQ ID NO: 54). In some cases, a plasmid may comprise a nucleic acid sequence encoding a polypeptide that has 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 comprise 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 comprise a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity to 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 IGFBP-2(SEQ ID NO: 54). In some cases, the 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, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to 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 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 100 to at least 163 contiguous 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).

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 contiguous 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).

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 contiguous 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).

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 contiguous 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).

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 contiguous 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).

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 contiguous 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 contiguous amino acids of IGFBP-2(SEQ ID NO: 54).

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, a 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). In some cases, the plasmid may comprise a nucleic acid sequence that has at least 95% sequence identity to IGFBP-2(SEQ ID NO: 43). In some cases, the plasmid may comprise a nucleic acid sequence having at least 99% sequence identity to IGFBP-2(SEQ ID NO: 43). In some cases, the plasmid may comprise a nucleic acid sequence having 100% sequence identity to IGFBP-2(SEQ ID NO: 43). In some cases, the plasmid may comprise a nucleic acid sequence having 100% sequence identity to IGFBP-2(SEQ ID NO: 43).

In some cases, the plasmid can 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 can 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 can comprise a nucleic acid sequence encoding a polypeptide having at least 90% 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 95% 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 99% sequence identity to survivin (SEQ ID NO: 85). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to survivin (SEQ ID NO: 85). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having 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 70% sequence identity to at least 10 to at least 38 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can 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 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide that has 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).

In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having at least 80% sequence identity to at least 10 to at least 38 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can 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 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide that has 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).

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 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can 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 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide that has 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).

In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to at least 10 to at least 38 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can 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 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide that has 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).

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 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can 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 contiguous 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).

In some cases, the plasmid can comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to at least 10 to at least 38 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can 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 contiguous amino acids of survivin (SEQ ID NO: 85). In some cases, the plasmid can 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).

In some cases, the plasmid can comprise a nucleic acid sequence having at least 50% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid can 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 can comprise a nucleic acid sequence having at least 70% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid can comprise a nucleic acid sequence having at least 80% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid can comprise a nucleic acid sequence having at least 90% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence having at least 95% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence having at least 99% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence having 100% sequence identity to survivin (SEQ ID NO: 86). In some cases, the plasmid may comprise a nucleic acid sequence having 100% sequence identity to survivin (SEQ ID NO: 86).

In some cases, a plasmid can comprise a nucleic acid sequence encoding a polypeptide having at least 70% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid can comprise a nucleic acid sequence that encodes a polypeptide that has 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, a plasmid can comprise a nucleic acid sequence that encodes a polypeptide that has at least 80% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid can comprise a nucleic acid sequence that encodes a polypeptide that has at least 90% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid can comprise a nucleic acid sequence that encodes a polypeptide having at least 95% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid can comprise a nucleic acid sequence that encodes a polypeptide having at least 99% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid can comprise a nucleic acid sequence that encodes a polypeptide having 100% sequence identity to HIF-1A (SEQ ID NO: 87). In some cases, a plasmid can comprise a nucleic acid sequence encoding a polypeptide having 100% sequence identity to HIF-1A (SEQ ID NO: 87).

In some cases, a plasmid can 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, a 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 contiguous amino acids of HIF-1A (SEQ ID NO: 87). In some cases, a plasmid may 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).

In some cases, a plasmid can 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, a 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 contiguous amino acids of HIF-1A (SEQ ID NO: 87). In some cases, a plasmid may 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).

In some cases, a plasmid can 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, a 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 contiguous amino acids of HIF-1A (SEQ ID NO: 87). In some cases, a plasmid may 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).

In some cases, a plasmid can 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, a 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 contiguous amino acids of HIF-1A (SEQ ID NO: 87). In some cases, a plasmid may 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).

In some cases, a plasmid can comprise a nucleic acid sequence that encodes 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, a plasmid may comprise a nucleic acid sequence that encodes 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 contiguous amino acids of HIF-1A (SEQ ID NO: 87). In some cases, a plasmid can comprise a nucleic acid sequence that encodes a polypeptide that has 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).

In some cases, a plasmid can 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, a plasmid can comprise a nucleic acid sequence that encodes 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 contiguous amino acids of 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 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).

In some cases, a plasmid can comprise a nucleic acid sequence that has at least 50% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a 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, a plasmid can comprise a nucleic acid sequence that has at least 70% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid can comprise a nucleic acid sequence that has at least 80% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid can comprise a nucleic acid sequence that has at least 90% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid can comprise a nucleic acid sequence that has at least 95% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid can comprise a nucleic acid sequence that has at least 99% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid can comprise a nucleic acid sequence that has 100% sequence identity to HIF-1A (SEQ ID NO: 88). In some cases, a plasmid can comprise a nucleic acid sequence having 100% sequence identity to HIF-1A (SEQ ID NO: 88).

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 that has 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). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having at least 95% 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 99% sequence identity to IGF-IR (SEQ ID NO: 73). In some cases, the plasmid may comprise a nucleic acid sequence encoding a polypeptide having 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 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 70% sequence identity to at least 50 to at least 104 contiguous 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 contiguous 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 50 to at least 104 contiguous 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 contiguous 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 50 to at least 104 contiguous 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 contiguous 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 50 to at least 104 contiguous 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 contiguous amino acids of IGF-IR (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 50 to at least 104 contiguous 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 contiguous 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 50 to at least 104 contiguous 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 contiguous amino acids of IGF-1R (SEQ ID NO: 73).

In some cases, the plasmid may comprise a nucleic acid sequence having 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 having at least 70% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence having at least 80% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence having at least 90% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence having at least 95% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence having at least 99% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence having 100% sequence identity to IGF-IR (SEQ ID NO: 63). In some cases, the plasmid may comprise a nucleic acid sequence having 100% sequence identity to IGF-IR (SEQ ID NO: 63).

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

Sometimes, the isolated and purified plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from the group consisting of SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 70% sequence identity to the epitope sequence. The plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 80% sequence identity to the epitope sequence. The plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 90% sequence identity to the epitope sequences of the polypeptides. The plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 95% sequence identity to the epitope sequences of the polypeptides. The plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 99% sequence identity to the epitope sequences of the polypeptides. The plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. polypeptides having 100% sequence identity to epitope sequences 73, 85, and 87. The plasmid may comprise at least 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, and 100% sequence identity. In some cases, at least 4 nucleic acid sequences independently encode a polypeptide directed to a sequence selected from SEQ ID NOs: 54. 73, 85, and 87. In other cases, at least 4 of the nucleic acid sequences encode a nucleic acid sequence directed against a nucleic acid sequence selected from SEQ ID NOs: 54. 73, 85, and 87.

In some cases, the plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 70% sequence identity to the epitope sequence. The plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 80% sequence identity to the epitope sequence. The plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 90% sequence identity to the epitope sequences of the polypeptides. The plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 95% sequence identity to the epitope sequences of the polypeptides. The plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, or a polypeptide having at least 99% sequence identity to the epitope sequences of the polypeptides. The plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. polypeptides having 100% sequence identity to epitope sequences 73, 85, and 87. The plasmid may comprise 4 nucleic acid sequences, wherein each of the 4 nucleic acid sequences encodes a polypeptide that differs from a sequence selected from SEQ ID NOs: 54. 73, 85, and 87, and 100% sequence identity. In some cases, the 4 nucleic acid sequences independently encode a polypeptide directed to a sequence selected from SEQ ID NOs: 54. 73, 85, and 87. In other cases, the 4 nucleic acid sequences encode a nucleic acid sequence directed against a nucleic acid sequence selected from SEQ ID NOs: 54. 73, 85, and 87.

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

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

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

Sometimes, a plasmid comprising more than one epitope sequence(s) may comprise a spacer between each epitope sequence. In some cases, the table bit sequence may be encoded in series without using spacers. In some cases, the table bit sequence may be encoded using spacer sub-concatenation. In some cases, the spacer may comprise a sequence encoding from about 1 to about 50, from about 3 to about 40, from about 5 to about 35, or from about 10 to about 30 amino acid residues. In some cases, the spacer may comprise 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.

In some cases, the plasmid may contain a nucleic acid sequence encoding at least one tag. In some cases, the tag may 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 may be a histidine tag having 3 histidine residues, a histidine tag having 4 histidine residues, a histidine tag having 5 histidine residues, a histidine tag having 6 histidine residues, or the like. Any suitable technique known to those skilled in the art may be used to determine the expression of a tag in a subject.

In some cases, the plasmid may be sequenced using any sequencing technique known to those of ordinary skill in the art such that the results of the sequencing provide nucleotide level resolution of the entire plasmid.

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

Nucleic acids

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

An isolated nucleic acid molecule can be DNA, including genomic and cDNA, RNA, or hybrids thereof, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acid may be obtained by chemical synthesis methods or by recombinant methods. Although the term "nucleic acid molecule" mainly refers to a physical nucleic acid molecule and the term "nucleic acid sequence" mainly refers to a nucleotide sequence on a nucleic acid molecule, the two terms are used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, capable of encoding a protein or a protein domain.

A nucleic acid molecule may refer to at least 2 nucleotides covalently linked together. Although the Nucleic Acids described herein may contain phosphodiester linkages in many cases (e.g., in the construction of primers and probes such as labeled probes), Nucleic acid analogs may be included which may have alternating backbones, including, for example, phosphoramides (Beaucage et al, Tetrahedron 49 (10): 1925(1993) and references thereto; 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, Chemicals 26: 141: 1986), phosphorothioate et al, (1986), USA phosphate et al, (19819; see, USA for phosphorothioate analogue, USA) and oligonucleotide linker 3519 (1989, USA) and oligonucleotide linker for oligonucleotide linker 1(1989, USA) and oligonucleotide linker for oligonucleotide linker (1989, S. (1988, USA) and oligonucleotide linker for oligonucleotide linker for oligonucleotide linker for oligonucleotide linker for oligonucleotide for, 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), which are all incorporated herein by reference). Other similar nucleic acids include those having a bicyclic 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, Nucleotide & Nucleotide 13: 1597 (1994); chapters 2 and 3, ASC symphosseries 580, "sugar Modifications in Antisense Research"; Carbohydrate modification in Antisense Research ", Sanghui et al and P ok Con coding"; modifier et al, Bioorganic chemistry & modification in peptide Research "; Sanghui et al, and P ok coding"; sugar Modifications in Meismaker et al, Biochemical chemistry & molecular chemistry & modification, Sanghui et al, Sanghui, and S.7, Sanghui, S.7, and S.7, S.11, and S.5,32, S.5, S. 5,32, et al, Biochemical modification of sugar Modifications in Biocoding, Biochemical engineering, and S.5,32, S.5, et al, S. 5,32, et al, S.5,32, et al, S. 5,32, et al, Nucleotide Modifications, see, Nucleotide, et al, see, Nucleotide, et al, Nucleotide, see, Nucleotide, see, Nucleotide. 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, 1997, 6.2.1997, page 35. "locked nucleic acids" are also included in the definition of nucleic acid analogs. LNA is a class of nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge linking 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 made to increase the stability and half-life of such molecules in physiological environments. For example, PNA: DNA and LNA-DNA hybrids may exhibit greater stability and thus may be used in some embodiments. The target nucleic acid may be single-stranded or double-stranded, as specifically shown herein, or contain portions of both double-stranded or single-stranded sequences. Depending on the application, the nucleic acid can be DNA (including, for example, genomic DNA, mitochondrial DNA, and cDNA), RNA (including, for example, mRNA and rRNA), or hybrids, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides, any combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, hypoxanthine, isocytosine, and isoguanine, and the like.

A recombinant nucleic acid molecule is a molecule comprising at least one of any nucleic acid sequence encoding any one or more of the proteins described herein operably linked to at least one of any transcriptional control sequence effective to modulate the expression of the nucleic acid molecule in the cell to be transfected. Although the term "nucleic acid molecule" mainly refers to a physical nucleic acid molecule and the term "nucleic acid sequence" mainly refers to a nucleotide sequence on a nucleic acid molecule, the two terms are used interchangeably, in particular with respect to a nucleic acid molecule, or a nucleic acid sequence, capable of encoding a protein. In addition, the term "recombinant molecule" refers primarily to a nucleic acid molecule operably linked to a transcriptional control sequence, but is used interchangeably with the term "nucleic acid molecule" administered to an animal.

Recombinant nucleic acid molecules include recombinant vectors, which are any nucleic acid sequence, typically a heterologous sequence, which is operably linked to an isolated nucleic acid molecule encoding a fusion protein of the invention, which enables the recombinant production of the fusion protein, and which is capable of delivering the nucleic acid molecule into a host cell according to the invention. Such vectors may contain nucleic acid sequences that are not naturally found adjacent to the isolated nucleic acid molecule into which the vector is to be inserted. The vector may be eukaryotic or prokaryotic RNA or DNA, and is preferably a virus or plasmid in the present invention. Recombinant vectors can be used for the cloning, sequencing, and/or manipulation of nucleic acid molecules, and can be used to deliver such molecules (e.g., in DNA compositions or viral vector-based compositions). Recombinant vectors are preferably used for the expression of nucleic acid molecules and may also be referred to as expression vectors. Preferred recombinant vectors are capable of expression in transfected host cells.

In the recombinant molecules of the invention, the nucleic acid molecule is operably linked to an expression vector containing regulatory sequences, such as transcriptional control sequences, translational control sequences, origins of replication, and other regulatory sequences, which are compatible with the host cell and which control the expression of the nucleic acid molecule of the invention. In particular, recombinant molecules of the invention include nucleic acid molecules operably linked to one or more expression control sequences. The term "operably linked" refers to the linkage of a nucleic acid molecule to an expression control sequence in a manner that causes the molecule to be expressed upon transfection (i.e., transformation, transduction, or transfection) into a host cell.

Pharmaceutical composition

The immunogenic compositions of the invention are preferably formulated as vaccines for in vivo administration to a subject such that they confer antibody titers that are superior to the seroprotection standard for an acceptable percentage of the antigenic components of the subject. It is well known that the associated antibody titers of an antigen above which a subject is considered to have seroconverted against said antigen are published by tissues such as WHO. Preferably more than 80% of the subject samples with statistical significance are seroconverted, more preferably more than 90%, more preferably more than 93%, most preferably 96-100%.

Adjuvant

The immunogenic compositions of the invention are preferably adjuvanted. Adjuvants may be used to enhance the immune response (humoral and/or cellular) elicited in a patient receiving the vaccine. Sometimes, adjuvants can elicit a TH 1-type response. Sometimes, adjuvants can elicit a TH 2-type response. A TH 1-type response may be characterized by the production of cytokines such as IFN- γ, as opposed to a TH 2-type response, which may be characterized by the production of cytokines such as IL-4, IL-5, and IL-10.

Adjuvants may include stimulating molecules, such as cytokines. Non-limiting examples of cytokines include: CCL20, interferon-alpha (IFN-. alpha.), interferon-beta (IFN-. beta.), interferon-gamma, Platelet Derived Growth Factor (PDGF), TNFa, granulocyte-macrophage colony stimulating factor (GM-CSF), Epidermal Growth Factor (EGF), cutaneous T cell attracting chemokine (CTACK), epidermal thymus-expressing 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-la, MIP-1-, IL-8, L-selectin, P-selectin, E-selectin, MIP-6, and the like, 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 factors, fibroblast growth factors, IL-7, nerve growth factors, vascular epidermal growth factors, Fas, TNF receptors, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARECRD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase, Fos, c-jun, Sp-1, Ap-2, p38, p65, RekD 539 7, MyK-539 7, SAK-SAP-2, SAP-LSA-1, SAP-2, SAP-K-response genes, SAP-685, SAP-2, SAP-K-2, SAP-I, and DNA, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, and TAP 2. In some cases, the adjuvant is GM-CSF.

Other adjuvants include: MCP-1, MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutated forms of IL-18, CD40, CD40L, vascular growth factors, fibroblast growth factors, IL-7, IL-22, nerve growth factors, vascular epidermal growth factors, Fas, TNF receptors, Fit, Apo-1, P55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, TRAIL 5, KIER 2, TRICK-6, caspase 2, FOJUNC-2, FOUN-1, ICE-3, FORD, Sp-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactivated NIK, SAP K, SAP-1, JNK, interferon-responsive 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.

In some aspects, the adjuvant may 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 may be used in conjunction with the vaccines described herein may include, and are not limited to, saponins, CpG ODNs, and the like.

Sometimes, the adjuvant may comprise an aluminium salt, such as an aluminium hydroxide gel (alum), aluminium phosphate, calcium, iron or zinc salt, or may be an insoluble suspension of acylated tyrosine, or an acylated sugar, a cationically or anionically derivatised polysaccharide, or a polyphosphazene.

Sometimes suitable adjuvant systems to promote a major Th1 response include monophosphoryl lipid a or derivatives thereof, especially 3-de-O-acylated monophosphoryl lipid a, and combinations of monophosphoryl lipid a, preferably 3-de-O-acylated monophosphoryl lipid a (3D-MPL), with aluminium salts. The potentiating system comprises a combination of monophosphoryl lipid a and a saponin derivative, particularly QS21 and 3D-MPL, as described in WO 94/00153, or a less reactogenic composition 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 QS 21. The formulation may also comprise an oil-in-water emulsion and tocopherol (WO 95/17210). Unmethylated CpG-containing oligonucleotides (WO 96/02555) are also potential inducers of Th1 responses and are suitable for use in the present invention.

In some cases, aluminum salts are used. Sometimes, polysaccharide conjugates may be unadjuvanted in order to minimize the level of adjuvant in the compositions of the invention.

In some cases, 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 TH 1-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, bacterial endotoxin Immunology and humoral pharmacology of bacterial endotoxins), Plenum pub. Corp., NY, p.407-419, have described a non-toxic derivative of LPS, monophosphoryl lipid A (MPL), which is produced by removal of the core glycosyl group and phosphate from the terminally shortened glucosamine.

Further detoxified forms of MPL are produced by removing the acyl chain from the 3-position of the disaccharide backbone, and are referred to as 3-O-deacylated monophosphoryl lipid A (3D-MPL). It has been purified and prepared by the method described in GB 2122204B, the reference also discloses the preparation of diphosphoryl lipid A and 3-O-deacylated variants thereof.

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 its preparation is described in WO 94/21292. Aqueous formulations comprising monophosphoryl lipid a and a surfactant have been described in WO9843670a 2. 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 is described in GB 2220211 and US 4912094. Other purified and synthetic lipopolysaccharides have been described (Hilgers et al, 1986, int. ArchAllergy. Immunol, 79 (4): 392-6; Hilgers et al, 1987, Immunology, 60 (1): 141-6; and EP 0549074B 1). A particularly preferred bacterial lipopolysaccharide adjuvant is 3D-MPL.

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

Saponins are described in Lacaille-Dubois, M and Wagner H. (1996. review of saponins biological and pharmaceutical activities of saponarins. phytomedine, Vol.2, pp.363-. Saponins are steroid or triterpene saponins widely distributed in the plant and marine animal kingdoms. Saponins are used to form a colloidal solution in water, which forms a foam upon shaking, and to precipitate cholesterol. When saponins approach the cell membrane, they create a porous structure in the membrane, which leads to membrane rupture. Hemolysis of red blood cells is an example of this phenomenon, which is a property of some, but not all, saponins.

Saponins are known as adjuvants in systemically administered vaccines. The adjuvant and hemolytic activity of individual saponins has been extensively studied in the art (Lacaille-Dubois and Wagner, supra). For example, Quil a (bark derived from quillaja saponaria molina) and parts thereof are described in US 5,057,540 and "Saponins as vaccine adjuvants" (saponin adjuvants), "Kensil, c.r., CritRev TherDrug Carrier Syst, 1996, 12 (1-2): 1 to 55; and EP 0362279B 1.

The particle structure comprising the Quil a moiety, known as the Immune Stimulating Complex (ISCOMS), is hemolytic and has been used for the preparation of vaccines (Morein, B., EP 0109942B 1; WO 96/11711; WO 96/33739). Hemolytic saponins QS21 and QS17 (HPLC purified fraction of Quil a) have been described as potent systemic adjuvants and methods for their production are described in us patent nos. 5,057,540 and EP 0362279B 1. Other saponins that have been used in systemic immunization studies include those derived from other plant species such as carnation and soapwort (Bomford et al, Vaccine, 10 (9): 572. sup. 577, 1992).

The potentiating system comprises a combination of a non-toxic lipid a derivative and a saponin derivative, in particular a combination of QS21 and 3D-MPL, as described in WO 94/00153, or a less reactogenic composition in which QS21 is quenched with cholesterol, as described in WO 96/33739.

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.

Sometimes, the adjuvant is an oil-in-water emulsion. An 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 sub-micron diameter, this small size being achieved by the microfluidisation bed to provide a stable emulsion. Droplets with a size of less than 220nm are preferred because they can be filter sterilized.

The oils used may include, for example, oils of animal (e.g., fish) or vegetable origin. Sources of vegetable oil may include nuts, seeds and grains. Examples of the most common nut oils are peanut oil, soybean oil, coconut oil and olive oil. Jojoba oil, for example, available from jojoba may be employed. The seed oil includes safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, etc. The group of grains may comprise: corn oil and oils of other cereals such as wheat, oat, rye, rice, teff, triticale, etc. The 6-10 carbon fatty acid esters of glycerol and 1, 2-propanediol, while not naturally present in seed oils, can be prepared by hydrolysis, isolation and esterification of suitable materials, starting from nuts and seed oils. Fats and oils from mammalian emulsions can be metabolizable and thus can be used in the vaccines described herein. The procedures of separation, purification, saponification and other methods necessary to obtain pure oils of animal origin are well known in the art. Fish may contain readily recoverable metabolizable oil. For example, several examples of fish oils useful herein are cod liver oil, shark liver oil, and whale oil (such as spermaceti wax). A number of branched-chain oils, collectively referred to as terpenes, can be synthesized biochemically in 5-carbon isoprene units. Shark liver oil contains a branched chain unsaturated terpenoid called squalene, 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-tetracosahexaene. The saturated analog of squalene, squalane, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art.

Other useful oils include tocopherol, which may be included in a vaccine for elderly patients (e.g., patients over the age of 60) because vitamin E is reported to have a positive effect on immune responses in this patient population. Additionally, tocopherol has antioxidant properties which help to stabilize the emulsion. There are various tocopherols (alpha, beta, gamma, delta, epsilon, or zeta), but alpha-tocopherol is commonly used. An example of alpha-tocopherol is DL-alpha-tocopherol. Alpha-tocopherol succinate is compatible with cancer vaccines and is a useful preservative to replace mercury-containing compounds.

Oil mixtures may be used, for example, squalene and alpha-tocopherol. The oil content which can be used is from 2 to 20% by volume.

Surfactants can be classified by their "HLB" (hydrophilic/lipophilic balance). In some cases, the surfactant has an HLB of at least 10, at least 15, and/or at least 16. Surface active agentMay include, but is not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as tweens), particularly polysorbate 20 and polysorbate 80; under the trade name DOWFAX^TMCopolymers of Ethylene Oxide (EO), Propylene Oxide (PO) and/or Butylene Oxide (BO) sold, such as linear EO/PO block copolymers; octoxynol with different numbers of repeating ethoxy groups (oxy-1, 2-ethanediyl), of particular interest is octoxynol 9 (Triton X-100 or t-octylphenoxypolyethoxyethanol); (octylphenoxy) polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids, such as phosphatidylcholine (lecithin); esters of nonyl phenols, e.g. Tergitol ^TMNP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as SPAN) such as sorbitan trioleate (SPAN 85) and sorbitan monolaurate. Nonionic surfactants may be used herein.

Mixtures of surfactants may 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 sorbitol ester and/or octoxynol.

The amount of surfactant (% by weight) may be: polyoxyethylene sorbitan esters (e.g., tween 80)0.01 to 1%, particularly about 0.1%; octyl-or nonyl-phenoxypolyethoxyethanols (such as triton X100 or other detergents of the triton series) 0.001-0.1%, in particular 0.005-0.02%; polyoxyethylene ethers (e.g. laureth 9) are present in an amount of 0.1 to 20%, preferably 0.1 to 10%, especially 0.1 to 1% or about 0.5%.

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

a submicron emulsion of squalene, polysorbate 80 and sorbitol trioleate. The composition of the emulsion by volume may be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% span 85. These proportions are 4.3% squalene, 0.5% polysorbate 80 and 0.48% span 85 by weight. This adjuvant is referred to as "MF 59". The MF59 emulsion preferably contains citrate ions, such as 10mM sodium citrate buffer.

A submicron emulsion of squalene, tocopherol, and polysorbate 80. These emulsions may contain 2-10% squalene, 2-10% tocopherol and 0.3-3% polysorbate 80, and the ratio of squalene: the weight ratio of tocopherol is preferably ≦ 1 (e.g., 0.90) because this provides a more stable emulsion. The volume ratio of squalene to polysorbate 80 can be about 5: 2, or the weight ratio can be about 11: 5. One such emulsion can be prepared by the following method: tween 80 was dissolved in PBS to give a 2% solution, and then 90ml of this solution was mixed with a mixture of 5g of DL-a-tocopherol and 5ml of squalene, and then the mixture was microfluidized. The resulting emulsion contains submicron oil droplets having, for example, an average diameter of 100-250nm, preferably about 180 nm. The emulsion may also contain 3-de-O-acylated monophosphoryl lipid A (3 d-MPL). Another useful emulsion of this type may contain (per human dose) 0.5-10mg squalene, 0.5-11mg tocopherol and 0.1-4mg polysorbate 80.

An emulsion of squalene, tocopherol and a triton detergent (e.g. triton X-100). The emulsion may also comprise 3d-MPL (see below). The emulsion may comprise a phosphate buffer.

An emulsion comprising a polysorbate (e.g., polysorbate 80), a triton detergent (e.g., triton X-100), and a tocopherol (e.g., alpha-tocopherol succinate). The emulsion may contain these three components in a mass ratio of about 75: 11: 10 (e.g., 750. mu.g/ml polysorbate 80, 110. mu.g/ml triton X-100, and 100. mu.g/ml alpha-tocopherol succinate), these concentrations should include the contribution of these components in the antigen. The emulsion may further comprise squalene. The emulsion may also comprise 3 d-MPL. The aqueous phase may comprise a phosphate buffer.

Squalane, polysorbate 80 and Poloxamer 401 ("Pluronic^TML121 ") of a latex. The emulsion can be formulated with phosphate buffered saline at pH 7.4. This emulsion is a useful muramyl dipeptide delivery vehicle and can be used with a "SAF-1" adjuvant containing threonyl-MDP (0.05-1% Thr-MDP, 5% squalene, 2.5% pluronic L121 and 0.2% polysorbate 80). It can also be used without Thr-MDP, for example with "AF" adjuvant (5% squalene, 1.25% pluronic L121 and 0.2% polysorbate)80)。

An emulsion comprises squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (such as polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (such as sorbitan ester or mannide ester, such as sorbitan monooleate or "span 80"). The emulsion may be thermally reversible and/or wherein at least 90% of the oil droplets (by volume) are less than 200nm in size. The emulsion may also comprise one or more of the following: a sugar alcohol; cryoprotectants (e.g., sugars such as dodecyl maltoside and/or sucrose); and/or alkyl polyglycosides. The emulsion may comprise a TLR4 agonist. Such emulsions may be lyophilized.

Emulsions of squalene, poloxamer-105 and Abil-Care. The final concentration (by weight) of these components in the adjuvanted vaccine may be 5% squalene, 4% poloxamer-105 (pluronic polyol) and 2% Abil-Care 85 (bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone; caprylic/capric triglyceride).

An emulsion comprising 0.5-50% oil, 0.1-10% phospholipid and 0.05-5% nonionic surfactant. The phospholipid component may include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin, and cardiolipin. Submicron droplet sizes are preferred.

A submicron oil-in-water emulsion of a non-metabolizable oil (such as light mineral oil) and at least one surfactant (such as lecithin, tween 80 or span 80). Additives may include, for example, QuilA saponin, cholesterol, saponin-lipophilic conjugates (such as GPI-0100 produced by the addition of an aliphatic amine to a deacylated saponin via the carboxyl group of glucuronic acid), dimethyldioctadecylammonium bromide and/or N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine.

Vehicles and excipients

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, bacteriostats, chelating agents, suspending agents, thickeners, and/or preservatives), water, oils, including oils from petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, saline solutions, aqueous dextrose and glycerol solutions, flavors, colors, detackifiers, and other acceptable additives, adjuvants, or binders, other pharmaceutically acceptable auxiliary substances required to mimic physiological conditions, such as pH buffers, tonicity adjusting agents, emulsifiers, wetting agents, and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk (chalk), silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. In other cases, the pharmaceutical preparation is preferably preservative-free. In other cases, the pharmaceutical preparation may contain at least one preservative. General approaches to Drug Dosage Forms are found in Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems (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 may be used to administer the pharmaceutical compositions described herein, the type of carrier will vary depending on the mode of administration.

In some cases, the composition may comprise a surfactant. Exemplary surfactants can include octylphenoxypolyethoxyethanol and polyoxyethylene sorbitan esters, as described in Attwood and Florence, Surfactant Systems (1983, Chapman and Hall). Octylphenoxy polyoxyethanols (octoxyols) include t-octylphenoxy polyethoxyethanols (triton X-100)^TM) Also described in Merck Index Entry 6858 (page 1162, 12 th edition, Merck)&Inc., Whitehouse Station, n.j., USA; ISBN 0911910-12-3). The polyoxyethylene sorbitan ester comprises polyoxyethylene sorbitan monoester (Tween 80)^TM) Also described in Merck Index Entry 7742 (page 1308, 12 th edition, Merck)&Inc., Whitehouse Station, n.j., USA; ISBN 0911910-12-3). Both can be prepared using the methods described herein or purchased from commercially available sources such as Sigma (Sigma Inc))。

Exemplary nonionic surfactants can include Triton X-45, t-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 (laureth 9), and polyoxyethylene-9-stearyl ether (steareth 9). The polyoxyethylene ethers may include polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

Other terms or names of polyoxyethylene lauryl ether 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 (12 th edition: entry 7717, Merck & Co.Inc., Whitehouse Station, N.J., USA; ISBN 0911910-12-3). Laureth 9 is formed by reacting ethylene oxide with dodecanol and has an average of 9 ethylene oxide units.

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

Within the experimental error inherent in such biological assays, 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 formula (I) of the present invention preferably has hemolytic activity. Ideally, the polyoxyethylene ether or ester should have a similar hemolytic activity (i.e., within a 10-fold difference) to polyoxyethylene-9 lauryl ether or polyoxyethylene-8 stearyl ether.

2 or more nonionic surfactants from different groups of said surfactants may be present in the vaccine formulations described herein. In particular, polyoxyethylene sorbitan esters such as polyoxyethylene sorbitan monooleate (Tween 80) are preferred^TM) And octylbenzene polysaccharide such as t-octylphenoxy polyethoxyethanol (triton) X-100^TMA mixture of (a). Another particularly preferred combination of nonionic surfactants comprises laureth 9 plus polyoxyethylene sorbitan ester or octoxynol or both.

Preferably, each nonionic surfactant is present in the final vaccine formulation at a concentration of from 0.001 to 20%, more preferably from 0.01 to 10%, and most preferably up to 2% (w/v). Where one or two surfactants are present, they are generally present in the final formulation at concentrations of up to about 2% each, typically up to about 0.6% each. One or more other surfactants may be present, typically each up to a concentration of about 1%, and typically each up to a trace amount of about 0.2% or 0.1%. Any mixture of surfactants may be present in the vaccine formulations of the present invention. The non-ionic surfactants such as those described above have the following preferred concentrations in the final vaccine composition: polyoxyethylene sorbitan esters such as Tween 80 ^TM: 0.01 to 1%, most preferably about 0.1% (w/v); octyl-or nonylphenoxy polyoxyethanols such as triton X-100^TMOr 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).

The compositions may also be encapsulated in liposomes using well known techniques. Biodegradable microspheres may also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. 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.

The composition may be administered into liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are well known to those skilled in the art. U.S. Pat. No. 4,789,734, the contents of which are incorporated herein by reference, describes a method of encapsulating biological material into liposomes. Specifically, the material is dissolved in an aqueous solution, appropriate phospholipids and lipids are added, and surfactants are required, and the material is dialyzed or sonicated as necessary. Gregoriadis, chapter 14, "liposomes", Drug Carriers in Biology and Medicine (drugs in Biology and Medicine), page 2. sup.87-341 (academic Press, 1979) provides an overview of known methods.

Microspheres formed from polymers or proteins are well known to those skilled in the art and may be adapted for direct access to the blood stream via the gastrointestinal tract. Alternatively, the compound may be incorporated and the microspheres or microsphere composites may be implanted for sustained release over a period of from 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.

The composition may contain a preservative such as thimerosal or 2-phenoxyethanol. In some cases, the vaccine is substantially free (e.g., < 10 μ g/ml) of mercury-containing substances, e.g., free of thimerosal. Alpha-tocopherol succinate can be used as a substitute for mercury-containing compounds.

To control tonicity, physiological salts such as sodium salts may be included in the vaccine. Other salts may include potassium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, and/or magnesium chloride, among others.

The composition may have an osmolality in the range of 200 to 400mOsm/kg, 240 to 360mOsm/kg or 290 to 310 mOsm/kg.

The composition may comprise one or more buffers, such as Tris buffer; a borate buffer; a succinate buffer; histidine buffer (particularly with aluminum hydroxide adjuvant); or citrate buffers. In some cases, buffers are included at concentrations generally in the range of 5-20 mM.

The pH of the composition may 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.

The composition may be sterile. The vaccine can be pyrogen-free, e.g., less than 1 EU/dose (endotoxin unit, standard measure), and can be less than 0.1 EU/dose. The composition may be gluten free.

The composition may contain a detergent such as a polyoxyethylene sorbitan ester surfactant (known as "tween"), octoxynol (such as octoxynol-9 (triton X-100) or t-octylphenoxypolyethoxyethanol), cetyltrimethylammonium bromide ("CTAB") or sodium deoxycholate, particularly suitable for use in split or surface antigen vaccines. The detergent may be present in only trace amounts. Thus, the vaccine may comprise octoxynol-10 and polysorbate 80 each in an amount of less than 1 mg/ml. Other trace residual components may be antibiotics (e.g. neomycin, kanamycin, polymyxin B).

The compositions may be formulated as sterile solutions or suspensions, suitable carriers, as are well known in the art. The pharmaceutical compositions may be sterilized by conventional sterilization techniques, which are well known, or may be sterile filtered. The resulting aqueous solution may be packaged for use as is (as-is), or lyophilized, the lyophilized formulation being combined with a sterile solution prior to administration. Suitable formulations and other carriers are described in remington: pharmaceutical sciences and practices (Remington: The Science and Practice of Pharmacy) (20 th edition, Lippincott Williams & Wilkins, Baltimore Md.), which is incorporated herein by reference in its entirety.

The compositions may 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, and the like. Such salts may include salts of inorganic or organic acids, such as hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, p-toluenesulfonic, acetic, fumaric, succinic, lactic, mandelic, malic, citric, tartaric, or maleic acid. Additionally, if the agent contains a carboxyl or other acidic group, it may be converted to a pharmaceutically acceptable addition salt with an inorganic or organic base. Examples of suitable bases include sodium hydroxide, potassium hydroxide, ammonia, cyclohexylamine, dicyclohexyl, ethanolamine, diethanolamine, triethanolamine, and the like.

The addition salt may comprise a bile acid or derivative thereof. These include cholic acid derivatives and salts thereof, especially the sodium salts of cholic acid or cholic acid derivatives. Examples of bile acids and derivatives thereof include cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid and derivatives such as the sugar-, tauro-, aminopropyl-1-propanesulfonic acid-, aminopropyl-2-hydroxy-1-propanesulfonic acid derivatives of the aforementioned bile acids, or N, N-bis (3D glycosaminopropyl) deoxycholamine. A particularly preferred example is sodium deoxycholate (NaDOC), which may be present in the final vaccine formulation.

The compositions described herein comprising an active adjuvant, such as a peptide or nucleic acid, and one or more adjuvants may be formulated in conventional manner using one or more physiologically acceptable carriers, including excipients, diluents, and/or adjuvants, for example, which facilitate processing of the active agent into an administrable formulation. Suitable agents may depend, at least in part, on the route of administration chosen. A variety of routes or modes of administration may be used to deliver the agents described herein, including oral, buccal, topical, rectal, transdermal, mucosal, subcutaneous, intravenous, and intramuscular applications, as well as by inhalation.

The active agent may also be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion bottles or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, for example, solutions in aqueous polyethylene glycol.

For injectable formulations, the carrier may be selected from those known in the art to be suitable, including aqueous or oil suspensions, or emulsions, as well as sesame, corn, cottonseed or peanut oil, as well as elixirs, mannitol, dextrose or sterile aqueous solutions, and similar pharmaceutical carriers. The formulation may also comprise a polymer composition that is biocompatible or biodegradable, such as poly (lactic-co-glycolic acid). These materials can be made into microspheres or nanospheres, loaded with drug and further coated or derivatized to produce superior sustained release properties. Suitable carriers for periocular or intraocular injection include, for example, suspensions of the therapeutic agents in injection-grade water, liposomes, and carriers for lipophilic materials. Other vehicles for periocular or intraocular injection are well known in the art.

In some cases, the compositions are formulated in accordance with conventional methods into pharmaceutical compositions suitable for intravenous administration to humans. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. If desired, the composition may also contain a solubilizing agent and a local anesthetic (e.g., lidocaine) to relieve pain at the site of injection. Typically, the ingredients are provided separately or mixed together in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in a sealed container (e.g., ampoule or sachet) indicating the active agent content. When the composition is administered by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampule of sterile water for injection or saline may be provided for mixing with the pharmaceutical ingredient prior to administration.

When administered by injection, the active agent may be formulated in aqueous solution, particularly 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, stabilizing and/or dispersing agents. Alternatively, the active compound may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. In another embodiment, the pharmaceutical composition does not comprise an adjuvant or any other substance for addition to enhance the immune response stimulated by the peptide. In another embodiment, the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide. Methods of formulation are known in the art, for example, as described in latest edition of Remington pharmaceutical sciences, Mark Publishing Co., Easton P.

In addition to the above formulations, the active agents may also be formulated as depot preparations. Such long acting formulations may be administered by implantation or transdermal delivery (e.g., subcutaneous or intramuscular), intramuscular injection, or using a transdermal patch. Thus, for example, the reagents may also be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

In some cases, compositions comprising one or more agents exhibit local and regional effects when administered topically or injected at or near a particular injection site. Direct topical application of viscous liquids, solutions, suspensions, dimethyl sulfoxide (DMSO) -based solutions, liposomal formulations, gels, jellies, creams, emulsions, ointments, suppositories, foams, or aerosol sprays can be used for topical administration to produce, for example, a topical and/or regional effect. Suitable pharmaceutically acceptable carriers for such formulation include, for example, lower aliphatic alcohols, polyols (e.g., glycerol, or polyethylene glycol), esters of fatty acids, oils, fats, silicones, and the like. Such preparations may also include preservatives (e.g., parabens) and/or antioxidants (e.g., ascorbic acid and tocopherol). See also dermatological preparations: percutaneous absorption (transdermal Formulations: Percutaneous absorption), Barry (eds.), Marcel Dekker Inc, 1983. In another embodiment, a topical formulation comprising a transport agent, vehicle, or ion channel inhibitor may be used to treat an epidermal or mucosal viral infection.

The composition may contain a cosmetically or dermatologically acceptable carrier. Such vehicles are compatible with the skin, nails, mucous membranes, tissues, and/or hair, and may include any commonly used cosmetic or dermal vehicle that meets these requirements. Such carriers can be readily selected by one of ordinary skill in the art. In formulating a skin ointment, the agent or combination of agents may be formulated in an oily hydrocarbon base, an aqueous absorption base, a water-in-oil absorption base, an oil-in-water removable base, and/or an aqueous 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), pyrrolidones, glycerol 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.

Ointments and creams may be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsed or on-demand delivery of pharmaceutical agents.

Lubricants that may 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, a condensed aerosol of silica gel, synthetic silica, or mixtures thereof. The lubricant may optionally be added in an amount of less than about 1% by weight of the pharmaceutical composition.

The composition may be in any form suitable for topical administration, including aqueous, aqueous-alcoholic or oily solutions, emulsions or serum dispersions, aqueous, anhydrous or oily gels, emulsions obtained by dispersing the fatty phase into an aqueous phase (O/W or oil-in-water), or inverse (W/O or water-in-oil), microemulsions or dispersions of microcapsules, microparticles or lipid carriers of ionic and/or non-ionic type. These compositions can be prepared according to conventional methods. The amounts of the various components of the compositions of the present invention, other than the agents of the present invention, are selected as is conventional in the art. These compositions constitute in particular protective, therapeutic or care ointments, milks, lotions or foams for the face, hands, body and/or mucous membranes, or for cleansing the skin. The composition may also consist of a solid preparation constituting a soap or a cleansing bar.

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 amounts of these adjuvants are 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.

For oral administration, the active agent can be readily formulated by combining the active agent with pharmaceutically acceptable carriers well known in the art. The agents of the present invention can be formulated, for example, as tablets (including chewable tablets), pills, dragees, capsules, lozenges, hard candies, liquids, gels, syrups, slurries, powders, suspensions, elixirs, wafers, and the like, to be ingested orally by the patient to be treated. Such formulations may contain pharmaceutically acceptable carriers comprising solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents. A solid carrier can be one or more substances that also act as diluents, flavoring agents, stabilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is typically a finely divided solid which is in admixture with the finely divided active component. In tablets, the active ingredient is generally mixed with a carrier having the required binding capacity in suitable proportions and compacted in the shape and size desired. 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, methyl cellulose, sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and the like. Generally, 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 of the oral dosage form is included in an amount sufficient to provide the desired unit dose.

Aqueous suspensions for oral use can contain the active agent in association with 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 coloring, preserving, flavoring, and the like.

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, e.g., a liposome preparation, may be used. For liposome preparation, any known method of preparing liposomes for treating a disorder can be used. See, e.g., Bangham et al, j.mol.biol.23: 238-: 4194 and 4198(1978), incorporated herein by reference. Ligands may also be attached to the liposomes to direct these compositions to a particular site of action.

Pharmaceutical preparations for oral use can be obtained by: with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; flavour elements, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, for example cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. The agents may also be formulated as sustained release formulations.

Dragee cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain acacia, talc, polyvinyl pyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identification or characterization of different combinations of active agents.

Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active agent may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All dosages of orally administered formulations should be adapted to the mode of administration.

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 may be prepared as solutions, for example, aqueous polyethylene glycol solutions or may contain emulsifying agents, for example, lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickeners. Aqueous suspensions may be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. Suitable fillers or carriers with which the compositions can be administered include sugars, alcohols, fats, lactose, starch, cellulose derivatives, polysaccharides, polyvinylpyrrolidone, silica, sterile saline, and the like, or mixtures thereof, used in suitable amounts. Solid form preparations include solutions, suspensions, and emulsions in addition to the active ingredient and may contain coloring agents, flavoring agents, stabilizing agents, buffering agents, artificial and natural sweeteners, dispersing agents, thickening agents, solubilizing agents, and the like.

A syrup or suspension may be prepared by adding the active compound to a concentrated aqueous solution of a sugar, for example sucrose, to which any adjuvants may be added. Such adjuvants may include flavoring agents, agents that prevent crystallization of the sugar or agent to increase the solubility of any other ingredient, for example, polyols, such as glycerol or sorbitol.

When formulating compounds for oral administration, it is desirable to employ a gastric retentive formulation to enhance absorption in the Gastrointestinal (GI) tract. Formulations that stay in the stomach for several hours can slowly release the compounds of the invention and provide sustained release that can be used herein. The content of such gastroretentive formulations is found in Klausner, e.a.; lavy, e.; barta, m.; cserepes, e.; friedman, m.; hoffman, a.2003 "new gastroretentive dosage forms: evaluation of gastric retention and its effect on levodopa in humans ("Novel gastrointestinal nasal passages for evaluation of gastrointestinal and its effects 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 (pharmacological and pharmacological aspects of gastric retentive forms)" int.j.pharm.11, 141-53, Streubel, a.; siepmann, J; bodmeier, r.; 2006 "gastric retentive drug delivery systems (gastric retentive drug delivery systems)" Expert opin. drug delivery.3, 217-3, and charanpatil, m.d.; jain, P.; chaudhari, s.; shear, r.; vavia, p.r. "new slow release swallowable and bioadhesive gastric retentive drug delivery system for ofloxacin (Novel sustained release, swellable and bioadhesive gastric retentive system for olfoxacin)" int.j.pharm.2006epub.3 months 24. Swellable floating bioadhesive technology can be employed to maximize the absorption of the compounds of the invention.

The solubility of the components of the composition may be enhanced by surfactants or other suitable co-solvents in the composition. Such co-solvents include polysorbate 20, 60 and 80, pluronic F68, F-84 and P-103, cyclodextrins, or other agents known to those skilled in the art.

These co-solvents are typically used at levels of about 0.01 to 2 weight percent.

The composition may be packaged in multiple doses. Preservatives may preferably be used to prevent microbial contamination during use. Suitable agents include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, disodium edetate, sorbic acid, Onamer M, or other agents known to those of skill in the art. In prior art ophthalmic products, such preservatives can be employed at levels of 0.004% to 0.02%. In the compositions of the present invention, preservatives, preferably benzalkonium chloride, may be used at levels of from 0.001% to less than 0.01% by weight, for example from 0.001% to 0.008% by weight, preferably about 0.005% by weight. It has been found that a benzalkonium chloride concentration of 0.005% is sufficient to protect the compositions of the present invention from microbial attack.

In connection with topical administration, the composition may include one or more penetration enhancers. For example, the formulation may comprise a suitable solid or gel phase carrier or excipient that increases permeation and facilitates delivery of the agent or combination of agents of the invention across a permeation 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, alpha-bisabolol, d-limonene, urea, and N, N-diethyl-m-toluamide, and the like. Other examples include humectants (e.g., urea), glycols (e.g., propylene glycol and polyethylene glycol), glycerol monolaurate, alkanes, alkanols, water, orgealase, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and/or other polymers. In another embodiment, the composition may comprise one or more such penetration enhancers.

The topically applied compositions may contain one or more antimicrobial preservatives, such as quaternary ammonium compounds, organic mercurials, p-hydroxybenzoic acid, aromatic alcohols, chlorobutanol, and the like.

The compositions may be formulated as aerosol solutions, suspensions or dry powders. The aerosol may be administered through the respiratory system or nasal passages. For example, one skilled in the art will recognize that the compositions of the present invention may be suspended or dissolved in a suitable carrier, e.g., a pharmaceutically acceptable propellant, and administered directly into the lungs using a nasal spray or inhaler. For example, aerosol formulations comprising a transport agent, carrier, or ion channel inhibitor may be dissolved, suspended, or emulsified in a propellant or a mixture of a solvent and a propellant, e.g., for administration as a nasal spray or inhalation. The aerosol formulation may contain any propellant acceptable under pressure, such as a cosmetically or dermatologically or pharmaceutically acceptable propellant, as is conventionally used in the art.

Aerosol formulations for nasal administration are typically aqueous solutions designed to be administered to the nasal passages as droplets or sprays. Nasal solutions may 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 may additionally be used. Antimicrobial agents or preservatives may also be included in the formulations.

Aerosol formulations or inhalants for inhalation may be designed such that the agent or combination of agents is carried into the respiratory tract of a subject when administered by the nasal or oral respiratory route. The inhalation solution can be administered, for example, by nebulizer. Inhalation or insufflation of a drug containing a fine powder or liquid can deliver a pharmaceutical aerosol of a solution or suspension of the agent or combination of agents in a propellant to the respiratory system, e.g., to aid in compensation. The propellant may be a liquefied gas including halogenated hydrocarbons, for example, fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

Halogenated hydrocarbon propellants may include fluorocarbon propellants in which all hydrogens are replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Halogenated hydrocarbon propellants are described in Johnson, U.S. patent No. 5,376,359, issued 12, 27, 1994; byron et al, U.S. Pat. No. 5,190,029 issued on 3/2/1993; and U.S. Pat. No. 5,776,434 issued to Purewal et al, 7.7.1998. 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 ethers. The aerosol formulations of the present invention may also comprise more than one propellant. For example, the aerosol formulation may contain more than one propellant of the same class, such as 2 or more fluorocarbon-containing hydrocarbons; or more than one, more than 2, more than 3 propellants from different classes, such as fluorocarbons and hydrocarbons. The pharmaceutical compositions of the present invention may also be dispersed with a compressed gas, for example, an inert gas such as carbon dioxide, nitric oxide or nitrogen.

The aerosol formulation may also include other components, for example, ethanol, isopropanol, propylene glycol, and surfactants or other components such as oils and detergents. These components may be used to stabilize formulations and/or lubricating value components.

Aerosol formulations can be packaged under pressure and can be formulated as aerosols using solutions, suspensions, emulsions, powders, and semi-solid preparations. For example, a solution aerosol formulation may comprise a solution of an agent of the invention, such as a transport agent, carrier or ion channel inhibitor, in a (substantially) pure propellant, or a mixture of a propellant and a solvent. The solvent may be used to dissolve the reagent and/or retard propellant evaporation. Solvents may include, for example, water, ethanol, and glycols. Any combination of suitable solvents may optionally be combined with the preservative, antioxidant, and/or other aerosol components.

Aerosol formulations may be dispersions or suspensions. A suspended aerosol formulation may comprise a suspension of an agent or combination of agents of the invention, e.g., a transport agent, carrier, or ion channel inhibitor, and a dispersing agent. Dispersants may include, for example, sorbitan trioleate, oleoyl alcohol, oleic acid, lecithin, and corn oil. The suspension aerosol formulation may also include lubricants, preservatives, antioxidants, and/or other aerosol components.

Aerosol formulations may be formulated similarly to emulsions. Emulsion aerosol formulations may 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, e.g., a transport agent, a carrier, or an ion channel. The surfactants used may be nonionic, anionic or cationic. One 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, vegetable oil, glyceryl monostearate, and propane.

The compounds may also be formulated for administration as suppositories. Low melting waxes, such as triglycerides, fatty acid glycerides, Witepsol S55 (trademark of Dynamite Nobel Chemical, Germany) or mixtures of edible oils are first melted and the active ingredient is dispersed homogeneously, for example by stirring. The molten homogeneous mixture is then poured into a mold of conventional size, allowed to cool, and thereby solidified.

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

The compounds may be releasably attached to biocompatible polymers for sustained release formulations, or to inert objects for local, intraocular, periocular or systemic administration. Controlled release from biocompatible polymers may also be used for water soluble polymers to form pourable formulations. Controlled release from biocompatible polymers such as PLGA microspheres or nanospheres can be used in formulations that are injected using intraocular implants or for sustained release administration. Any suitable biodegradable and biocompatible polymer may be used.

Dosage, route of administration and treatment regimen

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 may 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 may be a multiple antigen ovarian cancer vaccine.

In some cases, the subject may carry a tumor prior to administration of the vaccine. In other cases, the subject may not carry a tumor prior to administration of the vaccine. In other cases, the subject does not carry a tumor prior to administration of the vaccine, but carries a tumor after administration of the vaccine. In other cases, the subject does not carry a tumor prior to administration of the vaccine and may not carry a tumor after administration of the vaccine. In some cases, the tumor may be a breast cancer tumor. In some cases, the breast cancer tumor in the rodent is a DMBA-induced tumor. For example, breast cancer tumors in rodents may be derived from M6 or MMC cells. Typically, breast cancer tumors in humans are triple negative tumors in humans.

The compositions described herein can be administered as a vaccine to a subject in need thereof. In some cases, a subject may be immunized with a multiple antigen breast cancer vaccine or a multiple antigen ovarian cancer vaccine. For example, the vaccine may be a breast cancer vaccine (e.g., a multiple antigen vaccine) or an ovarian cancer vaccine (e.g., a multiple antigen vaccine).

The vaccines described herein can be delivered by a variety of routes. Delivery routes may include oral (including buccal and sublingual), rectal, nasal, topical, transdermal patch, pulmonary, vaginal, suppository or parenteral (including intramuscular, intraarterial, intrathecal, intradermal, intraperitoneal, subcutaneous and intravenous) administration or a form suitable for administration by aerosolization, inhalation or insufflation. General details of Drug Delivery Systems are found in Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems (Pharmaceutical Dosage Forms and Drug Delivery Systems) (Lippencott Williams & Wilkins, Baltimore Md. (1999)). The vaccines described herein may be administered intramuscularly, or may be administered by intradermal or subcutaneous injection, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine may be employed.

In some cases, the vaccine may also be formulated for administration through the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, include coarse powders having a particle size in the range of about 10 to about 500 microns which are administered in the form of snuff, i.e., by rapid inhalation through the nasal passage from a powder container held close to the nose. The formulation may be administered as a nasal spray, nasal drops, or as an aerosol by nebulizer. The formulation may comprise an aqueous or oily solution of the vaccine.

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 (e.g., administration by injection), such as a sterile suspension or emulsion.

Vaccines may contain a single immunisation agent, or may contain multiple immunisation agents (i.e. a "multi-dose" kit). The multi-dose configuration preferably contains a preservative. As an alternative (or complement) to including a preservative in a multi-dose composition, the composition may be contained in a container equipped with a sterile connector to remove the substance.

The vaccine may 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, vaccines can be administered at higher doses, e.g., in excess of 1 ml.

In some cases, a subject may be immunized with a dose of vaccine. In other cases, more than one dose of vaccine may be used to immunize a subject. For example, a subject can be immunized with more than 1 dose, more than 2 doses, more than 3 doses, more than 4 doses, more than 5 doses, more than 6 doses, more than 7 doses, more than 8 doses, more than 9 doses, more than 10 doses, more than 11 doses, more than 12 doses, more than 13 doses, more than 14 doses, more than 15 doses, more than 16 doses, more than 17 doses, more than 18 doses, more than 19 doses, or more than 20 doses of the vaccine. In an exemplary case, the subject is immunized with 3 doses of the vaccine.

In the case where a subject receives more than 1 dose of the vaccine, time may elapse between the first dose of the vaccine and each subsequent dose. In some cases, the time elapsed between the first dose and each subsequent dose of the vaccine may be seconds, minutes, hours, days, weeks, months, or years. For example, the subject may be administered more than 1 dose at intervals. In some cases, the interval may be seconds, minutes, hours, days, weeks, months, or years. In some cases, the subject may receive a booster. For example, a booster can be administered to a subject with more than 1 dose, more than 2 doses, more than 3 doses, more than 4 doses, more than 5 doses, more than 6 doses, more than 7 doses, more than 8 doses, more than 9 doses, more than 10 doses, more than 11 doses, more than 12 doses, more than 13 doses, more than 14 doses, more than 15 doses, more than 16 doses, more than 17 doses, more than 18 doses, more than 19 doses, or more than 20 doses of the booster vaccine. In an exemplary case, the subject may receive up to 3 booster vaccines.

In some cases, the interval may be the same between vaccine doses. In some cases, the interval may be the same between boosters of the vaccine. In some cases, the interval may vary between vaccine doses. In some cases, the spacing may vary between boosters of the vaccine.

In an exemplary case, the subject is administered more than 1 dose at intervals of at least 1 day. In some cases, the interval can be a 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 day interval. In other instances, the interval can be in the range of days, e.g., 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, 20-35 days, 20-40 days, 20-50 days, 25-50 days, 30-50 days, 35-50 days, or 40-50 days.

The subject may be evaluated after administration of the vaccine. In some cases, the subject may be evaluated within 1 month (e.g., short term) of the last vaccine administration. For example, the 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 the last administration of the vaccine. In some cases, subjects can be evaluated within 4 months (e.g., a long term) of the last vaccine administered. For example, the 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 vaccine administration.

In some cases, the subject may receive at least 1 booster of vaccine after the last vaccine dose administered. For example, at least 1 booster can 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 administration. In some cases, a subject may receive 1 boost, 2 boost, 3 boost, 4 boost, 5 boost, 6 boost, 7 boost, 8 boost, 9 boost, 10 boost, 11 boost, 12 boost, 13 boost, 14 boost, 15 boost, 16 boost, 17 boost, 18 boost, 19 boost, 20 boost, 21 boost, 22 boost, 23 boost, 24 boost, 25 boost, 26 boost, 27 boost, 28 boost, 29 boost, or 30 boost.

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

Suitable devices for use in the intradermal vaccines described herein include short needle devices such as those described in US 4,886,499, US5,190,521, US5,328,483, US5,527,288, US 4,270,537, US5,015,235, US5,141,496, US5,417,662. Intradermal vaccines may also be administered by means of devices and functional equivalents thereof which limit the effective penetration length of the needle into the skin, such as those described in WO99/34850, which is incorporated herein by reference. Jet injection devices are also suitable which deliver liquid vaccine to the dermis either by a liquid jet injector or by a needle which penetrates the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described, for example, in US5,480,381, US5,599,302, US5,334,144, US5,993,412, US5,649,912, US5,569,189, US5,704,911, US5,383,851, US5,893,397, US5,466,220, US5,339,163, US5,312,335, US5,503,627, US5,064,413, US5,520,639, US 4,596,556, US 4,790,824, US 4,941,880, US 4,940,460, WO 97/37705 and WO 97/13537. Also suitable are ballistic powder/particle delivery systems which use compressed gas to accelerate the vaccine in powder form through the outer layers of the skin to the dermis. In addition, conventional syringes may be used in the classical mantoux test for intradermal administration. However, the use of conventional syringes requires highly skilled operators and preferably devices capable of accurate delivery without the need for highly skilled users.

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

The compositions of one aspect of the present invention may be administered using techniques well known to those skilled in the art. Preferably, the compounds are formulated and administered by genetic immunization. Techniques for formulation and administration may be found in Remington's pharmaceutical sciences, 18 th edition, 1990, Mark publishing Co., Iston, Bin, Md. Suitable routes may include parenteral delivery, such as intramuscular, intradermal, subcutaneous, intramedullary, and intrathecal, direct intraventricular, intravenous, intraperitoneal, or intraocular injection, and the like. Other routes include oral or transdermal delivery. For injection, the composition of one aspect of the invention may be formulated in an aqueous solution, preferably a physiologically compatible buffer, such as hanks 'solution, ringer' solution or physiological saline buffer.

For parenteral administration, including intramuscular, intradermal, subcutaneous, intranasal, intracapsular, intraspinal, intrasternal, and intravenous injections, especially suitable are injectable sterile solutions, preferably oily or aqueous solutions, and suspensions, or implants, including suppositories. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. These compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

For enteral administration, particularly suitable are tablets, dragees, liquids, drops, suppositories or capsules. The pharmaceutical compositions may be formulated in conventional manner with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (such as lactose, microcrystalline cellulose or dibasic calcium phosphate); lubricants (such as magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Can be administered by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous carriers (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); such liquid formulations are formulated with preservatives (e.g., methyl or propyl parabens, or sorbic acid). The formulation may also suitably contain buffer salts, flavouring agents, colouring agents and sweetening agents. Syrups, elixirs and the like may be employed wherein a sweetening carrier is employed.

Sustained or directed release compositions can be formulated, e.g., liposomes or where the active compound is protected by a differentially degradable coating, e.g., microencapsulation, multicoating, etc. It is also possible to lyophilize the novel compounds and to use the lyophilizates obtained, for example, for preparing products for injection.

For administration by inhalation, the compounds used in one aspect of the invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a metered amount may be delivered through a valve to determine the dosage unit. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. For topical or transdermal administration, non-sprayable forms, viscous to semi-solid or solid forms, comprising a carrier compatible with topical administration and having a dynamic viscosity preferably greater than water, may be employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, salves, powders, liniments, ointments, aerosols, and the like, which can be sterile if desired and mixed with excipients (e.g., preservatives, stabilizers, humectants, buffers, or salts) to affect osmotic pressure, and the like. For topical application, sprayable aerosol preparations are also suitable, wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or mixed with a compressed volatile, typically a gaseous propellant, e.g., freon. If desired, the composition may be enclosed in a packaging or dispensing device which may contain one or more unit dosage forms containing the active ingredient. For example, the package may comprise a metal or plastic sheet, such as a blister pack. The packaging or dispensing device may be accompanied by instructions for administration.

According to one aspect of the invention, the composition may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material well known to those skilled in the art. These materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g., intravenous, cutaneous or subcutaneous, intra-mucosal (e.g., intestinal), intranasal, intramuscular, or intraperitoneal routes.

In general, the term "biological activity" means that a compound (including a protein or peptide) has at least one detectable activity that has an effect on the metabolism or other processes of a cell or organism, as measured or observed in vivo (i.e., in a neutral physiological environment) or in vitro (i.e., under laboratory conditions).

Immunogenicity of compositions

The compositions described herein can be evaluated for immunogenicity in a subject. In some cases, epitopes encoded by a composition (e.g., a plasmid-based vaccine) can be evaluated in a recipient subject. For example, the recipient may be a rodent, a non-human primate, or a human. In some cases, the rodent is a mouse. For example, the mouse may be a neu-TG mouse, a C3 mouse, or an FVB mouse.

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 may 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 may be a multiple antigen ovarian cancer vaccine.

The immune response may be a type I immune response, a type II immune response, or both a type I and type II immune response. In some cases, a type I immune response can result in the secretion of inflammatory cytokines (e.g., IFN γ, TNF α) by antigen-specific T-cells. Inflammatory cytokines (e.g., type I cytokines) can activate cytotoxic T-cells, which can, for example, kill cells expressing at least one epitope encoded by the vaccine (e.g., nucleic acids, plasmids) or delivered (e.g., peptides, proteins). In some cases, the Th1 cytokine may activate other immune cells. In some cases, a type II immune response may result in the secretion of immunosuppressive cytokines (e.g., IL-10, IL-4, and IL-5) by regulatory T-cells. Immunosuppressive cytokines (e.g., type II cytokines) can activate cytotoxic T-cells, which, for example, may not kill cells expressing at least one epitope encoded by the vaccine (e.g., nucleic acids, plasmids) or delivered (e.g., peptides, proteins) but only suppress the Th1 immune response.

Either the Th1 or Th2 immune responses, or both, that may occur in a subject may be the result of the affinity between the epitope and the MHC-T cell receptor interaction. In some cases, the affinity of the binding peptide for MHC molecules may be high. In other cases, the affinity of the binding peptide for MHC molecules may be low. In some cases, low affinity binding peptides can induce a Th2 response. In other cases, high affinity binding peptides can induce a Th1 response. Candidate binding peptides can be screened for affinity to MHC molecules. For example, IFN γ and IL-10 secretion induced by a candidate binding peptide can be determined as described herein or using techniques known to those of ordinary skill in the art.

Any of a variety of methods known to those of ordinary skill in the art can be used to analyze the immunogenicity of a vaccine in a subject. In some cases, immunogenicity can be assayed by detecting expression in a subject of a peptide encoded by a vaccine administered to the subject. For example, detection methods can include ELISPOT, ELISA, Western blot, flow cytometry, histology, chromatography, mass spectrometry, and the like. Generally, immunogenicity against an isolated peptide produced in response to a vaccine in a subject can be analyzed. In some cases, a sample of tumor cells, cancer cells, spleen cells, or normal cells taken from a subject may be analyzed.

In some cases, lymphocytes can be isolated from a subject for analysis of immunogenicity. For example, lymphocytes may be isolated from the spleen, from lymph nodes, and/or from draining lymph nodes. In some cases, lymphocytes may be isolated after administration of a single dose of vaccine. In other cases, lymphocytes may be isolated after the last dose of a multi-dose vaccine is administered. For example, lymphocytes may be isolated 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 after the last single dose of vaccine.

In some cases, lymphocytes may be isolated after the last dose of a multi-dose vaccine is administered. In other cases, lymphocytes may be isolated after the last dose of a multi-dose vaccine is administered. For example, lymphocytes may be isolated 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 after the last dose of the multi-dose vaccine was administered.

In some cases, protein detection methods can be used to determine the amount of each peptide produced by a subject that is encoded by the nucleic acid of a composition (e.g., a plasmid-based vaccine). For example, ELISPOT can be performed and ELISPOT can detect IFN γ. For another example, a differential ELISPOT can be performed and the ELISPOT can detect granzyme B. In some cases, protein detection methods can be used to determine the presence of protein-specific T-cells of a responsive composition (e.g., a plasmid-based vaccine) produced by a subject. For example, ELISPOT can be performed and ELISPOT can detect IFN γ. For another example, a differential ELISPOT can be performed and the ELISPOT can detect granzyme B.

Immunogenicity of a peptide encoded by a vaccine can be determined by comparing the results of a subject administered the composition (e.g., vaccine) to the results of the methods described herein for subjects administered a control composition (e.g., plasmid does not encode any substance or peptide). In some cases, the control may be adjuvant only. In other cases, the control can be a negative control (e.g., a blank plasmid lacking antigenic peptide epitopes). Immunogenicity can be determined by an increase in the amount of IFN γ produced (IFN γ positive spot on ELISPOT) or an increase in the amount of tumor specific granzyme B produced (granzyme B positive spot on ELISPOT). An increase can be observed in the subject following administration of the composition (e.g., vaccine) compared to the subject administered the control composition. In some cases, the increase may be statistically different from the control, as indicated by a P value (e.g., P < 0.05). Typically, statistical differences at p < 0.05 are statistically significant.

For example, statistical significance of immunogenicity can be determined by comparing 2 groups (n ═ 10 subjects/group) at 98% potency, where at least bilateral levels can be 0.05 and true effector volume can be 2.0. In some cases, the effector amount may be defined as the difference between the average specific T-cell response levels divided by the common standard deviation. The amount of true effect of about 1.5 or less will be insignificant.

Other parameters may be analyzed after administration of at least one dose of vaccine. In some cases, blood may be isolated from a subject and subjected to a variety of tests known to those of ordinary skill in the art. For example, basal metabolome and/or complete blood counts are performed. In some cases, other tissues may be examined. For example, the spleen, skin, skeletal muscle, lymph nodes, bone marrow, ovary, fallopian tube, uterus, peripheral nerve, brain, heart, thymus, lung, kidney, liver, and/or pancreas can be examined after administration of at least one dose of the vaccine.

Composition efficacy Using model System

The compositions described herein can be used in a variety of mouse model systems. In some cases, the mouse model can include a genetically diverse mouse model. In some cases, the mouse model may be a tumor transplant model. For example, mice may include TgMMTV-neu (neu-TG) and TgC3(I) -Tag (C3). In some cases, genetically similar mouse models can be used. For example, the neu-TG mouse model system may have a genotype similar to the following 2 different types of human cancers: (1) human adenocarcinoma and is estrogen receptor negative (ER-) and (2) HER2+ human breast cancer and overexpresses the neu oncogene. In other cases, C3 mice have a genotype that may be similar to basal breast cancer and/or triple negative breast cancer. A mouse model of DMBA-induced breast cancer in FVB mice may be xenogeneic and may have tumors comparable to multiple subtypes of human breast cancer. For example, a genetically similar mouse model may be medroxyprogesterone-DMBA-induced tumors (DMBA) in FVB mice.

In some cases, the mouse model may be a tumor transplant model. For example, a tumor transplantation model as shown in fig. 35 can be used to analyze the therapeutic efficacy of the compositions described herein. For example, the combination may be a breast cancer vaccine. In some cases, tumor cells can be implanted subcutaneously in mice. For example, at least 1,000, 2,500, 5,000, 7,500, 10,000, 12,500, 15,000, 17,500, 20,000, 22,500, 25,000, 27,500, 30,000, 35,000, 40,000, 45,000, 50,000, 75,000, 100,000, 125,000, 150,000, 175,000, 200,000, 225,000, 250,000, 275,000, 300,000, 350,000, 400,000, 450,000, 500,000, 750,000, 1,000,000, 1,250,000, 1,500,000, 1,750,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000, 6,500,000, 7,000, 35000, 8,000, 9,000, or less cells may be implanted subcutaneously in a mouse. In some cases, the tumor cell may be an MMA cell.

Tumor growth can be measured using methods known to those of ordinary skill in the art. For example, the measurement methods may include tumor diameter, tumor volume, tumor mass, and the like. In some cases, imaging, extraction, or histological techniques may be used. For example, any technique may include the use of a contrast agent.

In some cases, vaccine efficacy can be determined by the size of tumor growth relative to a control (e.g., an unimmunized mouse or a mouse treated with a control vaccine). For example, in the absence of immunization, more than 90% of mice can develop tumors, and in the presence of immunization, a 60% inhibition of tumor growth can be observed. In some cases, the immunity can inhibit tumor growth by at least 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 99%.

After administration of the vaccine, the subject may be 100% tumor-free. In other cases, the subject may be less than 100% tumor free after administration of the vaccine. For example, a subject may be less than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or less than 10% tumor free after administration of the vaccine. In some cases, the subject may become tumor-free hours after administration of the vaccine. For example, a subject can become tumor-free 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, or more after administration of a vaccine. In other cases, the subject may become tumor-free days after administration of the vaccine. For example, a subject can become tumor-free 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, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, or more after administration of a vaccine. In other cases, the subject may become tumor-free weeks after administration of the vaccine. For example, a subject can become tumor-free 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, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks or more after administration of a vaccine. In other cases, the subject may become tumor-free months after administration of the vaccine. For example, a subject can become tumor-free 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, or more after administration of a vaccine. In other cases, the subject may become tumor-free years after administration of the vaccine. For example, a subject may become tumor-free 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, or more after vaccine administration.

In some cases, the efficacy of a vaccine can be determined by the amount of IFN γ produced in an immunized subject (e.g., mouse) relative to a control (e.g., an unimmunized mouse). In some cases, the efficacy of a vaccine can be determined by the amount of IL-10 produced in an immunized subject (e.g., mouse) relative to a control (e.g., an unimmunized mouse).

In some aspects, the polyclonality of an epitope-specific immune response can be evaluated. In some aspects, polyclonality can be assessed by estimating IgG antibodies produced in response to epitopes of the vaccine administered. In some cases, IgG can be elicited against xenogeneic antigens. In other cases, IgG can be elicited against multiple antigens. In some cases, lysates may be prepared from samples taken from a subject and evaluated from the subject's pre-and post-immune sera. For example, the subject may be a mouse of a neu-TG mouse model, and IgG is detected using a peptide detection method such as ELISA or ELISPOT.

In some cases, statistical methods can be used to analyze the response to each antigen between a pre-immune subject (e.g., a mouse) and a post-immune subject (e.g., a mouse). For example, statistical methods may include analysis using one-way ANOVA. In some cases, analysis of the number of antigens that developed immunity against a subject (e.g., a mouse) during the course of immunization can be performed.

Toxicity and safety profiles of compositions

The toxicity and safety of the compositions described herein can be evaluated. Methods known to those of ordinary skill in the art for assessing toxicity and safety can be used with the compositions described herein. In some cases, dose escalation studies can be performed. In some cases, toxicity and safety studies can be screened for disease development in a subject, organ destruction in a subject, tissue destruction in a subject, cell destruction in a subject, blood disorders, and the like. For example, the disease may include an autoimmune disease.

Preparation and quality control of compositions

Testing for the preparation of the compositions described herein (e.g., plasmid-based vaccines) can be performed according to existing standards for cGMP Biological Production Facilities (BPF). Process development may include transformation of each candidate cell (e.g., cell line) containing the appropriate plasmid construct with a kanamycin selection marker for cGMP BPF. In some cases, a study library can be generated from a bacterial reservoir. For example, pilot production matching the production of newer cGMP can be used to assess plasmid yield and purity. In some cases, an initial production lot record and quality control test protocol may be established. For example, a master cell bank can be generated from each bacterial reservoir. In some cases, quality control tests may be performed, including: plasmid and host cell identity, plasmid copy number, purity, viability, and resistance to biological retention (plasmid retention).

In some cases, final and approved production lot records and standard procedures can be in accordance with cGMP production and purification of vaccine plasmids, and can open up mass release standards. In some cases, the final total/pooled purified product may be quality control tested according to existing regulatory guidelines and then bottled as a single unit dosage form according to validated fill and refine standard operating procedures. According to cGMP regulations, bottled products may be subjected to quality control testing prior to final dosing.

Clinical trials for ovarian cancer

The compositions and methods described herein can be administered to a human subject in need thereof. The compositions and methods described herein can be administered to human subjects in accordance with established guidelines, although for standard times in accordance with the regulations and rules of the federal agricultural administration (FDA) and any other relevant government. In some cases, a pre-study new drug (IND) package was prepared. In some cases, the standard procedures (SOP) for producing the plasmids described herein can be used for production purposes. For example, production may occur in a Good Manufacturing Practice (GMP) facility. In some cases the GMP facility may include a master cell bank.

Clinical trials can be conducted to assess safety and determine the dose of optimal immunogenicity of a multi-antigen DNA plasmid based vaccine. In some cases, clinical trials may be conducted to determine the safety of multiple antigen vaccine (e.g., three doses) administration (e.g., intradermal) in a subject (e.g., patient) likely to have ovarian cancer and to determine the immunogenic dose of a composition (e.g., multiple antigen vaccine) in the subject. In some cases, clinical trials may be conducted to determine the safety of multiple antigen vaccine (e.g., three doses) administration (e.g., intradermal) in a subject (e.g., patient) likely to have ovarian cancer and to determine the immunogenic dose of a composition (e.g., multiple antigen vaccine) in the subject.

The clinical trial can be a phase I trial for the safety and immunogenicity of the compositions (e.g., vaccines) described herein. In some cases, subjects with non-metastatic ovarian cancer may be recruited. In some cases, the patient may have been treated to complete remission. In some cases, the patient may have been treated with primary or rehabilitation therapy. In some cases, patients may have been fully chemo-treated, radiotherapy and/or use systemic steroids prior to enrollment into clinical trials. For example, the patient may 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, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days or more after any of the last cytotoxic chemotherapy and/or radiation therapy and systemic steroid. In an exemplary case, the patient may be 28 days after the last cytotoxic chemotherapy and/or radiotherapy and any use of systemic steroids.

In some cases, phase I can evaluate 3 dosage levels of a composition (e.g., a vaccine). For example, a subject may be assigned one of three doses: group 1(150mcg), group 2(300mcg), and group 3(600 mcg). In some cases, no more than 10 subjects may be recruited in each group. For example, three doses of vaccine may be administered to each subject such that a month elapses between each dose. In some cases, a booster may be administered to the subject after the third dose of vaccine. For example, boosters can be administered such that 1 booster is administered 2 months after the third dose of vaccine and 1 booster is administered 4 months after the booster.

In some cases, 10 subjects of group 2 may be enrolled in a phase I trial if the dose is safe in group 1. In some cases, if group 2 is safe, the immunogenic efficacy of groups 1 and 2 can be tested. For example, if the group 1 dose is more effective, the trial may end. In some cases, subjects may be enrolled into a group 3 dose regimen if the efficacy of the group 2 dose is greater compared to the group 1 dose. In some cases, if group 3 doses appear to be safe, the immune efficacy between groups 2 and 3 will be tested. For example, security can be evaluated as CTEP CTCAE v.4.0. In some cases, the safety benchmark moving to the next group may be 15% or less grade 3 toxicity rate and 15% or less grade 4 toxicity rate.

The efficacy of the immunization at 3 doses can be assessed by assessing T cell production. In some cases, immune efficacy can be defined as achieving enhanced IFN-g T-cellular immunity to an antigen in a vaccine.

The 2 endpoints of the phase I clinical trial of the design and recruitment program may be (1) to determine the safety of intradermal administration of GM-CSF and adjuvant vaccines and (2) to determine the immunogenicity of the vaccine. In some cases, a subject may be immunized with a plasmid-based vaccine (e.g., 100 μ g total plasmid/subject). In some cases, subjects may receive 3 vaccines between intervals (30 days). For example, a vaccine can be administered to a subject at the deltoid region of a draining lymph node (fig. 5).

Subjects with stage IIb, III, and IV ovarian cancer who have been treated to complete remission may be enrolled in the trial. In some cases, the trial may accumulate a target number of subjects (e.g., 22 subjects). Subjects may undergo leukopheresis (e.g., for baseline immune evaluation) and blood draw (e.g., for baseline toxicity evaluation) at the start of the study and draw 180cc of blood at 1, 6, and 12 months after the last immunization. In some cases, blood can be analyzed for changes in serum chemistry as an assessment of potential toxicity. Peripheral blood mononuclear cells and serum collected at these time points can be evaluated for the development of tumor-specific immunity, as described herein. Leukapheresis products can be obtained after the last immunization (e.g., 3 months) for further studies.

And (4) safety. In some cases, the vaccine may elicit an immediate allergic response. In some cases, vaccines can cause side effects as a result of immunization targeting other unrelated tissues. In some cases, after evaluating these toxicities, criteria can be established for discontinuing treatment and removing subjects from the study.

Over 200 subjects received ovarian cancer vaccine during phase I. In some cases, the composition may comprise GM-CSF (100-150. mu.g) in admixture with the ovarian cancer vaccine. In some cases, the composition may be administered using a monthly intradermal injection method for 3-6 months. For example, intradermal administration can result in allergic reactions of the skin. In some cases, the subject may be monitored after immunization (e.g., for 1 hour).

Subjects can be examined at regular visits following administration of the vaccine. In some cases, subjects can be evaluated at each visit based on improved national cancer institute toxicity criteria. In some cases, the subject may undergo a full physical examination. In addition, each subject can be evaluated for serum chemistry, including renal function tests, uric acid, blood counts, serum glucose, and liver function tests. In some cases, the development of connective tissue disorders and laboratory self-antibody responses can be evaluated as potential immunotoxicities associated with immunization with DNA. In some cases, the development of anti-DNA antibodies can be evaluated, e.g., anti-ANA, anti-C3, anti-thyroid, and ds-DNA antibodies. In some cases, the evaluation may be performed at the end of the immunization schedule, and at 12 months thereafter.

A sample size of 22 subjects may be enrolled in each group of the trial. In some cases, if toxicity is not produced, the probability of producing toxicity may be at least 90%, if any grade 3 or 4 toxicity is 10% or less. For example, generating toxicity may represent a toxicity rate of less than 10%. In some cases, the test will continue and may be considered sufficiently safe as long as the observed toxicity rate is consistent with a true grade 3 rate of 15% or less and a true grade 4 rate of 5% or less. In some cases, a stopping rule will be employed such that if there is sufficient evidence that the true toxicity rate exceeds these thresholds, the study is stopped. For example, sufficient evidence may be a lower one-sided confidence limit that exceeds a suitable threshold. For level 3, this limit may be reached if such toxicity levels occur in 2 or less of the first 3, 3 or less of the first 7, 4 or less of the first 12, 5 or less of the first 17, or 6 or less of the first 22 recruits. For level 4, one of the following may cause the test to stop: 2 or less of the first 10, and 3 or less of the first 22 enrolled subjects experienced grade 4 toxicity. For example, if the true probability of grade 3 toxicity is 10% or 30%, then the probability of study discontinuation may be about.06 and.76, respectively. If the true probability of grade 4 toxicity is 3% or 23%, then the probability of cessation is roughly.05 and.93, respectively (estimated from 5000 simulations).

And (3) immunogenicity. The vaccine may elicit an immune response in a subject. In some cases, the type of immune response elicited after immunization can be determined. For example, the compositions described herein may elicit a Th1 immune response when administered to a subject. In some cases, a Th1 immune response may include the formation and maintenance of antigen-specific T cells that recognize at least one peptide of a vaccine. For example, the peptide may be a stem cell and/or EMT antigen.

The type of immune response can be determined by assessing the type of cytokine secreted by the antigen-specific T cells. In some cases, the ELISPOT assay can be used to identify the type of cytokine. For example, the ELISPOT method can include analysis of sample supernatants after antigen stimulation (e.g., 72 hours). In some cases, the sample supernatant may be evaluated for a set of cytokines. In some cases, the evaluation may be a multiplex analysis. For example, multiple assays for cytokines may include cytokines from Th1 (e.g., IFN-g, IL-2, TNF-a, IL-1b, GM-CSF), Th17(IL-17), and Th2 (e.g., IL-6, IL-4, IL-10, IL-13). An exemplary data set is shown in fig. 37. In some cases, the sample supernatant may also be analyzed for the presence of TGF- β. For example, TGF-. beta.may be assayed using ELISA methods. In some cases, the pattern or magnitude of secretion may be used as a biomarker for post-immune clinical outcome.

A heat map can be generated from the multiple cytokine data. In some cases, the heatmap is color-coded according to the magnitude of increase or decrease in the inoculated antigen-specific cytokine. In some cases, the heatmap may show a specific pattern of magnitude and type of immune response to at least one immune antigen.

In some cases, subjects may be classified as immunized by the development of a protein-specific precursor frequency stronger than 1: 20,000PBMC against most immunizing antigens. In some cases, the response may be enhanced by more than 2-fold of the baseline response if the subject is pre-immunized against any antigen.

In some cases, analysis of immunogenicity can determine the magnitude of Th1 antigen-specific immune responses. For example, Th1 responses can be determined by performing IFN-g ELISPOT, at 2.0 and 3.5x10⁵PBMC/well are linear and accurate with a 1: 60,000 monitoring limit and 93% detection efficiency. In some cases, pre-vaccine and post-vaccine samples can be analyzed simultaneously to correct for differences. For example, cryopreservation methods that preserve antigen-specific T cell responses in frozen cells compared to freshly isolated PBMCs can be used. In some cases, the sample may include 1 μ g/ml protein antigen (e.g., recombinant protein accessible on all proposed candidate antigens, human myoglobin (negative control)) or 1 μ g/ml CMV lysate and 0.5U/ml tt (positive control) and peptide antigen included in the vaccine at 10 μ g/ml).

Ovarian cancer vaccines may show immune success, which may be analyzed using statistical methods. In general, the success of immunization of a vaccine may be the development of an immune response against more than 50% of the antigens expressed by the plasmids within the vaccine (e.g., Th 1). In some cases, a vaccine may be administered to a group of subjects (e.g., 22 subjects) such that the probability of more than 50% success rate observed at a true success rate of 40% is below 0.1. For example, the observed success rate may be.06. In some cases, a vaccine may be administered to a group of subjects (e.g., 22 subjects) such that a probability of more than 50% success rate is observed at a true success rate of 70% is greater than 0.7.

For example, using a group of 22 patients, it can be demonstrated with 80% confidence that the estimated immune response can be within a true response rate of at least.14. In some cases, if half of the subjects elicited an immune response, the efficacy may be at least 91% for statistically significant (on a bilateral level of.05) and differences in continuous measurements (if the true effector dose is 1.5). For example, the correlation between 2 consecutive measurements can be estimated using spearman correlation coefficients. In some cases, the data may estimate an expected response rate in a larger population.

As another example, 25% of subjects can elicit a good response against the vaccine. In some cases, 25% of subjects with good response may be baseline to evaluate vaccine efficacy. In some cases, the true response rate may be 60%, where using a set of 22 subjects may provide 97% efficacy for a statistically significant response rate compared to a fixed rate of 25% (one-sided significance level.05).

Clinical trials for breast cancer

In some cases, the clinical trial can be a phase I trial of the safety and immunogenicity of a subject with non-metastatic breast cancer against a composition (e.g., vaccine) described herein. In some cases, the breast cancer may be a positive triple negative (ER (-) PR (-) HER2/neu (-)) breast cancer (TNBC). In some cases, the patient has been treated to complete remission. In some cases, the patient has been treated with primary or rehabilitation therapy. In some cases, patients may have been fully chemo-treated, radiotherapy and/or use systemic steroids prior to enrollment into clinical trials. For example, the patient may 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, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days or more after any of the last cytotoxic chemotherapy and/or radiation therapy and systemic steroid. In an exemplary case, the patient may be 28 days after the last cytotoxic chemotherapy and/or radiotherapy and any use of systemic steroids.

In some cases, as described above, phase I can evaluate 3 dose levels of a composition (e.g., a vaccine), e.g., group 1(150mcg), group 2(300mcg), and group 3(600 mcg). In some cases, no more than 10 subjects may be recruited in each group. For example, three doses of vaccine may be administered to each subject such that a month elapses between each dose. In some cases, a booster may be administered to the subject after the third dose of vaccine. For example, boosters can be administered such that 1 booster is administered 2 months after the third dose of vaccine and 1 booster is administered 4 months after the booster.

In some cases, 10 subjects of group 2 may be enrolled in a phase I trial if the dose is safe in group 1. In some cases, if group 2 is safe, the immunogenic efficacy of groups 1 and 2 can be tested. For example, if the group 1 dose is more effective, the trial may end. In some cases, subjects may be enrolled into a group 3 dose regimen if the efficacy of the group 2 dose is greater compared to the group 1 dose. In some cases, if the group 3 dose appears to be safe, the immune efficacy between groups 2 and 3 will be tested. For example, security can be evaluated as CTEP CTCAE v.4.0. In some cases, the safety benchmark moving to the next group may be 15% or less grade 3 toxicity rate and 15% or less grade 4 toxicity rate.

The efficacy of the immunization at 3 doses can be assessed by assessing T cell production. In some cases, immune efficacy can be defined as achieving enhanced IFN γ T-cell immunity against an antigen in a vaccine.

Plans are designed and recruited. The 2 endpoints of the phase I clinical trial may be (1) to determine the safety of intradermal administration of the GM-CSF and adjuvant vaccine and (2) to determine the immunogenicity of the vaccine. In some cases, a subject may be immunized with a plasmid-based vaccine (e.g., 100 μ g total plasmid/subject). In some cases, subjects may receive 3 vaccines between intervals (30 days). For example, a vaccine can be administered to a subject at the deltoid region of a draining lymph node (fig. 5).

Subjects with stage IIb, III, and IV breast cancer (e.g., triple negative) who have been treated to complete remission may be enrolled in the trial. In some cases, the trial may accumulate a target number of subjects (e.g., 22 subjects). Subjects may undergo leukopheresis (e.g., for baseline immune evaluation) and blood draw (e.g., for baseline toxicity evaluation) at the start of the study and draw 180cc of blood at 1, 6, and 12 months after the last immunization. In some cases, blood can be analyzed for changes in serum chemistry as an assessment of potential toxicity. Peripheral blood mononuclear cells and serum collected at these time points can be evaluated for the development of tumor-specific immunity, as described herein. Leukapheresis products can be obtained after the last immunization (e.g., 3 months) for further studies.

And (4) safety. In some cases, the vaccine may elicit an immediate allergic response. In some cases, vaccines can cause side effects as a result of immunization targeting other unrelated tissues. In some cases, after evaluating these toxicities, criteria can be established for discontinuing treatment and removing subjects from the study.

During phase I, more than 200 subjects received a breast cancer vaccine. In some cases, the composition may comprise GM-CSF (100-150. mu.g) in admixture with a HER2 peptide/protein and/or DNA-based vaccine. In some cases, the composition may be administered using a monthly intradermal injection method for 3-6 months. For example, intradermal administration can result in allergic reactions of the skin. In some cases, the subject may be monitored after immunization (e.g., for 1 hour).

Subjects can be examined at regular visits following administration of the vaccine. In some cases, subjects can be evaluated at each visit based on improved national cancer institute toxicity criteria. In some cases, the subject may undergo a full physical examination. In addition, each subject can be evaluated for serum chemistry, including renal function tests, uric acid, blood counts, serum glucose, and liver function tests. In some cases, the development of connective tissue disorders and laboratory self-antibody responses can be evaluated as potential immunotoxicities associated with immunization with DNA. In some cases, the development of anti-DNA antibodies can be evaluated, e.g., anti-ANA, anti-C3, anti-thyroid, and ds-DNA antibodies. In some cases, the evaluation may be performed at the end of the immunization schedule, and at 12 months thereafter.

A sample size of 22 subjects may be enrolled in each group of the trial. In some cases, if toxicity is not produced, the probability of producing toxicity may be at least 90%, if any grade 3 or 4 toxicity is 10% or less. For example, generating toxicity may represent a toxicity rate of less than 10%. In some cases, the test will continue and may be considered sufficiently safe as long as the observed toxicity rate is consistent with a true grade 3 rate of 15% or less and a true grade 4 rate of 5% or less. In some cases, a stopping rule will be employed such that if there is sufficient evidence that the true toxicity rate exceeds these thresholds, the study is stopped. For example, sufficient evidence may be a lower one-sided confidence limit that exceeds a suitable threshold. For level 3, this limit can be reached as follows: such toxicity levels occur in recruits of 2 or fewer of the first 3, 3 or fewer of the first 7, 4 or fewer of the first 12, 5 or fewer of the first 17, or 6 or fewer of the first 22. For level 4, one of the following may cause the test to stop: 2 or less of the first 10, and 3 or less of the first 22 enrolled subjects experienced grade 4 toxicity. For example, if the true probability of grade 3 toxicity is 10% or 30%, then the probability of study discontinuation may be about.06 and.76, respectively. If the true probability of grade 4 toxicity is 3% or 23%, then the probability of cessation is roughly.05 and.93, respectively (estimated from 5000 simulations).

And (3) immunogenicity. The vaccine may elicit an immune response in a subject. In some cases, the type of immune response elicited after immunization can be determined. For example, the compositions described herein may elicit a Th1 immune response when administered to a subject. In some cases, a Th1 immune response may include the formation and maintenance of antigen-specific T cells that recognize at least one peptide of a vaccine. For example, the peptide may be a stem cell and/or EMT antigen.

The type of immune response can be determined by assessing the type of cytokine secreted by the antigen-specific T cells. In some cases, the ELISPOT assay can be used to identify the type of cytokine. For example, the ELISPOT method can include analysis of sample supernatants after antigen stimulation (e.g., 72 hours). In some cases, the sample supernatant may be evaluated for a set of cytokines. In some cases, the evaluation may be a multiplex analysis. For example, multiple assays for cytokines may include cytokines from Th1 (e.g., IFN γ, IL-2, TNF α, IL-1b, GM-CSF), Th17(IL-17), and Th2 (e.g., IL-6, IL-4, IL-10, IL-13). An exemplary data set is shown in fig. 37. In some cases, the sample supernatant may also be analyzed for the presence of TGF- β. For example, TGF-. beta.may be assayed using ELISA methods. In some cases, the pattern or magnitude of secretion may be used as a biomarker for post-immune clinical outcome.

A heat map can be generated from the multiple cytokine data. In some cases, the heatmap is color-coded according to the magnitude of increase (e.g., red, see fig. 37) or decrease (e.g., blue, see fig. 37) in the inoculated antigen-specific cytokine. For example, the color intensity may represent the lowest (e.g., light, see fig. 37) to highest (e.g., dark, see fig. 37) quartile of the response. In some cases, the heatmap may show a specific pattern of magnitude and type of immune response to at least one immune antigen. For example, the magnitude of cytokine secretion.

In some cases, subjects may be classified as immunized by the development of a protein-specific precursor frequency stronger than 1: 20,000PBMC against most immunizing antigens. In some cases, the response may be enhanced by more than 2-fold of the baseline response if the subject is pre-immunized against any antigen.

In some cases, analysis of immunogenicity can determine the magnitude of Th1 antigen-specific immune responses. For example, Th1 responses can be determined by performing IFN γ ELISPOT, at 2.0 and 3.5x10⁵PBMC/well are linear and accurate with a 1: 60,000 monitoring limit and 93% detection efficiency. In some cases, pre-vaccine and post-vaccine samples can be analyzed simultaneously to correct for differences. For example, cryopreservation methods that preserve antigen-specific T cell responses in frozen cells compared to freshly isolated PBMCs can be used. In some cases, the sample may include 1 μ g/ml protein antigen (e.g., recombinant protein accessible over all of the proposed candidate antigens, human myoglobin (negative control)) or 1 μ g/ml CMV lysate and 0.5U/ml tt (positive control) and peptide antigen included in the vaccine at 10 μ g/ml).

Breast cancer vaccines can show immune success, which can be analyzed using statistical methods. In general, the immune success of a vaccine may be the development of an immune response against more than 50% of the antigens expressed by plasmids within the vaccine (e.g., Th 1). in some cases, a vaccine may be administered to a group of 22 subjects such that the probability of more than 50% success observed with a true success rate of 40% is less than 0.1. For example, the observed success rate may be.06. In some cases, a vaccine may be administered to a group of 22 subjects such that a probability of more than 50% success rate observed at a true success rate of 70% is greater than 0.7.

For example, using a group of 22 patients, it can be demonstrated with 80% confidence that the estimated immune response can be within a true response rate of at least.14. In some cases, if half of the subjects elicit an immune response, the efficacy may be at least 91% for statistically significant (on a bilateral level of 0.05) and differences in continuous measurements (if the true effector dose is 1.5). For example, the correlation between 2 consecutive measurements can be estimated using spearman correlation coefficients. In some cases, the data may estimate an expected response rate in a larger population.

As another example, 25% of subjects can elicit a good response against the vaccine. In some cases, 25% of subjects with good response may be baseline to evaluate vaccine efficacy. In some cases, the true response rate may be 60%, where using a set of 22 subjects may provide 97% efficacy for a statistically significant response rate compared to a fixed rate of 25% (unilateral significance level 0.05).

Applications of

The compositions described herein can be administered to a subject in need of a vaccine to prevent breast or ovarian cancer. In some cases, the cancer is breast cancer. In some cases, the cancer is ovarian cancer. The methods described herein can be combined with the compositions described herein to administer to a subject in need of a vaccine to prevent breast or ovarian cancer. In some cases, administration of the vaccine can trigger cell elimination and the cells begin to express increased levels of proteins as components of the vaccine. In some cases, the protein may be stem cell/EMT-related. For example, increased levels of a protein may be expressed during malignant transformation of normal cells into cancer cells, such as breast cancer cells or ovarian cancer cells. In some cases, elimination of breast or ovarian cancer cells before the disease is clinically evident can prevent the subject from developing cancer. In some cases, elimination of breast or ovarian cancer cells before the disease is clinically evident may prevent the subject from developing breast or ovarian cancer.

A vaccine for preventing breast or ovarian cancer may be administered to a subject in a single dose at a dose of at least 10 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g, 35 μ g, 40 μ g, 45 μ g, 50 μ g, 55 μ g, 60 μ g, 65 μ g, 70 μ g, 75 μ g, 80 μ g, 85 μ g, 86 μ g, 87 μ g, 88 μ g, 89 μ g, 90 μ g, 91 μ g, 92 μ g, 93 μ g, 4 μ g, 95 μ g, 96 μ g, 97 μ g, 98 μ g, 99 μ g, 100 μ g, 101 μ g, 102 μ g, 103 μ g, 104 μ g, 105 μ g, 106 μ g, 107 μ g, 108 μ g, 109 μ g, 110 μ g, 111 μ g, 112 μ g, 113 μ g, 114 μ g, 115 μ g, 116 μ g, 117 μ g, 118 μ g, 119 μ g, 120 μ g, 125 μ g, 155 μ g, 145 μ g, 140 μ g, 145 μ g, 150 μ g, 145 μ g, 95 μ g, 96 μ g, 95 μ g, 2 μ g, 1 μ g, 2 μ g, 1 μ g, 2 μ g, 1 μ g, 2 μ g, 1 μ g, 2 μ g, 1 μ g, 2 μ g, 1 μ g, 2 μ g, 23 μ g, 2 μ g, 1 μ g, 2 μ g, 23 μ g, 2 μ g, 1 μ g, 2 μ g, 23 μ g, 1 μ g, 2 μ g, 23 μ g, 1 μ g, 23 μ g, 1 μ g, 23 μ g, 160 μ g, 165 μ g, 170 μ g, 175 μ g, 180 μ g, 185 μ g, 190 μ g, 195 μ g, or at least 200 μ g per plasmid. In an exemplary case, the single dose administered to a subject is 100 μ g/plasmid.

The vaccine for preventing breast cancer or ovarian cancer may be administered to a subject in more than one dose, each dose being at least 10. mu.g, 15. mu.g, 20. mu.g, 25. mu.g, 30. mu.g, 35. mu.g, 40. mu.g, 45. mu.g, 50. mu.g, 55. mu.g, 60. mu.g, 65. mu.g, 70. mu.g, 75. mu.g, 80. mu.g, 85. mu.g, 86. mu.g, 87. mu.g, 88. mu.g, 89. mu.g, 90. g, 91. mu.g, 92. g, 93. mu.g, 4. g, 95. mu.g, 96. g, 97. g, 98. g, 99. g, 100. g, 101. mu.g, 102. mu.g, 103. g, 104. mu.g, 105. g, 106. g, 107. g, 108. g, 109. g, 110. mu.g, 111. g, 112. mu.g, 113. g, 114. mu.g, 115. g, 116. g, 117. mu.g, 119. g, 130. g, 145. mu.g, 140. mu.g, 145. g, 150. mu.g, 150. g, 70. g, 95. mu.g, 100. g, 1. g, 100. g, 1. mu.g, 100. g, 1. g, 100. g, 1. g, 23. g, 1 g, 20. g, 1 g, 20. g, 23. g, 20. g, 23. g, 1. g, 23. g, 20. g, 23. g, 23. g, 23, 155. mu.g, 160. mu.g, 165. mu.g, 170. mu.g, 175. mu.g, 180. mu.g, 185. mu.g, 190. mu.g, 195. mu.g, or at least 200. mu.g per plasmid. In some cases, each dose administered to a subject may be greater than or less than the dose previously administered to the subject.

The compositions described herein can be administered to a subject in need of a vaccine to treat breast or ovarian cancer. The methods described herein can be combined with the compositions described herein to administer to a subject in need of a vaccine to treat breast or ovarian cancer. In some cases, administration of the vaccine can elicit the elimination of cells that express increased levels of protein elimination as a component of the vaccine. In some cases, the protein may be stem cell/EMT-related. For example, a cancer cell, such as a breast cancer cell or an ovarian cancer cell, may express increased levels of a protein. In some cases, elimination of cancer cells after clinical evidence of disease may prevent the persistence and progression of breast or ovarian cancer in a subject. In some cases, elimination of breast or ovarian cancer cells after clinical evidence of disease may prevent the persistence and progression of breast or ovarian cancer in a subject.

Object

The compositions described herein can be administered to a subject in need of a vaccine against breast or ovarian cancer. The methods described herein can be administered to a subject in need of a vaccine against breast or ovarian cancer in combination with the compositions described herein. In some cases, the vaccine may be administered to a subject that does not have breast or ovarian cancer. In other cases, the vaccine may be administered to a subject who has had breast or ovarian cancer. In other cases, the vaccine may be administered to a subject having breast or ovarian cancer.

In some cases, the subject may be a healthy individual. In some cases, the subject may be an individual with breast or ovarian cancer. For example, the individual may be a patient. In some cases, the subject is a human individual. In other cases, the subject is a non-human individual. For example, the non-human individual may be a non-human primate, including, for example, gorilla and other apes and monkeys; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese and the like. The term "subject" does not denote a particular age. Thus, it is intended to cover both adult and newborn individuals.

Breast and ovarian cancer

In certain embodiments, described herein are vaccines for treating breast cancer or ovarian cancer. In some cases, the breast cancer is a relapsed or refractory breast cancer. In some cases, the ovarian cancer is a relapsed or refractory ovarian cancer. In some cases, the breast cancer is metastatic breast cancer. In some cases, the ovarian cancer is metastatic ovarian cancer.

Breast cancer type

The compositions described herein can be administered to a subject in need of a vaccine against a cancer, typically a breast cancer. The methods described herein can be administered to a subject in need of a vaccine against cancer in combination with the compositions described herein. In general, the breast cancer can be any type of breast cancer, for example, the breast cancer can be ductal carcinoma in situ, phyllodes carcinoma in situ, invasive ductal carcinoma, inflammatory breast cancer, triple negative breast cancer, Paget's disease of the nipple, phyllodes tumor, angiosarcoma, adenoid cystic carcinoma of the tongue, adenoid cystic carcinoma, low-grade adenocarcinoma, medullary carcinoma, mucinous carcinoma, glioma, papillary carcinoma, tubular carcinoma, anaplasia, spindle cell carcinoma, squamous cell carcinoma, papillary carcinoma, and mixed carcinoma.

In some cases, a subject may be classified with a particular grade of breast cancer. For example, the breast cancer may be grade X, 1, 2, 3 or 4. For another example, breast cancer can be expressed as a classification of tubule formation, nuclear grade, and/or mitotic rate. Each category may also assign a specific score between 1 and 3. In some cases, a subject may have a particular stage of breast cancer. In some cases, stages may be assigned based on tumor, regional lymph node, and/or peripheral metabolism. For example, the stage assigned to a tumor may be TX, T0, Tis, T1, T2, T3, or T4. For example, the stage assigned to a regional lymph node may be NX, N0, N1, N2, or N3. For example, the stage assigned to peripheral metabolism may be MX, M0, or M1. In some cases, stage may be stage 0, stage I, stage II, stage III, or stage IV. Typically, breast cancer is classified as being of more than 1 grade or stage of cancer.

Other therapeutic agents

In some cases, the breast or ovarian cancer vaccines described herein can be administered to a patient with other therapeutic agents. In some cases, the other therapeutic agent is a chemotherapeutic agent, a steroid, an immunotherapeutic agent, a targeted therapy, or a combination thereof.

In some embodiments, the additional therapeutic agent is selected from: doxorubicin, actinomycin D, bleomycin, vinblastine, cisplatin, acivicin, aclarubicin, alcodazole hydrochloride, aclonimine, aldeoxine, aldesleukin, altretamine, ambroxycin, amethone acetate, aminoglutethimide, amsacrine, anastrozole, amtricin, asparaginase, clindamin, azacitidine, azatepa, azomycin, batimastat, zotepa, bicalutamide, bisazidoide hydrochloride, bisnefad dimesylate, bizelesin, bleomycin sulfate, brequina sodium, bripirimid, busulfan, actinomycin C, carpoterone, carbinamine, cabetimer, carboplatin, carmustine, camocicin hydrochloride, camabine, cedarvatine, chlorambucil, cerocidine, cerubicin, drobin, drobineA, mechlorethamine, cyclophosphamide, acartisone, dacarbazine hydrochloride, dacarbazine, daclizine hydrochloride, dacarbazine, sildenafine, ciprofloxacin, sildenafine, daclizine, meclizine hydrochloride, daclizine, and other, Daunorubicin hydrochloride, decitabine, dexomalatine, dizaoguaning mesylate, disazoquinone, doxorubicin hydrochloride, droloxifene citrate, drostandrosterone propionate, daptomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, empipridine, epirubicin hydrochloride, ebuzole, esorubicin hydrochloride, estramustine sodium phosphate, etanidazole, etoposide phosphate, chlorphenethamine, fazole hydrochloride, fazabine, fenviramide, azauridine, fludarabine phosphate, fluorouracil, flurocitabine, foquacin sodium, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, imofovir, interleukin 2 (including interleukin 2 or rIL2), interferon alpha-2 a, Interferon alpha-2 b, interferon alpha-n 1, interferon alpha-n 3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liazole hydrochloride, lometrexol sodium, lomustine, losoxantrol hydrochloride, maxolone, maytansine, mechlorethamine hydrochloride, acetate progesterone, melengestrol acetate, melphalan, melnoluril, mercaptopurine, methotrexate sodium, chlorpheniramine, metoteracil, mitotic, mitocaicin, mitorubicin, mitomycin, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, norramycin, omaplatin, oxiran, sulbactam, pemetrexenase, pemetrexen, phosphoamide sulfate, lomustine, pemetrexen, lomustine hydrochloride, lomustine, loxacin, interferon gamma-Ib-B, and other compounds, Pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, pluriment, porfil sodium, pofilomycin, poitemustine, procarbazine hydrochloride, puromycin hydrochloride, pyrazolofuranin, lybodenosine, roguinine, safrog hydrochloride, semustine, octreozine, sodium phosphonoaspartamate, sparcomycin, germanospiramine hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulfochlorpheniramine, talithromycin, tegafur, tilloanthraquinone hydrochloride, temoporfin, teniposide, tirocine, testolactone, thioprim, thioguanine, thiotepa, thiazolfuroline, tirapazamine, toremifene citrate, tritulone acetate, troxitabine phosphate, trimetrexadine, tricresyl, triptorelin hydrochloride, trogliptin, troxerutin hydrochloride, troloxacin hydrochloride, trexone hydrochloride, trematodine hydrochloride, lox, Ulitepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinrolfine sulfate, vinorelbine tartrate, vinrosine sulfate, vinzolidine sulfate, vorozole, zeniplatin, setastatin, and zorubicin hydrochloride.

In some embodiments, the additional therapeutic agent is selected from: 20-epi-1, 25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecanol, aldeoxine, aldesleukin, ALL-TK antagonist, altretamine, ammostatin, amidox (amidox), amifostine, aminoacetylpropionic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, enriches, anti-dorsal chemoattractant protein-1, antiandrogen, antiestrogen, antineoplast (antineoplaton), alfadimycin glycine, apoptotic gene modulator, apoptotic modulator, apurinic nucleic acid, ara-CDP-DL-PTBA, arginine deaminase, asulamydike (ascroline), atamestane, amoxastine, amoxicillin, adrol, adriamycin, adriaminolide, adriaminosine, adriacin, adriaminosine, adriacin, adriakob, adriab, and adriab, and adriab, adria, Asistatin (axinstatin) 1, asistatin 2, asistatin 3, azasetron, azatoxin, diazotyrosine, baccatin III derivatives, balanol (balanol), batimastat, BCR/ABL antagonists, benzodichlorin (benzochlorin), benzoyl staurosporine, beta lactam derivatives, beta-auricine (beta-alethine), betataraxamycin (betaalamin) B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, diazirin spermine, bisnefad, bistratane a (bizelesin) a, bizelesin, befluvalinate (brefellate), bripirimidine, brazidine titanium, butylthionine, calcipotriol, cavidine (calphostin) C, camptothecin derivatives, hypericin-2, canapotabine, carboxyamide-amino-triazole, carboxy-triazole, 3, cartilage derivatives of cartilage-derived n, 89700, Carzelin, Casein kinase Inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chloroquinoxalinesulfonamide, cicaprostil, cis-porphyrin, cladribine, Enclomiphene analogs, clotrimazole, clindamycin (collimycin) A, clindamycin B, combretastatin A4, combretastatin analogs, clonidine (conagenin), Colebesin (Crambescidin)816, Cllinacet, Cryptophycin (Cryptophycin)8, Cryptophycin A derivatives, Jatrophin (curcin) A, cyclopentaquinone (cycloparaquinone), cyclopropanlatam (cyclopendazole), Serpentamycin (dehydropenem), Cytostatin (sodium Astaradelphosphate), Cytosine, Cytostatin (Cytostatin), Dexibin B, dexamethasone, Detrovazamide, dexrazoxane, closterin, clozapine, and a method for example, C-D-S-D-S-D-S-D-S-D, Dabeninin B, dihydroxybenzohydroxamic acid (didox), diethylspermine, dihydro-5-azacytidine, dioxacin (dioxamycin), diphenylspiromustine, docetaxel, dolasetron, doxifluridine, droloxifene, dronabinol, dactinomycin SA, ebselen, etokacin, edelimumab, eflomaniine, elemene, ethimidifluoride, epirubicin, epristeride, estramustine analogs, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastimide, finasteride, clausenone (flavopiridol), fludrostine, luculilone (flusterone), fludarabine, fludaunorubicin hydrochloride (fluorodaunomycin), fotemicin, fotemustine, fopristine, gadolinitum, gadolinium (gadolstatin), gadolzine (gadolzine), Gallium nitrate, galocitabine, ganirelix, gelatinase inhibitor, gemcitabine, glutathione inhibitor, heshum (hepsulfam), heregulin, cyclohexylglycamide, hypericin, ibandronic acid, flavobilin, idoxifene, idomenone, imofovir, ilomastat, imidazidone, imiquimod, immunostimulatory peptides, insulin-like growth factor-1 receptor inhibitor, interferon agonist, interferon, interleukin, iobenguane, iomycin, Ipomoeal, 4-, iloprazine, isosorrel, isobbenazol (isobbenazol), isohalichondrin (isohomohalilondrin) B, itasetron, desmetide (jasplakinolide), carhalelalide (kahalalide) F, lamellarin (lamellarrin) -N triacetate, lanreotide, ranicin (leiampelinocin), polysaccharide, castastine sulfate (1-saratin), saratin, and amatin, Letrozole, leukemia inhibitory factor, leukocyte interferon, leuprorelin + estrogen + progesterone, leuprorelin, levamisole, linazole, linear polyamine analogs, lipophilic glycopeptides, lipophilic platinum compounds, lisocinamide 7, lobaplatin, guanidinoethylserine phosphate serine, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribin, lurtotecan, desporphyrin lutetium (lutetium texaphyrin), lysophenanthroline (lysofylline), lytic peptides, maytansine, minostatin (manostatin) A, marimastat, masoprocol, maspin (maspin), matrilysin inhibitors, matrix metalloproteinase inhibitors, melanolide, thiobarbital, metreleline, methioninase, metoclopramide, MIF inhibitors, mifepristosone, miltefosine, mibexazone, mitoxantrone, RNA mismatch RNA, mismatching RNA, mikava, mitoxanol, leucinolone, mitoxantrone, leucinolone, mibepotriol, mikava, and mismatching RNA, Mitomycin analogs, mitonaphthylamine, mitotoxin (mitoxin) fibroblast growth factor-saporin, mitoxantrone, mofetil, moraxetin, monoclonal antibodies, human chorionic gonadotropin, monophosphoryl lipid a + mycobacterial cell wall backbone, mopidanol, multidrug resistance gene inhibitors, multiple tumor inhibitor 1-based therapies, mustard anticancer agents, Indian sponge (mycaperoxide) B, mycobacterial cell wall extracts, miazinone (myciapponone), N-acetyldinaline, N-substituted benzamides, nafarelin, narestop (nagarethip), naloxone + analgesia, naparin (napavin), napthalene terpene diol (napterin), nartostatin, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamide, nisacicin, antioxidant modulators, nitric oxide, Nidoline (nitrulyn), O6-benzylguanine, octreotide, oxycodone (okicenone), oligonucleotides, onapristone, ondansetron, olanexin (oracin), oral cytokine inducers, ormaplatin, oxaliplatin, oxaonomycin (oxaauromicin), pamolamine (palauaamine), palmitoylrhizomycin, pamidronic acid, panaxatriol, panomifene, paraferin (paraabacin), pazeocin, pemetrexed, pedexrazine, pentostatin, pentrozole (pentoxazole), perfluorobrome, phosphoramide, perillyl alcohol, phenamycin (phenazinomocin), phenyl acetate (phenacylacetate), phosphatase inhibitors, picibacil (pivanil), pivalcaine, pirukacin, piritin, pirbutrine (pterosin) A, a (paclitaxel), a-platinum complex, a-platinum (platinum) complex, platinum (platinum-A) complex, platinum (platinum-platinum (platinum) complex, platinum (platinum) complex), platinum (platinum) and platinum (platinum) complex), or (platinum) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (or) or (, Porphin sodium, poisofycin, prednisone, propylbisacridone, prostaglandin J2, proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors, microalgae (microalgal), protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoline acridines, pyridoxalized hemoglobin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras-GAP inhibitors, demethylated reteplatin, etidronate rhenium Re186, rhizomycin, ribozymes, RII vecarboxamide (retinamide), luovine, Roxitucine (rohituine), romopeptide, Roquinacre, Lubiginone (rubiginone) B1, Lubose (ruboxyl), safrole, ruscogen, saratropine (saidopin), Sarcopherol (CNafei) A, Saxagrastim, Sdi 1 mimetic, semustine, senescence-derived inhibitor 1, sense oligonucleotide, signal transduction inhibitor, signal transduction modulator, single-chain antigen binding protein, cezopyran, sobuzosin, sodium boro-catenate, sodium phenylacetate, solenol (solverol), growth regulator binding protein, sodamine, phosphonomenacin, scamycin (spicamycin) D, spiromustine, splenic pentapeptide (spleenetin), spongistatin (spongistatin)1, squalamine, stem cell inhibitor, stem cell division inhibitor, stillamide (stiiamide), matriptamin inhibitor, semaphorin (sufinistine), potent vasoactive intestinal peptide antagonist, surada (suradita), suramin, swainsonine, synthetic mucopolysaccharide, tamoxifen methiodide, taurinine, tazarotene, teolane, tegafur (tellurium), tellurium (tellurium), and combinations thereof, Telomerase inhibitors, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide (tetrachlordecaoxide), tezoamine (tetrazomine), teriparatine (thalistatin), thiocoraline, thrombopoietin mimetics, thymalfasin, thymopoietin receptor agonists, thymotreonam, thyroid stimulating hormones, betarubicin, tirapazamine, dichlorocyclopentadienium titanium, topostin, toremifene, totipotent stem cell factor, translation inhibitors, tretinoin, triacetyluridine, tricitabine, trimetrexate, triptorelin, tropisetron, tolteromide, tyrosine kinase inhibitors, tyrosine phosphorylation inhibitors (tyrphostin), UBC inhibitors, ubenimex, urogenital derived growth inhibitory factor, urea kinase receptor antagonists, pravastatin, orilin (variolin) B, therapeutic carrier systems, erythrocytic gene, urogenital tract, Veratrix, veratramine, walnuts (verdins), verteporfin, vinorelbine, veclosartan (vinxaline), vitaxine (Vitaxin), vorozole, zanoteron, zeniplatin, benzalvin C, and netstastin ester.

In some embodiments, the additional therapeutic agent is selected from: agents which act by blocking cells in the G2-M phase by virtue of stabilized microtubules, for example, erbuzole (also known as R-55104), dolastatin 10 (also known as DLS-10 and NSC-376128), mevalonine isethionate (also known as CI-980), vincristine, NSC-639829, peridermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also known as E-7010), Otorlin (such as Otorlin A and Otorlin C), Spongastine (such as Spongastine 1, Spongastine 2, Spongastine 3, Spongastine 4, Spongastine 5, Spongastine 6, Spongastine 7, Spongastine 8 and Spongastine 9), Cyindatine hydrochloride (also known as LU-103793 and NSC-669356), Spongastine LU A-1, and Spongastine 9), Epothilones (e.g., epothilone A, epothilone B, epothilone C (also known as desoxyepothilone A or dEpoA), epothilone D (also known as KOS-862, dEpoB and desoxyepothilone B), epothilone E, epothilone F, epothilone B N-oxide, epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (also known as BMS-310705), 21-hydroxyepothilone D (also known as desoxyepothilone F and dEpoF), 26-fluoroepothilone, Orisidine PE (also known as NSC-654663), Sopholide pavilion (also known as TZT-1027), LS-4559-P (Pharmacia, also known as LS-4557), LS-4578(Pharmacia, also known as LS-477-P), and the like, LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), vincristine sulfate, DZ-3358 (Daiichi), FR-182877(Fujisawa, also known as "9885B"), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungary academy of sciences), BSF-223651(BASF, also known as "ILX-651" and "LU-223651"), SAH-49960 (Li/Nowa), SDZ-268970 (Li/Nowa), AM-97(Armad/Kyowa Hakko), AM-132 (Armad), AM-138(Armad/Kyowa Hakko), IDN-5005 (Indena), Cleistanol 52 (also known as "355703), Ajo-39 (Ajo), AVO-7739 (AVO) and SAH-7739 (AVO-7739A), AC-7700(Ajinomoto, also known as AVE-8062, AVE-8062A and CS-39-L-Ser. HCl and RPR-258062A), Veltitrexamide, tobramycin A, Canavanosol, cornasterxanthin (also known as NSC-106969), T-138067(Tularik, also known as T-67, TL-138067, TI-138067), COBRA-1 (Parke-Houss research center, also known as DDE-261 and WHI-261), H10 (university of Kitas), H16 (university of Kansasii), Onculi A1 (also known as BTO-956 and DIME), DDE-313 (Parke-Houss research center), Nondelia Kanlie B, labour Marie, SPA-2 (Parke-Houss research center), SPA-1 (Spke-Houss research center, also known as SPP-IK-research center), Sp-Hous research center, Sp-K-261, and so on, 3-IAABU (cytoskeleton corporation/Cinesian institute of medicine, also known as MF-569), Nacorcine (also known as NSC-5366), Nasicabine, D-24851(Asta medicine corporation), A-105972 (Atpezil corporation), Fenoxanthin, 3-BAABU (cytoskeleton corporation/Cinesian institute of medicine, also known as MF-191), TMPN (university of Arizona), Vanadosin acetylacetonate, T-138026(Tularik corporation), Munserol, Innovoxin (also known as NSC-698666), 3-IAABE (cytoskeleton corporation/Cinesian institute of medicine), A-204197 (Atpeyer corporation), T-607(Tularik corporation, also known as T-900607), RPR-115781(Aventis corporation), abrins (such as demethyl, deacetyl, isodurin-A and Z-arg), Cariboside, Caribelin, Halichondol B, D-64131(Asta medical Corp.), D-68144(Asta medical Corp.), diazoamide A, A-293620 (Yapek), NPI-2350 (Nereus), Tacarnolol A, TUB-245 (Aventis), A-259754 (Yapek), Diazostadine, (-) -phenylassistine (also known as NSCL-96F037), D-68838(Asta medical Corp.), D-68836(Asta medical Corp.), saratin B, D-43411(Zentaris Corp., also known as D-81862), A-289099 (Yapek.), A-318315 (Yapek.), HTI-286 (also known as SPA-110, trifluoroacetate) (Whityashi pharmaceutical Co.), D-82317(Zentaris Corp.), D-82318(Zentaris Corp.), and, SC-12983(NCI), Levostaudin sodium phosphate, BPR-OY-007 (national institutes of health), and SSR-250411(Sanofi corporation).

In some embodiments, the additional therapeutic agent is selected from: agents that affect the tumor microenvironment (e.g., the cell signal transduction network, e.g., the phosphatidylinositol 3-kinase (PI3K) signal transduction pathway, signal transduction from B-cell receptors and IgE receptors). Examples of agents that affect the tumor microenvironment include PI3K signal transduction inhibitors, syc kinase inhibitors, protein kinase inhibitors such as dasatinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, nilotinib, pazoninib, sorafenib, sunitinib, temsirolimus; other angiogenesis inhibitors such as GT-111, JI-101, R1530; other kinase inhibitors such as AC220, AC480, ACE-041, AMG 900, AP 245634, Arry-614, AT7519, AT9283, AV-951, axitinib, AZD1152, AZD7762, AZD8055, AZD8931, Bafitinib, BAY 73-4506, BGJ398, BGT226, BI 811283, BI6727, BIBF 1120, BIBW 2992, BMS-690154, BMS-777607, BMS 5631-461364, CAL-101, CEP-11981, CYC116, DCC-2036, Didizhicilib, polyvirtinib lactate, E7050, EMD 1214063, ENMD-2076, foscamtinib disodium, GSK 6098, GSK690693, INCB18424, INNO-406, JJ-26483327, JX 594-594, Kneiny 391-391, NMS-NMS 8, NMS-36391, NMS 8-Na-367354, MLN-36 1286937, MK-36493, OSI-36 1286937, OSI-3647, IBM-353, IBM-353, NMS-IBM-3, IBM-3, IBM-3, IBM-3, IBM-3, IBM-3, IBM-3, IBM-3, NAK-3, IBM-, PF-02341066, PF-03814735, PF-04217903, PF-04554878, PF-04691502, PF-3758309, PHA-739358, PLC3397, hematopoietic growth factor, R547, R763, ramucirumab, regorafenib, RO5185426, SAR103168, SCH727965, SGI-1176, SGX523, SNS-314, TAK-593, TAK-901, TKI258, TLN-232, TTP607, XL147, XL228, XL281RO5126766, XL418, XL 765.

In some embodiments, the additional therapeutic agent is selected from: mitogen-activated protein kinase signal transduction inhibitors, such as U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin or LY 294002; (ii) a Syk inhibitor; an mTOR inhibitor; and antibodies (e.g., rituximab).

In some embodiments, the additional therapeutic agent is selected from: interferons, interleukins, tumor necrosis factors, growth factors, and the like.

In some embodiments, the additional therapeutic agent is selected from: ansisitine, filgrastim, lengrastim, morastim, pegfilgrastim, sargrastim; interferons such as natural interferon alpha, interferon alpha-2 a, interferon alpha-2 b, interferon alfacon-1, interferon alpha-n 1, natural interferon beta, interferon beta-1 a, interferon beta-1 b, interferon gamma, pegylated interferon alpha-2 a, pegylated interferon alpha-2 b; interleukins such as aldesleukin, opper interleukin; other immunostimulants such as bcg, glatiramer acetate, histamine dihydrochloride, immune cyanines, lentinan, melanoma vaccine, mivawood peptide, pegase, pidotimod, plexafof, poly I: C. poly ICLC, loquimect, tasolomine, thymopentin; immunosuppressants such as abacavir, abelimus, exemestane, anti-lymphocyte immunoglobulin (horse), anti-thymocyte immunoglobulin (rabbit), eculizumab, efavirenzumab, everolimus, guanlimus, leflunomide, molomab-CD 3, mycophenolic acid, natalizumab, sirolimus; TNF- α inhibitors such as adalimumab, afiumumab, certolizumab ozogamicin, exemestane, golimumab, infliximab; interleukin inhibitors such as anakinra, basiliximab, cananouumab, daclizumab, meperilizumab, linacetp, tositumumab, ubeskinumab; calcineurin inhibitors such as cyclosporine, tacrolimus; other immunosuppressive agents such as azathioprine, methotrexate, lenalidomide, thalidomide.

In some embodiments, the additional therapeutic agent is selected from: adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab, daclizumab, eculizumab, efletuzumab, gemuzumab, ibritumomab, infliximab, moruzumab-CD 3, natalizumab, panitumumab, rituximab, tositumomab, trastuzumab, ranibizumab, and the like, or a combination thereof.

In some embodiments, the additional therapeutic agent is selected from: monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, eltromumab, edrecolomab, gemuzumab, panitumumab, rituximab, trastuzumab; immunosuppressants, eculizumab, efletuzumab, morronizumab-CD 3, natalizumab; TNF- α inhibitors such as adalimumab, alfumomab, golimumab, infliximab; interleukin inhibitors, basiliximab, kanamyudumab, daclizumab, meperfolizumab, tuzumab, ubschumab; radiopharmaceuticals, ibritumomab, tositumomab, monoclonal antibody; other monoclonal antibodies such as abamectin, adalimumab, alemtuzumab, anti-CD 30 monoclonal antibody Xmab2513, anti-Met monoclonal antibody MetMAb, aprezumab, arabizumab, alemtuzumab, basiliximab, bispecific antibody 2B1, bonarezumab, bretuximab, carpolizumab, cetuximab, clausizumab, conatuzumab, daclizumab, dinolizumab, ecumab, epratuzumab, ermatuzumab, ertuzumab, fituzumab, frietuzumab, galiximab, ganitumumab, gemtuzumab, greumumab, ibritumomab, oimtuzumab, ipilimumab, rituximab, lintuzumab, lutumuzumab, matuzumab, milatuzumab, CC49, monoclonal antibody CC49, Niximumab, nimotuzumab, agovacizumab, pertuzumab, ramumab, ranibizumab, cetilizumab, matuzumab, tanlizumab, tositumomab, trastuzumab, tremelimumab, simon interleukin mab, vituzumab, vislizumab, floriximab, zalutumumab.

In some embodiments, the additional therapeutic agent is selected from: nitrogen mustards such as bendamustine, chlorambucil, nitrogen mustards, cyclophosphamide, ifosfamide, melphalan, prednimustine, chloroacetohydroxamide; alkyl sulfonates such as busulfan, mannosuman, trooshusan; ethyleneimines such as carboquinone, thiotepa, and triaminoquinone; nitrosoureas like carmustine, fotemustine, lomustine, nimustine, ranimustine, semustine, streptozotocin; epoxy compounds such as ethydine; other alkylating agents, such as dacarbazine, dibromomannitol, methylphenidate, temozolomide; folic acid analogs such as methotrexate, pemetrexed, pralatrexate, raltitrexed; purine analogs such as cladribine, clofarabine, fludarabine, mercaptopurine, nelarabine, thioguanine; pyrimidine analogues such as lenazacitidine, capecitabine, carmofur, cytarabine, decitabine, fluorouracil, gemcitabine, tegafur; vinca alkaloids such as vinblastine, vincristine, vinblastine, vinflunine, and canosine; podophyllotoxin derivatives such as etoposide, teniposide; colchicine derivatives such as colchicine; taxoids such as docetaxel, paclitaxel, and polyglutamic acid paclitaxel; other plant alkaloids and natural products such as trabectedin; actinomycin compounds such as dactinomycin; anthracyclines such as daunorubicin, doxorubicin, aclarubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, valrubicin, zurubicin; other cytotoxic antibiotics such as bleomycin, ixabepilone, mitomycin, plicamycin; platinum compounds such as carboplatin, cisplatin, oxaliplatin, satraplatin; methylhydrazines such as procarbazine; sensitizers such as aminolevulinic acid, sensitizers, methylaminolevulinic acid, porfimer sodium, temoporfin; protein kinase inhibitors such as erlotinib, gefitinib, dasatinib, everolimus, imatinib, lapatinib, nilotinib, pazoninib, sorafenib, sunitinib, temsirolimus; other antineoplastic agents such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, bexarotene, bortezomib, celecoxib, dinil, estramustine, hydroxyurea, irinotecan, lonidamine, maxorinol, miltefosine, propimidrazone, mitotane, orlistat, pemetrexed, romidepsin, sisimal, thiazoline, topotecan, tretinoin, vorinostat; estrogens such as diethylstilbene, ethinylestradiol, fosfestrol, polyestradiol; progestogens such as norgestrel, megestrol; gonadotropin releasing hormone analogues such as buserelin, goserelin, leuprorelin, triptorelin; antiestrogens such as fulvestrant, tamoxifen, toremifene; antiandrogens such as bicalutamide, flutamide, nilutamide, enzyme inhibitors, aminoglutethimide, anastrozole, letrozole, exemestane, formestane, vorozole; other hormone antagonists such as abarelix, degarelix; immunostimulants such as histamine dihydrochloride, mivaxatide, pidotimod, plexafot, roquinacre, thymopentin; immunosuppressants such as everolimus, guanolimus, leflunomide, mycophenolic acid, sirolimus; calcineurin inhibitors such as cyclosporine, tacrolimus; other immunosuppressive agents such as azathioprine, methotrexate, thalidomide, lenalidomide; and radiopharmaceuticals such as iodobenzylguanidine.

In some embodiments, the additional therapeutic agent is selected from checkpoint inhibitors. Exemplary checkpoint inhibitors include:

PD-L1 inhibitors such as MPDL3280A (RG7446) from Genentech, anti-mouse PD-L1 antibody clone 10F.9G2 (catalog number BE0101) from BioXcell, anti-PD-L1 monoclonal antibody MDX-1105(BMS-936559) and BMS-935559 from Bristol-Meyer's Squibb, MSB0010718C, mouse anti-PD-L1 clone 29E.2A3, and MEDI4736 from AstraZeneca;

PD-L2 inhibitors such as AMP-224(Amplimmune) from Kulanbin Stecke, and rHIgM12B 7;

PD-1 inhibitors from BioXcell such as anti-mouse PD-1 antibody clone J43 (catalog number BE0033-2), anti-mouse PD-1 antibody clone RMP1-14 from BioXcell (catalog number BE0146), mouse anti-PD-1 antibody clone EH12, Merck MK-3475 anti-mouse PD-1 antibody (Keytruda, Pamumab, Lamblizumab), anti-PD-1 antibody of AnatypsBio designated ANB011, antibody MDX-1106(ONO-4538), human IgG4 monoclonal antibody of Bristol-Myers Squibb Nanwumab (Nonwuzab) (Biokum-K)BMS-936558, MDX1106), AMP-514 and AMP-224 from AstraZeneca, Pidilizumab (Pidilizumab) from curative technology Ltd (CureTech Ltd) (CT-011);

anti-CTLA-4 antibody ipilimumab (also known as Bristol Meyers Squibb) to CTLA-4 inhibitors such as Bristol Meyers Squibb (see also for MDX-010, BMS-734016, and MDX-101), anti-CTLA 4 antibody clone 9H10 from milbebo, tremelimumab (CP-675, 206, tixelimumab) from paris, and anti-CTLA 4 antibody clone BNI3 from Abcam;

LAG3 inhibitors such as the anti-LAG-3 antibody clone eBioC9B7W (C9B7w) from ebiosciences, the anti-LAG 3 antibody LS-B2237 from LifeSpan Biosciences, IMP321(ImmuFact) from Immutep, the anti-LAG 3 antibody BMS-986016, and the LAG-3 chimeric antibody A9H 12;

B7-H3 inhibitors such as MGA 271;

KIR inhibitors such as rituximab (Lirilumab) (IPH 2101);

CD137 inhibitors such as Ulumamab (urelumab) (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1 BB, PF-2566, pyroxene), or XmAb-5592 (Xencor);

PS inhibitors such as baveximab;

and inhibitors, such as antibodies or fragments thereof (e.g., monoclonal, human, humanized, or chimeric antibodies), RNAi molecules, or small molecules against TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

Sample (I)

Samples for analysis of immunogenicity, safety and/or toxicity may be isolated from an individual. In some cases, the sample may be selected from the group consisting of: whole blood, isolated blood, serum, plasma, sweat, tear fluid, ear fluid, sputum, lymph, bone marrow suspension, lymph, urine, saliva, semen, vaginal fluid, stool, transcervical lavage, cerebrospinal fluid, cerebral fluid, ascites fluid, breast milk, vitreous fluid, aqueous humor, sebum, endolymph (endolymph), peritoneal fluid, pleural fluid, cerumen, epicardial fluid, and secretions of the respiratory, intestinal, and genitourinary tracts. In some cases, the sample may be a tissue, typically a biopsy sample. For example, a biopsy may contain skin tissue, heart tissue, gland tissue, skeletal muscle tissue, and/or adipose tissue.

Reagent kit

Kits and articles of manufacture are also provided herein for use in one or more of the methods described herein. The kit may contain one or more of the nucleic acid molecules and/or one or more of the polypeptides described herein, such as SEQ ID NO: 1-91, or a polypeptide or nucleic acid having an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 1-91, or a nucleic acid, having a sequence of at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more sequence homology. The kit may also contain a nucleic acid encoding one or more of the polypeptides described herein. The kit may also contain adjuvants, reagents, and buffers necessary for the preparation and delivery of the vaccine.

The kit may comprise a carrier, package, or container that is compartmentalized to receive in one or more containers, e.g., vials, tubes, and the like, each container comprising a separate element, e.g., a polypeptide and an adjuvant, for use in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be made of various materials such as glass or plastic.

The articles provided by the present invention comprise packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for the desired formulation and desired mode of administration and treatment.

The kit halves contain labels for the listed components and/or instructions for use, and package inserts with instructions for use. A set of instructions is typically provided.

Examples

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The scope of the invention is determined by the following claims, and methods and structures within the scope of these claims and their equivalents are intended to be covered by this invention.

Example 1

Identification of breast cancer antigens and determination of epitopes eliciting Th1 CD4+ T-cell responses

Sera from 224 individuals were used for antibody analysis. Antibody responses of 124 patients with stage I/II breast cancer (BrCA) were evaluated against each candidate protein: 78ER + (63%), 31HER2+ (25%), and 15TNBC (12%). The control group consisted of 100 age-matched women. Candidate proteins for analysis of immunogenicity are shown in the following figures using a mouse model. In addition, candidate proteins for analysis of immunogenicity are listed in FIG. 39.

Indirect ELISA using recombinant proteins. Recombinant proteins can be used for all proposed candidate antigens. Subject sera were analyzed in duplicate or triplicate at titers of 1: 100 and 1: 200. Western blot analysis demonstrated that the sensitivity and specificity of the ELISA assay was 70% or higher, except for CD105, which was not verified by Western blot.

Breast cancer pathology and controls were screened. All tested proteins were immunogenic, i.e., at least 1 individual analyzed demonstrated detectable IgG antibody immunity to a particular antigen, as demonstrated by Western blotting with an appropriate specificity control (e.g., CDH3 in fig. 3). Antibody responses can be identified in volunteer controls and cancer patients. To illustrate the immunity rate against a particular antigen, a control population of average ug/ml and 2 standard deviations was used to determine a cutoff value at 95% confidence that the response was considered positive. The antibody immunity rates of the tested antigens ranged from 2% (BrCA patients against Yb-1) to 13% (BrCA patients against CDC25 b) positive. Demonstrating a more than 2-fold difference in response rate between patients and donors for BrCA over 6 antigens: CDC25b, CDH3, survivin, MDM2, SATB1, and ID 1. It should be noted that SATB1 and ID1 responses were found in a greater number of volunteer donors compared to cancer patients. Fig. 4 panel B shows the results for 9 antigens with a 2-fold or less difference in incidence between BrCA patients and donors.

Statistical analysis of cancer specificity for immune response. Four of the antigens demonstrated statistically significant differences in immunity between cancer patients and controls using the welch's corrected unpaired T test. BrCA patients had higher levels of antibody immunity against CDC25B (p 0.02), CDH3(p 0.0002), survivin (p 0.09) than volunteer controls.

Candidate antigens are identified for epitope mapping. Identification of 15 stem cell/EMT-related immunogenic proteins mapped to epitopes.

For example, see FIG. 28, which shows that T-cells responding to an epitope from the C-terminus of IGFBP-2 secrete IFN γ and IL-10. Specifically, FIG. 28 shows in section (A) ELISPOT of IFN γ (white) and IL-10 (black) in breast cancer patient PBMCs against IGFBP-2 peptide, shown as a quartile box plot with Duke whiskers (Tukey whisker). The median correction point per well (CSPW) is represented by a horizontal line. In part (B), PBMCs stimulated with IGFBP-2 peptides in ELISPOT induced a percentage of IFN γ (white bars) responses, IL-10 (black bars) responses, or both (gray bars).

In another example, see FIG. 29, which shows tumor growth inhibition and Th1 immune responses against the N-terminus, but not the C-terminus, of IGFBP-2. (A) IFN γ ELISPOT from splenocytes of mice immunized with the indicated vaccine. Data are expressed as per-well calibration points (CSPW). Horizontal lines represent mean CSPW ± SEM. n-10 mice/group; p < 0.01. (B) Mean tumor volume (mm) of mice injected with only pUVC 3(●), pUVC 3-hIGFBP2(1-328) (■), pUVC 3-hIGFBP2 (164. ang. 328) (. tangle-solidup.) or pUVC 3-hIGFBP2(1-163) (o) ³± SEM). n-5 mice/group; p < 0.001.

In another example, see FIG. 30, which shows the anti-tumor effect of IGFBP-2 vaccine-induced Th2 on elimination of IGFBP-2-specific Th 1. Amplification of secretion of type II cytokines IL-4 and IL-10(A) and type I cytokines TNF α and IFN γ (B) from T-cell lines (mean ng/mI. + -. SD) with peptides from IGFBP2(1-163) or IGFBP2 (164-328); p < 0.001, # p < 0.01 and # p < 0.05. (C) Mean tumor volume (mm) of CD3+ T-cell infused mice³. + -. SEM), the CD3+ T-cells were expanded in mice immunized with pUMVC3-hIGFBP2(1-163) (o), pUMVC3-hIGFBP2 (164. ang. 328) (. tangle-solidup) or natural T-cells (●). n-4 mice/group; p < 0.01. (D) From the plants consisting of only pUMVC3(●), pUMVC3-hIGFBP2(164 + 328) (. tangle-solidup.), pUMVC3-hIGFBP2(1-163) (o) or pUMVC3-hIGFBP2(1-163) + pUMVC3-hIGFBP2(164 + 328)Mean tumor volume (mm) of injected mice³± SEM). n-5 mice/group; p < 0.01. Fig. 1 demonstrates that the Th1 and Th2 epitopes differ in functional affinity.

For another example, see fig. 16, which shows a Western blot analysis of HIF1 α expression in a single plasmid, pHIF1 α and a plasmid encoding 5 antigens, BCMA 5. After cleavage along the dotted line shown, the blot was probed using a 10-fold dilution of anti-HIF α (0.1-10 μ g/. mu.L). Only one lysate loaded lane was used per antibody titer.

Example 2

Identification of promiscuous high affinity binding class II epitopes derived from stem cells/EMT antigens

Archived leukopheresis from 20 BrCA patients and 20 control donors were used. The mean age of BRCA patients was 63 (range 49-87) and the mean age of controls was 38 (range 29-65). In BrCA patients, 81% are phase I or II at diagnosis, 10% are unknown stages and the remainder are phase III.

Peptides were constructed according to computer mapping. An algorithmic approach was used to predict class II epitopes of the first 6 immunogenic BCS/EMT proteins. Briefly, 3 published web-based algorithms were used to screen candidate antigens for potential MHC II epitopes in their protein sequences. Each protein was screened between the most common 14HLA-DR alleles. A maximum of 20 epitopes per allele (allele) were selected based on estimated binding affinity. Each amino acid (aa) in each predicted epitope gives a score (total score) based on the number of times aa appears in the predicted high binding epitope. The total score is multiplied by the number of alleles of the epitope containing that particular amino acid, and thus, each amino acid is assigned a number of scores that are a result of increasing the frequency of binding affinity over a broad population and the clutter of the predicted response. 49 peptides derived from all 6 candidate proteins selected based on the multiple scores of each peptide were constructed for screening. The peptides were selected to allow coverage of at least 25% of the full-length protein. The median peptide coverage of the candidate proteins was 28% (range 26-45%) of the total sequence.

Samples were screened by IFN γ ELISPOT. Figure 6 shows the cumulative data of all donors' IFN γ responses to all peptides/antigens in response to antigen. Generally, IFN γ responses were higher in volunteer donors compared to BrCA patients, and statistically higher for: survivin (BIRC 5); p < 0.001, MDM 2; p is less than 0.001, SOX-2; p ═ 0.01, and Yb-1; p is less than 0.001.

Positive responses were defined as the mean of experimental replicates being statistically greater (p < 0.05) than the mean of antigen-free wells. The percentage incidence was similar between donor types, with 24-38% of subjects corresponding to a particular antigen above the median response. Two-way ANOVA to detect differences between experimental groups or peptides resulted in a 2.82% difference in response donor type (p-0.0265) and 71.33% difference in response to the particular peptide tested (p-0.003) for IFN γ responses. Although the donor type is less significant, the differences produced are small.

Identification and screening of peptides from each candidate antigen. For the IFN γ response, 4 epitopes(s) from each candidate antigen were identified.

Example 3

The stem cell/EMT antigen derived epitopes are evaluated preferably against T-cells that elicit secretion of IFN γ or IL-10, and peptides are selected as candidate vaccine epitopes with low immunosuppressive potential, samples are screened by IL-10 ELISPOT.

Figure 7 shows the cumulative data of all donors' IL-10 responses to all peptides/antigens in response to antigen. In general, the IL-10 response is at a lower magnitude than the IFN γ response, and volunteer donors show a higher median response statistically significant against most antigens: survivin (birc 5); p < 0.0001, CDC25 b; p ═ 0.044, CDH 3; p ═ 0.037, FOXQ 1; p ═ 0.0014, HIF1 α; p ═ 0.017, MDM 2; p is 0.0039, SOX-2; p ═ 0.044, and Yb-1; p is 0.0007.

Similar to IFN γ data, donor types accounted for less than 0.01% of the difference between antigens (p ═ 0.911) and individual peptides accounted for 90.51% (p < 0.0001).

Peptides were identified that induced antigen-specific IFN γ secreting T-cells compared to IL-10 secreting T-cells. A matrix scoring system of antigens, preferably for in vivo evaluation of extended epitopes, is used which demonstrates IFN γ -specific activity in the absence of IL-10 activity between populations. Extended epitopes are preferred over shorter class II epitopes because longer epitopes elicit a diverse immune response consisting of T and B cells and the anti-tumor response is dependent on both CD4 and CD 8T-cells. Regions in candidate antigens containing multiple epitopes preferably induce greater magnitude and frequency of IFN γ and little or no IL-10 inducing activity is identified. The ratio of the occurrence of X-order antigen-specific IL-10 induction/the occurrence of X-order antigen-specific IFN γ induction (IFN γ/IL-10 activity ratio) was evaluated (see FIGS. 10 and 11).

For example, figure 10 shows the IFN γ dominated by higher magnitude and incidence. The ratio of IFN γ/IL-10 activity of the antigen was selected. The IFN γ/IL-10 ratio, defined as the mean cSPW x for each peptide, is shown by donor type. The incidence of IFN γ cSPW x is shown on the positive y-axis, white bars are volunteer donors, and black-bottom white bars show cancer donors. The incidence of IL-10cSPW x is shown on the negative y-axis, black bars are volunteer donors, and white-backed black bars show cancer donors. The figure shows CDH 3.

For example, figure 11 shows lower magnitude and incidence IFN γ predominance. The ratio of IFN γ/IL-10 activity of the antigen was selected. The IFN γ/IL-10 ratio, defined as the mean cSPW x for each peptide, is shown by donor type. The incidence of IFN γ cSPW x is shown on the positive y-axis, white bars are volunteer donors, and black-bottom white bars show cancer donors. The incidence of IL-10cSPW x is shown on the negative y-axis, black bars are volunteer donors, and white-backed black bars show cancer donors. The figure shows HIF1 α.

The antigens evaluated were divided into 4 main groups based on the IFN γ/IL-10 activity ratio. The first group of CDH3 (fig. 10) showed a high incidence/magnitude of IFN γ responses and very little IL-10 activity. This pattern suggests top-grade (top tier) antigens, including CDH3, SOX2, MDM2, and Yb-1. The secondary antigen demonstrated a similar dominant IFN γ response in the selected epitopic region and little to no IL-10 induction, however the magnitude of the immune response was more than one log lower than the top candidate. FIG. 11, HIF1 α shows this separation, which also includes CD105, CDC25B, and SATB 1. Vaccine candidates were derived from these categories (see figures 10 and 11).

The other 2 groups had less desirable characteristics for vaccine immunogens. Although there are epitopes that stimulate high magnitude and incidence of IFN γ, these sequences equally induce high magnitude IL-10 immunity at equal incidence in the individuals tested. Antigens such as c-met, IGF-1R, PRL3, and SIX1 were classified into this category. Finally, some candidate antigens were not immunogenic, as indicated by the low incidence and low magnitude of any immune response, as shown by SOX-2 in figure 19. ID1 and SNAIL were also associated with a very low incidence and magnitude of immune responses. Immunogenic and induce high magnitude and incidence of IL-10 responses, or weak immunogenicity, the latter two classes of antigens will be excluded from further consideration for the final vaccine formulation. In figure 9, based on IFN gamma/IL-10 activity ratio extension epitope list. See also fig. 17-20.

Example 4

Identification of epitope-specific Th1 cell response to proteins presented on endogenous APCs

Generating a T cell line against the candidate epitope. PBMCs derived from 3 donors who have demonstrated a positive response in the ELISPOT assay were stimulated with 10ug/ml of the antigenic peptide. IL-12(10ng/ml) and IL-2(10u/ml) were added to the cultures on day 5. Peptide stimulation and cytokine addition were repeated at 7-10 day intervals. The cultures were then expanded with CD3/CD28 beads. IL-2(30u/ml) was added to the cultures every 2-3 days for 10-11 days. The established peptide-specific T-cell lines were then evaluated using a 3-day IFN γ ELISPOT assay. Cultured T-cells were stimulated with varying concentrations of peptides and commercially available recombinant proteins loaded onto autologous APCs. Irrelevant peptides and proteins were used as negative controls. The response to peptide and protein antigens was significantly increased (p < 0.05) in at least one donor compared to the no antigen control, as shown by a positive response. MHC II and MHC I blocking antibodies (10 μ g/ml) were added to some antigen-stimulated wells to verify whether the epitope was MHC restricted. In addition, a fraction of T-cells were stained with CD3 FITC, CD4 PE-Cy7, CD45RO APC, and CD62L PE or CCR7 PE to assess central memory cell populations in T-cell lines compared to cells before antigen stimulation (PBMC).

T-cell lines were screened for specificity for peptides and recombinant proteins against positive and negative controls. FIG. 15A shows validation data for the HIF1 α peptide p60-82 as a representative example. The HIF 1. alpha.p 60-82 specific T-cell line responds to peptides presented on endogenous APCs and to recombinant HIF 1. alpha.protein. Both peptides (10ug/ml and 50ug/ml) and proteins (1ug/ml) significantly stimulated the IFN γ response from peptide-specific T-cell lines. This was not the case for the unrelated peptide (HIV p52-68) and protein (CD105) (FIG. 15A). MHC II significantly inhibited peptide and protein induced IFN γ responses, but MHC I antibodies did not (fig. 15A). CD45RO + CD62L + CD4+ central memory cells (73%) were expanded in cultured T-cell lines compared to baseline levels (9%) in pre-culture PBMCs.

For example, see fig. 8, which shows that the peptide in the extended sequence is validated as a native epitope. CD105 extended epitope (52aa) QNGTWPREVLLVLSVNS SVFLHL QALGI PLHLAYNSSLVTFQEPPGVNTTEL (SEQ ID NO: 1) (one of 2 epitopes in the sequence).

Similarly, the validated peptides were identified and diffracted from CD105, SATB1, CDH3, SOX2, YB1, and MDM 2. Of the peptides validated, all peptide-specific T-cell lines demonstrated statistically positive peptide responses. In 75% of the peptide-specific lines evaluated, MHC II antibodies inhibited more than 70% of the peptide-specific responses, and in 20% of the lines, MHC I antibodies inhibited more than 70% of the peptide responses. Among all positive protein responders (90%), MHC II antibodies inhibited > 70% of protein specific responses. The central memory T-cell population increased significantly after culture.

For the top and second-level groups of 6 antigens, CDH3, SOX2, MDM2, YB1, HIF1 α, and CD105, regions of extended epitopes suitable for immunization with high to moderately high IFN γ ratios and low to very low IL-10 ratios were identified. Each sequence includes 2 or more short (15-22aa) validated class II epitopes. The length of the extended sequence ranged from 32-aa to 90-aa (see Table 6).

Sequences derived from CDH3, Yb-1, MDM2, SOX2, and CD105 were ligated together and encoded a unique fusion peptide called "STEMVAC".

Candidate epitopes from each candidate antigen for inclusion in a multiple antigen vaccine are identified. The 5 extended epitopes are derived from 5 of 15 vaccine-suitable candidate antigens which preferentially induce IFN γ secretion with little identification of Th2 (IL-10). These sequences are combined into a unique fusion protein STEMVAC, which is the basis of a breast cancer vaccine.

Example 5

Construction of vaccine targeting stem cell/EMT antigen based on multi-antigen Th1 multi-epitope plasmid and determination of safety and immunogenicity

The immunogenicity and efficacy of plasmid-based vaccine constructs containing either or both short Th epitopes or extended Th epitopes were determined using a TgMMTVneu mouse model and IGF-1R antigen. To directly compare the ability of short and extended epitope plasmid vaccines to control tumor growth, a syngeneic tumor suppression model was used. Mice (TgMMTVneu) were divided into 4 immunization groups (pIGF-IRexep, pIGF-IRshep, vector and IGF-IR peptide) and implanted with syngeneic breast cancer cells (MMC) 7 days after the third immunization. The dosage is as described above. The ability of MMC cells to form tumors, and the tumor growth rate were measured. The IGF-IR peptide vaccine, the short epitope plasmid vaccine, and the extended epitope plasmid vaccine all significantly controlled tumor growth compared to the group immunized with vector only (p < 0.0001 from day 14-31). Mice immunized with pIGF-IRexep had the slowest growing tumors, but they did not differ significantly in tumor growth in animals immunized with pIGF-IRshep.

For example, in fig. 2, the Th2 immune response abrogated the anti-tumor efficacy of the Th1 immune response.

For example, figure 21, the immunogenicity and efficacy of HIF1a peptides and plasmids in mice was determined. (A) DTH responses were measured by ear thickness (mm) change 24 hours after application of HIF1 alpha peptide cocktail in 50% DMSO. The response of individual FVB/NJ mice from different immunization groups is plotted: controls (adjuvant and vector group only, see methods), HIF1 α Peps (peptide vaccine) and pHifla (plasmid vaccine). The dashed line indicates a 0.0mm change in ear thickness from baseline. P < 0.001 control. (B) DTH responses measured 24 hours after the application of HIF1 alpha peptide mixture in 50% DMSO. The response of a single MMTV-C3(1) -tagged transgenic mouse from the different immunization groups described above is plotted. P < 0.001 control. (C) Tumor volume (mm) over time (days) following implantation in MMTV-C3(1) -tagged transgenic mice was measured³) To evaluate the efficacy of the vaccine in controlling the growth of M6 tumors. The error bars for the immunization groups were adjuvant only (. smallcircle.), vector (□), HIF 1. alpha. peptide (. tangle-solidup.), or pHIF 1. alpha. (α 0) show the SEM for each group. Compared to mice immunized with HIF1 α peptide and HIF1 α DNA by 24 days post-implantation, there was significantly less tumor burden. P < 0.0001 vs. adjuvant only group. (D) IFN γ ELISPOT evaluates T-cell responses to peptide or control stimuli. Each plotted point represents each well point of a single FVB/NJ mouse in the immunised group treated with adjuvant alone (o), vector (□), HIF1a peptide (a), or phi 1a (■). Line shows the response Average and SEM. P < 0.001HIF1 α peptide versus no antigen response. While the HIF1 α plasmid generated a DTH response, IFN γ ELISPOT was low.

As another example, figure 22, the immunogenicity and efficacy of CD105 peptides and plasmids in mice were determined. (A) DTH responses were measured by ear thickness (mm) change 24 hours after application of CD105 peptide mixture in 50% DMSO. The response of individual FVB/NJ mice from different immunization groups is plotted: controls (adjuvant and vector group only, see methods), CD105 Peps (peptide vaccine) and pCD105 (plasmid vaccine). The dashed line indicates a 0.0mm change in ear thickness from baseline. P < 0.05, p < 0.001 control. (B) DTH responses measured 24 hours after application of CD105 peptide mixture in 50% DMSO. The response of a single MMTV-C3(1) -tagged transgenic mouse from the different immunization groups described above is plotted. P < 0.001 control. (C) Tumor volume (mm) over time (days) following implantation in MMTV-C3(1) -tagged transgenic mice was measured³) To evaluate the efficacy of the vaccine in controlling the growth of M6 tumors. The immunization groups were adjuvant only (. smallcircle.), vector (□), CD105 peptide (. tangle-solidup.), or pCD105(■). Error bars show SEM for each group. There was significantly less tumor burden in mice immunized with CD105 peptide compared to mice immunized with CD105 DNA 24 days after implantation. P < 0.0001 vs. adjuvant only group. (D) IFN γ ELISPOT evaluates T-cell responses to peptide or control stimuli. Each plotted point represents a point per well of a single FVB/NJ mouse in the immunization group treated with adjuvant alone (o), vector (□), CD105 peptide (a), or pCD105(■). The line shows the mean and SEM of the response. There was no significant finding of CD105 peptide response in any group.

As another example, figure 23, the immunogenicity and efficacy of CDH3 peptide and plasmid in mice were determined. (A) DTH responses were measured by ear thickness (mm) change 24 hours after application of CDH3 peptide mixture in 50% DMSO. The response of individual FVB/NJ mice from different immunization groups is plotted: controls (see methods), CDH3 Peps (peptide vaccine), pCDH3 (plasmid vaccine) and pUbVV-CDH 3. The dashed line indicates a 0.0mm change in ear thickness from baseline. P < 0.05, p < 0.01 control. (B) DTH responses measured 24 hours after application of CDH3 peptide mixture in 50% DMSO. Drawn from the different immunization groupsResponse of a single FVB/N/Tg-neu transgenic mouse. P < 0.05, p < 0.01 control. (C) By measuring tumor volume (mm) over time (days) following implantation in FVB/N/Tg-neu transgenic mice³) To evaluate the efficacy of the vaccine in controlling MMC tumor growth. Immunization groups were adjuvant only (. smallcircle.), vector (□), CDH3 peptide (. tangle-solidup.), pCDH3(■), or pUBHV-CDH 3 (. diamond-solid.). Error bars show SEM for each group. None of the CDH3 peptide or DNA immunized mice had a significantly smaller tumor burden than the control mice. (D) IFN γ ELISPOT evaluates T-cell responses to peptide or control stimuli. Each plotted point represents each well point of a single FVB/NJ mouse in the immunised group treated with adjuvant only (. smallcircle.), CDH3 peptide (. tangle-solidup.), pCDH3(■), or pUBVV-CDH3 (. diamond-solid.). The line shows the mean and SEM of the response. Peptides < 0.0001CDH3 vs no antigen response.

As another example, figure 24, the immunogenicity and efficacy of SOX2 peptides and plasmids in mice were determined. (A) DTH responses were measured by ear thickness (mm) change 24 hours after application of SOX2 peptide mixture in 50% DMSO. The response of individual FVB/NJ mice from different immunization groups is plotted: controls (see methods), SOX2 Peps (peptide vaccine), pSOX2 (plasmid vaccine) and pUbVV-SOX 2. The dashed line indicates a 0.0mm change in ear thickness from baseline. No significance was found compared to the control. (B) DTH responses measured 24 hours after application of the SOX2 peptide mixture in 50% DMSO. The response of individual FVB/N/Tg-neu transgenic mice from the different immunization groups described above is plotted. P < 0.05 control. (C) By measuring tumor volume (mm) over time (days) following implantation in FVB/N/Tg-neu transgenic mice³) To evaluate the efficacy of the vaccine in controlling MMC tumor growth. Immunization groups were adjuvant only (. smallcircle.), vector (□), SOX2 peptide (. tangle-solidup.), pSOX2(■), or pUBVV-SOX2 (. diamond-solid.). Error bars show SEM for each group. P < 0.001, p < 0.0001 vs. adjuvant only group. (D) IFN γ ELISPOT evaluates T-cell responses to peptide or control stimuli. Each plotted point represents each well point of a single FVB/NJ mouse in the immunization group treated with adjuvant only (. smallcircle.), SOX2 peptide (. tangle-solidup.), pSOX2(■), or pUBVV-SOX2 (. diamond-solid.). The line shows the mean and SEM of the response. Peptides of p < 0.001SOX2, p < 0.0001pSOX2, p < 0.0001 pruvv-SOX 2 versus no antigen response. Although the SOX2 peptide and plasmid produced IFN γ E LISPOT responses, but DTH responses were low or not significant in plasmids and peptides.

As another example, figure 25, the immunogenicity and efficacy of MDM2 peptide and plasmid in mice were determined. (A) DTH responses were measured by changes in ear thickness (mm) 24 hours after application of MDM2 peptide mixture in 50% DMSO. The response of individual FVB/NJ mice from different immunization groups is plotted: controls (see methods), MDM2 Peps (peptide vaccine), pMDM2 (plasmid vaccine) and pUbVV-MDM 2. The dashed line indicates a 0.0mm change in ear thickness from baseline. P < 0.001 control. (B) DTH responses measured 24 hours after application of MDM2 peptide mixture in 50% DMSO. The response of individual FVB/N/Tg-neu transgenic mice from the different immunization groups described above is plotted. P < 0.001 control. (C) IFN γ ELISPOT evaluates T-cell responses to peptide or control stimuli. Each plot represents the per-well spot of individual FVB/NJ mice in the immunization groups treated with adjuvant only (. smallcircle.), vector (□), MDM2 peptide (. tangle-solidup.), pMDM2(■), or pUBVV-MDM2 (. diamond-solid.). The line shows the mean and SEM of the response. P < 0.001pMDM2 vs no antigen response.

After immunization, mouse weight was determined. For example, figure 26 shows the quality of mice 3 months after the last vaccine. Mice (n-5) were left untreated with either only pUMCV 3, pUMCV 3-hHif1a (30-119), or pUMCV 3-hCD105(87-138), x-axis, CFA/IFA as adjuvant. The weight of each mouse, y-axis, (mean ± SEM) was recorded 3 months after the last vaccine. Mice weights were also determined 10 days after the last vaccine. Referring to fig. 27, mice (n ═ 5) were left untreated with either pUMVC3 only, pUMVC3-hHif1a (30-119), or pUMVC3-hCD105(87-138), x-axis, CFA/IFA as adjuvant. The weight of each mouse, y-axis, (mean ± SEM) was recorded 10 days after the last vaccine.

The sequence of short and extended epitopes was determined and constructed from IGF-1R. 2 plasmids were constructed, the DNA sequence verified, and used for immunization experiments. A short epitope plasmid, pIGF-IRshep, expresses a protein having a tandem MHC II epitope corresponding to human IGF-IR. In addition, 4 amino acids (MAVP) at the N-terminus and 3 amino acids (AAA) at the C-terminus are not related to the IGF-IR sequence. The extended epitope plasmid, pIGF-IRexep, expressed a protein with 2 1360. In addition, 4 amino acids (MAVP) at the N-terminus and 3 amino acids (AAA) at the C-terminus are not related to the IGF-IR sequence. The vector backbone of each plasmid was pUMCV 3, which contains a CMV promoter, directing constitutive expression of genes in mammalian cells. The vector is suitable for clinical application. Synthetic peptides were tested for selected epitopes in the C-terminal region of IGF-IR and demonstrated a propensity to induce greater stimulation of Th1(IFN γ) compared to Th2(IL-10) cells in an ELISPOT assay of human PBMC samples (described in the original proposal).

Immunogenicity of short and extended IGF-1R epitopes was assessed by IFN γ ELISPOT in TgMMTVneu mice. An immunization experiment was designed to test the immunogenicity of short and extended IGF-IR epitope plasmids in TgMMTVneu mice in adoptive cell transformation assays. 8 mice per group were immunized 5 times at 2 week intervals with pIGF-IRexep, pIGF-IRshep, IGF-IR peptides (p1196-1210, p1242-1256, p1332-1351, and p1341-1355) or pUMCV 3 (vector only). Plasmids and peptides were formulated with CFA-IFA adjuvant at 50 ug/injection. At 2 weeks after the last immunization, splenocytes from each immunization group were isolated and separated into CD3+ T-cells and CD 3-negative fractions using magnetic beads of negatively selected mouse T-cells (MACS). Each cell fraction was monitored by flow cytometry to determine the percentage of T and B cells in each fraction. For all groups, the CD3+ fraction contained > 96% T-cells and < 3.3% B-cells. For all groups, the CD 3-negative fraction contained 71-75% B cells and 2.4-6% T-cells. Splenocytes fractions were then injected 5 days before MMC tumor cells (2x 10) ⁵MMC/mouse) in tail vein of non-immunized TgMMTVneu mice (10⁶Individual cells/mouse). Tumor volume was measured. CD3+ T-cells from peptide immunized mice significantly inhibited tumor growth (p < 0.001) compared to vehicle controls, but CD3+ T-cells from either IGF-IR plasmid vaccine group did not promote significant tumor protection. CD 3-negative splenocytes (majority of B cells) from pIGF-IRexep immunized animals did not significantly inhibit tumor growth (p < 0.001) compared to vehicle controls. Neither CD 3-negative cells from pIFG-IRshep nor peptide-immunized mice controlled tumor growth.

Since T-cell immunity is essential for the production of anti-tumor antibodies, a delayed hypersensitivity (DTH) assay was performed to show that antigen-specific reactive T-cells are produced by pIGF-IRexep immunization. FVB mice received 3 injections at 2 week intervals: pIGF-IRexep, pUMCV 3 vector, IGF-IR peptide, or adjuvant only (plasmid and peptide were formulated in 50 ug/CFA/IFA adjuvant for injection). The DTH assay was performed by vigorous rubbing of PBS or IGF-IR peptide mixtures on the mouse ear 2 weeks after the third immunization and ear swelling was monitored for 3 days. The results demonstrate that significant DTH responses (p < 0.05, 4-48 hours, one-way ANOVA) against IGF-IR peptide occurred in peptide-immunized and pIGF-IRexep-immunized mice compared to PBS-treated ears compared to vehicle and adjuvant controls. Neither the vehicle nor the adjuvant control had a significant DTH response compared to PBS treatment.

Short contrast extension of IGF-IR epitopes in TgMMTVneu mice for clinical efficacy evaluation. To directly compare the ability of short and extended epitope plasmid vaccines to control tumor growth, a syngeneic tumor suppression model was used. Mice (TgMMTVneu) were divided into 4 immunization groups (pIGF-IRexep, pIGF-IRshep, vector and IGF-IR peptide) and implanted with syngeneic breast cancer cells (MMC) 7 days after the third immunization. The dosage is as described above. The ability of MMC cells to form tumors, and the tumor growth rate were measured. The IGF-IR peptide vaccine, the short epitope plasmid vaccine, and the extended epitope plasmid vaccine all significantly controlled tumor growth compared to the group immunized with vector only (p < 0.0001 from day 14-31). Mice immunized with pIGF-IRexep had the slowest growing tumors, but they did not differ significantly in tumor growth in animals immunized with pIGF-IRshep.

The mechanism of action of the therapeutic efficacy was determined by a blocking study. To further describe the role of B-cells and T-cells in pIGF-IRexep and IGF-IR peptide vaccine mediated tumor protection, depleting antibodies specific for T and B cells were used to block important effectors. Mice are immunized and then depleted of lymphocytes with specific antibodies. MMC tumor growth after immunization was measured in animals that consumed T cells or B cells. The pIGF-IRexep vaccine is tumor protective (p < 0.01) compared to vehicle immunized animals except in groups where B cells or T cells have been depleted. This result suggests a role for both lymphocyte classes in protective immune responses. Apart from the group in which the T cells had been depleted, the pIGF-IR peptide vaccine was tumor protective (p < 0.01) compared to vehicle immunized animals. B cell depletion had no significant effect on tumor protection of the peptide vaccine. The extended epitope plasmid vaccine can induce tumor protective immunity through B cells and T-cells, but the short epitope peptide only induces tumor protective T-cell immunity.

Example 6

Determination of safety and immunogenicity of individual antigen vaccines

Stem cell/EMT related proteins elicit IgG antibody immunity. Figure 4 shows the incidence of antibody immune stem cells/EMT antigens in BrCA patients and volunteer donors. The% donor positive volunteers are shown as white bars, while positive BrCA patients are shown as black bars. (1) Antigens with an incidence of more than 2-fold difference between 2 groups, (2) positive incidence of 2-fold or less between 2 groups. Y-axis, positive%, X-axis, antigen.

Figure 5 shows the results of a population-based epitope screening experiment. Figure 6 shows in more detail the antigen specific IFN γ response against stem cell/EMT proteins. IFN γ ELISPOT response against stem cell/EMT antigen. The Y-axis shows the corrected points/wells (for background correction) and the X-axis shows the antigens tested against two volunteer donors (white bars) and BrCA patients (gray bars). Data are presented as a quartile box with duck whiskers. The median CSPW is represented by the horizontal line.

Figure 7 also shows antigen specific IL-10 responses against stem cell/EMT proteins. IL-10ELISPOT response to stem cell/EMT antigens. The Y-axis shows the corrected points/wells (for background correction) and the X-axis shows the antigens tested against two volunteer donors (white bars) and BrCA patients (gray bars). Data are presented as a quartile box with duck whiskers. The median CSPW is represented by the horizontal line.

All rodent experiments were performed from the same Standard Operating Protocol (SOP), which is consistent with the requirements submitted by the research new drug (IND) application of FDS. For immunization studies, FVB/NJ mice (n ═ 7/group) were immunized intradermally in the ear 4 times every 7-10 days with the following: CFA/IFA + PBS (control), CFA/IFA + 50. mu.g of pUMMVC 3 (vector control), CFA/IFA + 50. mu.g of each validated peptide derived from a single antigen mixed, or CFA/IFA + 50. mu.g of pUMMVC 3 encoding an extended epitope from a single antigen. Delayed allergy test (DTH) was performed 3 days after the fourth immunization. Peptide and DNA immunized mice were given 50 μ g of each antigenic peptide in equal volume of DMSO, which was rubbed on their non-immunized ears. Control mice CFA/IFA and CFA/IFA + 50. mu.g of pUMMVC 3 were treated with: immunopeptide (n ═ 4), control peptide (n ═ 4), or DMSO + PBS (n ═ 6), were rubbed onto the nonimmunized ears. The experimental groups were tested with a mixture of antigen-specific peptides in DMSO. The ears were measured before treatment (0 hours) and 24 hours, and the overall change in ear thickness between 2 time points was compared. Significance was measured by one-way ANOVA with dukeley's post hoc test at p-0.05 level to determine differences between each pair of treatment groups. 10 days after DTH, mice were sacrificed and spleens were treated for IFN γ ELISPOT assay, 300,000 cells/well in 2 day culture with antigenic stimuli or controls. Significant differences in IFN γ responses were measured at the p 0.05 level by two-way ANOVA post pennflanni test to determine differences within each treatment group comparing no antigen stimulation (NoAg) to each other stimulation.

For clinical efficacy studies, MMTV-neutg (neutg) or MMTV-C3(1) Tg (C3T) transgenic mice (n ═ 6/group) were immunized intradermally in the ear four times every 7-10 days with the following: CFA/IFA + PBS, CFA/IFA + 50. mu.g of pUMVC3, CFA/IFA + 50. mu.g of each of the identified short peptide epitopes mixed, or CFA/IFA + 50. mu.g of PUMVC3 encoding the selected extended epitope sequence. 7-10 days after the fourth immunization, 5x10 was subcutaneously implanted⁵One for each syngeneic tumor cell of the particular model. After tumor development, measurements were taken 2-3 times per week. Two-way ANOVA tested after bangfrony considered significant differences in tumor volume at the p 0.05 level to determine the differences between treatment groups at the time points of measurement. DTH was performed 28 days after tumor implantation. Peptide and DNA immunized mice were given 50 μ g of each immunizing peptide + an equal volume of DMSO, which was rubbed on their non-immunized ears. Control mice CFA/IFA and CFA/IFA + 50. mu.g of pUMMVC 3 were treated with: immunopeptide (n ═ 3), control peptide (n ═ 3), or DMSO + PBS (n ═ 5), were rubbed onto the nonimmunized ears. The ears were measured before treatment (0 hours) and 24 hours, and the overall change in ear thickness between 2 time points was compared. Significance was measured at the p-0.05 level by one-way ANOVA with dukay's post hoc test to determine differences between pairs of treatment groups 。

IFN γ ELISPOT was performed on murine cryopreserved cells and the validated DTH response was correlated with IFN γ ELISPOT. Of the matching analysis of DTH with IFN γ ELISPOT, 97 matching trials and analysis of 7 different antigens, there was a statistically significant correlation between 2 tests with p < 0.0001.

Safety studies were performed in mice. FIG. 35 shows that the polyepitope IGF-1R vaccine inhibits growth of implanted breast cancer. Mice were immunized every other week with IGF-1R peptide, followed by subcutaneous injection of 1X10⁶MMC cells. Data show mean implanted tumor measurements + -SEM (PBS only (●); IGF-1R vaccine (. smallcircle.)) for 8 mice.

In addition, FIG. 13 shows the immunogenicity and efficacy of Yb-1 plasmid based vaccines. Immunogenicity and efficacy of Yb-1 peptides and plasmids in mice. (1) DTH responses were measured by changes in ear thickness (mm) 24 hours after application of Yb-1 peptide mixtures in 50% DMSO. The response of individual FVB/NJ mice from different immunization groups is plotted: controls (see methods), YB1 Peps (peptide vaccine), pYB1 (plasmid vaccine) and pUbVV-YB 1. The dashed line indicates a 0.0mm change in ear thickness from baseline. P < 0.05, p < 0.01, p < 0.001 control. (2) DTH responses measured 24 hours after application of YB1 peptide mixtures in 50% DMSO. The response of individual FVB/N/Tg-neu transgenic mice from the different immunization groups described above is plotted. P < 0.05, p < 0.01 control. (3) IFN γ ELISPOT evaluates T-cell responses to peptide or control stimuli. Each plotted point represents each well point of a single FVB/NJ mouse in the immunization group treated with adjuvant alone (o), vector (□), YB1 peptide (a), pYB1(■), or pUBVV-YB1 (diamond-solid). The line shows the mean and SEM of the response. There was no significant finding of YB1 peptide response in any group.

In vivo evaluations were also performed simultaneously in TgMMTNneu mice (see figure 12).

And (5) constructing a vaccine. Each extension epitope is encoded in pucvc 3. Once the monoclonal antigen toxicity study was completed, multiple constructs containing multiple antigens were generated in the preparations used for the test.

Expression of all constructs was tested by transfection into HEK293 cells and antigen expression of the resulting cell lysates in the presence or absence of the proteasome inhibitor MG132 was assessed. The expression proteins of the extended epitopes transfected in all the resulting constructs have been tested. Primary antibodies against extended epitopes were affinity purified from rabbit antisera obtained from rabbits vaccinated with extended epitopes of long peptides or commercially available antibodies. When multiple epitopes were encoded in a single plasmid, the protein expression of a single antigen in HEK293 cells was significantly increased compared to the single antigen construct (see figure 16). Western blot analysis in plasmid-transfected HEK293 cell lysates demonstrated HIF1 α extended epitope expression. Each plasmid was expressed for the expected size protein, which was not present in the vector transfection control. The expression of HIF1 α fragments of HIF1 α peptide (10.4kDa) and BCMA5 fusion protein (43.2kDa) was verified in phi 1 α (single antigen vaccine) and pBCMA5(5 antigen vaccine) -transfected HEK293 lysates, respectively.

To address the therapeutic efficacy of multiple antigens or 3 single antigen-encoding plasmids encoded in one vector, neuTG mice were immunized with vaccines against neu, IGF-IR, and IGFBP-2 at 18 weeks. Adjuvant-only control groups, groups of peptides derived from the antigens, groups of 3 plasmids (50 ug/each) encoding epitopes from each single antigen, and groups of single plasmids encoding epitopes from 3 antigens linked together were treated. All vaccines significantly delayed breast tumor development in transgenic animals compared to controls, peptides; p ═ 0.0004, 3 plasmid; p, 0.0001 and single particles with 3 antigens; p is 0.0003. There were no statistical differences in efficacy between each of the 3 vaccine approaches.

With the exception of CDH3, all antigens tested to date showed immunogenic and anti-tumor effects in rodent models by either DTH and ELIPOST. For example, figure 3 demonstrates antigen-specific IgG immunization. Western blot validation of IgG antibodies responding to CDH 3. Western blot of probed recombinant human CDH3 using (I) polyclonal anti-CDH 3 Ab, representative ELISA-positive BrCA patient samples (II) and ELISA-negative subject samples (III). Molecular weight scale (leftmost). The size of CDH3 is labeled 175 kDa.

The toxicity of the immunity was determined by blood chemistry and histological studies. No undesired effects of immunization were observed in any mice immunized with any antigen. Mice did not experience weight loss or loss of hairiness prior to final analysis. No toxicity was seen in acute and chronic toxicity studies for single antigen vaccines; blood, chemical or histological. Acute and chronic toxicity reports (against SATB1 and CDC 25B). Acute toxicity studies were performed 1 week after the fourth immunization and chronic toxicity studies were performed 3 months after the fourth immunization.

As shown in fig. 36, the multiple antigen multiple epitope vaccine prevented breast cancer development in neu-TG mice. (A) Disease-free survival (B) overall survival (n 15/group).

Toxicity results of SATB1 and CDC25B

Chronic toxicity studies. Mice were injected 4 times with 100mcg of only pUMVC3, pUMVC3-hSATB1(387-450) or pUMVC3-hCDC25B (124-164) every 7-10 days using CFA/IFA. Control mice were untreated (see tables 1 and 3).

Table 1: immunization and tissue/blood Collection time

Serum chemistry and CBC. All serum chemistry and complete blood counts of all groups were not significantly different from control (untreated) mice, except HCT in the CDC25B group, MCH in the SATB1 group, and poly in the pUMVC3 and SATB1 groups (see tables 5 and 6). Note that all sets of HCT values are within the established range limits. In addition, although the MCH values of the SATB1 group were significantly different, they were closer to the established range of values obtained from the jackson laboratory than the control group (15.2-15.6 pg). The pluvc 3 vehicle also achieved a significance of the poly%, indicating that the difference was likely a direct result of vehicle action rather than SATB1 insertion (see tables 5-16).

Pathology. There were no treatment-related lesions, which could be considered consistent with toxicity responses between the divided groups (see tables 13-16).

And (4) weight. 3 months after immunization, the animals receiving pUMCV 3-hSATB1(387-450) weighed 23.4 + -2.3 grams (g), 23.4 + -1.3 grams receiving pUMCV 3-hCDC25B (124-164), 23.6 + -1.4 grams receiving pUMCV 3 and 21.5 + -1.1 healthy untreated controls. There were no statistically significant differences between any of the groups (see tables 17-24).

Acute toxicity studies. Mice were injected 4 times with 100mcg of only pUMVC3, pUMVC3-hSATB1(387-450) or pUMVC3-hCDC25B (124-164) every 7-10 days using CFA/IFA. Control mice were untreated (see table 2).

Table 2: immunization and tissue/blood Collection time

Serum chemistry and CBC. All serum chemistry and complete blood counts of all groups were not significantly different from control (untreated) mice, except for chloride in the SATB1 and CDC25B groups, calcium, Osm, ALT, poly in the CDC25B group, and lymph in the pUMVC3 group (see tables 7 and 8). It should be noted that the chlorine in the SATB1 and CDC25B groups was closer to the established range (110- > 204meq/l) than the control group. Similarly, Osm in the CDC25B group was closer to the established range (321- & 330 arbitrary units) than the control group. ALT for all groups was within established range limits (see tables 5-16).

Pathology. There were no treatment-related lesions, which could be considered consistent with toxicity responses between the divided groups (tables 17-24).

And (4) weight. 10 days after immunization, the animals receiving pUVC 3-hSATB1(387-450) weighed 20.9. + -. 0.7 g (g), the animals receiving pUVC 3-hDCC 25B (124-164) 21. + -. 1.2g, the animals receiving pUVC 3 21.3. + -. 1.0g and the healthy untreated control 21.4. + -. 1.5 g. There were no statistically significant differences between any of the groups.

Hifla and CD105 toxicity results

Chronic toxicity studies. Mice were injected 4 times with 100mcg of either pUVC 3 only, pUVC 3-hHifla (30-119) or pUVC 3-hCD105(87-138) every 7-10 days using CFA/IFA. Control mice were untreated (see table 3).

Table 3: immunization and tissue/blood Collection time

Serum chemistry and CBC. All serum chemistry and complete blood count values of all groups were not significantly different from control (untreated) mice, except BUN in the pUMVC3 and Hif1a groups, anion space in all groups, osmolarity in the pUMVC3 group, and cholesterol in the Hifla and CD105 groups (see tables 9 and 10). It should be noted that BUN and anion gaps in the distinct groups were much closer to the established range (18-28 mg/dl and 23.3-27 arbitrary units, respectively) than the control group. The range of osmolarity establishment was 321- > 330, but none of the groups were within this range and the treated groups were not significantly different from untreated mice. Although cholesterol was significantly different in the treated groups compared to the control group, the values for all groups were within the established range limits of 50-138 (see tables 5-16).

Pathology. Organs were collected for this study, but have not been analyzed by a pathologist (see tables 17-24).

And (4) weight. 3 months after immunization, animals receiving pUMCV 3-hHifla (30-119) weighed 23.4 + -2.4 grams (g), 22.3 + -1.6 g received pUMCV 3-hCD105(87-138), 21.5 + -1.4 g received pUMCV 3 and 23.1 + -1.7 g of healthy untreated controls. There were no statistically significant differences between any of the groups.

Acute toxicity studies. Mice were injected 4 times with 100mcg of either pUVC 3 only, pUVC 3-hHifla (30-119) or pUVC 3-hCD105(87-138) every 7-10 days using CFA/IFA. Control mice were untreated (see table 4).

Table 4: immunization and tissue/blood Collection time

Serum chemistry and CBC. All serum chemistry and complete blood count values of all groups were not significantly different from control (untreated) mice, except cholesterol and globulin in the Hif1a group, phosphorus in all groups, calcium in the pUMVC3 group, chlorine in the CD105 group, and lymphocytes and leukocytes in the pUMVC3 and Hifla groups (see tables 5-16). Although cholesterol, leukocytes and calcium were significantly different in the Hif1a group compared to the control group, the values for all groups were within the established range limits of 50-138mg/dl, 2-15K/. mu.L and 8-14meq/L, respectively. It should be noted that globulin, chlorine and phosphorus in the treatment group were closer to the established ranges (1.9-2.4 g/dL, 110-204meq/l and 4.6-10.8mg/dL, respectively) than in the control group (see tables 5-16).

Pathology. There were no treatment-related lesions, which could be considered consistent with toxicity responses between the divided groups (tables 17-24).

And (4) weight. 10 days after immunization, animals receiving pUMCV 3-hHif1a (30-119) weighed 20.8 + -1.6 grams (g), 20.5 + -1.4 grams receiving pUMCV 3-hCD105(87-138), 21.5 + -2.2 grams receiving pUMCV 3 and 21.1 + -1.1 grams of healthy untreated controls. There were no statistically significant differences between any of the groups (see tables 5-16).

Table 5: summary of (Chronic) serum chemistry for all groups (median and Range)

P < 0.05 compared to control (untreated)

Table 6: summary of (Chronic) CBCs for all groups (median and Range)

P < 0.05 compared to control (untreated)

Table 7: summary of (acute) serum chemistry for all groups (median and range)

P < 0.05 compared to control (untreated)

Table 8: summary of (acute) CBCs (median and Range) for all groups

P < 0.05 compared to control (untreated)

Table 9: summary of (Chronic) serum chemistry for all groups (median and Range)

P < 0.01 compared to control (untreated)

P < 0.05 compared to control (untreated)

Table 10: summary of (Chronic) serum chemistry for all groups (median and Range)

Table 11: summary of (acute) serum chemistry for all groups (median and range)

P < 0.05 compared to control (untreated)

Table 12: summary of (acute) serum chemistry for all groups (median and range)

P < 0.05 compared to control (untreated)

Table 13: control (untreated) mouse (chronic) histology

NSL: no obvious focus of infection

MF: multiple focus

MG: granuloma miniaturus

NA: tissue unavailability

Table 14: pUMVC3 Immunized mouse (Chronic) histology

FE: enlargement of focus

LH: lymphoproliferation

MZ: edge zone

EMH: extramedullary hematopoiesis

PV: collateral blood vessel

PB: auxiliary bronchus

LA: lymph collection

Table 15: pUMVC3-hSATB1(387-450) Immunity in mouse histology (Chronic)

Table 16: pUMVC3-hCDC25B (124-164) immunized mice (Chronic) histology

Table 17: control (untreated) mouse histology

Table 18: histology of pUMCC 3 immunized mice

Table 19: histology of pUMVC3-hSATB1(387-450) immunized mice

Table 20: histology of pUMVC3-hCDC25B (124-164) immunized mice

ISCH: excess cells in sinus

Table 21: untreated (control) mouse histology

Table 22: histology of pUMCC 3 immunized mice

Table 23: histology of pUMVC3-hHifla (30-119) immunized mice

Table 24: histology of pUMVC3-hHif1a (87-138) immunized mice

DLN: draining lymph node

An exemplary list of plasmid-based vaccines, some of which are administered to a subject as described herein, is provided in table 25.

Table 25: plasmid-based vaccines now constructed

Example 7

Determination of safety and immunogenicity of multi-epitope antigen vaccines in humans

An exemplary composition is shown in figure 14, the composition is a vaccine and is referred to as STEMVAC.

FIG. 15A shows peptide-specific T-cells verified as native epitopes. Peptide (a) and HIF1 α protein (B) induced an IFN γ response. Denotes p < 0.05 vs. no antigen (white bars). C-D, peptide (10 ug/ml; C) and protein (1 ug/ml; D) (black bars) induced responses that were suppressed by MHC II Ab but not MHC 1 Ab (dark grey). P < 0.05 control peptide or protein. Light gray, MHC II and MHC I Ab only.

Another exemplary composition is a vaccine comprising the HER2 epitope. Figure 31 shows that vaccines based on the HER2 Th1 epitope can increase survival rates, which correlates with epitope spreading in advanced HER2+ breast cancer patients. (A) At median follow-up 8.5 years, the overall survival rate for patients with stage IV breast cancer after HER2 Th1 immunization was 38%. Time 0 represents the start of immunization. (B) Kaplan-meier curves of immunized patients; overall survival between epitope spreading positive (ES +, solid line) (median 84 months) and negative (ES-, dashed line) patients (median 24 months).

Figure 32 shows the results of comparing plasmid and peptide based vaccines. Vaccines based on extended Th plasmids are more effective than peptide-based vaccines in generating tumor antigen-specific Th1 immunity. Post-immunization ELISPOT responses of group 1(∘) and group 2(●) are shown. ELISPOT responses against HER2 ICD Th peptide 776 and HER2 ICD or ECD protein domains are reported as antigen-specific T-cells/10 ⁶PBMCs, each circle represents a single patient response; and the bold line represents the median of all groups.

Persistent HER2 ICD specific immunity for more than one year after termination of plasmid DNA-based immunity. 10 patients showed HER2 ICD specific IFN γ ELISPOT responses to HER2 ICD. See fig. 33. The values plotted for each subject were pre-immunization (pre), maximal response during immunization (max), and post-1 year response (LTFU).

Supernatants from ELISPOT trials were used during phase II studies on HER2 peptide vaccines from 8 patients receiving immunized advanced HER2+ breast cancer. In fig. 37, the values collected by cytokine multiplexing were color-coded as the magnitude of increase (red) or decrease (blue) of the inoculated antigen-specific cytokine (shown as cytokine "heat map"). The intensity of the color indicates the lowest (light) to highest (dark) quartile of the response. The data show a specific pattern of Th responses against HER2 ICD protein (immune antigen); th1/17, Th2, and "mixed". Patients 12 and 17 were immunized to increase HER 2-specific type I cytokine and IL-17 secretion. Th1 type responses were similar to those expected after immunization with a vaccine designed to elicit Th1 immunity. Patient 16 reduced HER 2-specific Th1 and Th17 cytokine production simultaneously. This phenomenon may limit the development or residence of tumor antigen-specific immunity. Data are expressed as post-vaccine minus pre-vaccine cytokine levels (1 month post-final vaccine). Deep red: the fourth (highest) quartile showed increased cytokine levels, reflecting the decreasing red color of the third, second and first quartile increases, respectively. Deep blue: the fourth (highest) quartile showed a decrease in cytokine levels, reflecting the decreasing blue color of the third, second and first quartile decreases, respectively.

Example 8

TABLE 26 epitope and construct sequences

Example 9

Multiple antigen construct design and expression

pUMVC3-IGFBP 2-survivin-HIF 1a-IGF1R fusion constructs are according to FIG. 40 and Table 27. The fusion protein was expressed in HEK-293 cells and protein expression was examined by Western blot analysis. Protein expression bands were further quantified using NIH Image J software. FIG. 41 shows protein expression of IGFBP-2, survivin, HIF-1A, and IGF-1R.

Table 27.

Tumor antigens	RefSeq numbering	Amino acids	Homology in mice
				IGFBP-2	NP_000588	1-163	72％
Survivin	NP_001159	83-120	87％
				HIF1a	NP_001521	30-119	94％
IGF-IR	NP_000866	1196-1261	100％
						1323-1360	97％

Example 10

Infection of ID8 with luciferase vector by lentivirus and selection of cell lines

Ovarian epidermal cells derived from C57B6 mice were infected with 2 mL/well of supernatant of pLentiIII-Luc2 viral vector (applied biomaterials) in the presence of 8. mu.g/mL polybrene (EMD Mirabbo Co.)ID8 cells. At 37 deg.C/5% CO₂After the next overnight incubation, the virus supernatant and polybrene-containing medium were removed and washed with PBS before adding the pre-warmed medium. Cells were cultured in growth medium for 72 hours and then placed under drug selection with 1 μ g/mL puromycin (invitrogen) added daily. 100 μ l of cells were added per well in a white 96-well plate (EMD Millipore) and an equal volume of 3000 μ g/ml d-fluorescein was added just prior to reading. Cell lines were selected based on the maximum relative light units measured as functional luciferase expression after addition of substrate.

In vivo proliferation of ID8-luc2 tumor.

Albino C57/BL/6/BrdCsHsd-Tyr^c(Harlan Laboratories) was dosed with 200. mu.L of 5X10⁶Intraperitoneal injection of individual ID8 cells. For mice in the supine position, half of the dose was injected using a 25 gauge needle in the lower left quadrant and the other half in the lower right quadrant. At predetermined intervals after tumor implantation, mice were weighed, bled, and imaged to monitor tumor proliferation.

Bioluminescence/imaging.

Bioluminescent images were taken with Xenogen IVIS (in vivo imaging protocol) using D-fluorescein (fig. 42A-42B). Images were normalized using Living Image software (parkinson's). The total photon flow per second and the maximum luminous intensity of the upper abdominal (metastatic) or lower abdominal (primary) region of each mouse were calculated and reported.

Fig. 42A shows the total flow (photons/sec) of the primary implant and fig. 42B shows the total flow at the site of the transfer. In addition, the corresponding animals were shown to be adjuvant only (fig. 42C), and three antigen immunized (fig. 42D).

Example 11

A method for identifying promiscuous MHC class II epitopes for the development of human vaccines.

The predicted MHC II epitopes of 14 of the common HLA-DR proteins were identified using a compilation of 3 different published algorithms. An algorithm is used to generate epitope binding scores to map epitopes within a protein sequence predicted to contain epitopes that interact with a variety of HLA-DR proteins, these epitopes being referred to as "promiscuous epitopes". The 14 HLA-DR proteins screened by the algorithm were: HLA-DRB1 x 0101, HLA-DRB1 x 0301, HLA-DRB1 x 0401, HLA-DRB1 x 0404, HLA-DRB1 x 0405, HLA-DRB1 x 0701, HLA-DRB1 x 0802, HLA-DRB1 x 0901, HLA-DRB1 x 1101, HLA-DRB1 x 1201, HLA-DRB1 x 1302, HLA-DRB1 x 1501, HLA-DRB4 x 0101, and HLA-DRB 5x 0101. The web-based algorithms used are SYFPEITHI (http:// www.syfpeithi.de/Scripts/MHCServer.dll/EpitopReduction. htm), PROPRED (http:// www.imtech.res.in/raghava/PROPRED /), and RANKPEP (http:// affected. med. um. ues/Tools/RANKPEP. html). The number of HLA-DR proteins available for screening varies among sites (6, 11 or 13 out of the 14 above). The reference protein sequences were obtained from the NCBI database and copied into the fast format for entry into the algorithmic search engine and the first 20 scoring epitopes of each HLA-DR protein were used to generate the "MHC II heatmap" of the reference protein. To compile and analyze epitope prediction data, a workbook based on MS Excel, called "MHC II heatmap template", was developed. Since each of the three algorithms has a different scoring system to identify epitopes, the epitope scores are first normalized before compiling the results from the different search methods. To normalize the epitope scores, all scores are divided by the top score obtained by each algorithm, so that the epitope with the highest predicted affinity should have a normalized score of 1.0. The normalized scores were then pasted into an MHC II heatmap template, which used a number of embedded equations and functions to perform the following tasks: (i) assigning a normalized score for an epitope to each amino acid of a particular epitope, (ii) calculating and plotting the number of different HLA-DR proteins/alleles having an epitope at each amino acid position, (iii) calculating and plotting the sum of the normalized scores for each epitope at each amino acid position, and (iv) calculating and plotting a "multi-score" which is the product of the normalized sum of the scores and the HLA-DR allele. The multiscale represents the epitope binding strength and epitope clutter and this value can be used to generate an MHC II heatmap of the reference protein. Mapping of amino acid positions (x-axis) against multiple scores (y-axis) allows for easy visualization of regions of the protein predicted to contain promiscuous epitopes. In addition, MS Access applications were generated to simplify the input of FASTA protein sequences into columns of MHC II heatmap templates. Once a protein sequence has been entered, peptide selection for use in immunoassays can be aided by generating MHC II heatmaps based on multi-score amino acid color coding. Generally, color coding represents multiple scores of 75-100%, 50-75%, 25-50%, and 10-25%.

Peptides were constructed based on composite scores. Peripheral Blood Mononuclear Cells (PBMC) from 40 human donors were evaluated by ELISPOT against antigen-specific IFN-. gamma. (g) and IL-10 production induced by predicted epitopes covering a minimum of 25% of the protein. For IFN-g ELISPOT, cells were at 2X10⁵Per well (96 well plates) were inoculated into medium containing 10. mu.g/mL of each peptide or HIVp17, PHA (1. mu.g/mL), CEF (2.5. mu.g/mL) or medium alone at 37 ℃ with 5% CO₂For 7 days. On day 5, recombinant human IL-2(10U/ml) was added. By adding 2X10 to the original culture on day 8⁵Peptide-loaded (same concentration as above) autologous irradiated (3000rads) human PBMC for second In Vitro Stimulation (IVS) and incubated for 24 hours. A96-well nitrocellulose plate was coated with 10ug/ml anti-human IFN-g. Washed nitrocellulose plates were blocked by incubation with PBMC cultures for 24 hours after 2% bovine serum albumin in DPBS. After thorough washing, biotinylated anti-human IFN-g was added for 2 hours. For IL-10ELISPOT, anti-human IL-10 coated (2. mu.g/ml) nitrocellulose 96-well plates were blocked as described above. PBMC concentrations and peptide stimulation were as described above except PHA was used at 20. mu.g/ml. After extensive washing, biotinylated anti-human IL-10 was added for 2 hours. After extensive washing, 1. mu.g/mL streptavidin-AP was added for 45 min. Spots were visualized by incubating the plates with BCIP and NBT solution spots were counted on a c.t.l.elispot plate reader. Raw data were entered into TVG database ELISPOT tool and positive responses were defined by statistically significant differences (p < 0.05) between the mean of 5 replicate spots in the experimental wells and the mean of no antigen control wells. A TH1/TH2 ratio was generated that analyzed the magnitude and frequency of ELISPOT responses for each predicted class II specific peptide using the following algorithm: (mean point corrected per well) x (percent responding donors). The TH1/TH2 activity ratio is also derived from EL against type I and type II cytokines using antigen-specific T-cell stimulation media and by multiplex assays against a complex TH1/TH2 phenotype And (5) performing ISA test. Incidence and magnitude were incorporated into these analyses in a similar manner.

Fig. 43A and 43B show TH1 response as a function of protein sequence. Selective TH1 inducible sequences were identified in the C-terminus of survivin and the N-terminus of HIF1 a. The average cSPW x incidence for each peptide is shown by donor type. The incidence of IFN-g cSPW x is shown on the positive y-axis for volunteer donors (n ═ 20) (white) and cancer donors (n ═ 20) (grey). The incidence of IL-10cSPW x is shown on the negative y-axis for volunteer donors (solid black) and cancer donors (dotted black). FIG. 43A shows TH1 responses corresponding to HIF-1A peptide. Fig. 43B shows TH1 response corresponding to survivin peptide. The vertical lines show the selected sequence.

Example 12

Antibody immunization studies showed that IGF-IR, HIF1a and survivin are also ovarian cancer antigens (FIG. 44). IGFBP-2 has previously been shown to be immunogenic in ovarian cancer patients. Serum samples were taken at the initial surgery of 120 ovarian cancer patients and analyzed by indirect ELISA using commercially available recombinant proteins. Control sera from 100 age-matched volunteers were analyzed in a similar manner. IgG antibodies were evaluated as indicators of cellular immunity, since syngeneic CD4+ T-cells were required to convert immunoglobulin classes from IgM to IgG. The results were confirmed by Western blotting. Ovarian cancer patients had significantly higher levels of antibodies to antigenic proteins than illumination (FIG. 44). Patients also demonstrated significantly higher antibody incidence than controls (range 1-6%), ranging from 8-25% positive (median 15%).

Sequence listing

<110> Washington UNIVERSITY Business center (unity OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIRCIALIZATION)

<120> Breast and ovarian cancer vaccine

<130> 41299-768.602

<150> US 61/972,176

<151> 2014-03-28

<160> 91

<170> PatentIn version 3.5

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Ala Tyr Asn Ser Ser Leu Val Thr Phe Gln Glu Pro Pro Gly Val Asn

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Thr Thr Glu Leu

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ttccaggagc cccccggcgt gaacaccacc gagctg 156

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accgtgttca tgcgcctgaa catcatctcc cccgacctgt ccggctgcac ctccaagggc 60

ctggtgctgc ccgccgtgct gggcatcacc ttcggcgcct tcctgatcgg cgccctgctg 120

accgccgccc tgtggtacat ctactcccac acccgctccc cctccaagcg cgagcccgtg 180

gtggccgtgg ccgcccccgc ctcctccgag tcctcctcca ccaaccactc catcggctcc 240

acccagtcca ccccctgctc cacctcctcc atggcc 276

<210> 4

<211> 267

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 4

accgtgtcca tgcgcctgaa catcgtgtcc cccgacctgt ccggcaaggg cctggtgctg 60

ccctccgtgc tgggcatcac cttcggcgcc ttcctgatcg gcgccctgct gaccgccgcc 120

ctgtggtaca tctactccca cacccgcggc ccctccaagc gcgagcccgt ggtggccgtg 180

gccgcccccg cctcctccga gtcctcctcc accaaccact ccatcggctc cacccagtcc 240

accccctgct ccacctcctc catggcc 267

<210> 5

<211> 267

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 5

accgtgtcca tgcgcctgaa catcgtgtcc cccgacctgt ccggcaaggg cctggtgctg 60

ccctccgtgc tgggcatcac cttcggcgcc ttcctgatcg gcgccctgct gaccgccgcc 120

ctgtggtaca tctactccca cacccgcgcc ccctccaagc gcgagcccgt ggtggccgtg 180

gccgcccccg cctcctccga gtcctcctcc accaaccact ccatcggctc cacccagtcc 240

accccctgct ccacctcctc catggcc 267

<210> 6

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 6

Glu Ala Arg Met Leu Asn Ala Ser Ile Val Ala Ser Phe Val Glu Leu

1 5 10 15

Pro Leu

<210> 7

<211> 52

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 7

Gln Asn Gly Thr Trp Pro Arg Glu Val Leu Leu Val Leu Ser Val Asn

1 5 10 15

Ser Ser Val Phe Leu His Leu Gln Ala Leu Gly Ile Pro Leu His Leu

20 25 30

Ala Tyr Asn Ser Ser Leu Val Thr Phe Gln Glu Pro Pro Gly Val Asn

35 40 45

Thr Thr Glu Leu

50

<210> 8

<211> 92

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 8

Thr Val Phe Met Arg Leu Asn Ile Ile Ser Pro Asp Leu Ser Gly Cys

1 5 10 15

Thr Ser Lys Gly Leu Val Leu Pro Ala Val Leu Gly Ile Thr Phe Gly

20 25 30

Ala Phe Leu Ile Gly Ala Leu Leu Thr Ala Ala Leu Trp Tyr Ile Tyr

35 40 45

Ser His Thr Arg Ser Pro Ser Lys Arg Glu Pro Val Val Ala Val Ala

50 55 60

Ala Pro Ala Ser Ser Glu Ser Ser Ser Thr Asn His Ser Ile Gly Ser

65 70 75 80

Thr Gln Ser Thr Pro Cys Ser Thr Ser Ser Met Ala

85 90

<210> 9

<211> 89

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 9

Thr Val Ser Met Arg Leu Asn Ile Val Ser Pro Asp Leu Ser Gly Lys

1 5 10 15

Gly Leu Val Leu Pro Ser Val Leu Gly Ile Thr Phe Gly Ala Phe Leu

20 25 30

Ile Gly Ala Leu Leu Thr Ala Ala Leu Trp Tyr Ile Tyr Ser His Thr

35 40 45

Arg Gly Pro Ser Lys Arg Glu Pro Val Val Ala Val Ala Ala Pro Ala

50 55 60

Ser Ser Glu Ser Ser Ser Thr Asn His Ser Ile Gly Ser Thr Gln Ser

65 70 75 80

Thr Pro Cys Ser Thr Ser Ser Met Ala

85

<210> 10

<211> 89

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 10

Thr Val Ser Met Arg Leu Asn Ile Val Ser Pro Asp Leu Ser Gly Lys

1 5 10 15

Gly Leu Val Leu Pro Ser Val Leu Gly Ile Thr Phe Gly Ala Phe Leu

20 25 30

Ile Gly Ala Leu Leu Thr Ala Ala Leu Trp Tyr Ile Tyr Ser His Thr

35 40 45

Arg Ala Pro Ser Lys Arg Glu Pro Val Val Ala Val Ala Ala Pro Ala

50 55 60

Ser Ser Glu Ser Ser Ser Thr Asn His Ser Ile Gly Ser Thr Gln Ser

65 70 75 80

Thr Pro Cys Ser Thr Ser Ser Met Ala

85

<210> 11

<211> 96

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 11

ggagtgccag tgcagggctc caagtacgct gccgaccgca accactaccg ccgctaccca 60

cgccgtcgcg gcccaccccg caactaccag cagaac 96

<210> 12

<211> 96

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 12

ggcgtgcccg tgcagggctc caagtacgcc gccgaccgca accactaccg ccgctacccc 60

cgccgccgcg gccccccccg caactaccag cagaac 96

<210> 13

<211> 96

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 13

ggcgtgcccg tgcagggctc caagtacgcc gccgaccgca accactaccg ccgctacccc 60

cgccgccgcg gccccccccg caactaccag cagaac 96

<210> 14

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 14

Glu Asp Val Phe Val His Gln Thr Ala Ile Lys Lys Asn Asn Pro Arg

1 5 10 15

Lys

<210> 15

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 15

Tyr Arg Arg Asn Phe Asn Tyr Arg Arg Arg Arg Pro Glu Asn

1 5 10

<210> 16

<211> 32

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 16

Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala Asp Arg Asn His Tyr

1 5 10 15

Arg Arg Tyr Pro Arg Arg Arg Gly Pro Pro Arg Asn Tyr Gln Gln Asn

20 25 30

<210> 17

<211> 177

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 17

ggcctcaatg cgcacggcgc agcgcagatg cagcccatgc accgctacga cgtgagcgcc 60

ctgcagtaca actccatgac cagctcgcag acctacatga acggctcgcc cacctacagc 120

atgtcctact cgcagcaggg cacccctggc atggctcttg gctccatggg ttcggtg 177

<210> 18

<211> 177

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 18

ggcctgaacg cccacggcgc cgcccagatg cagcccatgc accgctacga cgtgtccgcc 60

ctgcagtaca actccatgac ctcctcccag acctacatga acggctcccc cacctactcc 120

atgtcctact cccagcaggg cacccccggc atggccctgg gctccatggg ctccgtg 177

<210> 19

<211> 177

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 19

ggcctgaacg cccacggcgc cgcccagatg cagcccatgc accgctacga cgtgtccgcc 60

ctgcagtaca actccatgac ctcctcccag acctacatga acggctcccc cacctactcc 120

atgtcctact cccagcaggg cacccccggc atggccctgg gctccatggg ctccgtg 177

<210> 20

<211> 59

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 20

Gly Leu Asn Ala His Gly Ala Ala Gln Met Gln Pro Met His Arg Tyr

1 5 10 15

Asp Val Ser Ala Leu Gln Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr

20 25 30

Met Asn Gly Ser Pro Thr Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr

35 40 45

Pro Gly Met Ala Leu Gly Ser Met Gly Ser Val

50 55

<210> 21

<211> 240

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 21

aggtcactga aggaaaggaa tccattgaaa atcttcccat ccaaacgtat cttacgaaga 60

cacaagagag attgggtggt tgctccaata tctgtccctg aaaatggcaa gggtcccttc 120

ccacagagac tgaatcagct caagtctaat aaagatagag acaccaagat tttctacagc 180

atcacggggc cgggtgcaga cagcccacct gagggtgtct tcgctgtaga gaaggagaca 240

<210> 22

<211> 216

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 22

ttgaaaatct tcccatccaa acgtatctta cgaagacaca agagagattg ggtggttgct 60

ccaatatctg tccctgaaaa tggcaagggt cccttcccac agagactgaa tcagctcaag 120

tctaataaag atagagacac caagattttc tacagcatca cggggccggg tgcagacagc 180

ccacctgagg gtgtcttcgc tgtagagaag gagaca 216

<210> 23

<211> 216

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 23

ttgaaaatct tcccatccaa acgtatctta cgaagacaca agagagattg ggtggttgct 60

ccaatatctg tccctgaaaa tggcaagggt cccttcccac agagactgaa tcagctcaag 120

tctaataaag atagagacac caagattttc tacagcatca cggggccggg tgcagacagc 180

ccacctgagg gtgtcttcgc tgtagagaag gagaca 216

<210> 24

<211> 216

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 24

gtgatgaact cccccccctc ccgcatcctg cgccgccgca agcgcgagtg ggtgatgccc 60

cccatctccg tgcccgagaa cggcaagggc cccttccccc agcgcctgaa ccagctgaag 120

tccaacaagg accgcggcac caagctgttc tactccatca ccggccccgg cgccgactcc 180

ccccccgagg gcgtgttcac catcgagaag gagacc 216

<210> 25

<211> 80

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 25

Arg Ser Leu Lys Glu Arg Asn Pro Leu Lys Ile Phe Pro Ser Lys Arg

1 5 10 15

Ile Leu Arg Arg His Lys Arg Asp Trp Val Val Ala Pro Ile Ser Val

20 25 30

Pro Glu Asn Gly Lys Gly Pro Phe Pro Gln Arg Leu Asn Gln Leu Lys

35 40 45

Ser Asn Lys Asp Arg Asp Thr Lys Ile Phe Tyr Ser Ile Thr Gly Pro

50 55 60

Gly Ala Asp Ser Pro Pro Glu Gly Val Phe Ala Val Glu Lys Glu Thr

65 70 75 80

<210> 26

<211> 72

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 26

Leu Lys Ile Phe Pro Ser Lys Arg Ile Leu Arg Arg His Lys Arg Asp

1 5 10 15

Trp Val Val Ala Pro Ile Ser Val Pro Glu Asn Gly Lys Gly Pro Phe

20 25 30

Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp Arg Asp Thr Lys

35 40 45

Ile Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu Gly

50 55 60

Val Phe Ala Val Glu Lys Glu Thr

65 70

<210> 27

<211> 72

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 27

Ala Met His Ser Pro Pro Thr Arg Ile Leu Arg Arg Arg Lys Arg Glu

1 5 10 15

Trp Val Met Pro Pro Ile Phe Val Pro Glu Asn Gly Lys Gly Pro Phe

20 25 30

Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp Arg Gly Thr Lys

35 40 45

Ile Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu Gly

50 55 60

Val Phe Thr Ile Glu Lys Glu Ser

65 70

<210> 28

<211> 72

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 28

Val Met Asn Ser Pro Pro Ser Arg Ile Leu Arg Arg Arg Lys Arg Glu

1 5 10 15

Trp Val Met Pro Pro Ile Ser Val Pro Glu Asn Gly Lys Gly Pro Phe

20 25 30

Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp Arg Gly Thr Lys

35 40 45

Leu Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu Gly

50 55 60

Val Phe Thr Ile Glu Lys Glu Thr

65 70

<210> 29

<211> 231

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 29

acctacacca tgaaggaggt gctgttctac ctgggccagt acatcatgac caagcgcctg 60

tacgacgaga agcagcagca catcgtgtac tgctccaacg acctgctggg cgacctgttc 120

ggcgtgccct ccttctccgt gaaggagcac cgcaaaatct acaccatgat ctaccgcaac 180

ctggtggtgg tgaaccagca ggagtcctcc gactccggca cctccgtgtc c 231

<210> 30

<211> 222

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 30

acctacacca tgaaggagat catcttctac atcggccagt acatcatgac caagcgcctg 60

tacgacgaga agcagcagca catcgtgtac tgctccaacg acctgctggg cgacgtgttc 120

ggcgtgccct ccttctccgt gaaggagcac cgcaagatct acgccatgat ctaccgcaac 180

ctggtggccg tgtcccagca ggactccggc acctccctgt cc 222

<210> 31

<211> 222

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 31

atctacacca tgaaggagat catcttctac atcggccagt acatcatgac caagcgcctg 60

tacgacgaga agcagcagca catcgtgtac tgctccaacg acctgctggg cgacgtgttc 120

ggcgtgccct ccttctccgt gaaggagcac cgcaagatct acgccatgat ctaccgcaac 180

ctggtggtgg tgtcccagca ggactccggc acctccccct cc 222

<210> 32

<211> 76

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 32

Thr Tyr Thr Met Lys Glu Val Leu Phe Tyr Leu Gly Gln Tyr Ile Met

1 5 10 15

Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser

20 25 30

Asn Asp Leu Leu Gly Asp Leu Phe Gly Val Pro Ser Phe Ser Val Lys

35 40 45

Glu His Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val Val

50 55 60

Asn Gln Gln Glu Ser Ser Asp Ser Gly Thr Ser Val

65 70 75

<210> 33

<211> 74

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 33

Thr Tyr Thr Met Lys Glu Ile Ile Phe Tyr Ile Gly Gln Tyr Ile Met

1 5 10 15

Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser

20 25 30

Asn Asp Leu Leu Gly Asp Val Phe Gly Val Pro Ser Phe Ser Val Lys

35 40 45

Glu His Arg Lys Ile Tyr Ala Met Ile Tyr Arg Asn Leu Val Ala Val

50 55 60

Ser Gln Gln Asp Ser Gly Thr Ser Leu Ser

65 70

<210> 34

<211> 74

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 34

Ile Tyr Thr Met Lys Glu Ile Ile Phe Tyr Ile Gly Gln Tyr Ile Met

1 5 10 15

Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser

20 25 30

Asn Asp Leu Leu Gly Asp Val Phe Gly Val Pro Ser Phe Ser Val Lys

35 40 45

Glu His Arg Lys Ile Tyr Ala Met Ile Tyr Arg Asn Leu Val Val Val

50 55 60

Ser Gln Gln Asp Ser Gly Thr Ser Pro Ser

65 70

<210> 35

<211> 987

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 35

atggcggtac ccatgcaact gtcctgctct agacagaacg gcacctggcc ccgcgaggtg 60

ctgctggtgc tgtccgtgaa ctcctccgtg ttcctgcacc tacaggccct gggcatcccc 120

ctgcacctgg cctacaactc ctccctggtg accttccagg agccccccgg cgtgaacacc 180

accgagctga gatccaccgg tggagtgcca gtgcagggct ccaagtacgc tgccgaccgc 240

aaccactacc gccgctaccc acgccgtcgc ggcccacccc gcaactacca gcagaacacg 300

cgtggcctca atgcgcacgg cgcagcgcag atgcagccca tgcaccgcta cgacgtgagc 360

gccctgcagt acaactccat gaccagctcg cagacctaca tgaacggctc gcccacctac 420

agcatgtcct actcgcagca gggcacccct ggcatggctc ttggctccat gggttcggtg 480

agatcccaat tgaggtcact gaaggaaagg aatccattga aaatcttccc atccaaacgt 540

atcttacgaa gacacaagag agattgggtg gttgctccaa tatctgtccc tgaaaatggc 600

aagggtccct tcccacagag actgaatcag ctcaagtcta ataaagatag agacaccaag 660

attttctaca gcatcacggg gccgggtgca gacagcccac ctgagggtgt cttcgctgta 720

gagaaggaga caagatccgc cggcgaaacc tacaccatga aggaggtgct gttctacctg 780

ggccagtaca tcatgaccaa gcgcctgtac gacgagaagc agcagcacat cgtgtactgc 840

tccaacgacc tgctgggcga cctgttcggc gtgccctcct tctccgtgaa ggagcaccgc 900

aaaatctaca ccatgatcta ccgcaacctg gtggtggtga accagcagga gtcctccgac 960

tccggcacct ccgtgtccag atcttag 987

<210> 36

<211> 1059

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 36

atggcggtac ccatgaccgt gttcatgcgc ctgaacatca tctcccccga cctgtccggc 60

tgcacctcca agggcctggt gctgcccgcc gtgctgggca tcaccttcgg cgccttcctg 120

atcggcgccc tgctgaccgc cgccctgtgg tacatctact cccacacccg ctccccctcc 180

aagcgcgagc ccgtggtggc cgtggccgcc cccgcctcct ccgagtcctc ctccaccaac 240

cactccatcg gctccaccca gtccaccccc tgctccacct cctccatggc caccggtgga 300

gtgccagtgc agggctccaa gtacgctgcc gaccgcaacc actaccgccg ctacccacgc 360

cgtcgcggcc caccccgcaa ctaccagcag aacacgcgtg gcctcaatgc gcacggcgca 420

gcgcagatgc agcccatgca ccgctacgac gtgagcgccc tgcagtacaa ctccatgacc 480

agctcgcaga cctacatgaa cggctcgccc acctacagca tgtcctactc gcagcagggc 540

acccctggca tggctcttgg ctccatgggt tcggtgagat cccaattgtt gaaaatcttc 600

ccatccaaac gtatcttacg aagacacaag agagattggg tggttgctcc aatatctgtc 660

cctgaaaatg gcaagggtcc cttcccacag agactgaatc agctcaagtc taataaagat 720

agagacacca agattttcta cagcatcacg gggccgggtg cagacagccc acctgagggt 780

gtcttcgctg tagagaagga gacaagatcc gccggcgaaa cctacaccat gaaggaggtg 840

ctgttctacc tgggccagta catcatgacc aagcgcctgt acgacgagaa gcagcagcac 900

atcgtgtact gctccaacga cctgctgggc gacctgttcg gcgtgccctc cttctccgtg 960

aaggagcacc gcaaaatcta caccatgatc taccgcaacc tggtggtggt gaaccagcag 1020

gagtcctccg actccggcac ctccgtgtcc agatcttag 1059

<210> 37

<211> 1041

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 37

atggcggtac ccatgaccgt gtccatgcgc ctgaacatcg tgtcccccga cctgtccggc 60

aagggcctgg tgctgccctc cgtgctgggc atcaccttcg gcgccttcct gatcggcgcc 120

ctgctgaccg ccgccctgtg gtacatctac tcccacaccc gcggcccctc caagcgcgag 180

cccgtggtgg ccgtggccgc ccccgcctcc tccgagtcct cctccaccaa ccactccatc 240

ggctccaccc agtccacccc ctgctccacc tcctccatgg ccaccggtgg cgtgcccgtg 300

cagggctcca agtacgccgc cgaccgcaac cactaccgcc gctacccccg ccgccgcggc 360

cccccccgca actaccagca gaacacgcgt ggcctgaacg cccacggcgc cgcccagatg 420

cagcccatgc accgctacga cgtgtccgcc ctgcagtaca actccatgac ctcctcccag 480

acctacatga acggctcccc cacctactcc atgtcctact cccagcaggg cacccccggc 540

atggccctgg gctccatggg ctccgtgaga tcccaattgg ccatgcactc cccccccacc 600

cgcatcctgc gccgccgcaa gcgcgagtgg gtgatgcccc ccatcttcgt gcccgagaac 660

ggcaagggcc ccttccccca gcgcctgaac cagctgaagt ccaacaagga ccgcggcacc 720

aagatcttct actccatcac cggccccggc gccgactccc cccccgaggg cgtgttcacc 780

atcgagaagg agtccagatc cgccggcgaa acctacacca tgaaggagat catcttctac 840

atcggccagt acatcatgac caagcgcctg tacgacgaga agcagcagca catcgtgtac 900

tgctccaacg acctgctggg cgacgtgttc ggcgtgccct ccttctccgt gaaggagcac 960

cgcaagatct acgccatgat ctaccgcaac ctggtggccg tgtcccagca ggactccggc 1020

acctccctgt ccagatctta g 1041

<210> 38

<211> 1041

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 38

atggcggtac ccatgaccgt gtccatgcgc ctgaacatcg tgtcccccga cctgtccggc 60

aagggcctgg tgctgccctc cgtgctgggc atcaccttcg gcgccttcct gatcggcgcc 120

ctgctgaccg ccgccctgtg gtacatctac tcccacaccc gcgccccctc caagcgcgag 180

cccgtggtgg ccgtggccgc ccccgcctcc tccgagtcct cctccaccaa ccactccatc 240

ggctccaccc agtccacccc ctgctccacc tcctccatgg ccaccggtgg cgtgcccgtg 300

cagggctcca agtacgccgc cgaccgcaac cactaccgcc gctacccccg ccgccgcggc 360

cccccccgca actaccagca gaacacgcgt ggcctgaacg cccacggcgc cgcccagatg 420

cagcccatgc accgctacga cgtgtccgcc ctgcagtaca actccatgac ctcctcccag 480

acctacatga acggctcccc cacctactcc atgtcctact cccagcaggg cacccccggc 540

atggccctgg gctccatggg ctccgtgaga tcccaattgg tgatgaactc ccccccctcc 600

cgcatcctgc gccgccgcaa gcgcgagtgg gtgatgcccc ccatctccgt gcccgagaac 660

ggcaagggcc ccttccccca gcgcctgaac cagctgaagt ccaacaagga ccgcggcacc 720

aagctgttct actccatcac cggccccggc gccgactccc cccccgaggg cgtgttcacc 780

atcgagaagg agaccagatc cgccggcgaa atctacacca tgaaggagat catcttctac 840

atcggccagt acatcatgac caagcgcctg tacgacgaga agcagcagca catcgtgtac 900

tgctccaacg acctgctggg cgacgtgttc ggcgtgccct ccttctccgt gaaggagcac 960

cgcaagatct acgccatgat ctaccgcaac ctggtggtgg tgtcccagca ggactccggc 1020

acctccccct ccagatctta g 1041

<210> 39

<211> 328

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 39

Met Ala Val Pro Met Gln Leu Ser Cys Ser Arg Gln Asn Gly Thr Trp

1 5 10 15

Pro Arg Glu Val Leu Leu Val Leu Ser Val Asn Ser Ser Val Phe Leu

20 25 30

His Leu Gln Ala Leu Gly Ile Pro Leu His Leu Ala Tyr Asn Ser Ser

35 40 45

Leu Val Thr Phe Gln Glu Pro Pro Gly Val Asn Thr Thr Glu Leu Arg

50 55 60

Ser Thr Gly Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala Asp Arg

65 70 75 80

Asn His Tyr Arg Arg Tyr Pro Arg Arg Arg Gly Pro Pro Arg Asn Tyr

85 90 95

Gln Gln Asn Thr Arg Gly Leu Asn Ala His Gly Ala Ala Gln Met Gln

100 105 110

Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn Ser Met Thr

115 120 125

Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser Met Ser Tyr

130 135 140

Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly Ser Met Gly Ser Val

145 150 155 160

Arg Ser Gln Leu Arg Ser Leu Lys Glu Arg Asn Pro Leu Lys Ile Phe

165 170 175

Pro Ser Lys Arg Ile Leu Arg Arg His Lys Arg Asp Trp Val Val Ala

180 185 190

Pro Ile Ser Val Pro Glu Asn Gly Lys Gly Pro Phe Pro Gln Arg Leu

195 200 205

Asn Gln Leu Lys Ser Asn Lys Asp Arg Asp Thr Lys Ile Phe Tyr Ser

210 215 220

Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu Gly Val Phe Ala Val

225 230 235 240

Glu Lys Glu Thr Arg Ser Ala Gly Glu Thr Tyr Thr Met Lys Glu Val

245 250 255

Leu Phe Tyr Leu Gly Gln Tyr Ile Met Thr Lys Arg Leu Tyr Asp Glu

260 265 270

Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp Leu Leu Gly Asp Leu

275 280 285

Phe Gly Val Pro Ser Phe Ser Val Lys Glu His Arg Lys Ile Tyr Thr

290 295 300

Met Ile Tyr Arg Asn Leu Val Val Val Asn Gln Gln Glu Ser Ser Asp

305 310 315 320

Ser Gly Thr Ser Val Ser Arg Ser

325

<210> 40

<211> 352

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 40

Met Ala Val Pro Met Thr Val Phe Met Arg Leu Asn Ile Ile Ser Pro

1 5 10 15

Asp Leu Ser Gly Cys Thr Ser Lys Gly Leu Val Leu Pro Ala Val Leu

20 25 30

Gly Ile Thr Phe Gly Ala Phe Leu Ile Gly Ala Leu Leu Thr Ala Ala

35 40 45

Leu Trp Tyr Ile Tyr Ser His Thr Arg Ser Pro Ser Lys Arg Glu Pro

50 55 60

Val Val Ala Val Ala Ala Pro Ala Ser Ser Glu Ser Ser Ser Thr Asn

65 70 75 80

His Ser Ile Gly Ser Thr Gln Ser Thr Pro Cys Ser Thr Ser Ser Met

85 90 95

Ala Thr Gly Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala Asp Arg

100 105 110

Asn His Tyr Arg Arg Tyr Pro Arg Arg Arg Gly Pro Pro Arg Asn Tyr

115 120 125

Gln Gln Asn Thr Arg Gly Leu Asn Ala His Gly Ala Ala Gln Met Gln

130 135 140

Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn Ser Met Thr

145 150 155 160

Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser Met Ser Tyr

165 170 175

Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly Ser Met Gly Ser Val

180 185 190

Arg Ser Gln Leu Leu Lys Ile Phe Pro Ser Lys Arg Ile Leu Arg Arg

195 200 205

His Lys Arg Asp Trp Val Val Ala Pro Ile Ser Val Pro Glu Asn Gly

210 215 220

Lys Gly Pro Phe Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp

225 230 235 240

Arg Asp Thr Lys Ile Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser

245 250 255

Pro Pro Glu Gly Val Phe Ala Val Glu Lys Glu Thr Arg Ser Ala Gly

260 265 270

Glu Thr Tyr Thr Met Lys Glu Val Leu Phe Tyr Leu Gly Gln Tyr Ile

275 280 285

Met Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys

290 295 300

Ser Asn Asp Leu Leu Gly Asp Leu Phe Gly Val Pro Ser Phe Ser Val

305 310 315 320

Lys Glu His Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val

325 330 335

Val Asn Gln Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser Arg Ser

340 345 350

<210> 41

<211> 346

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 41

Met Ala Val Pro Met Thr Val Ser Met Arg Leu Asn Ile Val Ser Pro

1 5 10 15

Asp Leu Ser Gly Lys Gly Leu Val Leu Pro Ser Val Leu Gly Ile Thr

20 25 30

Phe Gly Ala Phe Leu Ile Gly Ala Leu Leu Thr Ala Ala Leu Trp Tyr

35 40 45

Ile Tyr Ser His Thr Arg Gly Pro Ser Lys Arg Glu Pro Val Val Ala

50 55 60

Val Ala Ala Pro Ala Ser Ser Glu Ser Ser Ser Thr Asn His Ser Ile

65 70 75 80

Gly Ser Thr Gln Ser Thr Pro Cys Ser Thr Ser Ser Met Ala Thr Gly

85 90 95

Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala Asp Arg Asn His Tyr

100 105 110

Arg Arg Tyr Pro Arg Arg Arg Gly Pro Pro Arg Asn Tyr Gln Gln Asn

115 120 125

Thr Arg Gly Leu Asn Ala His Gly Ala Ala Gln Met Gln Pro Met His

130 135 140

Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn Ser Met Thr Ser Ser Gln

145 150 155 160

Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser Met Ser Tyr Ser Gln Gln

165 170 175

Gly Thr Pro Gly Met Ala Leu Gly Ser Met Gly Ser Val Arg Ser Gln

180 185 190

Leu Ala Met His Ser Pro Pro Thr Arg Ile Leu Arg Arg Arg Lys Arg

195 200 205

Glu Trp Val Met Pro Pro Ile Phe Val Pro Glu Asn Gly Lys Gly Pro

210 215 220

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

225 230 235 240

Lys Ile Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu

245 250 255

Gly Val Phe Thr Ile Glu Lys Glu Ser Arg Ser Ala Gly Glu Thr Tyr

260 265 270

Thr Met Lys Glu Ile Ile Phe Tyr Ile Gly Gln Tyr Ile Met Thr Lys

275 280 285

Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp

290 295 300

Leu Leu Gly Asp Val Phe Gly Val Pro Ser Phe Ser Val Lys Glu His

305 310 315 320

Arg Lys Ile Tyr Ala Met Ile Tyr Arg Asn Leu Val Ala Val Ser Gln

325 330 335

Gln Asp Ser Gly Thr Ser Leu Ser Arg Ser

340 345

<210> 42

<211> 346

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 42

Met Ala Val Pro Met Thr Val Ser Met Arg Leu Asn Ile Val Ser Pro

1 5 10 15

Asp Leu Ser Gly Lys Gly Leu Val Leu Pro Ser Val Leu Gly Ile Thr

20 25 30

Phe Gly Ala Phe Leu Ile Gly Ala Leu Leu Thr Ala Ala Leu Trp Tyr

35 40 45

Ile Tyr Ser His Thr Arg Ala Pro Ser Lys Arg Glu Pro Val Val Ala

50 55 60

Val Ala Ala Pro Ala Ser Ser Glu Ser Ser Ser Thr Asn His Ser Ile

65 70 75 80

Gly Ser Thr Gln Ser Thr Pro Cys Ser Thr Ser Ser Met Ala Thr Gly

85 90 95

Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala Asp Arg Asn His Tyr

100 105 110

Arg Arg Tyr Pro Arg Arg Arg Gly Pro Pro Arg Asn Tyr Gln Gln Asn

115 120 125

Thr Arg Gly Leu Asn Ala His Gly Ala Ala Gln Met Gln Pro Met His

130 135 140

Arg Tyr Asp Val Ser Ala Leu Gln Tyr Asn Ser Met Thr Ser Ser Gln

145 150 155 160

Thr Tyr Met Asn Gly Ser Pro Thr Tyr Ser Met Ser Tyr Ser Gln Gln

165 170 175

Gly Thr Pro Gly Met Ala Leu Gly Ser Met Gly Ser Val Arg Ser Gln

180 185 190

Leu Val Met Asn Ser Pro Pro Ser Arg Ile Leu Arg Arg Arg Lys Arg

195 200 205

Glu Trp Val Met Pro Pro Ile Ser Val Pro Glu Asn Gly Lys Gly Pro

210 215 220

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

225 230 235 240

Lys Leu Phe Tyr Ser Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu

245 250 255

Gly Val Phe Thr Ile Glu Lys Glu Thr Arg Ser Ala Gly Glu Ile Tyr

260 265 270

Thr Met Lys Glu Ile Ile Phe Tyr Ile Gly Gln Tyr Ile Met Thr Lys

275 280 285

Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp

290 295 300

Leu Leu Gly Asp Val Phe Gly Val Pro Ser Phe Ser Val Lys Glu His

305 310 315 320

Arg Lys Ile Tyr Ala Met Ile Tyr Arg Asn Leu Val Val Val Ser Gln

325 330 335

Gln Asp Ser Gly Thr Ser Pro Ser Arg Ser

340 345

<210> 43

<211> 489

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 43

atgctgccga gagtgggctg ccccgcgctg ccgctgccgc cgccgccgct gctgccgctg 60

ctgccgctgc tgctgctgct actgggcgcg agtggcggcg gcggcggggc gcgcgcggag 120

gtgctgttcc gctgcccgcc ctgcacaccc gagcgcctgg ccgcctgcgg gcccccgccg 180

gttgcgccgc ccgccgcggt ggccgcagtg gccggaggcg cccgcatgcc atgcgcggag 240

ctcgtccggg agccgggctg cggctgctgc tcggtgtgcg cccggctgga gggcgaggcg 300

tgcggcgtct acaccccgcg ctgcggccag gggctgcgct gctatcccca cccgggctcc 360

gagctgcccc tgcaggcgct ggtcatgggc gagggcactt gtgagaagcg ccgggacgcc 420

gagtatggcg ccagcccgga gcaggttgca gacaatggcg atgaccactc agaaggaggc 480

ctggtggag 489

<210> 44

<211> 423

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 44

atgctgcccc gcctgggcgg ccccgccctg cccctgctgc tgccctccct gctgctgctg 60

ctgctgctgg gcgccggcgg ctgcggcccc ggcgtgcgcg ccgaggtgct gttccgctgc 120

cccccctgca cccccgagcg cctggccgcc tgcggccccc cccccgacgc cccctgcgcc 180

gagctggtgc gcgagcccgg ctgcggctgc tgctccgtgt gcgcccgcca ggagggcgag 240

gcctgcggcg tgtacatccc ccgctgcgcc cagaccctgc gctgctaccc caaccccggc 300

tccgagctgc ccctgaaggc cctggtgacc ggcgccggca cctgcgagaa gcgccgcgtg 360

ggcaccaccc cccagcaggt ggccgactcc gacgacgacc actccgaggg cggcctggtg 420

gag 423

<210> 45

<211> 423

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 45

atgctgcccc gcctgggcgg ccccgccctg cccctgctgc tgccctccct gctgctgctg 60

ctgctgctgg gcgccggcgg ctgcggcccc ggcgtgcgcg ccgaggtgct gttccgctgc 120

cccccctgca cccccgagcg cctggccgcc tgcggccccc cccccgacgc cccctgcgcc 180

gagctggtgc gcgagcccgg ctgcggctgc tgctccgtgt gcgcccgcca ggagggcgag 240

gcctgcggcg tgtacatccc ccgctgcgcc cagaccctgc gctgctaccc caaccccggc 300

tccgagctgc ccctgaaggc cctggtgacc ggcgccggca cctgcgagaa gcgccgcgtg 360

ggcaccaccc cccagcaggt ggccgactcc gaggacgacc actccgaggg cggcctggtg 420

gag 423

<210> 46

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 46

Asn His Val Asp Ser Thr Met Asn Met Leu Gly Gly Gly Gly Ser

1 5 10 15

<210> 47

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 47

Glu Leu Ala Val Phe Arg Glu Lys Val Thr Glu Gln His Arg Gln

1 5 10 15

<210> 48

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 48

Leu Gly Leu Glu Glu Pro Lys Lys Leu Arg Pro Pro Pro Ala Arg

1 5 10 15

<210> 49

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 49

Asp Gln Val Leu Glu Arg Ile Ser Thr Met Arg Leu Pro Asp Glu

1 5 10 15

<210> 50

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 50

Gly Pro Leu Glu His Leu Tyr Ser Leu His Ile Pro Asn Cys Asp

1 5 10 15

<210> 51

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 51

Lys His Gly Leu Tyr Asn Leu Lys Gln Cys Lys Met Ser Leu Asn

1 5 10 15

<210> 52

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 52

Pro Asn Thr Gly Lys Leu Ile Gln Gly Ala Pro Thr Ile Arg Gly

1 5 10 15

<210> 53

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 53

Pro Glu Cys His Leu Phe Tyr Asn Glu Gln Gln Glu Ala Arg Gly

1 5 10 15

<210> 54

<211> 163

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 54

Met Leu Pro Arg Val Gly Cys Pro Ala Leu Pro Leu Pro Pro Pro Pro

1 5 10 15

Leu Leu Pro Leu Leu Pro Leu Leu Leu Leu Leu Leu Gly Ala Ser Gly

20 25 30

Gly Gly Gly Gly Ala Arg Ala Glu Val Leu Phe Arg Cys Pro Pro Cys

35 40 45

Thr Pro Glu Arg Leu Ala Ala Cys Gly Pro Pro Pro Val Ala Pro Pro

50 55 60

Ala Ala Val Ala Ala Val Ala Gly Gly Ala Arg Met Pro Cys Ala Glu

65 70 75 80

Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Ser Val Cys Ala Arg Leu

85 90 95

Glu Gly Glu Ala Cys Gly Val Tyr Thr Pro Arg Cys Gly Gln Gly Leu

100 105 110

Arg Cys Tyr Pro His Pro Gly Ser Glu Leu Pro Leu Gln Ala Leu Val

115 120 125

Met Gly Glu Gly Thr Cys Glu Lys Arg Arg Asp Ala Glu Tyr Gly Ala

130 135 140

Ser Pro Glu Gln Val Ala Asp Asn Gly Asp Asp His Ser Glu Gly Gly

145 150 155 160

Leu Val Glu

<210> 55

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 55

Met Leu Pro Arg Leu Gly Gly Pro Ala Leu Pro Leu Leu Leu Pro Ser

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Ala Gly Gly Cys Gly Pro Gly Val

20 25 30

Arg Ala Glu Val Leu Phe Arg Cys Pro Pro Cys Thr Pro Glu Arg Leu

35 40 45

Ala Ala Cys Gly Pro Pro Pro Asp Ala Pro Cys Ala Glu Leu Val Arg

50 55 60

Glu Pro Gly Cys Gly Cys Cys Ser Val Cys Ala Arg Gln Glu Gly Glu

65 70 75 80

Ala Cys Gly Val Tyr Ile Pro Arg Cys Ala Gln Thr Leu Arg Cys Tyr

85 90 95

Pro Asn Pro Gly Ser Glu Leu Pro Leu Lys Ala Leu Val Thr Gly Ala

100 105 110

Gly Thr Cys Glu Lys Arg Arg Val Gly Thr Thr Pro Gln Gln Val Ala

115 120 125

Asp Ser Asp Asp Asp His Ser Glu Gly Gly Leu Val Glu

130 135 140

<210> 56

<211> 141

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 56

Met Leu Pro Arg Leu Gly Gly Pro Ala Leu Pro Leu Leu Leu Pro Ser

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Ala Gly Gly Cys Gly Pro Gly Val

20 25 30

Arg Ala Glu Val Leu Phe Arg Cys Pro Pro Cys Thr Pro Glu Arg Leu

35 40 45

Ala Ala Cys Gly Pro Pro Pro Asp Ala Pro Cys Ala Glu Leu Val Arg

50 55 60

Glu Pro Gly Cys Gly Cys Cys Ser Val Cys Ala Arg Gln Glu Gly Glu

65 70 75 80

Ala Cys Gly Val Tyr Ile Pro Arg Cys Ala Gln Thr Leu Arg Cys Tyr

85 90 95

Pro Asn Pro Gly Ser Glu Leu Pro Leu Lys Ala Leu Val Thr Gly Ala

100 105 110

Gly Thr Cys Glu Lys Arg Arg Val Gly Ala Thr Pro Gln Gln Val Ala

115 120 125

Asp Ser Glu Asp Asp His Ser Glu Gly Gly Leu Val Glu

130 135 140

<210> 57

<211> 1101

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 57

acgatgcgga gactgctgca ggaaacggag ctggtggagc cgctgacacc tagcggagcg 60

atgcccaacc aggcgcagat gcggatcctg aaagagacgg agctgaggaa ggtgaaggtg 120

cttggatctg gcgcttttgg cacagtctac aagggcatct ggatccctga tggggagaat 180

gtgaaaattc cagtggccat caaagtgttg agggaaaaca catcccccaa agccaacaaa 240

gaaatcttag acgaagcata cgtgatggct ggtgtgggct ccccatatgt ctcccgcctt 300

ctgggcatct gcctgacatc cacggtgcag ctggtgacac agcttatgcc ctatggctgc 360

ctcttagacc atgtccggga aaaccgcgga cgcctgggct cccaggacct gctgaactgg 420

tgtatgcaga ttgccaaggg gatgagctac ctggaggatg tgcggctcgt acacagggac 480

ttggccgctc ggaacgtgct ggtcaagagt cccaaccatg tcaaaattac agacttcggg 540

ctggctcggc tgctggacat tgacgagaca gagtaccatg cagatggggg caaggtgccc 600

atcaagtgga tggcgctgga gtccattctc cgccggcggt tcacccacca gagtgatgtg 660

tggagttatg gtgtgactgt gtgggagctg atgacttttg gggccaaacc ttacgatggg 720

atcccagccc gggagatccc tgacctgctg gaaaaggggg agcggctgcc ccagcccccc 780

atctgcacca ttgatgtcta catgatcatg gtcaaatgtt ggatgattga ctctgaatgt 840

cggccaagat tccgggagtt ggtgtctgaa ttctcccgca tggccaggga cccccagcgc 900

tttgtggtca tccagaatga ggacttggct cccggagctg gcggcatggt gcaccacagg 960

caccgcagct catctcctct gcctgctgcc cgacctgctg gtgccactct ggaaaggccc 1020

aagactctct ccccagggaa gaatggggtc gtcaaagacg tttttgcctt tgggggtgcc 1080

gtggagaacc ccgagtactt g 1101

<210> 58

<211> 1101

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 58

accatgcgcc gcctgctgca ggagaccgag ctggtggagc ccctgacccc ctccggcgcc 60

gtgcccaacc aggcccagat gcgcatcctg aaggagaccg agctgcgcaa gctgaaggtg 120

ctgggctccg gcgccttcgg caccgtgtac aagggcatct ggatccccga cggcgagaac 180

gtgaagatcc ccgtggccat caaggtgctg cgcgagaaca cctcccccaa ggccaacaag 240

gagatcctgg acgaggccta cgtgatggcc ggcgtgggct ccccctacgt gtcccgcctg 300

ctgggcatct gcctgacctc caccgtgcag ctggtgaccc agctgatgcc ctacggctgc 360

ctgctggacc acgtgcgcga gcaccgcggc cgcctgggct cccaggacct gctgaactgg 420

tgcgtgcaga tcgccaaggg catgtcctac ctggaggagg tgcgcctggt gcaccgcgac 480

ctggccgccc gcaacgtgct ggtgaagtcc cccaaccacg tgaagatcac cgacttcggc 540

ctggcccgcc tgctggacat cgacgagacc gagtaccacg ccgacggcgg caaggtgccc 600

atcaagtgga tggccctgga gtccatcctg cgccgccgct tcacccacca gtccgacgtg 660

tggtcctacg gcgtgaccgt gtgggagctg atgaccttcg gcgccaagcc ctacgacggc 720

atccccgccc gcgagatccc cgacctgctg gagaagggcg agcgcctgcc ccagcccccc 780

atctgcacca tcgacgtgta catgatcatg gtgaagtgct ggatgatcga ctccgagtgc 840

cgcccccgct tccgcgagct ggtgtccgag ttctcccgca tggcccgcga cccccagcgc 900

ttcgtggtga tccagaacga ggacctggcc ctgggcaccg gctccaccgc ccaccgccgc 960

caccgctcct cctccccccc cccccccatc cgccccgccg gcgccaccct ggagcgcccc 1020

aagaccctgt cccccggcaa gaacggcgtg gtgaaggacg tgttcgcctt cggcggcgcc 1080

gtggagaacc ccgagtacct g 1101

<210> 59

<211> 1101

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 59

accatgcgcc gcctgctgca ggagaccgag ctggtggagc ccctgacccc ctccggcgcc 60

atgcccaacc aggcccagat gcgcatcctg aaggagaccg agctgcgcaa ggtgaaggtg 120

ctgggctccg gcgccttcgg caccgtgtac aagggcatct ggatccccga cggcgagaac 180

gtgaagatcc ccgtggccat caaggtgctg cgcgagaaca cctcccccaa ggccaacaag 240

gagatcctgg acgaggccta cgtgatggcc ggcgtgggct ccccctacgt gtcccgcctg 300

ctgggcatct gcctgacctc caccgtgcag ctggtgaccc agctgatgcc ctacggctgc 360

ctgctggacc acgtgcgcga gcaccgcggc cgcctgggct cccaggacct gctgaactgg 420

tgcgtgcaga tcgccaaggg catgtcctac ctggaggacg tgcgcctggt gcaccgcgac 480

ctggccgccc gcaacgtgct ggtgaagtcc cccaaccacg tgaagatcac cgacttcggc 540

ctggcccgcc tgctggacat cgacgagacc gagtaccacg ccgacggcgg caaggtgccc 600

atcaagtgga tggccctgga gtccatcctg cgccgccgct tcacccacca gtccgacgtg 660

tggtcctacg gcgtgaccgt gtgggagctg atgaccttcg gcgccaagcc ctacgacggc 720

atccccgccc gcgagatccc cgacctgctg gagaagggcg agcgcctgcc ccagcccccc 780

atctgcacca tcgacgtgta catgatcatg gtgaagtgct ggatgatcga ctccgagtgc 840

cgcccccgct tccgcgagct ggtgtccgag ttctcccgca tggcccgcga cccccagcgc 900

ttcgtggtga tccagaacga ggacctgacc cccggcaccg gctccaccgc ccaccgccgc 960

caccgctcct cctcccccct gccccccgtg cgccccgccg gcgccaccct ggagcgcccc 1020

aagaccctgt cccccggcaa gaacggcgtg gtgaaggacg tgttcgcctt cggcggcgcc 1080

gtggagaacc ccgagtacct g 1101

<210> 60

<211> 367

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 60

Thr Met Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr

1 5 10 15

Pro Ser Gly Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu

20 25 30

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

35 40 45

Val Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro

50 55 60

Val Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys

65 70 75 80

Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr

85 90 95

Val Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val

100 105 110

Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn

115 120 125

Arg Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile

130 135 140

Ala Lys Gly Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp

145 150 155 160

Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile

165 170 175

Thr Asp Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr

180 185 190

His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser

195 200 205

Ile Leu Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly

210 215 220

Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly

225 230 235 240

Ile Pro Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu

245 250 255

Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys

260 265 270

Cys Trp Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val

275 280 285

Ser Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile

290 295 300

Gln Asn Glu Asp Leu Ala Pro Gly Ala Gly Gly Met Val His His Arg

305 310 315 320

His Arg Ser Ser Ser Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr

325 330 335

Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys

340 345 350

Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu

355 360 365

<210> 61

<211> 367

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 61

Thr Met Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr

1 5 10 15

Pro Ser Gly Ala Val Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu

20 25 30

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

35 40 45

Val Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro

50 55 60

Val Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys

65 70 75 80

Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr

85 90 95

Val Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val

100 105 110

Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu His

115 120 125

Arg Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Val Gln Ile

130 135 140

Ala Lys Gly Met Ser Tyr Leu Glu Glu Val Arg Leu Val His Arg Asp

145 150 155 160

Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile

165 170 175

Thr Asp Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr

180 185 190

His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser

195 200 205

Ile Leu Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly

210 215 220

Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly

225 230 235 240

Ile Pro Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu

245 250 255

Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys

260 265 270

Cys Trp Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val

275 280 285

Ser Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile

290 295 300

Gln Asn Glu Asp Leu Ala Leu Gly Thr Gly Ser Thr Ala His Arg Arg

305 310 315 320

His Arg Ser Ser Ser Pro Pro Pro Pro Ile Arg Pro Ala Gly Ala Thr

325 330 335

Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys

340 345 350

Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu

355 360 365

<210> 62

<211> 367

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 62

Thr Met Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr

1 5 10 15

Pro Ser Gly Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu

20 25 30

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

35 40 45

Val Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro

50 55 60

Val Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys

65 70 75 80

Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr

85 90 95

Val Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val

100 105 110

Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu His

115 120 125

Arg Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Val Gln Ile

130 135 140

Ala Lys Gly Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp

145 150 155 160

Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile

165 170 175

Thr Asp Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr

180 185 190

His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser

195 200 205

Ile Leu Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly

210 215 220

Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly

225 230 235 240

Ile Pro Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu

245 250 255

Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys

260 265 270

Cys Trp Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val

275 280 285

Ser Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile

290 295 300

Gln Asn Glu Asp Leu Thr Pro Gly Thr Gly Ser Thr Ala His Arg Arg

305 310 315 320

His Arg Ser Ser Ser Pro Leu Pro Pro Val Arg Pro Ala Gly Ala Thr

325 330 335

Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys

340 345 350

Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu

355 360 365

<210> 63

<211> 312

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 63

tggtccttcg gcgtggtgct gtgggagatc gccaccctgg ccgagcagcc ctaccagggc 60

ctgtccaacg agcaggtgct gcgcttcgtg atggagggcg gcctgctgga caagcccgac 120

aactgccccg acatgctgtt cgagctgatg cgcatgtgct ggcagtacaa ccccaagatg 180

cgcccctcct tcctggagca caaggccgag aacggccccg gccccggcgt gctggtgctg 240

cgcgcctcct tcgacgagcg ccagccctac gcccacatga acggaggccg caagaacgag 300

cgcgccctgc cc 312

<210> 64

<211> 312

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 64

tggtccttcg gcgtggtgct gtgggagatc gccaccctgg ccgagcagcc ctaccagggc 60

ctgtccaacg agcaggtgct gcgcttcgtg atggagggcg gcctgctgga caagcccgac 120

aactgccccg acatgctgtt cgagctgatg cgcatgtgct ggcagtacaa ccccaagatg 180

cgcccctcct tcctggagca caaggccgag aacggccccg gccccggcgt gctggtgctg 240

cgcgcctcct tcgacgagcg ccagccctac gcccacatga acggcggccg cgccaacgag 300

cgcgccctgc cc 312

<210> 65

<211> 306

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 65

tggtccttcg gcgtggtgct gtgggagatc gccaccctgg ccgagcagcc ctaccagggc 60

ctgtccaacg agcaggtgct gcgcttcgtg atggagggcg gcctgctgga caagcccgac 120

aactgccccg acatgctgtt cgagctgatg cgcatgtgct ggcagtacaa ccccaagatg 180

cgcccctcct tcctggagca caaggccgag aacggccccg gcgtgctggt gctgcgcgcc 240

tccttcgacg agcgccagcc ctacgcccac atgaacggcg gccgcgccaa cgagcgcgcc 300

ctgccc 306

<210> 66

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 66

Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val Ile Thr Glu

1 5 10 15

<210> 67

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 67

Ile Arg Gly Trp Lys Leu Phe Tyr Asn Tyr Ala Leu Val Ile Phe

1 5 10 15

<210> 68

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 68

Val Val Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val

1 5 10 15

<210> 69

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 69

Phe Phe Tyr Val Gln Ala Lys Thr Gly Tyr Glu Asn Phe Ile His

1 5 10 15

<210> 70

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 70

Leu Ile Ile Ala Leu Pro Val Ala Val Leu Leu Ile Val Gly Gly

1 5 10 15

<210> 71

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 71

Leu Val Ile Met Leu Tyr Val Phe His Arg Lys Arg Asn Asn Ser

1 5 10 15

<210> 72

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 72

Asn Cys His His Val Val Arg Leu Leu Gly Val Val Ser Gln Gly

1 5 10 15

<210> 73

<211> 104

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 73

Trp Ser Phe Gly Val Val Leu Trp Glu Ile Ala Thr Leu Ala Glu Gln

1 5 10 15

Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val Leu Arg Phe Val Met Glu

20 25 30

Gly Gly Leu Leu Asp Lys Pro Asp Asn Cys Pro Asp Met Leu Phe Glu

35 40 45

Leu Met Arg Met Cys Trp Gln Tyr Asn Pro Lys Met Arg Pro Ser Phe

50 55 60

Leu Glu His Lys Ala Glu Asn Gly Pro Gly Pro Gly Val Leu Val Leu

65 70 75 80

Arg Ala Ser Phe Asp Glu Arg Gln Pro Tyr Ala His Met Asn Gly Gly

85 90 95

Arg Lys Asn Glu Arg Ala Leu Pro

100

<210> 74

<211> 104

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 74

Trp Ser Phe Gly Val Val Leu Trp Glu Ile Ala Thr Leu Ala Glu Gln

1 5 10 15

Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val Leu Arg Phe Val Met Glu

20 25 30

Gly Gly Leu Leu Asp Lys Pro Asp Asn Cys Pro Asp Met Leu Phe Glu

35 40 45

Leu Met Arg Met Cys Trp Gln Tyr Asn Pro Lys Met Arg Pro Ser Phe

50 55 60

Leu Glu His Lys Ala Glu Asn Gly Pro Gly Pro Gly Val Leu Val Leu

65 70 75 80

Arg Ala Ser Phe Asp Glu Arg Gln Pro Tyr Ala His Met Asn Gly Gly

85 90 95

Arg Ala Asn Glu Arg Ala Leu Pro

100

<210> 75

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 75

Trp Ser Phe Gly Val Val Leu Trp Glu Ile Ala Thr Leu Ala Glu Gln

1 5 10 15

Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val Leu Arg Phe Val Met Glu

20 25 30

Gly Gly Leu Leu Asp Lys Pro Asp Asn Cys Pro Asp Met Leu Phe Glu

35 40 45

Leu Met Arg Met Cys Trp Gln Tyr Asn Pro Lys Met Arg Pro Ser Phe

50 55 60

Leu Glu His Lys Ala Glu Asn Gly Pro Gly Val Leu Val Leu Arg Ala

65 70 75 80

Ser Phe Asp Glu Arg Gln Pro Tyr Ala His Met Asn Gly Gly Arg Ala

85 90 95

Asn Glu Arg Ala Leu Pro

100

<210> 76

<211> 1947

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 76

atggcggtac caatgctgcc gagagtgggc tgccccgcgc tgccgctgcc gccgccgccg 60

ctgctgccgc tgctgccgct gctgctgctg ctactgggcg cgagtggcgg cggcggcggg 120

gcgcgcgcgg aggtgctgtt ccgctgcccg ccctgcacac ccgagcgcct ggccgcctgc 180

gggcccccgc cggttgcgcc gcccgccgcg gtggccgcag tggccggagg cgcccgcatg 240

ccatgcgcgg agctcgtccg ggagccgggc tgcggctgct gctcggtgtg cgcccggctg 300

gagggcgagg cgtgcggcgt ctacaccccg cgctgcggcc aggggctgcg ctgctatccc 360

cacccgggct ccgagctgcc cctgcaggcg ctggtcatgg gcgagggcac ttgtgagaag 420

cgccgggacg ccgagtatgg cgccagcccg gagcaggttg cagacaatgg cgatgaccac 480

tcagaaggag gcctggtgga gcaattgacg atgcggagac tgctgcagga aacggagctg 540

gtggagccgc tgacacctag cggagcgatg cccaaccagg cgcagatgcg gatcctgaaa 600

gagacggagc tgaggaaggt gaaggtgctt ggatctggcg cttttggcac agtctacaag 660

ggcatctgga tccctgatgg ggagaatgtg aaaattccag tggccatcaa agtgttgagg 720

gaaaacacat cccccaaagc caacaaagaa atcttagacg aagcatacgt gatggctggt 780

gtgggctccc catatgtctc ccgccttctg ggcatctgcc tgacatccac ggtgcagctg 840

gtgacacagc ttatgcccta tggctgcctc ttagaccatg tccgggaaaa ccgcggacgc 900

ctgggctccc aggacctgct gaactggtgt atgcagattg ccaaggggat gagctacctg 960

gaggatgtgc ggctcgtaca cagggacttg gccgctcgga acgtgctggt caagagtccc 1020

aaccatgtca aaattacaga cttcgggctg gctcggctgc tggacattga cgagacagag 1080

taccatgcag atgggggcaa ggtgcccatc aagtggatgg cgctggagtc cattctccgc 1140

cggcggttca cccaccagag tgatgtgtgg agttatggtg tgactgtgtg ggagctgatg 1200

acttttgggg ccaaacctta cgatgggatc ccagcccggg agatccctga cctgctggaa 1260

aagggggagc ggctgcccca gccccccatc tgcaccattg atgtctacat gatcatggtc 1320

aaatgttgga tgattgactc tgaatgtcgg ccaagattcc gggagttggt gtctgaattc 1380

tcccgcatgg ccagggaccc ccagcgcttt gtggtcatcc agaatgagga cttggctccc 1440

ggagctggcg gcatggtgca ccacaggcac cgcagctcat ctcctctgcc tgctgcccga 1500

cctgctggtg ccactctgga aaggcccaag actctctccc cagggaagaa tggggtcgtc 1560

aaagacgttt ttgcctttgg gggtgccgtg gagaaccccg agtacttggg ccggccggta 1620

ccttggtcct tcggcgtggt gctgtgggag atcgccaccc tggccgagca gccctaccag 1680

ggcctgtcca acgagcaggt gctgcgcttc gtgatggagg gcggcctgct ggacaagccc 1740

gacaactgcc ccgacatgct gttcgagctg atgcgcatgt gctggcagta caaccccaag 1800

atgcgcccct ccttcctgga gcacaaggcc gagaacggcc ccggccccgg cgtgctggtg 1860

ctgcgcgcct ccttcgacga gcgccagccc tacgcccaca tgaacggagg ccgcaagaac 1920

gagcgcgccc tgcccgcggc cgcatag 1947

<210> 77

<211> 1881

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 77

atggcggtac caatgctgcc ccgcctgggc ggccccgccc tgcccctgct gctgccctcc 60

ctgctgctgc tgctgctgct gggcgccggc ggctgcggcc ccggcgtgcg cgccgaggtg 120

ctgttccgct gccccccctg cacccccgag cgcctggccg cctgcggccc cccccccgac 180

gccccctgcg ccgagctggt gcgcgagccc ggctgcggct gctgctccgt gtgcgcccgc 240

caggagggcg aggcctgcgg cgtgtacatc ccccgctgcg cccagaccct gcgctgctac 300

cccaaccccg gctccgagct gcccctgaag gccctggtga ccggcgccgg cacctgcgag 360

aagcgccgcg tgggcaccac cccccagcag gtggccgact ccgacgacga ccactccgag 420

ggcggcctgg tggagcaatt gaccatgcgc cgcctgctgc aggagaccga gctggtggag 480

cccctgaccc cctccggcgc cgtgcccaac caggcccaga tgcgcatcct gaaggagacc 540

gagctgcgca agctgaaggt gctgggctcc ggcgccttcg gcaccgtgta caagggcatc 600

tggatccccg acggcgagaa cgtgaagatc cccgtggcca tcaaggtgct gcgcgagaac 660

acctccccca aggccaacaa ggagatcctg gacgaggcct acgtgatggc cggcgtgggc 720

tccccctacg tgtcccgcct gctgggcatc tgcctgacct ccaccgtgca gctggtgacc 780

cagctgatgc cctacggctg cctgctggac cacgtgcgcg agcaccgcgg ccgcctgggc 840

tcccaggacc tgctgaactg gtgcgtgcag atcgccaagg gcatgtccta cctggaggag 900

gtgcgcctgg tgcaccgcga cctggccgcc cgcaacgtgc tggtgaagtc ccccaaccac 960

gtgaagatca ccgacttcgg cctggcccgc ctgctggaca tcgacgagac cgagtaccac 1020

gccgacggcg gcaaggtgcc catcaagtgg atggccctgg agtccatcct gcgccgccgc 1080

ttcacccacc agtccgacgt gtggtcctac ggcgtgaccg tgtgggagct gatgaccttc 1140

ggcgccaagc cctacgacgg catccccgcc cgcgagatcc ccgacctgct ggagaagggc 1200

gagcgcctgc cccagccccc catctgcacc atcgacgtgt acatgatcat ggtgaagtgc 1260

tggatgatcg actccgagtg ccgcccccgc ttccgcgagc tggtgtccga gttctcccgc 1320

atggcccgcg acccccagcg cttcgtggtg atccagaacg aggacctggc cctgggcacc 1380

ggctccaccg cccaccgccg ccaccgctcc tcctcccccc ccccccccat ccgccccgcc 1440

ggcgccaccc tggagcgccc caagaccctg tcccccggca agaacggcgt ggtgaaggac 1500

gtgttcgcct tcggcggcgc cgtggagaac cccgagtacc tgggccggcc ggtaccttgg 1560

tccttcggcg tggtgctgtg ggagatcgcc accctggccg agcagcccta ccagggcctg 1620

tccaacgagc aggtgctgcg cttcgtgatg gagggcggcc tgctggacaa gcccgacaac 1680

tgccccgaca tgctgttcga gctgatgcgc atgtgctggc agtacaaccc caagatgcgc 1740

ccctccttcc tggagcacaa ggccgagaac ggccccggcc ccggcgtgct ggtgctgcgc 1800

gcctccttcg acgagcgcca gccctacgcc cacatgaacg gcggccgcgc caacgagcgc 1860

gccctgcccg cggccgcata g 1881

<210> 78

<211> 1875

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 78

atggcggtac caatgctgcc ccgcctgggc ggccccgccc tgcccctgct gctgccctcc 60

ctgctgctgc tgctgctgct gggcgccggc ggctgcggcc ccggcgtgcg cgccgaggtg 120

ctgttccgct gccccccctg cacccccgag cgcctggccg cctgcggccc cccccccgac 180

gccccctgcg ccgagctggt gcgcgagccc ggctgcggct gctgctccgt gtgcgcccgc 240

caggagggcg aggcctgcgg cgtgtacatc ccccgctgcg cccagaccct gcgctgctac 300

cccaaccccg gctccgagct gcccctgaag gccctggtga ccggcgccgg cacctgcgag 360

aagcgccgcg tgggcgccac cccccagcag gtggccgact ccgaggacga ccactccgag 420

ggcggcctgg tggagcaatt gaccatgcgc cgcctgctgc aggagaccga gctggtggag 480

cccctgaccc cctccggcgc catgcccaac caggcccaga tgcgcatcct gaaggagacc 540

gagctgcgca aggtgaaggt gctgggctcc ggcgccttcg gcaccgtgta caagggcatc 600

tggatccccg acggcgagaa cgtgaagatc cccgtggcca tcaaggtgct gcgcgagaac 660

acctccccca aggccaacaa ggagatcctg gacgaggcct acgtgatggc cggcgtgggc 720

tccccctacg tgtcccgcct gctgggcatc tgcctgacct ccaccgtgca gctggtgacc 780

cagctgatgc cctacggctg cctgctggac cacgtgcgcg agcaccgcgg ccgcctgggc 840

tcccaggacc tgctgaactg gtgcgtgcag atcgccaagg gcatgtccta cctggaggac 900

gtgcgcctgg tgcaccgcga cctggccgcc cgcaacgtgc tggtgaagtc ccccaaccac 960

gtgaagatca ccgacttcgg cctggcccgc ctgctggaca tcgacgagac cgagtaccac 1020

gccgacggcg gcaaggtgcc catcaagtgg atggccctgg agtccatcct gcgccgccgc 1080

ttcacccacc agtccgacgt gtggtcctac ggcgtgaccg tgtgggagct gatgaccttc 1140

ggcgccaagc cctacgacgg catccccgcc cgcgagatcc ccgacctgct ggagaagggc 1200

gagcgcctgc cccagccccc catctgcacc atcgacgtgt acatgatcat ggtgaagtgc 1260

tggatgatcg actccgagtg ccgcccccgc ttccgcgagc tggtgtccga gttctcccgc 1320

atggcccgcg acccccagcg cttcgtggtg atccagaacg aggacctgac ccccggcacc 1380

ggctccaccg cccaccgccg ccaccgctcc tcctcccccc tgccccccgt gcgccccgcc 1440

ggcgccaccc tggagcgccc caagaccctg tcccccggca agaacggcgt ggtgaaggac 1500

gtgttcgcct tcggcggcgc cgtggagaac cccgagtacc tgggccggcc ggtaccttgg 1560

tccttcggcg tggtgctgtg ggagatcgcc accctggccg agcagcccta ccagggcctg 1620

tccaacgagc aggtgctgcg cttcgtgatg gagggcggcc tgctggacaa gcccgacaac 1680

tgccccgaca tgctgttcga gctgatgcgc atgtgctggc agtacaaccc caagatgcgc 1740

ccctccttcc tggagcacaa ggccgagaac ggccccggcg tgctggtgct gcgcgcctcc 1800

ttcgacgagc gccagcccta cgcccacatg aacggcggcc gcgccaacga gcgcgccctg 1860

cccgcggccg catag 1875

<210> 79

<211> 648

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 79

Met Ala Val Pro Met Leu Pro Arg Val Gly Cys Pro Ala Leu Pro Leu

1 5 10 15

Pro Pro Pro Pro Leu Leu Pro Leu Leu Pro Leu Leu Leu Leu Leu Leu

20 25 30

Gly Ala Ser Gly Gly Gly Gly Gly Ala Arg Ala Glu Val Leu Phe Arg

35 40 45

Cys Pro Pro Cys Thr Pro Glu Arg Leu Ala Ala Cys Gly Pro Pro Pro

50 55 60

Val Ala Pro Pro Ala Ala Val Ala Ala Val Ala Gly Gly Ala Arg Met

65 70 75 80

Pro Cys Ala Glu Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Ser Val

85 90 95

Cys Ala Arg Leu Glu Gly Glu Ala Cys Gly Val Tyr Thr Pro Arg Cys

100 105 110

Gly Gln Gly Leu Arg Cys Tyr Pro His Pro Gly Ser Glu Leu Pro Leu

115 120 125

Gln Ala Leu Val Met Gly Glu Gly Thr Cys Glu Lys Arg Arg Asp Ala

130 135 140

Glu Tyr Gly Ala Ser Pro Glu Gln Val Ala Asp Asn Gly Asp Asp His

145 150 155 160

Ser Glu Gly Gly Leu Val Glu Gln Leu Thr Met Arg Arg Leu Leu Gln

165 170 175

Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Ala Met Pro Asn

180 185 190

Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu Arg Lys Val Lys

195 200 205

Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile Trp Ile

210 215 220

Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg

225 230 235 240

Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr

245 250 255

Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile

260 265 270

Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu Met Pro Tyr Gly

275 280 285

Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg Leu Gly Ser Gln

290 295 300

Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly Met Ser Tyr Leu

305 310 315 320

Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu

325 330 335

Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu Ala Arg

340 345 350

Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly Gly Lys Val

355 360 365

Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg Phe Thr

370 375 380

His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met

385 390 395 400

Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arg Glu Ile Pro

405 410 415

Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr

420 425 430

Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ser Glu

435 440 445

Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg Met Ala

450 455 460

Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp Leu Ala Pro

465 470 475 480

Gly Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Pro Leu

485 490 495

Pro Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

500 505 510

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly Gly

515 520 525

Ala Val Glu Asn Pro Glu Tyr Leu Gly Arg Pro Val Pro Trp Ser Phe

530 535 540

Gly Val Val Leu Trp Glu Ile Ala Thr Leu Ala Glu Gln Pro Tyr Gln

545 550 555 560

Gly Leu Ser Asn Glu Gln Val Leu Arg Phe Val Met Glu Gly Gly Leu

565 570 575

Leu Asp Lys Pro Asp Asn Cys Pro Asp Met Leu Phe Glu Leu Met Arg

580 585 590

Met Cys Trp Gln Tyr Asn Pro Lys Met Arg Pro Ser Phe Leu Glu His

595 600 605

Lys Ala Glu Asn Gly Pro Gly Pro Gly Val Leu Val Leu Arg Ala Ser

610 615 620

Phe Asp Glu Arg Gln Pro Tyr Ala His Met Asn Gly Gly Arg Lys Asn

625 630 635 640

Glu Arg Ala Leu Pro Ala Ala Ala

645

<210> 80

<211> 626

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 80

Met Ala Val Pro Met Leu Pro Arg Leu Gly Gly Pro Ala Leu Pro Leu

1 5 10 15

Leu Leu Pro Ser Leu Leu Leu Leu Leu Leu Leu Gly Ala Gly Gly Cys

20 25 30

Gly Pro Gly Val Arg Ala Glu Val Leu Phe Arg Cys Pro Pro Cys Thr

35 40 45

Pro Glu Arg Leu Ala Ala Cys Gly Pro Pro Pro Asp Ala Pro Cys Ala

50 55 60

Glu Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Ser Val Cys Ala Arg

65 70 75 80

Gln Glu Gly Glu Ala Cys Gly Val Tyr Ile Pro Arg Cys Ala Gln Thr

85 90 95

Leu Arg Cys Tyr Pro Asn Pro Gly Ser Glu Leu Pro Leu Lys Ala Leu

100 105 110

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

115 120 125

Gln Gln Val Ala Asp Ser Asp Asp Asp His Ser Glu Gly Gly Leu Val

130 135 140

Glu Gln Leu Thr Met Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu

145 150 155 160

Pro Leu Thr Pro Ser Gly Ala Val Pro Asn Gln Ala Gln Met Arg Ile

165 170 175

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

180 185 190

Phe Gly Thr Val Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val

195 200 205

Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys

210 215 220

Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly

225 230 235 240

Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val

245 250 255

Gln Leu Val Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val

260 265 270

Arg Glu His Arg Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys

275 280 285

Val Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Glu Val Arg Leu Val

290 295 300

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His

305 310 315 320

Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu

325 330 335

Thr Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala

340 345 350

Leu Glu Ser Ile Leu Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp

355 360 365

Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro

370 375 380

Tyr Asp Gly Ile Pro Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly

385 390 395 400

Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile

405 410 415

Met Val Lys Cys Trp Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg

420 425 430

Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe

435 440 445

Val Val Ile Gln Asn Glu Asp Leu Ala Leu Gly Thr Gly Ser Thr Ala

450 455 460

His Arg Arg His Arg Ser Ser Ser Pro Pro Pro Pro Ile Arg Pro Ala

465 470 475 480

Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly

485 490 495

Val Val Lys Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu

500 505 510

Tyr Leu Gly Arg Pro Val Pro Trp Ser Phe Gly Val Val Leu Trp Glu

515 520 525

Ile Ala Thr Leu Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn Glu Gln

530 535 540

Val Leu Arg Phe Val Met Glu Gly Gly Leu Leu Asp Lys Pro Asp Asn

545 550 555 560

Cys Pro Asp Met Leu Phe Glu Leu Met Arg Met Cys Trp Gln Tyr Asn

565 570 575

Pro Lys Met Arg Pro Ser Phe Leu Glu His Lys Ala Glu Asn Gly Pro

580 585 590

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

595 600 605

Tyr Ala His Met Asn Gly Gly Arg Ala Asn Glu Arg Ala Leu Pro Ala

610 615 620

Ala Ala

625

<210> 81

<211> 624

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 81

Met Ala Val Pro Met Leu Pro Arg Leu Gly Gly Pro Ala Leu Pro Leu

1 5 10 15

Leu Leu Pro Ser Leu Leu Leu Leu Leu Leu Leu Gly Ala Gly Gly Cys

20 25 30

Gly Pro Gly Val Arg Ala Glu Val Leu Phe Arg Cys Pro Pro Cys Thr

35 40 45

Pro Glu Arg Leu Ala Ala Cys Gly Pro Pro Pro Asp Ala Pro Cys Ala

50 55 60

Glu Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Ser Val Cys Ala Arg

65 70 75 80

Gln Glu Gly Glu Ala Cys Gly Val Tyr Ile Pro Arg Cys Ala Gln Thr

85 90 95

Leu Arg Cys Tyr Pro Asn Pro Gly Ser Glu Leu Pro Leu Lys Ala Leu

100 105 110

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

115 120 125

Gln Gln Val Ala Asp Ser Glu Asp Asp His Ser Glu Gly Gly Leu Val

130 135 140

Glu Gln Leu Thr Met Arg Arg Leu Leu Gln Glu Thr Glu Leu Val Glu

145 150 155 160

Pro Leu Thr Pro Ser Gly Ala Met Pro Asn Gln Ala Gln Met Arg Ile

165 170 175

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

180 185 190

Phe Gly Thr Val Tyr Lys Gly Ile Trp Ile Pro Asp Gly Glu Asn Val

195 200 205

Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Asn Thr Ser Pro Lys

210 215 220

Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Gly Val Gly

225 230 235 240

Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val

245 250 255

Gln Leu Val Thr Gln Leu Met Pro Tyr Gly Cys Leu Leu Asp His Val

260 265 270

Arg Glu His Arg Gly Arg Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys

275 280 285

Val Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Asp Val Arg Leu Val

290 295 300

His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His

305 310 315 320

Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu

325 330 335

Thr Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile Lys Trp Met Ala

340 345 350

Leu Glu Ser Ile Leu Arg Arg Arg Phe Thr His Gln Ser Asp Val Trp

355 360 365

Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Lys Pro

370 375 380

Tyr Asp Gly Ile Pro Ala Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly

385 390 395 400

Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile

405 410 415

Met Val Lys Cys Trp Met Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg

420 425 430

Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Phe

435 440 445

Val Val Ile Gln Asn Glu Asp Leu Thr Pro Gly Thr Gly Ser Thr Ala

450 455 460

His Arg Arg His Arg Ser Ser Ser Pro Leu Pro Pro Val Arg Pro Ala

465 470 475 480

Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly

485 490 495

Val Val Lys Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu

500 505 510

Tyr Leu Gly Arg Pro Val Pro Trp Ser Phe Gly Val Val Leu Trp Glu

515 520 525

Ile Ala Thr Leu Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn Glu Gln

530 535 540

Val Leu Arg Phe Val Met Glu Gly Gly Leu Leu Asp Lys Pro Asp Asn

545 550 555 560

Cys Pro Asp Met Leu Phe Glu Leu Met Arg Met Cys Trp Gln Tyr Asn

565 570 575

Pro Lys Met Arg Pro Ser Phe Leu Glu His Lys Ala Glu Asn Gly Pro

580 585 590

Gly Val Leu Val Leu Arg Ala Ser Phe Asp Glu Arg Gln Pro Tyr Ala

595 600 605

His Met Asn Gly Gly Arg Ala Asn Glu Arg Ala Leu Pro Ala Ala Ala

610 615 620

<210> 82

<211> 89

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 82

Asp Ser Lys Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser

1 5 10 15

Tyr Cys Asp Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu

20 25 30

Leu Leu Gly Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp

35 40 45

His Leu Thr Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr

50 55 60

Thr Gly Gln Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp Val

65 70 75 80

Glu Thr Gln Ala Thr Val Ile Tyr Asn

85

<210> 83

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 83

Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr

1 5 10 15

Gly His Ser Val Phe Asp Phe Thr His Pro

20 25

<210> 84

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 84

Gly Gly Tyr Val Trp Val Glu Thr Gln Ala Thr Val Ile Tyr Asn Thr

1 5 10 15

Lys Asn Ser Gln

20

<210> 85

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 85

Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu Thr Leu

1 5 10 15

Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys Ile Ala

20 25 30

Lys Glu Thr Asn Asn Lys

35

<210> 86

<211> 114

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 86

ggctgcgcct tcctgtccgt gaagaagcag ttcgaggagc tgaccctggg cgagttcctg 60

aagctggacc gcgagcgcgc caagaacaag atcgccaagg agaccaacaa caag 114

<210> 87

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 87

Arg Ser Lys Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro

1 5 10 15

Leu Pro His Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg

20 25 30

Leu Thr Ile Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp Ala Gly Asp

35 40 45

Leu Asp Ile Glu Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys

50 55 60

Ala Leu Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile

65 70 75 80

Tyr Ile Ser Asp Asn Val Asn Lys

85

<210> 88

<211> 267

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 88

cgctccaagg agtccgaggt gttctacgag ctggcccacc agctgcccct gccccacaac 60

gtgtcctccc acctggacaa ggcctccgtg atgcgcctga ccatctccta cctgcgcgtg 120

cgcaagctgc tggacgctgg cgacctggac atcgaggacg acatgaaggc ccagatgaac 180

tgcttctacc tgaaggccct ggacggcttc gtgatggtgc tgaccgacga cggcgacatg 240

atctacatct ccgacaacgt gaacaag 267

<210> 89

<211> 415

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 89

Met Ala Val Pro Met Leu Pro Arg Val Gly Cys Pro Ala Leu Pro Leu

1 5 10 15

Pro Pro Pro Pro Leu Leu Pro Leu Leu Pro Leu Leu Leu Leu Leu Leu

20 25 30

Gly Ala Ser Gly Gly Gly Gly Gly Ala Arg Ala Glu Val Leu Phe Arg

35 40 45

Cys Pro Pro Cys Thr Pro Glu Arg Leu Ala Ala Cys Gly Pro Pro Pro

50 55 60

Val Ala Pro Pro Ala Ala Val Ala Ala Val Ala Gly Gly Ala Arg Met

65 70 75 80

Pro Cys Ala Glu Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Ser Val

85 90 95

Cys Ala Arg Leu Glu Gly Glu Ala Cys Gly Val Tyr Thr Pro Arg Cys

100 105 110

Gly Gln Gly Leu Arg Cys Tyr Pro His Pro Gly Ser Glu Leu Pro Leu

115 120 125

Gln Ala Leu Val Met Gly Glu Gly Thr Cys Glu Lys Arg Arg Asp Ala

130 135 140

Glu Tyr Gly Ala Ser Pro Glu Gln Val Ala Asp Asn Gly Asp Asp His

145 150 155 160

Ser Glu Gly Gly Leu Val Glu Gln Leu Gly Cys Ala Phe Leu Ser Val

165 170 175

Lys Lys Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu Asp

180 185 190

Arg Glu Arg Ala Lys Asn Lys Ile Ala Lys Glu Thr Asn Asn Lys Gly

195 200 205

Ser Glu Phe Arg Ser Lys Glu Ser Glu Val Phe Tyr Glu Leu Ala His

210 215 220

Gln Leu Pro Leu Pro His Asn Val Ser Ser His Leu Asp Lys Ala Ser

225 230 235 240

Val Met Arg Leu Thr Ile Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp

245 250 255

Ala Gly Asp Leu Asp Ile Glu Asp Asp Met Lys Ala Gln Met Asn Cys

260 265 270

Phe Tyr Leu Lys Ala Leu Asp Gly Phe Val Met Val Leu Thr Asp Asp

275 280 285

Gly Asp Met Ile Tyr Ile Ser Asp Asn Val Asn Lys Tyr Arg Ser Gly

290 295 300

Arg Pro Val Pro Trp Ser Phe Gly Val Val Leu Trp Glu Ile Ala Thr

305 310 315 320

Leu Ala Glu Gln Pro Tyr Gln Gly Leu Ser Asn Glu Gln Val Leu Arg

325 330 335

Phe Val Met Glu Gly Gly Leu Leu Asp Lys Pro Asp Asn Cys Pro Asp

340 345 350

Met Leu Phe Glu Leu Met Arg Met Cys Trp Gln Tyr Asn Pro Lys Met

355 360 365

Arg Pro Ser Phe Leu Glu His Lys Ala Glu Asn Gly Pro Gly Pro Gly

370 375 380

Val Leu Val Leu Arg Ala Ser Phe Asp Glu Arg Gln Pro Tyr Ala His

385 390 395 400

Met Asn Gly Gly Arg Lys Asn Glu Arg Ala Leu Pro Ala Ala Ala

405 410 415

<210> 90

<211> 1248

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 90

atggcggtac caatgctgcc gagagtgggc tgccccgcgc tgccgctgcc gccgccgccg 60

ctgctgccgc tgctgccgct gctgctgctg ctactgggcg cgagtggcgg cggcggcggg 120

gcgcgcgcgg aggtgctgtt ccgctgcccg ccctgcacac ccgagcgcct ggccgcctgc 180

gggcccccgc cggttgcgcc gcccgccgcg gtggccgcag tggccggagg cgcccgcatg 240

ccatgcgcgg agctcgtccg ggagccgggc tgcggctgct gctcggtgtg cgcccggctg 300

gagggcgagg cgtgcggcgt ctacaccccg cgctgcggcc aggggctgcg ctgctatccc 360

cacccgggct ccgagctgcc cctgcaggcg ctggtcatgg gcgagggcac ttgtgagaag 420

cgccgggacg ccgagtatgg cgccagcccg gagcaggttg cagacaatgg cgatgaccac 480

tcagaaggag gcctggtgga gcaattgggc tgcgccttcc tgtccgtgaa gaagcagttc 540

gaggagctga ccctgggcga gttcctgaag ctggaccgcg agcgcgccaa gaacaagatc 600

gccaaggaga ccaacaacaa gggatccgaa ttccgctcca aggagtccga ggtgttctac 660

gagctggccc accagctgcc cctgccccac aacgtgtcct cccacctgga caaggcctcc 720

gtgatgcgcc tgaccatctc ctacctgcgc gtgcgcaagc tgctggacgc tggcgacctg 780

gacatcgagg acgacatgaa ggcccagatg aactgcttct acctgaaggc cctggacggc 840

ttcgtgatgg tgctgaccga cgacggcgac atgatctaca tctccgacaa cgtgaacaag 900

tacagatccg gccggccggt accttggtcc ttcggcgtgg tgctgtggga gatcgccacc 960

ctggccgagc agccctacca gggcctgtcc aacgagcagg tgctgcgctt cgtgatggag 1020

ggcggcctgc tggacaagcc cgacaactgc cccgacatgc tgttcgagct gatgcgcatg 1080

tgctggcagt acaaccccaa gatgcgcccc tccttcctgg agcacaaggc cgagaacggc 1140

cccggccccg gcgtgctggt gctgcgcgcc tccttcgacg agcgccagcc ctacgcccac 1200

atgaacggag gccgcaagaa cgagcgcgcc ctgcccgcgg ccgcatag 1248

<210> 91

<211> 5248

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesis of a polynucleotide or polypeptide

<400> 91

tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca 60

acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg 120

tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 180

cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 240

gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 300

cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 360

ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 420

cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 480

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 540

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 600

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 660

cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 720

agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc 780

cgtgccaaga gtgacgtaag taccgcctat agactctata ggcacacccc tttggctctt 840

atgcatgcta tactgttttt ggcttggggc ctatacaccc ccgcttcctt atgctatagg 900

tgatggtata gcttagccta taggtgtggg ttattgacca ttattgacca ctccaacggt 960

ggagggcagt gtagtctgag cagtactcgt tgctgccgcg cgcgccacca gacataatag 1020

ctgacagact aacagactgt tcctttccat gggtcttttc tgcagtcacc gtcgtcgacg 1080

gtatcgataa gcttgatatc gaattgccgc caccatggcg gtaccaatgc tgccgagagt 1140

gggctgcccc gcgctgccgc tgccgccgcc gccgctgctg ccgctgctgc cgctgctgct 1200

gctgctactg ggcgcgagtg gcggcggcgg cggggcgcgc gcggaggtgc tgttccgctg 1260

cccgccctgc acacccgagc gcctggccgc ctgcgggccc ccgccggttg cgccgcccgc 1320

cgcggtggcc gcagtggccg gaggcgcccg catgccatgc gcggagctcg tccgggagcc 1380

gggctgcggc tgctgctcgg tgtgcgcccg gctggagggc gaggcgtgcg gcgtctacac 1440

cccgcgctgc ggccaggggc tgcgctgcta tccccacccg ggctccgagc tgcccctgca 1500

ggcgctggtc atgggcgagg gcacttgtga gaagcgccgg gacgccgagt atggcgccag 1560

cccggagcag gttgcagaca atggcgatga ccactcagaa ggaggcctgg tggagcaatt 1620

gggctgcgcc ttcctgtccg tgaagaagca gttcgaggag ctgaccctgg gcgagttcct 1680

gaagctggac cgcgagcgcg ccaagaacaa gatcgccaag gagaccaaca acaagggatc 1740

cgaattccgc tccaaggagt ccgaggtgtt ctacgagctg gcccaccagc tgcccctgcc 1800

ccacaacgtg tcctcccacc tggacaaggc ctccgtgatg cgcctgacca tctcctacct 1860

gcgcgtgcgc aagctgctgg acgctggcga cctggacatc gaggacgaca tgaaggccca 1920

gatgaactgc ttctacctga aggccctgga cggcttcgtg atggtgctga ccgacgacgg 1980

cgacatgatc tacatctccg acaacgtgaa caagtacaga tccggccggc cggtaccttg 2040

gtccttcggc gtggtgctgt gggagatcgc caccctggcc gagcagccct accagggcct 2100

gtccaacgag caggtgctgc gcttcgtgat ggagggcggc ctgctggaca agcccgacaa 2160

ctgccccgac atgctgttcg agctgatgcg catgtgctgg cagtacaacc ccaagatgcg 2220

cccctccttc ctggagcaca aggccgagaa cggccccggc cccggcgtgc tggtgctgcg 2280

cgcctccttc gacgagcgcc agccctacgc ccacatgaac ggaggccgca agaacgagcg 2340

cgccctgccc gcggccgcat agtgatagat ctttttccct ctgccaaaaa ttatggggac 2400

atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca 2460

atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat 2520

ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca ttcttccgct 2580

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 2640

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 2700

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 2760

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 2820

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 2880

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 2940

ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 3000

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 3060

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 3120

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 3180

ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 3240

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 3300

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 3360

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 3420

ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 3480

taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 3540

atctcagcga tctgtctatt tcgttcatcc atagttgcct gactcggggg gggggggcgc 3600

tgaggtctgc ctcgtgaaga aggtgttgct gactcatacc aggcctgaat cgccccatca 3660

tccagccaga aagtgaggga gccacggttg atgagagctt tgttgtaggt ggaccagttg 3720

gtgattttga acttttgctt tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc 3780

tgatccttca actcagcaaa agttcgattt attcaacaaa gccgccgtcc cgtcaagtca 3840

gcgtaatgct ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga 3900

gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa 3960

gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct 4020

ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt 4080

caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg 4140

gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat 4200

caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa 4260

atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga 4320

acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga 4380

atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa 4440

aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat 4500

ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg 4560

gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt 4620

tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt 4680

cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta 4740

ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa 4800

cgtggctttc cccccccccc cattattgaa gcatttatca gggttattgt ctcatgagcg 4860

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 4920

gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 4980

ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac 5040

acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 5100

cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat 5160

cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa 5220

ggagaaaata ccgcatcaga ttggctat 5248