Individualized cancer epitopes and methods of using the same

Abstract

The present disclosure relates to methods of treating cancer or preventing metastasis of cancer in a subject in need thereof. The present disclosure further relates to compositions useful for adoptive immunotherapy, comprising a heterogeneous population of T cells having reactivity against an individualized cancer epitope or neoantigen, and methods for making such T cell compositions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 63 / 342,608, filed May 16, 2022, and U.S. Application No. 63 / 340,058, filed May 10, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference The Sequence Listing, submitted as an XML file entitled "GENE-001-PCT_SL," created on May 10, 2023, and having a size of 569,374 bytes, is incorporated herein by reference in accordance with 37 C.FR § 1.52(e)(5).

[0003] Field The present disclosure relates to methods of treating cancer or preventing metastasis of cancer in a subject in need of therapy or prevention. The present disclosure further relates to compositions useful for adoptive immunotherapy comprising heterogeneous populations of T cells with reactivity to individualized cancer epitopes or neoantigens, and methods for making such T cell compositions. [Background technology]

[0004] After the human immune system first detects an antigen, a population of antigen-recognizing T cells is generated over several days, and these T cells subsequently determine the nature of the response to that antigen. Antigen recognition and specificity by T cells are conferred by the structural characteristics of the T cell receptor (TCR) expressed on the T cell surface. Thus, the antigen specificity of a T cell is characterized by the presence and function of a specific TCR displayed by the T cell. A single T cell possesses a TCR that can bind to a single antigen presented in combination with a specific major histocompatibility complex molecule, i.e., MHC.

[0005] Adoptive transfer of ex vivo expanded antigen-specific T cells was shown to confer immunity against CMV and EBV as early as the 1990s. See Riddell et al., Science, 1992, 257:238 (Non-Patent Document 1); Rooney et al., Blood, 1998, 92:1549-1555 (Non-Patent Document 2). Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) or cells genetically engineered to express anti-cancer antigen TCRs has also been shown to produce positive clinical responses in some cancer patients. However, during tumor progression, the immune response to tumors becomes focused on a few "dominant" antigens that are ineffective at promoting tumor regression. Previous attempts to use ex vivo expanded T cells in immunotherapy have inadvertently expanded tumor-associated dominant antigen-reactive T cells, resulting in inconsistent results. Furthermore, obstacles remain to successfully use adoptive cell therapy for the widespread treatment of cancer and other diseases. For example, T cells and TCRs that specifically recognize cancer antigens can be difficult to identify and / or isolate from patients. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Riddell et al.,Science,1992,257:238 [Non-patent document 2] Rooney et al.,Blood,1998,92:1549-1555 Summary of the Invention

[0007] The present disclosure relates to methods of treating cancer or preventing metastasis of cancer in a subject in need of such therapy or prevention.

[0008] In one aspect, the present disclosure relates to a method of treating cancer involving one or more neoantigens in a subject in need thereof, the method comprising: (a) administering to a subject in need thereof one or more nucleic acid sequences encoding the one or more neoantigens; (b) allowing a period of time sufficient for clonal T cells primed against the one or more neoantigens in the subject to expand to biologically significant numbers; (c) isolating the clonal T cells from the subject; (d) identifying one or more nucleotide sequences encoding subsets of T cell receptors (TCRs) that are highly immunogenic in response to the one or more neoantigens in the subject; and (e) administering to a subject in need thereof a therapeutically effective amount of T cells comprising nucleic acid molecules encoding the one or more TCR subsets.

[0009] In some embodiments, clonal T cells are isolated by taking a blood sample from a subject and sorting peripheral blood mononuclear cells (PBMCs) from the sample according to receptor expression on the surface of the PBMCs.

[0010] In some embodiments, step (d) comprises performing an assay that measures one or a combination of: (i) the avidity or affinity with which cells expressing the TCR bind to cells in vitro; and (ii) the percentage of CD8+ and / or CD4+ on cells expressing the TCR.

[0011] In some embodiments, the method further comprises sequencing one or more nucleotide sequences encoding a subset of TCRs that are highly immunogenic from T cells that express the TCRs.

[0012] In some embodiments, the method further comprises identifying one or more antigens from a tissue sample removed from the subject.

[0013] In some embodiments, the tissue sample comprises tissue from a brushing, biopsy, or surgical resection of a subject.

[0014] In some embodiments, the methods do not include in vitro expansion of PBMCs and / or tumor-infiltrating lymphocytes.

[0015] In some embodiments, the total number of clonal T cells primed against one or more antigens in a subject comprises about 0.01% to about 10% CD8+ reactivity against one or more neo-antigens.

[0016] In some embodiments, step (a) comprises administering a nucleic acid molecule comprising one or more nucleotide sequences encoding one or more neoantigens. In some embodiments, the nucleic acid molecule encodes from about 10 to about 55 neoantigens. In some embodiments, each neoantigen encoded by the nucleic acid molecule is separated from another by one or more linkers. In some embodiments, the one or more linkers comprise a furin protease cleavage site or a porcine teschovirus-1 2A (P2A) cleavage site.

[0017] In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the expressible nucleic acid sequence is located within a multiple cloning site of a plasmid selected from (i) pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES, or (ii) a plasmid containing at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES.

[0018] In some embodiments, the nucleic acid molecule is GNOS-PV02.

[0019] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell carcinoma or bone and soft tissue sarcoma, hematologic neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma soft tissue sarcoma, and small cell lung cancer.

[0020] In another aspect, the present disclosure relates to a method of treating cancer that expresses one or more neoantigens in a subject in need thereof, comprising: (a) administering to the subject one or more nucleic acid sequences encoding the one or more neoantigens; and (b) administering to the subject a therapeutically effective amount of T cells from the subject that comprise one or more nucleic acid sequences encoding one or more T cell receptors (TCRs) or functional fragments thereof, wherein the T cells are highly immunogenic in response to the one or more neoantigens.

[0021] In some embodiments, the methods do not include in vitro expansion of PBMCs and / or tumor-infiltrating lymphocytes.

[0022] In some embodiments, the method further comprises allowing the subject to mount an immune response against one or more neo-antigens.

[0023] In some embodiments, the method further comprises, after step (a) but before step (b), sequencing one or more nucleic acid sequences encoding one or more TCRs or functional fragments thereof from T cells isolated from the subject.

[0024] In some embodiments, the method includes, after step (a), allowing the subject a period of time sufficient to expand a clonal T cell population primed against the one or more neo-antigens, wherein the clonal T cell population comprises about 25% to about 50% CD8+ reactivity against the one or more neo-antigens.

[0025] In some embodiments, the method comprises a step comprising administering a nucleic acid molecule comprising one or more nucleic acid sequences encoding one or more neo-antigens.

[0026] In some embodiments, the expressible nucleic acid sequence encodes about 10 to about 55 neoantigens. In some embodiments, each neoantigen encoded by the nucleic acid molecule is separated from another by one or more linkers. In some embodiments, the one or more linkers comprise a furin protease cleavage site or a porcine teschovirus-1 2A (P2A) cleavage site.

[0027] In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the nucleic acid molecule is GNOS-PV02.

[0028] In some embodiments, the expressible nucleic acid sequence is located within a multiple cloning site of (i) a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES, GNOS-PV02, or (ii) a plasmid containing at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), GNOS-PV02, phCMV1, pTCP, and pIRES.

[0029] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell carcinoma or bone and soft tissue sarcoma, hematologic neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma soft tissue sarcoma, and small cell lung cancer.

[0030] In yet another aspect, the present disclosure relates to a method for producing a population of T cells expressing one or more TCRs or functional fragments thereof that recognize one or more neo-antigens, the method comprising: (a) administering to a subject comprising one or more cells that express the one or more neo-antigens one or more nucleic acid sequences encoding the one or more neo-antigens; and (b) isolating from the subject clonally derived T cells that express the one or more TCRs or functional fragments thereof.

[0031] In some embodiments, the method does not include in vitro expansion of PBMCs and / or tumor-infiltrating lymphocytes. In some embodiments, the method further comprises, after step (b), sequencing one or more nucleic acid sequences encoding one or more TCRs or functional fragments thereof.

[0032] In some embodiments, the method further comprises, after step (a), allowing the subject a period of time sufficient to expand the clonal T cell population primed against the one or more neo-antigens.

[0033] In some embodiments, the clonal T cell population comprises about 25% to about 50% CD8+ reactivity to one or more neo-antigens.

[0034] In some embodiments, the method further comprises transferring one or more nucleic acid sequences encoding one or more TCRs or functional fragments thereof into T cells obtained from the subject.

[0035] In yet another aspect, the present disclosure relates to a method of preventing metastasis of a cancer comprising one or more neoantigens in a subject, the method comprising: (a) administering to the subject one or more nucleic acid sequences encoding the one or more neoantigens; (b) allowing a period of time sufficient for clonal T cells primed against the one or more neoantigens in the subject to expand to biologically significant numbers; (c) isolating the clonal T cells from the subject; (d) identifying one or more nucleotide sequences encoding a subset of TCRs that are highly immunogenic in response to the one or more neoantigens in the subject; and (e) administering to a subject in need thereof a therapeutically effective amount of T cells comprising nucleic acid molecules encoding the one or more TCR subsets.

[0036] In some embodiments, clonal T cells are isolated by taking a blood sample from a subject and sorting peripheral blood mononuclear cells (PBMCs) from the sample according to receptor expression on the surface of the PBMCs.

[0037] In some embodiments, step (d) comprises performing an assay that measures one or a combination of: (i) the avidity or affinity with which cells expressing the TCR bind to cells in vitro; and (ii) the percentage of CD8+ and / or CD4+ on cells expressing the TCR.

[0038] In some embodiments, the method further comprises sequencing one or more nucleotide sequences encoding a subset of TCRs that are highly immunogenic from T cells that express the TCRs.

[0039] In some embodiments, the method further comprises identifying one or more antigens from a tissue sample removed from the subject.

[0040] In some embodiments, the tissue sample comprises tissue from a brushing, biopsy, or surgical resection of a subject.

[0041] In some embodiments, the methods do not include in vitro expansion of PBMCs and / or tumor-infiltrating lymphocytes.

[0042] In some embodiments, the total number of clonal T cells primed against one or more antigens in a subject comprises about 25% to about 50% CD8+ reactivity against one or more neo-antigens.

[0043] In some embodiments, step (a) comprises administering a nucleic acid molecule comprising one or more nucleotide sequences encoding one or more neo-antigens.

[0044] In some embodiments, the nucleic acid molecule encodes about 10 to about 55 neoantigens. In some embodiments, the disclosure relates to a composition comprising a nucleic acid molecule encoding about 10 to about 55 neoantigens. In some embodiments, the disclosure relates to a composition comprising a nucleic acid molecule encoding about 19 to about 60 neoantigens. In some embodiments, the disclosure relates to a composition comprising a nucleic acid molecule encoding about 20 to about 60 neoantigens. In some embodiments, the disclosure relates to a composition comprising a nucleic acid molecule encoding about 20 to about 65 neoantigens.

[0045] In some embodiments, each neo-antigen encoded by the nucleic acid molecule is separated from another by one or more linkers, in some embodiments, the one or more linkers comprise a furin protease cleavage site or a porcine teschovirus-1 2A (P2A) cleavage site.

[0046] In some embodiments, the nucleic acid molecule is a plasmid.

[0047] In some embodiments, the nucleic acid molecule is placed within a multiple cloning site of (i) a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES, or (ii) a plasmid containing at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES.

[0048] In some embodiments, the nucleic acid molecule is GNOS-PV02.

[0049] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell carcinoma or bone and soft tissue sarcoma, hematologic neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma soft tissue sarcoma, and small cell lung cancer. [Brief explanation of the drawings]

[0050] [Figure 1]Schematic diagram of the disclosed method for developing personalized T cell vaccines based on tumor-specific neoantigens. Because the majority of neoantigens are unique to each patient's cancer, the "mutanome" of each patient's tumor is determined, allowing for the identification of candidate neoantigens for vaccine targeting. Neoantigen-specific T cells are activated and expanded in vivo by administering one or more DNA or RNA vaccines encoding one or more patient-specific neoantigens. T cells are then isolated from the patient and assayed to identify T cell receptors (TCRs) that are highly immunogenic in response to one or more patient-specific neoantigens. Once identified, engineered T cells expressing such TCRs can be generated and administered to the patient. [Figure 2] 1 shows the predicted MHC class I / II binding affinity (nM) of the pooled composition for immunoassays with the GEN-PV-001 vaccine. [Figure 3] We show that using a 33-amino acid sequence for each neoantigen in a vaccine allows for the encoding of potential CD8 and CD4 epitopes encompassing the neoantigen, resulting in a more effective adaptive immune response. CD8 epitopes are more commonly 9 amino acids long but can range from 8 to 14 amino acids, and CD4 epitopes are generally 15 amino acids long but can range from 9 to 25 amino acids. See Chong et al., Mol. Cell Proteomics, 2018, 17(3):533-548. This means that the 33-amino acid sequence should encompass all predicted epitopes in the neoantigen, including overlapping CD8 and CD4 epitopes. [Figure 4A]Figures 4A-4B show the immune responses to the tested neoantigens. Figure 4A: The immune responses (IFNγ ELISpot) to the tested neoantigens (marked with an asterisk in the schematic) are not significantly different when located at positions 1, 10, 20, 30, or 40 of the 40-epitope neoantigen DNA vaccine (4806 nucleotide insert). Figure 4B: The immune responses (IFNγ ELISpot) to a vaccine containing 30 epitopes (27 neoantigens and 3 tumor-associated antigens) divided into four pools: Pool 1 contains vaccine epitopes 1-8, Pool 2 contains vaccine epitopes 9-16, Pool 3 contains vaccine epitopes 17-23, and Pool 4 contains vaccine epitopes 24-30. After vaccination of patients with anaplastic astrocytoma, responses were observed to all four pools, demonstrating that the DNA neoantigen vaccines are expressed as protein antigens present across the entire length of the DNA sequence. [Figure 4B] See legend to Figure 4A. [Figure 5] Figure 1 shows that T cell responses (IFNγ ELISpot) were detected against 17 of the 30 neoantigens encoded by the GEN-PV-001 vaccine. [Figure 6] Heatmap of intracellular cytokine staining (ICS) analysis. Both CD8+ and CD4+ T cell responses were detected against epitopes encoded by the GEN-PV-001 vaccine, and responses were directed against multiple markers. Heatmap ranges are in % and represent the % of CD4+ or CD8+ T cells expressing the indicated marker in a peptide-specific manner (peptide stimulation minus vehicle control). [Figure 7A] Figures 7A-7B show a neoantigen DNA vaccine that generated CD8 and CD4 responses to multiple epitopes in cancer patients. Figure 7A: Heatmap ranges are in % and represent the % of CD4+ or CD8+ T cells expressing the indicated markers in a peptide-specific manner (peptide stimulation - vehicle control). Figure 7B: Representative flow plots showing the presence of CD8+, CD4+ T cells producing IFNγ, TNFα, or both in a peptide-specific manner. [Figure 7B] See the legend to Figure 7A. [Figure 8A] Figure 8A is a non-limiting example of a manufacturing process for a personalized DNA vaccine. Needle-to-needle development has been achieved in as little as 6 weeks and can routinely be achieved in 6-8 weeks. Figure 8B is a non-limiting example of a clinical trial. [Figure 8B] See the legend to Figure 8A. [Figure 9A] Figure 9A shows a spider plot depicting the first 12 patients in the clinical trial at the time of data cut. Figure 9B shows a waterfall plot depicting the best overall response achieved by the first 12 subjects in the clinical trial at the time of data cut. The best overall response represents a 25% partial response rate and a 67% disease control rate. Figure 9C shows tumor imaging scans (day 0 vs. week 27 post-treatment) of a patient classified as a PR. The red arrow points to the tumor. [Figure 9B] See the legend to Figure 9A. [Figure 9C] See the legend to Figure 9A. [Figure 10A] Figure 10A shows a bar graph showing that all patients analyzed to date (n = 10) had newly detected and expanded T cell clones after treatment with GNOS-PV02. Figure 10B shows the cumulative frequency of expanded clones pre-vaccination versus post-vaccination (week 9) in peripheral blood (PBMC, left) and tumor tissue (right) per patient. Figure 10C shows the expansion of a pre-vaccination clone (points along the X-axis) and the detection of multiple new T cell clones (points along the Y-axis) post-vaccination in blood and tumor tissue from subject Pt7. Arrows highlight the infiltration of frequent clones from blood to tumor 9 weeks post-vaccination (only the top six clones are shown for clarity). Most abundant clones exhibit an active phenotype (CD8+CD69+) as assessed by TCRβ and RNA sequencing. Approximately 75% of new TIL clones were not detected in the blood before vaccination. [Figure 10B] See the legend to Figure 10A. [Figure 10C] See the legend to Figure 10A. [Figure 11A] Figure 11A shows that patient-specific clonal TCR sequences were gene-optimized using the GOAL algorithm and inserted into the pMXs-IRES-GFP retroviral plasmid vector, which contains viral packaging signals, transcription and processing elements, and a GFP reporter gene. Figure 11B shows an example of post-vaccination anti-tumor-specific T cell reactivity assessed by ELISpot (subject Pt8). PBMCs were stimulated with a pool of peptides or individual peptides encoded in the personalized GNOS-PV02 treatment. Figure 11C shows representative images of activated GFP-positive CD8 and CD4 TCR-engineered T cells (subject Pt8) stimulated with ATP1A1-ALB (10 μg / mL). TNTC: too numerous to count; EOT: end of treatment. [Figure 11B] See the legend to Figure 11A. [Figure 11C] See the legend to Figure 11A. [Figure 12A]Figure 12 shows that PTCV drives neoantigen-specific responses detected in the blood. Figure 12A shows the results of ELISpot assays from PBMC samples for the presence of vaccine-induced neoantigen-specific responses before and after personalized GNOS-PV02 vaccination without cytokine stimulation. The post-vaccination response is each patient's "best" (maximum) response across time points. Figure 12B shows the positive neoantigens (black and red bars, respectively) pre- and post-vaccination as defined by IFNγ ELISpot assay relative to the total number contained in each patient's PTCV (gray bars). Figure 12C shows the percentage of positive response epitopes by clinical response group and by time points before and during treatment. Figure 12D shows the Spearman correlation between positive epitopes and the total number of neoantigens contained in each patient's PTCV. Figure 12E shows neoantigen-specific T cell activation assessed by ex vivo stimulation of patient-derived PBMCs (weeks 9 or 12) with DMSO or patient-specific epitope pools using intracellular cytokine staining. Figure 12F shows polyfunctional assessment by Boolean gating on the CD4+ or CD8+ cytokine+ population. To assess the cytolytic potential of neoantigen-reactive T cells, T cell activation (CD69 and CD107a) and proliferation (Ki67) were assessed along with double-positive expression of granzyme A (GrzA) and perforin (Prf). Results are expressed as the % of the positive cell population (parental frequency). Black circles represent individual patients; boxes extend from the 25th to the 75th percentile; the line within the box is the median; and whiskers extend from the minimum to the maximum. Four patients (one CR, three PR) were analyzed. Figure 12G shows polyfunctional assessment by Boolean gating on the CD4+ or CD8+ cytokine+ population. To assess the cytolytic potential of neoantigen-reactive T cells, T cell activation (CD69 and CD107a) and proliferation (Ki67) were assessed, along with double-positive expression of granzyme A (GrzA) and perforin (Prf). Results are expressed as the % of the positive cell population (parental frequency). Black circles represent individual patients, boxes extend from the 25th to the 75th percentile, the line within the box is the median, and whiskers extend from the minimum to the maximum.Four patients (1 CR, 3 PR) were analyzed. [Figure 12B] See the legend to Figure 12A. [Figure 12C] See the legend to Figure 12A. [Figure 12D] See the legend to Figure 12A. [Figure 12E] See the legend to Figure 12A. [Figure 12F] See the legend to Figure 12A. [Figure 12G] See the legend to Figure 12A. [Figure 13A]Figure 13 shows that GNOS-PV02 leads to the expansion of new T cell clones that traffic to tumors. Figure 13A shows that in 14 of 14 subjects, T cell clones expanded in the periphery, and new or expanded clones were enriched in each patient's matched tumor sample. The sum of PBMC and tumor-associated T cell expansions was calculated by comparing post-treatment PBMC or tumor samples with pre-treatment PBMC or tumor samples, respectively (differential abundance statistical analysis). Figure 13B shows that GNOS-PV02 leads to the expansion of new T cell clones that traffic to tumors. The cumulative frequency of peripherally expanded TCR rearrangements tracked in pre- and post-treatment tumor biopsies is shown. Figure 13C shows that GNOS-PV02 leads to the expansion of new T cell clones that traffic to tumors. The number of significantly expanded clones found in pre- and post-treatment tumor biopsies is shown. Circles represent individual patients, boxes extend from the 25th to 75th percentile, the line within the box is the median, and whiskers extend from the minimum to the maximum. Figure 13D shows that GNOS-PV02 leads to the expansion of new T cell clones that traffic to tumors. TCR clonality and TCR repertoire richness are shown in tumor biopsies from 14 evaluated patients (bar graphs and stacked bar graphs). Error bars correspond to the upper SE for each group. Simpson clonality reports the distribution of TCR rearrangements in the samples, with 0 indicating an even distribution of frequencies and 1 indicating an asymmetric distribution. Lower numbers indicate more focused TCR diversity. Figure 13E shows that GNOS-PV02 leads to the expansion of new T cell clones that traffic to tumors. TCR clonality and TCR repertoire richness are shown in tumor biopsies from 14 evaluated patients (bar graphs and stacked bar graphs). Error bars correspond to the upper SE for each group. Simpson clonality reports the distribution of TCR rearrangements in a sample, with 0 indicating an even distribution of frequencies and 1 indicating an asymmetric distribution. Lower numbers indicate more focused TCR diversity. [Figure 13B] See the description of Figure 13A. [Figure 13C] See the description of Figure 13A. [Figure 13D] See the description of Figure 13A. [Figure 13E] See the description of Figure 13A. [Figure 14A] Figure 14 shows that post-vaccination expanded TCR clones identified in tumors are reactive to PTCV-encoded antigens. Figure 14A shows the most frequent TCRs identified by TCRβ and RNA sequencing in patients. Pre-vaccination vs. 9 weeks post-vaccination (pairwise scatter plot). Blue asterisks indicate selected high-frequency new T cell clones detected in PBMCs after vaccination and their abundance in tumors. Orange, green, and gray circles represent expanded, contracted, and unchanged T cell clones, respectively. Figure 14B shows evaluated TCRs selected for cloning based on their high frequency occurrence in PBMCs and trafficking to tumors after vaccination. Figure 14C shows the results of patient-specific clonal TCR sequences optimized and inserted into the pMXs-IRES-GFP retroviral plasmid vector, which contains viral packaging signals, transcription and processing elements, and a GFP reporter gene. Figures 14D and 14E show that TCR-engineered T cells (GFP-positive) derived from unvaccinated PBMCs were stimulated with increasing concentrations of epitope pools (2, 10, and 25 μg / mL) for 6 hours, and CD69 expression was assessed by flow cytometry. Peptide pools 1 and 2 contain either the first or second half of the neoantigen contained in PTCV. [Figure 14B] See the legend to Figure 14A. [Figure 14C] See the legend to Figure 14A. [Figure 14D] See the legend to Figure 14A. [Figure 14E] See the legend to Figure 14A. DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description The disclosed methods and compositions may be understood more readily by reference to the following detailed description of specific embodiments and the examples included therein, as well as the drawings and their preceding and subsequent descriptions.

[0052] It is to be understood that the disclosed methods and compositions are not limited to particular synthetic methods, specific analytical techniques, or particular reagents, unless otherwise specified, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0053] It is understood that the disclosed methods and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0054] definition It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a nucleic acid sequence" includes a plurality of nucleotides formed, and a reference to "the nucleic acid sequence" is a reference to one or more nucleic acid sequences and equivalents thereof known to those skilled in the art.

[0055] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value, such as a quantity, "about" is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, or ±0.1% from the specified value, when such variations are appropriate for performing the disclosed methods. When "about" is present before a series of numbers or ranges, it is understood that "about" can modify each of the numbers in the series or range.

[0056] As used herein, the terms "activate," "stimulate," "enhance," "increase," and / or "induce" (and similar terms) are generally used interchangeably to refer to the act of directly or indirectly improving or increasing a concentration, level, function, activity, or behavior relative to natural, expected, or average, or relative to a control condition. "Activate," in the context of immunotherapy, refers to a primary response induced by binding of a cell surface moiety. For example, in the context of a receptor such as a TCR, such stimulation involves receptor binding and a subsequent signaling event. In some embodiments, activating means that a TCR engages a tumor-specific epitope or antigen, and a TCR-containing cell responds by releasing an immunostimulatory agent in response to the engagement. Furthermore, a stimulatory event can activate a cell and upregulate or downregulate the expression or secretion of molecules. Thus, binding of a cell surface moiety, even in the absence of a direct signaling event, can result in rearrangements of cytoskeletal structure or coalescence of cell surface moieties, each of which can be useful in enhancing, modifying, or altering subsequent cellular responses. In some embodiments, an epitope is activated or highly immunogenic if, upon engagement with the neoantigen or tumor-specific epitope, cells release IFN-gamma, TNF-alpha, or IL-2. In some embodiments, a neoantigen or TCR is highly immunogenic if cells comprising the TCR are clonally expanded and exposed to the neoantigen and about 20%, 25%, 30%, 35%, or about 40% or more of the population secrete IFN-gamma, TNF-alpha, or IL-2. In some embodiments, a neoantigen or TCR is highly immunogenic against or with the neoantigen if cells comprising the TCR are clonally expanded and exposed to the neoantigen and about 20%, 25%, 30%, 35%, or about 40% or more of the cell population express one or more immunostimulatory agents in response to the presence of or engagement with the neoantigen. In some embodiments, highly immunogenic TCRs are those TCRs with respect to binding or association with neo-antigens:

[0057] As used herein, the term "CD8+ T cell activation" or "CD8+ T cell activation" refers to a process (e.g., a signaling event) that causes or results in one or more cellular responses selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers of CD8+ T cells (CTLs). As used herein, "activated CD8+ T cells" refer to CD8+ T cells that have received an activation signal and therefore exhibit one or more cellular responses selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. Suitable assays for measuring CD8+ T cell activation are known in the art and are described herein.

[0058] Activation can be defined as: >50 IFNγ spots / 1×10 PBMCs as assessed by ELISpot; >0.05% IFNγ-positive T cells as assessed by FACS; >100 pg / mL as assessed by ELISA; and >2-fold IFNγ mRNA expression. Furthermore, activation can be defined by CD137 and / or CD69 expression, which can be measured by RNA sequencing, flow cytometry, or ELISA. Alternatively, cells can be sorted using bead- or column-bound antibodies against CD137, CD69, CD25, and / or CD38.

[0059] As used herein, the term "adjuvant" is meant to refer to any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigen encoded by the DNA plasmid and the encoding nucleic acid sequence described below.

[0060] As used herein, the term "allogeneic" refers to medical therapy in which the donor and recipient are different individuals of the same species.

[0061] As used herein in the specification and claims, the term "and / or" should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Other elements other than those specifically identified by the "and / or" clause may optionally be present, whether related or not to the elements specifically identified, unless clearly indicated to the contrary. Thus, as a non-limiting example, when used in combination with open-ended language such as "comprising," a reference to "A and / or B" may refer, in some embodiments, to A without B (optionally including elements other than B); in other embodiments, to B without A (optionally including elements other than A); in yet other embodiments, to both A and B (optionally including other elements), etc.

[0062] As used herein, "antigen" or "Ag" refers to a molecule that elicits an immune response, which may include antibody production, activation of specific immunologically competent cells (e.g., T cells), or both. Antigens may be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. Antigens can be synthesized using methods known in the art, recombinantly produced, or derived from biological samples. For example, novel antigens can be generated using methods known in the art, such as chromosomal rearrangements or breakages. Exemplary biological samples that may contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens may be produced by cells that have been modified or genetically engineered to express the antigen. The term "antigen" includes antigenic determinants that are 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acid residues in length, that bind to an MHC molecule, that form part of an MHC class I or II complex, or that are recognized when complexed with such a molecule, etc.

[0063] As used herein, the term "antigen-presenting cell (APC)" refers to a class of cells that can present one or more antigens in the form of peptide-MHC complexes that can be recognized by specific effector cells of the immune system, thereby inducing an effective cellular immune response against the presented antigen or antigens. Examples of professional APCs are dendritic cells and macrophages, but any cell that expresses MHC class I or II molecules can potentially present peptide antigens.

[0064] As used herein, the term "anti-tumor response" refers to an immune system response that includes, but is not limited to, activating T cells to attack antigens or antigen-presenting cells.

[0065] As used herein, the term "autologous" refers to a medical therapy in which the donor and recipient are the same human being.

[0066] As used herein, the term "cancer" refers to any disease caused by or resulting from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. Examples of cancer include, but are not limited to, non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell carcinoma or bone and soft tissue sarcoma, hematological neoplasms, multiple myeloma, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma, soft tissue sarcoma, and small cell lung cancer.

[0067] As used herein, the term "checkpoint inhibitor" is meant to refer to any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof, that inhibits an inhibitory pathway and allows broader immune activity. In certain embodiments, the checkpoint inhibitor is an inhibitor of the programmed death-1 (PD-1) pathway, for example, but not limited to, an anti-PD1 antibody, such as niboiumab. In other embodiments, the checkpoint inhibitor is an anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) antibody. In still additional embodiments, the checkpoint inhibitor targets a member of the TNF superfamily, such as CD40, OX40, CD137, GITR, CD27, or TIM-3. In some cases, targeting of the checkpoint inhibitor is achieved with an inhibitory antibody or similar molecule. In other cases, it is achieved with an agonist of the target. Examples of this class include the stimulatory targets OX40 and GITR.

[0068] As used herein, the term "combination therapy" refers to the sequential administration of one or more therapeutic agents, i.e., each agent is administered at a different time, as well as the substantially simultaneous administration of these therapeutic agents, or at least two therapeutic agents. For example, sequential administration includes separately administering (i) a pharmaceutical composition comprising an effective amount of a pooled sample of tumor-specific neoantigens or DNA / RNA encoding same and a pharmaceutically acceptable salt, carrier, or diluent, (ii) an effective amount of IL-12 or DNA / RNA encoding same, and (iii) an effective amount of a checkpoint inhibitor and a pharmaceutically acceptable salt, carrier, or diluent. Substantially simultaneous administration can be achieved, for example, by administering to a subject a single capsule having a fixed ratio of each therapeutic agent, or by administering multiple single capsules for each therapeutic agent. For example, one combination therapy of the present disclosure may include a pooled sample of tumor-specific neoantigens or DNA / RNA encoding same, IL-12 or DNA / RNA encoding same, and a checkpoint inhibitor, and pharmaceutically acceptable salts, carriers, or diluents, administered simultaneously or at different times. In some embodiments, the pharmaceutical compositions of the present disclosure may be formulated as a single co-formulated pharmaceutical composition containing two or three compounds. As another example, in some embodiments, the combinations of the present disclosure (e.g., a DNA neo-antigen vaccine, IL-12, and a checkpoint inhibitor) are formulated as separate pharmaceutical compositions that can be administered at the same time or at different times. As used herein, the term "concurrently" refers to the simultaneous administration of one or more agents. For example, in certain embodiments, a cancer vaccine or immunogenic composition and a checkpoint inhibitor are administered simultaneously. "Concurrently" includes administration "contemporaneously," i.e., for the same period of time. In certain embodiments, one or more agents are administered simultaneously at the same time or on the same day. Sequential or substantially simultaneous administration of each therapeutic agent may be achieved by any suitable route, including, but not limited to, oral, intravenous, subcutaneous, intramuscular, direct absorption via mucosal tissues (e.g., nose, mouth, vagina, and rectum), and ocular routes (e.g., intravitreal, intraocular, etc.).Therapeutic agents can be administered by the same route or by different routes. For example, in some embodiments, one component of a particular combination is administered by intravenous injection, while the other component(s) of the combination are administered orally. The components can be administered in any therapeutically effective order. A "combination" encompasses groups of compounds or non-drug therapies useful as part of a combination therapy.

[0069] As used herein, the term "cytotoxic T cells" or "cytotoxic T lymphocytes" refers to a type of immune cell that contains CD receptors capable of recognizing one or more antigens and can kill certain cells, including foreign cells, tumor cells, and cells expressing the antigen. Cytotoxic T cells can be separated from other blood cells, grown ex vivo, and then administered to a patient to kill tumor or viral cells that express the antigen recognizable by the cell. Cytotoxic T cells are a subset of white blood cells and a type of lymphocyte.

[0070] As used herein, the term "dendritic cell" or "DC" describes a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues. See Steinman, Ann. Rev. Immunol. 9:271-296 (1991).

[0071] As used herein, the terms "electroporation," "electropermeabilization," or "electrokinetic enhancement" ("EP") are used interchangeably and are meant to refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes whose presence allows biomolecules, such as plasmids, oligonucleotides, siRNA, drugs, ions, and / or water, to pass from one side of the cell membrane to the other.

[0072] As used herein, the terms "endogenous" or "native" refer to a gene, protein, or activity present in an in vivo host cell. A gene, protein, or activity that is mutated, overexpressed, shuffled, duplicated, or otherwise altered compared to the normal, non-mutated gene, protein, or activity is still considered endogenous or native to that particular in vivo host cell. For example, endogenous control sequences from a first gene (e.g., promoter, translational attenuation sequence) can be used to alter or regulate expression of a second native gene or nucleic acid molecule, where the expression or regulation of the second native gene or nucleic acid molecule differs from its normal expression or regulation in the parent cell.

[0073] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion preferably comprises at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may comprise 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more nucleotides or amino acids.

[0074] The term "functional fragment" refers to any portion of a polypeptide of sufficient length to retain at least partial biological function similar or substantially similar to the biological function of the wild-type polypeptide on which the fragment is based. A functional fragment of a TCR disclosed herein is a fragment of a TCR disclosed herein that maintains at least partial binding affinity for its target. In some embodiments, the functional fragment has a length of at least about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 contiguous amino acids. In some embodiments, the functional fragment has a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the functional fragment has a length of about 25 amino acids to about 35 amino acids. In some embodiments, the functional fragment has a length of about 27 amino acids to about 35 amino acids. In some embodiments, the functional fragment has a length of about 29 amino acids to about 35 amino acids. In some embodiments, the functional fragment has a length of about 31 amino acids to about 35 amino acids. In some embodiments, the functional fragment is a fragment of a TCR disclosed herein and has a length of at least about 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids.

[0075] As used herein, the term "genetic construct" is meant to refer to a DNA or RNA molecule comprising a nucleotide or ribonucleotide sequence encoding one or more amino acid sequences. In some embodiments, the amino acid sequence is a protein, a protein fragment, or an antigen. In some embodiments, the genetic construct comprises one or more coding sequences and one or more regulatory sequences. In some embodiments, the coding sequence comprises an initiation sequence and a termination sequence operably linked to a regulatory element. In some embodiments, the regulatory element comprises a promoter and a polyadenylation signal capable of directing expression in cells of an individual to whom the DNA or RNA molecule is administered.

[0076] As used herein, a "heterologous" or "exogenous" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to the host cell but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of a heterologous or exogenous nucleic acid molecule, construct, or sequence may be from a different genus or species. In some embodiments, a heterologous or exogenous nucleic acid molecule is added to a host cell or host genome (i.e., is not endogenous or native) by, for example, conjugation, transformation, transfection, electroporation, etc., and the added molecule may be integrated into the host genome or may exist as extrachromosomal genetic material (e.g., a plasmid or other form of self-replicating vector), and may exist in multiple copies. Additionally, "heterologous" or "exogenous" refers to a non-native enzyme, protein, or other activity encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity.

[0077] The term "host cell," as used herein, refers to a cell that can be used to express a genetic construct, such as a nucleic acid, of the present disclosure. The cell may 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" may be used to refer to a host cell that has been transformed or transfected with an expressing nucleic acid. A host cell may also be a cell that contains a claimed nucleic acid sequence but does not express it at a level sufficient to elicit an immune response unless a regulatory sequence is introduced into the host cell so that it is operably linked to the nucleic acid. It is understood that the term host cell, in some embodiments, refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still within the scope of the term as used herein.

[0078] As used herein, the term "hybridize" refers to the pairing of a complementary polynucleotide sequence (e.g., a gene described herein) or a portion thereof to form a double-stranded molecule under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507.)

[0079] As used herein, "immune cell" or "immune system cell" refers to any cell of the immune system that originates from hematopoietic stem cells in the bone marrow and gives rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, mast cells, platelets, erythrocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells, or "lymphocytes"). As used herein, the term "lymphocyte" refers to a subtype of white blood cell of the vertebrate immune system, characterized by its predominant presence in lymph and generally large nuclei. Lymphocytes include, for example, T cells (CD4+ T cells, CD8+ T cells, CD4-CD8- double-negative T cells, γδ T cells, regulatory T cells), B cells, and natural killer (NK) cells. Other exemplary immune system cells include macrophages and dendritic cells, as well as other myeloid cells described herein. Macrophages and dendritic cells may be referred to as "professional antigen-presenting cells" (or "professional APCs"), which are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of the APC interact with TCRs on the surface of the T cell. Alternatively, any hematopoietic stem cell or immune system cell can be converted into an APC by introducing a nucleic acid molecule that expresses an antigen recognized by a TCR or another antigen-binding protein (e.g., a chimeric antigen receptor or antibody). The immune cells or lymphocytes used in the vaccine compositions or treatment methods of the present disclosure can be autologous, allogeneic, or syngeneic to the subject receiving the composition or treatment method.

[0080] As used herein, the term "immune checkpoint" is meant to refer to an inhibitory pathway that slows or stops the immune response and prevents excessive tissue damage due to uncontrolled activity of immune cells.

[0081] As used herein, the term "immune response" is meant to refer to the activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of a nucleic acid molecule comprising a nucleotide sequence encoding a neoantigen as described herein.

[0082] As used herein, an "immunogenicity enhancer" includes a molecule encoded by a polynucleotide contained in a host cell, such as a T cell, that enhances the immunogenicity of an exogenous neoantigen encoded by a polynucleotide contained in the cell. Host cell-encoded immunogenicity enhancers can provide localized and concentrated adjuvant activity that improves the immune response to the neoantigen. Exemplary immunogenicity enhancers include IL-12 (such as membrane-bound IL-12), GM-CSF, inducible cell death factor, bacterial flagellin, CD80, CD137L, CD40L, secreted IL-2, secreted IL-2 that binds to T cells independently of CD25, secreted IL-15, secreted IL-15-IL-15Rα complex, secreted IFNβ, secreted IFN-α1, secreted IL-7, or any combination thereof. In some embodiments, the immunogenic enhancer is endogenously expressed by the host cell (e.g., the host cell endogenously expresses, for example, GM-CSF), in which case the host cell is engineered to increase expression of the immunogenic enhancer, or the immunogenic enhancer is exogenous to the host cell.

[0083] As used herein, the phrase "in need of" means that an animal or mammal has been identified as having or is suspected of having a need for a particular method or treatment. In some embodiments, identification can be by any means of diagnosis or observation. An animal or mammal may be in need of any of the methods and treatments described herein. In some embodiments, a subject in need thereof is a human seeking cancer prevention. In some embodiments, a subject in need thereof is a human diagnosed with cancer. In some embodiments, a subject in need thereof is a human seeking cancer treatment. In some embodiments, a subject in need thereof is a human undergoing cancer treatment. In some embodiments, a subject in need thereof is a healthy subject.

[0084] The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection," or "transformation," or "transduction," and includes reference to incorporating a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be integrated into the cell's genome (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon contained in an episomal expression vector (see, e.g., Van Caenenbroeck et al., Eur. J. Biochem. 267:5665 (2000)), or transiently expressed (e.g., transfected mRNA).

[0085] As used herein, the term "isolated" means that a polynucleotide or polypeptide, or a fragment, variant, or derivative thereof, is essentially removed from other biological material with which it is naturally associated, or from, for example, recombinant host cells that have been genetically engineered to express the polypeptide of the invention.

[0086] As used herein, the term "ligand" is meant to refer to a molecule that has a structure complementary to that of a receptor and can form a complex with this receptor. According to embodiments of the present disclosure, a ligand is specifically understood to mean a peptide or peptide fragment that has a suitable length and a suitable binding motif in its amino acid sequence, such that the peptide or peptide fragment can form a complex with an MHC class I or MHC class II protein.

[0087] As used herein, the terms "MHC molecule," "MHC protein," or "HLA protein" refer to proteins that bind to peptides resulting from proteolytic cleavage of protein antigens, represent potential T cell epitopes, and transport them to the cell surface, where they can be presented to specific cells, specifically cytotoxic T lymphocytes or T helper cells. The major histocompatibility complex in the genome contains gene regions whose expressed gene products on the cell surface are important for binding and presenting endogenous and / or foreign antigens and are therefore important for regulating immunological processes. The major histocompatibility complex is divided into two gene groups encoding different proteins: MHC class I molecules and MHC class II molecules. The two MHC class molecules are specialized for 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 distinct roles. Structurally, class I MHC molecules consist of heavy and light chains and can bind and present peptides of approximately 8 to 11 amino acids, usually 9 or 10 amino acids, to cytotoxic T lymphocytes if the peptide has a suitable binding motif. The heavy chain of a class I MHC molecule is preferably an HLA-A, HLA-B, or HLA-C monomer, and the light chain is β-2-microglobulin. Class II MHC molecules consist of α and β chains and can bind and present peptides of approximately 15 to 24 amino acids to T helper cells if the peptide has a suitable binding motif. The α and β chains are specifically HLA-DR, HLA-DQ, and HLA-DP monomers.

[0088] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference, wild-type, or endogenous nucleic acid sequence or polypeptide sequence, respectively. Mutations can result in several different types of changes in sequence, including substitution, insertion, or deletion of nucleotide(s) or amino acid(s). In some embodiments, the mutation is a substitution of one or more codons or amino acids. In some embodiments, the mutation is an insertion of one or more codons or amino acids. In some embodiments, the mutation is a deletion of one or more codons or amino acids. In some embodiments, the mutation is any combination of substitution of one or more codons or amino acids, insertion of one or more codons or amino acids, and deletion of one or more codons or amino acids.

[0089] As used herein, "neoantigen" refers to a host cell product that contains a structural change, alteration, or mutation that creates a new antigen or antigenic epitope not previously observed in a subject's genome (i.e., in a sample of healthy tissue from the subject) or not "seen" or recognized by the host's immune system. Neoantigens can arise, for example, from encoding a polynucleotide with an alteration (substitution, addition, deletion) that results in an altered or variant product, or from the insertion of an exogenous nucleic acid molecule or protein into a cell, or from exposure to an environmental agent (e.g., chemical, radiological) that results in a genetic change. Neoantigens can arise separately from, or from, or associated with tumor antigens. A "tumor neoantigen" (or "tumor-specific neoantigen") refers to a protein containing a neoantigenic determinant associated with, arising from, or arising within a tumor cell or multiple cells within a tumor. Tumor neo-antigenic determinants are found, for example, on antigenic tumor proteins or peptides containing one or more somatic mutations encoded by the DNA of tumor cells, as well as proteins or peptides from viral open reading frames associated with virus-associated tumors (e.g., cervical cancer, some head and neck cancers). For example, tumor neo-antigens can arise within or from any of the exemplary tumor or other antigens, as well as from "driver" cancer antigens (e.g., the G12D neo-antigen from KRAS described in Tran et al., N. Eng. J. Med. 375:2255-2262 (2016)), as well as mutant B-Raf, SF31, MYD88, DDX3X, MAPK1, GNB1, etc.).

[0090] As used herein, the term "neo-antigenic mutation" refers to a mutation that is predicted to encode a neo-antigenic peptide that can be expressed on the surface of cancer cells of a subject.

[0091] As used herein, the term "nucleic acid molecule" includes one or more nucleotide sequences encoding one or more proteins. In some embodiments, the nucleic acid molecule includes initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in cells of an individual to whom the nucleic acid molecule is administered. In some embodiments, the nucleic acid molecule also includes a plasmid containing one or more nucleotide sequences encoding one or more neo-antigens. In some embodiments, the present disclosure relates to pharmaceutical compositions comprising first, second, third, or more nucleic acid molecules, each of which, independently or concomitantly, encodes one or more neo-antigens, and at least one of the plasmids includes one or more of the formulas disclosed herein.

[0092] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of that nucleotide sequence (e.g., the level, timing, or location of expression). 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 location of expression). Regulatory sequences can exert their effect, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). For further examples of regulatory sequences, see, e.g., 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, incorporated herein by reference.

[0093] 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 separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., the inclusion of at least one of a number or list of elements, but more than one, and, optionally, additional unlisted items. Only terms clearly indicated otherwise, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. Generally, as used herein, the term "or" should be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by the terms of exclusivity "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0094] The "percent identity" or "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. As used herein, "identical" or "identity" in the context of two or more nucleic acid or amino acid sequences can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions at which identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths or the alignment results in one or more staggered ends, and a specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm stands for Basic Local Alignment Search Tool and is suitable for determining sequence similarity. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). This algorithm involves first obtaining a query sequence that matches or meets a positive threshold score T when aligned with words of the same length in database sequences, which identifies high-scoring sequence pairs (HSPs) by identifying short words. T is referred to as the neighborhood word score threshold (Altschul et al., supra).These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is stopped when: 1) the cumulative alignment score drops by a quantity X from its maximum achieved value, 2) the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below 0, 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 as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, incorporated herein by reference in its entirety), alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered to be similar to another nucleic acid if the smallest sum probability in a comparison of the test nucleic acid with the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. Two single-stranded polynucleotides are "complements" of each other if their sequences can be aligned in an antiparallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without the introduction of gaps and without unpaired nucleotides at the 5' or 3' end of either sequence.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.

[0095] As used herein, "peripheral blood mononuclear cells" or "PBMCs" are any peripheral blood cells with a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes. In humans, the majority of the PBMC population is made up of lymphocytes, followed by monocytes, and a small proportion of dendritic cells.

[0096] As used herein, the term "pharmaceutically acceptable" refers to approved or deemed approvable by a regulatory agency of a federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, including humans.

[0097] As used herein, the term "pharmaceutically acceptable excipient, carrier, or diluent" is meant to refer to an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy its pharmacological activity, and is not toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0098] As used herein, the term "pharmaceutically acceptable salts" of tumor-specific neoantigens can be acid or base salts generally considered in the art to be suitable for use in contact with human or animal tissues without undue 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 or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, maleic, glycolic, fumaric, sulfuric, sulfamic, suifanilic, formic, toluenesulfonic, methanesulfonic, benzenesulfonic, ethanedisulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic acids such as acetic, HOOC-(CH)-COOH (n is 0-4), etc. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize, from this disclosure and knowledge in the art, additional pharmaceutically acceptable salts for the pooled tumor-specific neoantigens provided herein, including those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.

[0099] The term "pharmaceutical composition" includes, but is not limited to, (i) a pharmaceutical composition comprising an effective amount of a pooled sample of tumor-specific neoantigens or DNA / RNA encoding same and a pharmaceutically acceptable salt, carrier, or diluent, (ii) an effective amount of IL-12 or DNA / RNA encoding same, and (iii) an effective amount of a checkpoint inhibitor and a pharmaceutically acceptable salt, carrier, or diluent, separately administered. This further includes any composition comprising two or three of these components and a pharmaceutically acceptable salt, carrier, or diluent.

[0100] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), 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, nucleic acid molecules of the present disclosure comprise a contiguous open reading frame encoding an antibody or fragment thereof as described herein. As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" can mean at least two nucleotides covalently linked to each other. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, nucleic acids also encompass 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 hybrids. Nucleic acids can contain combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods. Nucleic acids generally contain phosphodiester bonds, but can also include nucleic acid analogs that can have at least one different bond type, such as phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bonds, and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those containing normal backbones, non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated by reference in their entireties.Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included in one definition of nucleic acid. Modified nucleotide analogs can be located, for example, at the 5'-end and / or 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs can be selected from ribonucleotides modified at the sugar or backbone. However, nucleobase-modified ribonucleotides, i.e., ribonucleotides containing non-naturally occurring nucleobases instead of naturally occurring nucleobases such as uridine or cytidine modified at the 5-position, for example, 5-(2-amino)propyluridine, 5-bromouridine, adenosine and guanosine modified at the 8-position, for example, 8-bromoguanosine, deazanucleotides, for example, 7-deaza-adenosine, and O- and N-alkylated nucleotides, for example, N6-methyladenosine, are also of note. The 2'-OH group can be replaced with a group selected from H, OR, R, halo, SH, SR, NH, NHR, N, or CN, where R is C-C alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I. Modified nucleotides also include nucleotides conjugated to cholesterol via a hydroxyproline bond, as described, for example, in Krutzfeldt et al., Nature (October 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Application Publication No. 20050107325, the entire contents of which are incorporated herein by reference. Modified nucleotides and nucleic acids can also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, the entire contents of which are incorporated herein by reference. Further modified nucleotides and nucleic acids are described in U.S. Patent Application Publication No. 20050182005, which is incorporated herein by reference in its entirety. Modifications of the ribose-phosphate backbone can be made for a variety of reasons, such as 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 made. Alternatively, mixtures of different nucleic acid analogs and mixtures of naturally occurring nucleic acids and analogs can be made.

[0101] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by unnatural amino acids or non-amino acid chemical groups. The terms also encompass modified amino acid polymers, including, 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 unnatural or synthetic amino acids, including glycine and both D- or L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0102] As used herein, the terms "prevent", "preventing", or "prevention", "prophylactic treatment", and the like are meant to refer to reducing the likelihood of developing a disease or condition in a subject who does not have the disease or condition, but who is at risk of or susceptible to developing the disease or condition.

[0103] As used herein, the term "purified" refers to a polynucleotide or polypeptide, or a fragment, variant, or derivative thereof, that is substantially free from other biological materials with which it is naturally associated, or from other biological materials, e.g., from a recombinant host cell genetically engineered to express the polypeptide of the invention. For example, a purified polypeptide of the present disclosure is a polypeptide that is at least about 70 to about 100% pure, i.e., the polypeptide is present in a composition in which the polypeptide constitutes about 70 to about 100% by weight of the total composition. In some embodiments, a purified polypeptide of the present disclosure 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. Similarly, a purified polypeptide of the present disclosure is a polynucleotide that is at least about 70 to about 100% pure, i.e., the polynucleotide is present in a composition in which the polynucleotide constitutes about 70 to about 100% by weight of the total composition. In some embodiments, the purified polynucleotides of the present disclosure are 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.

[0104] As used herein, the term "receptor" refers to a group of molecules that can bind to a biological molecule or ligand. Receptors can play a role in transmitting information in cells, cell formation, or organisms. A receptor includes at least one receptor unit, preferably two receptor units, and each receptor unit can be composed of a protein molecule, specifically a glycoprotein molecule. A receptor has a structure complementary to that of a ligand and can complex with the ligand as a binding partner. Information is transmitted specifically by a conformational change of the receptor after complexation of the ligand on the surface of a cell. According to embodiments of the present disclosure, receptors should be understood to specifically mean MHC class I and II proteins that can form receptor / ligand complexes with ligands, particularly peptides or peptide fragments of a suitable length.

[0105] As used herein, the term "sample" generally refers to a limited amount of something similar and intended to represent a larger quantity. In the present disclosure, a sample is a collection, swab, brushing, scraping, biopsy, tissue removal, or surgical resection to test for the absence, presence, or grading of hyperproliferative tissue, possibly cancerous tissue, or one or more cells. In some embodiments, the sample is taken from a patient or subject suspected of having cancer, hyperplasia, precancerous disease, or one or more tumor cells. In some embodiments, a sample suspected of containing one or more hyperproliferative cells is compared to a "control sample" known to contain no one or more hyperproliferative cells. The present disclosure contemplates using any one or more samples disclosed herein to identify, detect, sequence, and / or quantify the amount of (highly or minimally immunogenic) neoantigens within a particular sample. In some embodiments, a method involves exposing a swab, brushing, or other sample from the environment to a set of reagents sufficient to isolate and / or sequence DNA and RNA of one or more cells in the sample.

[0106] As used herein, the term "small molecule" refers to a low molecular weight (<900 daltons) organic compound, on the order of 1 nm in size, that can help regulate biological processes. Most drugs are small molecules.

[0107] As used herein, "specifically binds" or "specific for" means that the affinity or Ka (i.e., the equilibrium association constant of a specific binding interaction (units: 1 / M)) of a binding protein (e.g., receptor, antibody, CAR, or TCR) or binding component (or fusion protein thereof) for a target molecule is 10 M or greater (equal to the ratio of the on rate [k] to the off rate [k] of this association reaction), but does not significantly associate or bind with other molecules or components in a sample. Binding proteins or binding domains (or fusion proteins thereof) may be classified as "high affinity" binding proteins or binding domains (or fusion proteins thereof) and "low affinity" binding proteins or binding domains (or fusion proteins thereof). A "high affinity" binding protein or binding domain refers to a binding protein or binding domain having a K a of at least about 10 M, at least about 10 M, at least about 10 M, at least about 10 M, at least about 10 M, at least about 10 M, or at least about 10 M. A "low affinity" binding protein or binding domain refers to a binding protein or binding domain having a K a of up to about 10 M, up to about 10 M, or up to about 10 M. In some embodiments, affinity is defined as the equilibrium dissociation constant (K) of a particular binding interaction (units: M (e.g., 10 M to 10 M)). Various assays for identifying binding domains that specifically bind to particular targets and for determining the affinity of binding domains or fusion proteins are known, such as Western blots, ELISAs, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore®) analysis (e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents, all of which are incorporated herein by reference). In some embodiments, one or more TCRs bind one or more respective neo-antigens with high affinity.High affinity binding means, in some embodiments, that the TCR binds to the neo-antigen with a Ka or Kd of about 500 nM or less.

[0108] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a nucleic acid molecule hybridizes to another nucleic acid molecule but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of a 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 generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions are conditions in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salt), at pH 7.0 to 8.3, and a temperature of at least about 30° C. for short probes, primers, or oligonucleotides (e.g., 10 to 50 nucleotides), and at least about 60° C. for longer probes, primers, or oligonucleotides. Stringent conditions can also be achieved by adding destabilizing agents such as formamide.

[0109] As used herein, the terms "subject," "individual," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. The methods described herein are applicable to both human therapeutic applications and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments, the subject is a human. In some embodiments, the subject is a dog, horse, pig, sheep, cat, cow, donkey, llama, emu, or goat.

[0110] "Substantially identical" refers to a nucleic acid molecule or polypeptide that exhibits at least 50% sequence identity to a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein) or amino acid sequence (e.g., any one of the amino acid sequences described herein). In some embodiments, such a sequence is at least about 60% sequence identical to the reference sequence used for comparison. In some embodiments, such a sequence is at least about 70% sequence identical to the reference sequence used for comparison. In some embodiments, such a sequence is at least about 80% sequence identical to the reference sequence used for comparison. In some embodiments, such a sequence is at least about 85% sequence identical to the reference sequence used for comparison. In some embodiments, such a sequence is at least about 90% sequence identical to the reference sequence used for comparison. In some embodiments, such a sequence is at least about 95% sequence identical to the reference sequence used for comparison. In some embodiments, such a sequence is at least about 99% sequence identical to the reference sequence used for comparison.

[0111] "T cells" (or "T lymphocytes") are immune system cells that mature in the thymus and produce T cell receptors (TCRs), which can be obtained (enriched or isolated) from, for example, peripheral blood mononuclear cells (PBMCs) and are referred to herein as "bulk" T cells. After T cell isolation, both cytotoxic (CD8+) and helper (CD4+) T cells can be sorted into naive, memory, and effector T cell subpopulations, either before or after expansion. T cells can be naive (not exposed to antigen; have increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO, compared to central memory T cells (TCM), memory T cells (TM) (antigen-experienced, long-lived), and effector cells (antigen-experienced, cytotoxic)). TM can be further divided into subsets of central memory T cells (TCM; increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA, compared with naive T cells) and effector memory T cells (TEM; decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127, compared with naive T cells or TCM). Effector T cells (TE) are antigen-experienced CD8+ cytotoxic T lymphocytes that express decreased CD62L, CCR7, and CD28, and are positive for granzymes and perforin, compared with TCM. Helper T cells (Th) are CD4+ cells that affect the activity of other immune cells by releasing cytokines. CD4+ T cells can both activate and suppress adaptive immune responses, depending on the presence of other cells and signals. T cells can be collected according to known techniques, and various subpopulations or combinations thereof can be enriched or depleted by known techniques such as affinity binding to antibodies, flow cytometry, or immunomagnetic selection.

[0112] As used herein, the term "T cell epitope" is meant to refer to a peptide sequence that can be bound by class I or II MHC molecules in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, recognized and bound by cytotoxic T lymphocytes or T helper cells, respectively.

[0113] A "T cell population" can include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, and activated T lymphocytes. A T cell population can include αβ T cells, including CD4+ T cells, CD8+ T cells, γδ T cells, natural killer T cells, or any other subset of T cells.

[0114] As used herein, the term "T cell receptor" (TCR) refers to a member of the immunoglobulin superfamily that has a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997), and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. TCRs can be found on the surface of cells or in soluble form, and generally consist of a heterodimer having an α chain and a β chain (also known as TCRα and TCRβ, respectively) or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively). Similar to immunoglobulins, the extracellular portions of TCR chains (e.g., α chain, β chain) contain two immunoglobulin domains: a variable domain at the N-terminus (e.g., α chain variable domain or Va; β chain variable domain or Vβ, typically amino acids 1-116 according to Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.)) and one constant domain adjacent to the cell membrane (e.g., α chain constant domain or Ca, typically amino acids 117-259 according to Kabat; β chain constant domain or Cp, typically amino acids 117-295 according to Kabat). Similar to immunoglobulins, variable domains comprise complementarity-determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBOJ. 7:3745, 1988; Lefranc et al., Dev. Comp. Immunol. 27:55, 2003).In certain embodiments, TCRs are found on the surface of T cells (or "T lymphocytes") and are associated with the CD3 complex. The source of the TCRs used in this disclosure can be from various animal species, such as human, mouse, rat, rabbit, or other mammals. In some embodiments, the source of the TCR, as used in this disclosure, is from a subject in which neoantigens used for in vivo T cell activation and expansion are identified.

[0115] As used herein, the term "therapeutic agent" means an agent utilized to treat, combat, mitigate, prevent, or ameliorate an unwanted condition or disease in a patient.

[0116] As used herein, the term "therapeutic effect" refers to some degree of alleviation of one or more symptoms of a disorder (e.g., a neoplasm or tumor) or its associated pathology. As used herein, a "therapeutically effective amount" refers to an amount of an agent that, upon single or multiple administration to a cell or subject, is effective in prolonging survival of a patient with such disorder, or in reducing, preventing, or delaying one or more signs or symptoms of the disorder beyond that expected in the absence of such treatment. A "therapeutically effective amount" is intended to identify the amount necessary to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the required "therapeutically effective amount" (e.g., ED50) of the pharmaceutical composition. For example, a physician or veterinarian can start administering a compound of the present disclosure used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In some embodiments, a therapeutically effective amount is an amount effective to shrink a solid tumor by about 2% total mass compared to its mass or estimated mass before treatment, or by about 4% total mass, about 6% total mass, about 8% total mass, about 10% total mass, about 15% total mass, about 20% total mass, about 25% total mass, about 30% total mass, about 35% total mass, about 40% total mass, about 45% total mass, or about 50% total mass compared to the total mass of the solid tumor before treatment. In some embodiments, for any therapeutic agent described herein, a therapeutically effective amount is initially determined from preliminary in vitro studies and / or animal models. In some embodiments, a therapeutically effective dose is determined from human data. In some embodiments, the applied dose is adjusted based on the relative bioavailability and efficacy of the administered agent. Adjusting the dose to achieve maximum efficacy based on the above methods and other well-known methods is well within the capabilities of one of ordinary skill in the art.General principles for determining therapeutic efficacy 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.

[0117] As used herein, the terms "treat," "treated," "treating," "treatment," and the like are meant to refer to the alleviation or amelioration of a disorder and / or its associated symptoms (e.g., cancer or tumor). "Treating" may refer to the administration of a neo-antigen vaccine described herein to a subject after the onset or suspected onset of cancer. "Treating" may also refer to the administration of an engineered TCR transgenic T cell described herein to a subject after the onset or suspected onset of cancer. "Treating" encompasses the concept of "alleviating," which refers to reducing the frequency or severity of the occurrence or recurrence of symptoms or other adverse effects associated with cancer and / or side effects associated with cancer treatment. The term "treating" also encompasses the concept of "managing," which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., extending the remission period of a patient suffering from a disease. It is understood, although not excluded, that treating a disorder or condition does not require the complete elimination of the associated disorder, condition, or symptoms. Thus, as used herein, the term "treating cancer" is not intended to be an absolute term. In some embodiments, the compositions and methods of the present disclosure seek to reduce tumor size or cancer cell number, induce cancer remission, or prevent cancer cells from growing in size or number. In some situations, treatment with the compositions and methods of the present disclosure results in an improved prognosis.

[0118] As used herein, "tumor antigen" or "tumor-associated antigen" or "TAA" refers to a mutant protein found in oncogenic or tumor cells that elicits a humoral immune response, a cellular immune response, or both, and may be found only in tumor cells or in tumor cells and other normal cells. In some embodiments, a TAA is the product of a mutant oncogene (e.g., p53, raf, ras, myc, EGFR). In some embodiments, a TAA is a mutant tumor suppressor gene (e.g., pRb, TP53, PTEN, CD95). In some embodiments, a TAA is a mutant gene that overexpresses or aberrantly expresses a cellular protein, etc.

[0119] As used herein, the term "vaccine" is meant to refer to a composition for generating immunity for the prevention and / or treatment of disease (e.g., cancer). A vaccine is thus a pharmaceutical comprising an antigen and intended for use in humans or animals to generate specific defense and protection by vaccination. A "vaccine composition" or "neo-antigen vaccine composition" may include a pharmaceutically acceptable excipient, adjuvant, or diluent.

[0120] "Tumor infiltrating lymphocytes" or "TILs," as used herein, refer to a type of immune cell that migrates from the blood to a tumor. Tumor infiltrating lymphocytes can recognize and kill cancer cells. In some embodiments for cancer treatment, tumor infiltrating lymphocytes are removed from a patient's tumor, grown in large numbers in vitro, and then readministered to the patient to help the immune system treat the subject and kill one or more cancer cells.

[0121] A "vector" is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which allows additional DNA segments to be introduced into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide. The present disclosure relates to any one or more vectors containing a nucleic acid sequence encoding any one or more amino acid sequences disclosed herein.

[0122] The vector can include a heterologous nucleic acid encoding a neoantigen and can further include an initiation codon, which can be upstream of the neoantigen coding sequence, and a stop codon, which can be downstream of the neoantigen coding sequence. The initiation and stop codons can be in-frame with the neoantigen coding sequence. The vector can also include a promoter operably linked to the neoantigen coding sequence. The promoter operably linked to the neoantigen coding sequence can be a promoter derived from Simian Virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter derived from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can 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. 2004 / 0175727, the contents of which are incorporated herein in their entirety.

[0123] The vector may also contain a polyadenylation signal 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 the polyadenylation signal from the pCEP4 vector (Invitrogen, San Diego, Calif.).

[0124] The vector may also contain an enhancer upstream of the neoantigen coding sequence. 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 those derived from CMV, HA, RSV, or EBV. Polynucleotide function enhancement is described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94 / 016737, the contents of each of which are incorporated by reference in their entirety. The vector may also contain a mammalian origin of replication to maintain the vector extrachromosomally and produce multiple copies of the vector within a cell.

[0125] The present disclosure relates to paramceitics.

[0126] In some embodiments, the nucleic acid molecule or pharmaceutical composition comprises a DNA backbone comprising all of the lowercase base pairs from any of the above-identified plasmids, wherein a first lowercase backbone sequence and a second lowercase backbone sequence flank an expressible nucleic acid sequence encoding multiple tumor-specific antigen sequences, such as Formula I, Formula I(a), Formula II, or Formula III(a).

[0127] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising the formula I([(AEDn)-(linker)]n-[AEDn+1]), where each linker can be independently selected from natural or non-natural nucleic acids of a length from about 0 to about 25. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising the formula I([(AEDn)-(linker)]n-[AEDn+1]), where each linker can be independently selected from natural or non-natural nucleic acids of a length from about 0 to about 25, from about 0 to about 25, from about 1 to about 25, from about 2 to about 25, from about 3 to about 25, from about 4 to about 25, from about 5 to about 25, from about 6 to about 25, from about 7 to about 25, from about 8 to about 25, from about 9 to about 25, from about Each linker can be independently selected from natural or unnatural nucleic acids having a length of 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, or about 24 to about 25. In some embodiments, each linker is a natural or unnatural nucleic acid 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, or about 25. In some embodiments, each linker is a natural or non-natural nucleic acid of about 21 in length. In certain embodiments, two linkers can be fused and used together. Thus, in some embodiments, the first linker can be independently selected from natural or unnatural nucleic acids of about 0 to about 25 in length, natural or unnatural nucleic acids 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, and about 24 to about 25 in length.In some embodiments, the second linker can be independently selected from natural or unnatural nucleic acids having 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, or about 24 to about 25. In some embodiments, the first linker can be independently selected from linkers that are natural or non-natural nucleic acids 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, or about 25 in length. In some embodiments, the second linker can be independently selected from linkers that are natural or non-natural nucleic acids 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, or about 25 in length.

[0128] In some embodiments, at least one linker comprises about 15 to about 300 nucleotides and encodes an amino acid cleavage site, and in some embodiments, each linker disposed between each AED comprises about 15 to about 120 nucleotides and is the same nucleotide sequence encoding an amino acid cleavage site.

[0129] In some embodiments, the formula (e.g., [(AEDn)-(linker)]n-[AEDn+1]) includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more linkers.

[0130] In some embodiments, the vector can be LLC, TC1, ID8, pGX0001, pGX4501, pGX4503, pGX4504, pGX4505, pGX4506, and / or pGX6001, or 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 sequences that are pVAX1. The vector backbone can be pAV0242. The vector can be a replication-deficient adenovirus type 5 (Ad5) vector.

[0131] In some embodiments, the vector comprises regulatory sequences that may be well suited for gene expression in mammalian or human cells to which the vector is administered. The neo-antigen coding sequence may include codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0132] In some embodiments, the vector is pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli) further comprising an expressible nucleic acid. The vector can also be pYES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in Saccharomyces cerevisiae strains of yeast. The vector can also be the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The vector can 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. Vectors can be readily available starting materials, including expression vectors or systems for producing proteins by routine techniques and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is fully incorporated by reference.

[0133] Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria); such controls are generally available in expression vectors. The vector is then introduced into the host bacterium for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The vector of the present disclosure is a polynucleotide sequence, such as a plasmid, that contains a nucleic acid sequence encoding one or more neoantigens or a TCR sequence that highly associates with one or more neoantigens.

[0134] In some embodiments, where the nucleic acid molecule comprises an expressible nucleic acid sequence, the expressible nucleic acid sequence of Formula I is placed in a multiple cloning site of a plasmid selected from the group consisting of LLC, TC1, ID8, pGX0001, pGX4501, pGX4503, pGX4504, pGX4505, pGX4506, and / or pGX6001. In some embodiments, the nucleic acid sequence of Formula I is placed in the multiple cloning site of LLC. In some embodiments, the nucleic acid sequence of Formula I is placed in the multiple cloning site of TC1. In some embodiments, the nucleic acid sequence of Formula I is placed in the multiple cloning site of ID8. In some embodiments, the nucleic acid sequence of Formula I is placed in the multiple cloning site of pGX0001. In some embodiments, the nucleic acid sequence of Formula I is placed in the multiple cloning site of pGX4501. In some embodiments, the nucleic acid sequence of Formula I is placed in the multiple cloning site of pGX4503. In some embodiments, the nucleic acid sequence of Formula I is located in 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 in the multiple cloning site of pGX4506. In some embodiments, the plasmid is a sequence that is 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to pGX4505 or each of the nucleotide sequences identified above. In some embodiments, the plasmid is a sequence that comprises about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to pGX0001 or SEQ ID NO: 356. In some embodiments, the plasmid is a sequence that contains about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to pGX6001 or SEQ ID NO: 360.

[0135] (Table W) Plasmid backbone sequence TIFF2025516676000002.tif232167TIFF2025516676000003.tif168167TIFF2025516676000004.tif227167

[0136] The present disclosure also relates to nucleic acid molecules comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is a DNA backbone domain of the nucleic acid molecule and the second nucleic acid sequence is an expressible nucleic acid sequence, the expressible nucleic acid sequence comprising, in a 5' to 3' direction, multiple antigen-expressing domains. In some embodiments, the expressible nucleic acid sequence comprises a nucleic acid sequence encoding a linker at the 5' end of the first antigen-expressing domain. In some embodiments, the expressible nucleic acid sequence encodes a linker between each of the antigen-expressing domains. In some embodiments, the expressible nucleic acid sequence encodes a leader sequence, multiple antigen-expressing domains, each antigen-expressing domain separated by a linker sequence. In some embodiments, there are at least 20 antigen-expressing domains. In some embodiments, there are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 or more antigen-expressing 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 linked to an 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:356, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:356. 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:360, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:360.

[0137] The present disclosure also relates to nucleic acid molecules comprising first, second, and third nucleic acid sequences, wherein the first nucleic acid sequence is a first DNA backbone domain of the nucleic acid molecule, the second nucleic acid sequence is a 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, in a 5' to 3' direction, multiple antigen-expressing domains. In some embodiments, the expressible nucleic acid sequence comprises a nucleic acid sequence encoding a linker at the 5' end of the first antigen-expressing domain. In some embodiments, the expressible nucleic acid sequence encodes a linker between each of the antigen-expressing domains. In some embodiments, the expressible nucleic acid sequence encodes a leader sequence, multiple antigen-expressing domains, each antigen-expressing domain separated by a linker sequence. In some embodiments, there are at least 20 antigen-expressing domains. In some embodiments, there are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 or more antigen-expressing 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 linked to the expressible nucleic acid sequence.

[0138] 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:357, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:357. 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:358, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:358.

[0139] In some embodiments, the composition or pharmaceutical composition one or more neo-antigens administered to a subject to re-elicit an antigen-specific immune response comprises a nucleic acid comprising: (i) a first DNA backbone domain comprising 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: 357, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 357; and (ii) a second 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: 358, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 358; and (iii) a third nucleic acid sequence located between each of the first and second scaffold sequences, wherein the third nucleic acid sequence is an expressible nucleic acid sequence encoding a plurality of neo-antigens. In some embodiments, the third nucleic acid sequence comprises expressible nucleic acid sequences encoding at least about 20, 25, 30, 35, 40, 45, 50, 55, or 60 or more neo-antigens. In some embodiments, the expressible nucleic acid sequence comprises, in a 5' to 3' orientation, a nucleic acid sequence encoding an Ig leader sequence and multiple antigen-expressing domains, where the antigen-expressing domains are separated by linkers. In some embodiments, the antigen-expressing domains consist of a single antigen sequence encoding a tumor-specific neo-antigen of interest. In some embodiments, each antigen-expressing domain is about 25 to about 40 nucleotides in length. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising the formula I([(AEDn)-(linker)]n-[AEDn+1]), where each linker is independently selectable from natural or non-natural nucleic acids of length from about 0 to about 25, and n is a positive integer from about 10 to about 40.

[0140] 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 has Formula IV: [(AEDn)-(linker)]n-(AEDn+1)-(linker)n+1]n-AED(3' end) Including, wherein each AED is independently selectable from any one or more tumor-associated antigens from the subject, n is any positive integer from about 1 to about 50, and each "linker" is a nucleic acid sequence encoding one or more amino acid cleavage sites. Each linker can be the same or independently selectable to include one or more linkers disclosed herein. In some embodiments, the linker is a furin cleavage site about 9 to about 105 nucleotides in length and encodes an amino acid sequence that is 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 one, two, three, four, five, or more nucleic acid molecules, each of which expresses any of the patterns or formulas of the AEDs disclosed herein.

[0141] In some embodiments, the expressible 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 has the formula III(a): Leader sequence-[(AEDn)-(linker)]n-(AEDn+1)-linkern+1-(AEDn+2)]n Including, wherein each AED is independently selectable from any 20 or more tumor-associated antigens from a subject, n is any positive integer from about 1 to about 50, and each "linker" is a nucleic acid sequence encoding one or more amino acid cleavage sites. Each linker can be the same or independently selectable to include one or more linkers disclosed herein, and each "-" represents a bond between each subunit. In some embodiments, the linker is a furin cleavage site about 9 to about 105 nucleotides in length and encodes an amino acid sequence that is 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) includes a third linker attached to the 3' end of the third AED sequence. In some embodiments, the last AED sequence in the 5' to 3' orientation is not attached to a linker at its 3' end.

[0142] The present disclosure also relates to nucleic acid sequences comprising coding and non-coding regions, wherein the coding region has the structure of Formula I(b): [(AED1)-(linker)-(AED2)-(linker)]n-[(AED3)]n+1, It consists of wherein n is a positive integer from about 1 to about 30, each "linker" encodes one or more amino acid cleavage sequences, the non-coding region comprises at least one regulatory sequence operably linked to one or more AEDs, and in the 5' to 3' orientation, AED3 is the terminal antigen-expressing domain in the sequence of the AED. In some embodiments, n is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more, and wherein AED1 and AED2 are each antigen-expressing domains independently selectable as antigen sequences. In some embodiments, the regulatory sequence is any of the regulatory sequences shown in the figures or a functional fragment comprising at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a regulatory sequence shown in the figures.

[0143] The present disclosure also relates to nucleic acid sequences comprising coding and non-coding regions, wherein the coding region has the structure of Formula I(b): Ig leader sequence-[(AED)-(linker)-(AED)-(linker)]n-[(AED)]n+1 It consists of wherein n is a positive integer from about 1 to about 30, each "linker" encodes one or more amino acid cleavage sequences, the non-coding region comprises at least one regulatory sequence operably linked to one or more AEDs, and in the 5' to 3' orientation, AED3 is the terminal antigen-expressing domain in the sequence of the AED. In some embodiments, n is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more, and wherein AED1 and AED2 are each antigen-expressing domains independently selectable as antigen sequences. In some embodiments, the regulatory sequence is any of the regulatory sequences shown in the figures or a functional fragment comprising at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a regulatory sequence shown in the figures.

[0144] In some embodiments, the compositions of the present disclosure relate to any nucleic acid molecule comprising: (i) an expressible nucleic acid sequence having at least 70%, 80%, or 90% sequence identity to any of the plasmid backbones identified herein; and (ii) one or more antigen expression domains separated by nucleic acid sequences encoding proteolytic cleavage sites. In some embodiments, the expressible nucleic acid sequence encodes about 19 to about 60 distinct amino acid sequences that are antigens. In some embodiments, the present disclosure relates to a cell comprising a nucleic acid molecule comprising: (i) an expressible nucleic acid sequence having at least 70%, 80%, or 90% sequence identity to any of the plasmid backbones identified herein; and (ii) one or more antigen expression domains separated by nucleic acid sequences encoding proteolytic cleavage sites. In some embodiments, the present disclosure relates to a cell comprising one or more amino acid sequences encoded by one or more antigen expression domains, wherein the amino acid sequences are cleaved upon expression and exposure to intracellular protease activity.

[0145] Identification of neoantigens The present disclosure relates to methods for treating a cancer comprising one or more neoantigens in a subject in need thereof. The present disclosure further relates to methods for preventing metastasis of a cancer comprising one or more neoantigens in a subject in need thereof. Because tumor neoantigens often arise from somatic "passenger" mutations in the DNA of tumor cells, many tumor neoantigens are unique to an individual patient's cancer. Therefore, for the disclosed methods to develop one or more cancer-specific neoantigens, it is necessary to determine the "mutanome" of the subject's tumor and identify candidate tumor neoantigens. Thus, in some embodiments, the disclosed methods include identifying one or more neoantigens unique to the subject. In some embodiments, the neoantigen comprises an antigenic peptide or epitope derived from a protein encoded by a nucleic acid molecule having a missense mutation, a nonstop mutation, a splice mutation, a gene fusion, a frameshift mutation (e.g., an addition or deletion), or a combination thereof, compared to the wild-type nucleic acid molecule.

[0146] Neoantigens can be identified using any of several well-known techniques (see, e.g., Rajasagi et al., Blood 124:453, 2014). For example, in some embodiments, a population of tumor-specific neoantigens is identified by sequencing tumor DNA (or RNA) and DNA (or RNA) from normal tissues of each patient to identify tumor-specific mutations and determine the patient's HLA allotype. In some embodiments, the population of tumor-specific neoantigens and their cognate native antigens is subjected to bioinformatic analysis using a validated algorithm to predict which tumor-specific mutations create epitopes capable of binding to the patient's HLA allotype, specifically, which tumor-specific mutations create epitopes capable of binding to the patient's HLA allotype more effectively than the cognate native antigen. Based on this analysis, identified nucleotide sequences corresponding to these mutations are designed for each patient and, in some embodiments, used together as a cancer vaccine in immunizing the subject.

[0147] In some embodiments, the methods of the disclosure include identifying one or more subject-specific neo-antigen mutations in a subject, the subject having been diagnosed with, suspected of having, or containing one or more hyperproliferative cells (e.g., tumors), the steps including: (a) sequencing a nucleic acid sample from the subject's tumor and a nucleic acid sample from the subject's non-tumor sample; (b) analyzing the sequences to determine coding and non-coding regions; (c) identifying sequences containing tumor-specific non-synonymous or non-silent mutations that are not present in the non-tumor sample; (d) identifying single nucleotide mutations, as well as single nucleotide insertions and deletions; (e) producing subject-specific peptides encoded by sequences containing tumor-specific non-synonymous or non-silent mutations that are not present in the non-tumor sample; and (f) measuring binding properties of the subject-specific peptides, wherein each subject-specific peptide is an expression product of a subject-specific DNA neo-antigen that is not present in the non-tumor sample, thereby identifying one or more subject-specific DNA neo-antigens in the subject.

[0148] Any cell type or tissue can be used to obtain a nucleic acid sample for use in the sequencing methods described herein. In some embodiments, DNA or RNA samples are obtained from neoplasms, tumors, or bodily fluids, such as blood, obtained by known techniques (e.g., venipuncture) or saliva. In other embodiments, nucleic acid testing can be performed on dry samples (e.g., hair or skin).

[0149] A subject-specific neoantigenic mutation can be any mutation in any gene that encodes a mutant amino acid sequence (also referred to as a "non-silent mutation") and is expressed in cancer cells but not in normal, non-cancer cells. Non-limiting examples of cancer-specific mutations that can be identified in the disclosed methods include missense, nonsense, insertion, deletion, duplication, frameshift, and repeat expansion mutations. In some embodiments, the disclosed methods include identifying at least one gene containing a cancer-specific mutation that encodes a mutant amino acid sequence. However, the number of genes containing such cancer-specific mutations that can be identified in the disclosed methods is not limited and can include two or more genes (e.g., about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). Similarly, in some embodiments, the disclosed methods include identifying at least one cancer-specific mutation that encodes a mutant amino acid sequence. However, the number of such cancer-specific mutations that can be identified in the disclosed methods is not limited and can include two or more genes (e.g., about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). In some embodiments in which two or more cancer-specific mutations are identified, the cancer-specific mutations are located in the same gene. In other embodiments in which two or more cancer-specific mutations are identified, the cancer-specific mutations are located in different genes.

[0150] In some embodiments, identifying one or more cancer-specific mutations in nucleic acids of a cancer cell includes sequencing the complete or substantially complete exome, whole genome, or whole transcriptome of the cancer cell. Sequencing can be performed by any suitable method known in the art. Examples of sequencing techniques that may be useful in the disclosed methods include, but are not limited to, next-generation sequencing (NGS) (also referred to as "massively parallel sequencing technology") or third-generation sequencing. NGS refers to high-throughput DNA sequencing technologies that are not based on the Sanger method. In NGS, millions or billions of DNA strands can be sequenced in parallel, resulting in substantially greater throughput and minimizing the need for fragment cloning methods often used in Sanger sequencing of genomes. In NGS, nucleic acid templates can be randomly read in parallel along the entire genome by breaking it down into small pieces. NGS can advantageously provide nucleic acid sequence information for the entire genome, exome, or transcriptome in a very short period of time, for example, within about 1 to about 2 weeks, within about 1 to about 7 days, or within less than about 24 hours. Multiple NGS platforms, commercially available or described in the literature, can be used in the context of the disclosed methods, e.g., the methods described in Zhang et al., J. Genet. Genomics, 38(3):95-109 (2011) and Voelkerding et al., Clinical Chemistry, 55:641-658 (2009).

[0151] Non-limiting examples of NGS technologies and platforms include sequencing-by-synthesis (also known as "pyrosequencing") (e.g., implemented using the GS-FLX 454 Genome Sequencer, 454 Life Sciences (Branford, Conn.), the ILLUMINA SOLEXA Genome Analyzer (Illumina Inc., San Diego, Calif.), or the ILLUMINA HISEQ 2000 Genome Analyzer (Illumina), or as described, for example, in Ronaghi et al., Science, 281(5375): 363-365 (1998)), sequencing-by-ligation (e.g., implemented using the SOLID platform (Life Technologies Corporation, Carlsbad, Calif.) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)), single molecule sequencing (e.g., implemented using the PACBIO RS system (Pacific Biosciences (Menlo Park, Calif.)) or the HELISCOPE platform (Helicos These include nanotechnology for single molecule sequencing (e.g., implemented using the GRIDON platform from Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nanopore sequencing (HANS) platform developed by Nabsys (Providence, RI), and a ligase-based DNA sequencing platform with DNA nanoball (DNB) technology called probe-anchor ligation (cPAL)), electron microscope-based techniques for single molecule sequencing, and ion semiconductor sequencing.

[0152] Thus, in some embodiments, a population of cancer-specific neoantigens is identified by sequencing a patient's cancer / tumor and normal DNA to identify cancer-specific mutations and determining the patient's HLA allotype. In some embodiments, the population of cancer-specific neoantigens and their cognate natural antigens is subjected to bioinformatic analysis using a validated algorithm to predict which cancer-specific mutations create epitopes that can bind to the patient's HLA allotype, specifically, which cancer-specific mutations create epitopes that can bind to the patient's HLA allotype more effectively than the cognate natural antigen. Based on this analysis, in some embodiments, the identified nucleotide sequences corresponding to these mutations are designed for each patient and used together as a personalized cancer vaccine in immunizing the patient.

[0153] In some embodiments, the methods of the disclosure include identifying one or more subject-specific neo-antigenic mutations in a subject, wherein the subject has been diagnosed with, is suspected of having, or comprises one or more hyperproliferative cells (e.g., tumors, etc.). In some embodiments, the methods of the disclosure include identifying one or more subject-specific neo-antigenic mutations in a subject, wherein the subject has been diagnosed with, is suspected of having, or comprises one or more hyperproliferative cells (e.g., tumors, etc.). In some embodiments, a method of the disclosure features a step of identifying one or more subject-specific neo-antigenic mutations in a subject, the subject having been diagnosed with, suspected of having, or containing one or more hyperproliferative cells (e.g., tumors) characterized by the presence or amount of multiple neo-antigenic mutations, the step of sequencing nucleic acid samples from the subject's tumor and a non-tumor sample from the subject; analyzing the sequences to determine coding and non-coding regions; identifying sequences containing tumor-specific non-synonymous or non-silent mutations that are not present in the non-tumor sample; identifying single nucleotide mutations, as well as single nucleotide insertions and deletions; producing subject-specific peptides encoded by the sequences containing tumor-specific non-synonymous or non-silent mutations that are not present in the non-tumor sample; and measuring binding properties of the subject-specific peptides, wherein each subject-specific peptide is an expression product of a subject-specific DNA neo-antigen that is not present in the non-tumor sample, thereby identifying one or more subject-specific DNA neo-antigens in the subject. Measuring the binding characteristics of the subject-specific peptide can be performed by one or more of measuring binding of the subject-specific peptide to a T cell receptor, measuring binding of the subject-specific peptide to an HLA protein of the subject, or measuring binding of the subject-specific peptide to a transporter associated with antigen processing (TAP).

[0154] Efficient selection of specific mutations to utilize as immunogens requires the ability to identify the patient's HLA type and predict which mutant peptides will efficiently bind to the patient's HLA alleles. Thus, in some embodiments, measuring binding of a subject-specific peptide to a T cell receptor comprises measuring binding of the subject-specific peptide to an HLA protein of the subject.

[0155] In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 10 nM to about 550 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 20 nM to about 500 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 30 nM to about 450 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 40 nM to about 400 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 50 nM to about 350 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 60 nM to about 300 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 70 nM to about 250 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 80 nM to about 200 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 90 nM to about 200 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 100 nM to about 150 nM.

[0156] In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 10 nM to about 100 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 20 nM to about 150 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 30 nM to about 175 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 40 nM to about 200 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 50 nM to about 225 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 60 nM to about 250 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 70 nM to about 275 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 80 nM to about 300 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 90 nM to about 325 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 100 nM to about 350 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 110 nM to about 375 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 120 nM to about 400 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 130 nM to about 425 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 140 nM to about 450 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 150 nM to about 475 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of about 175 nM to about 500 nM.

[0157] In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 550 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 500 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 450 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 400 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 350 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 300 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 250 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 200 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 150 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 100 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 90 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 80 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 70 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 60 nM. In some embodiments, the subject-specific peptide binds to the subject's HLA protein with an IC50 of less than about 50 nM.

[0158] In some embodiments, the disclosed methods further comprise ranking the subject-specific peptides based on their binding characteristics. In some embodiments, the disclosed methods further comprise measuring the CD8+ T cell immune response generated by the subject-specific peptides. Methods for measuring CD8+ T cell responses are known in the art and described herein.

[0159] In some embodiments, the disclosed methods further comprise formulating the subject-specific neo-antigen into an immunogenic composition for administration to a subject. In some embodiments, the subject-specific neo-antigen is formulated into the immunogenic composition in the form of DNA. In some embodiments, the subject-specific neo-antigen is formulated into the immunogenic composition in the form of RNA. In some embodiments, the subject-specific neo-antigen is formulated into the immunogenic composition in the form of a protein. In embodiments where the subject-specific neo-antigen is formulated into the immunogenic composition in the form of DNA, the DNA neo-antigen can be subcloned into one or more vectors, which in some embodiments are one or more plasmids. Methods of administering DNA, RNA, or protein vaccines are known in the art.

[0160] In some embodiments, the top 200 neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 150 neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 100 neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 50 neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 25 neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 10 neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 5 neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, about 5 to about 20 of the top neo-antigenic variations (by frequency) are included in an immunogenic composition for administration to a subject. In some embodiments, about 10 to about 20 of the top neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, about 25 to about 100 of the top neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, about 50 to about 100 of the top neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, about 100 to about 200 of the top ranked neo-antigenic variations are included in an immunogenic composition for administration to a subject. In some embodiments, the ranking is based on the lowest IC 50 Highest IC from the value 50 The value is determined by ordering the neoantigens on the list up to the

[0161] Thus, in some embodiments, the methods of the present disclosure include identifying or selecting one or more cancer-specific neo-antigens from a subject, the steps comprising: (a) sequencing DNA and / or RNA from a cancer / tumor sample from the subject; (b) measuring binding of the subject-specific neo-antigen to a T cell receptor; measuring binding of the subject-specific neo-antigen to an HLA protein of the subject; and (c) determining whether the subject-specific neo-antigen has an IC50 of less than about 500 nM, 400 nM, 300 nM, 200 nM, or 100 nM. 50and optionally (d) selecting one or more neo-antigens from the sample that bind to the HLA protein of interest at the lowest IC 50 Highest IC from the value 50 and ordering the neo-antigens in order of value.

[0162] Administering neoantigen(s) to a subject The present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of a nucleic acid molecule disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the disclosure relates to compositions comprising: a first pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid molecule disclosed herein and a pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid molecule encoding IL-12 or a functional fragment thereof and a pharmaceutically acceptable carrier. In some embodiments, the nucleic acid molecule disclosed herein comprises at least one expressible nucleic acid sequence encoding about 19 to about 50 neo-antigens from a subject, or functional fragments thereof. In some embodiments, the neo-antigens, or nucleic acid sequences encoding them, are selected by administering a vaccine to the subject, isolating the subject's PBMCs or antigen-presenting cells, exposing the subject's PBMCs or antigen-presenting cells to cells comprising one or more nucleic acid molecules encoding one or more TCRs, and selecting one or more TCRs that bind to the neo-antigens based on the quantified strength of binding between the TCRs and the neo-antigens.

[0163] To activate the subject's immunity to the cancer-specific neoantigens, the disclosed method further includes administering the cancer-specific neoantigens and the subject-specific neoantigens to the subject. The administration results in a subject-specific immune response elicited in response to the neoantigens. Immune cells from the subject can be isolated, modified, and then re-administered to the subject as a treatment. The cancer-specific neoantigens and the subject-specific neoantigens can be administered to the subject in the form of peptides, DNA, and / or RNA. In some embodiments, the cancer-specific neoantigens and the subject-specific neoantigens are administered to the subject in the form of peptides in a composition comprising one or more pharmaceutically acceptable carriers. In some embodiments, the cancer-specific neoantigens and the subject-specific neoantigens are administered to the subject in the form of DNA in a composition comprising one or more pharmaceutically acceptable carriers. In some embodiments, the cancer-specific neoantigens and the subject-specific neoantigens are administered to the subject in the form of RNA in a composition comprising one or more pharmaceutically acceptable carriers. In some embodiments, the cancer-specific neoantigens and the subject-specific neoantigens are administered to the subject in the form of a mixture of DNA and RNA in a composition comprising one or more pharmaceutically acceptable carriers.

[0164] In embodiments in which the cancer-specific neo-antigens and the subject-specific neo-antigens are administered to the subject in the form of DNA, the DNA neo-antigens can be subcloned into one or more vectors, exemplified by the vectors disclosed herein, which in some embodiments are one or more plasmids. Thus, in some embodiments, the disclosed methods further comprise the step of producing a DNA vaccine or manufacturing a pharmaceutical composition comprising such a DNA vaccine, which comprises performing one or more of the steps described above, further comprising subcloning the nucleic acid sequence encoding the one or more neo-antigens into one or more nucleic acid molecules, and optionally suspending the resulting nucleic acid molecules in one or more pharmaceutically acceptable carriers.

[0165] Any method for generating DNA vaccines expressing neoantigens or short peptides can be used in the disclosed methods. DNA vaccines are disclosed in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are incorporated herein by reference in their entireties. As an example, a DNA vaccine suitable for the disclosed methods can be generated using the method disclosed in International Application Publication No. 2019 / 227106, which is incorporated herein by reference. Briefly, one or more nucleotide sequences encoding one or more cancer / tumor-specific neoantigens can be subcloned into a nucleic acid molecule. In some embodiments, only one nucleotide sequence encoding one single cancer / tumor-specific neoantigen is subcloned into a nucleic acid molecule. In some embodiments, two or more nucleotide sequences encoding the same cancer / tumor-specific neoantigen are subcloned into a nucleic acid molecule. In some embodiments, two or more nucleotide sequences encoding two or more cancer / tumor-specific neoantigens are subcloned into a nucleic acid molecule.

[0166] Thus, in some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 1 to about 100 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 2 to about 95 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 3 to about 90 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 4 to about 85 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 5 to about 80 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 6 to about 75 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 7 to about 70 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 8 to about 65 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 9 to about 60 neoantigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes from about 10 to about 55 neoantigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about 20 to about 65 neoantigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes more than 20 neoantigens. In some embodiments, all neoantigens encoded by the nucleic acid molecule or resulting DNA vaccine are the same neoantigen. In some embodiments, at least about two or more of the neoantigens encoded by the nucleic acid molecule or resulting DNA vaccine are different from one another.

[0167] In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes one single neo-antigen. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about two different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about three different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about four different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about five different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about six different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about seven different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about eight different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about nine different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about 10 different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes about 20 different neo-antigens. In some embodiments, the nucleic acid molecule or resulting DNA vaccine encodes more than about 20 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 30 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 40 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 50 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 55 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 60 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 65 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 70 different neo-antigens.In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 75 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 80 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 85 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 90 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 95 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes about 100 different neo-antigens. In some embodiments, the nucleic acid molecule, or resulting DNA vaccine, encodes more than about 100 different neo-antigens.

[0168] In embodiments in which the nucleic acid molecule or resulting DNA vaccine encodes more than one neo-antigen, each neo-antigen encoded by the nucleic acid molecule can be separated from another by one or more linkers. Thus, in some embodiments, the nucleic acid molecule or resulting DNA vaccine of the present disclosure further comprises one or more nucleotide sequences encoding one or more linkers. In some embodiments, each linker can be independently selected from a length of about 0 to about 30, 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, or about 24 to about 25 amino acids. In some embodiments, each linker can be independently selected from 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, or about 25 amino acids in length.

[0169] In embodiments in which two or more linkers are encoded by the nucleic acid molecules or resulting DNA vaccines of the present disclosure, the lengths of each linker can be the same or different. In some embodiments, the lengths of each linker are the same. In some embodiments, the lengths of each linker are 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, or about 25 amino acids in length, 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, or about 25 amino acids in length, and the length of the first linker is different from the length of the second linker. Various configurations are contemplated by the present disclosure, in which the nucleic acid molecules or DNA vaccines of the present disclosure comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more linkers, and the linkers are of similar or different lengths. In some embodiments, the linkers are all the same length, and there are at least about 19 linkers.

[0170] In some embodiments, two linkers can be used together as a fusion peptide encoded by a single nucleotide sequence. Thus, in some embodiments, the first linker can be independently selected from a range of amino acids in length from about 0 to about 30, 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, or about 24 to about 25. In some embodiments, the second linker can be independently selected from a length of about 0 to about 30, 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, or about 24 to about 25 amino acids. In some embodiments, the first linker can be independently selected from 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, or about 25 amino acids in length. In some embodiments, the second linker can be independently selected from 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, or about 25 amino acids in length.

[0171] The present disclosure relates to nucleic acid molecules comprising at least one expressible nucleic acid sequence comprising any of the formulas contained herein and encoding any of the antigens identified in Tables M, N, and / or O, or a functional fragment of a sequence that is at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an antigen identified in Tables M, N, and / or O. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding at least about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 or more different antigen domains, wherein at least one of the antigen domains is selected from an antigen identified in Tables M, N, and / or O, or a functional fragment of a sequence that is at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an antigen identified in Tables M, N, and / or O. The present disclosure relates to cells comprising a nucleic acid molecule comprising at least one expressible nucleic acid sequence comprising any of the formulas contained herein and encoding any of the antigens identified in Tables M, N, and / or O, or a functional fragment of a sequence that is at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an antigen identified in Tables M, N, and / or O.In some embodiments, a composition of the disclosure comprises a cell comprising a nucleic acid molecule comprising at least one expressible nucleic acid sequence comprising any of the formulas contained herein and encoding any of the antigens identified in Tables M, N, and / or O, or a functional fragment of a sequence that is at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an antigen identified in Tables M, N, and / or O, and at least one cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR sequence. In some embodiments, the cells comprise a plasmid encoding one or more TCR sequences selected from any one or more of Tables Q, R, X, Y or Z, or a functional fragment thereof that is at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a TCR sequence in Tables Q, R, X, Y and Z.

[0172] In some embodiments, one or more linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure contain an amino acid cleavage site. Any amino acid cleavage site may be used. One non-limiting example is the furin protease cleavage site recognized by a protease known as furin, which resides in the trans-Golgi network of eukaryotic cells. Furin functions to cleave proteins at the step immediately preceding delivery to their final cellular destination. Furin recognizes the consensus amino acid sequences RXRR, RXRK, or KXKR (where X is any amino acid; Moehring et al., 1993, incorporated herein by reference in its entirety) and cleaves proteins containing these sequences upon reaching the trans-Golgi network. Furin is a Ca2+-dependent serine endoprotease that cleaves protein precursors with high specificity following several basic motifs, as shown in Table 1 below.

[0173] (Table 1) Basic motifs of furin cleavage sites. TIFF2025516676000005.tif36142

[0174] Another non-limiting example of an amino acid cleavage site is the cleavage site recognized by the 2A peptide, a small, "self-cleaving" peptide. The average length of 2A peptides is 18-22 amino acids. The "2A" designation refers to a specific region of the picornavirus polyprotein. Of the 2A peptides identified to date, four have been widely used in research: FMDV 2A (abbreviated herein as F2A), Equine Rhinitis A Virus (ERAV) 2A (E2A), Porcine Ovarian Cancer Virus-1 2A (P2A), and Thoseaasigna Virus 2A (T2A). The former three viruses belong to picornaviruses, while the latter is an insect virus. The DNA and corresponding amino acid sequences of various 2A peptides are shown in Table 2 below. The underlined sequences encode the amino acid GSG, which was added to improve cleavage efficiency. P2A indicates porcine teschovirus-1 2A, T2A indicates Thoseaasigna virus 2A, E2A indicates equine rhinitis A virus (ERAV) 2A, and F2A indicates FMDV2A.

[0175] Table 2: DNA and corresponding amino acid sequences of various 2A peptides. The table discloses SEQ ID NOs: 595-602, respectively, in order of appearance. TIFF2025516676000006.tif123158

[0176] In some embodiments, one or more of the linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise a furin protease cleavage site. In some embodiments, one or more of the linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise a furin protease cleavage site comprising the sequence RX-[R / K]-R, where R represents arginine, X is any amino acid, and K is lysine. "R / K" indicates that the amino acid is either arginine or lysine.

[0177] In some embodiments, one or more of the linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise a 2A cleavage site. In some embodiments, one or more of the linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise a porcine teschovirus-1 2A (P2A) cleavage site. In some embodiments, one or more of the linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise a Thosea asigna virus 2A (T2A) cleavage site. In some embodiments, one or more of the linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise an equine rhinitis A virus (ERAV) 2A 9 (E2A) cleavage site. In some embodiments, one or more of the linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise an FMDV 2A (F2A) cleavage site.

[0178] In some embodiments, the nucleic acid molecule or DNA vaccine of the present disclosure further comprises a nucleotide sequence encoding a leader sequence. A "leader sequence" (or sometimes referred to as a "signal peptide") is an amino acid sequence that typically directs the localization of a protein. As used herein, a leader sequence preferably facilitates secretion of the protein from the cell in which it is produced. The leader sequence is often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. When present, the leader sequence is linked at the N-terminus of the protein. A non-limiting example of a leader sequence is the IgE leader sequence described in U.S. Application Publication No. 2016 / 0175427, which is incorporated herein by reference in its entirety.

[0179] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure may also include one or more regulatory sequences. If present, such one or more regulatory sequences are operably linked to the nucleotide sequence encoding the neoantigen, linker, and / or leader sequence. Examples of regulatory sequences include, but are not limited to, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in 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.

[0180] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are contained in a vector or plasmid. In some embodiments, the vector is a plasmid. The plasmid may be useful for transfecting cells with the nucleic acid molecules or DNA vaccines of the present disclosure, such that the transformed cells express the neoantigens and elicit an immune response. In some embodiments, the compositions of the present disclosure relate to nucleic acid molecules comprising a nucleic acid sequence encoding multiple neoantigens. In some embodiments, the plasmid further comprises a start codon, which may be upstream or downstream of the neoantigen coding sequence, and a stop codon. In some embodiments, the start codon and stop codon are in frame with the neoantigen coding sequence.

[0181] In some embodiments, the plasmid also includes a promoter operably linked to the coding sequence. In some embodiments, the promoter operably linked to the coding sequence is a promoter derived from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. In some embodiments, the promoter is a promoter derived from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. In some embodiments, the promoter is 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. 2004 / 0175727, the entire contents of which are incorporated herein. In some embodiments, the plasmid also includes a polyadenylation signal, which may be downstream of the coding sequence. In some embodiments, the polyadenylation signal is 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. In some embodiments, the SV40 polyadenylation signal may be a polyadenylation signal from the pCEP4 plasmid (Invitrogen, San Diego, CA).

[0182] In some embodiments, the plasmid also contains an enhancer upstream of the coding sequence. In some embodiments, the enhancer is human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as those derived from CMV, FMDV, RSV, or EBV. Polynucleotide function enhancers are described, for example, in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94 / 016737, the contents of each of which are incorporated herein by reference in their entireties. In some embodiments, the plasmid also contains a mammalian origin of replication for extrachromosomal maintenance of the plasmid and for producing multiple copies of the plasmid within the cell. In some embodiments, the plasmid is pVAX1, pCEP4, or pREP4 from ThermoFisher Scientific (San Diego, CA), which contains the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, which produces high-copy episomal replication without integration.

[0183] In some embodiments, the vector is pVAX1 or a pVax1 variant with changes such as the variant plasmids described herein. The variant pVax1 plasmid is a 2998 base pair variant of the backbone vector plasmid pVAX1 (Invitrogen, Carlsbad CA). The CMV promoter is located at bases 137-724. The T7 promoter / priming site is at bases 664-683. The multiple cloning site is at bases 696-811. The bovine GH polyadenylation signal is at bases 829-1053. The kanamycin resistance gene is at bases 1226-2020. The pUC origin is at bases 2320-2993. The nucleic acid sequence of the pVAX1 (SEQ ID NO: 361) backbone sequence is as follows:

[0184] In some embodiments, the nucleic acid molecule comprises at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the pVAX1 backbone or a functional fragment thereof, and the nucleic acid sequence further comprises an expressible nucleic acid sequence in a multiple cloning site.

[0185] Other vectors or plasmids that can be used herein to produce the vaccines of the present disclosure include, but are not limited to, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES.

[0186] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to a subject in an amount sufficient to elicit a cellular immune response. A "cellular immune response" is meant to include a cellular response directed against cells characterized by antigen presentation with class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes, which function as either "helpers" or "killers." Helper T cells (also called CD4+ T cells) play a central role by regulating the immune response, while killer cells (also called cytotoxic T cells, cytotoxic T cells, CD8+ T cells, or CTLs) kill diseased cells, such as cancer cells, and prevent the production of more diseased cells. In some embodiments, the present disclosure involves stimulating an anti-cancer CTL response against cancer cells that express one or more cancer-specific neoantigens, preferably presenting such cancer-specific neoantigens with class I MHC.

[0187] In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to elicit a CD8+ T cell response against any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to elicit a CD8+ T and / or CD4+ T cell response against any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to elicit a CD4+ T cell response against any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine.

[0188] In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 25% or more CD4+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 30% or more CD4+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 40% or more CD4+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 50% or more CD4+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to a subject in an amount sufficient to elicit a subpopulation of T cells comprising greater than about 60% CD4+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine.

[0189] In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 25% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 30% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 40% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 50% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to a subject in an amount sufficient to elicit a subpopulation of T cells comprising greater than about 60% CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine.

[0190] In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 25% or more CD4+ T cells and at least about 25% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 30% or more CD4+ T cells and at least about 30% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 40% or more CD4+ T cells and at least about 40% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 50% or more CD4+ T cells and at least about 50% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine. In some embodiments, a nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount sufficient to induce a subpopulation of T cells comprising at least about 60% or more CD4+ T cells and at least about 60% or more CD8+ T cells in response to any one or more neo-antigens encoded by the nucleic acid molecule or DNA vaccine.

[0191] In some embodiments, the nucleic acid molecule or DNA vaccine of the present disclosure is administered to a subject in an amount of about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 microgram to about 10 milligrams, or about 1 milligram to about 2 milligrams. In some embodiments, a pharmaceutical composition comprising the nucleic acid molecule or DNA vaccine of the present disclosure in an amount of about 1 nanogram to about 1000 micrograms of DNA is used for administration.

[0192] The nucleic acid molecules or DNA vaccines of the present disclosure, or vectors or plasmids comprising same, can be administered or delivered to a subject by several well-known techniques, including, but not limited to, in vivo electroporation, liposome-mediated, nanoparticle-facilitated, DNA injection (also called DNA vaccination) with or without recombinant vectors (such as recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant vaccines). In some embodiments, neoantigens are delivered via DNA injection and in conjunction with in vivo electroporation.

[0193] The nucleic acid molecules or DNA vaccines of the present disclosure, or vectors or plasmids containing same, can be administered by electroporation. This can be achieved using an electroporation device configured to deliver a pulse of energy to the desired mammalian tissue effective to form reversible pores in the cell membrane, preferably a constant current similar to a preset current input by the user. The electroporation device can include an electroporation component and an electrode assembly or handle assembly. The electroporation component can include or incorporate one or more of the various elements of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. Electroporation can be achieved using an in vivo electroporation device, such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.), to facilitate transfection of cells with the plasmid.

[0194] The electroporation component may function as one element of an electroporation device, with the other element being a separate element (or components) that communicates with the electroporation component. The electroporation component may function as more than one element of an electroporation device, which may communicate with yet other elements of the electroporation device that are separate from the electroporation component. The elements of an electroporation device that exist as part of a single electromechanical or mechanical device may be unlimited, as the elements can function as a single device or as separate elements that communicate with each other. The electroporation component may be capable of delivering a pulse of energy that generates a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly may include an electrode array having multiple electrodes in a spatial arrangement, which receives the pulse of energy from the electroporation component and delivers it to the desired tissue through the electrodes. At least one of the multiple electrodes is neutral during delivery of the pulse of energy, measures impedance at the desired tissue, and communicates the impedance to the electroporation component. A feedback mechanism can receive the measured impedance and adjust the pulses of energy delivered by the electroporation component to maintain a constant current.

[0195] The plurality of electrodes can deliver pulses of energy in a distributed pattern. The plurality of electrodes can deliver pulses of energy in a distributed pattern through control of the electrodes under a programmed sequence, the programmed sequence being input into the electroporation component by a user. The programmed sequence can include multiple pulses delivered sequentially, each pulse of the plurality of pulses being delivered by at least two active electrodes with one indifferent electrode measuring impedance, and each subsequent pulse of the plurality of pulses being delivered by a different one of the at least two active electrodes with one indifferent electrode measuring impedance.

[0196] In some embodiments, the feedback mechanism is implemented by either hardware or software. In some embodiments, the feedback mechanism is implemented by an analog closed-loop circuit. Feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous, as determined by available techniques for determining response time). The indifferent electrode may measure the impedance in the desired tissue and communicate the impedance to the feedback mechanism, which responds to the impedance and adjusts the pulse of energy to maintain a constant current at a value similar to the preset current. The feedback mechanism may maintain a constant current continuously and instantaneously during delivery of the pulse of energy.

[0197] Examples of electroporation devices and methods that can enhance delivery of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 to Draghia-Akli et al. and U.S. Patent Publication No. 2005 / 0052630 to Smith et al., the entire contents of which are incorporated herein by reference. Other electroporation devices and methods that can be used to enhance delivery of DNA vaccines include those provided in co-pending and co-owned U.S. patent application Ser. No. 11 / 874,072, filed October 17, 2007, which claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Application Ser. No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein in their entireties.

[0198] U.S. Patent No. 7,245,963 to Draghia-Akli et al. describes a modular electrode system and its use for promoting the introduction of biomolecules into cells of selected tissues in living organisms or plants. The controlled electrode system may include multiple needle electrodes, a hypodermic needle, an electrical connector providing conductive connection from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and securely insert them into selected tissues within the body or plant. The biomolecules are then delivered to the selected tissue via the hypodermic needle. A programmable constant current pulse controller is activated, applying constant current electrical pulses to the multiple needle electrodes. The applied constant current electrical pulses promote the introduction of biomolecules into cells between the multiple electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference in their entirety.

[0199] U.S. Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues in living organisms or plants. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant-current pulse patterns between electrodes in an array based on user control and input of pulse parameters, and allows for the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk with an array of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of U.S. Patent Publication No. 2005 / 0052630 are incorporated herein by reference in their entirety. The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 are suitable for deep penetration of tissues such as muscle, as well as other tissues or organs. The electrode array configuration also allows the injection needle (for delivering the selected biomolecule) to be fully inserted into the target organ, and the injection is administered perpendicular to the target tissue in the area pre-delineated by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0200] Additionally, in some embodiments incorporating electroporation devices and their uses, the electroporation devices described in the following patents are contemplated: U.S. Patent No. 5,273,525, issued December 28, 1993; U.S. Patent No. 6,110,161, issued August 29, 2000; U.S. Patent No. 6,261,281, issued July 17, 2001; U.S. Patent No. 6,958,060, issued October 25, 2005; and U.S. Patent No. 6,939,862, issued September 6, 2005. Also contemplated herein are patents covering subject matter provided in U.S. Patent No. 6,697,669, issued February 24, 2004, relating to delivery of DNA using any of various devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, relating to methods of injecting DNA. The above patents are incorporated herein by reference in their entireties.

[0201] In some embodiments, the cancer-specific neoantigens and subject-specific neoantigens are administered to the subject in the form of RNA. In such embodiments, the neoantigen-encoding RNA can be delivered as a transgene in an RNA vaccine (such as Moderna's mRNA platform). RNA vaccines are non-infectious, non-integrating, and naturally degradable. RNA vaccines have been shown to stimulate strong B-cell responses against the encoded transgene. Naked RNA can be formulated with lipid nanoparticles (LNPs) to encapsulate the RNA, protecting the transgene from degradation.

[0202] The composition comprising the RNA nucleic acid sequence can be delivered via lipid-containing nanoparticles. In some embodiments, the composition comprises at least one RNA polynucleotide having an open reading frame encoding one or more cancer-specific neoantigens with at least one modification, at least one 5'-end cap, and is formulated in lipid nanoparticles. In some embodiments, the at least one chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, 5'-capping of polynucleotides is completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). In other embodiments, 5'-capping of modified RNAs is completed post-transcriptionally using vaccinia virus capping enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). The Cap 1 structure can be generated using both vaccinia virus capping enzyme and 2'-O methyl-transferase to generate m7G(5')ppp(5')G-2'-O-methyl.The Cap 2 structure can be generated from the Cap 1 structure, followed by 2'-O-methylation of the 5'-antepenultimate nucleotide using a 2'-O-methyl-transferase. The Cap 3 structure can be generated from the Cap 2 structure, followed by 2'-O-methylation of the 5'-preantepenultimate nucleotide using a 2'-O-methyl-transferase. The enzyme is preferably derived from a recombinant source. The modified mRNA, when transfected into mammalian cells, has a stability of about 12 to about 18 hours or greater than about 18 hours, e.g., 24, 36, 48, 60, 72 hours, or greater than about 72 hours.

[0203] In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid, in some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), and N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530).

[0204] In some embodiments, the neoantigen-encoding RNA of the present disclosure is formulated into lipid nanoparticles. In some embodiments, the neoantigen-encoding RNA of the present disclosure is formulated into lipid-polycation complexes called cationic lipid nanoparticles. The formation of lipid nanoparticles can be achieved by methods known in the art and / or described in U.S. Publication No. 2012 / 0178702, which is incorporated herein by reference in its entirety. By way of non-limiting example, polycations include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine, and the cationic peptides described in WO 2012 / 013326 or U.S. Publication No. 2013 / 0142818, each of which is incorporated herein by reference in its entirety. In some embodiments, the neoantigen-encoding RNA of the present disclosure is formulated into lipid nanoparticles comprising a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0205] Lipid nanoparticle formulations can be influenced by biophysical parameters, including but not limited to, the selection of cationic lipid components, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all components, and size.In one example by Semple et al. (Nature Biotech.2010 28:172-176; the entirety of which is incorporated herein by reference), lipid nanoparticle formulations are composed of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.In another example, changing the composition of cationic lipids has been shown to more effectively deliver siRNA to various antigen-presenting cells (Basha et al.Mol Ther.2011 19:2186-2200; the entirety of which is incorporated herein by reference).

[0206] In some embodiments, the lipid nanoparticle formulation may comprise 35%-45% cationic lipid, 40%-50% cationic lipid, 50%-60% cationic lipid, and / or 55%-65% cationic lipid. In some embodiments, the lipid to RNA (e.g., mRNA) ratio in the lipid nanoparticle is 5:1-20:1, 10:1-25:1, 15:1-30:1, and / or at least 30:1.

[0207] In some embodiments, the proportion of PEG in the lipid nanoparticle formulation is increased or decreased, and / or the carbon chain length of the PEG lipid is modified from C14 to C18, to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, in some embodiments, the lipid nanoparticle formulation contains a lipid molar ratio of about 0.5% to about 3.0%, about 1.0% to about 3.5%, about 1.5% to about 4.0%, about 2.0% to about 4.5%, about 2.5% to about 5.0%, and / or about 3.0% to about 6.0% PEG-c-DOMG (R-3-[(co-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In some embodiments, the PEG-c-DOMG is replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol), and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). In some embodiments, the cationic lipid is selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200, and DLin-KC2-DMA.

[0208] In some embodiments, the neoantigen-encoding RNA of the present disclosure is formulated as a nanoparticle comprising at least one lipid, in some embodiments, the lipid is selected from, but not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine (L608), N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine (L530), PEGylated lipids, and aminoalcohol lipids.

[0209] In some embodiments, the lipid nanoparticle formulation comprises about 25% to about 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di(Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319) (e.g., about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50%, or about 40% on a molar basis). In some embodiments, the lipid nanoparticle formulation comprises about 0.5% to about 15% neutral lipid on a molar basis (e.g., about 3% to about 12%, about 5% to about 10%, or about 15%, about 10%, or about 7.5% on a molar basis). Non-limiting examples of neutral lipids include DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises about 5% to about 50% sterol on a molar basis (e.g., about 15% to about 45%, about 20% to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a molar basis). A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises about 0.5% to about 20% PEG or PEG-modified lipid on a molar basis (e.g., about 0.5% to about 10%, about 0.5% to about 5%, about 0.5%, about 1.0%, about 1.5%, about 3.5%, or about 5% on a molar basis). In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of about 2,000 Da. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than about 2,000, e.g., about 1,500 Da, about 1,000 Da, or about 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), and PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), which is incorporated herein by reference in its entirety).

[0210] In some embodiments, the lipid nanoparticle formulation comprises about 25-75% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 0.5-15% neutral lipid, about 5-50% sterol, and about 0.5-20% PEG or PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation comprises about 35-65% by molar amount of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 3-12% neutral lipid, about 15-45% sterol, and about 0.5-10% PEG or PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation comprises about 45-65% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 5-10% neutral lipid, about 25-40% sterol, and about 0.5-10% PEG or PEG-modified lipid.In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, about 60% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% neutral lipid, about 31% sterol, and about 1.5% PEG or PEG-modified lipid.

[0211] In some embodiments, the neoantigen-encoding RNA of the present disclosure comprises at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding one or more cancer-specific neoantigens, wherein at least about 80% of the uracils in the open reading frame have chemical modifications, and optionally, the neoantigen-encoding RNA is formulated into a lipid nanoparticle. In some embodiments, the neoantigen-encoding RNA is formulated into liposomes, such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); incorporated herein by reference in its entirety), and hyaluronan-coated liposomes (Quiet Therapeutics, Israel). In some embodiments, the neoantigen-encoding RNA may be formulated into lyophilized gel-phase liposome compositions, such as those described in U.S. Publication No. 2012 / 060293, incorporated herein by reference in its entirety.

[0212] In some embodiments, the nanoparticle formulation comprises a phosphate conjugate. The phosphate conjugate may increase the in vivo circulation time of the nanoparticle and / or increase the targeted delivery of the nanoparticle. In some embodiments, the conjugate for use with the present disclosure is made by the methods described in WO 2013 / 033438 or U.S. Publication No. 2013 / 0196948, the contents of each of which are incorporated herein by reference in their entirety. As a non-limiting example, the phosphate conjugate may comprise a compound of any one of the formulas described in WO 2013 / 033438, the contents of which are incorporated herein by reference in their entirety. Specifically, the present disclosure relates to pharmaceutical compositions comprising nanoparticles comprising RNA encoding one or more cancer-specific neoantigens, (i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles, and / or (ii) the nanoparticles have a neutral or net negative charge, and / or (iii) the charge ratio of positive to negative charges in the nanoparticle is 1.4:1 or less; and / or (iv) The zeta potential of the nanoparticles is about 0 or less.

[0213] In some embodiments, the nanoparticles described herein are colloidally stable for at least about 2 hours, meaning that they do not aggregate, precipitate, or increase in size or polydispersity index by more than about 30%, as measured by dynamic light scattering. In some embodiments, the charge ratio of positive to negative charges in the nanoparticles is about 1.4:1 to about 1:8, about 1.2:1 to about 1:4, about 1:1 to about 1:3, about 1:1.2 to about 1:2, about 1:1.2 to about 1:1.8, about 1:1.3 to about 1:1.7, about 1:1.4 to about 1:1.6, or about 1:1.5. In some embodiments, the zeta potential of the nanoparticles is about -5 or less, about -10 or less, about -15 or less, about -20 or less, or about -25 or less. In various embodiments, the zeta potential of the nanoparticles is about -35 or more, about -30 or more, or about -25 or more. In some embodiments, the nanoparticles have a zeta potential of about 0 mV to about −50 mV, about 0 mV to about −40 mV, or about −10 mV to about −30 mV.

[0214] In some embodiments, the pharmaceutical compositions of the present disclosure comprise nanoparticles or liposomes encapsulating DNA, RNA, or DNA / RNA hybrids containing at least one expressible nucleic acid sequence. Liposomes are microscopic lipid vesicles, often containing one or more bilayers of vesicle-forming lipids such as phospholipids, and are capable of encapsulating drugs. In the context of the present disclosure, different types of liposomes may be used, including, but not limited to, multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), sterically stabilized liposomes (SSLs), multivesicular vesicles (MVs), and large multivesicular vesicles (LMVs), as well as other bilayer forms known in the art. The size and lamellar structure of liposomes depend on the mode of preparation, and the choice of vesicle type used depends on the preferred method of administration. There are several other forms of supramolecular organizations in which lipids can exist in aqueous media, including lamellar phases, hexagonal and reverse hexagonal phases, cubic phases, micelles, and reverse micelles composed of monolayers. These phases may also be obtained in combination with DNA or RNA, and interactions with RNA and DNA may substantially affect the phase state. In some embodiments, one or more of the above-mentioned phases are present in the nanoparticle RNA formulations of the present disclosure.

[0215] For the formation of RNA lipoplexes from RNA and liposomes, any suitable method for forming liposomes can be used as long as it provides the intended RNA lipoplexes. In some embodiments, liposomes are formed using standard methods such as reverse evaporation (REV), ethanol injection, dehydration-rehydration (DRV), sonication, or other suitable methods.

[0216] After liposome formation, the liposomes can be sized to obtain a population of liposomes having a substantially uniform size range.

[0217] Bilayer-forming lipids typically have two hydrocarbon chains, particularly acyl chains, and either polar or nonpolar head groups. Bilayer-forming lipids can be either natural lipids or synthetic, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, and sphingomyelin, where the two hydrocarbon chains are typically about 14 to 22 carbon atoms in length and have varying degrees of unsaturation. Other suitable lipids for use in the compositions of the present invention include glycolipids and sterols, such as cholesterol and its various analogs, which can also be used in liposomes.

[0218] Cationic lipids typically have a lipophilic moiety, such as a sterol, acyl, or diacyl chain, and an overall net positive charge. The head group of the lipid typically carries the positive charge. Cationic lipids preferably have 1 to 10 positive charges, more preferably 1 to 3 positive charges, and more preferably a single positive charge. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkoxy-3-dimethylammonium propane, dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA).In some embodiments, the cationic lipid is DOTMA, DOTAP, DODAC, and DOSPA. In some embodiments, the neutral lipid is DOTMA.

[0219] In addition, the nanoparticles described herein preferably further contain a neutral lipid in terms of structural stability, etc. The neutral lipid can be appropriately selected taking into account the delivery efficiency of the RNA-lipid complex. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, sterol, and cerebroside. In some embodiments, the neutral lipid is DOPE and / or DOPC. In some embodiments, the neutral lipid is DOPE. When the cationic liposome contains both a cationic lipid and a neutral lipid, the molar ratio of the cationic lipid to the neutral lipid can be appropriately determined taking into account the stability of the liposome, etc.

[0220] In some embodiments, the nanoparticles described herein comprise a phospholipid. In some embodiments, the phospholipid is a glycerophospholipid. Examples of glycerophospholipids include, but are not limited to, three types of lipids: (i) amphoteric phospholipids, such as phosphatidylcholine (PC), egg yolk phosphatidylcholine, soybean-derived PC in native, partially hydrogenated, or fully hydrogenated forms, dimyristoylphosphatidylcholine (DMPC), sphingomyelin (SM), etc.; (ii) negatively charged phospholipids, such as phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), dipalmipoyl PG, dimyristoylphosphatidylglycerol (DMPG); synthetic derivatives in which the conjugate renders the zwitterionic phospholipid negatively charged, such as in the case of methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE); and (iii) cationic phospholipids, such as phosphatidylcholine or sphingomyelin in which the phosphomonoester has been O-methylated to form a cationic lipid.

[0221] Association of RNA with lipid carriers can occur, for example, by the RNA-filled interstitial spaces of the carrier, so that the carrier physically entraps the RNA, or by adsorption through covalent, ionic, or hydrogen bonds, or nonspecific binding. Regardless of the method of association, the RNA must retain its therapeutic, i.e., antigen-coding, properties.

[0222] In some embodiments, the nanoparticles comprise at least one lipid. In some embodiments, the nanoparticles comprise at least one cationic lipid. The cationic lipid can be mono- or polycationic. Any cationic amphiphilic molecule, e.g., a molecule comprising at least one hydrophilic and lipophilic portion, is a cationic lipid within the meaning of the present invention. In some embodiments, the positive charge is contributed by at least one cationic lipid, and the negative charge is contributed by RNA. In some embodiments, the nanoparticles comprise at least one helper lipid. The helper lipid can be a neutral or anionic lipid. In some embodiments, the helper lipid is a natural lipid, such as a phospholipid or an analog of a natural lipid. In some embodiments, the helper lipid is a completely synthetic lipid or lipid-like molecule, and does not resemble a natural lipid. In some embodiments, the cationic lipid and / or the helper lipid are bilayer forming lipids.

[0223] In some embodiments, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) or an analog or derivative thereof, and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or an analog or derivative thereof. In some embodiments, at least one helper lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or an analog or derivative thereof, cholesterol (Chol) or an analog or derivative thereof, and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or an analog or derivative thereof. In some embodiments, the molar ratio of at least one cationic lipid to at least one helper lipid is 10:0 to 3:7, preferably 9:1 to 3:7, 4:1 to 1:2, 4:1 to 2:3, 7:3 to 1:1, or 2:1 to 1:1, preferably about 1:1. In some embodiments, in this ratio, the molar amount of cationic lipid is obtained by multiplying the molar amount of cationic lipid by the number of positive charges in the cationic lipid. In various embodiments, the lipid is not functionalized, e.g., functionalized with mannose, histidine, and / or imidazole, the nanoparticle does not include a targeting ligand such as a mannose-functionalized lipid, and / or the nanoparticle does not include one or more of the following: a pH-dependent compound, a cationic polymer such as a polymer containing histidine and / or polylysine (the polymer may optionally be PEGylated and / or histidylated), or a divalent ion such as Ca2+.

[0224] In various embodiments, the RNA nanoparticles may comprise peptides that preferentially have a molecular weight of up to 2500 Da.

[0225] In the nanoparticles described herein, the lipid may complex with and / or encapsulate the RNA. In some embodiments, the nanoparticle comprises a lipoplex or a liposome. In some embodiments, the lipid is contained in a vesicle that encapsulates the RNA. In some embodiments, the vesicle is a multilamellar vesicle, a unilamellar vesicle, or a mixture thereof. In some embodiments, the vesicle is a liposome. In some embodiments, the nanoparticle is a lipoplex comprising DOTMA and DOPE in a molar ratio of about 10:0 to about 1:9, about 8:2 to about 3:7, or about 7:3 to about 5:5, and the charge ratio of positive charges in DOTMA to negative charges in the RNA is about 1.8:2 to about 0.8:2, about 1.6:2 to about 1:2, about 1.4:2 to about 1.1:2, or about 1.2:2.

[0226] In some embodiments, the nanoparticles are lipoplexes comprising DOTMA and cholesterol in a molar ratio of about 10:0 to about 1:9, about 8:2 to about 3:7, or about 7:3 to about 5:5, and the charge ratio of positive charges in DOTMA to negative charges in RNA is about 1.8:2 to about 0.8:2, about 1.6:2 to about 1:2, about 1.4:2 to about 1.1:2, or about 1.2:2. In some embodiments, the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of about 10:0 to about 1:9, about 8:2 to about 3:7, or about 7:3 to about 5:5, and the charge ratio of positive charges in DOTMA to negative charges in RNA is about 1.8:2 to about 0.8:2, about 1.6:2 to about 1:2, about 1.4:2 to about 1.1:2, or about 1.2:2. In some embodiments, the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of about 2:1 to about 1:2, or about 2:1 to about 1:1, wherein the charge ratio of positive charges in DOTMA to negative charges in RNA is about 1.4:1 or less. In some embodiments, the nanoparticles are lipoplexes comprising DOTMA and cholesterol in a molar ratio of about 2:1 to about 1:2, or about 2:1 to about 1:1, wherein the charge ratio of positive charges in DOTMA to negative charges in RNA is about 1.4:1 or less. In some embodiments, the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of about 2:1 to about 1:2, or about 2:1 to about 1:1, wherein the charge ratio of positive charges in DOTAP to negative charges in RNA is about 1.4:1 or less. In some embodiments, the nanoparticles have an average diameter ranging from about 50 nm to about 1000 nm, from about 50 nm to about 400 nm, from about 100 nm to about 300 nm, or from about 150 nm to about 200 nm. In some embodiments, the nanoparticles have a diameter ranging from about 200 to about 700 nm, from about 200 to about 600 nm, from about 250 to about 550 nm, from about 300 to about 500 nm, or from about 200 to about 400 nm.

[0227] In some embodiments, the polydispersity index of the nanoparticles described herein, as measured by dynamic light scattering, is about 0.5 or less, about 0.4 or less, or about 0.3 or less. In some embodiments, the nanoparticles described herein are obtained by one or more of the following: (i) incubation of liposomes in an aqueous phase with RNA in the aqueous phase, (ii) incubation of lipids dissolved in an organic water-miscible solvent such as ethanol with RNA in an aqueous solution, (iii) reverse phase evaporation techniques, (iv) freezing and thawing the product, (v) dehydrating and rehydrating the product, (vi) lyophilizing and rehydrating the product, or (vii) spray drying and rehydrating the product.

[0228] In some embodiments, the nanoparticle formulation comprises a polymer conjugate. In some embodiments, the polymer conjugate is a water-soluble conjugate. In some embodiments, the polymer conjugate has a structure described in U.S. Publication No. 2013 / 0059360, the entire contents of which are incorporated herein by reference. In some embodiments, the polymer conjugates with polynucleotides of the present disclosure are made using the methods and / or segmented polymer reagents described in U.S. Publication No. 2013 / 0072709, the entire contents of which are incorporated herein by reference. In other embodiments, the polymer conjugate has pendant side groups comprising ring moieties, such as, but not limited to, the polymer conjugates described in U.S. Publication No. 2013 / 0196948, the entire contents of which are incorporated herein by reference.

[0229] In some embodiments, the nanoparticle formulation comprises a conjugate that enhances delivery of the nanoparticles of the present invention in a subject. Furthermore, the conjugate can inhibit phagocytic clearance of the nanoparticles in a subject. In some embodiments, the conjugate is a "self" peptide designed from the human membrane protein CD47 (e.g., the "self" particle described by Rodriguez et al. (Science 2013, 339, 971-975), the entire contents of which are incorporated herein by reference). As shown by Rodriguez et al., the self peptide delayed macrophage-mediated clearance of the nanoparticles, which enhanced the delivery of the nanoparticles. In other embodiments, the conjugate is the membrane protein CD47 (e.g., see Rodriguez et al. Science 2013, 339, 971-975, the entire contents of which are incorporated herein by reference). Rodriguez et al. showed that, like the "self" peptide, CD47 can increase the circulating particle ratio in a subject compared to nanoparticles coated with scrambled peptides and PEG.

[0230] In some embodiments, about 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5' position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, about 100% of the uracils in the open reading frame have N1-methylpseudouridine at the 5' position of the uracil.

[0231] In some embodiments, the neoantigen-encoding RNA of the present disclosure is administered to a subject in the form of an RNA vaccine. In some embodiments, the efficacy of the RNA vaccine of the present disclosure can be significantly enhanced when combined with a flagellin adjuvant, particularly when one or more RNA vaccines are combined with mRNA encoding flagellin.

[0232] RNA vaccines combined with flagellin adjuvants (e.g., mRNA-encoded flagellin adjuvants) have superior properties in that they can generate much greater antibody titers and generate earlier responses than commercially available vaccine formulations. Without wishing to be bound by theory, it is believed that RNA vaccines, e.g., as mRNA polynucleotides, are better designed to generate the appropriate protein conformations during translation for both antigens and adjuvants because RNA vaccines share natural cellular machinery. Unlike conventional vaccines, which are produced ex vivo and can trigger undesirable cellular responses, RNA vaccines are presented to cellular systems in a more natural manner.

[0233] In some embodiments, the RNA vaccines of the present disclosure comprise at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding one or more cancer-specific neoantigens and subject-specific neoantigens, and at least one RNA (e.g., mRNA polynucleotide) having an open reading frame encoding a flagellin adjuvant. In some embodiments, the at least one flagellin polypeptide (e.g., the encoded flagellin polypeptide) is a flagellin protein. In some embodiments, the at least one flagellin polypeptide (e.g., the encoded flagellin polypeptide) is an immunogenic flagellin fragment. In some embodiments, the at least one flagellin polypeptide and the at least one antigen polypeptide are encoded by a single RNA (e.g., mRNA) polynucleotide. In other embodiments, the at least one flagellin polypeptide and the at least one antigen polypeptide are each encoded by a different RNA polynucleotide.

[0234] In some embodiments, the method further comprises formulating the subject-specific DNA neo-antigens into an immunogenic composition for administration to the subject. In some embodiments, the cancer-specific neo-antigens and the subject-specific neo-antigens are formulated in the immunogenic composition in the form of DNA. In some embodiments, the cancer-specific neo-antigens and the subject-specific neo-antigens are formulated in the immunogenic composition in the form of RNA. In some embodiments, the cancer-specific neo-antigens and the subject-specific neo-antigens are formulated in the immunogenic composition in the form of protein. In embodiments where the cancer-specific neo-antigens and the subject-specific neo-antigens are formulated in the immunogenic composition in the form of DNA and / or RNA, the DNA / RNA neo-antigens may be subcloned into one or more vectors, which in some embodiments are one or more plasmids. Methods for administering DNA, RNA, or protein vaccines are known in the art. One of skill in the art can determine which treatment regimen is appropriate for each subject, depending, for example, on their cancer and their immune status (e.g., T cell, B cell, or NK cell activity and / or number).

[0235] Routes of administration include, but are not limited to, intramuscular, intranasal, intradermal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral, as well as topical, transdermal, inhalation, or suppository, or mucosal tissues, such as by lavage to vaginal, rectal, urethral, ​​buccal, and sublingual tissues. Preferred routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injection. In some embodiments, the genetic construct is administered by means including, but not limited to, a conventional syringe, a needleless injection device, a "microprojectile bombardment gun," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0236] Nucleic acid molecules encoding one or more neoantigens can also be administered to a subject for therapeutic or immunization purposes. Numerous methods are conveniently used to deliver nucleic acids to a subject. For example, nucleic acids can be delivered directly as "naked DNA." This approach is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, as described, for example, in U.S. Patent No. 5,204,253. Particles consisting solely of DNA can be administered. Alternatively, DNA can be attached to particles, such as gold particles.

[0237] Nucleic acids can also be delivered by complexing them with cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO1996 / 18372, WO1993 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691(1988), U.S. Patent No. 5,279,833, WO1991 / 06309, and Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414(1987).

[0238] RNA encoding neoantigens can also be used for delivery (see, for example, Kiken et al., 2011; Su et al., 2011).

[0239] Pharmaceutically acceptable carriers or excipients can include functional molecules such as vehicles, adjuvants, carriers, or diluents, which are known and generally readily available. In some embodiments, the pharmaceutically acceptable carrier is an adjuvant. In some embodiments, the pharmaceutically acceptable excipient is a transfection-facilitating agent. In some embodiments, the transfection-facilitating agent is a polyanion, polycation, or lipid, more preferably poly-L-glutamic acid. In some embodiments, the nucleic acid molecule or DNA plasmid is delivered to cells in combination with administration of a polynucleotide function-enhancing agent or a genetic vaccine-facilitating agent (or transfection-facilitating agent). Polynucleotide function-enhancing agents are described in U.S. Pat. No. 5,593,972, U.S. Pat. No. 5,962,428, and International Patent Application No. PCT / US94 / 00899, filed Jan. 26, 1994, each of which is incorporated herein by reference in its entirety. Genetic vaccine facilitating agents are described in U.S. Patent Application Serial No. 021,579, filed April 1, 1994, which is incorporated herein by reference in its entirety. The transfection facilitating agent can be administered in combination with the nucleic acid molecule as a mixture with the nucleic acid molecule, or can be administered separately and simultaneously, before or after administration of the nucleic acid molecule. Examples of transfection facilitating agents include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene; hyaluronic acid can also be used and administered in combination with the genetic construct. In some embodiments, DNA plasmid vaccines may also include transfection facilitating agents such as lipids, liposomes including lecithin liposomes or other liposomes known in the art, e.g., DNA-liposome mixtures (see, e.g., WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.Preferably, the transfection-enhancing agent is a polyanion, a polycation, including poly-L-glutamic acid (LGS), or a lipid.

[0240] In some embodiments, the DNA plasmids are delivered with genes for proteins that further enhance the immune response, such as those encoding other cytokines and lymphokines, such as alpha interferon, gamma interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and IL-15 (including IL-15 with its signal sequence deleted and optionally containing a signal peptide from IgE). Other genes that may be useful include those encoding: MCP-1, MIP-1α, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD4 0L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0241] When the agents described herein are administered to humans or animals as pharmaceuticals, they can be administered per se or as a pharmaceutical composition comprising the active ingredient in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

[0242] The actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied to provide an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without causing toxicity to the patient. Generally, the medicaments or pharmaceutical compositions of the present disclosure are administered in an amount sufficient to induce an immune gene response in the subject.

[0243] One or more nucleic acid molecules encoding cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions comprising same, for administration to a subject can contain amounts of DNA ranging from about 1 nanogram to about 10 milligrams, about 1 microgram to about 10 milligrams, about 0.1 microgram to about 10 milligrams, or about 100 micrograms to about 1 milligram. In some embodiments, one or more nucleic acid molecules encoding cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions comprising same, for administration to a subject, contain about 5 nanograms to about 1000 micrograms of DNA. In some embodiments, one or more nucleic acid molecules encoding cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions comprising same, for administration to a subject, contain about 10 nanograms to about 800 micrograms of DNA. In some embodiments, one or more nucleic acid molecules encoding cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions comprising same, for administration to a subject, contain about 0.1 to about 500 micrograms of DNA. In some embodiments, one or more nucleic acid molecules encoding the cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions comprising the same, for administration to a subject, comprise about 1 to about 350 micrograms of DNA. In some embodiments, one or more nucleic acid molecules encoding the cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions comprising the same, for administration to a subject, comprise about 25 to about 250 micrograms of DNA. In some embodiments, one or more nucleic acid molecules encoding the cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions comprising the same, for administration to a subject, comprise about 100 micrograms to about 1 milligram of DNA.

[0244] One or more nucleic acid molecules encoding the cancer-specific neoantigens and subject-specific neoantigens described herein, or compositions containing the same, for administration to a subject according to the present disclosure are formulated according to the mode of administration to be used. If they are injectable pharmaceutical compositions, they are sterile, pyrogen-free, and particulate-free. Isotonic formulations can be used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0245] In some embodiments, DNA formulations for use with muscle or skin EP devices have high DNA concentrations, e.g., concentrations containing microgram to tens of milligram or milligram amounts of DNA in a small injection volume, ideally 25-200 microliters (μL), optimal for delivery to the skin. In some embodiments, the DNA formulations have high DNA concentrations, such as 1 mg / mL or more (mg DNA / volume of formulation). In some embodiments, the DNA formulations have DNA concentrations that provide gram quantities of DNA in 200 μL of formula. In some embodiments, the DNA formulations have DNA concentrations that provide gram quantities of DNA in 100 μL of formula.

[0246] The DNA plasmids of the present disclosure for use with electroporation devices can be formulated or manufactured using a combination of known devices and techniques, for example, they are manufactured using the optimized plasmid manufacturing techniques described in U.S. Patent Application Publication No. 2009 / 0004716, the entire contents of which are incorporated herein by reference. In some embodiments, the DNA plasmids used can be formulated at a concentration of 10 mg / mL or greater. Manufacturing techniques also include or incorporate various devices and protocols commonly known to those skilled in the art, in addition to those described in U.S. Patent Application Publication No. 2009 / 0004716 and U.S. Patent No. 7,238,522, the entire contents of which are incorporated herein by reference. The high concentrations of plasmids used in the skin electroporation devices and delivery techniques described herein allow for the administration of plasmids to the ID / SC space in reasonably small volumes, helping to enhance expression and immune effects.

[0247] The amount and dosage regimen administered to a subject will depend on many factors, including the method of administration, the nature of the condition being treated, the weight of the subject being treated, and the judgment of the prescribing physician. The amount of nucleic acid molecule (DNA or RNA) included in the therapeutically active formulations according to the present disclosure is an amount effective to induce an immunogenic response to one or more neoantigens in a subject. Determination of an effective amount is within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, an efficacious or effective amount of an agent is determined by initially administering low doses of the agent(s) and then gradually increasing the dosage until the desired effect (e.g., induction of an immunogenic response) is observed in the treated subject with minimal or tolerable toxic side effects. Applicable methods for determining appropriate doses and dosing schedules for administration of the pharmaceutical compositions of the present disclosure are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005), each of which is incorporated herein by reference.

[0248] Activation, expansion, and isolation of neoantigen-specific T cells When administered to a subject, the cancer-specific neoantigens and subject-specific neoantigens activate the subject's immune response to produce T cells specific for the cancer-specific neoantigens and subject-specific neoantigens, hereinafter referred to as "clonal T cells." Such clonal T cells primed against one or more cancer-specific neoantigens and subject-specific neoantigens can then be expanded in vivo in the subject after a certain period of time. Primed T cells are those that respond to epitopes such that the T cell population expands. This typically takes about 3-5 days, peaking at about 7-10 days.

[0249] In some embodiments, the subject is diagnosed with cancer, hi some embodiments, the subject is suspected of having cancer, hi some embodiments, the subject has been previously treated and has not responded to checkpoint inhibitor therapy.

[0250] In some embodiments, the nucleic acid molecule is administered to the subject by electroporation. In some embodiments, the method does not include using electroporation to administer the nucleic acid sequence.

[0251] In some embodiments, the cancer-specific neo-antigens and the subject-specific neo-antigens activate a CD8+ T cell immune response in the subject. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.01% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.05% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.1% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.2% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.3% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.4% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, activating the CD8+ T cell immune response comprises activating about 0.5% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.6% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.7% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.8% to about 50% of the CD8+ T cells reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.9% to about 50% of CD8+ T cells that are reactive to one or more neo-antigens.In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 1% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 2% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 3% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 4% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 5% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 6% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 7% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 8% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 9% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 10% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 15% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 20% to about 50% of the CD8+ T cells that are reactive to one or more neo-antigens.In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 25% to about 50% of CD8+ T cells that are reactive to one or more neo-antigens.

[0252] In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.01% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.05% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.1% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.2% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.3% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.4% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating 0.5% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.6% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.7% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.8% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive.In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 0.9% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 1% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 2% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 3% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 4% to about 50% of CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 5% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 6% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 7% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 8% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 9% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive.In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 10% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 15% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 20% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive. In some embodiments, enhancing the CD8+ T cell immune response comprises activating about 25% to about 50% of the CD8+ T cells reactive to one or more neoantigens that are IFN-γ positive.

[0253] In some embodiments, activation of CD8+ T cells is achieved within about 1 hour of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 2 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 3 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 4 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 5 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 6 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 7 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 8 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 9 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject. In some embodiments, activation of CD8+ T cells is achieved within about 10 hours of contact with antigen-presenting cells that express or comprise nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject.In some embodiments, activation of CD8+ T cells is achieved after more than about 10 hours of contact with antigen-presenting cells that express or contain nucleic acid sequences encoding cancer-specific neo-antigens and subject-specific neo-antigens in the subject.

[0254] In some embodiments, PBMCs from a subject are sampled and then run through an activation assay to identify a T cell population that is activated in the presence of a neoantigen.

[0255] In some embodiments, activating a CD8+ T cell immune response comprises expanding CD8+ T cells in culture specific for cancer-specific neoantigens and subject-specific neoantigens in a subject to a biologically significant number or concentration in a fluid. In some embodiments, a biologically significant number of neoantigen-specific CD8+ T cells is from about 100 cells per mL of blood in the subject to about 200 cells per mL of blood in the subject. In some embodiments, when the cells are clonally isolated and expanded, a biologically significant number of neoantigen-specific CD8+ T cells is from about 100 cells per mL of pharmaceutically acceptable carrier to about 200 cells per mL of pharmaceutically acceptable carrier. In some embodiments, when the cells are clonally isolated and expanded, a biologically significant number of neoantigen-specific CD8+ T cells is from about 75 cells per mL of pharmaceutically acceptable carrier to about 150 cells per mL of pharmaceutically acceptable carrier. In some embodiments, the CD8+ T cells are suspended in sterile buffered saline. In some embodiments, the CD8+ cells are in a pharmaceutically acceptable carrier, such as sterile saline, to allow for readministration to the subject.

[0256] T cell activation can be detected and measured in a variety of ways. Methods for detecting specific T cell activation include detecting T cell proliferation, cytokine (e.g., lymphokine) production, or generation of cytolytic activity. Methods for measuring T cell activity include, but are not limited to, inducing T cell proliferation, inducing signal transduction in T cells, inducing expression of activation markers in T cells, such as interferon gamma (IFN-γ), inducing cytokine secretion by T cells, and T cell cytotoxic activity. For example, in some embodiments, CD8+ T cell activation is measured by a proliferation assay. In other embodiments, activation is measured after stimulation of cells or a cell sample with the encoded nucleic acid sequence after the cells are isolated from a subject.

[0257] In some embodiments, CD8+ T cell activation is assessed or measured by determining the secretion of cytokines such as gamma interferon (IFN-γ), tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), or interleukin-2 (IL-2). In some embodiments, ELISA is used to determine cytokine secretion, e.g., gamma interferon (IFN-γ), tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), or interleukin-2 (IL-2). In some embodiments, ELISPOT (enzyme-linked immunospot) technology is used to detect T cells that secrete a given cytokine (e.g., gamma interferon (IFN-γ)) in response to stimulation with cancer-specific neoantigens and target-specific neoantigens, or any composition comprising same. T cells are plated in wells coated with anti-IFN-γ antibody. Secreted IFN-γ is captured by the coated antibody and revealed with a secondary antibody conjugated to a chromogenic substrate. Thus, locally secreted cytokine molecules form spots, each corresponding to one IFN-γ-secreting cell. The number of spots allows the frequency of IFN-γ-secreting cells in the analyzed sample to be determined. ELISPOT assays have also been described for the detection of tumor necrosis factor α, interleukin-4 (IL-4), IL-5, IL-6, IL-10, IL-12, granulocyte-macrophage colony-stimulating factor, and granzyme B-secreting lymphocytes (Klinman D, Nutman T. Current protocols in immunology. New York, NY: John Wiley & Sons, Inc.; 1994. pp. 6.19.1-6.19.8, the entire contents of which are incorporated herein by reference).

[0258] Flow cytometric analysis of intracellular cytokines can also be used to measure cytokine content, but does not provide information about the number of T cells that actually secrete cytokines. If T cells are treated with secretion inhibitors such as monensin or brefeldin A, they will accumulate cytokines in their cytoplasm upon activation (e.g., along with a nucleic acid molecule of the present invention). After fixation and permeabilization of lymphocytes, intracellular cytokines can be quantified by cytometry. This technique allows for the determination of the cytokines produced, the type of cells producing these cytokines, and the amount of cytokine produced per cell.

[0259] In some embodiments, activation of CD8+ T cells is determined by assaying the cytotoxic activity of CD8+ T cells. The cytotoxic activity of T cells can be assessed by any suitable technique known to those of skill in the art. For example, a sample containing T cells exposed to one or more neoantigens according to the present disclosure can be assayed for cytotoxic activity in a standard cytotoxicity assay. Such assays can include, but are not limited to, chromium release CTL assays and Alamar Blue™ fluorescence assays, both of which are known in the art.

[0260] In some embodiments, the activation and expansion of neoantigen-specific T cells disclosed herein enhances the expression of certain key molecules in T cells that reprotect against apoptosis or otherwise prolong survival in vivo or in vitro. Apoptosis typically results from the induction of specific signals in T cells. Thus, neoantigens can provide protection to T cells from cell death due to T cell stimulation. Thus, the disclosed methods also include protection from premature death or the absence or depletion of recognized T cell growth markers, such as Bcl-xL, growth factors, cytokines, or lymphokines, normally required for T cell survival, as well as protection from crosslinking of Fas or tumor necrosis factor receptors (TNFRs), or enhanced T cell growth due to exposure to certain hormones or stresses.

[0261] Neoantigen-specific T cells produced by a subject can be isolated in a variety of ways. In some embodiments, neoantigen-specific T cells are isolated by collecting a blood sample from the subject and sorting peripheral blood mononuclear cells (PBMCs) from the sample according to receptor expression on the surface of the PBMCs. In some embodiments, isolating neoantigen-specific T cells further comprises removing a human tissue sample from the subject. In some embodiments, the human tissue sample removed for isolation of neoantigen-specific T cells comprises tissue from a brushing, biopsy, or surgical resection of the subject. In some embodiments, neoantigen-specific T cells are isolated based on expression of T cell activation markers by cell sorting or other suitable techniques known in the art. In some embodiments, the disclosed methods further comprise determining which neoantigens are immunologically recognized by the T cells (e.g., by a depletion process).

[0262] In some embodiments, selecting neoantigen-specific T cells includes (i) selecting T cells that secrete a greater amount of one or more cytokines compared to the amount of one or more cytokines secreted by a negative control, or (ii) selecting T cells in which at least twice the number of T cells secrete one or more cytokines compared to the number of negative control T cells that secrete the one or more cytokines. The one or more cytokines can include any cytokine whose secretion by T cells is characteristic of T cell activation (e.g., a TCR expressed by a T cell that specifically binds and immunologically recognizes the neoantigen). Non-limiting examples of cytokines characteristic of T cell activation include IFN-γ, IL-2, tumor necrosis factor alpha (TNF-α), granulocyte / monocyte colony-stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

[0263] In some embodiments, T cells are considered to have antigen specificity for a neoantigen, and thus neoantigen-specific T cells, if the T cells secrete at least twice as much IFN-γ as the amount of IFN-γ secreted by a negative control. In some embodiments, the negative control is, for example, autologous T cells (e.g., derived from PBMCs). IFN-γ secretion can be measured by methods known in the art, for example, enzyme-linked immunosorbent assay (ELISA).

[0264] In some embodiments, the disclosed methods further comprise isolating a nucleotide sequence encoding a T cell receptor (TCR) or an antigen-binding portion thereof from the selected neoantigen-specific T cells, wherein the TCR or antigen-binding portion thereof has antigen specificity for the neoantigen. In some embodiments, the disclosed methods further comprise identifying one or more nucleotide sequences encoding a subset of TCRs or antigen-binding portions thereof that are highly immunogenic in response to one or more neoantigens of interest. In some embodiments, the one or more nucleotide sequences encoding the highly immunogenic subset of TCRs are identified by performing an assay that measures the avidity or affinity of cells expressing the TCR to bind to cells in vitro. In some embodiments, the one or more nucleotide sequences encoding the highly immunogenic subset of TCRs are identified by performing an assay that measures the percentage of CD8+ and / or CD4+ on cells expressing the TCR or antigen-binding portion thereof. In some embodiments, the method may further comprise expanding the cells expressing the TCRs or antigen-binding portions thereof in culture prior to identifying one or more nucleotide sequences encoding a subset of TCRs or antigen-binding portions thereof that are highly immunogenic in response to one or more neo-antigens in the subject. In some embodiments, the method may further comprise sequencing the nucleotide sequences encoding the one or more TCRs or antigen-binding portions thereof that are highly immunogenic from the cells expressing the TCRs or antigen-binding portions thereof.

[0265] The term "highly immunogenic," as used herein, means that a T cell, TCR, or antigen-binding portion thereof expressed by a T cell is capable of specifically binding to and immunologically recognizing cancer-specific neoantigens and subject-specific neoantigens to an extent that stimulates a biological response, such as the secretion of cytokines, following exposure to the neoantigens.

[0266] As used herein, the term "antigen-binding portion" of a TCR refers to any portion comprising consecutive amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to cancer-specific neo-antigens and subject-specific neo-antigens. The term "antigen-binding portion" refers to any portion or fragment of a TCR that retains the biological activity of the TCR of which it is a part (the parent TCR). An antigen-binding portion encompasses, for example, a portion of a TCR that retains the ability to specifically bind to cancer-specific neo-antigens and subject-specific neo-antigens, or the ability to detect, treat, or prevent cancer, to a similar extent, the same extent, or a greater extent than the parent TCR. With respect to the parent TCR, a functional portion can include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the length of the parent TCR.

[0267] The antigen-binding portion can comprise the antigen-binding portion of either or both of the α and β chains of a TCR, for example, a portion comprising one or more of complementarity-determining regions (CDRs) 1, CDR2, and CDR3 of the variable region(s) of the α and / or β chain of a TCR. In some embodiments, the antigen-binding portion can comprise the amino acid sequence of CDR1 of the α chain (CDR1α), CDR2 of the α chain (CDR2α), CDR3 of the α chain (CDR3α), CDR1 of the β chain (CDR1β), CDR2 of the β chain (CDR2β), CDR3 of the β chain (CDR3β), or any combination thereof. In some embodiments, the antigen-binding portion comprises the amino acid sequences of CDR1α, CDR2α, and CDR3α, the amino acid sequences of CDR1β, CDR2β, and CDR3β, or all of the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of a TCR.

[0268] In some embodiments, the antigen-binding portion may comprise a combination of variable and constant regions, hi some embodiments, the antigen-binding portion may comprise the full-length α or β chain of a TCR, or both the α and β chains.

[0269] Isolating the nucleotide sequence encoding the TCR or antigen-binding portion thereof from the selected T cells can be done by any suitable method known in the art. For example, the method can include isolating RNA from the selected T cells and sequencing the TCR or antigen-binding portion thereof using TCR-α and TCR-β chain constant primers using established molecular cloning techniques and reagents such as, for example, 5' Rapid Amplification of cDNA Ends (RACE) polymerase chain reaction (PCR).

[0270] The present disclosure relates to a nucleic acid molecule comprising first, second, and third nucleic acid sequences, wherein the first nucleic acid sequence is a first DNA backbone domain of the nucleic acid molecule, the second nucleic acid sequence is a 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, in a 5' to 3' orientation, a plurality of antigen-expressing domains, each antigen-expressing domain having at least about 70 amino acid sequences corresponding to SEQ ID NOs: 1-128. %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 1-128, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 1-128.

[0271] The present disclosure relates to a nucleic acid molecule comprising first, second, and third nucleic acid sequences, wherein the first nucleic acid sequence is a first DNA backbone domain of the nucleic acid molecule, the second nucleic acid sequence is a 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, in a 5' to 3' orientation, a plurality of antigen-expressing domains, each antigen-expressing domain having at least about 70 amino acid sequences corresponding to SEQ ID NOs: 397-494. %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 397-494, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 397-494.

[0272] The present disclosure relates to a cell comprising a TCR or antigen-binding fragment thereof 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 NOs: 161-167, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 161-167.

[0273] The present disclosure relates to a cell comprising a TCR or antigen-binding fragment thereof 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 NOs: 367-394, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 367-394.

[0274] The present disclosure relates to a cell comprising a TCR or antigen-binding fragment thereof 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 NOs: 495-501, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 495-501.

[0275] The present disclosure relates to a cell comprising a TCR or antigen-binding fragment thereof 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 NOs: 168-174, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 168-174.

[0276] The present disclosure relates to a cell comprising a TCR or antigen-binding fragment thereof 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 NOs: 331-355, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 331-355.

[0277] The present disclosure relates to a cell comprising a TCR or antigen-binding fragment thereof 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 NOs: 255-329, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 255-329.

[0278] The present disclosure relates to a cell comprising a TCR or antigen-binding fragment thereof 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 NOs: 179-253, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOs: 179-253.

[0279] The present disclosure relates to nucleic acid molecules encoding a TCR or antigen-binding fragment thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 179-253, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 179-253.

[0280] The present disclosure relates to nucleic acid molecules encoding a TCR or antigen-binding fragment thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SEQ ID NOs: 509-588, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 509-588.

[0281] The present disclosure relates to nucleic acid molecules encoding a TCR or antigen-binding fragment thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the sequence identifiers in Tables S, T, and / or U, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the sequence identifiers in Tables S, T, and / or U.

[0282] The present disclosure relates to nucleic acid molecules encoding a TCR or antigen-binding fragment thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the sequence identifiers of Table Z, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the sequence identifiers of Table Z.

[0283] TCR-expressing T cells Disclosed are cells comprising a TCR comprising one alpha and one beta subunit, wherein the alpha and beta subunits are those disclosed in Table Z. In some embodiments, the TCR comprising one alpha and one beta subunit comprises one alpha and one beta subunit that have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the alpha and beta subunits disclosed in Table Z.

[0284] Disclosed are cells comprising a TCR comprising one alpha and one beta subunit, wherein the alpha subunit is selected from the group consisting of SEQ ID NOs: 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, an amino 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 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, or 587; and the beta subunit is selected from one of SEQ ID NOs: 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636 68, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, or a combination thereof.

[0285] Methods for TCR-Expressing T Cells In some embodiments, the method can include cloning a nucleotide sequence encoding a TCR or an antigen-binding portion thereof from clonally expanded T cells active against one or more neoantigens from a subject into a recombinant expression vector using established molecular cloning techniques, for example, as described in Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012). For purposes of this specification, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that allows for the expression of an mRNA, protein, polypeptide, or peptide by a host cell, where the construct contains a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed in the cell. The vectors of the present disclosure are not naturally occurring in their entirety. However, portions of the vector may be naturally occurring. Recombinant expression vectors can comprise any type of nucleotide, including, but not limited to, DNA (e.g., complementary DNA (cDNA)) and RNA, which can be single-stranded or double-stranded, synthetic, or derived in part from natural sources, and can contain natural, non-natural, or altered nucleotides. Recombinant expression vectors can include naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. In some embodiments, the non-naturally occurring or altered nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

[0286] The recombinant expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include vectors designed for propagation and expansion, or for expression, or both, such as plasmids and viruses. The vector can be selected from the group consisting of transposon / transposase, pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, the recombinant expression vector is a viral vector, e.g., a retroviral vector.

[0287] TCRs or antigen-binding portions thereof isolated by the disclosed methods can be useful in preparing cells for adoptive cell therapy. In some embodiments, the present disclosure provides methods for preparing a population of cells expressing TCRs or antigen-binding portions thereof with high immunogenic specificity for cancer-specific neoantigens and subject-specific neoantigens identified by the disclosed methods, the method comprising isolating a TCR or antigen-binding portion thereof as described herein and introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into one or more host cells to obtain cells expressing the TCR or antigen-binding portion thereof.

[0288] Introduction of a nucleotide sequence (e.g., a recombinant expression vector) encoding an isolated TCR or antigen-binding portion thereof into a host cell can be accomplished by any of a variety of different methods known in the art, for example, as described in Green et al., supra. Non-limiting examples of techniques useful for introducing nucleotide sequences into a host cell include transformation, transduction, transfection, and electroporation.

[0289] The host cell into which the nucleotide sequence encoding the TCR or antigen-binding portion thereof is introduced can be any type of cell capable of containing a recombinant expression vector disclosed herein. In some embodiments, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae. In some embodiments, the host cell is a prokaryotic cell, such as a bacterium or protozoan. In some embodiments, the host cell is a cultured cell. In other embodiments, the host cell is a primary cell, i.e., isolated directly from an organism, e.g., a human. In some embodiments, the host cell is an adherent cell. In some embodiments, the host cell is a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. In some embodiments, for purposes of amplifying or replicating the recombinant expression vector, the host cell is a prokaryotic cell, e.g., a DH5α cell. For purposes of producing the TCR or antigen-binding portion thereof, the host cell is, in some embodiments, a mammalian cell. In some embodiments, the host cell is a human cell. The host cell may be of any cell type, may be derived from any type of tissue, and may be at any stage of development, although in some embodiments the host cell is preferably a PBL or PBMC. In some embodiments, the host cell is a T cell.

[0290] In some embodiments, the PBMCs comprise T cells. The T cells can be any type of T cell. Without being bound by a particular theory or mechanism, it is believed that less differentiated, "younger" T cells may be associated with any one or more of greater survival, proliferation, and anti-tumor activity compared to more differentiated, "older" T cells. Thus, the disclosed methods advantageously identify and isolate TCRs or antigen-binding portions thereof that are highly immunogenic in response to one or more cancer-specific and target-specific neo-antigens, and introduce the TCRs or antigen-binding portions thereof into "younger" T cells, which may provide any one or more of greater survival, proliferation, and anti-tumor activity compared to "older" T cells (e.g., effector cells in a patient's tumor).

[0291] In some embodiments, the host cells are autologous to the subject. In some embodiments, the TCRs or antigen-binding portions thereof identified and isolated by the disclosed methods are individualized for each subject or patient. In some embodiments, the disclosed methods identify and isolate TCRs or antigen-binding portions thereof that are highly immunogenic in response to one or more cancer-specific neo-antigens and subject-specific neo-antigens encoded by recurrent (also referred to as "hotspot") cancer-specific mutations. In some embodiments, the method comprises introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into a host cell that is allogeneic to the patient. For example, in some embodiments, the method comprises introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into a host cell from another patient whose tumor expresses the same mutation in the context of the same MHC molecule.

[0292] In some embodiments, the disclosed methods further include expanding the number of host cells, e.g., T cells, that express the TCR or antigen-binding portion thereof. Expanding the number of T cells can be achieved by any of a number of methods known in the art, for example, as described in U.S. Pat. No. 8,034,334, U.S. Pat. No. 8,383,099, U.S. Patent Application Publication No. 2012 / 0244133, Dudley et al., J. Immunother., 26:332-42 (2003), and Riddell et al., J. Immunol. Methods, 128:189-201 (1990), the contents of each of which are incorporated herein by reference. In some embodiments, expanding the number of T cells is achieved by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs). In some embodiments, the disclosed methods generate large numbers of T cells that are highly immunogenic in response to one or more cancer-specific neo-antigens and target-specific neo-antigens.

[0293] T cells expressing a TCR or antigen-binding portion thereof can be formulated into a composition, such as a pharmaceutical composition. Thus, in some embodiments, the present disclosure provides a pharmaceutical composition comprising any of T cells comprising a nucleic acid molecule encoding one or more TCRs or antigen-binding portions thereof and a pharmaceutically acceptable carrier. In some embodiments, such pharmaceutical compositions further comprise another pharmaceutically active agent(s) or drug(s), for example, a chemotherapeutic agent such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, or the like.

[0294] In some embodiments, the carrier in such pharmaceutical compositions is a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are readily available to the public. In some embodiments, a pharmaceutically acceptable carrier is one that has no adverse side effects or toxicity under the conditions of use.

[0295] In some embodiments, T cells comprising nucleic acid molecules encoding one or more TCRs or antigen-binding portions thereof, or pharmaceutical compositions comprising same, are re-administered to a subject by injection (e.g., intravenously). When such T cells are administered, pharmaceutically acceptable carriers for injectable cells include any isotonic carrier, such as, for example, saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In some embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0296] It is contemplated that T cells comprising nucleic acid molecules encoding one or more TCRs or antigen-binding portions thereof disclosed herein, or pharmaceutical compositions comprising the same, can be used in methods of treating or preventing cancer in a subject. Without being bound to a particular theory or mechanism, it is believed that the TCRs or antigen-binding portions thereof expressed by such T cells specifically bind to neoantigens encoded by cancer-specific mutations found in the subject, such that the TCRs or antigen-binding portions thereof, when expressed by cells in the subject, can mediate an immune response against target cells expressing the neoantigen. Thus, in some embodiments, the present disclosure provides methods of treating or preventing cancer in a patient, comprising administering any of the pharmaceutical compositions, TCRs, antigen-binding portions thereof, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations described herein in an amount effective to treat or prevent cancer in the subject.

[0297] The present disclosure provides a method of treating a subject in need thereof, comprising: (i) activating a first population of T cells from the sample; (ii) sequencing nucleic acid expression in T cells; and (iii) generating a vector comprising one or more nucleic acid sequences encoding a TCR from the sample; and (iv) transducing the vector into a second population of T cells; (v) administering to the subject a therapeutically effective amount of a second population of T cells.

[0298] In some embodiments, the methods of the present disclosure include activating a first population of T cells from a sample by exposing the isolated T cells to one or more neoantigens from the subject. Exposure can be achieved by incubating the cells in the presence of one or more neoantigens.

[0299] In some embodiments, the step of sequencing nucleic acid expression of the T cell comprises sequencing nucleic acid sequences encoding one or more TCRs or antigen-binding fragments thereof from the T cell.

[0300] The disclosed methods can be carried out by using autologous T cells from the patient for cell administration or by using isolated T cells cultured from another source. In some embodiments in which autologous T cells are administered to the subject in step (v), the method further comprises isolating the T cells from the sample prior to the sequencing step. In some embodiments, the isolating step comprises one or a combination of flow cytometry, cell filtration, exposure to a column containing T cell-specific antibodies, and / or exposure to a magnetic surface containing T cell-specific antibodies. In some embodiments, the isolated T cells from the subject are cultured with a nucleic acid molecule comprising an expressible nucleic acid sequence encoding one or more TCR sequences or antigen-binding fragments thereof. In some embodiments, the methods disclosed herein further comprise transducing the nucleic acid molecule into the T cells by transfection or lipofection. In some embodiments, the isolated T cells are cultured with the nucleic acid molecule encoding one or more TCRs or antigen-binding fragments thereof for about 3 to about 5 days prior to administration.

[0301] For methods involving the administration of non-autologous T cell transfer, activated T cells from a sample from a subject can be used to simply identify TCR sequences by sequencing the TCR sequences or antigen-binding fragments from the activated sample. RNA or DNA can be isolated from the T cells and sequenced using known sequencing techniques. These identified nucleic acid sequences can then, in some embodiments, be synthesized de novo from the isolated DNA or subcloned to generate one or more inserts. Such nucleic acid inserts can be further subcloned into one of the disclosed plasmids and resuspended in a buffer sufficient for transduction of the isolated non-autologous T cells. After transduction, the resulting T cells can be used for therapy.

[0302] In some embodiments, the nucleic acid molecule transduced in the T cell comprises an expressible nucleic acid sequence encoding one or more TCRs or antigen-binding fragments thereof. In some embodiments, the nucleic acid sequence encoding the one or more TCRs or antigen-binding fragments thereof comprises about 15 to about 50 nucleotides and encodes an antigen-binding fragment selected from one or a combination of the sequences provided in Tables S, T, U, or W.

[0303] The amount or dose of T cells comprising nucleic acid molecules encoding one or more TCRs or antigen-binding portions thereof disclosed herein, or pharmaceutical compositions comprising the same (e.g., the number of cells when a population of T cells is administered), should be sufficient to effect, for example, a therapeutic or prophylactic response in a subject over a reasonable time frame. For example, the dose of T cells or pharmaceutical compositions comprising the same should be sufficient to bind to a TCR or antigen-binding portion thereof that is immunogenic for cancer, or to detect, treat, or prevent cancer, for a period of about 2 hours or more, e.g., 12 to 24 hours or more, from the time of administration. In some embodiments, the period may be even longer. The dose is determined by the efficacy of the particular T cells or pharmaceutical compositions comprising the same administered, the condition of the patient, and the weight of the patient being treated.

[0304] Many assays for determining the dosage to be administered are known in the art. For example, an assay involving comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a TCR or an antigen-binding portion thereof can be used to determine the starting dose to be administered to a subject when a given dose of such T cells is administered to a set of mammals, each receiving a different dose of cells. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a dose can be assayed by methods known in the art.

[0305] The dose of T cells comprising nucleic acid molecules encoding one or more TCRs, or antigen-binding portions thereof, will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular T cell population or pharmaceutical composition comprising same. Typically, the attending physician will determine the dose of T cells or pharmaceutical compositions comprising same for treating each individual patient, taking into account various factors such as age, weight, general health, diet, sex, the T cells or pharmaceutical compositions comprising same being administered, the route of administration, and the severity of the condition being treated.

[0306] In some embodiments, the number of T cells administered per infusion can vary, for example, within the range of 1 million to 200 billion cells. However, amounts below or above this exemplary range are within the scope of the present disclosure. In some embodiments, the daily dose of TCR-expressing T cells administered is about 1 million to about 200 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 10 million to about 200 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 100 million to about 200 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 5 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 10 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 20 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 20 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 30 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 40 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 50 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 60 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 70 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 80 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 90 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 100 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 250 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 350 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 450 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 500 million cells.In some embodiments, the daily dose of TCR-expressing T cells administered is about 650 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 800 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 900 million cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 1 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 5 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 20 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 30 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 40 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 60 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 80 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 100 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 120 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 130 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 150 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 160 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 170 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 180 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 190 billion cells. In some embodiments, the daily dose of TCR-expressing T cells administered is about 200 billion cells.

[0307] In some embodiments, when a population of T cells is administered, the cells can be allogeneic or autologous to the subject. In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are allogeneic to the subject.

[0308] How to Treat Cancer The present disclosure relates to a method of treating cancer or preventing cancer metastasis in a subject in need thereof, comprising first inducing an immune response in the subject against one or more neo-antigens produced by the cancer in the subject, as disclosed elsewhere herein, and subsequently identifying one or more nucleotide sequences encoding a subset of TCRs that are highly immunogenic in response to the one or more neo-antigens in the subject, as disclosed elsewhere herein, such that a therapeutically effective amount of T cells comprising one or more such nucleotide sequences can be readministered to the subject, as disclosed elsewhere herein. The immune response induced in the subject can provide a functional T cell response against the one or more neo-antigens. Neo-antigens have the advantage that they are found only in one or a few specific individuals and not in normal tissues (thus reducing off-target immunogenicity) and are not subject to central tolerance mechanisms.

[0309] In some embodiments, the neo-antigen used to induce an immune response in a subject is associated with a hyperproliferative disease or disorder (e.g., cancer), such as a tumor neo-antigen or a cancer neo-antigen. The identified tumor neo-antigen can then be introduced into the subject to activate neo-antigen-specific immune cells in the subject.

[0310] In some embodiments, treatment is determined by clinical outcome, an increase, enhancement, or prolongation of anti-tumor activity by T cells, an increase in the number of anti-tumor T cells or activated T cells compared to pre-treatment numbers, or a combination thereof. In some embodiments, the clinical outcome is selected from the group consisting of tumor regression, tumor shrinkage, tumor necrosis, an anti-tumor response by the immune system, tumor expansion growth, recurrence, or spread, or a combination thereof.

[0311] In some embodiments, the disclosed methods are used to treat patients diagnosed with cancer or at risk of developing cancer. In some embodiments, the subject has been previously treated and has not responded to checkpoint inhibitor therapy. In some embodiments, the subject has no detectable neoplasia but is at high risk of disease recurrence. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer has a high mutational burden. In other embodiments, the cancer has a moderate mutational burden. In some embodiments, the cancer has been shown to have an inadequate or poor response to checkpoint inhibitor therapy.

[0312] In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell carcinoma or bone and soft tissue sarcoma, hematologic neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma soft tissue sarcoma, and small cell lung cancer.

[0313] In certain embodiments, the cancer is non-small cell lung cancer or melanoma, both of which have been shown to have a high mutational burden, while in other embodiments, the cancer is ovarian cancer or glioblastoma multiforme, both of which exhibit an intermediate mutational burden and have been shown to respond poorly or poorly to checkpoint inhibitor therapy.

[0314] In some embodiments, the disclosed methods are of sufficient magnitude or efficacy to inhibit or slow tumor growth, induce tumor cell death, induce tumor regression, prevent or slow tumor recurrence, prevent tumor growth, prevent tumor spread, and / or induce tumor elimination.

[0315] In some embodiments, the disclosed methods include administering one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are biotherapeutic agents or small molecules. In some embodiments, the therapeutic agent is (i) a checkpoint inhibitor or a functional fragment thereof, or (ii) a nucleic acid molecule encoding a checkpoint inhibitor or a functional fragment thereof.

[0316] Checkpoint inhibitors include any agent that blocks or inhibits an inhibitory pathway of the immune system. Such inhibitors may include small molecule inhibitors, which may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. In some embodiments, the checkpoint inhibitor targets or inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (a member of the CD2 family of molecules expressed on all NK, gamma delta, and memory CD8+ (alpha beta) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands, or combinations thereof. Checkpoint inhibitors include CTLA-4, PDLl, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2,

[0317] Included are antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (a PD-1 blocker), nivolumab (an anti-PD1 antibody), CT-011 (an anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (an anti-PDL1 antibody), BMS-936559 (an anti-PDL1 antibody), MPLDL3280A (an anti-PDL1 antibody), MSB0010718C (an anti-PDL1 antibody), and Yervoy / ipilimumab (an anti-CTLA-4 checkpoint inhibitor). In some embodiments, the checkpoint inhibitor is a checkpoint inhibitor listed in Table 4.

[0318] Table 4: List of checkpoint inhibitors TIFF2025516676000007.tif60167

[0319] Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0320] In some embodiments, the checkpoint inhibitor is an inhibitor of the programmed death-1 (PD-1) pathway. In some embodiments, the checkpoint inhibitor is an anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-PDL1 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-PDL2 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-PD1 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-B7-H3 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-B7-H4 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-BTLA antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-HVEM antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-TIM3 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-Gal9 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-LAG3 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-VISTA antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-KIR antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-2B4 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-CD160 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-CHK1 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-CHK2 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-A2aR antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-B-7 antibody or a functional fragment thereof. In some embodiments, the checkpoint inhibitor is an anti-CGEN-15049 antibody or a functional fragment thereof.

[0321] (Table 3) Sequences of checkpoint proteins. TIFF2025516676000008.tif133166

[0322] In some embodiments, the therapeutic agent is a checkpoint inhibitor that is any of the full-length amino acid sequences identified above, or a fragment of any of the above full-length amino acid sequences that comprises about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequences identified above.

[0323] In some embodiments, the therapeutic agent is an adjuvant. The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies produced against the antigen, and an increase in T cell activity is typically manifested as an increase in cell proliferation, cytotoxicity, or cytokine secretion. An adjuvant can also alter the immune response, for example, by shifting a predominantly humoral or Th2 response to a predominantly cellular or Th1 response. In some embodiments, the adjuvant can be another gene expressed on an alternative plasmid or delivered as a protein in combination with the above-mentioned plasmid in the vaccine.

[0324] In some embodiments, the adjuvant may be selected from the group consisting of alpha interferon (IFN-α), beta interferon (IFN-β), gamma interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with the signal sequence deleted and optionally containing the signal peptide from IgE. In some embodiments, the adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNF-alpha, TNF-beta, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0325] Other genes that may be useful adjuvants include those encoding: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40 , CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR 4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0326] Other exemplary adjuvants include poly-ICLC (see Pharmacol Ther. 2015 Feb;146:120-31, which is incorporated herein by reference in its entirety), 1018ISS (see Vaccine. 2003 Jun 2;21(19-20):2461-7, which is incorporated herein by reference in its entirety), aluminum salts, Amplivax AS15, Bacillus Colmette-Guerin (BCG) (see Clin Immunol. 2000, which is incorporated herein by reference in its entirety). Jan;94(1):64-72), CP-870,893, CpG7909 (GenBank Accession No. CS576603.1), CyaA (GenBank Accession No. KP670536.1), GM-CSF (GenBank Accession No. M11220.1), IC30 (see Expert Review Vaccines. 2007 Oct;6(5):741-6, which is incorporated herein by reference in its entirety), IC31 (see Expert Review Vaccines. 2007 Oct;6(5):741-6, which is incorporated herein by reference in its entirety), imiquimod (Vaccine.2006 Mar 10;24(11):1958-6), ImuFact 1MP321, IS Patch, ISS, ISCOMATRIX, Julvlmmune, LipoVac, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector systems, PLGA microparticles, resiquimod, S L172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, acrylic or methacrylic polymers, maleic anhydride and Aquila Copolymers of QS21 stimulon, and functional fragments of any thereof, or (ii) (i) Poly ICLC, 1018 ISS, aluminum salts, Amplivax AS15, BCG, CP-870,893, CpG7909, CyaA, GM-CSF, IC30, IC31, imiquimod, ImuFact 1MP321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, monophosphoryllipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, Vector System, PLGA microparticles, resiquimod, S These include, but are not limited to, nucleic acid molecules encoding adjuvants selected from the group consisting of L172, virosomes an...

Claims

1. A method for treating cancer comprising one or more neoantigens in a subject in need thereof, comprising: (a) administering to the subject in need thereof one or more nucleic acid sequences encoding the one or more neoantigens; (b) allowing a sufficient period for the clonal T cells primed against the one or more antigens in the subject to expand to a biologically significant number; (c) isolating the clonal T cells from the subject; (d) identifying one or more nucleotide sequences encoding a subset of T cell receptors (TCRs) that are highly immunogenic in response to the one or more antigens in the subject; and (e) administering to the subject in need thereof a therapeutically effective amount of T cells comprising a nucleic acid molecule encoding one or more subsets of the TCRs. A method comprising the above steps.

2. The method according to claim 1, wherein the clonal T cells are isolated by collecting a blood sample from the subject and sorting peripheral blood mononuclear cells (PBMCs) from the sample according to receptor expression on the surface of the PBMCs.

3. Step (d) comprises (i) measuring the avidity or affinity of cells expressing the TCR for binding to cells in vitro, and (ii) the percentage of CD8+ and / or CD4+ on cells expressing the TCR, by performing an assay for measuring one or a combination thereof. The method according to claim 1 or 2.

4. The method according to claim 3, further comprising sequencing the one or more nucleotide sequences encoding the subset of TCRs that are highly immunogenic from the T cells expressing the TCR.

5. The method according to any one of claims 1 to 4, further comprising identifying the one or more antigens from a tissue sample removed from the subject.

6. The method according to claim 5, wherein the tissue sample comprises tissue from brushing, biopsy, or surgical resection of the subject.

7. The method according to any one of claims 1 to 6, not including in vitro expansion of PBMCs and / or tumor infiltrating lymphocytes.

8. The method according to any one of claims 1 to 7, wherein the total number of the clonal T cells primed against the one or more antigens in the subject comprises from about 0.01% to about 10% CD8+ reactivity against the one or more neoantigens.

9. The method according to any one of claims 1 to 8, wherein step (a) comprises administering a nucleic acid molecule comprising the one or more nucleotide sequences encoding the one or more neoantigens.

10. The method according to claim 9, wherein the nucleic acid molecule encodes from about 10 to about 55 neoantigens.

11. The method according to claim 10, wherein each neoantigen encoded by the nucleic acid molecule is separated from another by one or more linkers.

12. The method according to claim 11, wherein the one or more linkers comprise a furin protease cleavage site or a foot-and-mouth disease virus-1 2A (P2A) cleavage site.

13. The method according to any one of claims 9 to 12, wherein the nucleic acid molecule is a plasmid.

14. The nucleic acid molecule is (i) a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES, or (ii) a plasmid having at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES The method according to any one of claims 9 to 13, which is located within the multiple cloning site of.

15. The method according to any one of claims 9 to 14, wherein the nucleic acid molecule is GNOS-PV02.

16. The cancer is selected from the group consisting of non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell cancer or bone and soft tissue sarcoma, hematological neoplasm, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, cholangiocarcinoma, head and neck squamous cell carcinoma soft tissue sarcoma, and small cell lung cancer, the method according to any one of claims 1 to 15.

17. The method according to claim 16, wherein the cancer is hepatocellular carcinoma (HCC).

18. A method for treating cancer that expresses one or more antigens in a subject in need thereof, comprising: (a) administering to the subject in need thereof one or more nucleic acid sequences encoding the one or more antigens; and (b) administering to the subject a therapeutically effective amount of T cells comprising one or more nucleic acid sequences encoding one or more T cell receptors (TCRs) or functional fragments thereof that are highly immunogenic in response to the one or more neoantigens. A method comprising the above steps.

19. The method according to claim 17, which does not include in vitro expansion and proliferation of PBMC and / or tumor infiltrating lymphocytes.

20. The method according to claim 17 or 18, further comprising inducing an immune response in the subject against the one or more neoantigens.

21. The method according to any one of claims 17 to 19, further comprising sequencing the one or more nucleic acid sequences encoding the one or more TCRs or functional fragments thereof from T cells isolated from the subject, after step (a) and before step (b).

22. After step (a) and allowing the subject a sufficient period for the clonal T cell population primed against the one or more neoantigens to expand and proliferate, wherein the clonal T cell population comprises about 25% to about 50% CD8+ reactivity against the one or more neoantigens. The method according to any one of claims 17 to 20.

23. The method according to any one of claims 17 to 21, wherein step (a) comprises administering a nucleic acid molecule comprising the one or more nucleic acid sequences encoding the one or more antigens.

24. The method according to claim 22, wherein the nucleic acid molecule encodes about 10 to about 55 neoantigens.

25. The method according to claim 23, wherein each neoantigen encoded by the nucleic acid molecule is separated from another by one or more linkers.

26. The method according to claim 24, wherein the one or more linkers comprise a furin protease cleavage site or a foot-and-mouth disease virus-1 2A (P2A) cleavage site.

27. The method according to any one of claims 22 to 25, wherein the nucleic acid molecule is a plasmid.

28. The nucleic acid molecule is (i) A plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRESh, or (ii) A plasmid containing at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRESh The method according to any one of claims 22 to 26, which is arranged within the multiple cloning site of

29. The method according to any one of claims 22 to 27, wherein the nucleic acid molecule is GNOS-PV02.

30. The method according to any one of claims 17 to 28, wherein the cancer is selected from the group consisting of non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell cancer, or bone and soft tissue sarcoma, hematological neoplasm, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular cancer, cholangiocarcinoma, head and neck squamous cell cancer, soft tissue sarcoma, and small cell lung cancer.

31. The method according to claim 30, wherein the cancer is HCC.

32. A method for producing a population of T cells expressing one or more TCRs or functional fragments thereof that recognize one or more neoantigens, comprising: (a) Administering to a subject comprising one or more cells expressing the one or more neoantigens, one or more nucleic acid sequences encoding the one or more neoantigens; and (b) Isolating from the subject, clonal-derived T cells expressing the one or more TCRs or functional fragments thereof. A method comprising.

33. The method according to claim 30, which does not include in vitro expansion and proliferation of PBMC and / or tumor-infiltrating lymphocytes.

34. The method according to claim 30 or 31, further comprising sequencing one or more nucleic acid sequences encoding the one or more TCRs or functional fragments thereof after step (b).

35. The method according to any one of claims 30 to 32, further comprising, after step (a), allowing the subject a period sufficient for the clonal T cell population primed against the one or more neoantigens to expand and proliferate.

36. The method according to claim 33, wherein the clonal T cell population comprises from about 25% to about 50% CD8+ reactivity against the one or more neoantigens.

37. The method according to any one of claims 30 to 34, further comprising introducing one or more nucleic acid sequences encoding the one or more TCRs or functional fragments thereof into T cells obtained from the subject.

38. A method of preventing metastasis of cancer comprising one or more antigens in a subject, comprising: (a) administering to the subject one or more nucleic acid sequences encoding one or more neoantigens; (b) allowing the subject a period sufficient for the clonal T cells primed against the one or more neoantigens to expand and proliferate to a biologically significant number; (c) isolating the clonal T cells from the subject; (d) identifying one or more nucleotide sequences encoding a subset of TCRs that are highly immunogenic in response to the one or more neoantigens in the subject; (e) administering to the subject in need thereof a therapeutically effective amount of T cells comprising a nucleic acid molecule encoding the subset of the one or more TCRs. A method comprising.

39. The method according to claim 36, wherein the clonal T cells are isolated by collecting a blood sample from the subject and sorting peripheral blood mononuclear cells (PBMCs) from the sample according to receptor expression on the PBMC surface.

40. Step (d) comprises (i) the avidity or affinity of cells expressing the TCR to bind to cells in vitro, and (ii) the percentage of CD8+ and / or CD4+ on cells expressing the TCR. The method according to claim 36 or 37, comprising performing an assay for measuring one or a combination of the above.

41. The method according to claim 38, further comprising sequencing the one or more nucleotide sequences encoding the subset of TCRs that are highly immunogenic from the T cells expressing the TCR.

42. The method according to any one of claims 36 to 39, further comprising identifying the one or more neoantigens from a tissue sample removed from the subject.

43. The method according to claim 40, wherein the tissue sample comprises tissue from brushing, biopsy, or surgical resection of the subject.

44. The method according to any one of claims 36 to 41, wherein the method does not include in vitro expansion and proliferation of PBMC and / or tumor infiltrating lymphocytes.

45. The method according to any one of claims 36 to 42, wherein the total number of the clonal T cells primed against the one or more neoantigens in the subject comprises a CD8+ reactivity of about 25% to about 50% against the one or more neoantigens.

46. The method according to any one of claims 36 to 43, wherein step (a) comprises administering a nucleic acid molecule comprising the one or more nucleotide sequences encoding the one or more neoantigens.

47. The method according to claim 44, wherein the nucleic acid molecule encodes about 10 to about 55 neoantigens.

48. The method according to claim 45, wherein each neoantigen encoded by the nucleic acid molecule is separated from another by one or more linkers.

49. The method according to claim 46, wherein the one or more linkers comprise a furin protease cleavage site or a foot-and-mouth disease virus-1 2A (P2A) cleavage site.

50. The method according to any one of claims 45 to 47, wherein the nucleic acid molecule is a plasmid.

51. The nucleic acid molecule is (i) a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES, or (ii) a plasmid having at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES disposed within the multiple cloning site of. The method according to any one of claims 45 to 48.

52. The method according to any one of claims 45 to 49, wherein the nucleic acid molecule is GNOS-PV02.

53. The method according to any one of claims 36 to 50, wherein the cancer is selected from the group consisting of non-small cell lung cancer, melanoma, ovarian cancer, cervical cancer, glioblastoma, genitourinary cancer, gynecological cancer, lung cancer, gastrointestinal cancer, head and neck cancer, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel cell cancer or bone and soft tissue sarcoma, hematological neoplasm, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-resistant prostate cancer, colorectal cancer, ovarian cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma, cholangiocarcinoma, head and neck squamous cell carcinoma soft tissue sarcoma, and small cell lung cancer.

54. The method according to claim 53, wherein the cancer is HCC.

55. The method according to any one of claims 1 to 54, further comprising administering a checkpoint inhibitor to the subject. The method according to claim 55, wherein the checkpoint inhibitor is a PD-1 inhibitor.

56. The method according to claim 55, wherein the PD-1 inhibitor is pembrolizumab.

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