Individualized cancer epitopes and methods of use thereof

Abstract

The present disclosure relates to methods of treating cancer or preventing metastasis of cancer in a subject in need thereof. The disclosure further relates to compositions comprising heterogeneous populations of T cells reactive to individualized cancer epitopes or neoantigens useful for adoptive immunotherapy, as well as methods for preparing such T cell compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 63 / 342,608, filed on May 16, 2022, and U.S. Application No. 63 / 340,058, filed on May 10, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] References to sequence listings

[0004] The sequence listing, created on May 10, 2023 and having a size of 569,374 bytes, and filed on May 10, 2023 as an XML file named "GENE-001-PCT_SL," is hereby incorporated by reference pursuant to 37 CFR §1.52(e)(5). Technical Field

[0005] The present disclosure relates to methods of treating cancer in a subject in need of therapy or prevention or methods of preventing metastasis of cancer. The present disclosure further relates to compositions comprising a heterogeneous population of T cells reactive to individualized cancer epitopes or neoantigens useful for adoptive immunotherapy, and methods for preparing such T cell compositions. Background Art

[0006] After a person's immune system sees an antigen for the first time, a population of T cells that recognize the antigen will be generated within a few days, and these T cells then determine the nature of the response to the antigen. Antigen recognition and specificity of T cells are conferred by the structural characteristics of the T cell receptor (TCR) expressed on the surface of the T cells. Therefore, the antigen specificity of T cells is characterized by the presence and function of the specific TCR exhibited by the T cells. A single T cell has a TCR that can bind to a single antigen presented in combination with a specific major histocompatibility complex molecule or MHC.

[0007] As early as the 1990s, adoptive transfer of ex vivo expanded antigen-specific T cells was shown to confer immunity against CMV and EBV. See Riddell et al., Science, 1992, 257: 238; Rooney et al., Blood, 1998, 92: 1549-1555. Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) 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 in promoting tumor regression. In past attempts to use ex vivo expanded T cells for immunotherapy, tumor-associated dominant antigen-reactive T cells were unintentionally amplified, resulting in inconsistent results. In addition, obstacles to the successful use of adoptive cell therapy for the extensive treatment of cancer and other diseases still exist. For example, T cells and TCRs that specifically recognize cancer antigens may be difficult to identify and / or separate from patients. Summary of the invention

[0008] The present disclosure relates to methods of treating cancer in a subject in need of therapy or prevention or methods of preventing metastasis of cancer.

[0009] In one aspect, the present disclosure relates to a method of treating a cancer comprising one or more neoantigens in a subject in need thereof, the method comprising: (a) administering one or more nucleic acid sequences encoding one or more neoantigens to a subject in need thereof; (b) allowing a period of time sufficient to allow clonal T cells sensitized to the subject's one or more neoantigens to expand to biologically significant numbers; (c) isolating 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 subject's one or more neoantigens; and (e) administering to the subject in need thereof a therapeutically effective amount of T cells comprising nucleic acid molecules encoding one or more of the subsets of TCRs.

[0010] In some embodiments, clonal T cells are isolated by drawing a blood sample from a subject and sorting peripheral blood mononuclear cells (PBMCs) from the sample based on receptor expression on the surface of the PBMCs.

[0011] In some embodiments, step (d) comprises performing an assay to measure one or a combination of: (i) the affinity or avidity 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.

[0012] In some embodiments, the method further comprises sequencing one or more nucleotide sequences encoding a subset of TCRs from T cells expressing the TCRs that are highly immune.

[0013] In some embodiments, the method further comprises identifying one or more neo-antigens from a tissue sample removed from the subject.

[0014] In some embodiments, the tissue sample comprises brushing, biopsy, or surgically removed tissue from a subject.

[0015] In some embodiments, the method does not involve in vitro expansion of PBMCs and / or tumor infiltrating lymphocytes.

[0016] In some embodiments, the subject's total population of clonal T cells sensitized to one or more neoantigens comprises about 0.01% to about 10% CD8+ reactivity to the one or more neoantigens.

[0017] 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 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 joints. In some embodiments, one or more joints comprise a furin protease cleavage site or a porcine teschovirus-12A (P2A) cleavage site.

[0018] In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the expressible nucleic acid sequence is located within the multiple cloning site of: (i) a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES; or (ii) a plasmid comprising at least 70% sequence identity to a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP, and pIRES.

[0019] In some embodiments, the nucleic acid molecule is GNOS-PV02.

[0020] 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, hematological tumors, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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.

[0021] In another aspect, the present disclosure relates to a method for treating cancer expressing one or more neoantigens in a subject in need thereof, the method comprising: (a) administering one or more nucleic acid sequences encoding one or more neoantigens to a subject in need thereof; 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 from the subject, which T cell receptors or functional fragments thereof are highly immune in response to the one or more neoantigens.

[0022] In some embodiments, the method does not involve in vitro expansion of PBMCs and / or tumor infiltrating lymphocytes.

[0023] In some embodiments, the method further comprises allowing the subject to elicit an immune response to the one or more neo-antigens.

[0024] In some embodiments, the method further comprises sequencing one or more nucleic acid sequences encoding one or more TCRs or functional fragments thereof from T cells isolated from the subject after step (a) but before step (b).

[0025] In some embodiments, the method comprises, after step (a), allowing a period of time sufficient for the subject to expand a clonal T cell population sensitized to one or more neoantigens, wherein the clonal T cell population comprises about 25% to about 50% CD8+ reactivity to the one or more neoantigens.

[0026] In some embodiments, the methods include a step comprising administering a nucleic acid molecule comprising one or more nucleic acid sequences encoding one or more neo-antigens.

[0027] 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.

[0028] In some embodiments, the nucleic acid molecule is a plasmid. In some embodiments, the nucleic acid molecule is GNOS-PV02.

[0029] In some embodiments, the expressible nucleic acid sequence is located within the multiple cloning site of: (i) a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES, GNOS-PV02; or (ii) a plasmid comprising at least 70% sequence identity to a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), GNOS-PV02, phCMV1, pTCP and pIRES.

[0030] 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, hematological tumors, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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.

[0031] 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, which one or more TCRs or functional fragments thereof recognize one or more neo-antigens, the method comprising: (a) administering one or more nucleic acid sequences encoding one or more neo-antigens to a subject comprising one or more cells expressing one or more neo-antigens; and (b) isolating clonally derived T cells expressing one or more TCRs or functional fragments thereof from the subject.

[0032] In some embodiments, the method does not perform in vitro expansion of PBMCs and / or tumor infiltrating lymphocytes. In some embodiments, the method further comprises sequencing one or more nucleic acid sequences encoding one or more TCRs or functional fragments thereof after step (b).

[0033] In some embodiments, the method further comprises the step of allowing a period of time sufficient for the subject to expand a clonal T cell population sensitized to one or more neoantigens after step (a).

[0034] In some embodiments, the clonal T cell population comprises about 25% to about 50% CD8+ reactivity to one or more neoantigens.

[0035] 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.

[0036] In yet another aspect, the present disclosure relates to a method for preventing metastasis of a cancer comprising one or more neoantigens in a subject, the method comprising: (a) administering one or more nucleic acid sequences encoding one or more neoantigens to the subject; (b) allowing a period of time sufficient to allow clonal T cells sensitized to the subject's one or more neoantigens to expand to biologically significant numbers; (c) isolating 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 subject's one or more neoantigens; and (e) administering to a subject in need thereof a therapeutically effective amount of T cells comprising nucleic acid molecules encoding one or more of the subset of TCRs.

[0037] In some embodiments, clonal T cells are isolated by drawing a blood sample from a subject and sorting peripheral blood mononuclear cells (PBMCs) from the sample based on receptor expression on the surface of the PBMCs.

[0038] In some embodiments, step (d) comprises performing an assay to measure one or a combination of: (i) the affinity or avidity 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.

[0039] In some embodiments, the method further comprises sequencing one or more nucleotide sequences encoding a subset of TCRs from T cells expressing the TCRs that are highly immune.

[0040] In some embodiments, the method further comprises identifying one or more neo-antigens from a tissue sample removed from the subject.

[0041] In some embodiments, the tissue sample comprises brushing, biopsy, or surgically removed tissue from a subject.

[0042] In some embodiments, the method does not involve in vitro expansion of PBMCs and / or tumor infiltrating lymphocytes.

[0043] In some embodiments, the subject's population of clonal T cells sensitized to one or more neoantigens comprises about 25% to about 50% CD8+ reactivity to the one or more neoantigens.

[0044] In some embodiments, step (a) comprises administering a nucleic acid molecule comprising one or more nucleotide sequences encoding one or more neo-antigens.

[0045] In some embodiments, the nucleic acid molecule encodes about 10 to about 55 neoantigens. In some embodiments, the present disclosure relates to a composition comprising nucleic acid molecules encoding about 10 to about 55 neoantigens. In some embodiments, the present disclosure relates to a composition comprising nucleic acid molecules encoding about 19 to about 60 neoantigens. In some embodiments, the present disclosure relates to a composition comprising nucleic acid molecules encoding about 20 to about 60 neoantigens. In some embodiments, the present disclosure relates to a composition comprising nucleic acid molecules encoding about 20 to about 65 neoantigens.

[0046] In some embodiments, each neo-antigen encoded by a nucleic acid molecule is separated from another by one or more linkers.

[0047] In some embodiments, one or more linkers comprise a furin protease cleavage site or a porcine teschovirus-1 2A (P2A) cleavage site.

[0048] In some embodiments, the nucleic acid molecule is a plasmid.

[0049] In some embodiments, the nucleic acid molecule is located within the 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 comprising at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES.

[0050] In some embodiments, the nucleic acid molecule is GNOS-PV02.

[0051] 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, hematological tumors, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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 DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 A schematic diagram of a method for developing personalized T cell vaccines based on tumor-specific neoantigens disclosed herein is shown. Since most neoantigens are unique to individual patients' cancers, the "mutation group" of each patient's tumor is determined, allowing the identification of candidate neoantigens targeted by the vaccine. By administering one or more DNA or RNA vaccines encoding one or more patient-specific neoantigens, neoantigen-specific T cells are activated and amplified in vivo. 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 produced and administered to patients.

[0053] Figure 2 The predicted MHC class I / II binding affinities (nM) of the pool compositions immunized with the GEN-PV-001 vaccine are shown.

[0054] Figure 3 It is shown that each new antigen in the vaccine uses a 33 amino acid long sequence, allowing encoding of potential CD8 and CD4 epitopes covering the new antigen, which allows a more effective adaptive immune response. The length of the CD8 epitope is more usually 9 amino acids, but can be in the range of 8 to 14 amino acids, and the length of the CD4 epitope is usually 15 amino acids, but can be in the range of 9 to 25 amino acids. See Chong et al., Mol. Cell Proteomics, 2018, 17 (3): 533-548. This means that a 33 amino acid sequence should cover the entire predicted epitope in a new antigen with overlapping CD8 and CD4 epitopes.

[0055] FIG. 4A to FIG. 4B The immune responses to the tested neoantigens are shown. Figure 4A: There was no significant difference in the immune response (IFNγ ELISpot) when the tested neoantigen (marked with an asterisk in the schematic) was located at position 1, 10, 20, 30 or 40 of the neoantigen DNA vaccine of 40 epitopes (4806 nucleotide insertion). Figure 4B : The immune response (IFNγ ELISpot) to a vaccine containing 30 epitopes (27 neoantigens and 3 tumor-associated antigens) was divided into 4 pools: Pool 1 contained vaccine epitopes 1-8; Pool 2 contained vaccine epitopes 9-16; Pool 3 contained vaccine epitopes 17-23; and Pool 4 contained vaccine epitopes 24-30. After vaccination of patients with anaplastic astrocytomas, responses were found in all 4 pools, indicating that the DNA neoantigen vaccine is expressed as a protein antigen throughout the entire length of the DNA sequence.

[0056] Figure 5 Detection of T cell responses (IFNγ ELISpot) to 17 of the 30 neoantigens encoded by the GEN-PV-001 vaccine is shown.

[0057] Figure 6 Heatmaps of intracellular cytokine staining (ICS) analysis are shown. CD8+ and CD4+ T cell responses to epitopes encoded by the GEN-PV-001 vaccine were detected, and the responses were directed to multiple markers. The heatmap range is in % and represents the % of CD4+ or CD8+ T cells expressing the specified marker in a peptide-specific manner (peptide stimulated-vehicle control).

[0058] FIG. 7A to FIG. 7B Neoantigen DNA vaccines were shown to generate CD8 and CD4 responses against multiple epitopes in cancer patients. Fig. 7A : Heat map ranges are in % and represent the % of CD4+ or CD8+ T cells expressing the indicated marker in a peptide-specific manner (peptide stimulated - vehicle control). Figure 7B : Representative flow cytometry plots showing the presence of CD8+, CD4+ T cells producing IFNγ, TNFα, or both in a peptide-specific manner.

[0059] Fig. 8A A non-limiting example of a manufacturing process for a personalized DNA vaccine. Targeted treatment can be achieved in as little as 6 weeks and can be achieved regularly within 6-8 weeks. Figure 8B are non-limiting examples of clinical trials.

[0060] Fig.9A A spider plot is shown which displays the first 12 patients in the clinical trial at the time of data cutoff. Fig. 9BThe figure is a waterfall chart showing the best overall response achieved by the first 12 subjects in the clinical trial at the time of the data cut-off. The best overall response is shown as a partial response rate of 25% and a disease control rate of 67%. Fig. 9C Tumor imaging scans of patients classified as PR (day 0 and week 27 after treatment). Red arrows point to the tumor.

[0061] Fig. 10A Shown is a bar graph of newly detected and expanded T cell clones following treatment with GNOS-PV02 for all patients analyzed to date (n=10). Fig. 10B Cumulative frequencies of expanded clones in peripheral blood (PBMC, left) and tumor tissue (right) before and after vaccination (week 9) for each patient are shown. Fig. 10C The expansion of pre-vaccination clones (points along the X-axis) and the detection of multiple new T cell clones (points along the Y-axis) after vaccination in the blood and tumor tissue from subject Pt7 are shown. Arrows highlight that 9 weeks after vaccination, high-frequency clones infiltrated the tumor from the blood (only the top 6 clones are shown for clarity). As assessed by TCRβ and RNA sequencing, the most abundant clones showed an active phenotype (CD8+CD69+). Before vaccination, approximately 75% of the new TIL clones were undetectable in the blood.

[0062] Fig.11A Shown is the genetic optimization of patient-specific cloned TCR sequences using the GOAL algorithm and insertion into the pMXs-IRES-GFP retroviral plasmid vector containing viral packaging signals, transcription and processing elements, and a GFP reporter gene. Fig. 11B An example of anti-tumor specific T cell reactivity after vaccination assessed by ELISpot is shown (subject Pt 8). PBMCs were stimulated using pools of individual peptides or single peptides encoded in the personalized GNOS-PV02 treatment. Fig. 11C Representative images of activated, GFP-positive, CD8 and CD4 TCR-engineered T cells (subject Pt8) stimulated with ATP1A1-ALB (10 ug / mL) are shown. TNTC, too numerous to count; EOT, end of treatment.

[0063] FIG. 12 shows PTCV-driven neoantigen-specific responses detected in blood. Fig. 12A Results from ELISpot assays of PBMC samples are shown to determine the presence of vaccine-induced neoantigen-specific responses before and after personalized GNOS-PV02 vaccination without cytokine stimulation. The post-vaccination response was the 'best' (highest degree) response for each patient at different time points. Fig. 12BShown are the positive neoantigens pre- and post-vaccination (black and red bars, respectively) relative to the total number included in each patient's PTCV (grey bars), as defined by IFNγ ELISpot assay. Fig. 12C The percentages of positive responding epitopes for the clinical response groups and pre-treatment and on-treatment time points are shown. Fig.12D The Spearman correlation between the positive epitopes contained in the PTCV of each patient and the total number of neoantigens is shown. Fig.12E Neoantigen-specific T cell activation assessed by stimulation of patient-derived PBMCs with DMSO (week 9 or 12) or by intracellular cytokine staining of ex vivo patient-specific epitope pools is shown. Fig.12F and Figure 12G The multifunctionality evaluation via Boolean gating of CD4+ or CD8+ cytokines+ populations is shown. T cell activation (CD69 and CD107a) and proliferation (Ki67) are evaluated together with the double positive expression of granzyme A (GrzA) and perforin (Prf) to assess the cytolytic potential of neoantigen reactive T cells. The results are expressed as % positive cell population (frequency of parents). The solid circles represent individual patients, the boxes extend from the 25th percentile to the 75th percentile, the lines in the boxes are medians, and the bars extend from the minimum to the maximum. Four patients (1CR, 3PR) were analyzed.

[0064] FIG. 13 shows that GNOS-PV02 leads to expansion of new T cell clones that traffic to tumors. Fig.13A It was shown that in 14 of 14 subjects, T cell clones were expanded in peripheral blood, and new or expanded clones were enriched in matched tumor samples of each patient. The total number of PBMC and tumor-associated T cell expansions was calculated by comparing PBMC or tumor samples after treatment with those before treatment, respectively (differential abundance statistical analysis). Fig. 13B Shown are the cumulative frequencies of peripherally amplified TCR rearrangements tracked in pre- and post-treatment tumor biopsies. Fig. 13C 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 the 75th percentile, the line within the box is the median, and the whiskers extend from the minimum to the maximum value. Fig.13D and Fig.13ETCR clonality and TCR library richness in tumor biopsies of 14 evaluated patients are shown (bar lines and stacked bar graphs). Error bars correspond to the upper SE of each group. Simpson clonality reports the distribution of TCR rearrangements in a sample, where 0 indicates a uniform distribution of frequencies and 1 indicates an asymmetric distribution. Lower numbers indicate that TCR diversity is concentrated.

[0065] Figure 14 shows that TCR clones expanded after vaccination identified in tumors were reactive to PTCV-encoded antigens. Fig.14A The most common TCRs identified in patients by TCRβ and RNA sequencing are shown. Before vaccination and at week 9 after vaccination (paired scatter plots). Blue asterisks show selected high-frequency new T cell clones detected in PBMCs after vaccination and their abundance in tumors. Orange, green, and gray circles indicate T cell clones that expanded, shrank, and did not change significantly, respectively. Fig. 14B The evaluated TCRs selected for cloning based on their high frequency appearance in PBMCs and trafficking to tumors after vaccination are shown. Fig. 14C The results of gene optimization of patient-specific cloned TCR sequences and insertion into the pMXs-IRES-GFP retroviral plasmid vector containing viral packaging signals, transcription and processing elements, and a GFP reporter gene are shown. Fig.14D and Fig.14E Shown are TCR engineered T cells (GFP positive) from unvaccinated PBMCs stimulated for 6 hours with increasing concentrations of epitope pools (2 μg / mL, 10 μg / mL, 25 μg / mL) and expression of CD69 assessed by flow cytometry. Peptide pools 1 and 2 contained either the first or second half of the neoantigen contained in PTCV. DETAILED DESCRIPTION

[0066] The disclosed methods and compositions may be understood more readily by reference to the following detailed description of specific embodiments and examples included therein and the accompanying drawings and their previous and following descriptions.

[0067] It should be understood that, unless otherwise specified, the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques or specific reagents, and may vary accordingly. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0068] It should be understood that the disclosed methods and compositions are not limited to the specific methods, protocols and reagents described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims.

[0069] definition

[0070] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "nucleic acid sequence" includes a plurality of nucleotides formed, reference to a "nucleic acid sequence" is a reference to one or more nucleic acid sequences and their equivalents known to those skilled in the art, and so forth.

[0071] The term "about" as used herein means within the typical tolerance range of this field. For example, "about" can be understood as about 2 standard deviations of the mean value. According to certain embodiments, when referring to measurable values ​​such as amounts, "about" means to cover deviations 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 a specified value, because such deviations are suitable for performing the disclosed method. When "about" appears before a series of numbers or a range, it should be understood that "about" can modify each number in the numbers in the series or range.

[0072] As used herein, the terms "activate", "stimulate", "enhance", "increase" and / or "induce" (and similar terms) are used interchangeably and generally refer to the act of improving or increasing concentration, level, function, activity or characteristic, directly or indirectly, relative to natural, expected or average values ​​or relative to control conditions. In the context of immunotherapy, "activation" refers to the primary response induced by the binding of a cell surface portion. For example, in the context of a receptor such as a TCR, such stimulation requires the binding of the receptor and subsequent signal transduction events. In some embodiments, activation means that the TCR associates with a tumor-specific epitope or antigen, and the cell containing the TCR responds by releasing an immunostimulant in response to the association. In addition, the stimulation event can activate the cell and upregulate or downregulate the expression or secretion of the molecule. Therefore, even in the absence of a direct signal transduction event, the binding of the cell surface portion may also lead to the reorganization of the cytoskeletal structure, or in the case of aggregation of the cell surface portion, each portion is used to enhance, modify or change the subsequent cellular response. In some embodiments, if the cell releases IFNγ, TNFα or IL-2 after association with a new antigen or tumor-specific epitope, the epitope is activated or highly immunogenic. In some embodiments, if the cell containing the TCR is clonally expanded, exposed to the new antigen and greater than or equal to about 20%, 25%, 30%, 35% or about 40% of the population or secretes the new antigen IFNγ, TNFα or IL-2, the new antigen or TCR is highly immune. In some embodiments, if the cell containing the TCR is clonally expanded, exposed to the new antigen and greater than or equal to about 20%, 25%, 30%, 35% or about 40% of the cell population or expresses one or more immunostimulants in response to the presence of the new antigen or association with the new antigen, the new antigen or TCR is highly immune to the new antigen or to the new antigen. In some embodiments, highly immune TCRs are those TCRs with respect to binding or association with the new antigen,

[0073] As used herein, the term "activated CD8+T cells" or "CD8+T cell activation" refers to a process (e.g., signaling event) that causes or leads to one or more cellular responses of CD8+T cells (CTLs), which are selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. As used herein, "activated CD8+T cells" refer to CD8+T cells that have received activation signals and therefore exhibit one or more cellular responses selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays for measuring CD8+T cell activation are known in the art and are described herein.

[0074] Activation can be defined as: as assessed by ELISpot, >50 IFNγ spots / 1x106 PBMCs; by FACS, >0.05% IFNγ positive T cells, by ELISA, >100pg / mL and >2-fold IFNγ mRNA expression. In addition, activation can be defined by CD137 and / or CD69 expression, which can be measured by RNA sequencing, flow cytometry or ELISA. Alternatively, bead-bound or column-bound antibodies to CD137, CD69, CD25 and / or CD38 can be used to select sorted cells.

[0075] As used herein, the term "adjuvant" means any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigens encoded by the DNA plasmids described below and encoding the nucleic acid sequences.

[0076] As used herein, the term "allogeneic" refers to medical therapy in which the donor and recipient are different individuals of the same species.

[0077] As used herein, the phrase "and / or" in the specification and claims should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in conjunction in some cases and separately in other cases. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated to the contrary. Thus, as a non-limiting example, in some embodiments, when used in conjunction with open language such as "comprising," a reference to "A and / or B" may refer to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); and in yet another embodiment, to both A and B (optionally including other elements).

[0078] As used herein, "antigen" or "Ag" refers to a molecule that triggers an immune response, and the immune response may involve antibody production, activation of specific immunocompetent cells (e.g., T cells), or both. Antigens may be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, and the like. Antigens may be synthesized, recombinantly produced, or derived from biological samples using methods known in the art. For example, novel antigens may be produced using methods known in the art such as chromosome rearrangement or breakage. Exemplary biological samples that may contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or a combination thereof. Antigens may be produced by cells that have been modified or genetically engineered to express antigens. The term "antigen" includes antigenic determinants, such as peptides of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more amino acid residues in length, which are bound to MHC molecules, forming part of MHC class I or class II complexes, or recognized when complexed with such molecules.

[0079] As used herein, the term "antigen presenting cell (APC)" refers to a type of cell that is capable of presenting one or more antigens in the form of a peptide-MHC complex that can be recognized by specific effector cells of the immune system, and thereby induces 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 class II molecules may present peptide antigens.

[0080] As used herein, the term "anti-tumor response" refers to an immune system response, including but not limited to activation of T cells to attack antigens or antigen presenting cells.

[0081] As used herein, the term "autologous" refers to medical therapy in which the donor and recipient are the same individual.

[0082] As used herein, the term "cancer" means any disease caused 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 myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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.

[0083] As used herein, the term "check point inhibitor" means any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide or fragment thereof that inhibits inhibitory pathways and allows for a wider range of immune activity. In certain embodiments, the check point inhibitor is an inhibitor of programmed death 1 (PD-1) pathways, such as anti-PD1 antibodies, such as but not limited to nivolumab. In other embodiments, the check point inhibitor is an anti-cytotoxic T lymphocyte-associated antigen (CTLA-4) antibody. In other additional embodiments, the check point inhibitor targets members of the TNF superfamily, such as CD40, OX40, CD 137, GITR, CD27 or TIM-3. In some cases, targeted check point inhibitors are accomplished by inhibitory antibodies or similar molecules. In other cases, it is accomplished by agonists of the target; examples of this category include stimulatory targets OX40 and GITR.

[0084] As used herein, the term "combination therapy" means the administration of one or more therapeutic agents in a sequential manner, that is, each therapeutic agent is administered at different times, and the administration of these therapeutic agents, or at least two of the therapeutic agents, is administered in a substantially simultaneous manner. For example, sequential administration includes the separate administration of (i) a pharmaceutical composition comprising an effective amount of a tumor-specific neoantigen or a combined sample of DNA / RNA encoding it and a pharmaceutically acceptable salt, carrier, or diluent; (ii) a pharmaceutical composition comprising an effective amount of IL-12 or DNA / RNA encoding it 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 the subject a single capsule of each therapeutic agent having a fixed ratio or multiple, single capsules for each of the therapeutic agents. For example, a combination therapy of the present disclosure may include a combined sample of tumor-specific neoantigens or DNA / RNA encoding it, IL-12 or DNA / RNA encoding it, and a checkpoint inhibitor, and a pharmaceutically acceptable salt, carrier, or diluent administered simultaneously or at different times. In some embodiments, the pharmaceutical composition of the present disclosure may be formulated as a single, co-formulated pharmaceutical composition comprising two or three compounds. As another example, in some embodiments, the combination of the present disclosure (e.g., DNA neoantigen vaccine, IL-12 and checkpoint inhibitor) is formulated as a separate pharmaceutical composition that can be administered at the same or different times. As used herein, the term "simultaneously" means the simultaneous administration of one or more agents. For example, in certain embodiments, a cancer vaccine or an immunogenic composition and a checkpoint inhibitor are administered simultaneously. Simultaneously including administration in the same period, i.e., administration in the same time period. In certain embodiments, one or more agents are administered simultaneously at the same hour, or simultaneously on the same day. The order or substantially simultaneous administration of each therapeutic agent can be achieved by any appropriate route, including but not limited to oral routes, intravenous routes, subcutaneous routes, intramuscular routes, direct absorption through mucosal tissues (e.g., nose, mouth, vagina and rectum) and ocular routes (e.g., intravitreal, intraocular, etc.). The therapeutic agent 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 components of the combination are administered orally. These components can be administered in any therapeutically effective order. "Combination" encompasses groups of compounds or non-drug therapies that can be used as part of a combination therapy.

[0085] As used herein, the term "cytotoxic T cell" or "cytotoxic T lymphocyte" is a type of immune cell that contains a CD receptor that can recognize 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 cell's recognizable antigen. Cytotoxic T cells are a subset of white blood cells and are also a type of lymphocyte.

[0086] 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).

[0087] As used herein, "electroporation," "electroosmosis," or "electrokinetic enhancement" ("EP") are used interchangeably and refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; their presence permits the transfer of biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and / or water from one side of the cell membrane to the other.

[0088] As used herein, the term "endogenous" or "native" refers to a gene, protein or activity that is present in a host cell in vivo. A gene, protein or activity that has been mutated, overexpressed, reorganized, duplicated or otherwise altered compared to a normal, unmutated gene, protein or activity is still considered endogenous or native to a particular host cell in vivo. For example, an endogenous control sequence (e.g., a promoter, a translation attenuation sequence) from a first gene can be used to alter or regulate the expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule is different from the normal expression or regulation in the parent cell.

[0089] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment may contain 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.

[0090] The term "functional fragment" refers to any part of a polypeptide having a sufficient length to retain a biological function similar to or substantially similar to the wild-type polypeptide on which the fragment is based. The functional fragment of TCR disclosed herein is a fragment of TC disclosed herein and maintains at least a partial binding affinity with 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 adjacent amino acids. In some embodiments, the functional fragment has a length of 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids or 35 amino acids. In some embodiments, the functional fragment has a length between about 25 amino acids and about 35 amino acids. In some embodiments, the functional fragment has a length between about 27 amino acids and about 35 amino acids. In some embodiments, the functional fragment has a length between about 29 amino acids and about 35 amino acids. In some embodiments, the functional fragment has a length of between 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.

[0091] As used herein, the term "gene construct" means 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 gene construct comprises one or more coding sequences, and one or more regulatory sequences. In some embodiments, the coding sequence includes a start sequence and a termination sequence operably linked to a regulatory element. In some embodiments, the regulatory element comprises a promoter and a polyadenylation signal, which can direct expression in the cells of an individual to whom the DNA or RNA molecule is administered.

[0092] As used herein, a "heterologous" or "exogenous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or a portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or a portion of a nucleic acid molecule from a 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 (i.e., non-endogenous or natural) is added to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, etc., wherein the added molecule may be integrated into the host genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may exist in multiple copies. In addition, "heterologous" or "exogenous" refers to a non-natural 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.

[0093] As used herein, the term "host cell" refers to a cell that can be used to express a gene construct (such as a nucleic acid disclosed herein). It can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. The phrase "recombinant host cell" can be used to represent a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a claimed nucleic acid sequence but does not express it at a level sufficient to induce an immunogenic response, unless a regulatory sequence is introduced into the host cell so that it is operably connected to the nucleic acid. It should be understood that in some embodiments, the term host cell refers not only to a specific subject cell, but also to the progeny or potential progeny of this cell. Because some modifications may occur in subsequent generations due to, for example, mutations or environmental influences, such progeny may not actually be identical to the parental cell, but are still included in the scope of the term as used herein.

[0094] As used herein, the term "hybridization" means the formation of double-stranded molecules between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringent conditions (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0095] As used herein, "immune cell" or "immune system cell" means any cell of the immune system that originates from hematopoietic stem cells in the bone marrow, which give 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 cells of the vertebrate immune system, characterized in that they are primarily found in the lymph and typically have 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 as 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 the major histocompatibility complex (MHC) receptor on the surface of the APC interacts with the TCR 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 expressing an antigen recognized by the TCR or another antigen binding protein (e.g., a chimeric antigen receptor or an antibody). The immune cells or lymphocytes used in the vaccine composition or treatment method of the present disclosure may be autologous, allogeneic or isogenic to the subject receiving the composition or treatment method.

[0096] As used herein, the term "immune checkpoint" refers to inhibitory pathways that slow or stop the immune response and prevent the uncontrolled activity of immune cells from causing excessive tissue damage.

[0097] As used herein, the term "immune response" means that the host's immune system (eg, a mammal's immune system) is activated in response to the introduction of a nucleic acid molecule comprising a nucleotide sequence encoding a neoantigen as described herein.

[0098] As used herein, "immunogenic enhancers" include molecules encoded by polynucleotides contained in host cells such as T cells, which enhance the immunogenicity of exogenous neoantigens encoded by polynucleotides contained in the cells. Immunogenic enhancers encoded by host cells can provide local and concentrated adjuvant activity, which improves the immune response to neoantigens. Exemplary immunogenic enhancers include IL-12 (such as membrane-tethered IL-12), GM-CSF, inducible cell death factor, bacterial flagellin, CD80, CD137L, CD40L, secretory IL-2, secretory IL-2 independent of CD25 binding T cells, secretory IL-15, secretory IL-15-IL-15Rα complex, secretory IFNβ, secretory IFN-α1, secretory IL-7, or any combination thereof. In some embodiments, the immunogenicity 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 immunogenicity enhancer, or the immunogenicity enhancer is exogenous to the host cell.

[0099] As used herein, the phrase "in need thereof" means that an animal or mammal has been identified or is suspected of being in need of a particular method or treatment. In some embodiments, identification may be performed by any diagnostic or observational means. In any of the methods and treatments described herein, an animal or mammal may be in need thereof. In some embodiments, a subject in need thereof is a person seeking to prevent cancer. In some embodiments, a subject in need thereof is a person diagnosed with cancer. In some embodiments, a subject in need thereof is a person seeking treatment for cancer. In some embodiments, a subject in need thereof is a person who is currently receiving treatment for cancer. In some embodiments, a subject in need thereof is a healthy subject.

[0100] In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection" or "transformation" or "transduction", and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid molecule can be incorporated into the genome of the cell (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).

[0101] As used herein, the term "isolated" means that the polynucleotide or polypeptide, or fragment, variant or derivative thereof, has been substantially removed from other biological material with which it is naturally associated, or is substantially free of other biological material, for example, derived from a recombinant host cell that has been genetically engineered to express a polypeptide of the invention.

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

[0103] As used herein, the term "MHC molecule", "MHC protein" or "HLA protein" means a protein that is capable of binding peptides produced by proteolytic cleavage of protein antigens and represents potential T cell epitopes, transporting them to the cell surface and presenting them to specific cells, in particular cytotoxic T lymphocytes or T helper cells. The major histocompatibility complex in the genome includes genetic regions whose gene products expressed on the cell surface are important for binding and presenting endogenous and / or exogenous antigens and therefore for regulating immune processes. The major histocompatibility complex is divided into two gene sets encoding different proteins, namely MHC class I molecules and MHC class II molecules. The molecules of the two MHC classes are specifically directed against different antigen sources. MHC class I molecules present endogenously synthesized antigens, such as viral proteins and tumor antigens. MHC class II molecules present protein antigens derived from exogenous sources, such as bacterial products. The cell biology and expression patterns of the two MHC classes are adapted to these different roles. Structurally, class I MHC molecules consist of a heavy chain and a light chain, and are capable of binding to a peptide of about 8 to 11 amino acids, but usually 9 or 10 amino acids (if the peptide has a suitable binding motif), and presenting it to cytotoxic T lymphocytes. The heavy chain of class I MHC molecules is preferably an HLA-A, HLA-B or HLA-C monomer, and the light chain is beta-2-microglobulin. Class II MHC molecules consist of an alpha chain and a beta chain, and are capable of binding to a peptide of about 15 to 24 amino acids (if the peptide has a suitable binding motif), and presenting it to T helper cells. The alpha-chain and beta-chain are particularly HLA-DR, HLA-DQ and HLA-DP monomers.

[0104] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or a polypeptide molecule compared to a reference, wild-type or endogenous nucleic acid sequence or polypeptide sequence, respectively. Mutation can result in several different types of sequence changes, including substitution, insertion or deletion of nucleotides or amino acids. 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 a combination of any one 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.

[0105] As used herein, "neoantigen" refers to a host cell product containing structural changes, alterations or mutations, which produces a new antigen or antigenic epitope that was not previously observed in the subject's genome (i.e., in a sample of healthy tissue from the subject) or is "seen" or recognized by the host's immune system. The new antigen may be derived from, for example, a coding polynucleotide with a change (substitution, addition, deletion) of a product that causes a change or mutation, or from an exogenous nucleic acid molecule or protein inserted into a cell, or from exposure to environmental factors (e.g., chemistry, radiology) that causes genetic changes. The new antigen may be produced independently of the tumor antigen, or may be from a tumor antigen or associated with a tumor antigen. "Tumor neoantigen" (or "tumor-specific neoantigen") refers to a protein containing a new antigenic determinant associated with a tumor cell or multiple cells in a tumor, generated from a tumor cell or generated in a tumor cell. Tumor neoantigen determinants are present in, for example, antigenic tumor proteins or peptides containing one or more somatic mutations encoded by tumor cell DNA, and proteins or peptides from viral open reading frames associated with virus-related tumors (e.g., cervical cancer, certain head and neck cancers). For example, tumor neoantigens can arise within or be derived from any exemplary tumor or other antigen, as well as from "driver" cancer antigens (e.g., the G12D neoantigen from KRAS described in Tran et al., N. Eng. J. Med. 375:2255-2262 (2016)), as well as in mutant B-Raf, SF31, MYD88, DDX3X, MAPK1, GNB1, etc.

[0106] As used herein, the term "neoantigenic mutation" refers to a mutation predicted to encode a neoantigenic peptide. Such a neoantigenic peptide may be expressed on the surface of a cancer cell in a subject.

[0107] As used herein, the term "nucleic acid molecule" comprises one or more nucleotide sequences encoding one or more proteins. In some embodiments, the nucleic acid molecule comprises a start signal and a stop signal operably linked to a regulatory element, which comprises a promoter and a polyadenylation signal capable of being directly expressed in the 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 new antigens. In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a first, second, third or more nucleic acid molecules, wherein each nucleic acid molecule independently or simultaneously (e.g., in trans) encodes one or more new antigens, and at least one of each plasmid comprises one or more chemical formulas disclosed herein.

[0108] If the regulatory sequence affects the expression of the nucleotide sequence (e.g., the level, timing or position of expression), the nucleotide sequence is "operably linked" to the regulatory sequence. A "regulatory sequence" is a nucleic acid that affects the expression of a nucleic acid to which it is operably linked (e.g., the level, timing or position of expression). For example, a regulatory sequence may, for example, exert its effect directly on the regulated nucleic acid, or exert its effect through one or more other molecules (e.g., a polypeptide bound to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Other examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06, incorporated herein by reference.

[0109] As used herein, in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when the items in the list are separated, "or" or "and / or" should be interpreted as inclusive, that is, including many elements or at least one element in the list of elements, but also including more than one element and optionally other unlisted items. Only the terms clearly indicated on the contrary, such as "only one of..." or "exactly one of..." or "consisting of..." when used in the claims will refer to containing exactly one element in multiple elements or lists of elements. In general, when there is an exclusive term before, "any one", "one of...", "only one of..." or "exactly one of...", the term "or" as used herein should only be interpreted as indicating exclusive alternatives (that is, "one or the other, not two"). When used in the claims, "consisting essentially of..." should have its ordinary meaning used in the field of patent law.

[0110] The term "percent identity" or "percent homology" of two polynucleotides or two polypeptide sequences is determined by comparing sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. As used herein in the context of two or more nucleic acid or amino acid sequences, "identity" or "identity" may mean that the sequences have the same percentage of specified residues within a specified region. The percentage can be calculated by optimizing the alignment of the two sequences, comparing the two sequences within a specified region, determining the number of positions where the same residues occur in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to derive the percentage of sequence identity. In the case where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified comparison region contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Can manually or use computer sequence algorithm such as BLAST or BLAST 2.0 to carry out identity identification.In short, BLAST algorithm, represents local sequence alignment search basic tool, is suitable for determining sequence similarity.Software for carrying out BLAST analysis can be publicly obtained by National Center for Biotechnology Information (National Center for Biotechnology Information) (www.ncbi.nlm.nih.gov).This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, when comparing with words of the same length in the database sequence, the short words match or meet a certain positive threshold score T.T is referred to as the neighboring character score threshold (Altschul et al., the same).These initial neighborhood word hits serve as seeds for starting searches to find HSPs containing the initial neighborhood word hits.Word hits are expanded in both directions along each sequence until the cumulative comparison score can be increased. Extension of the word hit in each direction is terminated when: 1) the cumulative alignment score drops by the number X from its maximum achieved value; 2) the cumulative score drops to zero or less due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The Blast program uses the following defaults: word length (W) of 11, BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and comparison of both chains.BLAST algorithm (Karlin, Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, the document is incorporated herein by reference in its entirety) and Gapped BLAST perform statistical analysis on the similarity between two sequences. A similarity measure provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability of accidental matching between two nucleotide sequences. For example, if the minimum sum probability is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001 in the comparison of the test nucleic acid with other nucleic acids, the nucleic acid is considered to be similar to another nucleic acid. If the sequences of two single-stranded polynucleotides can be arranged in an antiparallel direction so that each nucleotide in one polynucleotide is opposite to the complementary nucleotide in another polynucleotide, there is no introduction of a gap, and there is no unpaired nucleotide at the 5' end or 3' end of any sequence, the two single-stranded polynucleotides are "complements" of each other. If two polynucleotides can hybridize with each other under moderately stringent conditions, the polynucleotide is "complementary" to another polynucleotide. Thus, a polynucleotide can be complementary to another polynucleotide rather than being its complement.

[0111] As used herein, "peripheral blood mononuclear cell" or "PBMC" is any peripheral blood cell with a round nucleus. These cells consist of lymphocytes (T cells, B cells, NK cells) and monocytes. In humans, lymphocytes make up the majority of the PBMC population, followed by monocytes and a small portion of dendritic cells.

[0112] As used herein, the term "pharmaceutically acceptable" means approved or approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0113] As used herein, the term "pharmaceutically acceptable excipient, carrier or diluent" means an excipient, carrier or diluent that can be administered to a subject together with a pharmaceutical agent and does not destroy its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the pharmaceutical agent.

[0114] As used herein, the term "pharmaceutically acceptable salt" of tumor-specific neoantigens may be an acid or base salt generally considered in the art to be suitable for contact with human or animal tissues without excessive toxicity, irritation, allergic response or other problems or complications. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali metal or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutically acceptable salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH2)n-COOH (wherein n is 0-4), etc. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that other pharmaceutically acceptable salts of the combined tumor-specific neoantigens are provided herein, including those listed in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, page 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from parent compounds containing a basic or acidic moiety by any conventional chemical method. In short, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in an appropriate solvent.

[0115] The term "pharmaceutical composition" includes, but is not limited to (i) a pharmaceutical composition comprising an effective amount of a tumor-specific neoantigen or a combined sample of DNA / RNA encoding the same and a pharmaceutically acceptable salt, carrier or diluent; (ii) a pharmaceutical composition comprising an effective amount of IL-12 or DNA / RNA encoding the same and (iii) an effective amount of a checkpoint inhibitor and a pharmaceutically acceptable salt, carrier or diluent. It further includes any composition comprising two or three of these ingredients and a pharmaceutically acceptable salt, carrier or diluent.

[0116] The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably throughout the text and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs) and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In some embodiments, the nucleic acid molecules of the present disclosure comprise adjacent open reading frames encoding antibodies or fragments thereof as described herein. As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions. Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acid can be DNA (both genomic and cDNA), RNA or hybrid, wherein nucleic acid can comprise a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acid can be obtained by chemical synthesis or recombinant methods. Nucleic acid generally contains phosphodiester bonds, although nucleic acid analogs that can have at least one different bonding (e.g., phosphoramidate, phosphorothioate, phosphorodithioate or O-methylphosphoramidate bonding and peptide nucleic acid backbone and bonding) can be included. Other similar nucleic acids include those with positive backbones, nonionic backbones and non-ribose backbones, including those described in the following: U.S. Patent Nos. 5,235,033 and 5,034,506, which are incorporated by reference in their entirety. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included in a definition of nucleic acid. Modified nucleotide analogs may be located at, for example, the 5'-end and / or 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar-modified or backbone-modified ribonucleotides. However, it should be noted that nucleobase-modified ribonucleotides, i.e., containing non-natural 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; denitronucleotides, such as 7-denitrogenation-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are also suitable. The 2'-OH-group may be substituted by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, N2 or CN, wherein R is a C1-C6 alkyl, alkenyl or alkynyl group and the halo is F, Cl, Br or I.Modified nucleotides also include nucleotides conjugated to cholesterol by, for example, hydroxyprolinol linkages, as described in Krutzfeldt et al., Nature (October 30, 2005), Soutschek et al., Nature 432: 173-178 (2004), and U.S. Patent Publication No. 20050107325, which are incorporated herein by reference in their entirety. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, which is incorporated herein by reference in its entirety. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No. 20050182005, which is incorporated herein by reference in its entirety. Modifications to the ribose-phosphate backbone may be performed for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs can be prepared, as well as mixtures of naturally occurring nucleic acids and analogs.

[0117] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may include modified amino acids, and it may be interrupted by non-natural amino acids or non-amino acid chemical groups. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptide mimetics.

[0118] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like mean reducing the likelihood of developing a disease or disorder in a subject who does not have the disease or disorder but is at risk or susceptible to the disease or disorder.

[0119] As used herein, the term "purified" means that a polynucleotide or polypeptide or a fragment, variant or derivative thereof is substantially free of other biological materials associated with it in nature, or free of other biological materials derived from, for example, a recombinant host cell that has been genetically engineered to express a polypeptide of the present invention. For example, a purified polypeptide of the present invention is a polypeptide that is at least about 70% to about 100% pure, that is, the polypeptide is present in a composition, wherein the polypeptide accounts for about 70% to about 100% of the total composition by weight. In some embodiments, the purified polypeptide of the present invention 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 polynucleotide of the present invention is a polynucleotide that is at least about 70% to about 100% pure, that is, the polynucleotide is present in a composition, wherein the polynucleotide accounts for about 70% to about 100% of the total composition by weight. 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.

[0120] As used herein, the term "receptor" means a biological molecule or group of molecules that can bind to a ligand. Receptors can be used to transmit information in cells, cell formations or organisms. The receptor comprises at least one receptor unit and preferably two receptor units, wherein each receptor unit can be composed of a protein molecule, in particular a glycoprotein molecule. The receptor has a structure that is complementary to the ligand structure and can complex the ligand into a binding partner. The information is transmitted in particular through conformational changes of the receptor after complexation with the ligand on the cell surface. According to embodiments of the present disclosure, receptors are understood to particularly mean MHC class I and class II proteins that can form receptor / ligand complexes with ligands, in particular peptides or peptide fragments of suitable length.

[0121] As used herein, the term "sample" generally refers to a limited amount of something that is intended to be similar to and represent a greater amount of that something. In the present disclosure, a sample is a collection, swab, brush, scrape, biopsy, removed tissue or surgical resection to test the absence, presence or grading of hyperproliferative tissue, which is in some cases cancerous tissue or one or more cells. In some embodiments, the sample is taken from a patient or subject who is believed to have cancer, hyperplasia, precancerous stage or comprises one or more tumor cells. In some embodiments, a sample that is believed to contain one or more hyperproliferative cells is compared with a "control sample" known not to contain one or more hyperproliferative cells. The present disclosure contemplates the use of any one or more samples disclosed herein to identify, detect, sequence and / or quantify the amount of new antigens (high or minimal immunogenicity) within a particular sample. In some embodiments, the method involves exposing a swab, brush or other sample from the environment to a set of reagents sufficient to separate and / or sequence the DNA and RNA of one or more cells in the sample.

[0122] As used herein, the term "small molecule" refers to an organic compound of low molecular weight (<900 Daltons) that is approximately 1 nm in size and that can help regulate biological processes. Most drugs are small molecules.

[0123] As used herein, "specific binding" or "specific for..." refers to the association or combination of a binding protein (e.g., a receptor, antibody, CAR or TCR) or a binding component (or its fusion protein) with a target molecule with an affinity or Ka (i.e., the equilibrium association constant for a specific binding interaction, in units of 1 / M) equal to or greater than 105M-1 (which is equal to the ratio of the association rate [kon] to the dissociation rate [koff] of this association reaction), while not significantly associated or combined with any other molecule or component in the sample. A binding protein or binding domain (or its fusion protein) can be classified as a "high affinity" binding protein or binding domain (or its fusion protein) or as a "low affinity" binding protein or binding domain (or its fusion protein). "High affinity" binding proteins or binding domains refer to those having a Ka of at least about 107M-1, at least about 108M-1, at least about 109M-1, at least about 1010M-1, at least about 1011M-1, at least about 1012M-1, or at least about 1013M-1. "Low affinity" binding proteins or binding domains refer to those having a Ka of up to about 107M-1, up to about 106M-1, or up to about 105M-1. In some embodiments, affinity can be defined as the equilibrium dissociation constant (Kd) for a specific binding interaction, in units of M (e.g., 10-5M to 10-13M). A variety of assays are known for identifying binding domains that specifically bind to a particular target, and for determining the affinity of a binding domain or fusion protein, such as Western blotting, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance. Analysis (see, 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 incorporated herein by reference). In some embodiments, one or more TCRs bind to one or more corresponding neoantigens with high affinity. Binding with high affinity means that, in some embodiments, the TCR binds to the neoantigen with a Ka or Kd of about 500 nM or less.

[0124] As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" means conditions under which a nucleic acid molecule will hybridize to another nucleic acid molecule but not to other sequences. Stringent conditions depend on the sequence and will be different in different situations. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequence is usually present in excess, 50% of the probes are occupied at equilibrium at Tm. Typically, stringent conditions are those in which the salt concentration is less than about 1.0 M sodium ions, typically about 0.01 M to 1.0 M sodium ions (or other salts) at pH 7.0 to 8.3, and for short probes, primers or oligonucleotides (e.g., 10 to 50 nucleotides), the temperature is at least about 30°C, and for longer probes, primers or oligonucleotides, the temperature is at least about 60°C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[0125] As used herein, the terms "subject," "individual," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments, the subject is a human. In some embodiments, the subject is a dog, horse, pig, sheep, cat, cow, donkey, llama, emu, or goat.

[0126] "Substantially identical" means that a nucleic acid molecule or polypeptide exhibits at least about 50% sequence identity with a reference nucleic acid sequence (e.g., any of the nucleic acid sequences described herein) or an amino acid sequence (e.g., any of the amino acid sequences described herein). In some embodiments, such a sequence has at least about 60% sequence identity with a reference sequence for comparison. In some embodiments, such a sequence has at least about 70% sequence identity with a reference sequence for comparison. In some embodiments, such a sequence has at least about 80% sequence identity with a reference sequence for comparison. In some embodiments, such a sequence has at least about 85% sequence identity with a reference sequence for comparison. In some embodiments, such a sequence has at least about 90% sequence identity with a reference sequence for comparison. In some embodiments, such a sequence has at least about 95% sequence identity with a reference sequence for comparison. In some embodiments, such a sequence has at least about 99% sequence identity with a reference sequence for comparison.

[0127] "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 separated) from, for example, peripheral blood mononuclear cells (PBMCs) and are referred to herein as "a large number of" T cells. After separation of T cells, before or after amplification, cytotoxic T cells (CD8+) and helper T cells (CD4+) can be sorted into naive T cell subsets, memory T cell subsets, and effector T cell subsets. T cells can be naive T cells (not exposed to antigens; compared with central memory T cells (TCM), the expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA increases, while the expression of CD45RO decreases), memory T cells (TM) (experienced with antigens and long lifespan) and effector cells (experienced with antigens, with cytotoxicity). 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 to naive T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA and increased expression of CD127 compared to naive T cells or TCM). Effector T cells (TE) refer to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28 compared to TCM and are positive for granzymes and perforins. Helper T cells (Th) are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate and suppress adaptive immune responses, and the effects they induce depend 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 by affinity binding to antibodies, flow cytometry, or immunomagnetic selection.

[0128] As used herein, the term "T cell epitope" means a peptide sequence that can be bound by a class I or class II MHC molecule in the form of a peptide presenting MHC molecule or MHC complex and then recognized and bound by a cytotoxic T lymphocyte or T helper cell, respectively, in this form.

[0129] "T cell population" may include thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes and activated T lymphocytes. T cell populations may include αβT cells, including CD4+T cells, CD8+T cells, γδT cells, natural killer T cells or any other subset of T cells.

[0130] As used herein, the term "T cell receptor" (TCR) refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail region) that is capable of specifically binding to an antigenic peptide bound to an MHC receptor; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd edition, Current Biology Publications, p. 4: 33, 1997). TCRs can be found on the surface of cells or in soluble form, and typically comprise heterodimers having α and β chains (also referred to as TCRα and TCRβ, respectively) or γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Like immunoglobulins, the extracellular portion of the TCR chain (e.g., α-chain, β-chain) contains two immunoglobulin domains at the N-terminus, a variable domain (e.g., α-chain variable domain or Va, β-chain variable domain or Vβ; generally amino acids 1 to 116, based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest," USDept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th edition), and a constant domain adjacent to the cell membrane (e.g., α-chain constant domain or Ca, generally amino acids 117 to 259, based on Kabat, β-chain constant domain or Cp, generally amino acids 117 to 295, based on Kabat). Likewise, like immunoglobulins, the variable domains contain 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., EMBO J. 7:3745, 1988; see also 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 associate with the CD3 complex. The source of TCRs as used in the present disclosure may be from various animal species, such as humans, mice, rats, rabbits, or other mammals. In some embodiments, the source of TCRs as used in the present disclosure is from a subject from which a new antigen for in vivo T cell activation and expansion is identified.

[0131] As used herein, the term "therapeutic agent" means an agent used to treat, combat, ameliorate, prevent or improve an unwanted condition or disease in a patient.

[0132] As used herein, the term "therapeutic effect" means a certain degree of relief of one or more symptoms of a disease (e.g., neoplasia or tumor) or its associated pathology. As used herein, "therapeutically effective amount" means effectively extending the survival of patients with this disease, alleviating one or more signs or symptoms of the disease, preventing or delaying the disease, etc. after a single dose or multiple doses are administered to cells or subjects, and exceeds the expected effect when this treatment is not received. "Therapeutically effective amount" is intended to quantify the amount required for the therapeutic effect. A doctor or veterinarian with ordinary skills in the art can easily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the desired pharmaceutical composition. For example, a doctor or veterinarian can start the dosage of the compound of the present disclosure used in the pharmaceutical composition at a level lower than the level required for achieving the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In some embodiments, the therapeutically effective amount is an amount that effectively reduces the total mass of the solid tumor by about 2% compared to its mass or estimated mass before treatment, and reduces the total mass by about 4%, about 6%, about 8%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% compared to the total mass of the solid tumor before treatment. In some embodiments, for any therapeutic agent described herein, the therapeutically effective amount is initially determined by preliminary in vitro studies and / or animal models. In some embodiments, the therapeutically effective dose is determined by human data. In some embodiments, the applied dose is adjusted based on the relative bioavailability and efficacy of the administered agent. It is entirely within the capabilities of ordinary technicians to adjust the dose to achieve maximum efficacy based on the above methods and other well-known methods. 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.

[0133] As used herein, the terms "treat / treated / treating / treatment" and the like mean to alleviate or improve a condition and / or symptoms associated therewith (e.g., cancer or tumor). "Treatment" may refer to administering a neoantigen vaccine described herein to a subject after an onset or suspected onset of cancer. "Treatment" may also refer to administering an engineered TCR transgenic T cell described herein to a subject after an onset or suspected onset of cancer. "Treatment" includes the concept of "alleviation", which refers to alleviating the frequency or severity of the occurrence or recurrence of any symptoms or other adverse effects associated with cancer and / or side effects associated with cancer therapy. The term "treatment" also encompasses the concept of "management", which refers to reducing the severity of a patient's particular disease or condition or delaying its recurrence, such as extending the remission period of a patient suffering from the disease. It should be understood that, although not excluded, treating a condition or illness does not require complete elimination of the condition, condition or symptom associated therewith. Therefore, as used herein, the term "treating cancer" is not intended to be an absolute term. In some aspects, the compositions and methods of the present disclosure seek to reduce the size of a tumor or the number of cancer cells, causing cancer to enter a state of remission, or preventing the growth of the size or number of cells of cancer cells. In some cases, treatment with the compositions and methods of the present disclosure results in an improved prognosis.

[0134] As used herein, "tumor antigen" or "tumor-associated antigen" or "TAA" refers to a mutant protein found in oncogenic or tumor cells that triggers a humoral immune response, a cellular immune response, or both, which may be present only in tumor cells or may be present in tumor cells and other normal cells. In some embodiments, TAA is the product of a mutated oncogene (e.g., p53, raf, ras, myc, EGFR). In some embodiments, TAA is a mutated tumor suppressor gene (e.g., pRb, TP53, PTEN, CD95). In some embodiments, TAA is a mutated gene that overexpresses or aberrantly expresses a cellular protein, etc.

[0135] As used herein, the term "vaccine" means a composition used to generate immunity for the prevention and / or treatment of a disease (e.g., cancer). Thus, a vaccine is a drug that includes an antigen and is intended for use in humans or animals to generate specific defense and protective substances through vaccination. A "vaccine composition" or "neoantigen vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent.

[0136] As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to a type of immune cell that has moved from the blood into a tumor. Tumor infiltrating lymphocytes are able to recognize and kill cancer cells. In some embodiments of cancer therapy, tumor infiltrating lymphocytes are removed from a patient's tumor, grown in large numbers outside the body, and then administered back to the patient to help the immune system treat the subject and kill one or more cancer cells.

[0137] "Vector" is a nucleic acid that can be used to introduce another nucleic acid connected thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule to which an additional nucleic acid segment can be joined. Another type of vector is a viral vector (e.g., replication-defective retrovirus, adenovirus, and adeno-associated virus), in which an additional DNA segment can be introduced into the viral genome. Some vectors can be autonomously replicated in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors comprising bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. "Expression vector" is a type of vector that can guide the expression of a selected polynucleotide. The present disclosure relates to any one or more vectors, which comprise a nucleic acid sequence encoding any one or more amino acid sequences disclosed herein.

[0138] The vector may comprise a heterologous nucleic acid encoding a neoantigen, and may further comprise a start codon (which may be located upstream of the neoantigen coding sequence) and a stop codon (which may be located downstream of the neoantigen coding sequence). The start and stop codons may be consistent with the neoantigen coding sequence. The vector may also comprise a promoter operably linked to the neoantigen coding sequence. The promoter operably linked to the neoantigen coding sequence may be a promoter from the following items: Simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter (such as bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter), Moloney virus promoter, avian leukosis virus (ALV) promoter, cytomegalovirus (CMV) promoter (such as CMV immediate early promoter), Epstein Barr virus (Epstein Barr virus) (EBV) promoter or Rous sarcoma virus (Rous sarcoma virus) (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a natural or synthetic muscle or skin-specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. US20040175727, the contents of which are incorporated herein in their entirety.

[0139] The vector may also include a polyadenylation signal, which may be located downstream of the HA coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, Calif.).

[0140] The vector may also include an enhancer upstream of the new antigen encoding. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer, such as one from CMV, HA, RSV or EBV. Polynucleotide functional enhancement is described in U.S. Patent Nos. 5,593,972, 5,962,428 and WO94 / 016737, the contents of each of which are fully incorporated by reference. The vector may also include a mammalian origin of replication so that the vector is maintained outside the chromosome and multiple copies of the vector are produced in the cell.

[0141] The present disclosure relates to drugs

[0142] In some embodiments, the nucleic acid molecule or pharmaceutical composition comprises a DNA backbone comprising all lowercase base pairs from any of the plasmids identified above, wherein a first lowercase backbone sequence and a second lowercase backbone sequence are flanked by expressible nucleic acid sequences encoding multiple tumor-specific antigen sequences, such as Formula I, Formula I(a), Formula II, or Formula III(a).

[0143] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]), wherein the length of each linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]), wherein the length of each linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids, about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25. In some embodiments, each adapter is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length. In some embodiments, each adapter is about 21 natural or non-natural nucleic acids in length. In certain embodiments, two adapters can be used together in a fusion. Thus, in some embodiments, the length of the first linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids, with a length of about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids. In some embodiments, the length of the second adapter can be independently selected from about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids.In some embodiments, the first adapter can be independently selected from adapters having a length of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids. In some embodiments, the second adapter can be independently selected from adapters having a length of about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids.

[0144] In some embodiments, at least one linker comprises about 15 to about 300 nucleotides and encodes n amino acid cleavage sites. In some embodiments, each linker between each AED is an identical nucleotide sequence comprising about 15 to about 120 nucleotides and encodes an amino acid cleavage site.

[0145] In some embodiments, the formula (eg, [(AEDn)-(Linker)]n-[AEDn+1]) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Linkers.

[0146] In some embodiments, the vector may 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 the sequence pVAX1. The backbone of the vector may be pAV0242. The vector may be a replication-deficient adenovirus type 5 (Ad5) vector.

[0147] 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 neoantigen coding sequence may comprise codons that may allow the coding sequence to be more efficiently transcribed in the host cell.

[0148] 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 a yeast strain of Saccharomyces cerevisiae. The vector can also be MAXBAC TM The vector may be a vector of the complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The vector may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells. The vector may be an expression vector or system to produce proteins by conventional techniques and readily available starting materials, including Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor (1989), which is fully incorporated by reference.

[0149] Expression vectors for different cell types are well known in the art and can be selected without excessive experimentation. Typically, DNA is inserted into expression vectors such as plasmids with appropriate orientation and correct reading frame for expression. If necessary, DNA can be connected to appropriate transcription and translation regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), but such controls are generally available in expression vectors. The vector is then introduced into the host bacteria to be cloned using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The vectors disclosed herein are those polynucleotide sequences, such as plasmids, which contain nucleic acid sequences encoding one or more new antigens or TCR sequences highly associated with one or more new antigens.

[0150] In some embodiments in which the nucleic acid molecule comprises an expressible nucleic acid sequence, the expressible nucleic acid sequence of Formula I is located within 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 located at the multiple cloning site of LLC. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of TC1. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of ID8. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX0001. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4501. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4503. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4504. In some embodiments, the nucleic acid sequence of Formula I is located within the multiple cloning site of pGX4505. In some embodiments, the nucleic acid sequence of Formula I is located at the multiple cloning site of pGX4506. In some embodiments, the plasmid is pGX4505 or a sequence that is 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to each of the nucleotide sequences identified above. In some embodiments, the plasmid is pGX0001 or a sequence that comprises about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 356. In some embodiments, the plasmid is pGX6001 or a sequence that comprises about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 360.

[0151] Table W - Plasmid backbone sequences

[0152]

[0153]

[0154]

[0155]

[0156] The present disclosure also relates to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is a DNA backbone domain of a nucleic acid molecule and the second nucleic acid sequence is an expressible nucleic acid sequence; wherein the expressible nucleic acid sequence comprises a plurality of antigen expression domains oriented 5' to 3'. In some embodiments, the expressible nucleic acid sequence comprises a nucleic acid sequence encoding a joint at the 5' end of the first antigen expression domain. In some embodiments, the expressible nucleic acid sequence encodes a joint between each of the antigen expression domains. In some embodiments, the expressible nucleic acid sequence encodes a leader sequence, a plurality of antigen expression domains, and each antigen expression domain is spaced apart by a joint sequence. In some embodiments, there are at least 20 antigen expression domains. In some embodiments, there are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or more antigen expression domains. In some embodiments, the nucleic acid molecule comprises formula I, Ia, II, IIa or IIIa. In some embodiments, the nucleic acid molecule comprises one or more regulatory sequences operably connected to the expressible nucleic acid sequence. In some embodiments, the first DNA backbone domain comprises a nucleic acid sequence comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:356, or a functional fragment comprising at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: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% or 100% sequence identity to SEQ ID NO:360.

[0157] The present disclosure also relates to nucleic acid molecules comprising the first, second and third nucleic acid sequences, wherein the first nucleic acid sequence is the first DNA backbone domain of the nucleic acid molecule, the second nucleic acid sequence is the second DNA backbone domain of the nucleic acid molecule and the third nucleic acid sequence is an expressible nucleic acid sequence; wherein the expressible nucleic acid sequence comprises a plurality of antigen expression domains oriented 5' to 3'. In some embodiments, the expressible nucleic acid sequence comprises a nucleic acid sequence encoding a joint at the 5' end of the first antigen expression domain. In some embodiments, the expressible nucleic acid sequence encodes a joint between each of the antigen expression domains. In some embodiments, the expressible nucleic acid sequence encodes a leader sequence, a plurality of antigen expression domains, and each antigen expression domain is spaced apart by a joint sequence. In some embodiments, there are at least 20 antigen expression domains. In some embodiments, there are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or more antigen expression domains. In some embodiments, the nucleic acid molecule comprises Formula I, Ia, II, IIa or IIIa. In some embodiments, the nucleic acid molecule comprises one or more regulatory sequences operably connected to the expressible nucleic acid sequence.

[0158] 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% or 100% 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% or 100% sequence identity to SEQ ID NO:358.

[0159] In some embodiments, the composition or pharmaceutical composition administered to a subject to re-prime an antigen-specific immune response to one or more neo-antigens comprises a nucleic acid molecule 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 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 NO: 357; (ii) a second 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; NO:358 comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or a functional fragment thereof having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity; and (iii) a third nucleic acid located between each of the first and second backbone sequences, wherein the third nucleic acid sequence is an expressible nucleic acid sequence encoding a plurality of new antigens. In some embodiments, the third nucleic acid sequence comprises an expressible nucleic acid sequence encoding at least about 20, 25, 30, 35, 40, 45, 50, 55 or 60 or more new antigens. In some embodiments, the expressible nucleic acid sequence comprises a nucleic acid sequence encoding an Ig leader sequence and a plurality of antigen expression domains in a 5' to 3' orientation, wherein the antigen expression domains are separated by linkers. In some embodiments, the antigen expression domains consist of a single antigen sequence encoding a tumor-specific neoantigen of the subject. In some embodiments, each antigen expression domain is about 25 to about 40 nucleotides in length. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising Formula I ([(AEDn)-(linker)]n-[AEDn+1]), wherein the length of each linker can be independently selected from about 0 to about 25 natural or non-natural nucleic acids and wherein n is a positive integer from about 10 to about 40.

[0160] In some embodiments, the nucleic acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more linker domains and the nucleic acid sequence comprises Formula IV:

[0161] [(AEDn)–(Linker)]n–(AEDn+1)-(Linker)n+1]n–AED(3' end)

[0162] Wherein each AED can be independently selected from any one or more tumor-associated antigens from a subject, and wherein n is any positive integer from about 1 to about 50 and wherein each "linker" is a nucleic acid sequence encoding one or more amino acid cleavage sites. Each linker can be the same or can be independently selected to contain one or more linkers disclosed herein. In some embodiments, the linker is a furin cleavage site of about 9 to about 105 nucleotides in length and encodes an amino acid sequence of an amino acid cleavage site. In some embodiments, the nucleic acid sequence is a component of a nucleic acid molecule. In some compositions contemplated herein, the composition comprises 1, 2, 3, 4, 5 or more nucleic acid molecules, each of which expresses any pattern or formula of an AED disclosed herein.

[0163] 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 comprises Formula III(a):

[0164] Leader sequence - [(AEDn) – (Linker)]n – (AEDn+1) – Linkern+1-(AEDn+2)]n

[0165] Wherein each AED can be independently selected from any one or more tumor-associated antigens from a subject, and wherein n is any positive integer from about 20 to about 50 and wherein each "linker" is a nucleic acid sequence encoding one or more amino acid cleavage sites. Each linker can be the same or can be independently selected to include one or more linkers disclosed herein; wherein each "-" represents a bond between each subunit. In some embodiments, the linker is a furin cleavage site of about 9 to about 105 nucleotides in length and is encoded as an amino acid sequence of an amino acid cleavage site. In some embodiments, the nucleic acid sequence is a component of a nucleic acid molecule. In some embodiments, Formula III (a) comprises a third linker bonded to the 3' end of the third AED sequence. In some embodiments, the last AED sequence is not bonded to the linker on its 3' end in a 5' to 3' orientation.

[0166] The present disclosure also relates to a nucleic acid sequence comprising a coding region and a non-coding region, wherein the coding region consists of Formula I(b):

[0167] [(AED1)–(connector)–(AED2)–(connector)]n–[(AED3)]n+1,

[0168] wherein n is a positive integer from about 1 to about 30, wherein each "linker" encodes one or more amino acid cleavage sequences, and wherein the non-coding region comprises at least one regulatory sequence operably linked to one or more AEDs; and wherein, in the 5' to 3' orientation, AED3 is the terminal antigen expression domain in the AED sequence. 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 independently selectable antigen expression domains as antigen sequences. In some embodiments, the regulatory sequence is each of the regulatory sequences depicted 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 the regulatory sequences depicted in the figures.

[0169] The present disclosure also relates to a nucleic acid sequence comprising a coding region and a non-coding region, wherein the coding region consists of Formula I(b):

[0170] Ig leader sequence-[(AED)–(linker)–(AED)–(linker)]n–[(AED)]n+1,

[0171] wherein n is a positive integer from about 1 to about 30, wherein each "linker" encodes one or more amino acid cleavage sequences, and wherein the non-coding region comprises at least one regulatory sequence operably linked to one or more AEDs; and wherein, in the 5' to 3' orientation, AED3 is the terminal antigen expression domain in the AED sequence. 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 independently selectable antigen expression domains as antigen sequences. In some embodiments, the regulatory sequence is each of the regulatory sequences depicted 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 the regulatory sequences depicted in the figures.

[0172] In some embodiments, the compositions of the present disclosure relate to any nucleic acid molecule comprising (i) at least 70%, 80% or 90% sequence identity to any plasmid backbone identified herein; and (ii) an expressible nucleic acid sequence comprising one or more antigen expression domains separated by a nucleic acid sequence encoding a protein cleavage site. In some embodiments, the expressible nucleic acid sequence encodes about 19 to about 60 different amino acid sequences that are antigens. In some embodiments, the present disclosure relates to a cell comprising a nucleic acid molecule comprising (i) at least 70%, 80% or 90% sequence identity to any plasmid backbone identified herein; and (ii) an expressible nucleic acid sequence comprising one or more antigen expression domains separated by a nucleic acid sequence encoding a protein cleavage site. 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 these amino acid sequences are cleaved after expression and exposure to intracellular protease activity.

[0173] Identification of neoantigens

[0174] The present disclosure relates to methods for treating cancers comprising one or more neoantigens in subjects in need thereof. The present disclosure further relates to methods for preventing metastasis of cancers comprising one or more neoantigens in subjects in need thereof. Since tumor neoantigens are often generated by somatic "passenger" mutations in the DNA of tumor cells, many tumor neoantigens are unique to the cancer of individual patients. Therefore, in order for the disclosed method to proceed, one or more cancer-specific neoantigens are necessary to determine the "mutation group" of the subject's tumor and identify candidate tumor neoantigens. Therefore, in some embodiments, the methods of the present disclosure include identifying one or more neoantigens that are unique to the subject. In some embodiments, compared to a wild-type nucleic acid molecule, the neoantigen comprises an antigenic peptide or epitope of a protein encoded by a nucleic acid molecule having a missense mutation, a non-interrupting mutation, a splice variant, a gene fusion, a frameshift mutation (e.g., an addition or deletion), or a combination thereof.

[0175] 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 each patient's tumor DNA (or RNA) and DNA (or RNA) from normal tissue to identify tumor-specific mutations and determine the patient's HLA allotype. In some embodiments, a population of tumor-specific neoantigens and their cognate natural antigens is bioinformatically analyzed using a validated algorithm to predict which tumor-specific mutations produce epitopes that can bind to the patient's HLA allotype, and in particular, which tumor-specific mutations produce epitopes that can bind to the patient's HLA allotype more effectively than the cognate natural antigen. Based on this analysis, in some embodiments, nucleotide sequences corresponding to the identification of these mutations are designed for each patient and used together as a cancer vaccine to immunize the subject.

[0176] In some embodiments, the methods of the present disclosure include the step of identifying one or more subject-specific neoantigen mutations in a subject, wherein the subject has been diagnosed with, suspected of having, or contains one or more hyperproliferative cells (e.g., such as a tumor), the step comprising (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 sequence to determine coding and non-coding regions; (c) identifying sequences for tumor-specific non-synonymous or non-silent mutations not present in non-tumor samples; (d) identifying single nucleotide variations and single nucleotide insertions and deletions; (e) generating subject-specific peptides encoded by sequences comprising tumor-specific non-synonymous or non-silent mutations not present in non-tumor samples; and (f) measuring the binding characteristics of the subject-specific peptides, wherein each subject-specific peptide is an expression product of a subject-specific DNA neoantigen not present in the non-tumor sample, thereby identifying one or more subject-specific DNA neoantigens of the subject.

[0177] Any cell type or tissue can be used to obtain nucleic acid samples for use in the sequencing methods described herein. In some embodiments, DNA or RNA samples are obtained from neoplasia, tumors, or body fluids (e.g., blood) or saliva obtained by known techniques (e.g., venipuncture). In other embodiments, nucleic acid testing can be performed on dried samples (e.g., hair or skin).

[0178] The subject-specific neoantigen mutation may be any mutation in any gene that encodes a mutated 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 may 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 that contains a mutated amino acid sequence encoding a cancer-specific mutation. However, the number of genes containing such cancer-specific mutations that can be identified in the disclosed methods is not limited and can include more than one gene (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). Likewise, in some embodiments, the disclosed methods include identifying at least one cancer-specific mutation of an amino acid sequence encoding a mutation. However, the number of such cancer-specific mutations that can be identified in the disclosed methods is not limited and can include more than one cancer-specific mutation (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 where more than one cancer-specific mutation is identified, the cancer-specific mutation is located in the same gene. In other embodiments where more than one cancer-specific mutation is identified, the cancer-specific mutation is located in different genes.

[0179] In some embodiments, one or more cancer-specific mutations in the nucleic acid of the cancer cell are identified, including sequencing the complete or substantially complete exome, whole genome or whole transcriptome of the cancer cell. Sequencing can be performed in any suitable manner known in the art. Examples of sequencing techniques that can be used in the disclosed method 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 a high-throughput DNA sequencing technology based on non-Sanger. Using NGS, millions or billions of DNA chains can be sequenced in parallel, thereby greatly improving throughput, and minimizing the need for the fragment cloning method often used in the Sanger sequencing of the genome. In NGS, by dividing the entire genome into small fragments, nucleic acid templates can be randomly read in parallel along the entire genome. NGS can advantageously provide nucleic acid sequence information of the whole genome, exome or transcriptome in a very short period of time (e.g., within about 1 to about 2 weeks, within about 1 to about 7 days, or within less than about 24 hours). A variety of NGS platforms that are commercially available or described in the literature can be used in the context of the disclosed methods, for example, pharmaceutically acceptable salts are described in detail in Zhang et al., J. Genet. Genomics, 38(3):95-109 (2011) and Voelkerding et al., Clinical Chemistry, 55:641-658 (2009).

[0180] Non-limiting examples of NGS techniques and platforms include sequencing-by-synthesis (also known as "pyrosequencing") (as performed, for example, using a GS-FLX 454 Genome Sequencer, 454 Life Sciences (Branford, Conn.), an ILLUMINA SOLEXA Genome Analyzer (Illumina Inc., San Diego, Calif.), or an ILLUMINA HISEQ 2000 Genome Analyzer (Illumina), or as performed, for example, in Ronaghi et al., Science, 281(5375):363-365 (1998)), sequencing-by-ligation (as performed, for example, using the SOLID platform (Life Technologies Corporation, Carlsbad, Calif.) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)), single molecule sequencing (as performed, for example, using the PACBIO RS system (Pacific Biosciences (Menlo Park, Calif.) or the HELISCOPE platform (Helicos Biosciences (Cambridge, Mass.)), nanotechnology for single-molecule sequencing (such as, for example, the GRIDON platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nanopore sequencing (HANS) platform developed by Nabsys (Providence, RI), and a ligase-based DNA sequencing platform using DNA nanoball (DNB) technology called probe anchor ligation (cPAL)), electron microscopy-based single-molecule sequencing technology, and ion semiconductor sequencing.

[0181] Thus, in some embodiments, a population of cancer-specific neoantigens is identified by sequencing the patient's cancer / tumor and normal DNA to identify cancer-specific mutations and determine the patient's HLA allotype. In some embodiments, a population of cancer-specific neoantigens and their cognate natural antigens are bioinformatically analyzed using validated algorithms to predict which cancer-specific mutations produce epitopes that can bind to the patient's HLA allotype, and in particular, which cancer-specific mutations produce epitopes that can bind to the patient's HLA allotype more effectively than the cognate natural antigen. Based on this analysis, in some embodiments, identified nucleotide sequences corresponding to these mutations are designed for each patient and used together as a personalized cancer vaccine to immunize the patient.

[0182] In some embodiments, the methods of the present disclosure include the step of identifying one or more neoantigenic mutations in a subject, wherein the subject has been diagnosed with, suspected of having, or comprising one or more hyperproliferative cells (e.g., such as a tumor). In some embodiments, the methods of the present disclosure include the step of identifying one or more subject-specific neoantigenic mutations in a subject, wherein the subject has been diagnosed with, suspected of having, or comprising one or more hyperproliferative cells (e.g., such as a tumor). In some embodiments, the method of the present disclosure includes the step of identifying one or more subject-specific neoantigen mutations in a subject, wherein the subject has been diagnosed with, suspected of having, or contains one or more hyperproliferative cells (e.g., such as a tumor) characterized by the presence or abundance of multiple neoantigen mutations, the method comprising sequencing a nucleic acid sample from the subject's tumor and a nucleic acid sample from a non-tumor sample of the subject; analyzing the sequence to determine coding and non-coding regions; identifying sequences of tumor-specific non-synonymous or non-silent mutations not present in non-tumor samples; identifying single nucleotide variations and single nucleotide insertions and deletions; generating subject-specific peptides encoded by sequences containing tumor-specific non-synonymous or non-silent mutations not present in non-tumor samples; and measuring the binding characteristics of the subject-specific peptides, wherein each subject-specific peptide is an expression product of a subject-specific DNA neoantigen not present in the non-tumor sample, thereby identifying one or more subject-specific DNA neoantigens of the subject. Measuring the binding characteristics of the subject-specific peptides can be performed by measuring one or more of the binding of the subject-specific peptides to a T cell receptor; measuring the binding of the subject-specific peptides to the subject's HLA protein; or measuring the binding of the subject-specific peptides to a transporter associated with antigen processing (TAP).

[0183] Effectively selecting which specific mutations to use as immunogens requires identifying the patient's HLA type and being able to predict which mutated peptides can effectively bind to the patient's HLA alleles. Therefore, in some embodiments, measuring the binding of a subject-specific peptide to a T cell receptor includes measuring the binding of a subject-specific peptide to the subject's HLA protein.

[0184] In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 10nM to about 550nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 20nM to about 500nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 30nM to about 450nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 40nM to about 400nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 50nM to about 350nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 60nM to about 300nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 70nM to about 250nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 80nM to about 200nM. 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.

[0185] In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 10nM to about 100nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 20nM to about 150nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 30nM to about 175nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 40nM to about 200nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 50nM to about 225nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 60nM to about 250nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 70nM to about 275nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 80nM to about 300nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 90nM to about 325nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 100nM to about 350nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 110nM to about 375nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 120nM to about 400nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 130nM to about 425nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 140nM to about 450nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 150nM to about 475nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 175nM to about 500nM.

[0186] In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 550nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 500nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 450nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 400nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 350nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 300nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 250nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 200nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 150nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject at an IC50 of about 100nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 90 nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 80 nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 70 nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 60 nM. In some embodiments, the subject-specific peptide binds to the HLA protein of the subject with an IC50 of about 50 nM.

[0187] In some embodiments, the disclosed methods further include the step of ranking the subject-specific peptides based on the binding characteristics. In some embodiments, the disclosed methods further include the step of 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.

[0188] In some embodiments, the disclosed methods further include formulating the subject-specific neoantigen into an immunogenic composition for administration to the subject. In some embodiments, the subject-specific neoantigen is formulated into an immunogenic composition in the form of DNA. In some embodiments, the subject-specific neoantigen is formulated into an immunogenic composition in the form of RNA. In some embodiments, the subject-specific neoantigen is formulated into an immunogenic composition in the form of protein. In embodiments where the subject-specific neoantigen is formulated into an immunogenic composition in the form of DNA, the DNA neoantigen may be subcloned into one or more vectors, which in some embodiments are one or more plasmids. Methods for administering DNA vaccines, RNA vaccines, or protein vaccines are known in the art.

[0189] In some embodiments, the top 200 neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 150 neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 100 neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 50 neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 25 neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 10 neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the top 5 neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, about 5 to about 20 top-ranked neoantigenic mutations (by frequency) are included in an immunogenic composition for administration to a subject. In some embodiments, about 10 to about 20 top-ranked neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the first about 25 to about 100 top-ranked neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, about 50 to about 100 of the top ranked neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, about 100 to about 200 of the top ranked neoantigenic mutations are included in an immunogenic composition for administration to a subject. In some embodiments, the neoantigens are ranked from the lowest IC 50 Value to the highest IC 50 The values ​​are sorted to determine the rank.

[0190] Thus, in some embodiments, the methods of the present disclosure include the step of identifying or selecting one or more cancer-specific neoantigens 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 neoantigen to a T cell receptor; and (c) measuring binding of the subject-specific neoantigen to an HLA protein of the subject; and (d) if the subject-specific neoantigen is expressed at an IC of less than about 500 nM, 400 nM, 300 nM, 200 nM, or 100 nM, the subject-specific neoantigen is expressed at an IC of less than about 500 nM, 400 nM, 300 nM, 200 nM, or 100 nM. 50 binds to the HLA protein of the subject, then one or more new antigens are selected from the sample; optionally (d) according to the lowest IC 50 Value to the highest IC 50 The order of the values ​​is used to sort the neoantigens.

[0191] Administration of neoantigens in subjects

[0192] The present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid molecule disclosed herein; and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure relates to a composition 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 nucleic acid molecule encoding a therapeutically effective amount of IL-12 or a functional fragment thereof; and a pharmaceutically acceptable carrier. In some embodiments, the nucleic acid molecules disclosed herein comprise at least one expressible nucleic acid sequence encoding about 19 to about 50 new antigens or their functional fragments from a subject. In some embodiments, the new antigen or the nucleic acid sequence encoding it is selected by: administering a vaccine to a subject, isolating the subject's PBMC or antigen presenting cells, exposing the subject's PBMC or antigen presenting cells to cells comprising one or more nucleic acid molecules encoding one or more TCRs, and selecting one or more TCRs bound to the new antigen based on the quantified binding strength between the TCR and the new antigen.

[0193] In order to activate the immunity of the subject to cancer-specific neoantigens, the method of the present disclosure further includes administering cancer-specific neoantigens and subject-specific neoantigens to the subject. Administration results in a subject-specific immune response triggered in response to the neoantigen. Immune cells from the subject can be separated, modified and then re-administered back into the subject as a treatment. Cancer-specific neoantigens and subject-specific neoantigens can be administered to the subject in the form of peptides, DNA and / or RNA. In some embodiments, cancer-specific neoantigens and 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, cancer-specific neoantigens and 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, cancer-specific neoantigens and 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, cancer-specific neoantigens and 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.

[0194] In embodiments where cancer-specific neoantigens and subject-specific neoantigens are administered to a subject in the form of DNA, the DNA neoantigens may be subcloned into one or more vectors, exemplified by those disclosed herein, which in some embodiments are one or more plasmids. Thus, in some embodiments, the disclosed methods further include the step of producing a DNA vaccine or manufacturing a pharmaceutical composition comprising such a DNA vaccine, which step includes performing any one or more of the foregoing steps and further includes subcloning a nucleic acid sequence encoding one or more neoantigens into one or more nucleic acid molecules; and optionally, suspending the resulting nucleic acid molecules in one or more pharmaceutically acceptable carriers.

[0195] Any method for producing a DNA vaccine expressing a new antigen or short peptide can be used in the disclosed method. DNA vaccines are disclosed in U.S. Patents 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 entirety. As an example, a DNA vaccine suitable for the method of the present disclosure can be produced using the method disclosed in International Application Publication No. WO2019 / 227106, which is incorporated herein by reference. In short, one or more nucleotide sequences encoding one or more cancer / tumor-specific new antigens can be subcloned into a nucleic acid molecule. In some embodiments, only one nucleotide sequence encoding a single cancer / tumor-specific new antigen 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.

[0196] Therefore, in some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 1 to about 100 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 2 to about 95 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 3 to about 90 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 4 to about 85 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 5 to about 80 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 6 to about 75 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 7 to about 70 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 8 to about 65 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 9 to about 60 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 10 to about 55 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 20 to about 65 new antigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes equal to or greater than 20 new antigens. In some embodiments, all new antigens encoded by the nucleic acid molecule or the resulting DNA vaccine are the same new antigens. In some embodiments, at least about two or more neo-antigens encoded by a nucleic acid molecule or resulting DNA vaccine are different from each other.

[0197] In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes a single neoantigen. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 2 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 3 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 4 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 5 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 6 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 7 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 8 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 9 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 10 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 20 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes equal to or greater than about 20 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 30 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 40 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 50 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 55 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 60 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 65 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 70 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 75 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 80 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 85 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 90 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 95 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes about 100 different neoantigens. In some embodiments, the nucleic acid molecule or the resulting DNA vaccine encodes more than about 100 different neoantigens.

[0198] In embodiments where the nucleic acid molecule or resulting DNA vaccine encodes more than one neoantigen, each neoantigen encoded by the nucleic acid molecule can be separated from another by one or more joints. Therefore, 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 joints. In some embodiments, the length of each joint can be independently selected 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 amino acids. In some embodiments, the length of 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.

[0199] In embodiments where more than one linker is encoded by a nucleic acid molecule or resulting DNA vaccine of the present disclosure, the length of each linker may be the same or different. In some embodiments, the length of each linker is the same. In some embodiments, the length of each linker is different. For example, in some embodiments, the length of the first linker is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, 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, wherein the length of the first linker is different from the length of the second linker. The present disclosure can envision various configurations, wherein 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, wherein these linkers have similar or different lengths. In some embodiments, the linkers all have the same length and there are at least about 19 linkers.

[0200] In some embodiments, two linkers can be used together as a fusion peptide encoded by a single nucleotide sequence. Thus, in some embodiments, the length of the first linker can be independently selected 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 amino acids. In some embodiments, the length of the second linker can be independently selected 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 amino acids. In some embodiments, the length of 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 some embodiments, the length of 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.

[0201] The present disclosure relates to nucleic acid molecules comprising at least one expressible nucleic acid sequence comprising any of the formulae contemplated herein and encoding any of the antigens identified in Tables M, N and / or O, or functional fragments of those sequences having at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to those antigens 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 antigenic domains, wherein at least one of the antigenic domains is selected from the antigens identified in Tables M, N, and / or O, or functional fragments of those sequences having at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to those antigens identified in Tables M, N, and / or O. The present disclosure relates to a cell comprising a nucleic acid molecule comprising at least one expressible nucleic acid sequence comprising any of the formulae contemplated herein and encoding any of the antigens identified in Tables M, N and / or O, or functional fragments of those sequences having at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to those antigens identified in Tables M, N and / or O. In some embodiments, the compositions of the present disclosure comprise cells containing nucleic acid molecules comprising at least one expressible nucleic acid sequence comprising any of the formulas included herein and encoding any of the antigens identified in Tables M, N and / or O, or functional fragments of those sequences having at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to those antigens identified in Tables M, N and / or O, and at least one cell containing 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 functional fragments having at least about 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to those TCR sequences in Tables Q, R, X, Y or Z.

[0202] In certain embodiments, one or more linkers encoded by nucleic acid molecules or DNA vaccines of the present disclosure include amino acid cleavage sites. Any amino acid cleavage site can be used. A non-limiting example is the furin cleavage site, which is identified by a protease called furin that remains in the trans-Golgi network of eukaryotic cells. The effect of furin is to cut proteins at the step before the protein is delivered to their final cell destination. Furin recognizes the consensus amino acid sequence RXRR, RXRK or KXKR (wherein X is any amino acid, Moehring et al., 1993, which is incorporated by reference in its entirety), and cuts off these proteins when the proteins containing these sequences arrive at the trans-Golgi network. Furin is a Ca2+ dependent serine endoprotease that can cut protein precursors with high specificity after the multiple basic motifs shown in Table 1 below.

[0203] Table 1. Basic motifs of furin cleavage sites.

[0204]

[0205] Another non-limiting example of an amino acid cleavage site is a cleavage site recognized by a 2A peptide, which is a "self-cleaving" small peptide. The average length of a 2A peptide is 18-22 amino acids. The designation "2A" refers to a specific region of a picornavirus polyprotein. Of the 2A peptides identified so far, four are widely used in research: FMDV 2A (abbreviated herein as F2A); Equine rhinitis virus (ERAV) 2A (E2A); Porcine Teschovirus-1 2A (P2A) and Thoseaasigna virus 2A (T2A). The first three viruses belong to picornaviruses, and the latter to insect viruses. The DNA and corresponding amino acid sequences of various 2A peptides are shown in Table 2 below. The underlined sequence encodes the amino acid GSG, which is added to improve cleavage efficiency. P2A indicates porcine teschovirus-1 2A; T2A, Echinops punctatus beta-tetrasomal virus 2A; E2A, equine rhinitis virus (ERAV) 2A; F2A, FMDV 2A.

[0206] Table 2. DNA and corresponding amino acid sequences of various 2A peptides. The table discloses SEQ ID NOs 595-602 in the order of appearance.

[0207]

[0208] In some embodiments, one or more linkers encoded by nucleic acid molecules or DNA vaccines of the present disclosure comprise a furin cleavage site. In some embodiments, one or more linkers encoded by nucleic acid molecules or DNA vaccines of the present disclosure comprise a furin sensitive cleavage site comprising the sequence RX-[R / K]-R, wherein R represents arginine, X is any arginine, and K is lysine. "R / K" indicates that the amino acid is arginine or lysine.

[0209] In some embodiments, one or more 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 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 linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise a Tetrasomal Virus of the Clear Veined Plankton Betavirus 2A (T2A) cleavage site. In some embodiments, one or more linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise an Equine Rhinitis Virus (ERAV) 2A 9 (E2A) cleavage site. In some embodiments, one or more linkers encoded by the nucleic acid molecules or DNA vaccines of the present disclosure comprise a FMDV 2A (F2A) cleavage site.

[0210] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure further comprise a nucleotide sequence encoding a leader sequence. A "leader sequence", or sometimes referred to as a "signal peptide", is an amino acid sequence that generally directs protein localization. The leader sequence used herein preferably causes the protein to be secreted from the cell producing the protein. After secretion from the cell, the leader sequence is generally cut from the remainder of the protein commonly referred to as the mature protein. The leader sequence (if present) is connected to 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.

[0211] In some embodiments, the nucleic acid molecules or DNA vaccines of the present invention may also include one or more regulatory sequences. When present, such one or more regulatory sequences are operably connected to the nucleotide sequence encoding the neoantigen, joint 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). Other examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06.

[0212] 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. Plasmids can be used to transfect cells with nucleic acid molecules or DNA vaccines of the present disclosure, wherein the transformed cells express neoantigens to elicit an immune response. In some embodiments, the compositions of the present disclosure relate to nucleic acid molecules comprising nucleic acid sequences encoding a variety of neoantigens. In some embodiments, the plasmid further comprises a start codon and a stop codon, which may be located upstream or downstream of the neoantigen coding sequence. In some embodiments, the start codon and the stop codon are in frame with the neoantigen coding sequence.

[0213] In some embodiments, the plasmid further comprises a promoter operably linked to the coding sequence. In some embodiments, the promoter operably linked to the coding sequence is a promoter from the following items: Simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter (such as bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter), Moloney virus promoter, avian leukosis virus (ALV) promoter, cytomegalovirus (CMV) promoter (such as CMV immediate early promoter), Epstein-Barr virus (EBV) promoter or Rous sarcoma virus (RSV) promoter. In some embodiments, the promoter is a promoter 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 muscle or skin-specific promoter, natural or synthetic. Examples of such promoters are described in U.S. Patent Application Publication No. US20040175727, the contents of which are incorporated herein in their entirety. In some embodiments, the plasmid further comprises a polyadenylation signal, which may be located 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 is a polyadenylation signal from a pCEP4 plasmid (Invitrogen, San Diego, CA).

[0214] In certain embodiments, plasmid also comprises the enhancer of coding sequence upstream.In certain embodiments, enhancer is human actin, human myosin, human hemoglobin, human muscle creatine or virus enhancer, such as one from CMV, FMDV, RSV or EBV.Polynucleotide functional enhancer is described in, for example, U.S. Patent No. 5,593,972,5,962,428 and WO94 / 016737, and its respective content is fully incorporated by reference.In certain embodiments, plasmid also comprises mammalian replication origin, so that plasmid is maintained outside chromosome and produces multiple copies of plasmid in cell.In certain embodiments, plasmid is pVAX1, pCEP4 or pREP4 from ThermoFisher Scientific (San Diego, CA), it comprises Epstein-Barr virus replication origin and nuclear antigen EBNA-1 coding region, and this produces high copy episomal replication in the case of non-integration.

[0215] In some embodiments, the vector may be pVAX1 or a pVax1 variant with variations, 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 located at bases 664-683. The multiple cloning site is located at bases 696-811. The bovine GH polyadenylation signal is located at bases 829-1053. The kanamycin resistance gene is located at bases 1226-2020. The pUC start is located at bases 2320-2993. The nucleic acid sequence of pVAX1 (SEQ ID NO: 361) backbone sequence is as follows:

[0216]

[0217] 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 within a multiple cloning site.

[0218] 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.

[0219] 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. "Cellular immune response" means a cellular response including cells characterized by presenting antigens with class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes, which act as "helpers" or "killers". Helper T cells (also known as CD4+T cells) play a central role by regulating immune responses, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+T cells or CTLs) kill diseased cells such as cancer cells, producing more diseased cells. In some embodiments, the present disclosure relates to stimulating anti-cancer CTL responses against cancer cells that express one or more cancer-specific neoantigens and preferably present such cancer-specific neoantigens with class I MHC.

[0220] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a CD8+T cell response to any one or more neoantigens encoded by the nucleic acid molecules or DNA vaccines. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a CD8+T and / or CD4+T cell response to any one or more neoantigens encoded by the nucleic acid molecules or DNA vaccines. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a CD4+T cell response to any one or more neoantigens encoded by the nucleic acid molecules or DNA vaccines.

[0221] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 25% CD4+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 30% CD4+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 40% CD4+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 50% CD4+T cells. 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 subpopulation of T cells that respond to any one or more neoantigens encoded by the nucleic acid molecule or DNA vaccine, comprising greater than at least about 60% CD4+ T cells.

[0222] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 25% CD8+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 30% CD8+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 40% CD8+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 50% CD8+T cells. 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 subpopulation of T cells that respond to any one or more neoantigens encoded by the nucleic acid molecule or DNA vaccine, comprising greater than at least about 60% CD8+ T cells.

[0223] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 25% CD4+T cells and greater than at least about 25% CD8+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 30% CD4+T cells and greater than at least about 30% CD8+T cells. In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to subjects in an amount sufficient to elicit a subpopulation of T cells that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 40% CD4+T cells and greater than at least about 40% CD8+T cells. 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 that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 50% CD4+T cells and greater than at least about 50% CD8+T cells. 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 that respond to any one or more new antigens encoded by the nucleic acid molecules or DNA vaccines, and these T cells contain greater than at least about 60% CD4+T cells and greater than at least about 60% CD8+T cells.

[0224] In some embodiments, the nucleic acid molecules or DNA vaccines of the present disclosure are administered to a subject in an amount of about 1 ng to 100 mg; about 1 μg to about 10 mg; about 0.1 μg to about 10 mg; or about 1 mg to about 2 mg. In some embodiments, pharmaceutical compositions of nucleic acid molecules or DNA vaccines of the present disclosure comprising an amount of DNA of about 1 ng to about 1000 μg are used for administration.

[0225] The nucleic acid molecules or DNA vaccines of the present disclosure, or vectors or plasmids comprising the same, can be administered or delivered to a subject by several well-known techniques, including but not limited to DNA injection with or without in vivo electroporation (also known as DNA vaccination), liposome mediation, nanoparticle promotion, recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. In some embodiments, the new antigens are delivered via DNA injection and in conjunction with in vivo electroporation.

[0226] The nucleic acid molecules or DNA vaccines disclosed herein, or vectors or plasmids containing the same, can be administered by electroporation. This can be accomplished using electroporation devices that can be configured to deliver energy pulses effective to cause reversible pores to form in the cell membrane to the desired tissue of a mammal, and preferably the energy pulses are a constant current similar to a preset current input by a user. The electroporation device may include an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include and be incorporated into one or more different elements of the electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. Electroporation can be accomplished using an in vivo electroporation device, for example The EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.) was used to facilitate cell transfection of plasmids.

[0227] The electroporation component can be used as an element of the electroporation device, and the other elements are separate elements (or components) that communicate with the electroporation component. The electroporation component can be used as more than one element of the electroporation device, which can communicate with other elements of the electroporation device separated from the electroporation component. Since the elements can be used as a device or separate elements that communicate with each other, there is no limitation on the elements of the electroporation device that exist as part of an electromechanical or mechanical device. The electroporation component is capable of delivering energy pulses that produce a constant current in the desired tissue and include a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives the energy pulse from the electroporation component and delivers it to the desired tissue through the electrode. At least one electrode of the plurality of electrodes is neutral during the delivery of the energy pulse, and measures the impedance in the desired tissue and transmits the impedance to the electroporation component. The feedback mechanism can accept the measured impedance and can adjust the energy pulse delivered by the electroporation component to maintain a constant current.

[0228] The plurality of electrodes may deliver energy pulses in a dispersed mode. The plurality of electrodes may deliver energy pulses in a dispersed mode by electrode control under a programmed sequence, and the programmed sequence is input into the electroporation component by a user. The programmed sequence may comprise a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes with a neutral electrode for measuring impedance, and wherein a subsequent pulse of the plurality of pulses is delivered by another of the at least two active electrodes with a neutral electrode for measuring impedance.

[0229] In some embodiments, the feedback mechanism is implemented by 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 real-time feedback or instantaneous feedback (i.e., substantially instantaneous as determined by available techniques for determining response time) is preferred. The neutral electrode can measure the impedance in the desired tissue and transmit the impedance to the feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the energy pulse to maintain the constant current at a value similar to the preset current. The feedback mechanism can maintain the constant current continuously and instantly during the delivery of the energy pulse.

[0230] Examples of electroporation devices and electroporation methods that can facilitate the delivery of the DNA vaccines of the present disclosure include those described in U.S. Patent No. 7,245,963 to Draghia-Akli et al., U.S. Patent Publication 2005 / 0052630 filed by Smith et al., the contents of which are hereby incorporated by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate the delivery of DNA vaccines include those provided in co-pending and co-owned U.S. Patent Application Serial No. 11 / 874072 filed on October 17, 2007, which claims the benefit of U.S. Provisional Application Serial No. 60 / 852,149 filed on October 17, 2006 and U.S. Provisional Application Serial No. 60 / 978,982 filed on October 10, 2007 under 35 USC 119(e), all of which are hereby incorporated by reference in their entirety.

[0231] U.S. Patent No. 7,245,963 to Draghia-Akli et al. describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues in the body or plants. The modular electrode system may include a plurality of needle electrodes; a hypodermic needle; an electrical connector that provides conductive connections from a programmable constant current pulse controller to the plurality of needle electrodes and a power source. An operator may hold the plurality of needle electrodes mounted on a support structure and firmly insert them into a selected tissue in the body or plant. The biomolecules are then delivered through the hypodermic needle that enters the selected tissue. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of biomolecules into cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are hereby incorporated by reference in their entirety.

[0232] US Patent Publication 2005 / 0052630 filed by Smith et al. describes an electroporation apparatus that can be used to effectively promote the introduction of biomolecules into cells of selected tissues in the body or plants. The electroporation apparatus includes an electrokinetic device ("EKD" device) whose operation is specified by software or hardware. The EKD device generates a series of programmable constant current pulse patterns between electrodes of the array based on user control and input of pulse parameters, and allows storage and acquisition of current waveform data. The electroporation apparatus also includes a replaceable electrode tray with a needle electrode array, a central injection channel for an injection needle, and a removable guide tray. The entire contents of US Patent Publication 2005 / 0052630 are hereby incorporated by reference in their entirety. The electrode array and method described in US Patent No. 7,245,963 and US Patent Publication 2005 / 0052630 can be used for deep penetration into not only tissues such as muscle, but also other tissues or organs. Due to the configuration of the electrode array, the injection needle (to deliver the selected biomolecule) is also fully inserted into the target organ and the injection is performed perpendicular to the target tissue in the area pre-demarcated by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0233] In addition, in some embodiments incorporating electroporation devices and their use, there are electroporation devices described in the following patents: U.S. Pat. No. 5,273,525 issued on December 28, 1993, U.S. Pat. No. 6,110,161 issued on August 29, 2000, U.S. Pat. No. 6,261,281 issued on July 17, 2001, and U.S. Pat. No. 6,958,060 issued on October 25, 2005, and U.S. Pat. No. 6,939,862 issued on September 6, 2005. In addition, patents covering subject matter in U.S. Pat. No. 6,697,669 issued on February 24, 2004 (which relates to delivery of DNA using any of a variety of devices) and U.S. Pat. No. 7,328,064 issued on February 5, 2008 (which relates to methods of injecting DNA) are contemplated herein. The above patents are incorporated by reference in their entirety.

[0234] In some embodiments, cancer-specific neoantigens and subject-specific neoantigens are administered to a subject in the form of RNA. In such embodiments, RNA encoding neoantigens can be delivered as a transgene in an RNA vaccine (such as Moderna's mRNA platform). RNA vaccines are non-infectious, non-integrative, and naturally degradable. RNA vaccines have been shown to stimulate B cells to respond strongly to the encoded transgene. Naked RNA can be formulated with lipid nanoparticles (LNPs) to encapsulate RNA and protect the transgene from degradation.

[0235] Compositions comprising RNA nucleic acid sequences 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 having at least one modification, at least one 5' end cap, and is formulated within a lipid nanoparticle. In some embodiments, 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 accomplished simultaneously during an in vitro transcription reaction using the following chemical RNA cap analogs to produce a 5'-guanosine cap structure: 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.) according to the manufacturer's protocol. In other embodiments, 5'-capping of modified RNAs is accomplished post-transcriptionally using a vaccinia virus capping enzyme to produce a "cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1 structures can be produced using a vaccinia virus capping enzyme and a 2'-O methyltransferase to produce 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'- third last nucleotide using a 2'-O methyltransferase. The cap 3 structure can be generated from the cap 2 structure, followed by 2'-O-methylation of the 5'- third last tetranucleotide using a 2'-O methyltransferase. The enzyme is preferably derived from a recombinant source. When transfected into mammalian cells, the modified mRNA has a stability of about 12 to about 18 hours or more than about 18 hours, such as 24, 36, 48, 60, 72 hours or more than about 72 hours.

[0236] In some embodiments, the lipid nanoparticle comprises 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 and 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-ene-1-yl) 9-((4-(dimethylamino)butanoyl)oxy) heptadecanedioate (L319), (12Z, 15Z)-N, N-dimethyl-2-nonyl heneicosadeca-12, 15-diene-1-amine (L608) and N, N-dimethyl-1-[(1S, 2R)-2-octylcyclopropyl] heptadecanedioate (L530).

[0237] In some embodiments, the RNA encoding the new antigen of the present disclosure is formulated in lipid nanoparticles. In some embodiments, the RNA encoding the new antigen of the present disclosure is formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle. The formation of lipid nanoparticles can be accomplished by methods known in the art and / or as described in U.S. Publication No. 20120178702, which is incorporated herein by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine and / or polyarginine and cationic peptides described in International Publication No. WO2012013326 or U.S. Publication No. US20130142818; each of which is incorporated herein by reference in its entirety. In some embodiments, the RNA encoding the new antigen of the present disclosure is formulated in a lipid nanoparticle, which includes a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0238] Lipid nanoparticle preparations may be subject to, but are 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 biophysical parameters (such as size). In one example of Semple et al. (Nature Biotech. 2010 28: 172-176; it is incorporated herein by reference in its entirety), lipid nanoparticle preparations are composed of 57.1% cationic lipids, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, changing the composition of cationic lipids can more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19: 2186-2200; it is incorporated herein by reference in its entirety).

[0239] In some embodiments, the lipid nanoparticle formulation may comprise 35% to 45% cationic lipids, 40% to 50% cationic lipids, 50% to 60% cationic lipids, and / or 55% to 65% cationic lipids. In some embodiments, the ratio of lipid to RNA (e.g., mRNA) in the lipid nanoparticle is 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1, and / or at least 30:1.

[0240] 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 change the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, in some embodiments, the lipid nanoparticle formulation contains 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% of the lipid molar ratio of PEG-c-DOMG (R-3-[(co-methoxy-poly(ethylene glycol) 2000) carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) compared to cationic lipids, DSPC and cholesterol. In some embodiments, PEG-c-DOMG is replaced by PEG lipids, 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.

[0241] In some embodiments, the RNA encoding the new antigen 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-nonyl heneicosane-12,15-diene-1-amine (L608), N, N-dimethyl-1-[(1S, 2R)-2-octylcyclopropyl] heptadecan-8-amine (L530), PEGylated lipids, and amino alcohol lipids.

[0242] In some embodiments, the lipid nanoparticle formulation comprises about 25% to about 75% by mole 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), such as about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50% or about 40%. In some embodiments, the lipid nanoparticle formulation comprises about 0.5% to about 15% by mole of a neutral lipid, such as about 3% to about 12%, about 5% to about 10% or about 15%, about 10% or about 7.5% by mole. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises sterols of about 5% to about 50% by mole (e.g., about 15% to about 45%, about 20% to about 40%, about 40%, about 38.5%, about 35%, or about 31% by mole). The non-limiting example of sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises PEG or PEG-modified lipids of about 0.5% to about 20% by mole (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% by mole). In some embodiments, PEG or PEG-modified lipids comprise PEG molecules having an average molecular weight of about 2,000Da. In some embodiments, PEG or PEG-modified lipids comprise PEG molecules having an average molecular weight of less than about 2,000, such as about 1,500Da, about 1,000Da, or about 500Da. 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 (discussed further in Reyes et al. J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety).

[0243] In some embodiments, the lipid nanoparticle formulation comprises about 25%-75% by mole 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% of a neutral lipid, about 5%-50% of a sterol, and about 0.5%-20% of a PEG or PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation comprises about 35%-65% by mole 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% of a neutral lipid, about 15%-45% of a sterol, and about 0.5%-10% of a PEG or PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation comprises about 45%-65% by mole 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% of a neutral lipid, about 25%-40% of a sterol, and about 0.5%-10% of a PEG or PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation comprises about 60% by mole 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 7.5% of a neutral lipid, about 31% of a sterol, and about 1.5% of a PEG or PEG-modified lipid.

[0244] In some embodiments, the RNA encoding the neoantigen 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 are chemically modified, and optionally wherein the RNA encoding the neoantigen is formulated in a lipid nanoparticle. In some embodiments, the RNA encoding the neoantigen is formulated in a liposome, such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., for siRNA delivery in ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); which is incorporated herein by reference in its entirety), and hyaluronan-coated liposomes (Quiet Therapeutics, Israel). In some embodiments, the RNA encoding the neoantigen may be formulated in a lyophilized gel phase liposome composition as described in U.S. Publication No. US2012060293, which is incorporated herein by reference in its entirety.

[0245] In some embodiments, the nanoparticle formulation comprises a phosphate conjugate. Phosphate conjugates can increase the in vivo circulation time and / or increase the targeted delivery of nanoparticles. In some embodiments, the conjugates used in the present disclosure are prepared by the methods described in International Publication No. WO2013033438 or U.S. Publication No. US20130196948, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, a phosphate conjugate may include a compound of any formula described in International Publication No. WO2013033438, which is incorporated herein by reference in its entirety. Specifically, the present disclosure relates to a pharmaceutical composition comprising nanoparticles, which nanoparticles comprise RNA encoding one or more cancer-specific neoantigens, wherein:

[0246] (i) the number of positive charges in the nanoparticle does not exceed the number of negative charges in the nanoparticle; and / or

[0247] (ii) the nanoparticles have a neutral or net negative charge; and / or

[0248] (iii) the charge ratio of positive to negative charges in the nanoparticles is 1.4:1 or less; and / or

[0249] (iv) The zeta potential of the nanoparticles is about 0 or less.

[0250] In some embodiments, the nanoparticles described herein are colloidally stable for at least about 2 hours, i.e., no aggregation, precipitation, or size and polydispersity index increase greater than about 30%, as measured by dynamic light scattering. In some embodiments, the charge ratio of positive charge to negative charge 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.

[0251] In some embodiments, the pharmaceutical composition of the present disclosure comprises a nanoparticle or liposome encapsulating a DNA, RNA or DNA / RNA hybrid comprising at least one expressible nucleic acid sequence. Liposomes are microscopic lipid vesicles, generally having a double layer of one or more lipids such as phospholipids that form vesicles, and are capable of encapsulating drugs. Different types of liposomes can be used in the context of the present disclosure, including but not limited to multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), spatially stable liposomes (SSL), multivesicular vesicles (MV) and multilamellar vesicles (LMV) and other double-layer forms known in the art. The size and lamellarity of the liposome will depend on the preparation method, and the selection of the vesicle type to be used will depend on the preferred mode of administration. There are several other forms of supramolecular organizations, in which lipids can be present in aqueous media, including lamellar phases, hexagonal phases and anti-hexagonal phases, cubic phases, micelles, reverse micelles composed of monolayers. These phases can also be obtained in combination with DNA or RNA, and the interaction with RNA and DNA can significantly affect the phase state. In some embodiments, one or more of the above phases are present in the nanoparticle RNA formulations of the present disclosure.

[0252] In order to form RNA lipid complexes from RNA and liposomes, any suitable method for forming liposomes can be used, as long as it provides the envisioned RNA lipid complexes. In some embodiments, liposomes are formed using standard methods such as reverse evaporation method (REV), ethanol injection method, dehydration-rehydration method (DRV), ultrasonic treatment or other suitable methods.

[0253] After liposome formation, the size of the liposomes can be adjusted to obtain a population of liposomes having a substantially uniform size range.

[0254] The lipids forming the bilayer usually have two hydrocarbon chains (particularly acyl chains) and a polar or nonpolar head group. The lipids forming the bilayer are composed of naturally occurring lipids or synthetic sources, including phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid (phosphatide acid), phosphatidylinositol and sphingomyelin, wherein the length of the two hydrocarbon chains is usually about 14-22 carbon atoms long, and has different degrees of unsaturation. Other suitable lipids for the compositions of the present invention include glycolipids and sterols, such as cholesterol and its multiple analogs that can also be used in liposomes.

[0255] Cationic lipids usually have a lipophilic portion, such as a sterol chain, an acyl chain or a diacyl chain, and have a total net positive charge. The head group of the lipid is usually positively charged. Cationic lipids preferably have a positive charge of 1 to 10 valencies, more preferably a positive charge of 1 to 3 valencies and more preferably a positive charge of 1 valency. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); dimethyldioctadecyl ammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-dimyristoyloxypropyl-1,3-dimethylhydroxyethylammonium (DMRIE) and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propylamine trifluoroacetate (DOSPA). In some embodiments, the cationic lipid is DOTMA, DOTAP, DODAC and DOSPA. In some embodiments, the neutral lipid is DOTMA.

[0256] In addition, considering structural stability, etc., the nanoparticles described herein preferably further include neutral lipids. Neutral lipids can be appropriately selected from the perspective of the delivery efficiency of RNA-lipid complexes. Examples of neutral lipids include, but are not limited to, 1,2-bis-(9Z-octadecanoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, sterols and cerebrosides. In some embodiments, the neutral lipid is DOPE and / or DOPC. In some embodiments, the neutral lipid is DOPE. In the case where the cationic liposomes include both cationic lipids and neutral lipids, the molar ratio of cationic lipids to neutral lipids can be appropriately determined from the perspectives of the stability of the liposomes.

[0257] In some embodiments, the nanoparticles described herein comprise phospholipids. In some embodiments, the phospholipids are glycerophospholipids. Examples of glycerophospholipids include, but are not limited to, three types of lipids: (i) zwitterionic phospholipids, including, for example, phosphatidylcholine (PC), egg yolk phosphatidylcholine, soy-derived PC (in natural, partially hydrogenated or fully hydrogenated form), dimyristoylphosphatidylcholine (DMPC) sphingomyelin (SM); (ii) negatively charged phospholipids: including, for example, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG) dipalmitoyl PG, dimyristoylphosphatidylglycerol (DMPG); synthetic derivatives in which the conjugates impart negative charges to zwitterionic phospholipids, such as methoxy-polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE); and (iii) cationic phospholipids, including, for example, phosphatidylcholine or sphingomyelin whose phosphate monoesters are O-methylated to form cationic lipids.

[0258] The association of RNA with the lipid carrier can be carried out, for example, by the RNA filling the pore space of the carrier so that the carrier physically restricts the RNA, or by covalent bonding, ionic bonding or hydrogen bonding, or by non-specific bonding adsorption. Regardless of the association mode, the RNA must maintain its therapeutic properties, i.e., antigen encoding properties.

[0259] In some embodiments, nanoparticles include at least one lipid. In some embodiments, nanoparticles include at least one cationic lipid. Cationic lipids can be monocationic or polycationic. Any cationic amphiphilic molecule (e.g., a molecule comprising at least one hydrophilic and lipophilic part) 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, nanoparticles include at least one helper lipid. Helper lipids can be neutral lipids or anionic lipids. In some embodiments, helper lipids are natural lipids, such as analogs of phospholipids or natural lipids. In some embodiments, helper lipids are completely synthetic lipids, or lipidoid molecules that have no similarity to natural lipids. In some embodiments, cationic lipids and / or helper lipids are lipids that form a double layer.

[0260] In some embodiments, at least one cationic lipid includes 1,2-di-O-octadecene-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 auxiliary lipid includes 1,2-di-(9Z-octadecene)-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 auxiliary 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 the cationic lipid is obtained by multiplying the molar amount of the cationic lipid by the number of positive charges in the cationic lipid. In various embodiments, the lipid is not functionalized, such as not functionalized by mannose, histidine and / or imidazole, the nanoparticle does not contain a targeting ligand, such as a mannose functionalized lipid, and / or the nanoparticle does not contain one or more of the following: a pH-dependent compound, a cationic polymer (such as a polymer containing histidine and / or polylysine, wherein the polymer may be optionally pegylated and / or histidine acylated) or a divalent ion (such as Ca 2+).

[0261] In various embodiments, the RNA nanoparticles may comprise peptides, preferably having a molecular weight of up to 2500 Da.

[0262] In the nanoparticles described herein, the lipids can form complexes with the RNA and / or can encapsulate the RNA. In some embodiments, the nanoparticles comprise lipid complexes or liposomes. In some embodiments, the lipids are contained in vesicles that encapsulate the RNA. In some embodiments, the vesicles are multilamellar vesicles, unilamellar vesicles, or mixtures thereof. In some embodiments, the vesicles are liposomes. In some embodiments, the nanoparticles are liposome complexes 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 wherein the charge ratio of the positive charge in the DOTMA to the negative charge 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.

[0263] In some embodiments, the nanoparticle is a liposome complex 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 wherein the charge ratio of the positive charges in DOTMA to the 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. In some embodiments, the nanoparticle is a liposome complex 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 wherein the charge ratio of the positive charges in DOTMA to the 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. In some embodiments, the nanoparticle is a liposome complex comprising DOTMA and DOPE in a molar ratio of about 2:1 to about 1:2 or about 2:1 to about 1:1, and wherein the charge ratio of the positive charge in DOTMA to the negative charge in RNA is about 1.4:1 or less. In some embodiments, the nanoparticle is a liposome complex comprising DOTMA and cholesterol in a molar ratio of about 2:1 to about 1:2 or about 2:1 to about 1:1, and wherein the charge ratio of the positive charge in DOTMA to the negative charge in RNA is about 1.4:1 or less. In some embodiments, the nanoparticle is a liposome complex comprising DOTAP and DOPE in a molar ratio of about 2:1 to about 1:2 or about 2:1 to about 1:1, and wherein the charge ratio of the positive charge in DOTAP to the negative charge in RNA is about 1.4:1 or less. In some embodiments, the nanoparticles have an average diameter in the range of about 50 nm to about 1000 nm, about 50 nm to about 400 nm, about 100 nm to about 300 nm, or about 150 nm to about 200 nm. In some embodiments, the nanoparticles have a diameter in the range of about 200 to about 700 nm, about 200 to about 600 nm, about 250 to about 550 nm, about 300 to about 500 nm, or about 200 to about 400 nm.

[0264] 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 can be obtained by one or more of the following: (i) incubating liposomes in an aqueous phase with RNA in an aqueous phase, (ii) incubating lipids dissolved in a water-miscible organic solvent such as, for example, 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.

[0265] 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 as described in U.S. Publication No. 20130059360, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the polymer conjugates with the polynucleotides of the present disclosure are prepared using the methods and / or segmented polymer reagents described in U.S. Publication No. 20130072709, which are incorporated herein by reference in their entirety. In other embodiments, the polymer conjugate has a side group comprising a ring portion, such as, but not limited to, the polymer conjugates described in U.S. Publication No. US20130196948, the contents of which are incorporated herein by reference in their entirety.

[0266] In some embodiments, the nanoparticle formulation comprises a conjugate to enhance the delivery of the nanoparticles of the present invention in a subject. In addition, the conjugate can inhibit the phagocytic clearance of nanoparticles in a subject. In some embodiments, the conjugate is a "self" peptide designed from the human membrane protein CD47 (e.g., "self" particles described in Rodriguez et al. (Science 2013, 339, 971-975), which are incorporated herein by reference in their entirety). As shown in Rodriguez et al., self-peptides delay macrophage-mediated nanoparticle clearance, thereby enhancing the delivery of nanoparticles. In other embodiments, the conjugate is a membrane protein CD47 (e.g., see Rodriguez et al. Science 2013, 339, 971-975, which are incorporated herein by reference in their entirety). Rodriguez et al. showed that, similar to the "self" peptide, CD47 can increase the circulating particle ratio in a subject compared to scrambled peptides and PEG-coated nanoparticles.

[0267] In some embodiments, about 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is in the 5'-position of the uracil. In some embodiments, the chemical modification is N1-methyl pseudouridine. In some embodiments, about 100% of the uracils in the open reading frame have N1-methyl pseudouridine in the 5'-position of the uracil.

[0268] In some embodiments, the RNA encoding the new antigen of the present disclosure is administered to a subject in the form of an RNA vaccine. In some embodiments, when combined with a flagellin adjuvant, particularly when one or more RNA vaccines are combined with an mRNA encoding flagellin, the efficacy of the RNA vaccine of the present disclosure can be significantly enhanced.

[0269] RNA vaccines combined with flagellin adjuvants (e.g., mRNA-encoded flagellin adjuvants) have superior properties because they can produce greater antibody titers and produce responses earlier than commercially available vaccine formulations. Although not wishing to be bound by theory, it is believed that RNA vaccines, such as mRNA polynucleotides, are better designed to produce appropriate protein conformations for both antigen and adjuvant upon translation because RNA vaccines co-select natural cellular machinery. Unlike traditional vaccines, which are made in vitro and may elicit unwanted cellular responses, RNA vaccines are presented to cellular systems in a more natural manner.

[0270] In some embodiments, the RNA vaccine of the present disclosure comprises at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding one or more cancer-specific 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, at least one flagellin polypeptide (e.g., an encoded flagellin polypeptide) is flagellin. In some embodiments, at least one flagellin polypeptide (e.g., an encoded flagellin polypeptide) is an immunogenic flagellin fragment. In some embodiments, at least one flagellin polypeptide and at least one antigenic polypeptide are encoded by a single RNA (e.g., mRNA) polynucleotide. In other embodiments, at least one flagellin polypeptide and at least one antigenic polypeptide are each encoded by different RNA polynucleotides.

[0271] In some embodiments, the disclosed methods further include formulating cancer-specific and subject-specific neoantigens into immunogenic compositions for administration to subjects. In some embodiments, cancer-specific and subject-specific neoantigens are formulated into immunogenic compositions in the form of DNA. In some embodiments, cancer-specific and subject-specific neoantigens are formulated into immunogenic compositions in the form of RNA. In some embodiments, cancer-specific and subject-specific neoantigens are formulated into immunogenic compositions in the form of proteins. In embodiments where cancer-specific and subject-specific neoantigens are formulated into immunogenic compositions in the form of DNA and / or RNA, DNA / RNA neoantigens can be subcloned into one or more vectors, which in some embodiments are one or more plasmids. Methods for administering DNA vaccines, RNA vaccines, or protein vaccines are known in the art. Those skilled in the art can determine which treatment regimen is suitable for a subject based on the subject, depending on, for example, their cancer and their immune status (e.g., T cell, B cell, or NK cell activity and / or quantity).

[0272] The route of administration includes, but is not limited to, intramuscular, intranasal, intradermal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular and oral, as well as topical, transdermal, by inhalation or suppository or mucosal tissue (such as by irrigation to the vagina, rectum, urethra, oral cavity and sublingual tissue). Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. In certain embodiments, the gene construct is administered by including, but not limited to, conventional syringes, needleless injection devices, "microprojectile bombardment gene guns (microprojectile bombardment gonegun)" or other physical methods such as electroporation ("EP"), "fluid dynamic methods" or ultrasound.

[0273] For therapeutic or immunization purposes, nucleic acid molecules encoding one or more new antigens can also be administered to a subject. Many methods can be conveniently used to deliver nucleic acids to a subject. For example, nucleic acids can be delivered directly as "naked DNA". This method is described in, for example, Wolff et al., Science 247:1465-1468 (1990) and those zirconium-containing pretreatment solutions described in U.S. Patents No. 5,580,859 and No. 5,589,466. Nucleic acids can also be administered using ballistic delivery, such as, for example, U.S. Patent No. 5,204,253. Particles containing only DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.

[0274] Nucleic acids can also be delivered in complex with cationic compounds such as cationic lipids. Lipid-mediated gene delivery methods are described in, for example, WO 1996 / 18372; WO 1993 / 24640; Mannino & Gould-Fogerite, BioTechniques 6 (7): 682-691 (1988); U.S. Patent No. 5,279,833; WO 1991 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), which describes pharmaceutically acceptable salts in detail.

[0275] RNA encoding neoantigens can also be used for delivery (see, eg, Kiken et al., 2011; Su et al., 2011).

[0276] Pharmaceutically acceptable carriers or excipients may include functional molecules, such as vehicles, adjuvants, carriers or diluents, which are known and readily available for public use. In certain embodiments, pharmaceutically acceptable carriers are adjuvants. In certain embodiments, pharmaceutically acceptable excipients are transfection facilitators. In certain embodiments, transfection facilitators are polyanions, polycations or lipids, and more preferably poly-L-glutamic acid. In certain embodiments, nucleic acid molecules or DNA plasmids are delivered to cells together with the administration of polynucleotide functional enhancers or gene vaccine facilitators (transfection facilitators). Polynucleotide functional enhancers are described in U.S. Patent No. 5,593,972, U.S. Patent No. 5,962,428 and International Patent Application No. PCT / US94 / 00899 filed on January 26, 1994, each of which is incorporated herein by reference in its entirety. Gene vaccine facilitators are described in U.S. Patent Application Serial No. 021,579 filed on April 1, 1994, which is incorporated herein by reference in its entirety. The transfection facilitator that can be co-administered with nucleic acid molecules can be used as a mixture with nucleic acid molecules, or when, before or after the nucleic acid molecules are used, they are used separately. The example of transfection facilitator includes surfactant, such as stimulating immune complex (ISCOMS), Freund's incomplete adjuvant, LPS analog, including monophosphoryl lipid A, muramyl peptide, quinone analog, vesicle such as squalene and squalene, and hyaluronic acid can be used for using with genetic construct. In certain embodiments, DNA plasmid vaccine can also include transfection facilitator, such as lipid, liposome, including lecithin liposome or other liposomes known in the art, such as DNA-liposome mixture (see, for example, WO9324640), calcium ion, viral protein, polyanion, polycation or nanoparticle or other known transfection facilitator. Preferably, transfection facilitator is polyanion, polycation, including poly-L-glutamic acid (LGS) or lipid.

[0277] In certain embodiments, the DNA plasmid is delivered together with the gene of the protein that further enhances immune response. Examples of such genes are genes 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 signal sequence with deletion and optionally including signal peptide from IgE. Other genes that may be useful include those encoding MCP-1, MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factor , fibroblast growth factor, IL-7, 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 their functional fragments.

[0278] When the agents described herein are administered to humans or animals as medicines, they may be administered per se or as a pharmaceutical composition containing an active ingredient in combination with a pharmaceutically acceptable carrier, excipient or diluent.

[0279] The actual dosage level and administration schedule of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. In general, the medicament or pharmaceutical composition of the present disclosure is administered in an amount sufficient to induce an immunogenic response in a subject.

[0280] One or more nucleic acid molecules encoding cancer-specific and subject-specific neoantigens described herein or compositions comprising them for administration to a subject may include about 1 ng to 10 mg; about 1 μg to about 10 mg; about 0.1 μg to about 10 mg; or about 100 μg to about 1 mg of DNA. In some embodiments, one or more nucleic acid molecules encoding cancer-specific and subject-specific neoantigens described herein or compositions comprising them for administration to a subject include about 5 ng to about 1000 μg of DNA. In some embodiments, one or more nucleic acid molecules encoding cancer-specific and subject-specific neoantigens described herein or compositions comprising them for administration to a subject include about 10 ng to about 800 μg of DNA. In some embodiments, one or more nucleic acid molecules encoding cancer-specific and subject-specific neoantigens described herein or compositions comprising them for administration to a subject include about 0.1 to about 500 μg of DNA. In some embodiments, one or more nucleic acid molecules encoding cancer-specific and subject-specific neoantigens described herein or compositions comprising them for administration to a subject include about 1 to about 350 μg of DNA. In some embodiments, one or more nucleic acid molecules encoding cancer-specific 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 cancer-specific 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.

[0281] One or more nucleic acid molecules encoding cancer-specific and subject-specific neoantigens described herein or compositions comprising the same for administration to a subject according to the present disclosure are formulated according to the mode of administration to be used. In the case where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particle-free. Isotonic formulations can be used. Typically, isotonic additives may include sodium chloride, glucose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, vasoconstrictors are added to the formulation.

[0282] In some embodiments, the DNA preparations used with the muscle or skin EP device have high DNA concentrations, such as concentrations including micrograms to tens of milligrams, or milligrams of small volumes of DNA, which are optimal for delivery to the skin, such as small injection volumes, ideally 25 microliters-200 microliters (μL). In some embodiments, the DNA preparations have high DNA concentrations, such as 1 mg / mL or higher (mg DNA / preparation volume). In some embodiments, the DNA preparations have a DNA concentration that provides a gram amount of DNA in a 200 μL preparation. In some embodiments, the DNA preparations have a DNA concentration that provides a gram amount of DNA in a 100 μL preparation.

[0283] The DNA plasmid of the present disclosure used with the electroporation device can be prepared or manufactured using a combination of known devices and techniques, such as using the optimized plasmid manufacturing technology described in U.S. Patent Application Publication No. 20090004716 to manufacture, which is incorporated herein by reference in its entirety. In certain embodiments, the DNA plasmid used can be prepared at a concentration greater than or equal to 10 mg / mL. In addition to those described in U.S. Patent Application Publication No. 20090004716 and those described in U.S. Patent No. 7,238,522, manufacturing technology also includes or incorporates various devices and schemes known to ordinary high-tech personnel in the art, which are disclosed in U.S. Patents and are incorporated herein by reference in their entirety. High-concentration plasmids used with the skin electroporation device and delivery technology described herein allow plasmids to be applied to the ID / SC space with reasonable low volumes and assist in enhancing expression and immune effects.

[0284] The amount administered to a subject and the dosage regimen will depend on many factors, such as the mode of administration, the nature of the condition being treated, the weight of the subject being treated and the judgment of the prescribing physician.

[0285] The amount of nucleic acid molecules (DNA or RNA) contained in the therapeutically active formulations according to the present disclosure is an effective amount for inducing an immunogenic response to one or more new antigens in a subject. The determination of an effective amount is entirely within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. In general, the effective amount or effective amount of a medicament is determined by first administering a low dose of the medicament, and then gradually increasing the dose or doses administered until the desired effect (e.g., inducing an immunogenic response) is observed in the treated subject, with minimal or acceptable toxic side effects. Useful methods for determining appropriate doses and dosing regimens for administering 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 ed., McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st ed., Gennaro and University of the Sciences in Philadelphia, Lippencott Williams & Wilkins, eds. (2003 and 2005), each of which is hereby incorporated by reference.

[0286] Activation, expansion and isolation of neoantigen-specific T cells

[0287] After administration to a subject, the cancer-specific and subject-specific neoantigens activate the subject's immune response to produce T cells specific for the cancer-specific and subject-specific neoantigens, hereinafter referred to as "clonal T cells". Then, after a certain period of time, such clonal T cells sensitized to one or more cancer-specific and subject-specific neoantigens can be expanded in vivo in the subject. A sensitized T cell is a cell in which a T cell responds to an epitope, resulting in an expansion of the number of T cells. This usually takes about 3-5 days, and the peak period is about 7-10 days.

[0288] In some embodiments, the subject is diagnosed with cancer. In some embodiments, the subject is suspected of having cancer. In some embodiments, the subject has been previously treated and is unresponsive to checkpoint inhibitor therapy.

[0289] In some embodiments, the nucleic acid molecule is administered to the subject by electroporation. In some embodiments, the method does not include the step of administering the nucleic acid sequence using electroporation.

[0290] In some embodiments, cancer-specific and subject-specific new antigens activate CD8+T cell immune responses in the subject. In some embodiments, activating CD8+T cell immune responses includes activating about 0.01% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating CD8+T cell immune responses includes activating about 0.05% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating CD8+T cell immune responses includes activating about 0.1% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating CD8+T cell immune responses includes activating about 0.2% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating CD8+T cell immune responses includes activating about 0.3% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating CD8+T cell immune responses includes activating about 0.4% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating 0.5% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 0.6% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 0.7% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 0.8% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 0.9% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 1% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 2% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response comprises activating about 3% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response comprises activating about 4% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response comprises activating about 5% to about 50% of CD8+T cells reactive to one or more new antigens.In some embodiments, activating a CD8+T cell immune response includes activating about 6% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 7% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 8% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 9% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 10% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 15% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+T cell immune response includes activating about 20% to about 50% of CD8+T cells reactive to one or more new antigens. In some embodiments, activating a CD8+ T cell immune response comprises activating about 25% to about 50% of CD8+ T cells reactive to one or more neoantigens.

[0291] In some embodiments, activating a CD8+T cell immune response includes activating about 0.01% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response includes activating about 0.05% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response includes activating about 0.1% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response includes activating about 0.2% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response includes activating about 0.3% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 0.4% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating 0.5% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 0.6% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 0.7% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 0.8% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 0.9% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response comprises activating about 1% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response comprises activating about 2% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response comprises activating about 3% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response comprises activating about 4% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ.In some embodiments, the activation of CD8+T cell immune response includes activating about 5% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 6% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 7% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 8% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 9% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, the activation of CD8+T cell immune response includes activating about 10% to about 50% of CD8+T cells that are reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response comprises activating about 15% to about 50% of CD8+T cells reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response comprises activating about 20% to about 50% of CD8+T cells reactive to one or more new antigens that are positive for IFN-γ. In some embodiments, activating a CD8+T cell immune response comprises activating about 25% to about 50% of CD8+T cells reactive to one or more new antigens that are positive for IFN-γ.

[0292] In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 1 hour. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 2 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 3 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 4 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 5 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 6 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 7 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 8 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 9 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for no more than about 10 hours. In some embodiments, the activation of CD8+T cells is completed after contacting with antigen-presenting cells expressing or comprising nucleic acid sequences encoding cancer-specific and subject-specific neoantigens in the subject for more than about 10 hours.

[0293] In some embodiments, PBMCs are sampled from a subject and then subjected to an activation assay to identify T cell populations that are activated in the presence of a neoantigen.

[0294] In some embodiments, the activation of CD8+T cell immune response includes amplifying CD8+T cells specific for cancer-specific and subject-specific neoantigens in the subject to a biologically significant number or concentration in the fluid. In some embodiments, the biologically significant number of neoantigen-specific CD8+T cells is about 100 cells per mL of blood of the subject to about 200 cells per mL of blood of the subject. In some embodiments, if the cells are cloned and amplified, the biologically significant number of neoantigen-specific CD8+T cells is about 100 cells per mL of pharmaceutically acceptable carrier to about 200 cells per mL of pharmaceutically acceptable carrier. In some embodiments, if the cells are cloned and amplified, the biologically significant number of neoantigen-specific CD8+T cells is about 75 cells per mL of pharmaceutically acceptable carrier to about 150 cells per mL of pharmaceutically acceptable carrier. In some embodiments, CD8+T cells are suspended in sterile buffered saline. In some embodiments, CD8+ cells are in a pharmaceutically acceptable carrier, such as sterile saline, so that they can be administered back into the subject.

[0295] The activation of T cells can be detected and measured in a variety of ways. The method for detecting specific T cell activation includes detecting the proliferation of T cells, the generation of cytokines (such as lymphokines) or the generation of cytolytic activity. The method for measuring T cell activity includes but is not limited to inducing T cell proliferation, inducing signal transduction in T cells, inducing the expression of activation markers in T cells, such as interferon-γ (IFN-γ), inducing T cells to secrete cytokines and the cytotoxic activity of T cells. For example, in some embodiments, CD8+T cell activation is measured by proliferation assay. In other embodiments, after separating cells from a subject, activation is measured after stimulating cells or cell samples by the encoded nucleic acid sequence.

[0296] In some embodiments, the activation of CD8+T cells is assessed or measured by determining the secretion of cytokines, such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNFa), interleukin-12 (IL-12), or interleukin 2 (IL-2). In some embodiments, ELISA is used to determine cytokine secretion, such as interferon gamma (IFN-γ), tumor necrosis factor alpha (TNFa), 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., interferon gamma (IFN-γ)) in response to stimulation of cancer-specific and subject-specific neoantigens, or any composition comprising it. T cells are placed in wells coated with anti-IFN-γ antibodies. The secreted IFN-γ is captured by the coated antibody and then displayed with a second antibody coupled to a chromogenic substrate. Therefore, locally secreted cytokine molecules form spots, each of which corresponds to an IFN-γ-secreting cell. The number of spots allows determination of the frequency of IFN-γ secreting cells in the sample analyzed. ELISPOT assays have also been described for detection of tumor necrosis factor alpha, 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. Pages 6.19.1–6.19.8, which are incorporated herein by reference in their entirety).

[0297] Flow cytometry analysis of intracellular cytokines can also be used to measure cytokine content, but it is not possible to provide information about the number of T cells that actually secrete cytokines. When T cells are treated with secretion inhibitors such as monensin or brefeldin A, they accumulate cytokines in their cytoplasm after activation (e.g., with nucleic acid molecules of the present invention). After lymphocyte fixation and permeabilization, intracellular cytokines can be quantified by cell counting. This technology allows the determination of the cytokines produced, the type of cells that produce these cytokines, and the number of cytokines produced by each cell.

[0298] In some embodiments, activation of CD8+ T cells is determined by measuring the cytotoxic activity of CD8+ T cells. The cytotoxic activity of T cells can be assessed by any suitable technique known to those skilled in the art. For example, the cytotoxic activity of a sample containing T cells that have been exposed to one or more new antigens according to the present disclosure can be measured in a standard cytotoxicity assay. Such assays may include, but are not limited to, chromium release CTL assays and Alamar Blue assays known in the art. TM Fluorescence assay.

[0299] In some embodiments, the activation and expansion of new antigen-specific T cells as disclosed herein enhance the expression of certain key molecules in T cells, which again protect apoptosis or otherwise prolong survival in vivo or in vitro. Apoptosis is usually caused by the induction of specific signals in T cells. Therefore, new antigens can protect T cells from cell death caused by stimulation of T cells. Therefore, the disclosed method also includes enhancing T cell growth by preventing premature death or preventing the absence or depletion of recognized T cell growth markers (such as Bcl-xL, growth factors, cytokines, or lymphokines that are usually necessary for T cell survival), and preventing Fas or tumor necrosis factor receptors (TNFR) from cross-linking or exposure to certain hormones or stresses.

[0300] The new antigen-specific T cells produced by the subject can be separated in a variety of ways. In some embodiments, the new antigen-specific T cells are separated by extracting a blood sample from the subject and sorting peripheral blood mononuclear cells (PBMC) from the sample according to the receptor expression on the surface of the PBMC. In some embodiments, the separation of new antigen-specific T cells further includes taking out a human tissue sample from the subject. In some embodiments, the human tissue sample taken out for separating new antigen-specific T cells includes tissue from a brush, biopsy or surgical resection of the subject. In some embodiments, new antigen-specific T cells are separated based on the expression of T cell activation markers by cell sorting or other appropriate techniques known in the art. In some embodiments, the disclosed method further includes determining which new antigens are recognized by T cell immunology (e.g., by an elimination process).

[0301] In some embodiments, selecting neoantigen-specific T cells includes selecting such T cells: (i) secreting a greater amount of one or more cytokines compared to the amount of one or more cytokines secreted by a negative control, or (ii) wherein the number of T cells secreting one or more cytokines is at least twice the number of negative control T cells secreting one or more cytokines. One or more cytokines may include any cytokine secreted by a T cell that is a characteristic of T cell activation (e.g., a TCR expressed by a T cell specifically binds to a neoantigen and immune recognizes the neoantigen). Non-limiting examples of cytokines whose secretion is a characteristic of T cell activation include IFN-γ, IL-2, and tumor necrosis factor α (TNF-α), granulocyte / monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

[0302] In some embodiments, if the amount of IFN-γ secreted by the T cell is at least twice the amount of IFN-γ secreted by the negative control, the T cell is considered to have antigen specificity for the new antigen, and therefore has a new antigen-specific T cell. In some embodiments, the negative control is, for example, an autologous T cell (e.g., derived from PBMC). IFN-γ secretion can be measured by methods known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0303] In some embodiments, the disclosed method further includes separating a nucleotide sequence encoding a T cell receptor (TCR) or an antigen binding portion thereof from a selected neoantigen-specific T cell, wherein the TCR or its antigen binding portion has antigen specificity for the neoantigen. In some embodiments, the disclosed method further includes identifying one or more nucleotide sequences encoding a subset of TCR or its antigen binding portion, which subsets of TCR or its antigen binding portion are highly immune in response to one or more neoantigens of the subject. In some embodiments, one or more nucleotide sequences encoding a subset of TCRs with high immunity are identified by measuring the affinity or affinity of cells expressing TCRs in vitro binding cells. In some embodiments, one or more nucleotide sequences encoding a subset of TCRs with high immunity are identified by measuring the percentage of CD8+ and / or CD4+ on cells expressing TCRs or their antigen binding portions. In some embodiments, the method may further include amplifying cells expressing TCRs or their antigen binding portions in culture, and then identifying one or more nucleotide sequences encoding a subset of TCRs or their antigen binding portions, which subsets of TCRs or their antigen binding portions are highly immune in response to one or more neoantigens of the subject. In some embodiments, the method may further include sequencing nucleotide sequences encoding one or more TCRs or antigen binding portions thereof from cells expressing the TCRs or antigen binding portions thereof and being highly immunogenic.

[0304] As used herein, the term "highly immune" means that T cells, TCRs expressed by T cells, or antigen-binding portions thereof can specifically bind to and immunologically recognize a certain degree of cancer-specific and subject-specific neoantigens, thereby stimulating a biological response, such as the secretion of cytokines after exposure to the neoantigens.

[0305] As used herein, the "antigen binding portion" of a TCR refers to any portion of the contiguous amino acids of the TCR of which it is a part, provided that the antigen binding portion specifically binds to cancer-specific and subject-specific neoantigens. The term "antigen binding portion" refers to any portion or fragment of a TCR that retains the biological activity of the TCR (parent TCR) of which it is a part. Antigen binding portions encompass, for example, those portions of a TCR that retain the ability to specifically bind to cancer-specific and subject-specific neoantigens, or the ability to detect, treat or prevent cancer to a similar degree, the same degree or a higher degree, with respect to a parent TCR. For example, the functional portion may comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or longer of the length of the parent TCR.

[0306] The antigen binding portion may include an antigen binding portion of one or both of the α chain and β chain of the TCR, such as a portion of one or more of the complementary determining regions (CDR) 1, CDR2, and CDR3 of the variable region of the α chain and / or β chain of the TCR. In some embodiments, the antigen binding portion may include the amino acid sequence of CDR 1 (CDR1α) of the α chain, CDR2 (CDR2α) of the α chain, CDR3 (CDR3α) of the α chain, CDR1 (CDR1β) of the β chain, CDR2 (CDR2β) of the β chain, CDR3 (CDR3β) of the β chain, or any combination thereof. In some embodiments, the antigen binding portion includes the amino acid sequence of CDR1α, CDR2α, and CDR3α; the amino acid sequence of CDR1β, CDR2β, and CDR3β; or the amino acid sequence of all of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the TCR.

[0307] In some embodiments, the antigen binding portion may comprise a combination of a variable region and a constant region. In some embodiments, the antigen binding portion may comprise the full length of the alpha chain or the beta chain or both the alpha chain and the beta chain of the TCR.

[0308] Isolation of nucleotide sequences encoding TCR or its antigen binding portion from selected T cells can be performed in any suitable manner known in the art. For example, the method may include isolating RNA from selected T cells and sequencing TCR or its antigen binding portion using established molecular cloning techniques and reagents (such as, for example, 5' rapid amplification (RACE) polymerase chain reaction (PCR) of cDNA ends using constant primers for TCR-α and TCR-β chains).

[0309] The present disclosure relates to a nucleic acid molecule comprising a first, a second and a third nucleic acid sequence, 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 a plurality of antigen expression domains in a 5' to 3' orientation; wherein each antigen expression domain comprises a nucleotide 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 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% or 100% sequence identity to any one of SEQ ID NOs: 1-128.

[0310] The present disclosure relates to a nucleic acid molecule comprising a first, a second and a third nucleic acid sequence, 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 a plurality of antigen expression domains in a 5' to 3' orientation; wherein each antigen expression domain comprises a nucleotide 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 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% or 100% sequence identity to any one of SEQ ID NOs: 397-494.

[0311] The present disclosure relates to a cell comprising a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID 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.

[0312] The present disclosure relates to a cell comprising a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 367-394, 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 NO: 367-394.

[0313] The present disclosure relates to a cell comprising a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: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 NO:495-501.

[0314] The present disclosure relates to a cell comprising a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 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 NO: 168-174.

[0315] The present disclosure relates to a cell comprising a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID 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.

[0316] The present disclosure relates to a cell comprising a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID 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.

[0317] The present disclosure relates to a cell comprising a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 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 NO: 179-253.

[0318] The present disclosure relates to a nucleic acid molecule encoding a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 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 NO: 179-253.

[0319] The present disclosure relates to a nucleic acid molecule encoding a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 509-588 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 one of SEQ ID NO: 509-588.

[0320] The present disclosure relates to a nucleic acid molecule encoding a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequence identifiers of 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% or 100% sequence identity to any of the sequence identifiers of Tables S, T and / or U.

[0321] The present disclosure relates to a nucleic acid molecule encoding a TCR or an antigen binding fragment thereof, which TCR or antigen binding fragment thereof comprises at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequence identifiers in 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 in Table Z.

[0322] T cells expressing TCR

[0323] Disclosed are cells comprising a TCR comprising one α and one β subunit, wherein the α and β subunits are those disclosed in Table Z. In some aspects, the TCR comprising one α and one β subunit comprises one α and one β subunit having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the α and β subunits disclosed in Table Z.

[0324] Disclosed is a cell comprising a TCR comprising an α and a β subunit, wherein the α subunit is selected from the group consisting of 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or a combination of amino acid sequences of SEQ ID NO:509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585 or 587; and wherein the beta subunit is selected from the group consisting of the amino acid sequences of SEQ ID NO:509, 511, NO: 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, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588 one or a combination of amino acid sequences having at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0325] Methods related to T cells expressing TCR

[0326] In some embodiments, the method may include cloning a nucleotide sequence encoding a TCR or an antigen-binding portion thereof from a cloned expanded T cell that is active against one or more new antigens from a subject into a recombinant expression vector using established molecular cloning techniques, such as described in, for example, Green et al. (ed.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th edition (2012). For the purposes of this article, when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows a host cell to express mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vector of the present disclosure is not naturally occurring as a whole. However, part of the vector may be naturally occurring. The recombinant expression vector may contain any type of nucleotide, including but not limited to DNA (e.g., complementary DCA (cDNA)) and RNA, which may be single-stranded or double-stranded, synthetic, or partially obtained from a natural source, and it may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may contain 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 hinder transcription or replication of the vector.

[0327] The recombinant expression vector can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host cell. Suitable vectors include those designed for breeding and amplification 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 LifeSciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage 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, such as a retroviral vector.

[0328] The TCR or antigen binding portion thereof isolated by the disclosed method can be used to prepare cells for adoptive cell therapy. In some embodiments, the present disclosure provides a method for preparing a population of cells expressing a TCR or antigen binding portion thereof, which TCR or antigen binding portion thereof has high immunological specificity for cancer-specific and subject-specific neoantigens identified by the disclosed method, 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.

[0329] The introduction of a nucleotide sequence encoding an isolated TCR or antigen binding portion thereof (e.g., a recombinant expression vector) into a host cell can be performed in any of a variety of different ways known in the art, such as described, for example, in Green et al., supra. Non-limiting examples of techniques that can be used to introduce a nucleotide sequence into a host cell include transformation, transduction, transfection, and electroporation.

[0330] The host cell to which the nucleotide sequence encoding TCR or its antigen binding portion is introduced can be any type of cell that can contain the recombinant expression vector disclosed herein. In some embodiments, the host cell is a eukaryotic cell, such as a plant, an animal, a fungus or an algae. In some embodiments, the host cell is a prokaryotic cell, such as a bacterium or a protozoan. In some embodiments, the host cell is a cultured cell. In other embodiments, the host cell is a primary cell, i.e., directly separated from an organism such as a human body. In some embodiments, the host cell is an adherent cell. In some embodiments, the host cell is a suspension cell, i.e., a cell grown in a suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, in some embodiments, the host cell is a prokaryotic cell, such as a DH5α cell. For the purpose of producing TCR or its antigen binding portion, in some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. Although the host cell may be of any cell type, may be derived from any type of tissue, and may be at any developmental stage, in some embodiments, the host cell is preferably a PBL or PBMC. In some embodiments, the host cell is a T cell.

[0331] In some embodiments, PBMCs include T cells. T cells may be any type of T cells. Without being bound by a particular theory or mechanism, it is believed that "young" T cells with a lower degree of differentiation may be associated with any one or more of higher in vivo persistence, proliferation, and anti-tumor activity compared to "old" T cells with a higher degree of differentiation. Therefore, the disclosed method can advantageously identify and separate TCRs or their antigen-binding portions with high immunity in response to one or more cancer-specific and subject-specific neoantigens, and introduce TCRs or their antigen-binding portions into "young" T cells, which can provide any one or more of higher in vivo persistence, proliferation, and anti-tumor activity compared to "old" T cells (e.g., effector cells in a patient's tumor).

[0332] In some embodiments, the host cell is autologous to the subject. In some embodiments, the TCR or its antigen binding portion identified and separated by the disclosed method is personalized for each subject or patient. In some embodiments, the disclosed method identifies and separates TCR or its antigen binding portion with high immunity in response to one or more cancer-specific and subject-specific neoantigens encoded by frequent (also referred to as "hot spots") cancer-specific mutations. In some embodiments, the method includes introducing a nucleotide sequence encoding a separated TCR or its antigen binding portion into a host cell allogeneic to the patient. For example, in some embodiments, the method includes introducing a nucleotide sequence encoding a separated TCR or its antigen binding portion into a host cell from another patient, whose tumor expresses the same mutation in the context of the same MHC molecule.

[0333] In some embodiments, the disclosed methods further include amplifying the number of host cells such as T cells expressing TCR or its antigen binding portion. The amplification of the number of T cells can be accomplished by any of a variety of methods known in the art, such as described in, for example, U.S. Patent No. 8,034,334; 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 these documents are incorporated herein by reference. In some embodiments, the amplification of the number of T cells is performed by culturing T cells with OKT3 antibodies, IL-2, and feeding PBMCs (e.g., irradiated allogeneic PBMCs). In some embodiments, the disclosed methods produce a large number of T cells with high immunity in response to one or more cancer-specific and subject-specific neoantigens.

[0334] The T cell expressing TCR or its antigen binding portion thereof can be formulated in a composition, such as a pharmaceutical composition. Therefore, in some embodiments, the present disclosure provides a pharmaceutical composition, which comprises any one of the T cells of the nucleic acid molecules encoding one or more TCR or its antigen binding portion thereof, and a pharmaceutically acceptable carrier. In certain embodiments, such pharmaceutical compositions further comprise another pharmaceutically active agent or drug, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0335] 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 a carrier that does not have harmful side effects or toxicity under the conditions of use.

[0336] In some embodiments, the T cell comprising the nucleic acid molecule encoding one or more TCR or its antigen binding portion thereof or the pharmaceutical composition comprising it is administered back to the subject by injection (e.g., intravenously). When such T cells are administered, the pharmaceutically acceptable carrier for the cell injected may include any isotonic carrier, such as, for example, normal saline (NaCl aqueous solution of about 0.90% w / v, NaCl aqueous solution of about 300mOsm / L, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, 111.), PLASMA-LYTE A (Baxter, Deerfield, 111.), about 5% glucose aqueous solution or Ringer's lactate. In some embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0337] It is expected that T cells or pharmaceutical compositions comprising nucleic acid molecules encoding one or more TCRs or their antigen binding portions disclosed herein can be used in methods for treating or preventing cancer in subjects. Without being bound by a particular theory or mechanism, it is believed that the TCR or its antigen binding portion expressed by such T cells specifically binds to a neoantigen encoded by a cancer-specific mutation found in the subject, so that when the TCR or its antigen binding portion is expressed by the subject's cells, it is possible to mediate an immune response to target cells expressing the neoantigen. Therefore, in some embodiments, the present disclosure provides a method for treating or preventing cancer in a patient, the method comprising administering to the subject an effective amount of a pharmaceutical composition, TCR, its antigen binding portion, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, or any one of a group of cells to treat or prevent cancer in the subject.

[0338] The present disclosure relates to a method of treating a subject in need thereof, comprising:

[0339] (i) activating a first population of T cells from a sample;

[0340] ii) sequencing nucleic acid expression in T cells;

[0341] (iii) generating a vector comprising one or more nucleic acid sequences encoding a TCR from a sample;

[0342] (iv) transducing the vector into a second population of T cells;

[0343] (v) administering to the subject a therapeutically effective amount of the second population of T cells.

[0344] 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 neo-antigens from a subject. The exposure can be accomplished by incubating the cells in the presence of the one or more neo-antigens.

[0345] In some embodiments, the step of sequencing the nucleic acid expression of the T cells comprises sequencing the nucleic acid sequence encoding one or more TCRs or antigen-binding fragments thereof from the T cells.

[0346] The method disclosed herein can be carried out by using autologous T cells for cell administration from patients or separated T cells cultivated from other sources. In some embodiments of applying autologous T cells to subjects in step (v), the method further includes the step of separating T cells from the sample before the sequencing step. In certain embodiments, the separation step includes one or a combination of the following: flow cytometry, cell filtration, exposure to a column comprising antibodies specific to T cells and / or exposure to a magnetic surface comprising antibodies specific to T cells. In certain embodiments, the T cells separated from the subject are cultivated with nucleic acid molecules, which comprise expressible nucleic acid sequences encoding one or more TCR sequences or their antigen-binding fragments. In certain embodiments, the method disclosed herein further includes transducing T cells with nucleic acid molecules by transfection or lipofection. In certain embodiments, the separated T cells are cultivated for about 3 to about 5 days together with nucleic acid molecules encoding one or more TCRs or their antigen-binding fragments before administration.

[0347] For methods involving administration of non-autologous T cell transfer, activated T cells from a sample of a subject can be used to identify TCR sequences simply by sequencing the TCR sequences or antigen binding fragments from the activated sample. RNA or DNA can be isolated from T cells and sequenced using known sequencing techniques. Then, in some embodiments, those identified nucleic acid sequences can be synthesized de novo or subcloned from isolated DNA to produce one or more inserts. Such nucleic acid inserts can be further subcloned into one of the disclosed plasmids and resuspended in a buffer sufficient to transduce isolated non-autologous T cells. After transduction, the resulting T cells can be used for therapy.

[0348] 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 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 those sequences provided in Tables S, T, U or W.

[0349] The amount or dosage of the T cells or pharmaceutical compositions comprising nucleic acid molecules encoding one or more TCRs or their antigen binding portions disclosed herein (e.g., the number of cells when a population of T cells is administered) administered should be sufficient to produce, for example, a therapeutic or preventive response in the subject within a reasonable time frame. For example, the dose of the T cells or pharmaceutical compositions comprising them should be sufficient to bind to a new antigen with an immune TCR or its antigen binding portion, or to detect, treat or prevent cancer within about 2 hours or longer (e.g., 12 to 24 hours or longer) after administration. In some embodiments, the time period may be even longer. The dosage will be determined by the efficacy of the specific T cells or pharmaceutical compositions administered and the patient's illness and the weight of the patient to be treated.

[0350] Many assays for determining the administered dose are known in the art. For example, a method can be used to determine the starting dose of a subject to be administered, which includes comparing the extent of target cell lysis or the extent of IFN-γ secretion by T cells expressing TCR or its antigen binding portion after administering a given dose of such T cells to a mammal in a group of mammals (wherein each mammal is given a different dose of cells). The extent of target cell lysis or IFN-γ secretion after administering a certain dose can be determined by methods known in the art.

[0351] The dosage of T cells comprising nucleic acid molecules encoding one or more TCRs or antigen-binding portions thereof will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular T cell population or a pharmaceutical composition comprising the same. Typically, the attending physician will determine the dosage of T cells or a pharmaceutical composition comprising the same for the treatment of each individual patient, taking into account a variety of factors such as age, weight, general health, diet, sex, the T cells to be administered or the pharmaceutical composition comprising the same, the route of administration, and the severity of the condition being treated.

[0352] In some embodiments, the number of T cells administered per infusion may vary, for example, in the range of one 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 T cells expressing TCR administered is about 1 million to about 200 billion cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 10 million to about 200 billion cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 100 million cells to about 200 billion cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 5 million cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 10 million cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 20 million cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 20 million cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 20 million cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 30 million cells. In some embodiments, the daily dose of T cells expressing TCR administered is about 40 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 50 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 60 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 70 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 80 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 90 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 100 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 250 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 350 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 450 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 500 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 650 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 800 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 900 million cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 1 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 5 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 20 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 30 billion cells.In some embodiments, the daily dose of the T cells expressing TCR administered is about 40 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 60 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 80 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 100 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 120 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 130 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 150 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 160 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 170 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 180 billion cells. In some embodiments, the daily dose of the T cells expressing TCR administered is about 190 billion cells. In some embodiments, the daily dose of T cells expressing the TCR administered is about 200 billion cells.

[0353] 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.

[0354] Methods of treating cancer

[0355] The present disclosure relates to methods for treating cancer in a subject in need thereof or preventing metastasis of cancer, which methods include first inducing an immune response in the subject to one or more neoantigens produced by the subject's cancer as disclosed elsewhere herein, followed by identifying one or more nucleotide sequences encoding a subset of TCRs that are highly immunogenic in response to one or more neoantigens of the subject as disclosed elsewhere herein, so that a therapeutically effective amount of T cells containing one or more such nucleotide sequences can be administered back to the subject as disclosed elsewhere herein. The immune response induced in the subject can provide a functional T cell response to one or more neoantigens. The advantages of neoantigens are that they are only found in one or a few specific individuals, are not found in normal tissues (and therefore, have reduced off-target immunogenicity), and are not affected by central tolerance mechanisms.

[0356] In some embodiments, the neoantigens used to induce an immune response in a subject are associated with a hyperproliferative disease or disorder (e.g., cancer), such as tumor neoantigens or cancer neoantigens. The identified tumor neoantigens can then be introduced into the subject to activate neoantigen-specific immune cells in the subject.

[0357] In some embodiments, treatment is determined by clinical outcome, increase, enhancement or prolongation of T cell anti-tumor activity, increase in the number of anti-tumor T cells or activated T cells compared to the number before treatment, or a combination thereof. In some embodiments, the clinical outcome is selected from the group consisting of tumor regression, tumor shrinkage, tumor necrosis, anti-tumor response of the immune system, tumor expansion, recurrence or spread, or a combination thereof.

[0358] In some embodiments, the disclosed methods are used to treat patients who have been diagnosed with cancer or are at risk of developing cancer. In some embodiments, the subject has been previously treated and does not respond to checkpoint inhibitor therapy. In some embodiments, the subject does not have detectable neoplasia but is at high risk for disease recurrence. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer has a high mutational load. In another embodiment, the cancer has a moderate mutational load. In some embodiments, the cancer has been shown to have a poor or low response to checkpoint inhibitor therapy.

[0359] 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, hematological neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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.

[0360] In certain embodiments, the cancer is non-small cell lung cancer or melanoma, both of which have been shown to have a high mutational load. In other embodiments, the cancer is ovarian cancer or glioblastoma multiforme, both of which show an intermediate mutational load and have been shown to have a poor or low response to checkpoint inhibitor therapy.

[0361] In some embodiments, the disclosed methods are of sufficient magnitude or efficacy to inhibit or delay tumor growth, induce tumor cell death, induce tumor regression, prevent or delay tumor recurrence, prevent tumor growth, prevent tumor spread, and / or induce tumor elimination.

[0362] 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 biological therapeutic 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.

[0363] Checkpoint inhibitors include any agent that blocks or suppresses the inhibitory pathway of the immune system.Such inhibitors may include small molecule inhibitors or may include antibodies or antigen binding fragments thereof that bind and block or suppress immune checkpoint receptors, or antibodies that bind and block or suppress immune checkpoint receptor ligands.In certain embodiments, checkpoint inhibitors target or suppress checkpoint proteins selected from the group consisting of: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (belonging to the CD2 molecule family and expressed on all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR and various B-7 family ligands or combinations thereof. Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics or small molecules that bind to and block or inhibit CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2,

[0364] Activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049. Illustrative immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody) and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor). In some embodiments, the checkpoint inhibitor is a checkpoint inhibitor listed in Table 4.

[0365] Table 4 – List of checkpoint inhibitors

[0366] FDA-approved checkpoint inhibitors Pembrolizumab Spartalizumab Nivolumab Camrelizumab Cimiprilimab Sintilimab Dostaslimab Tislelizumab Atezolizumab Toripalimab Avelumab Tremelimumab Durvalumab Ipilimumab Relatlimab

[0367] Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0368] 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 check point inhibitor is an anti-CD160 antibody or a functional fragment thereof. In some embodiments, the check point inhibitor is an anti-CHK1 antibody or a functional fragment thereof. In some embodiments, the check point inhibitor is an anti-CHK2 antibody or a functional fragment thereof. In some embodiments, the check point inhibitor is an anti-A2aR antibody or a functional fragment thereof. In some embodiments, the check point inhibitor is an anti-B-7 antibody or a functional fragment thereof. In some embodiments, the check point inhibitor is an anti-CGEN-15049 antibody or a functional fragment thereof.

[0369] Table 3. Sequences of checkpoint proteins.

[0370]

[0371] In some embodiments, the therapeutic agent is a checkpoint inhibitor which is any of the full-length amino acid sequences identified above, or any fragment thereof comprising 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.

[0372] In some embodiments, the therapeutic agent is an adjuvant. The ability of an adjuvant to increase the immune response to an antigen is generally manifested as a significant increase in immune-mediated reactions or the alleviation of disease symptoms. For example, an increase in humoral immunity is generally manifested as a significant increase in the antibody titer produced for the antigen, and an increase in T cell activity is generally manifested as an increase in cell proliferation or cellular cytotoxicity or cytokine secretion. An adjuvant can also change the immune response, for example by changing a major humoral or Th2 response to a major cell or Th1 response. In some embodiments, an adjuvant can be other genes expressed in a surrogate plasmid or delivered as a protein in combination with the above-mentioned plasmid in a vaccine.

[0373] 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), skin T cell attracting chemokine (CTACK), epithelial thymus expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 with a deleted signal sequence and optionally including a signal peptide from IgE. In some embodiments, the adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, 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, or a combination thereof.

[0374] 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, angiogenesis factor, and the like. , fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, 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, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[0375] Other exemplary adjuvants include, but are not limited to, poly-ICLC (see Pharmacol Ther. 2015 Feb;146:120-31, which is incorporated herein by reference in its entirety), 1018 ISS (see Vaccine. 2003 Jun 2;21(19-20):2461-7, which is incorporated herein by reference in its entirety), aluminum salts, Amplivax AS15, Bacillus Calmette-Guérin (BCG) (see Clin Immunol. 2000 Jan;94(1):64-72, which is incorporated herein by reference in its entirety), 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 Rev. Vaccines. 2007 Oct;6(5):741-6, which is incorporated herein by reference in its entirety), IC31 (see Expert Rev Vaccines. 2007 Oct;6(5):741-6, which is incorporated herein by reference in its entirety), Imiquimod (see Vaccine.2006 Mar 10;24(11):1958-6, which is incorporated herein by reference in its entirety), ImuFact1MP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, carrier systems, PLGA microparticles, resiquimod, SL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, acrylic or methacrylic acid polymers, maleic anhydride and Aquila's QS21 stimulator ( stimulon) and functional fragments of any of them; or (ii) a nucleic acid molecule encoding an adjuvant selected from the group consisting of: (i) polyICLC, 1018ISS, aluminum salts, Amplivax AS15, BCG, CP-870,893, CpG7909, CyaA, GM-CSF, IC30, IC31, Imiquimod, ImuFact 1MP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206...

Claims

1. A method of treating a cancer comprising one or more neoantigens in a subject in need thereof, the method comprising: (a) administering one or more nucleic acid sequences encoding the one or more neo-antigens to the subject in need thereof; (b) allowing a period of time sufficient to allow clonal T cells sensitized to the one or more antigens 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 T cell receptors (TCRs) that are hyperimmune 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 of the TCR subsets.

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

3. The method of claim 1 or 2, wherein step (d) comprises performing an assay to measure one or a combination of: (i) the affinity or avidity 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.

4. The method according to claim 3, further comprising sequencing the one or more nucleotide sequences encoding the TCR subset, wherein the TCR is derived from the T cell expressing the TCR and is highly immune.

5. The method of 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 of claim 5, wherein the tissue sample comprises brushing, biopsy, or surgically removed tissue from the subject.

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

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

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

11. The method of claim 10, wherein each neo-antigen encoded by the nucleic acid molecule is separated from another by one or more linkers.

12. The method of claim 11, wherein the one or more linkers comprise a furin cleavage site or a porcine teschovirus-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 method of any one of claims 9 to 13, wherein the nucleic acid molecule is located within a multiple cloning site of: (i) a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES; or (ii) a plasmid comprising at least 70% sequence identity to a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES.

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

16. The method of any one of claims 1 to 15, 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 carcinoma or bone and soft tissue sarcoma, hematological neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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.

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

18. A method of treating a cancer expressing one or more antigens in a subject in need thereof, the method comprising: (a) administering one or more nucleic acid sequences encoding said one or more antigens to said subject in need thereof; as well as (b) administering to the subject a therapeutically effective amount of T cells, wherein the T cells comprise one or more nucleic acid sequences encoding one or more T cell receptors (TCRs) or functional fragments thereof from the subject, wherein the T cell receptors or functional fragments thereof respond to the one or more new antigens and are highly immune.

19. The method of claim 17, wherein the method does not involve in vitro expansion of PBMCs and / or tumor infiltrating lymphocytes.

20. The method of claim 17 or 18, further comprising allowing the subject to elicit an immune response 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) but before step (b).

22. The method of any one of claims 17 to 20, wherein after step (a), a period of time sufficient to allow the subject to expand a clonal T cell population sensitized to the one or more neoantigens, wherein the clonal T cell population comprises about 25% to about 50% CD8+ reactivity to the one or more neoantigens.

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 neo-antigens.

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

25. The method of claim 23, wherein each neo-antigen encoded by the nucleic acid molecule is separated from another by one or more linkers.

26. The method of claim 24, wherein the one or more linkers comprise a furin cleavage site or a porcine teschovirus-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 method of any one of claims 22 to 26, wherein the nucleic acid molecule is located within a multiple cloning site of: (i) a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES; or (ii) a plasmid comprising at least 70% sequence identity to a plasmid selected from the group consisting of pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES.

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

30. The method of 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 carcinoma or bone and soft tissue sarcoma, hematological neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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.

31. The method of 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, wherein the one or more TCRs or functional fragments thereof recognize one or more new antigens, the method comprising: (a) administering one or more nucleic acid sequences encoding the one or more neo-antigens to a subject comprising one or more cells expressing the one or more neo-antigens; and (b) isolating clonally derived T cells expressing said one or more TCRs or functional fragments thereof from said subject.

33. The method of claim 30, wherein the method does not involve in vitro expansion of PBMCs and / or tumor infiltrating lymphocytes.

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

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

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

37. The method according to any one of claims 30 to 34, further comprising transferring 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 a cancer comprising one or more antigens in a subject, the method comprising: (a) administering to the subject one or more nucleic acid sequences encoding the one or more neo-antigens; (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 new 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 of the TCR subsets.

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

40. The method of claim 36 or 37, wherein step (d) comprises performing an assay to measure one or a combination of: (i) the affinity or avidity 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.

41. The method of claim 38, further comprising sequencing the one or more nucleotide sequences encoding the TCR subset, wherein the TCR is from the T cell expressing the TCR and is highly immune.

42. The method of 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 of claim 40, wherein the tissue sample comprises brushing, biopsy, or surgically resected tissue from the subject.

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

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

46. ​​The method of 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 neo-antigens.

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

48. The method of claim 45, wherein each neo-antigen encoded by the nucleic acid molecule is separated from another by one or more linkers.

49. The method of claim 46, wherein the one or more linkers comprise a furin cleavage site or a porcine teschovirus-1 2A (P2A) cleavage site.

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

51. A method according to any one of claims 45 to 48, wherein the nucleic acid molecule is located within the 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 comprising at least 70% sequence identity to a plasmid selected from pVAX1, pcDNA3.1(+), pCI mammalian expression vector, pSI vector, pZeoSV2(+), phCMV1, pTCP and pIRES.

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

53. The method of 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 carcinoma or bone and soft tissue sarcoma, hematological neoplasms, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome and acute lymphocytic leukemia, breast cancer, metastatic colorectal cancer, hormone-sensitive or hormone-refractory 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 of claim 53, wherein the cancer is HCC.

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

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

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