Vaccines and methods of their use in treatment of WNT-related cancers

Abstract

The present disclosure provides a composition comprising a nucleic acid sequence encoding from about 1 to about 100 amino acid sequences, the amino acid sequences being tumor-specific antigens wherein at least one tumor-specific antigen is an amino acid sequence associated with the WNT pathway. In some embodiments, the nucleic acid sequence encodes from about 20 to about 60 tumor specific antigens; wherein from about 1 to about 8 tumor specific antigens are selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3B. Also provided is a cell comprising any one or more compositions disclosed herein and a pharmaceutical composition comprising: (i) a therapeutically effective amount of one or more compositions as disclosed herein; and (ii) a pharmaceutically acceptable carrier. A method of treating cancer in a subject in need thereof is also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 63 / 342,605, filed on May 16, 2022, and U.S. Application No. 63 / 340,090, filed on May 10, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] References to sequence listings

[0004] The sequence listing, created on May 10, 2023 and having a size of 526,450 bytes and filed on May 10, 2023 as an XML file named “GENE-003-PCT_SL.xml,” is hereby incorporated by reference pursuant to 37 CFR §1.52(e)(5). Technical Field

[0005] The present disclosure generally relates to compositions and vaccines comprising antigens associated with the β-catenin pathway, and methods of treating β-catenin altered cancers in subjects having cancer by using cancer vaccines. Background Art

[0006] WNT is a secreted cysteine-rich and lipid-modified growth factor that binds to frizzled receptors and LRP5 / 6 co-receptors on the cell surface that receive Wnt signals. Intracellular signal transduction involves the stabilization of β-catenin. In the absence of Wnt, the β-catenin destruction complex resides in the cytoplasm, where it binds, phosphorylates and ubiquitinates β-catenin, leading to its degradation in the proteasome. Wnt induces the association of the complete complex with phosphorylated LRP5 / 6. After binding to LRP5 / 6, the destruction complex captures and phosphorylates β-catenin, but ubiquitination of βTrCP is blocked. Newly synthesized β-catenin accumulates, translocates to the nucleus and associates with TCF family transcription factors to initiate transcription of Wnt target genes (Clevers and Nusse, 2012). Summary of the invention

[0007] The present disclosure provides a composition comprising a nucleic acid sequence encoding about 1 to about 100 amino acid sequences, wherein the amino acid sequences are tumor-specific antigens, wherein at least one tumor-specific antigen is an amino acid sequence associated with the WNT pathway.

[0008] In some embodiments, the composition further comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen; wherein the nucleic acid molecule comprises a regulatory sequence operably linked to a nucleic acid sequence encoding a tumor-specific antigen; wherein the nucleic acid sequence encodes about 20 to about 60 tumor-specific antigens, each tumor-specific antigen is flanked by at least one joint on a continuous amino acid sequence, and wherein the nucleic acid sequence encodes a leader sequence on the 5' end of the first antigen sequence in a 5' to 3' orientation. In some embodiments, the composition further comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen; wherein the nucleic acid molecule comprises a regulatory sequence operably linked to a nucleic acid sequence encoding a tumor-specific antigen; wherein the nucleic acid sequence encodes about 1 to about 60 tumor-specific antigens, each antigen is flanked by at least one joint on a continuous amino acid sequence, and wherein the nucleic acid sequence encodes a leader sequence on the 5' end of the first antigen sequence in a 5' to 3' orientation. In some embodiments, the composition further comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen; wherein the nucleic acid molecule comprises a regulatory sequence operably linked to a nucleic acid sequence encoding a tumor-specific antigen; wherein the nucleic acid sequence encodes at least about 20 tumor-specific antigens, each antigen flanked by at least one linker on a contiguous amino acid sequence, and wherein the nucleic acid sequence encodes a leader sequence on the 5' end of the first antigen sequence in a 5' to 3' orientation.

[0009] In some embodiments, the tumor-specific antigen is selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3B.

[0010] In some embodiments, the nucleic acid sequence comprises a nucleic acid sequence encoding β-catenin or a fragment thereof, wherein the nucleic acid sequence encoding β-catenin or a fragment thereof comprises at least about 70% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[0011] In some embodiments, the antigen expression domain comprises a nucleic acid encoding β-catenin or a fragment thereof, wherein the nucleic acid sequence encoding β-catenin or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[0012] In some embodiments, the tumor-specific antigen does not contain 100% of a nucleic acid sequence comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0013] In some embodiments, the linker is a P2A linker sequence or a furin linker sequence.

[0014] In some embodiments, the nucleic acid sequence encodes about 20 to about 60 tumor-specific antigens; wherein about 1 to about 8 tumor-specific antigens are selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3B.

[0015] In some embodiments, the nucleic acid sequence encodes a leader sequence and one or more tumor specific antigens in a 5' to 3' initial orientation, wherein the one or more tumor antigens are flanked by at least one linker sequence.

[0016] Also provided is a cell comprising any one or more of the compositions disclosed herein.

[0017] Also provided is a nucleic acid molecule comprising any one or more of the compositions disclosed herein.

[0018] Also provided is a pharmaceutical composition comprising: (i) a therapeutically effective amount of one or more compositions as disclosed herein; and (ii) a pharmaceutically acceptable carrier.

[0019] In some embodiments, the composition comprises a plasmid comprising an expressible nucleic acid sequence encoding about 40 to about 60 tumor-specific antigens.

[0020] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of pVAX0001 comprising a nucleic acid sequence encoding about 1 to about 7 tumor-specific antigens selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid molecule disclosed herein comprising a nucleic acid sequence encoding about 20 to about 55 tumor-specific antigens selected from one or a combination of the epitopes disclosed in the examples, but wherein the tumor-specific epitope does not contain a WNT pathway epitope. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a nucleic acid molecule disclosed herein comprising a nucleic acid sequence encoding about 20 to about 75 tumor-specific antigens selected from one or a combination of the epitopes disclosed in the examples. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of pVAX0001 comprising a nucleic acid sequence encoding about 20 to about 55 tumor-specific antigens selected from one or a combination of the epitopes disclosed in the examples. In some embodiments, the tumor-specific antigen is encoded by one or more nucleic acid sequences encoding an amino acid sequence selected from one or a combination of SEQ ID NOs: 70 to 286, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid selected from one or a combination of SEQ ID NOs: 70 to 286.

[0021] Also provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (i) a pharmaceutical composition comprising a nucleic acid sequence encoding about twenty or more neoantigens or epitopes specific to neoantigens; or (ii) a pharmaceutical composition disclosed herein.

[0022] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a checkpoint inhibitor.

[0023] In some embodiments, the checkpoint inhibitor is selected from one or a combination of the checkpoint inhibitors in Table 1. In some embodiments, the therapeutically effective amount of the checkpoint inhibitor is about 150 mg to about 250 mg.

[0024] In some embodiments, the dosage of the pharmaceutical composition is about 0.3 to about 3 mg per subject.

[0025] In some embodiments, the administering step is accomplished by intravenous, intramuscular, intraperitoneal, or intradermal injection following electroporation of transfected cells.

[0026] In some embodiments, the pharmaceutical composition comprises an expressible nucleic acid sequence comprising Formula I: [(AEDn)-(linker)]n-[AEDn+1], wherein AED is an independently selected antigen expression domain, wherein AEDn is a first antigen expression domain and wherein AEDn+1 is a second antigen expression domain; wherein the length of each linker is independently selectable to be from about 0 to about 300 natural or non-natural nucleic acids, wherein the length of antigen expression domain 1 is independently selectable to be from about 12 to about 15,000 nucleotides and encodes a new antigenic epitope; wherein the length of antigen expression domain 2 is independently selectable to be from about 12 to about 15,000 nucleotides and encodes a new antigenic epitope; and wherein n is any positive integer from about 19 to about 500.

[0027] Also provided is a method for preventing resistance to checkpoint inhibitor therapy in a subject, the method comprising administering to a subject in need thereof: (i) a pharmaceutical composition as disclosed herein; (ii) a pharmaceutical composition comprising a nucleic acid sequence encoding about twenty or more neoantigens or epitopes specific to neoantigens.

[0028] In some embodiments, the composition comprises a plasmid comprising an expressible nucleic acid sequence encoding about 40 to about 60 tumor-specific antigens.

[0029] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of pGX0001 comprising a nucleic acid sequence encoding about 1 to about 7 tumor-specific antigens selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

[0030] In some embodiments, the subject has a cancer characterized by a high tumor burden.

[0031] In some embodiments, the subject has a cancer characterized by a mutation in one or more of WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

[0032] In some embodiments, the subject has a cancer characterized by one or a combination of aberrant regulation of expression of WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

[0033] In some embodiments, the subject has a cancer characterized by overamplification of one or a combination of amino acid sequences comprising at least 70% sequence identity to WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

[0034] In some embodiments, the dosage of the pharmaceutical composition is about 0.3 to about 3 mg per subject.

[0035] In some embodiments, the administering step is accomplished by intravenous, intramuscular, intraperitoneal, or intradermal injection following electroporation of transfected cells.

[0036] In some embodiments, the subject has a cancer characterized by dysfunction of the WNT pathway. In some embodiments, the subject has hepatocellular carcinoma.

[0037] Also provided is a method of inducing an immune response in a cell, the method comprising exposing the cell to one or more compositions disclosed herein. In some embodiments, the exposing step is performed in vivo.

[0038] Also provided is a method of enhancing a CD8+ T cell response in a subject, the method comprising administering to the subject a pharmaceutical composition disclosed herein. In some embodiments, the CD8+ T cell response is enhanced by about 10% to about 15% compared to the CD8+ T cell response of a subject not treated with the pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A is a diagram depicting elements of the pGX0001 plasmid, including the multiple cloning site. Figure 1B is a diagram depicting the elements of the pGX6001 plasmid containing the IL-12α and IL-12β subunits.

[0040] Figure 2 Activation of β-catenin is depicted.

[0041] Figure 3 is a diagram depicting the involvement of the immune system in hepatocellular carcinoma. The diagram shows that mutations in the WNT pathway lead to primary resistance to checkpoint inhibitors.

[0042] Figure 4 (Top) Depicts progression-free survival of HCC patients with intact WNT pathways and HCC patients with activated WNT pathways when treated with checkpoint inhibitors or sorafenib. WNT activation leads to resistance to checkpoint inhibitors. (Harding et al., Clin Cancer Res. 2019) Figure 4 (Bottom) is a dot plot showing progression-free survival or overall survival of patients treated with TKI or immunotherapy. Wnt activation can lead to resistance to immunotherapy.

[0043] Figure 5is a line graph plotting progression-free survival of HCC patients with WNT unchanged pathway and HCC patients with WNT activated pathway when treated with GNOS-PV02 and pembrolizumab. HCC patients with WNT activated pathway are not resistant to anti-PD1 therapy when treated in combination with GNS-PV02.

[0044] Fig. 6A The percentage change in lesion size over time for HCC patients with WNT unchanged pathways and HCC patients with WNT activated pathways is depicted. Figure 6B and Figure 6C Cancers with WNT activation (50%; Figure 6B ) and WNT-unaltered cancers (15.4%; Figure 6C ) in HCC patients treated with GNOS PV-02 in combination with anti-PD1 therapy over time and the overall response rate (ORR).

[0045] Figure 7 Table showing specific mutations in the WNT pathway genes CTNNB1 and AXIN1 found in 6 HCC patients treated with a combination of GNOS-PV02 and anti-PD1 and responses (PR, partial response) (SD, stable disease) (PD, progressive disease).

[0046] FIG. 8 is a graph showing the number of new antigens in HCC patients with activated WNT pathway or HCC patients with unchanged WNT pathway ( Fig. 8A ) or tumor mutation burden (TMB) ( Figure 8B ). WNT-activated tumors had a greater number of neoantigens and higher TMB compared with tumors with unchanged WNT.

[0047] Fig. 9 Gene expression (measured per million RNA transcripts) of AKR1C2, ABCC2, ALDH1L1, ALD3A2, GCLC, and GCLM is depicted. HCC patients with β-catenin and Axin1 mutations overexpress AKR1C2, ABCC2, ALDH1L1, ALD3A2, GCLC, and GCLM.

[0048] Fig.10 Depicted are the percentages of cancers (y-axis) with alterations (mutations, structural variants, amplifications, or deep deletions) of CTNNB1 in various tumor types (x-axis). Data from the CBIOportal database.

[0049] Fig.11The mutation counts of HCC patients with altered CTNNB1 or HCC patients with unaltered CTNNB1 are depicted ( Fig.11 A) or TMB( Fig.11 B).

[0050] Fig.12 Depicted are the effects of sorafenib treatment ( Fig.12 A) or immunotherapy ( Fig.12 B) Progression-free survival (PFS) after CTNNB1 mutation. HCC with CTNNB1 mutation responds to TKI therapy but not to CPI.

[0051] Fig.13 Depicted are the effects of sorafenib treatment ( Fig.13 A) or immunotherapy ( Fig.13 B) Overall survival rate after .

[0052] Fig.14 Depicted are the percentages of cancers (y-axis) with alterations (mutations, structural variants, amplifications, or deep deletions) of AXIN1 in various tumor types (x-axis). Data from the CBIOportal database.

[0053] Fig.15 Depicted are the percentages of cancers (y-axis) with alterations (mutations, structural variants, amplifications, or deep deletions) of AXIN2 in various tumor types (x-axis). Data from the CBIOportal database.

[0054] Fig.16 Depicted are the percentage of cancers (y-axis) with alterations (mutations, structural variants, amplifications, or deep deletions) of APC in various tumor types (x-axis). Data from the CBIOportal database.

[0055] Fig.17 Depicted are the percentage of cancers (y-axis) with alterations (mutations, structural variants, amplifications, or deep deletions) of CSNK1A1 in various tumor types (x-axis). Data from the CBIOportal database.

[0056] Fig.18 Depicted are the percentages of cancers (y-axis) with alterations (mutations, structural variants, amplifications, or deep deletions) of GSK3B in various tumor types (x-axis). Data from the CBIOportal database.

[0057] Fig.19is a bar graph showing Elispot responses at screening, week 3, week 6, and week 9 after administration of the neoantigen vaccine in patient PT1.

[0058] Fig. 20 is a bar graph showing Elispot responses at screening, week 3, week 6, week 9, and week 12 after administration of the neoantigen vaccine in patient PT6.

[0059] Fig.21 is a bar graph showing Elispot responses at screening, week 3, week 6, week 9, and week 12 after administration of the neoantigen vaccine in patient PT2.

[0060] Fig. 22 is a bar graph showing Elispot responses at screening, week 3, week 6, and week 9 after administration of the neoantigen vaccine in patient PT4.

[0061] Fig.23 is a flow cytometry plot showing a robust CD8 T cell response in patient PT6 (measured by CD69 and Ki67).

[0062] Fig.24 is a flow cytometry plot showing a robust CD4 T cell response in patient PT6 (measured by CD69 and Ki67).

[0063] Fig.25A is a dot plot showing overall response (Y-axis) versus number of neoantigens (X-axis) in 19 patients with advanced HCC treated with personalized cancer vaccine (PCV) + pIL12 + pembrolizumab in the second-line setting. Fig.25B Depicted digitally Fig.25A Graph of data from . Tumor size was significantly reduced after administration of PCVs encoding at least 20 neoantigens compared to plasmids encoding fewer than 20 neoantigens.

[0064] Fig.26A is a graph showing the overall change (%) in lesion size over time from baseline in 7 patients with advanced HCC treated with PCV (<20 neoantigens) + pIL12++pembrolizumab in the second-line setting. Fig.26B is a graph showing the overall change (%) in lesion size over time from baseline in 12 patients with advanced HCC treated with PCV (<20 neoantigens) + pIL12++pembrolizumab in the second-line setting.

[0065] Fig.27A A non-limiting example of a manufacturing process for a personalized DNA vaccine. Targeted treatment can be achieved in as little as 6 weeks and can be achieved regularly within 6-8 weeks. Fig.27BNon-limiting example designed for clinical trials.

[0066] Fig.28A A spider plot is shown which displays the first 12 patients in the clinical trial at the time of data cutoff. Fig.28B The figure is a waterfall chart showing the best overall response achieved by the first 12 subjects in the clinical trial at the time of the data cut-off. The best overall response is shown as a partial response rate of 25% and a disease control rate of 67%. Fig.28C Tumor imaging scans of patients classified as PR (day 0 and week 27 after treatment). Red arrows point to the tumor.

[0067] Fig.29A Shown is a bar graph of newly detected and expanded T cell clones following treatment with GNOS-PV02 for all patients analyzed to date (n=10). Fig.29B The cumulative frequency of expanded clones in peripheral blood (PBMC, left) and tumor tissue (right) before and after vaccination (week 9) for each patient is shown. Fig.29C The expansion of pre-vaccination clones (points along the X-axis) and the detection of multiple new T cell clones (points along the Y-axis) after vaccination in the blood and tumor tissue from subject Pt7 are shown. Arrows highlight that 9 weeks after vaccination, high-frequency clones infiltrated the tumor from the blood (only the top 6 clones are shown for clarity). As assessed by TCRβ and RNA sequencing, the most abundant clones showed an active phenotype (CD8+CD69+). Before vaccination, approximately 75% of the new TIL clones were undetectable in the blood.

[0068] Fig. 30A Shown is the genetic optimization of patient-specific cloned TCR sequences using the GOAL algorithm and insertion into the pMXs-IRES-GFP retroviral plasmid vector containing viral packaging signals, transcription and processing elements, and a GFP reporter gene. Fig. 30B An example of anti-tumor specific T cell reactivity after vaccination assessed by ELISpot is shown (subject PT 8). PBMCs were stimulated using pools of individual peptides or single peptides encoded in the personalized GNOS-PV02 treatment. Fig. 30C Representative images of activated, GFP-positive, CD8 and CD4 TCR-engineered T cells (subject PT8) stimulated with ATP1A1-ALB (10 ug / mL) are shown. TNTC, too numerous to count; EOT, end of treatment. DETAILED DESCRIPTION

[0069] The present disclosure relates to methods for treating cancer by using cancer vaccines. In some embodiments, the cancer is characterized by a dysfunction of the WNT phenotype. In some embodiments, the cancer is characterized by a dysfunction of WNT signaling. In some embodiments, the dysfunction of WNT signaling is caused by 1) cells expressing an axin instability regulator and / or TNKS pathway dysfunction. In some embodiments, the cancer is characterized by one or more mutations in one or more WNT pathway signaling molecules. In some embodiments, the cancer is characterized by one or more mutations in WNT, CTNNB1, Axin1, Axin2, APC, CK1, and / or GSK3B. Examples of the methods of the present disclosure are described in detail in the Examples and Figures section of the present disclosure.

[0070] Various terms related to the methods and other aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms should have their ordinary meanings in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0071] As used herein, when referring to a measurable value (such as an amount,

[0072] When used in conjunction with a method (e.g., a method for measuring a sample, a time duration, etc.), the term "about" is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value, as such variations are suitable for performing the disclosed methods.

[0073] As used in the specification and claims, the indefinite article "a / an"

[0074] Unless explicitly stated to the contrary, it should be understood to mean "at least one".

[0075] As used in the specification and claims, the phrase "and / or" should be understood as

[0076] means "either or both" of the elements so joined, i.e.

[0077] Elements that exist jointly in some cases and separately in other cases.

[0078] In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified, unless expressly stated to the contrary. Thus, as a non-limiting example, in one embodiment, when used in conjunction with open language such as "comprising," reference to "A and / or B" may refer to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0079] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when the items in the list are separated, "or" or "and / or" should be interpreted as inclusive, that is, including at least one element in multiple elements or element lists, but also including more than one element, and optionally other unlisted items. Only the terms clearly indicated on the contrary, such as "only one of..." or "exactly one of..." or "consisting of..." when used in the claims will refer to including exactly one element in multiple elements or element lists. In general, when there is an exclusive term before, "any one", "one of...", "only one of..." or "exactly one of...", the term "or" as used herein should only be interpreted as indicating exclusive alternatives (that is, "one or the other, not both"). When used in the claims, "essentially consisting of..." should have the common meaning as used in the field of patent law.

[0080] As used herein, the terms "activate," "stimulate," "enhance," "increase," and / or "induce" (and similar terms) are used interchangeably and generally refer to the act of improving or increasing a concentration, level, function, activity, or property, directly or indirectly, relative to a natural, expected, or average value, or relative to a control condition. "Activation" refers to the primary response induced by the engagement of a cell surface moiety. For example, in the context of a receptor, such stimulation requires engagement of the receptor and subsequent signal transduction events. In addition, the stimulation event can activate the cell and up-regulate or down-regulate the expression or secretion of a molecule. Thus, even in the absence of a direct signal transduction event, engagement of the cell surface moieties may result in the reorganization of the cytoskeletal structure, or in the case of aggregation of the cell surface moieties, each moiety is used to enhance, modify, or alter subsequent cellular responses.

[0081] As used herein, the term "activated CD8+T cells" or "CD8+T cell activation" refers to a process (e.g., signaling event) that causes or leads to one or more cellular responses of CD8+T cells (CTLs), which are selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. As used herein, "activated CD8+T cells" refer to CD8+T cells that have received activation signals and therefore exhibit one or more cellular responses selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring CD8+T cell activation are known in the art and are described herein.

[0082] As used herein, the term "adjuvant" means any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigens encoded by the DNA plasmids described below and encoding the nucleic acid sequences.

[0083] As used herein, "antigen" means any substance that will elicit an immune response when exposed to an antigen presenting cell or other immune cell capable of activation.

[0084] As used herein, the term "anti-tumor response" refers to an immune system response, including but not limited to activation of T cells to attack antigens or antigen presenting cells.

[0085] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition characterized by unregulated cell growth in a population of cells in mammals. Thus, the term "cancer" refers to a group of diseases involving abnormal cell growth that has the potential to invade or spread to other parts of the body. Examples of cancer include, but are not limited to, lung cancer, bone cancer, blood cancer, chronic myelomonocytic leukemia (CMML), bile duct cancer, cervical cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, eye cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, testicular cancer, gynecological tumors (e.g., uterine sarcoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, or vulvar cancer), Hodgkin's disease (Hodgkin's disease), esophageal cancer, small bowel cancer, cancer of the endocrine system (e.g., thyroid cancer, parathyroid cancer, or adrenal cancer), soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer (e.g., renal cell carcinoma, renal pelvis cancer), or central nervous system tumors (e.g., primary CNS lymphoma, spinal tumor, brain stem glioma, or pituitary adenoma).

[0086] Specific examples of cancer include, but are not limited to, acute lymphocytic leukemia in adults; acute lymphocytic leukemia in children; acute myeloid leukemia in adults; adrenocortical carcinoma; adrenocortical carcinoma in children; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma in children; cerebral astrocytoma in children; extrahepatic bile duct cancer; bladder cancer; bladder cancer in children; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma in children; brain tumors in adults; brain tumors in children, brain stem glioma; brain tumors in children, cerebellar astrocytoma; brain tumors in children, cerebral astrocytoma / malignant glioma; brain tumors in children, ependymoma; brain tumors in children, medulloblastoma; brain tumors in children, supratentorial primitive neuroectodermal tumors; brain tumors in children, visual pathway and hypothalamic gliomas; brain tumors in children (other); breast cancer; breast cancer and pregnancy; breast cancer in children; breast cancer in men; bronchial adenoma / carcinoid in children: carcinoid tumors in children; carcinoid tumors in the gastrointestinal tract ; Adrenocortical carcinoma; Islet cell carcinoma; Unknown primary cancer; Primary central nervous system lymphoma; Cerebellar astrocytoma, childhood; Brain astrocytoma / glioblastoma, childhood; Cervical cancer; Cancer, childhood; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorder; Clear cell sarcoma of the tenosynovium; Colon cancer; Colorectal cancer, childhood; Cutaneous T-cell lymphoma; Endometrial cancer; Ependymoma, childhood; Epithelial ovarian cancer; Esophageal cancer; Esophageal cancer, childhood; Ewing's family of tumors; Extracranial germ cell tumor, childhood; Extragonadal germ cell tumor; Extrahepatic bile duct cancer; Eye cancer, intraocular melanoma; Eye cancer, retinoblastoma; Gallbladder cancer; Gastric (Gastric / Stomach) cancer; Gastric (Gastric / Stomach) cancer, childhood; Gastrointestinal carcinoid tumors; Extracranial germ cell tumor, childhood; Extragonadal germ cell tumor; Ovarian germ cell tumor; Gestational trophoblastic tumor; Glioma. Brainstem gliomas, children; Gliomas, visual pathways and hypothalamus, children; Hairy cell leukemia; Head and neck cancer; Hepatocellular (liver) cancer (primary), adults; Hepatocellular (liver) cancer (primary), children; Hodgkin's lymphoma, adults; Hodgkin's lymphoma, children; Hodgkin's lymphoma during pregnancy; Hypopharyngeal cancer; Gliomas, hypothalamus and visual pathways, children; Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma Sarcoma); Kidney cancer; Laryngeal cancer; Laryngeal cancer in children; Acute lymphocytic leukemia in adults; Acute lymphocytic leukemia in children; Acute myeloid leukemia in adults; Acute myeloid leukemia in children; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cancer; Liver cancer (primary) in adults; Liver cancer (primary) in children; Non-small cell lung cancer; Small cell lung cancer; Acute lymphocytic leukemia in adults; Acute lymphocytic leukemia in children; Chronic lymphocytic leukemia; AIDS-related lymphoma; Central nervous system lymphoma (primary); Cutaneous T-cell lymphoma;Hodgkin's lymphoma in adults; Hodgkin's lymphoma in children; Hodgkin's lymphoma in pregnancy; Non-Hodgkin's lymphoma in adults; Non-Hodgkin's lymphoma in children; Non-Hodgkin's lymphoma in pregnancy; Primary central nervous system lymphoma; Waldenstrom's macroglobulinemia; Male breast cancer; Malignant mesothelioma in adults; Malignant mesothelioma in children; Malignant thymoma; Medulloblastoma in children; Melanoma; Intraocular melanoma; Merkel cell carcinoma Carcinoma); malignant mesothelioma; occult primary metastatic squamous neck cancer; multiple endocrine neoplasms syndrome, childhood; multiple myeloma / plasma cell neoplasms; mycosis fungoides; myelodysplastic syndrome; chronic myeloid leukemia; acute myeloid leukemia, childhood; acute myeloid leukemia, childhood; multiple myeloma; chronic myeloproliferative disorders; nasal cavity and paranasal sinus cancer; nasopharyngeal carcinoma; nasopharyngeal carcinoma, childhood; neuroblastoma; neurofibroma; non-Hodgkin's lymphoma, adult; non-Hodgkin's lymphoma, childhood; non-Hodgkin's lymphoma, pregnancy; non-small cell lung cancer; oral cancer, childhood; oral cavity and lip cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer, childhood; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; pancreatic cancer, childhood, islet cell Pancreatic cancer; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pheochromocytoma; Pineal and supratentorial primitive neuroectodermal tumors in children; Pituitary tumors; Plasma cell neoplasms / multiple myeloma; Pleuropulmonary blastoma; Pregnancy and breast cancer; Pregnancy and Hodgkin's lymphoma; Pregnancy and non-Hodgkin's lymphoma; Primary central nervous system lymphoma; Primary liver cancer in adults; Primary liver cancer in children; Prostate cancer; Rectal cancer; Renal cell (kidney) cancer; Renal cell cancer in children; Transitional cell carcinoma of the renal pelvis and ureter; Retinoblastoma; Rhabdomyosarcoma in children; Salivary gland cancer; Salivary gland cancer in children; Sarcoma, Ewing family of tumors; Kaposi's sarcoma; Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; Sarcoma, rhabdomyosarcoma in children; Soft tissue sarcoma in adults; Soft tissue sarcoma in children; Sezary syndrome Syndrome; Skin Cancer; Skin Cancer in Children; Skin Cancer (Melanoma); Merkel Cell Skin Cancer; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcomas in Adults; Soft Tissue Sarcomas in Children; Occult Primary Metastatic Squamous Neck Cancer; Stomach (Stomach / Gastric) Cancer; Stomach (Stomach / Gastric) Cancer in Children; Supratentorial Primitive Neuroectodermal Tumors in Children; Cutaneous T-Cell Lymphoma; Testicular Cancer; Thymoma in Children; Malignant Thymoma; Thyroid Cancer; Thyroid Cancer in Children; Transitional Cell Carcinoma of the Renal Pelvis and Ureter; Gestational Trophoblastic Tumor; Cancer of Unknown Primary Site in Children; Unusual Cancers in Children; Transitional Cell Carcinoma of the Ureter and Renal Pelvis; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma in Children; Vulvar Cancer; Waldenstrom's Macroglobulinemia; and Wilms' Tumor.

[0087] As used herein, the term "check point inhibitor" means any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide or fragment thereof that inhibits inhibitory pathways and allows for a wider range of immune activity. In some embodiments, the check point inhibitor is an inhibitor of programmed death 1 (PD-1) pathways, such as anti-PD1 antibodies, such as but not limited to nivolumab. In other embodiments, the check point inhibitor is an antibody that binds or associates with cytotoxic T lymphocyte-associated antigen (CTLA-4). In other additional embodiments, the check point inhibitor targets members of the TNF superfamily, such as CD40, OX40, CD 137, GITR, CD27 or TIM-3. In some embodiments, the targeted check point inhibitor is accomplished by inhibitory antibodies or similar molecules. In other cases, it is accomplished by an agonist of the target; examples of this category include stimulatory targets OX40 and GITR. In some embodiments, the check point inhibitor means any one or combination of the check point inhibitors from Table 1.

[0088] Table 1 – List of checkpoint inhibitors

[0089]

[0090]

[0091] As used herein, the term "combination therapy" means administering two or more therapeutic agents in a sequential manner, i.e., wherein each therapeutic agent is administered at a different time, as well as administering two or more therapeutic agents simultaneously or substantially simultaneously. In some embodiments, substantially simultaneously refers to administering the second agent within 120, 90, 60 minutes or less after administering the first agent.

[0092] As used herein, "electroporation," "electroosmosis," or "electrokinetic enhancement" ("EP") are used interchangeably and refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; their presence permits the transfer of biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and / or water from one side of the cell membrane to the other.

[0093] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, such as, but not limited to, a truncation mutant. This portion preferably contains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the full length of a reference nucleic acid molecule or polypeptide. A fragment may contain about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 or more nucleotides or amino acids of a nucleotide or amino acid sequence, respectively.

[0094] The term "functional fragment" means any part or fragment of a polypeptide or nucleic acid sequence associated with a corresponding full-length polypeptide or nucleic acid, which part or fragment has sufficient length and sufficient structure to confer a biological effect at least similar or substantially similar to the full-length polypeptide or nucleic acid on which the fragment is based. In some embodiments, a functional fragment is a portion of a full-length or wild-type nucleic acid sequence encoding any of the nucleic acid sequences disclosed herein, and the portion encodes a polypeptide of a certain length and / or structure, which is less than the full length, but encodes a domain that still has a biological function compared to the full-length or wild-type protein. In some embodiments, compared to the wild-type or full-length polypeptide sequence on which the fragment is based, the functional fragment may have reduced biological activity, approximately equivalent biological activity, or enhanced biological activity. In some embodiments, the functional fragment is derived from an organism, such as a human sequence. In such embodiments, the functional fragment may retain about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, or about 90% sequence identity with the wild-type or given sequence from which the sequence is derived. In some embodiments, a functional fragment may retain about 85%, about 80%, about 75%, about 70%, about 65%, or about 60% sequence homology to the wild-type sequence from which it was derived.

[0095] As used herein, the term "gene construct" means a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence comprises a start signal and a termination signal operably linked to regulatory elements, which comprise a promoter and a polyadenylation signal capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered.

[0096] As used herein, the term "host cell" means a cell that can be used to express a nucleic acid (such as the nucleic acid of the present disclosure). The host cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. The phrase "recombinant host cell" can be used to represent a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains nucleic acid but does not express it at a desired level, unless a regulatory sequence is introduced into the host cell so that it is operably connected to the nucleic acid. It should be understood that the term host cell refers not only to a specific subject cell, but also to the progeny or potential progeny of this cell. Because some modifications may occur in subsequent generations due to, for example, mutations or environmental influences, such progeny may not actually be identical to the parental cell, but are still included in the scope of the term as used herein.

[0097] As used herein, the term "hybridization" means the formation of double-stranded molecules between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringent conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0098] As used herein, the term "immune checkpoint" refers to inhibitory pathways that slow or stop the immune response and prevent the uncontrolled activity of immune cells from causing excessive tissue damage.

[0099] As used herein, the term "immune response" means that the host's immune system (eg, a mammal's immune system) is activated in response to the introduction of a nucleic acid molecule comprising a nucleotide sequence encoding a neoantigen as described herein.

[0100] As used herein, the term "isolated" means that a polynucleotide or polypeptide, or a fragment, variant or derivative thereof, has been substantially removed from other biological material with which it is naturally associated, or is substantially free of other biological material, for example, derived from a recombinant host cell that has been genetically engineered to express a polypeptide of the disclosure.

[0101] The term "isolated" means that for the purposes of this disclosure, a nucleic acid may not be a listed species. In other words, a nucleic acid may incorporate a combination of the above mutations and one or more other mutations, listed or not, but the nucleic acid may not be defined as a single species containing the listed nucleic acid mutations.

[0102] As used herein, the term "ligand" means a molecule having a structure complementary to that of a receptor and capable of forming a complex with the receptor. A ligand is understood to mean a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence so that the peptide or peptide fragment can form a complex with a protein of MHC I or MHC II class.

[0103] As used herein, the term "MHC molecule", "MHC protein" or "HLA protein" means a protein that is capable of binding peptides produced by proteolytic cleavage of protein antigens and represents potential T cell epitopes, transporting them to the cell surface and presenting them to specific cells, in particular cytotoxic T lymphocytes or T helper cells. The major histocompatibility complex in the genome includes genetic regions whose gene products expressed on the cell surface are important for binding and presenting endogenous and / or exogenous antigens and therefore for regulating immune processes. The major histocompatibility complex is divided into two gene sets encoding different proteins, namely MHC class I molecules and MHC class II molecules. The molecules of the two MHC classes are specifically directed against different antigen sources. MHC class I molecules present endogenously synthesized antigens, such as viral proteins and tumor antigens. MHC class II molecules present protein antigens derived from exogenous sources, such as bacterial products. The cell biology and expression patterns of the two MHC classes are adapted to these different roles.

[0104] In some embodiments, the class I MHC molecule comprises a heavy chain and a light chain and is capable of binding a peptide of about 8 to 11 amino acids, but typically 9 or 10 amino acids (if the peptide has a suitable binding motif), and presenting it to a cytotoxic T lymphocyte. The peptide bound by the class I MHC molecule is derived from an endogenous protein antigen. The heavy chain of the class I MHC molecule is preferably an HLA-A, HLA-B or HLA-C monomer, and the light chain is beta-2-microglobulin.

[0105] In some embodiments, the class II MHC molecule comprises an α chain and a β chain and is capable of binding a peptide of about 15 to 24 amino acids (if the peptide has a suitable binding motif) and presenting it to a T helper cell. The peptide bound by the class II MHC molecule is usually derived from an exogenous protein antigen outside the cell. The α-chain and β-chain are particularly HLA-DR, HLA-DQ and HLA-DP monomers.

[0106] As used herein, the term "neoantigen" refers to a class of tumor antigens that are generated by tumor-specific mutations in a subject's expressed protein. In some embodiments, the neoantigen is directly derived from a subject's tumor. This is in contrast to known tumor-associated antigens, which can be consensus sequences that are known to elicit an immune response against cells that express the tumor antigen but are not necessarily derived from a subject's tumor.

[0107] As used herein, the term "neoantigenic mutation" refers to a mutation predicted to encode a neoantigenic peptide.

[0108] As used herein, the term "pharmaceutically acceptable" means approved or approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0109] As used herein, the term "pharmaceutically acceptable excipient, carrier or diluent" means an excipient, carrier or diluent that can be administered to a subject together with a pharmaceutical agent and does not destroy its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the pharmaceutical agent.

[0110] As used herein, the term "pharmaceutically acceptable salt" of tumor-specific neoantigens may be an acid or base salt generally considered in the art to be suitable for contact with human or animal tissues without excessive toxicity, irritation, allergic response or other problems or complications. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali metal or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutically acceptable salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH2)n-COOH (wherein n is from about 0 to about 4), etc. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that other pharmaceutically acceptable salts of the combined tumor-specific neoantigens are provided herein, including those listed in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, page 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from parent compounds containing a basic or acidic moiety by any conventional chemical method. In short, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in an appropriate solvent.

[0111] As used herein, the terms "prevent", "preventing", "prevention", "prophylactic treatment" and the like mean reducing the likelihood of developing a disease or disorder in a subject who does not have the disease or disorder but is at risk or susceptible to the disease or disorder.

[0112] As used herein, the term "purified" means that a polynucleotide or polypeptide, or a fragment, variant, or derivative thereof, is substantially free of other biological materials with which it is naturally associated, or free of other biological materials derived from, for example, a recombinant host cell that has been genetically engineered to express the polypeptide. That is, for example, a purified polypeptide is a polypeptide that is at least about 70% to about 100% pure, i.e., the polypeptide is present in a composition wherein the polypeptide accounts for about 70% to about 100% of the total composition by weight. In some embodiments, the purified polypeptide is about 75% to about 99% pure by weight, about 80% to about 99% pure by weight, about 90% to about 99% pure by weight, or about 95% to about 99% pure by weight.

[0113] As used herein, the terms "subject," "individual," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments, the subject is a human.

[0114] As used herein, "a patient in need thereof" or "a subject in need thereof" refers to a living organism suffering from or susceptible to a disease or condition that can be treated by administering at least one composition, vaccine, or pharmaceutical composition disclosed herein, including, for example, a vaccine comprising a nucleic acid sequence encoding a neoantigen (such as a nucleic acid sequence encoding a β-catenin antigen according to the methods described herein). A "patient in need thereof" or "a subject in need thereof" may also refer to a living organism that is receiving a neoantigen DNA vaccine (or a pharmaceutical composition comprising a neoantigen DNA vaccine) or has received a neoantigen DNA vaccine (or a pharmaceutical composition comprising a neoantigen DNA vaccine); or a living organism suffering from a tumor or cancer. Non-limiting examples include humans, other mammals, such as cattle, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In embodiments, a patient in need thereof or a subject in need thereof is a human. In some embodiments, a subject in need thereof is a human patient suspected of having cancer or who has been diagnosed with cancer and exhibits cancer.

[0115] In some embodiments, the patient or subject in need thereof suffers from a cancer characterized by WNT dysfunction or WNT pathway abnormality. In some embodiments, the patient or subject in need thereof suffers from a cancer characterized by WNT signaling dysfunction. In some embodiments, the patient or subject in need thereof suffers from a cancer characterized by cells exhibiting axilin instability and / or TNKS pathway dysfunction. In some embodiments, the patient or subject in need thereof suffers from a cancer characterized by one or more mutations in one or more WNT pathway signaling molecules. In some embodiments, the patient or subject in need thereof suffers from a cancer characterized by one or more mutations in WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and / or GSK3B.

[0116] As used herein, the term "T cell epitope" means a peptide sequence that can be bound by a class I or class II MHC molecule in the form of a peptide presenting MHC molecule or MHC complex and then recognized and bound by a cytotoxic T lymphocyte or T helper cell, respectively, in this form.

[0117] As used herein, the term "therapeutic effect" means a certain degree of relief of one or more symptoms of a disease (e.g., neoplasia or tumor) or its associated pathology. As used herein, "therapeutically effective amount" means effectively extending the survival of patients with this disease, alleviating one or more signs or symptoms of the disease, preventing or delaying the disease, etc., after being administered to cells or subjects in a single dose or multiple doses, and exceeding the expected effect when this treatment is not received. "Therapeutically effective amount" is intended to quantify the amount required for the therapeutic effect. A doctor or veterinarian with ordinary skills in the art can easily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the desired pharmaceutical composition. For example, a doctor or veterinarian can start the dosage of the compound used in the pharmaceutical composition at a level lower than the level required for achieving the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0118] In some embodiments, a therapeutically effective amount is an amount that results in the prevention or amelioration or reduction of symptoms associated with a disease or disorder associated with Wnt signaling (ie, cancer). The disclosed compounds can be administered to a subject before or after the onset of a Wnt signaling-related disorder.

[0119] In some embodiments, the widest diameter of the tumor is reduced by about 2%, about 4%, about 6%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the widest tumor diameter of the solid tumor before treatment.

[0120] In some embodiments, the size of the tumor is measured by Response Evaluation Criteria in Solid Tumors ("RECIST"), wherein the longest diameter of a solid tumor as measured by radiological imaging (such as MRI or CT) is used as a proxy for the tumor. In some embodiments, a therapeutically effective amount is an amount effective to reduce the size of a solid tumor as measured by RECIST by about 2% compared to the size of the solid tumor as measured by RECIST before treatment, to reduce the size by about 4%, by about 6%, by about 8%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, or by about 100% compared to the size of the solid tumor as measured by RECIST before treatment.

[0121] In some embodiments, treatment results in a decrease in size of greater than 2% as measured by RECIST, a decrease in size of greater than about 5%, a decrease in size of greater than about 10%, a decrease in size of greater than about 15%, a decrease in size of greater than about 20%, a decrease in size of greater than about 25%, a decrease in size of greater than about 30%, a decrease in size of greater than about 35%, a decrease in size of greater than about 40%, a decrease in size of greater than about 45%, a decrease in size of greater than about 50%, a decrease in size of greater than about 60%, a decrease in size of greater than about 70%, a decrease in size of greater than about 90%, a decrease in size of greater than about 90%, or a decrease in size of about 100%, as compared to the size of the solid tumor as measured by RECIST before treatment. A decrease in size of 100% is designated as a complete clinical response (CR).

[0122] In some embodiments, the therapeutically effective amount is an amount effective to reduce the total mass of a solid tumor by about 2%, compared to its mass or estimated mass before treatment, by about 4%, by about 6%, by about 8%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, or by about 50%, compared to the total mass of the solid tumor before treatment.

[0123] As used herein, the terms "treat / treated / treating / treatment" and the like mean to alleviate or improve a condition and / or symptoms associated therewith (e.g., cancer or a tumor). "Treatment" may refer to administering the neoantigen vaccine described herein to a subject after an onset or suspected onset of cancer. "Treatment" includes the concept of "alleviation", which refers to reducing the frequency or severity of the occurrence or recurrence of any symptoms or other adverse effects associated with cancer and / or side effects associated with cancer therapy. The term "treatment" also encompasses the concept of "management", which refers to reducing the severity of a patient's particular disease or condition or delaying its recurrence, such as prolonging the remission period of a patient suffering from the condition. It should be understood that, although not excluded, the treatment of a condition or illness does not require the complete elimination of the condition, condition or symptom associated therewith.

[0124] As used herein, the term "treating cancer" is not intended to be an absolute term. In some aspects, the compositions and methods of the present disclosure seek to reduce the size of a tumor or the number of cancer cells, put a cancer into remission, or prevent the growth of the size or number of cancer cells in a subject in need of treatment. In some cases, treatment with the disclosed compositions results in an improved prognosis and / or an extended life expectancy.

[0125] The terms "prevent" or "preventing" mean to arrest the onset or recurrence of a disorder or one or more symptoms associated with a disorder.

[0126] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined by preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined based on human data. The dosage used can be adjusted according to the relative bioavailability and effectiveness of the administered medicament. It is entirely within the ability of ordinary technicians to adjust the dosage based on the above method and other well-known methods to achieve maximum efficacy. The general principles for determining the therapeutic effect (can be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference) are summarized as follows.

[0127] Pharmacokinetic principles provide the basis for modifying dosage regimens to achieve the desired degree of therapeutic efficacy while minimizing unacceptable side effects. Additional guidance for dosage modifications is available when plasma concentrations of a drug can be measured and correlated with the therapeutic window.

[0128] Drug products are considered pharmaceutically equivalent if they contain the same active ingredient and are the same in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent products are considered bioequivalent when there is no significant difference in the bioavailability and extent of the active ingredient in the two products under appropriate testing conditions.

[0129] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably throughout the text and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs) and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In some embodiments, the nucleic acid molecules of the present disclosure comprise adjacent open reading frames encoding antibodies or fragments thereof as described herein. As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions. Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA (both genomic and cDNA), RNA, or a hybrid, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[0130] Nucleic acids generally contain phosphodiester bonds, although in some embodiments may include nucleic acid analogs that may have at least one different bonding (e.g., phosphoramidate, phosphorothioate, phosphorodithioate or O-methyl phosphoramidate bonding and peptide nucleic acid backbones and bonding). Other similar nucleic acids include those with positive backbones, nonionic backbones, and non-ribose backbones, including those described in the following: U.S. Patent Nos. 5,235,033 and 5,034,506, which are incorporated by reference in their entirety. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included in a definition of nucleic acids. Modified nucleotide analogs may be located at, for example, the 5'-end and / or 3'-end of a nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar-modified or backbone-modified ribonucleotides. However, it should be noted that nucleobase-modified ribonucleotides, i.e. containing non-naturally occurring nucleobases instead of naturally occurring nucleobases, such as uridine or cytidine modified at the 5-position, e.g. 5-(2-amino)propyluridine, 5-bromouridine; adenosine and guanosine modified at the 8-position, e.g. 8-bromoguanosine; deazanucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6-methyladenosine are also suitable. The 2'-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH.sub.2, NHR, N.sub.2 or CN, wherein R is C.sub.1-C.sub.6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modified nucleotides also include nucleotides conjugated to cholesterol by, for example, hydroxyprolinol linkages, as described in Krutzfeldt et al., Nature (October 30, 2005), Soutschek et al., Nature 432: 173-178 (2004), and U.S. Patent Publication No. 20050107325, which are incorporated herein by reference in their entirety. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, which is incorporated herein by reference in its entirety. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No. 20050182005, which is incorporated herein by reference in its entirety. Modifications to the ribose-phosphate backbone may be performed for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs, as well as mixtures of naturally occurring nucleic acids and analogs can be prepared. In some embodiments, the nucleotide sequence encoding one or more antigens does not contain modified nucleotide analogs. In some embodiments, the nucleotide sequence encoding one or more antigens comprises about 1 to about 20 nucleic acid modifications. In some embodiments, the nucleotide sequence encoding one or more antigens comprises about 1 to about 50 nucleic acid modifications.In some embodiments, the nucleotide sequences encoding one or more antigens independently comprise about 1 to about 100 nucleic acid modifications.

[0131] As used herein, the term "nucleic acid molecule" comprises one or more nucleotide sequences encoding one or more proteins. In some embodiments, the nucleic acid molecule comprises a start signal and a stop signal operably linked to regulatory elements, which comprise a promoter and a polyadenylation signal capable of direct expression in the cells of an individual to whom the nucleic acid molecule is administered. In some embodiments, the nucleic acid molecule is also a plasmid comprising one or more nucleotide sequences encoding one or more new antigens. In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a first, second, third or more nucleic acid molecules, wherein each nucleic acid molecule encodes one or more new antigens and at least one of each plasmid comprises one or more of the formulas disclosed herein.

[0132] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may include modified amino acids, and it may be interrupted by non-natural amino acids or chemical groups other than amino acids. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and both the D or I optical isomers, as well as amino acid analogs and peptide mimetics.

[0133] As used herein, "conservative" amino acid substitutions may be defined as listed in Tables A, B or C below. Vaccines, compositions, pharmaceutical compositions and methods may include nucleic acid sequences containing one or more conservative substitutions. In some embodiments, vaccines, compositions, pharmaceutical compositions and methods include nucleic acid sequences that retain about 70% sequence identity to about 99% sequence identity with the sequence identifiers disclosed herein but contain one or more conservative substitutions. Conservative substitutions disclosed herein include those substitutions in which conservative substitutions (from nucleic acids or amino acid sequences) have been introduced by modification of polynucleotides encoding polypeptides. Amino acids can be classified according to physical properties and contributions to secondary and tertiary protein structures. Conservative substitutions are considered in the art to be substitutions of one amino acid for another amino acid with similar properties. In some embodiments, conservative substitutions are considered in the art to be substitutions of another nucleic acid with a nucleic acid having similar properties or having similar binding affinity to its target when encoded. In some embodiments, the target is a cell expressing β-catenin. Exemplary conservative substitutions are listed in Table A.

[0134] Table A - Conservative Substitutions I

[0135]

[0136] Alternatively, conservative amino acids may be described as in Lehninger (Biochemistry, 2nd ed.; Worth Publishers,

[0137] Inc. NY, NY (1975), pp. 71-77) and are grouped as listed in Table B.

[0138] Table B - Conservative Substitutions II

[0139]

[0140] Alternatively, exemplary conservative substitutions are listed in Table C.

[0141] Table C - Conservative Substitutions III Original Residues

[0142] Exemplary substitutions

[0143]

[0144] It is understood that the inhibitors described herein are intended to include nucleic acids, and wherein the inhibitors include polypeptides, polypeptides having one or more insertions, deletions or substitutions of amino acid residues or any combination thereof, and modified polypeptides other than insertions, deletions or substitutions of amino acid residues.

[0145] As used herein, "more than one" or "two or more" in the aforementioned amino acid substitutions means 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the listed amino acid or nucleic acid substitutions. In some embodiments, "more than one" means 2, 3, 4 or 5 of the listed amino acid substitutions or nucleic acid substitutions. In some embodiments, "more than one" means 2, 3, 4 or more of the listed amino acid substitutions or nucleic acid substitutions. In some embodiments, "more than one" means 2, 3 or 4 of the listed amino acid substitutions or nucleic acid substitutions. In some embodiments, "more than one" means 2 or more of the listed amino acid substitutions or nucleic acid substitutions. In some embodiments, "more than one" means 2 of the listed amino acid substitutions or nucleic acid substitutions.

[0146] The term "percent identity" or "percent homology" of two polynucleotides or two polypeptide sequences is determined by comparing sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. As used herein in the context of two or more nucleic acid or amino acid sequences, "identical" or "identity" may mean that the sequences have the same percentage of specified residues within a specified region. The percentage can be calculated by optimizing the alignment of the two sequences, comparing the two sequences within a specified region, determining the number of positions where the same residues occur in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to derive the percentage of sequence identity. In the case where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Can be manually or use computer sequence algorithm such as BLAST or BLAST2.0 to carry out identity identification.In short, BLAST algorithm, represents local sequence alignment search basic tool, is suitable for determining sequence similarity.Software for carrying out BLAST analysis can be publicly obtained by National Center for Biotechnology Information (National Center for Biotechnology Information) (http: / / www.ncbi.nlm.nih.gov).This algorithm first distinguishes high-scoring sequence pairs (HSPs) by distinguishing shorter words in the query sequence, when compared with words of the same length in the database sequence, the short word matches or satisfies a certain positive threshold score T.T is referred to as the neighboring character score threshold (Altschul et al., 1997).These initial neighborhood word hits serve as seeds for starting searches to find HSPs containing the initial neighborhood word hits.Word hits are extended in both directions along each sequence until the cumulative comparison score can be increased. Extension of the word hits in each direction is halted if: 1) the cumulative alignment score falls by the amount X from its maximum achieved value; 2) the cumulative score falls to zero or below due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.The Blast program uses the following default values: word length (W) is 11, BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignment (B) is 50, expectation (E) is 10, M=5, N=4, and comparison of both chains. The BLAST algorithm (Karlin, Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform statistical analysis of the similarity between two sequences. A similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match occurs by chance between two nucleotide sequences. For example, a nucleic acid is considered similar to another nucleic acid if the smallest sum probability in a comparison of the test nucleic acid to the other nucleic acids is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001.

[0147] If the sequences of two single-stranded polynucleotides can be arranged in an antiparallel orientation so that each nucleotide in one polynucleotide is opposite to a complementary nucleotide in the other polynucleotide, no gaps are introduced, and there are no unpaired nucleotides at the 5' or 3' ends of either sequence, then the two single-stranded polynucleotides are "complements" of each other. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to each other under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[0148] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" means conditions under which a nucleic acid molecule will hybridize to another nucleic acid molecule but not to other sequences. Stringent conditions depend on the sequence and will be different in different situations. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequence is usually present in excess, 50% of the probes are occupied at equilibrium at Tm. Typically, stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ions, typically about 0.01 M to 1.0 M sodium ions (or other salts) at pH 7.0 to 8.3, and for short probes, primers or oligonucleotides (e.g., 10 to 50 nucleotides), the temperature is at least about 30°C, and for longer probes, primers or oligonucleotides, the temperature is at least about 60°C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0149] "Substantially identical" means that the nucleic acid molecule (or polypeptide) exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, this sequence is at least about 60%, about 80% or about 85% and about 90%, about 95% or about 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0150] If the regulatory sequence affects the expression of the nucleotide sequence (e.g., the level, timing or position of expression), the nucleotide sequence is "operably linked" to the regulatory sequence. A "regulatory sequence" is a nucleic acid that affects the expression of a nucleic acid to which it is operably linked (e.g., the level, timing or position of expression). For example, a regulatory sequence may, for example, exert its effect directly on the regulated nucleic acid, or exert its effect through one or more other molecules (e.g., a polypeptide bound to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Other examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06.

[0151] As used herein, the term "sample" generally refers to a limited amount of something that is intended to be similar to and represent a greater amount of that something. In the present disclosure, a sample is a collection, swab, brush, scrape, biopsy, removed tissue or surgical resection to test the absence, presence or grading of a tissue, which in some cases is a cancerous tissue or one or more cells. In some embodiments, the sample is taken from a patient or subject who is believed to suffer from cancer, hyperplasia, precancerous stage or comprises one or more tumor cells. In some embodiments, a sample that is believed to contain one or more malignant, cancerous or precancerous cells is compared with a "control sample" that is known not to contain one or more malignant, cancerous or precancerous cells. The present disclosure contemplates the use of any one or more samples disclosed herein to identify, detect, sequence and / or quantify the amount of new antigens (high or minimal immunogenicity) within a particular sample. In some embodiments, the method involves exposing a swab, brush or other sample from the environment to a set of reagents sufficient to separate and / or sequence the DNA and RNA of one or more cells in the sample.

[0152] The present disclosure relates to a composition comprising a vector. "Vector" is a nucleic acid molecule that can be used to introduce a nucleic acid sequence subcomponent connected thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule to which an additional nucleic acid segment can be joined. Another type of vector is a viral vector (e.g., replication-defective retrovirus, adenovirus, and adeno-associated virus), which is in the form of RNA, DNA, or hybrid RNA / DNA molecules comprising a viral genome promoter sequence operably connected to an expressible nucleotide sequence. In certain embodiments, an expressible nucleotide sequence is introduced into the viral genome. Certain vectors can be autonomously replicated in a host cell into which they are introduced (e.g., a bacterial vector comprising a bacterial replication origin and an additional mammalian vector). Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and are thus replicated together with the host genome. "Expression vector" is a type of vector that can guide the expression of a selected polynucleotide. The present disclosure relates to any one or more vectors, which comprise a nucleic acid sequence encoding any one or more amino acid sequences disclosed herein.

[0153] As used herein, the term "vaccine" means a composition used to generate immunity for the prevention and / or treatment of a disease (e.g., cancer). Thus, a vaccine is a drug that includes an antigen and is intended for use in humans or animals to generate specific defense and protective substances through vaccination. A "vaccine composition" or "neoantigen vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent.

[0154] The present disclosure also relates to one or more amino acid sequences, which include any one or combination of the antigen amino acid sequences disclosed herein. In some embodiments, the expressible nucleotide sequence of the present disclosure encodes one or more amino acid sequences in the amino acid sequence. In some embodiments, the composition of the present disclosure comprises a chimeric protein or a fusion protein, wherein an amino acid sequence that is an antigen is fused to a second amino acid sequence continuously or non-continuously. As used herein, "chimeric protein" or "fusion protein" comprises all or part (preferably biologically active) of a polypeptide or compound of the present disclosure that is operably connected to a heterologous amino acid sequence (i.e., an amino acid sequence other than the compound of the present disclosure). In the fusion protein, the term "operably connected" is intended to indicate that the polypeptide of the present disclosure and the heterologous polypeptides are fused in frame with each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of any one or more polypeptides of the present disclosure. In some embodiments, the present disclosure relates to a composition comprising an amino acid sequence, the amino acid sequence comprising a first amino acid sequence (which is an antigen) fused to a second amino acid sequence (which is a protein tag), wherein the tag is a detectable marker after exposure to a stimulus or can be combined with one or more amino acids on a solid support so that the marker is conducive to the separation of the antigen.

[0155] A useful fusion protein is a GST fusion protein, in which the polypeptide of the present disclosure is fused to the C-terminus of one or more GST sequences. This fusion protein can facilitate the purification of the recombinant polypeptide of the present disclosure.

[0156] In some embodiments, the fusion protein contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of the polypeptide of the present disclosure can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretion sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., ed., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, Calif.). In another example, useful prokaryotic heterologous signal sequences include phoA secretion signals (Sambrook et al., supra) and protein A secretion signals (Pharmacia Biotech; Piscataway, NJ).

[0157] In some embodiments, the fusion protein is an immunoglobulin fusion protein, in which all or part of the polypeptide of the present disclosure is fused with a sequence derived from a member of the immunoglobulin family. The immunoglobulin fusion protein of the present disclosure can be incorporated into a pharmaceutical composition and administered to a subject to inhibit the interaction between a ligand (soluble or membrane-bound) and a protein (receptor) on the cell surface, thereby curbing in vivo signal transduction. The immunoglobulin fusion protein can be used to affect the bioavailability of the cognate ligand of the polypeptide of the present disclosure. The inhibition of ligand / receptor interactions can be useful in treatment, both for the treatment of proliferative and differentiation disorders, and for regulating (e.g., promoting or inhibiting) cell survival. In addition, the immunoglobulin fusion protein of the present disclosure can be used as an immunogen to produce antibodies to the polypeptide of the present disclosure in a subject, purify ligands, and identify molecules that inhibit the interaction of receptors with ligands in screening assays.

[0158] Chimeric proteins and fusion proteins of the present disclosure can be produced by standard recombinant DNA techniques. In certain embodiments, fusion genes can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that produce complementary overhangs between two consecutive gene fragments, which are then annealed and reamplified to produce chimeric gene sequences (see, e.g., Ausubel et al., supra). In addition, many expression vectors encoding fusion moieties (e.g., GST polypeptides) are commercially available. Nucleic acids encoding polypeptides of the present disclosure can be cloned into this expression vector so that the fusion moieties are connected in-frame with the polypeptides of the present disclosure.

[0159] Composition

[0160] The present disclosure relates to steps for identifying multiple mutations (e.g., translocations, inversions, large and small deletions and insertions, missense mutations, splice site mutations, etc.) within a cancer / tumor. Specifically, these mutations are present in the genome of the subject's cancer / tumor cells, but not in normal tissues from the subject. The present disclosure relates to the following innovative findings: administering a pharmaceutical composition comprising a nucleic acid sequence encoding about 1 to about 100 different amino acid sequences, which represent the mutational environment in several different cancer cells, wherein at least one or about one to about five mutations are amino acid sequences from cancers with WNT pathway dysfunction. If such mutations cause changes that alter the amino acid sequence of the protein, and such changes are unique to the patient's cancer / tumor (e.g., new antigens), then these mutations are particularly interesting. In some embodiments, the present disclosure relates to treating or preventing cancer in a subject in need of treatment, wherein the subject suffers from a cancer characterized by dysfunction of the WNT pathway.

[0161] A cancer with WNT pathway dysfunction is a cancer containing at least one mutation in at least one gene of an active molecule in the WNT pathway. In some embodiments, the mutation results in at least one change in the amino acid sequence of a Wnt pathway molecule. In some embodiments, the mutation is a substitution mutation, addition or deletion of any part of a Wnt pathway molecule. In some embodiments, the mutation is any mutation that results in no expression or limited expression of a Wnt pathway molecule, thereby causing a biological phenotype change due to low expression, limited expression or defective expression. In some embodiments, the mutation results in overexpression of a Wnt pathway molecule. In some embodiments, the mutation results in abnormal regulation of the expression of a Wnt pathway molecule. Wnt pathway molecules include but are not limited to WNT, CTNNB1 (or β-catenin), AXIN1, AXIN2, APC, CK1 and GSK3B. The mutations present in cancer can be determined by any method known in the art, including but not limited to DNA sequencing and determination of the presence or quantity of detection biomarkers. Abnormal regulation of the expression of WNT pathway molecules can be determined by any method known in the art, including but not limited to quantifying RNA and / or protein expression levels. Abnormal regulation of the expression of WNT pathway molecules means that their expression levels in non-tumor cells are about 10% higher (overexpressed) or about 10% lower (underexpressed). Tumor mutation burden or TMB can be measured as disclosed in N Engl J Med (Dec 21, 2017; 377:2500-250), which is incorporated by reference in its entirety, wherein mutation burden is quantified by examining the expression profiles of biopsy tumor tissues of 27 different cancer types.

[0162] In some embodiments, cancer is characterized by a high tumor mutation burden (TMB). Tumor mutation burden is the calculated frequency of certain mutations in tumor genes. To be included in TMB, mutations must change the protein expressed by the tumor. Each of these mutations will produce such a protein, which is an antigen and can be recognized and activated by the immune system. Methods for determining a high mutation burden are known in the art, and include, for example, next-generation whole exome sequencing, which sequences all protein-coding genes in the tumor, and sequences a gene panel that provides the sequence of the targeted genome. Tumors with a high mutation burden have at least about 10 mutations per million base pairs of tumor DNA.

[0163] In some embodiments, the cancer is characterized by resistance to a checkpoint inhibitor. Resistance (poor or low response) to checkpoint inhibitor therapy means less than about 2% reduction in tumor mass, no reduction in tumor mass or tumor growth following treatment with a checkpoint inhibitor.

[0164] In some embodiments, the compositions disclosed herein are used to treat a cancer selected from the group consisting of adrenocortical cancer / adrenocortical carcinoma, bladder cancer, bladder urothelial carcinoma, breast cancer, cervical adenocarcinoma, cervical squamous cell carcinoma, bile duct cancer, colorectal adenocarcinoma, colorectal cancer, diffuse glioma, endometrial cancer / endometrial carcinoma carcinoma), esophageal squamous cell carcinoma, esophagogastric adenocarcinoma, gastroesophageal junction adenocarcinoma, glioblastoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, invasive breast cancer, leukemia, mature B-cell neoplasms, melanoma, mesothelioma, miscellaneous neuroepithelial tumors, non-seminomatous tumors, non-small cell lung cancer, ocular melanoma, ovarian cancer, ovarian epithelial tumor, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, pheochromocytoma, pleural mesothelioma, prostate adenocarcinoma, prostate cancer, renal clear cell carcinoma, renal non-clear cell carcinoma, sarcoma, seminoma, thymic carcinoma, thymic epithelial tumor, thyroid cancer, undifferentiated gastric adenocarcinoma, or well-differentiated thyroid cancer.

[0165] The present disclosure features a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I:

[0166] [(AEDn)–(connector)]n–[AEDn+1],

[0167] Wherein AED is an independently selectable antigen expression domain comprising an expressible nucleic acid sequence, wherein AEDn is referred to as an antigen expression domain and wherein AEDn+1 is referred to as an antigen expression domain 2; wherein the length of each linker is independently selectable to be from about 0 to about 300 natural or non-natural nucleic acids, wherein the length of antigen expression domain 1 is independently selectable to be from about 12 to about 15,000 nucleotides and encodes a tumor-specific epitope of a subject; wherein the length of antigen expression domain 2 is independently selectable to be from about 12 to about 15,000 nucleotides and encodes a second tumor-specific epitope; and wherein n is any positive integer from about 1 to about 500. In some embodiments, n is equal to at least 19 or greater. In some embodiments, n is equal to about 19 to about 59.

[0168] In some embodiments, the length of each adapter is independently selected to be about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids. In some embodiments, each adapter is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length. In some embodiments, each adapter is independently selectable to be an adapter of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length. In some embodiments, each adapter is about 21 natural or non-natural nucleic acids in length.

[0169] In some embodiments, the length of each linker according to Formula I is different. For example, in some embodiments, the length of the first linker is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 lengths of natural or non-natural nucleic acids, and the length of the second linker is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 lengths of natural or non-natural nucleic acids, wherein the length of the first linker is different from the length of the second linker. The present disclosure can envision various configurations, wherein Formula I comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more joints, wherein these joints have similar or different lengths. In some embodiments, there is only a joint between each nucleic acid sequence encoding a tumor-specific antigen, and thus the 5' and 3' terminal tumor-specific nucleic acid sequences are each adjacent to only one joint. In some embodiments, the 5' terminal tumor-specific joint is located at the 3' of the Ig leader sequence (such as the IgE leader sequence). In some embodiments, the expressible nucleic acid sequence comprises about 20 to about 60 kinds of tumor-specific antigens, each having a joint encoding a tumor-specific antigen between the nucleic acid sequences, and the expressible nucleic acid sequence comprises a nucleic acid sequence at the 5' position of the nucleic acid sequence encoding the first tumor-specific antigen, the nucleic acid sequence encoding the leader sequence, and wherein the entire expressible nucleic acid sequence is operably connected to a promoter and / or enhancer sequence in the nucleic acid molecule.

[0170] In certain embodiments, two linkers can be used together in the nucleotide sequence encoding the fusion peptide. Thus, in some embodiments, the length of the first linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids, and the length is about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids. In some embodiments, the length of the second adapter can be independently selected from about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids. In some embodiments, the first adapter can be independently selected from adapters having a length of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids. In some embodiments, the second adapter can be independently selected from adapters having a length of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids.

[0171] In certain embodiments, antigen expression domain 1 and antigen expression domain 2 comprise a nucleic acid sequence encoding one or two epitopes of a specific tumor-specific neoantigen. In some embodiments, antigen expression domain 1 encodes a CD4 neoepitope. In some embodiments, antigen expression domain 1 encodes a CD8 neoepitope. In some embodiments, antigen expression domain 2 encodes a CD4 neoepitope. In some embodiments, antigen expression domain 2 encodes a CD8 neoepitope. In some embodiments, antigen domain 1 encodes a CD8 neoepitope and antigen expression domain 2 encodes a CD8 neoepitope. CD4 neoepitopes are epitopes recognized by CD4+T cells. CD8 neoepitopes are epitopes recognized by CD8+T cells.

[0172] The present disclosure also relates to a nucleic acid sequence comprising a plurality of antigen expression domains, which encode at least two new antigens separated by one or more joints. In some embodiments, the antigen expression domain encodes an amino acid sequence of about 3 to about 100 amino acids in length. In some embodiments, there is at least one joint encoding a joint of about 3 to about 25 amino acids in length. In some embodiments, the joint sequence is spaced apart from each antigen expression domain. In some embodiments, the nucleic acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more joints. In some embodiments, the nucleic acid sequence comprises about 10 to about 70 joints, about 15 to about 70 joints, about 20 to about 65 joints, about 25 to about 65 joints, about 30 to about 60 joints, about 35 to about 60 joints, about 40 to about 60 joints, about 45 to about 60 joints, about 50 to about 60 joints, or about 52 to about 58 joints. In some embodiments, the nucleic acid sequence comprises 52 adapters, 53 adapters, 54 adapters, 55 adapters, 56 adapters, 57 adapters, or 58 adapters. In some embodiments, the nucleic acid comprises 55 adapters.

[0173] In some embodiments, the nucleic acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more joints, wherein at least one or more joints comprise a furin joint. In some embodiments, the nucleic acid sequence comprises about 10 to about 70 joints, about 15 to about 70 joints, about 20 to about 65 joints, about 25 to about 65 joints, about 30 to about 60 joints, about 35 to about 60 joints, about 40 to about 60 joints, about 45 to about 60 joints, about 50 to about 60 joints or about 52 to about 58 joints, wherein at least one or more joints comprise a furin joint. In some embodiments, the nucleic acid sequence comprises 52 joints, 53 joints, 54 joints, 55 joints, 56 joints, 57 joints or 58 joints, wherein at least one or more joints comprise a furin joint. In some embodiments, the nucleic acid comprises 55 joints, wherein at least one or more joints comprise a furin joint.

[0174] In some embodiments, the nucleic acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more linker domains and the nucleic acid sequence comprises Formula I(a):

[0175] [(AEDn)–(connector)]n–(AEDn+1)]n

[0176] Wherein each AED can be independently selected from any one or more tumor-associated antigens from a subject, and wherein n is any positive integer from about 1 to about 50 and wherein each "linker" is a nucleic acid sequence encoding one or more amino acid cleavage sites. Each linker can be the same or can be independently selected to contain one or more linkers disclosed herein. In some embodiments, the linker is a furin cleavage site of about 9 to about 105 nucleotides in length and encodes an amino acid sequence of an amino acid cleavage site. In some embodiments, the nucleic acid sequence is a component of a nucleic acid molecule. In some compositions contemplated herein, the composition comprises 1, 2, 3, 4, 5 or more nucleic acid molecules, each of which expresses any pattern or formula of an AED disclosed herein.

[0177] In some embodiments, the nucleic acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more linker domains and the nucleic acid sequence comprises Formula III(a):

[0178] [(AEDn)–(connector)]n–(AEDn+1)–connectorn+1-(AEDn+2)]n

[0179] Wherein each AED can be independently selected from any one or more tumor-associated antigens from a subject, and wherein n is any positive integer from about 20 to about 50 and wherein each "linker" is a nucleic acid sequence encoding one or more amino acid cleavage sites. Each linker can be the same or can be independently selected to include one or more linkers disclosed herein; wherein each "-" represents a bond between each subunit. In some embodiments, the linker is a furin protease cleavage site having a length of about 9 to about 105 nucleotides and encoding an amino acid sequence of an amino acid cleavage site. In some embodiments, the nucleic acid sequence is a component of a nucleic acid molecule. In some embodiments, Formula III (a) comprises a third linker bonded to the 3' end of the third AED sequence. In some embodiments, the last AED sequence in a 5' to 3' orientation is not bonded to a linker. The present disclosure also relates to a nucleic acid sequence comprising a coding region and a non-coding region, the coding region consisting of Formula I (b): [(AED 1 )–(Connector)–(AED 2 )–(connector)] n .–[(AED 3 )] n+1 , wherein n is a positive integer from about 1 to about 30, wherein each "linker" encodes one or more amino acid cleavage sequences, and wherein the noncoding region comprises at least one regulatory sequence operably linked to one or more AEDs; and wherein, in the 5' to 3' orientation, the AED 3 is the terminal antigen expression domain in the AED sequence. In some embodiments, the regulatory sequence is each of the regulatory sequences depicted in the diagram or a functional fragment comprising at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the regulatory sequences depicted in the diagram.

[0180] In some embodiments, the nucleic acid molecules or sequences of the present disclosure include multiple antigen expression domains, which encode at least two new antigens separated by one or more joints. In some embodiments, the antigen expression domain encodes an amino acid sequence of about 3 to about 100 amino acids in length. In some embodiments, there is at least one joint encoding a joint of about 3 to about 25 amino acids in length. In some embodiments, the joint sequence is spaced apart from each antigen expression domain. In some embodiments, the nucleic acid sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more joints. In some embodiments, the nucleic acid sequence includes about 10 to about 70 joints, about 15 to about 70 joints, about 20 to about 65 joints, about 25 to about 65 joints, about 30 to about 60 joints, about 35 to about 60 joints, about 40 to about 60 joints, about 45 to about 60 joints, about 50 to about 60 joints, or about 52 to about 58 joints. In some embodiments, the nucleic acid sequence comprises 52 joints, 53 joints, 54 joints, 55 joints, 56 joints, 57 joints, or 58 joints, wherein each joint is located between at least two antigen expression domains encoding a tumor-specific antigen. In some embodiments, the nucleic acid comprises 55 joints. In some embodiments, the nucleic acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more joints, at least one or more of which comprises a furin joint.

[0181] In some embodiments, the nucleic acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more linker domains and the nucleic acid sequence comprises Formula I(a): (AED1)-(linker)-(AED2)]n, wherein each AED can be independently selected as any one or more tumor-associated antigens from a subject, and wherein n is any positive integer from about 1 to about 60. In some embodiments, n is about. In some embodiments, each "linker" is a nucleic acid sequence encoding one or more amino acid cleavage sites. Each linker can be the same or can be independently selected to include one or more linkers disclosed herein. In some embodiments, n is an integer value from about 20 to about 30.

[0182] In some embodiments, antigen expression domains 1 and / or 2 can be independently selected to be about 12 to about 15,000 nucleotides in length, about 50 to about 15,000 nucleotides in length, about 100 to about 15,000 nucleotides in length, about 500 to about 15,000 nucleotides in length, about 1,000 to about 15,000 nucleotides in length, about 5,000 to about 15,000 nucleotides in length, about 10,000 to about 15,000 nucleotides in length. In other embodiments, the antigen expression domain 1 is about 12, about 25, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 11,000, about 12,000, about 13,000, about 14,000, about 15,000 nucleotides in length. In some embodiments, the antigen expression domain 2 can be independently selected to be about 12 to about 15,000 nucleotides in length, about 50 to about 15,000 nucleotides in length, about 100 to about 15,000 nucleotides in length, about 500 to about 15,000 nucleotides in length, about 1,000 to about 15,000 nucleotides in length, about 5,000 to about 15,000 nucleotides in length, about 10,000 to about 15,000 nucleotides in length. In some embodiments, the length of the antigen expression domain 2 is about 12, about 25, about 50, about 75, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 11,000, about 12,000, about 13,000, about 14,000, about 15,000 nucleotides.

[0183] In some embodiments, the length of antigen expression domain 1 or antigen expression domain 2 can be independently selected to be about 20 to about 2,000 nucleotides. In some embodiments, the length of antigen expression domain 1 is about 20 to about 2,000 nucleotides, the length is about 50 to about 2,000 nucleotides, the length is about 100 to about 2,000 nucleotides, the length is about 500 to about 2,000 nucleotides, the length is about 1500 to about 2,000 nucleotides. In other embodiments, the length of antigen expression domain 1 is about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1900, about 2000 nucleotides. In some embodiments, the length of antigen expression domain 2 is about 20 to about 2,000 nucleotides, about 50 to about 2,000 nucleotides, about 100 to about 2,000 nucleotides, about 500 to about 2,000 nucleotides, about 1500 to about 2,000 nucleotides. In other embodiments, the length of the antigen expression domain 2 is about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1900, about 2000 nucleotides.

[0184] In some embodiments, antigen expression domain 1 and / or antigen expression domain 2 can be independently selected to be about 15 to about 150 nucleotides in length, for example, about 15 to about 150 nucleotides in length, about 15 to about 125 nucleotides in length, about 15 to about 100 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 80 nucleotides in length, about 15 to about 70 nucleotides in length, about 15 to about 60 nucleotides in length, about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0185] In some embodiments, antigen expression domain 1 and / or antigen expression domain 2 can be independently selected to be about 15 to about 100 nucleotides in length, such as about 3 to about 120 nucleotides in length, about 15 to about 100 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 80 nucleotides in length, about 15 to about 70 nucleotides in length, about 15 to about 60 nucleotides in length, about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0186] In some embodiments, antigen expression domain 1 and / or antigen expression domain 2 can be independently selected to be about 15 to about 50 nucleotides in length, for example, about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0187] In some embodiments, n is any positive integer from about 1 to about 500. In some embodiments, n is any positive integer from about 1 to about 500, from about 10 to about 500, from about 50 to about 500, from about 100 to about 500, from about 200 to about 500, from about 300 to about 500, from about 400 to about 500. In other embodiments, n is about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about 305, about 310, about 315, about 120, about 325, about 330, about 335, about 340, about 345, about 350, about 355, about 360, about 365, about 370, about 375, about 380, about any positive integer of about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, about 445, about 450, about 455, about 460, about 465, about 470, about 475, about 480, about 485, about 490, about 495, about 500.

[0188] In some embodiments, n is a positive integer from about 5 to about 30, from about 5 to about 25, from about 5 to about 20, from about 5 to about 15, from about 5 to about 10.

[0189] In some embodiments, n is a positive integer from about 2 to about 100, about 2 to about 90, about 2 to about 80, about 2 to about 70, about 2 to about 60, about 2 to about 50, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10.

[0190] In some embodiments, n is about 2 to about 58, about 3 to about 58, about 4 to about 58, about 5 to about 58, about 6 to about 58, about 7 to about 58, about 8 to about 58, about 9 to about 58, about 10 to about 58, about 11 to about 58, about 12 to about 58, about 13 to about 58, about 14 to about 58, about 15 to about 58, about 16 to about 58, about 17 to about 58, about 18 to about 58, about 19 to about 58, about 20 to about 58, about 21 to about 58, about 22 to about 58, about 23 to about 58, about 24 to about 58, about 25 to about 58, about 26 to about 58, about 27 to about 58, about 28 to about 58, about 29 to about 58, about The invention relates to a positive integer ranging from about 30 to about 58, about 31 to about 58, about 32 to about 58, about 33 to about 58, about 34 to about 58, about 35 to about 58, about 36 to about 58, about 37 to about 58, about 38 to about 58, about 39 to about 58, about 40 to about 58, about 41 to about 58, about 42 to about 58, about 43 to about 58, about 44 to about 58, about 45 to about 58, about 46 to about 58, about 47 to about 58, about 48 to about 58, about 49 to about 58, about 50 to about 58, about 51 to about 58, about 52 to about 58, about 53 to about 58, about 54 to about 58, about 55 to about 58, about 56 to about 58, and about 57 to about 58.

[0191] In some embodiments, n is a positive integer from about 2 to about 29, about 3 to about 29, about 4 to about 29, about 5 to about 29, about 6 to about 58, about 7 to about 29, about 8 to about 29, about 9 to about 29, about 10 to about 29, about 11 to about 29, about 12 to about 29, about 13 to about 29, about 14 to about 29, about 15 to about 29, about 16 to about 29, about 17 to about 29, about 18 to about 29, about 19 to about 29, about 20 to about 29, about 21 to about 29, about 22 to about 29, about 23 to about 29, about 24 to about 29, about 25 to about 29, about 26 to about 29, about 27 to about 29, or about 28 to about 29.

[0192] In some embodiments, n is any positive integer from about 30 to about 60. In some embodiments, n is any positive integer from about 35 to about 60. In some embodiments, n is any positive integer from about 40 to about 60. In some embodiments, n is any positive integer from about 45 to about 60. In some embodiments, n is any positive integer from about 50 to about 60. In some embodiments, n is 50. In some embodiments, n is 51. In some embodiments, n is 52. In some embodiments, n is 53. In some embodiments, n is 54. In some embodiments, n is 55. In some embodiments, n is 56. In some embodiments, n is 57. In some embodiments, n is 58. In some embodiments, n is 59. In some embodiments, n is 60.

[0193] In some embodiments, antigen expression domain 1 or antigen expression domain 2 can be independently selected to be about 50 to about 10,000 nucleotides in length, such as about 50 to about 15,000 nucleotides in length, about 100 to about 15,000 nucleotides in length, about 500 to about 15,000 nucleotides in length, about 1,000 to about 15,000 nucleotides in length, about 5,000 to about 15,000 nucleotides in length, about 10,000 to about 15,000 nucleotides in length, and n is any positive integer from about 6 to about 26, such as about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26.

[0194] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 1 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 1. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the nucleic acid sequence encoding β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the nucleic acid sequence encoding β-catenin or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 1.

[0195] In some embodiments, the antigen expression domain comprises a nucleic acid encoding β-catenin or a functional fragment thereof, wherein the nucleic acid sequence comprises RNA and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 2 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 2. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a fragment thereof, wherein the nucleic acid sequence encoding β-catenin or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a fragment thereof, wherein the nucleic acid sequence encoding β-catenin or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 2; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 2.

[0196] In some embodiments, the antigen expression domain comprises a nucleic acid encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 13 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 13. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 13; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 13.

[0197] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding AXIN1 or a functional fragment thereof, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 3 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 3. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a functional fragment thereof, wherein the nucleic acid sequence encoding AXIN1 comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a functional fragment thereof, wherein the nucleic acid sequence encoding AXIN1 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:3; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:3.

[0198] In some embodiments, the antigen expression domain comprises a nucleic acid encoding AXIN1 or a functional fragment thereof, wherein the nucleic acid sequence comprises RNA and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 4 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 4. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a fragment thereof, wherein the nucleic acid sequence encoding AXIN1 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a fragment thereof, wherein the nucleic acid sequence encoding AXIN1 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:4; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:4.

[0199] In some embodiments, the antigen expression domain comprises a nucleic acid encoding AXIN1 or a functional fragment thereof, wherein AXIN1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 14 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 14. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a functional fragment thereof, wherein AXIN1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a functional fragment thereof, wherein AXIN1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 14; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 14.

[0200] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding AXIN2 or a functional fragment thereof, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 5 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 5. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN2 or a functional fragment thereof, wherein the nucleic acid sequence encoding AXIN2 comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN2 or a functional fragment thereof, wherein the nucleic acid sequence encoding AXIN2 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:5.

[0201] In some embodiments, the antigen expression domain comprises a nucleic acid encoding AXIN2 or a functional fragment thereof, wherein the nucleic acid sequence comprises RNA and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 6 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 6. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN2 or a fragment thereof, wherein the nucleic acid sequence encoding AXIN2 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN2 or a fragment thereof, wherein the nucleic acid sequence encoding AXIN2 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:6; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:6.

[0202] In some embodiments, the antigen expression domain comprises a nucleic acid encoding AXIN2 or a functional fragment thereof, wherein AXIN2 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 15, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 15 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 15. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN2 or a functional fragment thereof, wherein AXIN2 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN2 or a functional fragment thereof, wherein AXIN2 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 15; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 15.

[0203] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding an APC or a functional fragment thereof, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 7 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 7. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding an APC or a functional fragment thereof, wherein the nucleic acid sequence encoding the APC comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding an APC or a functional fragment thereof, wherein the nucleic acid sequence encoding the APC or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:7; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:7.

[0204] In some embodiments, the antigen expression domain comprises a nucleic acid encoding an APC or a functional fragment thereof, wherein the nucleic acid sequence comprises RNA and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 8 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 8. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding an APC or a fragment thereof, wherein the nucleic acid sequence encoding the APC or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding an APC or a fragment thereof, wherein the nucleic acid sequence encoding the APC or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:8; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:8.

[0205] In some embodiments, the antigen expression domain comprises a nucleic acid encoding an APC or a functional fragment thereof, wherein the APC or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 16 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 16. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding an APC or a functional fragment thereof, wherein the APC or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding an APC or a functional fragment thereof, wherein the APC or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 16; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 16.

[0206] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding CSNK1A1 or a functional fragment thereof, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 9 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 9. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding CSNK1A1 or a functional fragment thereof, wherein the nucleic acid sequence encoding CSNK1A1 comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding CSNK1A1 or a functional fragment thereof, wherein the nucleic acid sequence encoding CSNK1A1 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:9; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:9.

[0207] In some embodiments, the antigen expression domain comprises a nucleic acid encoding CSNK1A1 or a functional fragment thereof, wherein the nucleic acid sequence comprises RNA and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 10 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 10. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding CSNK1A1 or a fragment thereof, wherein the nucleic acid sequence encoding CSNK1A1 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding CSNK1A1 or a fragment thereof, wherein the nucleic acid sequence encoding CSNK1A1 or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:10; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:10.

[0208] In some embodiments, the antigen expression domain comprises a nucleic acid encoding CSNK1A1 or a functional fragment thereof, wherein CSNK1A1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 17 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 17. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding CSNK1A1 or a functional fragment thereof, wherein CSNK1A1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding CSNK1A1 or a functional fragment thereof, wherein CSNK1A1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:17; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding amino acids comprising 100% sequence identity to SEQ ID NO:17.

[0209] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding GSK3B or a functional fragment thereof, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 11 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 11. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding GSK3B or a functional fragment thereof, wherein the nucleic acid sequence encoding GSK3B comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding GSK3B or a functional fragment thereof, wherein the nucleic acid sequence encoding GSK3B or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 11; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 11.

[0210] In some embodiments, the antigen expression domain comprises a nucleic acid encoding GSK3B or a functional fragment thereof, wherein the nucleic acid sequence comprises RNA and is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 12 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 12. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding GSK3B or a fragment thereof, wherein the nucleic acid sequence encoding GSK3B or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding GSK3B or a fragment thereof, wherein the nucleic acid sequence encoding GSK3B or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 12; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 12.

[0211] In some embodiments, the antigen expression domain comprises a nucleic acid encoding GSK3B or a functional fragment thereof, wherein GSK3B or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 18 and does not contain a nucleic acid sequence comprising 100% SEQ ID NO: 18. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding GSK3B or a functional fragment thereof, wherein GSK3B or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding GSK3B or a functional fragment thereof, wherein GSK3B or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 18; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 18.

[0212] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 19, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 19. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 19; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 19.

[0213] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 20; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 20.

[0214] In some embodiments, the nucleic acid molecule comprises an antigen expression domain, the antigen expression domain comprising a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 31. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 31; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 31.

[0215] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 21. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 21; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 21.

[0216] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 22. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 22; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 22.

[0217] In some embodiments, the nucleic acid molecule comprises an antigen expression domain, the antigen expression domain comprising a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 32. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 32; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 32.

[0218] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of AXIN1, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 23. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of AXIN1, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of AXIN1, wherein the nucleic acid sequence encoding the functional fragment of AXIN1 comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:23; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:23.

[0219] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of AXIN1, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of AXIN1, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of AXIN1, wherein the nucleic acid sequence encoding the functional fragment of AXIN1 comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:24; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO:24.

[0220] In some embodiments, the nucleic acid molecule comprises an antigen expression domain, the antigen expression domain comprising a nucleic acid sequence encoding a functional fragment of AXIN1, wherein the functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 33, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 33. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a functional fragment thereof, wherein AXIN1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding AXIN1 or a functional fragment thereof, wherein AXIN1 or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:33; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO:33.

[0221] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 25. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 25; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 25.

[0222] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 26. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 26; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 26.

[0223] In some embodiments, the nucleic acid molecule comprises an antigen expression domain, the antigen expression domain comprising a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 34, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 34. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 34; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 34.

[0224] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 27, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 27. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 27. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 27; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 27.

[0225] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 28; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 28.

[0226] In some embodiments, the nucleic acid molecule comprises an antigen expression domain, the antigen expression domain comprising a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 35, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 35. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 35. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 35; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 35.

[0227] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29, wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 29. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 29. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 29; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 29.

[0228] In some embodiments, the antigen expression domain or nucleic acid molecule comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 30. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the nucleic acid sequence encoding the functional fragment of β-catenin comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 30; and wherein the antigen expression domain does not contain a nucleic acid sequence comprising 100% sequence identity to SEQ ID NO: 30.

[0229] In some embodiments, the nucleic acid molecule comprises an antigen expression domain, the antigen expression domain comprising a nucleic acid sequence encoding a functional fragment of β-catenin, wherein the functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36, and wherein the functional fragment comprises at least about 70% sequence identity to SEQ ID NO: 36. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36. In some embodiments, the antigen expression domain comprises a nucleic acid sequence encoding β-catenin or a functional fragment thereof, wherein the β-catenin or a functional fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 36; and wherein the antigen expression domain does not contain a nucleic acid sequence encoding an amino acid comprising 100% sequence identity to SEQ ID NO: 36.

[0230] Embodiments of the present disclosure include compositions, pharmaceutical compositions and cells of the present disclosure, which include expressible nucleic acid sequences encoding multiple tumor-specific antigens, wherein the expressible nucleic acid sequences include about 15 to about 40 nucleotides encoding a portion of the tumor-specific antigen. Expressible nucleic acids may include any 15 to 40 continuous nucleic acids presented below to encode a fragment (or epitope) of an amino acid sequence. In some embodiments, the nucleic acid sequence includes one or more epitopes of about 30 to about 35 nucleotides of any one of the sequences selected from Table 2.

[0231] Table 2 – Full-length wild-type sequences of molecules in the Wnt pathway

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258] Table 3 – WT protein sequences

[0259]

[0260]

[0261]

[0262] Table 4 – Patient mutation sequences

[0263]

[0264] Table 5 - Patient amino acid mutation sequences

[0265]

[0266]

[0267] Table 6 – DNA insert sequences in pGX0001

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281] Table 7: Amino acid sequence

[0282]

[0283]

[0284]

[0285]

[0286] Table 4 – Patient mutation sequences

[0287]

[0288] Table 5 – Patient amino acid mutation sequences

[0289]

[0290] Table 6 – DNA insert sequences in pGX0001

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304] Table 7: Amino acid sequence

[0305]

[0306]

[0307]

[0308]

[0309] The present disclosure relates to nucleic acid molecules comprising the first, second and third nucleic acid sequences, wherein the first nucleic acid sequence is the first DNA backbone domain of the nucleic acid molecule, the second nucleic acid sequence is the second DNA backbone domain of the nucleic acid molecule and the third nucleic acid sequence is an expressible nucleic acid sequence; wherein the expressible nucleic acid sequence comprises a plurality of antigen expression domains oriented 5' to 3'. In some embodiments, the expressible nucleic acid sequence comprises a nucleic acid sequence encoding a joint at the 5' end of the first antigen expression domain. In some embodiments, the expressible nucleic acid sequence encodes a joint between each of the antigen expression domains. In some embodiments, the expressible nucleic acid sequence encodes a leader sequence, a plurality of antigen expression domains, and each antigen expression domain is spaced apart by a joint sequence. In some embodiments, there are at least 20 antigen expression domains. In some embodiments, there are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or more antigen expression domains. In some embodiments, the nucleic acid molecule comprises Formula I, Ia, II, IIa or IIIa. In some embodiments, the nucleic acid molecule comprises one or more regulatory sequences operably connected to the expressible nucleic acid sequence. In some embodiments, there are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 or more antigen expression domains comprising at least about 70%, 80%, 90%, 95%.

[0310] In some embodiments, the first DNA backbone domain comprises a nucleic acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:64, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:64. In some embodiments, the second DNA backbone domain comprises a nucleic acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:65, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:65.

[0311] The present disclosure also relates to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is a DNA backbone domain of a nucleic acid molecule and the second nucleic acid sequence is an expressible nucleic acid sequence; wherein the expressible nucleic acid sequence comprises a plurality of antigen expression domains oriented 5' to 3'. In some embodiments, the expressible nucleic acid sequence comprises a nucleic acid sequence encoding a joint at the 5' end of the first antigen expression domain. In some embodiments, the expressible nucleic acid sequence encodes a joint between each of the antigen expression domains. In some embodiments, the expressible nucleic acid sequence encodes a leader sequence, a plurality of antigen expression domains, and each antigen expression domain is spaced apart by a joint sequence. In some embodiments, there are at least 20 antigen expression domains. In some embodiments, there are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or more antigen expression domains. In some embodiments, the nucleic acid molecule comprises formula I, Ia, II, IIa or IIIa. In some embodiments, the nucleic acid molecule comprises one or more regulatory sequences operably connected to the expressible nucleic acid sequence. In some embodiments, the first DNA backbone domain comprises a nucleic acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:63, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:63. In some embodiments, the first DNA backbone domain comprises a nucleic acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:67, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:67.

[0312] Table 8 – Plasmid sequences

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331] In some embodiments, the nucleic acid molecule or pharmaceutical composition comprises a DNA backbone comprising all lowercase base pairs from any of the plasmids identified above, wherein a first lowercase backbone sequence and a second lowercase backbone sequence flank an expressible nucleic acid sequence encoding a plurality of tumor-specific antigen sequences, such as Formula I, Formula I(a), Formula II, or Formula III(a). In some embodiments, one or more DNA backbone segments of the nucleic acid molecule comprise at least one segment containing at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the lowercase sequences presented above.

[0332] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]), wherein the length of each linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]), wherein the length of each linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids, about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25. In some embodiments, each adapter is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length. In some embodiments, each adapter is about 21 natural or non-natural nucleic acids in length. In certain embodiments, two adapters can be used together in a fusion. Thus, in some embodiments, the length of the first linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids, with a length of about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids. In some embodiments, the length of the second adapter can be independently selected from about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids.In some embodiments, the first adapter can be independently selected from adapters having a length of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids. In some embodiments, the second adapter can be independently selected from adapters having a length of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids.

[0333] In some embodiments, at least one linker comprises about 15 to about 300 nucleotides and encodes an amino acid cleavage site. In some embodiments, each linker between each AED is an identical nucleotide sequence comprising about 15 to about 120 nucleotides and encodes an amino acid cleavage site.

[0334] In some embodiments, the formula (eg, [(AEDn)-(Linker)]n-[AEDn+1]) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Linkers.

[0335] In some embodiments, the formula comprises at least a first linker and a second linker.

[0336] In some embodiments, the formula comprises at least a first linker, a second linker, and a third linker. In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, and a fourth linker. In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, a fourth linker, and a fifth linker. In some embodiments, the formula comprises at least nineteen linkers flanked by at least twenty antigen AED domains.

[0337] In further embodiments, at least one linker comprises a furin cleavage site.

[0338] Furin is a protease that remains in the trans-Golgi network of eukaryotic cells. The effect of furin is to cut proteins at the step before proteins are delivered to their final cell destination. Furin recognizes the consensus amino acid sequence RXRR, RXRK or KXKR (wherein X is any amino acid, Moehring et al., 1993, which is incorporated by reference in its entirety), and cuts off these proteins when proteins containing these sequences arrive at the trans-Golgi network. Furin is a Ca2+ dependent serine endoprotease that can cut protein precursors with high specificity after the multiple basic motifs shown in the following table 9.

[0339] Table 9

[0340]

[0341] In certain embodiments, one or more nucleic acid molecules encode a furin-sensitive cleavage site selected from the sequence RX-[R / K]-R, wherein R represents arginine, X is any amino acid, and K is lysine. "R / K" indicates that the amino acid can be arginine or lysine.

[0342] In certain embodiments, a furin cleavage site is introduced after Antigenic Domain 1 and / or Antigenic Domain 2 (eg, [(AEDn)-(Linker)]n-[AEDn+1]).

[0343] In some embodiments, at least one linker comprises about 15 to about 300 nucleotides and encodes a cleavage site, wherein at least one linker comprises a 2A cleavage site. In some embodiments, at least one linker comprises about 15 to about 300 nucleotides and encodes a cleavage site, wherein at least one linker comprises a porcine teschovirus-1 2A (P2A) cleavage site.

[0344] The 2A peptide is a "self-cleaving" small peptide. The average length of the 2A peptide is 18-22 amino acids. The name "2A" refers to a specific region of the polyprotein of the picornavirus. Of the 2A peptides identified so far, four are widely used in research: FMDV 2A (abbreviated herein as F2A); Equine rhinitis virus (ERAV) 2A (E2A); Porcine Teschovirus-1 2A (P2A) and Thoseaasigna beta-tetrasomal virus 2A (T2A). The first three viruses belong to picornaviruses, and the latter belongs to insect viruses. The DNA and corresponding amino acid sequences of various 2A peptides are shown in Table 10 below. The underlined sequence encodes the amino acid GSG, which is added to improve the cleavage efficiency. P2A indicates porcine Teschovirus-1 2A; T2A, Thoseaasigna beta-tetrasomal virus 2A; E2A, Equine rhinitis virus (ERAV) 2A; F2A, FMDV 2A.

[0345] Table 10 discloses SEQ ID NOS 368-375, respectively, in order of appearance.

[0346]

[0347] In some embodiments, the formula comprises at least a first linker and a second linker, wherein the first and second linkers comprise a furin cleavage site.

[0348] In some embodiments, the formula comprises at least a first linker, a second linker, and a third linker, wherein the first, second, and third linkers comprise a furin cleavage site.

[0349] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, and a fourth linker, wherein the first, second, third, and fourth linkers comprise a furin cleavage site.

[0350] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, a fourth linker, and a fifth linker, wherein the first, second, third, fourth, and fifth linkers comprise a furin cleavage site.

[0351] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, a fourth linker, and a fifth linker, wherein the first, second, third, fourth, and fifth linkers comprise a furin cleavage site.

[0352] In some embodiments, the formula comprises at least a first linker and a second linker, wherein the first and second linkers comprise a P2A protease cleavage site.

[0353] In some embodiments, the formula comprises at least a first linker, a second linker, and a third linker, wherein the first, second, and third linkers comprise a P2A cleavage site.

[0354] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, and a fourth linker, wherein the first, second, third, and fourth linkers comprise a P2A cleavage site.

[0355] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, a fourth linker, and a fifth linker, wherein the first, second, third, fourth, and fifth linkers comprise a P2A cleavage site.

[0356] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, a fourth linker, a fifth linker, or more, wherein the first, second, third, fourth, fifth linker, or more linkers comprise a P2A protease cleavage site.

[0357] In some embodiments, the formula comprises at least a first linker and a second linker, wherein at least one of the first or second linker comprises a furin cleavage site.

[0358] In some embodiments, the formula comprises at least a first linker, a second linker, and a third linker, wherein at least one of the first, second, or third linkers comprises a furin cleavage site.

[0359] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, and a fourth linker, at least one of the first, second, third, or fourth linkers comprises a furin cleavage site.

[0360] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, a fourth linker, and a fifth linker, at least one of the first, second, third, fourth, or fifth linkers comprises a furin cleavage site.

[0361] In some embodiments, the formula comprises at least a first linker and a second linker, wherein at least one of the first or second linker comprises a P2A protease cleavage site.

[0362] In some embodiments, the formula comprises at least a first linker, a second linker, and a third linker, wherein at least one of the first, second, or third linkers comprises a P2A protease cleavage site.

[0363] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, and a fourth linker, and at least one of the first, second, third, or fourth linkers comprises a P2A protease cleavage site.

[0364] In some embodiments, the formula comprises at least a first linker, a second linker, a third linker, a fourth linker, and a fifth linker, and at least one of the first, second, third, fourth, or fifth linkers comprises a P2A protease cleavage site.

[0365] Nucleic acid molecules that can be used in the methods of the present disclosure include any nucleic acid molecule encoding a neoantigen or a fragment thereof; any nucleic acid encoding a linker, any nucleic acid encoding a regulatory sequence, any nucleic acid encoding a leader sequence. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides that are "substantially identical" to an endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. In some embodiments, the nucleic acid sequences or molecules of the present disclosure relate to nucleic acid sequences comprising nucleic acid sequences that are at least about 70%, 80%, 85, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequences identified in the diagrams.

[0366] In some embodiments, an exemplary leader sequence is an IgE leader amino acid sequence as shown in the following sequence and described in US20160175427, which is incorporated by reference in its entirety.

[0367] In some embodiments, the nucleic acid comprises a coding region consisting of any one of Formula I, I(a), I(b), II(a) and / or III(a) and one or more leader sequences. In some embodiments, the leader sequence is an IgE leader sequence: Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val (SEQ ID NO: 55) or a leader sequence as a functional fragment thereof, the functional fragment comprising at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the IgE leader sequence identified in the preceding sentence. In some embodiments, the nucleic acid sequence or molecule of the present disclosure relates to a nucleic acid sequence comprising a leader sequence having at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 55.

[0368] For example, stringent salt concentration is usually less than about 750mM NaCl and 75mM trisodium citrate, preferably less than about 500mM NaCl and 50mM trisodium citrate, and more preferably less than about 250mM NaCl and 25mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions usually include a temperature of at least about 30°C, more preferably at least about 37°C and most preferably at least about 42°C. Changing additional parameters such as the concentration of hybridization time, detergents such as sodium dodecyl sulfate (SDS) and the inclusion or exclusion of carrier DNA is well known to those skilled in the art. Various levels of stringency are achieved by combining these different conditions as needed. In certain embodiments, hybridization will occur at 750mM NaCl, 75mM trisodium citrate and 1% SDS at 30°C. In some embodiments, hybridization will occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In some embodiments, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations of these conditions will be apparent to those skilled in the art.

[0369] For most applications, the stringency of the washing step after hybridization will also be different. Washing stringency conditions can be defined by salt concentration and by temperature. As described above, washing stringency can be increased by reducing the salt concentration or by increasing the temperature. For example, the stringent salt concentration of the washing step is preferably less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The stringent temperature conditions of the washing step generally include a temperature of at least about 25°C, more preferably at least about 42°C and even more preferably at least about 68°C. In some embodiments, the washing step will occur at 25°C in 30mM NaCl, 3mM trisodium citrate and 0.1% SDS. In some embodiments, the washing step will occur at 42°C in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. In some embodiments, the washing step will occur at 68°C in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. Additional changes to these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0370] The nucleic acid sequence can be used in combination with other polynucleotide sequences encoding regulatory proteins that control the expression of the neoantigen sequence. For example, the nucleic acid molecule according to the present disclosure can additionally contain a recognition sequence, a regulatory sequence, a leader sequence, and a promoter sequence.

[0371] In some embodiments, the nucleic acid molecule further comprises at least one regulatory sequence, wherein the at least one nucleic acid sequence of Formula I is operably linked to the regulatory sequence.

[0372] In some embodiments, the nucleic acid molecule further comprises a leader sequence.

[0373] In some embodiments, an exemplary leader sequence is an IgE leader amino acid sequence as described in US20160175427, which is incorporated by reference in its entirety.

[0374] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]), wherein the length of Antigen Expression Domain 1 can be independently selected to be about 12 to about 15,000 nucleotides and encodes an epitope of one or more cancer cells from a subject; and the length of Antigen Expression Domain 2 can be independently selected to be about 12 to about 15,000 nucleotides and encodes an epitope of one or more cancer cells from a subject.

[0375] In some embodiments, the amount of a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]) is sufficient to elicit a cellular immune response. "Cellular immune response" means a cellular response that includes cells that present antigens with class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes, which act as "helpers" or "killers." Helper T cells (also known as CD4+T cells) play a central role by regulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+T cells, or CTLs) kill diseased cells such as cancer cells, producing more diseased cells. In some embodiments, the present disclosure relates to stimulating an anti-tumor CTL response against tumor cells that express one or more tumor-expressed antigens, and preferably presenting such tumor-expressed antigens with class I MHC.

[0376] In some embodiments, the amount of a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I ([(AEDn)-(Linker)]n-[AEDn+1]) is sufficient to elicit a CD8+ T cell response to any one or more amino acid sequences encoded by one or more antigen expression domains. In some embodiments, the amount of a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I ([(AEDn)-[AEDn+1]) is sufficient to elicit a CD8+ T and / or CD4+ T cell response to any one or more amino acid sequences encoded by one or more antigen expression domains.

[0377] In some embodiments, the amount of a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]) is sufficient to elicit a CD4+ T cell response to any one or more amino acid sequences encoded by one or more antigen expression domains. In some embodiments, the amount of a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]) is sufficient to elicit a subpopulation of T cells that respond to any one or more amino acid sequences encoded by one or more antigen expression domains, which is greater than at least about 40% CD4+ T cells, compared to a response produced without the nucleic acid sequence disclosed herein. In some embodiments, the amount of a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]) is sufficient to elicit a subpopulation of T cells that respond to any one or more amino acid sequences encoded by one or more antigen expression domains, which is greater than at least about 40% CD8+ T cells, compared to a response produced without the nucleic acid sequence disclosed herein. In some embodiments, the amount of a nucleic acid molecule comprising a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]) is sufficient to elicit a subpopulation of T cells responsive to any one or more amino acid sequences encoded by one or more antigen expression domains, the subpopulation of T cells comprising greater than at least about 40% CD4+ T cells and comprising approximately 40% CD8+ T cells, compared to a response produced without the nucleic acid sequence disclosed herein.

[0378] In yet other aspects, the nucleic acid molecule described in any one of the aspects and embodiments herein is a plasmid. In certain embodiments, the expression vector comprises a nucleic acid molecule described in any one of the aspects and embodiments. In certain embodiments, the nucleic acid expression vector is a plasmid. In some embodiments, the vector is capable of expressing one or more common new antigen sequences in a mammalian cell in an amount effective to elicit an antigen-specific immune response in a mammal against one or more common new antigens. In some embodiments, the vector is recombinant. In some embodiments, the vector comprises a heterologous nucleic acid encoding one or more new antigens. In some embodiments, the vector is a plasmid. In some embodiments, the vector can be used to transfect cells with nucleic acids encoding new antigens, and the transformed host cells are cultured and maintained under conditions where new antigen expression occurs. In some embodiments, the vector is capable of expressing one or more new antigen sequences in a mammalian cell in an amount effective to elicit an immune response in a mammal. In some embodiments, cells comprising nucleic acid molecules are capable of expressing one or more common new antigen sequences in a mammalian cell in an amount sufficient to elicit an immune response in a mammal, shrinking the tumor by greater than about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70% or more. In some embodiments, the cells comprising the nucleic acid molecule are capable of expressing one or more neo-antigen amino acid sequences in mammalian cells in an amount sufficient to induce clonal expansion of CD8+ T cells accounting for about 0.1% to about 50% of total T cell stimulation against the one or more neo-antigens.

[0379] In some embodiments, the vector comprises a heterologous nucleic acid encoding a neoantigen, and may further comprise a start codon (which may be located upstream of the neoantigen coding sequence) and a stop codon (which may be located downstream of the neoantigen coding sequence). The start and stop codons may be consistent with the neoantigen coding sequence. The vector may also comprise a promoter operably linked to the neoantigen coding sequence. The promoter operably linked to the neoantigen coding sequence may be a promoter from the following items: Simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter (such as bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter), Moloney virus promoter, avian leukosis virus (ALV) promoter, cytomegalovirus (CMV) promoter (such as CMV immediate early promoter), Epstein Barr virus (Epstein Barr virus) (EBV) promoter or Rous sarcoma virus (Rous sarcoma virus) (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a natural or synthetic muscle or skin-specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. US20040175727, the contents of which are incorporated herein in their entirety.

[0380] The vector may also include a polyadenylation signal, which may be located downstream of the HA coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, Calif.).

[0381] The vector may also include an enhancer upstream of the new antigen encoding. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as one from CMV, HA, RSV, or EBV. Polynucleotide function enhancement is described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94 / 016737, the contents of each of which are incorporated by reference in their entirety.

[0382] The vector may also include a mammalian origin of replication so that the vector is maintained outside the chromosome and multiple copies of the vector are produced in the cell. In some embodiments, the vector is LLC, TC1, ID8, pGX0001, pGX4501, pGX4503, pGX4504, pGX4505, pGX4506 and / or pGX6001 or includes any one or more regulatory or non-coding sequences of LLC, TC1, ID8, pGX0001, pGX4501, pGX4503, pGX4504, pGX4505, pGX4506 and / or pGX6001. In some embodiments, the vector comprises the sequence pVAX1. In some embodiments, the backbone of the vector is pAV0242. In some embodiments, the vector may be a replication-deficient adenovirus type 5 (Ad5) vector.

[0383] The vector may also contain regulatory sequences that may be well suited for gene expression in mammalian or human cells to which the vector is administered. The neoantigen coding sequence may contain codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0384] The vector may be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in E. coli. The vector may also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in a yeast strain of Saccharomyces cerevisiae. The vector may also be MAXBAC TM The vector may be a vector of the complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The vector may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. The vector may be an expression vector or system to produce proteins by conventional techniques and readily available starting materials, including Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor (1989), which is fully incorporated by reference.

[0385] The expression vector for different cell types is well known in the art and can be selected without excessive experiments. Usually, DNA is inserted into expression vectors such as plasmids with appropriate orientation and correct reading frame to express. If necessary, DNA can be connected with the appropriate transcription and translation regulation control nucleotide sequence recognized by the desired host (for example, bacteria), but such control is usually available in expression vectors. The vector is then introduced into the host bacteria to clone using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0386] In some embodiments, the nucleic acid sequence of Formula I is located within the multiple cloning site of a plasmid selected from the group consisting of LLC, TC1, ID8, pGX0001, pGX4501, pGX4503, pGX4504, pGX4505, pGX4506, and / or pGX6001.

[0387] In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of LLC. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of TC1. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of ID8. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX0001. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4501. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4503. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4504. In some embodiments, the nucleic acid sequence of Formula I is located within the multiple cloning site of pGX4505. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4506. In some embodiments, the plasmid is pGX4505 or a sequence that is 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to each of the nucleotide sequences identified above. In some embodiments, the plasmid is pGX0001, or a sequence comprising about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to each of the nucleotide sequences identified above. In some embodiments, the plasmid is pGX6001, or a sequence comprising about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to each of the nucleotide sequences identified above.

[0388] Table 11 – Plasmid backbone sequences

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395] The present disclosure also relates to cells comprising any one or more nucleic acid sequences disclosed herein. In some embodiments, the cell comprises one or more nucleic acid sequences disclosed herein. In some embodiments, the host cell comprises a plasmid disclosed herein or comprises any one or more plasmids encoding about 1 to about 100 tumor-specific antigens, wherein at least one tumor-specific antigen is an amino acid sequence associated with the WNT pathway. The host cell can be transfected in vivo (i.e., in an animal) or in vitro (i.e., outside the animal). Transfection of nucleic acid molecules into host cells can be accomplished by any method that can insert nucleic acid molecules into cells. Transfection techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion.

[0396] The present disclosure also provides that one or more new antigenic peptides of the present disclosure can be encoded by a single expression vector. The present disclosure also provides that one or more new antigenic peptides of the present disclosure can be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system).

[0397] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotides of the present disclosure may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded may be a coding strand or a non-coding (antisense) strand.

[0398] In some embodiments, the polynucleotide may include a coding sequence of a tumor-specific antigen peptide fused to a polynucleotide in the same reading frame, which facilitates, for example, expression and / or secretion of the peptide from a host cell (e.g., a leader sequence used as a secretory sequence to control transport of the polypeptide from a cell). A polypeptide with a leader sequence is a preprotein and may have a leader sequence cleaved by a host cell to form a mature form of the polypeptide.

[0399] In some embodiments, the polynucleotide may include a coding sequence for a tumor-specific antigen peptide fused to a marker sequence in the same reading frame, which allows, for example, purification of the encoded polypeptide, which can then be incorporated into a personalized neoplasia vaccine. For example, the marker sequence may be a hexa-histidine tag (SEQ ID NO: 367) provided by the pQE-9 vector to purify the mature polypeptide fused to the marker in the case of a bacterial host, or when a mammalian host (e.g., COS-7 cells) is used, the marker sequence may be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. Additional tags include, but are not limited to, calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tags, Xpress tags, Isoeptag, SpyTag, biotin carboxyl carrier protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep- tags, thioredoxin tags, TC tags, Ty tags, and the like.

[0400] In an embodiment, the polynucleotide may include coding sequences for one or more tumor-specific antigenic peptides in the tumor-specific antigenic peptide fused in the same reading frame to create a single tandem neo-antigenic peptide construct capable of producing multiple neo-antigenic peptides.

[0401] In an embodiment, the present disclosure provides an isolated nucleic acid molecule having a nucleotide sequence that is at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding a tumor specific neoantigen of the present disclosure.

[0402] A polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the amino-terminal or carboxyl-terminal position of the reference nucleotide sequence or at any position between these terminal positions, interspersed individually between nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence.

[0403] In practice, whether any particular nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98% or 99% identical to a reference sequence can be routinely determined using known computer programs such as, for example, the Bestfit program (Wisconsin Sequence Analysis Package, Unix version 8, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set so that the percent identity is calculated relative to the entire length of the reference nucleotide sequence, and homology gaps are allowed to account for up to 5% of the total number of nucleotides in the reference sequence.

[0404] The present disclosure also includes compositions comprising one or more nucleic acid molecules described herein.

[0405] The present disclosure also contemplates the use of nucleic acid molecules as vehicles for delivering neoantigens to subjects in vivo in the form of, for example, DNA / RNA vaccines (see, for example, WO2012 / 159643 and WO2012 / 159754, hereby incorporated by reference in their entireties).

[0406] In some embodiments, personalized neoplasia vaccines may include a separate DNA plasmid encoding, for example, one or more neoantigenic peptides / polypeptides identified according to the present disclosure. As discussed above, the accurate selection of expression vectors will depend on the peptide / polypeptide to be expressed, and is completely within the skill of ordinary technicians. The expected persistence of the DNA construct (e.g., in a free, non-replicating, non-integrated form in muscle cells) is expected to provide an increased protection duration. In some embodiments, the composition comprises a first, second, or third nucleic acid molecule, wherein at least the first nucleic acid molecule encodes one or more neoantigens. In some embodiments, the second nucleic acid molecule comprises one or more neoantigens. In some embodiments, the second nucleic acid molecule comprises a nucleic acid sequence encoding one or more adjuvants. In other embodiments, personalized neoplasia vaccines may include a separate RNA or cDNA molecule encoding a neoantigenic peptide / polypeptide of the present disclosure.

[0407] In some embodiments, personalized neoplasia or cancer vaccines may include viral-based vectors for use in human patients, such as, for example, an adenoviral system (see, e.g., Baden et al. First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). J Infect Dis. 2013 Jan 15;207(2):240-7, hereby incorporated by reference in its entirety).

[0408] Methods for identifying neoantigens

[0409] As described in more detail herein, a population of neoplasia / tumor-specific neoantigens can be identified by sequencing each patient's neoplasia / tumor and normal DNA to identify tumor-specific mutations and determine the patient's HLA allotype. The population of neoplasia / tumor-specific neoantigens and their cognate natural antigens can be bioinformatically analyzed using validated algorithms to predict which tumor-specific mutations produce epitopes that can bind to the patient's HLA allotype, and in particular, which tumor-specific mutations produce epitopes that can bind to the patient's HLA allotype more effectively than the cognate natural antigen. Based on this analysis, identified nucleotide sequences corresponding to these mutations can be designed for each patient and used together as a cancer vaccine to immunize subjects in need thereof.

[0410] The present disclosure relates to a method for identifying one or more subject-specific neoantigen mutations in a subject, wherein the subject has been diagnosed with, suspected of having, or contains one or more hyperproliferative cells (e.g., such as a tumor). In some embodiments, the present disclosure relates to a method for identifying one or more subject-specific neoantigen mutations in a subject, wherein the subject has been diagnosed with, suspected of having, or contains one or more hyperproliferative cells (e.g., such as a tumor) characterized by the presence or abundance of multiple neoantigen mutations, the method comprising sequencing a nucleic acid sample from a tumor of the subject and a nucleic acid sample from a non-tumor sample of the subject; analyzing the sequence to determine coding and non-coding regions; identifying sequences containing tumor-specific non-synonymous or non-silent mutations not present in non-tumor samples; identifying single nucleotide variations and single nucleotide insertions and deletions; generating subject-specific peptides encoded by sequences containing tumor-specific non-synonymous or non-silent mutations not present in non-tumor samples; and measuring the binding characteristics of subject-specific peptides, wherein each subject-specific peptide is an expression product of a subject-specific DNA neoantigen not present in a non-tumor sample, thereby identifying one or more subject-specific DNA neoantigens in the subject. In some embodiments, measuring the binding characteristics of the subject-specific peptide is performed by measuring one or more of binding of the subject-specific peptide to a T cell receptor; measuring binding of the subject-specific peptide to the subject's HLA protein; or measuring binding of the subject-specific peptide to a transporter associated with antigen processing (TAP).

[0411] Effectively selecting which specific mutations to use as immunogens requires identifying the patient's HLA type and being able to predict which mutated peptides can effectively bind to the patient's HLA alleles. Therefore, in some embodiments, measuring the binding of a subject-specific peptide to a T cell receptor includes measuring the binding of a subject-specific peptide to an HLA protein of the subject or sample.

[0412] In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 550nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 500nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 450nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 400nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 350nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 300nM.

[0413] In another embodiment, the method of identifying one or more subject-specific DNA neoantigen mutations in a subject further comprises the step of ranking the subject-specific peptides based on binding characteristics.

[0414] In another embodiment, the method of identifying one or more subject-specific DNA neoantigen mutations in a subject further comprises the step of measuring the CD8+ T cell immune response generated by the subject-specific peptide. Methods of measuring CD8+ T cell responses are known in the art and described herein.

[0415] In another embodiment, the method of identifying one or more subject-specific DNA neoantigen mutations in a subject further comprises formulating the subject-specific DNA neoantigen into an immunogenic composition for administration to the subject. In some embodiments, the top 200 neoantigen mutations are included or subcloned into an immunogenic composition, and in some embodiments, the immunogenic composition is one or more plasmids. In another embodiment, the top 150 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 100 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 50 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 25 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 10 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 5 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 5 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 5-20 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 10-50 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 25-100 neoantigen mutations are included in the immunogenic composition. In another embodiment, the top 50-100 neoantigenic mutations are included in the immunogenic composition. In another embodiment, the top 100-200 neoantigenic mutations are included in the immunogenic composition.

[0416] In another embodiment, the method of identifying one or more subject-specific DNA neoantigen mutations in a subject further comprises providing a culture comprising dendritic cells (DC) obtained from a subject; and contacting the dendritic cells with an immunogenic composition. DCs are potent antigen presenting cells that can initiate T cell immunity and can be used as cancer vaccines when loaded with one or more neoantigens of interest. In additional embodiments, the method further comprises administering dendritic cells to a subject; obtaining a population of CD8+T cells from a peripheral blood sample from a subject, wherein the CD8+ cells recognize at least one neoantigen; and expanding a population of CD8+T cells that recognize neoantigens.

[0417] In some embodiments, the population of expanded CD8+ T cells is administered to a subject.

[0418] Preferably, any suitable sequencing-by-synthesis platform can be used to identify mutations. There are currently four main sequencing-by-synthesis platforms: the genome sequencer of Roche / 454Life Sciences, the HiSeq analyzer of Illumina / Solexa, the SOLiD system of Applied BioSystems, and the Heliscope system of HelicosBiosciences. Pacific Biosciences and VisiGen Biotechnologies have also described sequencing-by-synthesis platforms. Each of these platforms can be used in the methods disclosed herein. In certain embodiments, the multiple nucleic acid molecules sequenced are bound to a support (e.g., a solid support). In order to fix nucleic acid on a support, capture sequences / universal priming sites can be added at the 3' and / or 5' ends of the template. By hybridizing the capture sequence with a complementary sequence covalently attached to the support, nucleic acid can be bound to the support. Capture sequence (also referred to as universal capture sequence) is a nucleic acid sequence complementary to the sequence attached to the support, which can double serve as a universal primer.

[0419] As an alternative to the capture sequence, a member of a coupling pair (such as, for example, an antibody / antigen, a receptor / ligand or an avidin-biotin pair, as described in, for example, U.S. Patent Application No. 2006 / 0252077) can be connected to each fragment to be captured on the surface of the corresponding second member coated with the coupling pair. After capture, the sequence can be analyzed, for example, by single molecule detection / sequencing, such as described in Examples and U.S. Patent No. 7,283,337, including template-dependent synthesis sequencing. In synthesis sequencing, the surface-bound molecules are exposed to a plurality of labeled triphosphate nucleotides in the presence of a polymerase. The sequence of the template is determined by the order of the labeled nucleotides incorporated into the 3' end of the growing chain. This can be done in real time or in a step-and-repeat mode. For real-time analysis, different optical labels for each nucleotide can be incorporated, and a plurality of lasers can be used to stimulate the incorporated nucleotides.

[0420] Any cell type or tissue can be used to obtain nucleic acid samples for use in the sequencing methods described herein. In some embodiments, DNA or RNA samples are obtained from neoplasia, tumors, or body fluids (e.g., blood) or saliva obtained by known techniques (e.g., venipuncture). Alternatively, nucleic acid testing can be performed on dry samples (e.g., hair or skin).

[0421] There are a variety of methods for detecting the presence of specific mutations or alleles in the DNA or RNA of an individual. Advances in this field provide accurate, simple and inexpensive large-scale SNP genotyping. For example, several new technologies have been described recently, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrophosphate sequencing, oligonucleotide specific engagement, TaqMan system and various DNA "chip" technologies such as Affymetrix SNP chips. These methods require amplification of the target gene region, usually by PCR. There are also other newly developed methods, based on the production of small signal molecules by invasive cutting, followed by mass spectrometry or fixed padlock probes and rolling circle amplification, which may ultimately eliminate the need for PCR. Several methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. The method disclosed herein should be understood to include all available methods.

[0422] PCR-based detection means may include multiplexed amplification of multiple markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously.

[0423] Alternatively, different markers can be amplified using differentially labeled primers, and therefore each marker can be subjected to differential detection. Of course, hybridization-based detection means allow differential detection of multiple PCR products in the sample. Other techniques known in the art allow multiple markers to be subjected to multiplex analysis.

[0424] Several methods have been developed to promote the analysis of single nucleotide polymorphisms in genomic DNA or cell RNA. In certain embodiments, single base polymorphisms can be detected by using special exonuclease-resistant nucleotides, such as, for example, U.S. Patent number 4,656,127. According to the method, a primer complementary to the allele sequence of the next-to-polymorphic site 3' is allowed to hybridize with a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to the specific exonuclease-resistant nucleotide derivative present, the derivative will be incorporated into the end of the hybridized primer. This incorporation makes the primer resistant to exonucleases, and thus allows its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the discovery that the primer is resistant to exonucleases shows that the nucleotides present in the polymorphic site of the target molecule are complementary to the nucleotides of the nucleotide derivatives used in the reaction. The advantage of the method is that a large amount of irrelevant sequence data does not need to be determined.

[0425] In another embodiment of the present disclosure, a solution-based method is used to determine the identity of the nucleotide at the polymorphic site. Cohen et al. (French Patent No. 2,650,840; PCT Application No. WO1991 / 02087). As in the method in U.S. Patent No. 4,656,127, a primer complementary to the allele sequence immediately 3' of the polymorphic site can be used. The method uses a labeled dideoxynucleotide derivative to determine the identity of the nucleotide at the site, and if the deoxynucleotide derivative is complementary to the nucleotide at the polymorphic site, the derivative will be incorporated into the end of the primer.

[0426] An alternative method called Genetic Bit Analysis or GBA is described in PCT Application No. WO1992 / 15712. GBA uses a mixture of labeled terminators and primers complementary to the sequence 3' of the polymorphic site. Thus, the incorporated labeled terminator is determined by and complementary to the nucleotide present in the polymorphic site of the target molecule being assessed. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Application No. WO1991 / 02087), the GBA method is preferably a heterogeneous analysis in which the primers or the target molecule are fixed to a solid phase.

[0427] Several primer-directed nucleotide incorporation procedures for determining polymorphic sites in DNA have been described (Komher, JS et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, BP, Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C, et al., Genomics 8:684-692 (1990); Kuppuswamy, MN et al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, TR et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA in that they all rely on the incorporation of labeled deoxynucleotides to distinguish the bases at the polymorphic site. In this format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms occurring in a run of the same nucleotide can result in a signal that is proportional to the run length (Syvanen, A.-C, et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0428] The present disclosure generally relates to a method for identifying or selecting one or more neoantigens from a sample, the method comprising (a) sequencing DNA / RNA from the sample, and (b) measuring binding of subject-specific peptides to T cell receptors, including measuring binding of subject-specific peptides to HLA proteins of the subject or the sample, and (c) selecting one or more neoantigens from the sample if the HLA proteins from the subject bind to the HLA proteins of the subject with an IC50 of less than about 500nM, 400nM, 300nM, 200nM or 100nM; and optionally (d) ranking the neoantigens in order of lowest IC50 value to highest IC50 value.

[0429] In some embodiments, the present disclosure relates to producing a vaccine or manufacturing a pharmaceutical composition, comprising performing any one or more of the aforementioned steps, and further comprising subcloning a nucleic acid sequence encoding one or more new antigens into one or more nucleic acid molecules; optionally, suspending the nucleic acid molecules in one or more pharmaceutically acceptable carriers.

[0430] In some embodiments, the nucleic acid sequence encoding the neoantigen further comprises a linker. In some embodiments, the nucleic acid molecule does not contain a nucleic acid sequence encoding a P2A linker. In some embodiments, the nucleic acid molecule does not contain a nucleic acid sequence encoding two different linkers. In some embodiments, the nucleic acid molecule does not contain a nucleic acid sequence encoding a linker such that at least two or more AED sequences in a 5' to 3' orientation are encoded as separate polypeptides or larger continuous fusion proteins.

[0431] In another embodiment, the method of identifying one or more subject-specific DNA neoantigen mutations in a subject further comprises providing a culture comprising dendritic cells (DC) obtained from a subject; and contacting the dendritic cells with an immunogenic composition. DCs are potent antigen presenting cells that can initiate T cell immunity and can be used as cancer vaccines when loaded with one or more neoantigens of interest. In additional embodiments, the method further comprises administering dendritic cells to a subject; obtaining a population of CD8+T cells from a peripheral blood sample from a subject, wherein the CD8+ cells recognize at least one neoantigen; and expanding a population of CD8+T cells that recognize neoantigens.

[0432] In some embodiments, the population of expanded CD8+ T cells is administered to a subject.

[0433] Preferably, any suitable sequencing-by-synthesis platform can be used to identify mutations. There are currently four main sequencing-by-synthesis platforms: the genome sequencer of Roche / 454Life Sciences, the HiSeq analyzer of Illumina / Solexa, the SOLiD system of Applied BioSystems, and the Heliscope system of HelicosBiosciences. Pacific Biosciences and VisiGen Biotechnologies have also described sequencing-by-synthesis platforms. Each of these platforms can be used in the methods disclosed herein. In certain embodiments, the multiple nucleic acid molecules sequenced are bound to a support (e.g., a solid support). In order to fix nucleic acid on a support, capture sequences / universal priming sites can be added at the 3' and / or 5' ends of the template. By hybridizing the capture sequence with a complementary sequence covalently attached to the support, nucleic acid can be bound to the support. Capture sequence (also referred to as universal capture sequence) is a nucleic acid sequence complementary to the sequence attached to the support, which can double serve as a universal primer.

[0434] As an alternative to the capture sequence, a member of a coupling pair (such as, for example, an antibody / antigen, a receptor / ligand or an avidin-biotin pair, as described in, for example, U.S. Patent Application No. 2006 / 0252077) can be connected to each fragment to be captured on the surface of the corresponding second member coated with the coupling pair. After capture, the sequence can be analyzed, for example, by single molecule detection / sequencing, such as described in Examples and U.S. Patent No. 7,283,337, including template-dependent synthesis sequencing. In synthesis sequencing, the surface-bound molecules are exposed to a plurality of labeled triphosphate nucleotides in the presence of a polymerase. The sequence of the template is determined by the order of the labeled nucleotides incorporated into the 3' end of the growing chain. This can be done in real time or in a step-and-repeat mode. For real-time analysis, different optical labels for each nucleotide can be incorporated, and a plurality of lasers can be used to stimulate the incorporated nucleotides.

[0435] Any cell type or tissue can be used to obtain nucleic acid samples for use in the sequencing methods described herein. In some embodiments, DNA or RNA samples are obtained from neoplasia / tumors or body fluids (e.g., blood) or saliva obtained by known techniques (e.g., venipuncture). Alternatively, nucleic acid testing can be performed on dry samples (e.g., hair or skin).

[0436] There are a variety of methods for detecting the presence of specific mutations or alleles in the DNA or RNA of an individual. Advances in this field provide accurate, simple and inexpensive large-scale SNP genotyping. For example, several new technologies have been described recently, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrophosphate sequencing, oligonucleotide specific engagement, TaqMan system and various DNA "chip" technologies such as Affymetrix SNP chips. These methods require amplification of the target gene region, usually by PCR. There are also other newly developed methods, based on the production of small signal molecules by invasive cutting, followed by mass spectrometry or fixed padlock probes and rolling circle amplification, which may ultimately eliminate the need for PCR. Several methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. The method disclosed herein should be understood to include all available methods.

[0437] PCR-based detection means may include multiplexed amplification of multiple markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously.

[0438] Alternatively, different markers can be amplified using differentially labeled primers, and therefore each marker can be subjected to differential detection. Of course, hybridization-based detection means allow differential detection of multiple PCR products in the sample. Other techniques known in the art allow multiple markers to be subjected to multiplex analysis.

[0439] Several methods have been developed to promote the analysis of single nucleotide polymorphisms in genomic DNA or cell RNA. In certain embodiments, single base polymorphisms can be detected by using special exonuclease-resistant nucleotides, such as, for example, U.S. Patent number 4,656,127. According to the method, a primer complementary to the allele sequence of the next-to-polymorphic site 3' is allowed to hybridize with a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to the specific exonuclease-resistant nucleotide derivative present, the derivative will be incorporated into the end of the hybridized primer. This incorporation makes the primer resistant to exonucleases, and thus allows its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the discovery that the primer is resistant to exonucleases shows that the nucleotides present in the polymorphic site of the target molecule are complementary to the nucleotides of the nucleotide derivatives used in the reaction. The advantage of the method is that a large amount of irrelevant sequence data does not need to be determined.

[0440] In another embodiment of the present disclosure, a solution-based method is used to determine the identity of the nucleotide at the polymorphic site. Cohen et al. (French Patent No. 2,650,840; PCT Application No. WO1991 / 02087). As in the method in U.S. Patent No. 4,656,127, a primer complementary to the allele sequence immediately 3' of the polymorphic site can be used. The method uses a labeled dideoxynucleotide derivative to determine the identity of the nucleotide at the site, and if the deoxynucleotide derivative is complementary to the nucleotide at the polymorphic site, the derivative will be incorporated into the end of the primer.

[0441] An alternative method called Genetic Bit Analysis or GBA is described in PCT Application No. WO1992 / 15712. GBA uses a mixture of labeled terminators and primers complementary to the sequence 3' of the polymorphic site. Thus, the incorporated labeled terminator is determined by and complementary to the nucleotide present in the polymorphic site of the target molecule being assessed. In contrast to the method of Cohen et al. (French Patent 2,650,840; PCT Application No. WO1991 / 02087), the GBA method is preferably a heterogeneous analysis in which the primers or the target molecule are fixed to a solid phase.

[0442] Several primer-directed nucleotide incorporation procedures for determining polymorphic sites in DNA have been described (Komher, JS et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, BP, Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C, et al., Genomics 8:684-692 (1990); Kuppuswamy, MN et al., Proc. Natl. Acad. Sci. (USA) 88:1143-1147 (1991); Prezant, TR et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem. 208:171-175 (1993)). These methods differ from GBA in that they all rely on the incorporation of labeled deoxynucleotides to distinguish the bases at the polymorphic site. In this format, since the signal is proportional to the number of deoxynucleotides incorporated, polymorphisms occurring in a run of the same nucleotide can result in a signal that is proportional to the run length (Syvanen, A.-C, et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

[0443] Pharmaceutical composition

[0444] The term "pharmaceutical formulation" or "pharmaceutical composition" includes formulations suitable for administration to mammals, such as humans. When the compounds of the present disclosure are administered to mammals (e.g., humans) as drugs, they can be administered alone or as a pharmaceutical composition containing, for example, about 0.1% to about 99.5% of an active ingredient in combination with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a nucleic acid molecule containing an expressible nucleic acid sequence disclosed herein; and a pharmaceutically acceptable carrier comprising a saline buffer. In some embodiments, the saline buffer is a saline sodium citrate buffer.

[0445] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions when administered to humans, such as stomach upset, dizziness, etc. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0446] The phrase "pharmaceutically acceptable carrier" is art-recognized and includes pharmaceutically acceptable materials, compositions or vehicles suitable for administering the compounds of the present disclosure to mammals. Carriers include liquid or solid fillers, diluents, excipients, solvents or encapsulating materials involved in carrying or transporting the subject agent from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic compatible substances employed in pharmaceutical formulations. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin, which is incorporated herein by reference in its entirety.

[0447] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0448] Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0449] The formulations disclosed herein include those suitable for oral, nasal, intravenous, intraperitoneal, intramuscular, topical, buccal, sublingual, rectal, vaginal and / or parenteral administration. The formulation can be conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces the therapeutic effect. Typically, in one hundred percent, the range of this amount will be about 1% to about ninety-nine percent of the active ingredient, preferably about 5% to about 70%, most preferably about 10% to about 30%.

[0450] The method for preparing these formulations or compositions comprises the step of associating the nucleic acid sequence or composition of the present disclosure with a carrier and optionally one or more auxiliary components. Generally, the formulation is prepared by uniformly and intimately combining the compound of the present disclosure with a liquid carrier or a finely dispersed solid carrier or both, and then, if necessary, shaping the product.

[0451] Formulations of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules; or as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; or as pastilles (using an inert base such as gelatin and glycerin or sucrose and acacia); and / or as a mouthwash, etc., each containing a predetermined amount of a compound of the present disclosure as the active ingredient. The compounds of the present disclosure may also be administered as a bolus, electuary, or paste.

[0452] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or any of the following: fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; humectants such as glycerol; disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents such as paraffin; absorption promoters such as quaternary ammonium compounds; wetting agents such as, for example, cetyl alcohol and glyceryl monostearate; adsorbents such as kaolin and bentonite; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard shell gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols.

[0453] Tablets can be prepared by compression or molding optionally with one or more auxiliary components. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be manufactured by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0454] Tablets and other solid dosage forms of pharmaceutical compositions of the present disclosure, such as dragees, capsules, pills and granules, can be optionally coated or prepared with coatings and shells (such as enteric coatings and other coatings well-known in the field of drug formulation). These dosage forms can also be formulated to use, for example, different proportions of hydroxypropyl methylcellulose to provide a sustained release or controlled release of the active ingredient therein to provide a desired release profile, other polymer matrices, liposomes, anionic vesicles and / or microspheres. The dosage form can be, for example, filtered through a bacteria retention filter before use or sterilized by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium. These compositions can also optionally contain an emulsifier and the composition of the composition can be such that it only or preferably releases one or more active ingredients in a delayed manner in a certain part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. If appropriate, the active ingredient can also be in a microencapsulated form together with one or more of the above-mentioned excipients.

[0455] Liquid dosage forms for oral administration of compounds of the present disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran methanol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof.

[0456] In addition to inert diluents, the oral composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, aromatics and preservatives. In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth gum and mixtures thereof, and the like.

[0457] The formulation of the pharmaceutical composition for rectal or vaginal administration can exist as a suppository, which can be prepared by mixing one or more compounds of the present disclosure with one or more suitable non-irritating excipients or carriers (including, for example, cocoa butter, polyethylene glycol, suppository wax or salicylate), and it is solid at room temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active compound. The formulation of the present disclosure suitable for vaginal administration also includes pessaries, tampons, creams, gels, pastes, foams or spray formulations, which contain suitable carriers as known in the art. The dosage form for topical or transdermal administration of the compound of the present disclosure includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed with a pharmaceutically acceptable carrier and any preservative, buffer or propellant that may be needed under aseptic conditions.

[0458] In addition to the active compounds, the ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0459] Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0460] Transdermal patches have the additional advantage of providing controlled delivery of the disclosed compounds to the body. Such dosage forms can be made by dissolving or dispersing the compound in an appropriate medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing the active compound in a polymer matrix or gel.

[0461] Ophthalmic formulations, eye ointments, powders, solutions and the like are also contemplated to be within the scope of this disclosure.

[0462] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and / or elixirs. In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as, for example, water or other solvents such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, solubilizers and emulsifiers, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral composition may also contain additional therapeutic and / or prophylactic agents, additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and / or aromatics. In certain embodiments for parenteral administration, the composition is mixed with a solubilizer such as Alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof are mixed.

[0463] Pharmaceutical compositions suitable for parenteral administration comprise a combination of one or more compounds of the present disclosure with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders to be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending agents which render the formulation isotonic with the blood of the intended recipient, or thickening agents.

[0464] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared using suitable dispersants, wetting agents and / or suspending agents according to known techniques. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in nontoxic parenteral acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be adopted are water, Ringer's solution, USP and isotonic sodium chloride solution. Sterile fixed oils are generally used as solvents or suspension media. For this purpose, any gentle fixed oil can be adopted, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injectables.

[0465] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0466] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by using coating materials, such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0467] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can ensure to prevent the action of microorganisms by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also possible to expect that isotonic agents, such as sugar, sodium chloride, etc. are included in these compositions. In addition, the extended absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0468] In some cases, in order to prolong the effect of the drug, it may be desirable to slow down the absorption of the drug injected subcutaneously or intramuscularly. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, and then may depend on the crystal size and crystalline form. Alternatively, the delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oily vehicle.

[0469] Injectable depot forms are prepared by forming a microcapsule matrix of the subject compound in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of the drug to the polymer and the properties of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot-type injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The preparations can be administered orally, parenterally, topically or rectally. They can be in a form suitable for each route of administration. For example, these preparations are administered in tablet or capsule form by injection, inhalation, eye lotion, ointment, suppository, etc.; administered by injection, infusion or inhalation; topically by lotion or ointment; and rectally by suppository. Oral and / or IV administration is preferred.

[0470] As used herein, the term "parenteral administration" or "administered parenterally" means modes of administration other than enteral administration and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0471] As used herein, the terms "systemic administration" or "administered systemically" and "peripheral administration" or "administered peripherally" mean administration of a compound, drug, or other material other than directly into the central nervous system so that it enters the patient's system and is thereby subject to metabolism and other similar processes, e.g., subcutaneous administration.

[0472] The compounds can be administered to humans and other animals for therapy by any suitable route of administration, including oral, nasal (such as by, for example, a spray), rectal, vaginal, parenteral, intracisternal, and topical (such as by powders, ointments or drops), including buccal and sublingual.

[0473] Regardless of the route of administration selected, the compounds of the present disclosure and / or the pharmaceutical compositions of the present disclosure, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0474] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0475] The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the disclosure employed, or its ester, salt or amide; the route of administration; the time of administration; the rate of absorption of the particular compound employed; the duration of the treatment; other drugs, compounds and / or materials used in combination with the particular compound employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and like factors well known in the medical arts.

[0476] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the compound of the present disclosure used in the pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0477] Typically, the appropriate daily dose of a compound of the present invention will be the amount of the compound at the lowest dose that effectively produces a therapeutic effect. Such an effective dose generally depends on the above factors. In general, when used for a specified anti-tumor effect, the subcutaneous dose range of a compound of the present invention for a patient will be about 0.0001 mg to about 100 mg, about 0.01 mg to about 50 mg, about 0.1 mg to about 5 mg, about 0.2 mg to about 5 mg, and about 0.3 mg to about 3 mg per dose. An effective amount is an amount for treating Wnt signaling-related disorders.

[0478] If desired, the effective daily dose of active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the year, optionally in unit dosage form. While it is possible for the compounds of the present disclosure to be administered alone, it is preferred to administer the compounds as a pharmaceutical composition in any of the methods disclosed herein.

[0479] The compounds of the present disclosure are prepared from commonly available compounds using procedures known to those skilled in the art, including but not limited to any one or more of the disclosed conditions.

[0480] Within the scope of this document, unless the context indicates otherwise, only readily removable groups which are not an integral part of the specific desired end product of the compounds of the present disclosure are designated as "protecting groups." The protection of functional groups by such protecting groups, the protecting groups themselves and their cleavage reactions are described, for example, in standard reference works, such as, for example, Science of Synthesis: Houben-Weyl Methods of Molecular Transformation. GeorgThieme Verlag, Stuttgart, Germany. 2005. p. 41627 (URL: http: / / www.science-of-synthesis.com (electronic version, Vol. 48)); JFW McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973, in TW Greene and PGM Wuts, "Protective Groups in Organic Synthesis", 3rd edition, Wiley, New York 1999, in "The Peptides"; Volume 3 (Editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in "Methoden der organischen Chemie", Chemie" (Methods of Organic Chemistry), Houben Weyl, 4th edition, Volume 15 / I, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jeschkeit, "Aminosauren, Peptide, Proteine" (Amino acids, Peptides, Proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate" (Chemistry of Carbohydrates: Monosaccharides and Derivatives), Georg Thieme Verlag, Stuttgart 1974.A characteristic of protecting groups is that they are readily removable (ie, without undesired secondary reactions), for example, by solvolysis, reduction, photolysis, or alternatively under physiological conditions (eg, by enzymatic cleavage).

[0481] Acid addition salts of the disclosed compounds are most preferably formed from pharmaceutically acceptable acids, and include, for example, acid addition salts formed from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid, and organic acids, such as succinic acid, malic acid, acetic acid or fumaric acid. Other non-pharmaceutically acceptable salts, such as oxalates, can be used, for example, to isolate the disclosed compounds for laboratory use, or for subsequent conversion to pharmaceutically acceptable acid addition salts. Solvates and hydrates are also included within the scope of the present disclosure.

[0482] In vivo hydrolyzable esters or amides of certain compounds of the present disclosure can be formed by treating those compounds having free hydroxyl or amino functional groups with the acid chloride of the desired ester in an inert solvent such as dichloromethane or chloroform in the presence of a base. Suitable bases include triethylamine or pyridine. Conversely, compounds having free carboxyl groups can be esterified using standard conditions, which may include activation, followed by treatment with the desired alcohol in the presence of a suitable base.

[0483] Examples of pharmaceutically acceptable addition salts include, but are not limited to, nontoxic inorganic and organic acid addition salts such as hydrochlorides from hydrochloric acid, hydrobromides from hydrobromic acid, nitrates from nitric acid, perchlorates from perchloric acid, phosphates from phosphoric acid, sulfates from sulfuric acid, formates from formic acid, acetates from acetic acid, aconitates from aconitic acid, ascorbates from ascorbic acid, benzenesulfonates from benzenesulfonic acid, benzoates from benzoic acid, cinnamates from cinnamic acid, citrates from citric acid, pamoates from methylene pamoic acid, The enanthate of enanthate, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the mesylate derived from methanesulfonic acid, the naphthalene-2-sulfonate derived from naphthalene-2-sulfonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid, the p-toluenesulfonate derived from p-toluenesulfonic acid, etc. In certain embodiments, the salt is the sodium salt, lysine salt and arginine salt of the compound of the present disclosure. Such salts can be formed by procedures well known in the art and described.

[0484] Other acids not considered pharmaceutically acceptable, such as oxalic acid, may be used to prepare salts useful as intermediates in obtaining the chemical compounds of the disclosure and their pharmaceutically acceptable acid addition salts.Metal salts of the compounds of the disclosure include alkali metal salts, such as sodium salts of the chemical compounds of the disclosure containing a carboxyl group.

[0485] The mixtures of isomers obtainable according to the present disclosure can be separated into the individual isomers in a manner known per se; diastereomers can be separated, for example, by partitioning between multiphase solvent mixtures, recrystallization and / or chromatographic separation, for example on silica gel or by medium pressure liquid chromatography, for example on a reverse phase column, and racemates can be separated, for example, by salt formation with optically pure salt-forming agents and separation of the mixtures of diastereomers thus obtainable, for example by fractional crystallization or by chromatography on optically active column materials.

[0486] Intermediates and final products can be worked up and / or purified according to standard methods, for example using chromatographic methods, distribution methods, (re)crystallization, etc. The following generally applies to all processes mentioned throughout this disclosure.

[0487] The compounds, including their salts, can also be obtained in the form of hydrates, or their crystals may, for example, include the solvent used for crystallization. Different crystalline forms are possible.

[0488] The disclosure also relates to those forms of the process in which compounds obtainable as intermediates at any stage of the process are used as starting materials and the remaining process steps are carried out, or in which starting materials are formed under the reaction conditions or are used in the form of derivatives, for example in protected form or in the form of salts, or compounds obtainable by the process according to the disclosure are generated under the process conditions and further processed in situ.

[0489] The present disclosure relates to the discovery of molecular targets that cancer cells rely on for survival, and thus can be used to kill cancer cells. Cancer cells in tumors exhibit considerable heterogeneity, which is partly due to (epi)genetic differences between cells, but also due to the variability of their interactions with other cells and the extracellular matrix, and the variability of obtaining oxygen, nutrients and endocrine signals. Cell heterogeneity poses a problem for cancer therapy because there may be subpopulations that do not rely on such survival signals, and they may survive in therapy and restore the tumor population (Greaves and Maley, 2012). Cells with such population recovery capabilities are sometimes referred to as cancer stem cells (CSC) (Lobo et al., 2007; Valent et al., 2012); the elimination or redifferentiation of CSC is an attractive therapeutic strategy. Although such therapy itself may be ineffective, it may "homogenize" the cancer cell phenotype within the tumor, and the "homogenization" of the cancer cell phenotype improves the therapeutic response to traditional cancer therapy.

[0490] Methods of treating cancer

[0491] The present disclosure provides a method for treating and / or preventing cancer in a subject, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of any of the nucleic acid molecules described herein (e.g., a nucleic acid molecule comprising an expressible nucleic acid sequence comprising Formula I: [[(AEDn)-(linker)]n-[AEDn+1]), any of the pharmaceutical compositions described herein, or any pharmaceutically acceptable salt thereof. In some embodiments, the expressible nucleic acid sequence comprises at least about twenty AED domains, and wherein the expressible nucleic acid sequence does not contain a nucleic acid sequence encoding a WNT pathway tumor-specific antigen. In some embodiments, the expressible nucleic acid sequence comprises at least about twenty AED domains, and wherein the expressible nucleic acid sequence encodes at least one WNT pathway tumor-specific antigen. In some embodiments, the expressible nucleic acid sequence comprises at least about twenty AED domains, and wherein the expressible nucleic acid sequence encodes at least one or a combination of any two or more WNT pathway tumor-specific antigens disclosed herein.

[0492] In order to achieve treatment or immunization purposes, the nucleic acid molecules of the present invention can also be applied to subjects. Many methods can be conveniently used to deliver nucleic acids to patients. For example, nucleic acids can be directly delivered as "naked DNA". This method is described in, for example, Wolff et al., Science 247:1465-1468 (1990) and those zirconium-containing pretreatment solutions described in U.S. Patents No. 5,580,859 and No. 5,589,466. Ballistic delivery can also be used to administer nucleic acids, such as, for example, U.S. Patent No. 5,204,253. Particles containing only DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.

[0493] Nucleic acids can also be delivered in complex with cationic compounds such as cationic lipids. Lipid-mediated gene delivery methods are described in, for example, WO 1996 / 18372; WO 1993 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833; WO 1991 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

[0494] RNA encoding a peptide of interest can also be used for delivery (see, eg, Kiken et al., 2011; Su et al., 2011).

[0495] In some embodiments, the nucleic acid molecule is administered to the subject by electroporation.

[0496] In some embodiments, treatment is determined by clinical outcome, increase, enhancement or prolongation of T cell anti-tumor activity, increase in the number of anti-tumor T cells or activated T cells compared to the number before treatment, or a combination thereof. In other embodiments, the clinical outcome is selected from the group consisting of tumor regression, tumor shrinkage, tumor necrosis, anti-tumor response of the immune system, tumor expansion, recurrence or spread, or a combination thereof.

[0497] Examples of cancers and cancerous conditions that can be treated with the combination therapies of the present document include, but are not limited to, patients in need thereof who have been diagnosed with cancer or are at risk of developing cancer. In some embodiments, the subject has previously been treated but is no longer responding to checkpoint inhibitor therapy.

[0498] The therapies described herein are also applicable in situations where the subject does not have detectable neoplasia but is at high risk for disease recurrence.

[0499] According to the present disclosure, the nucleic acid molecules described herein can be used in patients who have been diagnosed with cancer or are at risk of developing cancer.

[0500] In some embodiments, the method of treatment further comprises the step of administering a nucleic acid sequence encoding a cytokine simultaneously or sequentially in addition to the nucleic acid sequence encoding the disclosed plurality of antigens. In some embodiments, the method comprises administering about 0.5 to about 2 milligrams of nucleic acid encoding the cytokine and about 0.1 to about 4 milligrams of nucleic acid encoding one or more antigens. In some embodiments, the nucleic acid sequence is administered as a plasmid suspended in a sterile sodium phosphate buffer or a sterile sodium citrate buffer.

[0501] In some embodiments, the pharmaceutical composition is intradermally administered to each of the first and second deltoid regions of the subject in two volumes. In some embodiments, the method further includes exposing the subject to the step of electroporation events at one or more injection sites after administering the nucleic acid sequence. Electroporation can be accomplished by exposing the injection site to electric pulses in a local manner. Examples of electroporation devices and electroporation methods preferably used to promote the delivery of the DNA vaccine of the present invention include those described in U.S. Patent No. 7,245,963 of Draghia-Akli et al., U.S. Patent Application Publication No. 2005 / 0052630 submitted by Smith et al., the contents of which are hereby incorporated by reference in their entirety. Also preferred are the electroporation devices and electroporation methods for facilitating delivery of DNA vaccines provided in co-pending and co-owned U.S. patent application Ser. No. 11 / 874072, filed on Oct. 17, 2007, which claims the benefit under 35 USC 119(e) of U.S. Provisional Application Ser. No. 60 / 852,149, filed on Oct. 17, 2006, and U.S. Provisional Application Ser. No. 60 / 978,982, filed on Oct. 10, 2007, all of which are hereby incorporated in their entirety.

[0502] U.S. Patent No. 7,245,963 to Draghia-Akli et al. describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues in the body or plants. The modular electrode system includes a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides conductive connections from a programmable constant current pulse controller to the plurality of needle electrodes and a power source. An operator can hold the plurality of needle electrodes mounted on a support structure and firmly insert them into a selected tissue in the body or plant. The biomolecules are then delivered through the hypodermic needle that enters the selected tissue. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of biomolecules into cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are hereby incorporated by reference.

[0503] U.S. Patent Application Publication No. 2005 / 0052630, which is incorporated herein by reference in its entirety, describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues in the body or plants. The electroporation device includes an electrokinetic device ("EKD" device), the operation of which is specified by software or hardware. The EKD device generates a series of programmable constant current pulse patterns between electrodes of the array based on user control and input of pulse parameters, and allows storage and acquisition of current waveform data. The electroporation device also includes a replaceable electrode tray having an array of needle-shaped electrodes, a central injection channel for an injection needle, and a removable guide tray.

[0504] The electrode array and method described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 0052630 are not only suitable for deep penetration of tissues such as muscles, but also suitable for deep penetration of other tissues or organs. Due to the configuration of the electrode array, the injection needle (to deliver the selected biomolecule) is also fully inserted into the target organ, and the injection is performed perpendicular to the target tissue in the area pre-demarcated by the electrode. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0505] In certain exemplary embodiments, the electroporation device may be configured to deliver energy pulses to the desired tissue of a mammal, generating a constant current similar to a preset current input by a user. The electroporation device includes an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include and incorporate one or more different elements of the electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. The electroporation component may function as an element of the electroporation device, and the other elements are separate elements (or components) that communicate with the electroporation component. In some embodiments, the electroporation component may function as more than one element of the electroporation device, which may communicate with other elements of the electroporation device separated from the electroporation component. The present invention is not limited to the elements of the electroporation device that exist as part of an electromechanical or mechanical device, because these elements may function as a device or as separate elements that communicate with each other. The electroporation component is capable of delivering energy pulses that generate a constant current in the desired tissue and include a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives an energy pulse from an electroporation component and delivers it to a desired tissue through the electrode. At least one electrode of the plurality of electrodes is neutral during the delivery of the energy pulse and measures impedance in the desired tissue and transmits the impedance to the electroporation component. A feedback mechanism may receive the measured impedance and may adjust the energy pulse delivered by the electroporation component to maintain a constant current.

[0506] In some embodiments, the plurality of electrodes may deliver energy pulses in a dispersed mode. In some embodiments, the plurality of electrodes may deliver energy pulses in a dispersed mode by electrode control under a programmed sequence, and the programmed sequence is input into the electroporation component by a user. In some embodiments, the programmed sequence comprises a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with a neutral electrode for measuring impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by another of the at least two active electrodes with a neutral electrode for measuring impedance.

[0507] In some embodiments, the feedback mechanism is implemented by hardware or software. Preferably, the feedback mechanism is implemented by an analog closed loop circuit. In some embodiments, the feedback occurs once every 50 μs, 20 μs, 10 μs or 1 μs, but real-time feedback or instantaneous feedback (i.e., substantially instantaneous as determined by available techniques for determining response time) is preferred. In some embodiments, the neutral electrode measures the impedance in the desired tissue and transmits the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the energy pulse to maintain the constant current at a value similar to the preset current. In some embodiments, the feedback mechanism continuously and instantly maintains the constant current during the delivery of the energy pulse.

[0508] In certain embodiments, the method comprises treating the cancer as a solid tumor.In some embodiments, the cancer has a high mutational burden (HMB).

[0509] Tumor mutation burden is the frequency of certain mutations within tumor genes. To be included in TMB, mutations must change the proteins expressed by the tumor. Methods for determining high mutation burden are known in the art and include, for example, next-generation whole exome sequencing of tumor tissue, which sequences all protein-coding genes within the tumor, and sequencing a gene panel that provides the sequence of the targeted genome. In some embodiments, tumors with high mutation burden have at least about 10 mutations per million base pairs of tumor DNA.

[0510] In other embodiments, the cancer has been shown to have a poor or low response to check point inhibitor therapy. In some embodiments, the subject has developed tolerance to check point inhibitor therapy. Resistance (poor or low response) or tolerance to check point inhibitor therapy is defined as measured disease progression despite the subject having been administered check point inhibitor therapy. In some embodiments, the subject is resistant to check point inhibitor therapy. In some embodiments, after treatment with a check point inhibitor, the subject exhibits a reduction in tumor mass of less than about 2%, with no reduction in tumor mass or tumor growth.

[0511] In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is adrenocortical carcinoma. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is bladder urothelial carcinoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is cervical adenocarcinoma. In some embodiments, the cancer is cervical squamous cell carcinoma. In some embodiments, the cancer is bile duct carcinoma. In some embodiments, the cancer is colorectal adenocarcinoma. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is diffuse glioma. In some embodiments, the cancer is endometrial carcinoma. In some embodiments, the cancer is endometrial carcinoma. In some embodiments, the cancer is esophageal squamous cell carcinoma. In some embodiments, the cancer is esophageal gastric adenocarcinoma. In some embodiments, the cancer is gastroesophageal junction adenocarcinoma. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is invasive breast cancer. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is a mature B cell tumor. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is a miscellaneous neuroepithelial tumor. In some embodiments, the cancer is a non-seminomatous tumor. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is ocular melanoma. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is an ovarian epithelial tumor. In some embodiments, the cancer is a pancreatic adenocarcinoma. In some embodiments, the cancer is a pancreatic ductal adenocarcinoma. In some embodiments, the cancer is a pheochromocytoma. In some embodiments, the cancer is a pleural mesothelioma. In some embodiments, the cancer is a prostate adenocarcinoma. In some embodiments, the cancer is a prostate cancer. In some embodiments, the cancer is a renal clear cell carcinoma. In some embodiments, the cancer is a renal non-clear cell carcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is a seminoma. In some embodiments, the cancer is a thymic carcinoma. In some embodiments, the cancer is a thymic epithelial tumor. In some embodiments, the cancer is a thyroid cancer. In some embodiments, the cancer is an undifferentiated gastric adenocarcinoma. In some embodiments, the cancer is a well-differentiated thyroid cancer.

[0512] In some embodiments, the cancer is adrenocortical carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is adrenocortical carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is bladder cancer comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is urothelial carcinoma of the bladder comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is breast cancer comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is cervical adenocarcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is cervical squamous cell carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is bile duct carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is colorectal adenocarcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is colorectal carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is diffuse glioma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is endometrial carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is endometrial carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is esophageal squamous cell carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is esophagogastric adenocarcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a gastroesophageal junction adenocarcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a glioblastoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a head and neck squamous cell carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a hepatocellular carcinoma (HCC) comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is an invasive breast cancer comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a leukemia comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a mature B-cell tumor comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a melanoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a mesothelioma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a miscellaneous neuroepithelial tumor comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a non-seminomatous tumor comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a non-small cell lung cancer comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is an ocular melanoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is an ovarian cancer comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is an ovarian epithelial tumor comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is pancreatic adenocarcinoma comprising a malfunction of the WNT pathway. In some embodiments, the cancer is pancreatic ductal adenocarcinoma comprising a malfunction of the WNT pathway.In some embodiments, the cancer is a pheochromocytoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a pleural mesothelioma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a prostate adenocarcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a prostate cancer comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a renal clear cell carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a renal non-clear cell carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a sarcoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a seminoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a thymic carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a thymic epithelial tumor comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a thyroid carcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is an undifferentiated gastric adenocarcinoma comprising a dysfunction of the WNT pathway. In some embodiments, the cancer is a well-differentiated thyroid carcinoma comprising a dysfunction of the WNT pathway.

[0513] In some embodiments, the cancer is an adrenocortical carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is an adrenocortical carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a bladder cancer comprising an activated WNT pathway. In some embodiments, the cancer is a bladder urothelial carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a breast cancer comprising an activated WNT pathway. In some embodiments, the cancer is a cervical adenocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a cervical squamous cell carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a cholangiocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a colorectal adenocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a colorectal carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a diffuse glioma comprising an activated WNT pathway. In some embodiments, the cancer is an endometrial carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is an endometrial carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is an esophageal squamous cell carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is an esophagogastric adenocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a gastroesophageal junction adenocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a glioblastoma comprising an activated WNT pathway. In some embodiments, the cancer is a head and neck squamous cell carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a hepatocellular carcinoma (HCC) comprising an activated WNT pathway. In some embodiments, the cancer is an invasive breast cancer comprising an activated WNT pathway. In some embodiments, the cancer is a leukemia comprising an activated WNT pathway. In some embodiments, the cancer is a mature B cell tumor comprising an activated WNT pathway. In some embodiments, the cancer is a melanoma comprising an activated WNT pathway. In some embodiments, the cancer is a mesothelioma comprising an activated WNT pathway. In some embodiments, the cancer is a miscellaneous neuroepithelial tumor comprising an activated WNT pathway. In some embodiments, the cancer is a non-seminomatous tumor comprising an activated WNT pathway. In some embodiments, the cancer is a non-small cell lung cancer comprising an activated WNT pathway. In some embodiments, the cancer is an ocular melanoma comprising an activated WNT pathway. In some embodiments, the cancer is an ovarian cancer comprising an activated WNT pathway. In some embodiments, the cancer is an ovarian epithelial tumor comprising an activated WNT pathway. In some embodiments, the cancer is pancreatic adenocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is pancreatic ductal adenocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is pheochromocytoma comprising an activated WNT pathway. In some embodiments, the cancer is pleural mesothelioma comprising an activated WNT pathway. In some embodiments, the cancer is prostate adenocarcinoma comprising an activated WNT pathway.In some embodiments, the cancer is a prostate cancer comprising an activated WNT pathway. In some embodiments, the cancer is a renal clear cell carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a renal non-clear cell carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a sarcoma comprising an activated WNT pathway. In some embodiments, the cancer is a seminoma comprising an activated WNT pathway. In some embodiments, the cancer is a thymic carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a thymic epithelial tumor comprising an activated WNT pathway. In some embodiments, the cancer is a thyroid carcinoma comprising an activated WNT pathway. In some embodiments, the cancer is an undifferentiated gastric adenocarcinoma comprising an activated WNT pathway. In some embodiments, the cancer is a well-differentiated thyroid carcinoma comprising an activated WNT pathway.

[0514] In some embodiments, the cancer is adrenocortical carcinoma with a high mutational burden. In some embodiments, the cancer is adrenocortical carcinoma with a high mutational burden. In some embodiments, the cancer is bladder cancer with a high mutational burden. In some embodiments, the cancer is urothelial carcinoma of the bladder with a high mutational burden. In some embodiments, the cancer is breast cancer with a high mutational burden. In some embodiments, the cancer is cervical adenocarcinoma with a high mutational burden. In some embodiments, the cancer is cervical squamous cell carcinoma with a high mutational burden. In some embodiments, the cancer is bile duct carcinoma with a high mutational burden. In some embodiments, the cancer is colorectal adenocarcinoma with a high mutational burden. In some embodiments, the cancer is colorectal cancer with a high mutational burden. In some embodiments, the cancer is diffuse glioma with a high mutational burden. In some embodiments, the cancer is endometrial carcinoma with a high mutational burden. In some embodiments, the cancer is endometrial carcinoma with a high mutational burden. In some embodiments, the cancer is esophageal squamous cell carcinoma with a high mutational burden. In some embodiments, the cancer is esophagogastric adenocarcinoma with a high mutational burden. In some embodiments, the cancer is gastroesophageal junction adenocarcinoma with a high mutational burden. In some embodiments, the cancer is a glioblastoma with a high mutation burden. In some embodiments, the cancer is a squamous cell carcinoma of the head and neck with a high mutation burden. In some embodiments, the cancer is a hepatocellular carcinoma (HCC) with a high mutation burden. In some embodiments, the cancer is an invasive breast cancer with a high mutation burden. In some embodiments, the cancer is a leukemia with a high mutation burden. In some embodiments, the cancer is a mature B-cell tumor with a high mutation burden. In some embodiments, the cancer is a melanoma with a high mutation burden. In some embodiments, the cancer is a mesothelioma with a high mutation burden. In some embodiments, the cancer is a miscellaneous neuroepithelial tumor with a high mutation burden. In some embodiments, the cancer is a non-seminomatous tumor with a high mutation burden. In some embodiments, the cancer is a non-small cell lung cancer with a high mutation burden. In some embodiments, the cancer is an ocular melanoma with a high mutation burden. In some embodiments, the cancer is an ovarian cancer with a high mutation burden. In some embodiments, the cancer is an ovarian epithelial tumor with a high mutation burden. In some embodiments, the cancer is a pancreatic adenocarcinoma with a high mutation burden. In some embodiments, the cancer is a pancreatic ductal adenocarcinoma with a high mutation burden. In some embodiments, the cancer is a pheochromocytoma with a high mutation burden. In some embodiments, the cancer is pleural mesothelioma with a high mutation burden. In some embodiments, the cancer is prostate adenocarcinoma with a high mutation burden. In some embodiments, the cancer is prostate cancer with a high mutation burden. In some embodiments, the cancer is renal clear cell carcinoma with a high mutation burden. In some embodiments, the cancer is renal non-clear cell carcinoma with a high mutation burden. In some embodiments, the cancer is a sarcoma with a high mutation burden.In some embodiments, the cancer is a seminoma with a high mutation burden. In some embodiments, the cancer is a thymic carcinoma with a high mutation burden. In some embodiments, the cancer is a thymic epithelial tumor with a high mutation burden. In some embodiments, the cancer is a thyroid carcinoma with a high mutation burden. In some embodiments, the cancer is an undifferentiated gastric adenocarcinoma with a high mutation burden. In some embodiments, the cancer is a well-differentiated thyroid carcinoma with a high mutation burden.

[0515] In some embodiments, the cancer is adrenocortical carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is adrenocortical carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is bladder cancer that is unresponsive to immunotherapy. In some embodiments, the cancer is urothelial carcinoma of the bladder that is unresponsive to immunotherapy. In some embodiments, the cancer is breast cancer that is unresponsive to immunotherapy. In some embodiments, the cancer is cervical adenocarcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is cervical squamous cell carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is bile duct carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is colorectal adenocarcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is colorectal carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is diffuse glioma that is unresponsive to immunotherapy. In some embodiments, the cancer is endometrial carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is endometrial carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is esophageal squamous cell carcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is esophagogastric adenocarcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is a gastroesophageal junction adenocarcinoma that is not responsive to immunotherapy. In some embodiments, the cancer is a glioblastoma that is not responsive to immunotherapy. In some embodiments, the cancer is a head and neck squamous cell carcinoma that is not responsive to immunotherapy. In some embodiments, the cancer is a hepatocellular carcinoma (HCC) that is not responsive to immunotherapy. In some embodiments, the cancer is an invasive breast cancer that is not responsive to immunotherapy. In some embodiments, the cancer is a leukemia that is not responsive to immunotherapy. In some embodiments, the cancer is a mature B-cell tumor that is not responsive to immunotherapy. In some embodiments, the cancer is a melanoma that is not responsive to immunotherapy. In some embodiments, the cancer is a mesothelioma that is not responsive to immunotherapy. In some embodiments, the cancer is a miscellaneous neuroepithelial tumor that is not responsive to immunotherapy. In some embodiments, the cancer is a non-seminomatous tumor that is not responsive to immunotherapy. In some embodiments, the cancer is a non-small cell lung cancer that is not responsive to immunotherapy. In some embodiments, the cancer is an ocular melanoma that is not responsive to immunotherapy. In some embodiments, the cancer is an ovarian cancer that is not responsive to immunotherapy. In some embodiments, the cancer is an ovarian epithelial tumor that is not responsive to immunotherapy. In some embodiments, the cancer is a pancreatic adenocarcinoma that is not responsive to immunotherapy. In some embodiments, the cancer is pancreatic ductal adenocarcinoma that is unresponsive to immunotherapy. In some embodiments, the cancer is pheochromocytoma that is unresponsive to immunotherapy. In some embodiments, the cancer is pleural mesothelioma that is unresponsive to immunotherapy. In some embodiments, the cancer is prostate adenocarcinoma that is unresponsive to immunotherapy.In some embodiments, the cancer is a prostate cancer that is not responsive to immunotherapy. In some embodiments, the cancer is a renal clear cell carcinoma that is not responsive to immunotherapy. In some embodiments, the cancer is a renal non-clear cell carcinoma that is not responsive to immunotherapy. In some embodiments, the cancer is a sarcoma that is not responsive to immunotherapy. In some embodiments, the cancer is a seminoma that is not responsive to immunotherapy. In some embodiments, the cancer is a thymic carcinoma that is not responsive to immunotherapy. In some embodiments, the cancer is a thymic epithelial tumor that is not responsive to immunotherapy. In some embodiments, the cancer is a thyroid cancer that is not responsive to immunotherapy. In some embodiments, the cancer is an undifferentiated gastric adenocarcinoma that is not responsive to immunotherapy. In some embodiments, the cancer is a well-differentiated thyroid cancer that is not responsive to immunotherapy.

[0516] In some embodiments, the cancer is an adrenocortical carcinoma that is unresponsive to or resistant to check point inhibitor therapy. In some embodiments, the cancer is an adrenocortical carcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a bladder cancer that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a bladder urothelial carcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a breast cancer that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a cervical adenocarcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a cervical squamous cell carcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a bile duct carcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a colorectal adenocarcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a colorectal carcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a diffuse glioma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is an endometrial carcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is an endometrial carcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is an esophageal squamous cell carcinoma that does not respond to check point inhibitor therapy. In some embodiments, the cancer is an esophagogastric adenocarcinoma that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a gastroesophageal junction adenocarcinoma that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a glioblastoma that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a head and neck squamous cell carcinoma that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a hepatocellular carcinoma (HCC) that does not respond to check point inhibitor therapy. In some embodiments, the cancer is an invasive breast cancer that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a leukemia that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a mature B cell tumor that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a melanoma that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a mesothelioma that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a miscellaneous neuroepithelial tumor that does not respond to check point inhibitor therapy. In some embodiments, the cancer is a non-seminomatous tumor that does not respond to check point inhibitor therapy. In some embodiments, the cancer is non-small cell lung cancer that is unresponsive to checkpoint inhibitor therapy. In some embodiments, the cancer is ocular melanoma that is unresponsive to checkpoint inhibitor therapy. In some embodiments, the cancer is ovarian cancer that is unresponsive to checkpoint inhibitor therapy. In some embodiments, the cancer is an ovarian epithelial tumor that is unresponsive to checkpoint inhibitor therapy.In some embodiments, the cancer is a pancreatic adenocarcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a pancreatic ductal adenocarcinoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a pheochromocytoma that is unresponsive to check point inhibitor therapy. In some embodiments, the cancer is a pleural mesothelioma that is unresponsive to check point inhibitor therapy. In some embodiments, ...

Claims

1. A composition comprising a nucleic acid sequence encoding about 1 to about 100 amino acid sequences, wherein the amino acid sequences are tumor-specific antigens, wherein at least one tumor-specific antigen is an amino acid sequence associated with the WNT pathway.

2. The composition according to claim 1, further comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises the nucleic acid sequence encoding the tumor-specific antigen; wherein the nucleic acid molecule comprises a regulatory sequence operably linked to the nucleic acid sequence encoding one or more of the tumor-specific antigens; wherein the nucleic acid sequence encodes about 1 to about 60 tumor-specific antigens, each antigen being flanked by at least one linker on a contiguous amino acid sequence, and Wherein the nucleic acid sequence encodes a leader sequence on the 5' end of the first antigen sequence in a 5' to 3' orientation.

3. The composition according to claim 1 or 2, wherein the tumor-specific antigen is selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1 and GSK3B.

4. The composition of any one of claims 1 to 3, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding β-catenin or a fragment thereof, wherein the nucleic acid sequence encoding the β-catenin or a fragment thereof comprises at least about 70% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:

11.

5. The composition of any one of claims 1 to 4, wherein the antigen expression domain comprises a nucleic acid encoding β-catenin or a fragment thereof, wherein the nucleic acid sequence encoding the β-catenin or a fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:

11.

6. The composition according to any one of claims 1 to 5, wherein the tumor-specific antigen does not contain a nucleic acid sequence comprising 100% of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO:

11.

7. The composition according to any one of claims 1 to 6, wherein the linker is a P2A linker sequence or a furin linker sequence.

8. The composition of any one of claims 1 to 7, wherein the nucleic acid sequence encodes about 20 to about 60 tumor-specific antigens; and wherein about 1 to about 8 tumor-specific antigens are selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3B.

9. The composition according to any one of claims 1 to 8, wherein the nucleic acid sequence encodes a leader sequence and one or more tumor specific antigens in a 5' to 3' original orientation, wherein the one or more tumor antigens are flanked by at least one linker sequence.

10. A cell comprising any one or more compositions according to claims 1 to 9.

11. A composition comprising a nucleic acid molecule comprising any one or more nucleic acid sequences according to claims 1 to 9.

12. A pharmaceutical composition comprising: (i) a therapeutically effective amount of one or more compositions according to any one of claims 1 to 9 or 11; (ii) a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, wherein the composition comprises a plasmid comprising an expressible nucleic acid sequence encoding about 40 to about 60 tumor-specific antigens.

14. A pharmaceutical composition according to any one of claims 12 or 13, wherein the pharmaceutical composition comprises a therapeutically effective amount of plasmid pVAX0001, wherein the plasmid pVAX0001 comprises a nucleic acid sequence encoding about 1 to about 7 tumor-specific antigens, wherein the tumor-specific antigens are selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1 and GSK3Bh.

15. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (i) a pharmaceutical composition comprising nucleic acid sequences encoding about twenty or more neoantigens or epitopes specific for neoantigens; or (ii) The pharmaceutical composition according to any one of claims 12 to 14.

16. The method of claim 15, further comprising administering to the subject a therapeutically effective amount of a checkpoint inhibitor.

17. The method of claim 16, wherein the checkpoint inhibitor is selected from one or a combination of the checkpoint inhibitors in Table 1.

18. The method of any one of claims 15 to 17, wherein the therapeutically effective amount of a checkpoint inhibitor is about 150 mg to about 250 mg.

19. The method of any one of claims 15 to 18, wherein the dosage of the pharmaceutical composition is about 0.3 to about 3 mg per subject.

20. The method of any one of claims 15 to 19, wherein the administering step is accomplished by intravenous, intramuscular, intraperitoneal, or intradermal injection following electroporation of transfected cells.

21. The method according to any one of claims 15 to 20, wherein the pharmaceutical composition comprises an expressible nucleic acid sequence comprising Formula I: [(AED n )–(connector)] n –[AED n+1 ], wherein AED is an independently selectable antigen expression domain, wherein AED n is the first antigen expression domain and wherein AEDn+1 is the second antigen expression domain; wherein the length of each linker is independently selectable to be about 0 to about 300 natural or non-natural nucleic acids, wherein the length of antigen expression domain 1 is independently selectable to be about 12 to about 15,000 nucleotides and encodes a new antigenic epitope; wherein the length of antigen expression domain 2 is independently selectable to be about 12 to about 15,000 nucleotides and encodes a new antigenic epitope; and wherein n is any positive integer from about 19 to about 500.

22. A method of preventing resistance to checkpoint inhibitor therapy in a subject, the method comprising administering to the subject in need thereof: (i) a pharmaceutical composition according to any one of claims 12 to 14; or (ii) A pharmaceutical composition comprising nucleic acid sequences encoding about twenty or more neo-antigens or epitopes specific to neo-antigens.

23. The method of claim 22, wherein the composition comprises a plasmid comprising expressible nucleic acid sequences encoding about 40 to about 60 tumor-specific antigens.

24. The method of claim 22 or 23, wherein the pharmaceutical composition comprises a therapeutically effective amount of pGX0001, wherein the pGX0001 comprises a nucleic acid sequence encoding about 1 to about 7 tumor-specific antigens, wherein the tumor-specific antigens are selected from one or a combination of the following: WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

25. The method of any one of claims 22 to 24, wherein the subject has a cancer characterized by a high tumor burden.

26. The method of any one of claims 22 to 25, wherein the subject has a cancer characterized by one or more mutations in WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

27. The method of any one of claims 22 to 26, wherein the subject has a cancer characterized by one or a combination of aberrant regulation of expression of WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

28. The method of any one of claims 22 to 27, wherein the subject has a cancer characterized by overamplification of one or a combination of amino acid sequences comprising at least 70% sequence identity to WNT, CTNNB1, AXIN1, AXIN2, APC, CK1, and GSK3Bh.

29. The method of any one of claims 22 to 28, wherein the dosage of the pharmaceutical composition is about 0.3 to about 3 mg per subject.

30. The method of any one of claims 22 to 29, wherein the administering step is accomplished by intravenous, intramuscular, intraperitoneal, or intradermal injection following electroporation of transfected cells.

31. The method of any one of claims 22 to 30, wherein the subject has a cancer characterized by dysfunction of the WNT pathway.

32. The method of any one of claims 22 to 31, wherein the subject has hepatocellular carcinoma.

33. A method of inducing an immune response in a cell, the method comprising exposing the cell to a composition according to any one of claims 1 to 11.

34. The method of claim 33, wherein the exposing step is performed in vivo.

35. A method of enhancing a CD8+ T cell response in a subject, the method comprising administering to the subject a pharmaceutical composition according to any one of claims 12 to 14.

36. The method of claim 35, wherein the CD8+ T cell response is enhanced by about 10% to about 15% compared to the CD8+ T cell response of a subject not treated with the pharmaceutical composition.

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