Identification of neoantigen-reactive t cell receptors

Abstract

The present disclosure provides methods for identifying novel neoantigen-reactive T cell receptors (TCRs) by co-culturing a reporter T cell comprising a TCR expression cassette and an antigen presenting cell expressing a target neoantigen sequence and a matched human lymphocyte antigen (HLA) sequence. The present disclosure also provides novel neoantigen-reactive TCRs and the use thereof.

Description

[0001]Arnold & Porter Ref. P35502WO00 IDENTIFICATION OF NEOANTIGEN-REACTIVE T CELL RECEPTORS CROSS-REFERENCE TO RELATED APPLICATION AND INCORPORATION OF SEQUENCE LISTING This application claims the benefit of U.S. Provisional Application No.63 / 606,831, filed December 6, 2023, which is incorporated by reference herein in its entirety. A sequence listing contained in the file named “P35502WO00_ST26.xml” which is 664,173 bytes (measured in operating system MS- Windows®), created on December 5, 2024, containing a total number of 535 SEQ ID NOs, starting from SEQ ID NO:1 to SEQ ID NO:535, is filed electronically herewith and incorporated by reference in its entirety. FIELD The present disclosure relates to the identification of T cell receptors with defined antigen and HLA specificity and methods of using the same. BACKGROUND Rapid and accurate identification of T cell receptors (TCRs) with defined antigen and HLA specificity has the potential to enable the discovery of TCRs with therapeutic applications. Individual T cell receptors (TCRs) can generally be defined with three key pieces of information; 1) Full-length paired (e.g., α and β) TCR sequence, 2) Antigenic specificity and 3) HLA-restriction. Obtaining this information from a highly polyclonal population of T cells, such as those from peripheral blood or within tissues (e.g., tumor specimens) is challenging to do in an accurate and efficient manner. At the intersection of cutting-edge technologies and robust immunological assay systems, a platform for overcoming this challenge has been developed and is provided in the present disclosure. SUMMARY The present disclosure provides a neoantigen-reactive TCR, or an antigen-binding portion thereof, where the neoantigen-reactive TCR or the antigen-binding portion thereof can bind to a neoantigen / HLA complex disclosed herein. The present disclosure provides a method for identifying a neoantigen-reactive TCR, comprising: i) co-culturing a) a reporter T cell comprising a TCR expression cassette, and b) an antigen presenting cell (APC) that expresses a target neoantigen sequence and a matched human leukocyte antigen (HLA) sequence; and ii) identifying a positive reporter signal in the reporter T cell to identify a neoantigen-reactive TCR. In one aspect, the methods disclosed herein comprises identifying TCR sequences from tumor infiltrating lymphocytes (TILs) isolated from a tumor sample. In another aspect, the methods further comprise identifying somatic mutations in the tumor sample and determining the germline HLA typing of the tumor sample. The present disclosure provides a method of identifying a neoantigen-reactive T cell receptor (TCR), Arnold & Porter Ref. P35502WO00 comprising: i) obtaining TCR α and β chain sequences from TILs isolated from a tumor sample; ii) obtaining neoantigen sequences comprising somatic mutations present in the tumor sample, and the germline HLA typing of the tumor sample; iii) co-culturing a) a reporter T cell expressing a TCR sequence reconstructed from the TCR α and β chain sequences obtained in step i), and b) an antigen presenting cell (APC) that expresses a neoantigen sequence and a matched human leukocyte antigen (HLA) sequence obtained in step ii); and iv) evaluating the reporter activity in the reporter T cell to identify a neoantigen-reactive TCR. In one aspect, the present disclosure provides a method for identifying a neoantigen-reactive TCR, comprising: i) obtaining single-cell gene expression profiles from a population of tumor infiltrating lymphocytes (TIL) isolated from a patient sample, ii) performing bioinformatics analysis on the single cell gene expression data to identify TCR clonotypes, clustering the TCR clonotypes and to select a clonotype of interest, iii) creating recombinant alpha and beta TCR sequences in silico and preparing a reporter T cell comprising a TCR expression cassette encoding a TCR sequence reconstructed from paired TCR α and β chain sequences identified from the clonotype of interest in step ii), iv) preparing a tandem minigene (TMG) expression vector comprising nucleic acid sequences for the expression of concatenated amino acid sequences of non-synonymous single nucleotide variants (SNVs); v) analyzing the patient sequencing data to identify class I and class II HLA alleles and preparing HLA expression vectors comprising the class I HLA and class II HLA allele sequences; vi) preparing an APC comprising transfecting said TMG expression vector and up to four HLA expression vectors into a cell wherein each transfection condition comprises a TMG and one or two HLA types; vii) co-culturing the reporter T cell in step iii) with the APC of step vi), and viii) identifying a positive reporter activity in the reporter T cell to identify a neoantigen-reactive TCR. In certain aspect, the clustering comprises grouping the TCT clonotypes by CD8 or CD4 expression, gene function of differentially expressed genes, and the level of expression of each TCR. In one aspect, the present disclosure provides a method for identifying a neoantigen-reactive TCR, comprising: i) obtaining single-cell gene expression profiles from a population of tumor infiltrating lymphocytes (TIL) isolated from a patient sample, ii) performing bioinformatics analyses on the single cell gene expression data to identify TCR clonotypes, clustering the TCR clonotypes and to select a clonotype of interest, iii) creating recombinant alpha and beta TCR sequences in silico and preparing a reporter T cell comprising a TCR expression cassette encoding a TCR sequence reconstructed from paired TCR α and β chain sequences identified from the clonotype of interest in step ii), iv) preparing a tandem minigene (TMG) expression vector comprising nucleic acid sequences for the expression of concatenated amino acid sequences of non-synonymous single nucleotide variants (SNVs); v) analyzing the patient sequencing data to identify class I and class II Arnold & Porter Ref. P35502WO00 HLA alleles and preparing HLA expression vectors comprising the class I HLA and class II HLA allele sequences; vi) preparing an APC comprising transfecting said TMG expression vector and one or more HLA expression vectors into a cell wherein each transfection condition comprises a TMG and one or two HLA types; vii) co-culturing the reporter T cell in step iii) with the APC of step vi), and viii) identifying a positive reporter activity in the reporter T cell to identify a neoantigen-reactive TCR. In certain aspect, the clustering comprises grouping the TCT clonotypes by CD8 or CD4 expression, gene function of differentially expressed genes, and the level of expression of each TCR. In some aspects, the method comprises preparing an APC comprising transfecting the TMG expression vector and up to four, up to five, up to six, up to seven, up to eight, up to nine, up to ten, up to eleven, up to twelve, up to thirteen, up to fourteen, up to fifteen, up to sixteen, up to seventeen, up to eighteen, up to nineteen, or up to twenty HLA expression vectors into a cell. In one aspect, up to eight HLA expression vectors are transfected into a cell in the TCR screening protocol disclosed herein. In another aspect, up to seventeen HLA expression vectors are transfected into a cell in the TIL screening protocol disclosed herein. In some aspects, the method comprises pulsing neoantigen peptides into a cell instead of transfecting the cell with a TMG expression vector. In a further aspect, the present disclosure provides a method for identifying a neoantigen-reactive TCR, comprising: i) obtaining single-cell gene expression profiles from a population of tumor infiltrating lymphocytes (TIL) isolated from a patient sample and whole exome sequence (WES) data from the patient sample, ii) performing bioinformatics analysis on the single cell gene expression data to identify TCR clonotypes of interest, iii) creating recombinant TCR sequences, iv) preparing a reporter T cell comprising a TCR expression cassette encoding a TCR sequence reconstructed from paired TCR α and β chain sequences identified from the clonotype of interest in step ii), v) preparing a tandem minigene (TMG) expression vector; vi) identifying class I and class II HLA alleles and preparing HLA expression vectors comprising the class I HLA and class II HLA allele sequences; vii) preparing an APC comprising transfecting said TMG expression vector and up to four HLA expression vectors into a cell wherein each transfection condition comprises a TMG and one or two HLA types; viii)co-culturing the reporter T cell in step iii) with the APC of step vi), and ix) identifying a positive reporter activity in the reporter T cell to identify a neoantigen-reactive TCR. In certain aspect, the clustering comprises grouping the TCT clonotypes by CD8 or CD4 expression, gene function of differentially expressed genes, and the level of expression of each TCR. The present disclosure also provides a co-culture reporter system for identifying a T cell receptor (TCR) that recognizes a target neoantigen, comprising: i) a reporter T cell comprising a TCR expression cassette, co-cultured with ii) an antigen presenting cell (APC) that expresses a target neoantigen sequence and a matched human leukocyte antigen (HLA) sequence. Arnold & Porter Ref. P35502WO00 In one aspect, the TCR expression cassette as disclosed herein comprises a TCR sequence reconstructed from TCR α and β chain sequences identified from TILs isolated from a tumor sample, and wherein the target neoantigen sequence and the matched HLA sequence are identified from the same tumor sample. Methods of identifying TCR sequences, antigen or neoantigen sequences, or the HLA sequences from a tumor sample or a normal reference sample are known in the art. Some of the commonly used methods are also described herein. In one aspect, the isolated TILs are first expanded ex vivo and then co-cultured with APCs modified to express relevant HLA alleles and antigens obtained from the tumor sample. In a further aspect, a gene signature for identifying neoantigen reactive TCRs from ex vivo expanded TILs includes one or more gene(s) selected from the group consisting of XCL2, XCL1, IL2, CSF2, IFNG, CCL4, CCL4L2, TNF, CCL3, RGCC, TNFSF9, DUSP2, NFKBID, MIR155HG, NR4A3, EVI2A, CRTAM, ZBED2, FABP5, PIM3, NR4A1, IL10, TNFSF14, NR4A2, LINC00892, ZFP36L1, GZMB, MYC, SPRY1, KDM6B, EGR2, PHLDA1, PPP1R2, VSIR, REL, PRDX1, SLA, CYTOR, DDX21, IER3, PGAM1, NAMPT, HSP90AB1, IL23A, FAM107B, BCL2A1, ZEB2, ZBTB32, BTG2, GADD45B, RILPL2, SEMA7A, TGIF1, SRGN, RAN, CFLAR, MAT2A, SIAH2, PRNP, RNF19A, FASLG, NME1, EVI2B, HSPH1, NOP16, CSRNP1, and TAGAP. In one aspect, the reporter T cell disclosed herein is a primary T cell. In another aspect, the reporter T cell disclosed herein is from an immortalized T cell line. In a certain aspect, the reporter T cell disclosed herein is not a primary T cell. In certain aspects, the immortalized cell is a Jurkat cell or a SUP-T1 cell. In some aspects, the Jurkat cell is Jurkat NFAT. In one aspect, the endogenous T cell receptor of the cells is downregulated or knocked out, such as using routine methods in the art. In one aspect, the reporter T cell disclosed herein expresses any or all protein components of the TCR signaling complex or downstream signaling components. In a certain aspect, the reporter T cell expresses one or more components selected from the group consisting of CD3, CD4, CD8a, and CD8b. In further aspects, these protein components are modified, such as by mutation of one or more amino acids, to enhance their activities. In one aspect, the antigen presenting cell (APC) disclosed herein is a classical professional APC. In another aspect, the APCs disclosed herein are artificial APCs. In a certain aspect, the APC disclosed herein is not a professional APC. In certain aspects, the APC used in the methods or cell systems disclosed herein is a COS cell. In one aspect, the COS cell is a COS-7 cell. In one aspect, the APC is a 293-HEK cell. In another aspect, the APC is not a 293-HEK cell. In one aspect, the APC endogenously expresses an HLA allele. In another aspect, the APC does not express any endogenous HLA. In one aspect, the APC comprises one or more HLA expression plasmids. In one aspect, the APC expresses multiple HLA alleles in a single cell. Arnold & Porter Ref. P35502WO00 In one aspect, the APC expresses a co-stimulatory molecule. Examples of the co-stimulatory molecules include, but not limited to, 4-1BBL, CD40, CD80, CD86, or OX40L. In some aspects, the reporter T cell disclosed herein comprises a reporter system that is activated by the binding of a TCR to an antigen. Examples of the reporter systems are known in the art and include, but are not limited to, systems based on luciferase activity, fluorescence, or cytokine production. In one aspect of the present disclosure, the reporter T cells and the APCs are co-cultured at a ratio from about 16:1 to about 1:16. In one aspect, the reporter T cells and the APCs are co-cultured at a ratio of about 4:1. In another aspect, the reporter T cells and the APCs are co-cultured at a ratio of about 8:1. In certain aspect, the reporter T cells and the APCs are co-cultured at a ratio of about 1:16, 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, 8:1, or 16:1. In one aspect, the reporter T cells and the APCs are co-cultured for 1-48 hours. In another aspect, the reporter T cells and the APCs are co-cultured for about one hour, about 2 hours, about 3 hours, about hours, at least 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours. The present disclosure provides TCR sequences, or an antigen-binding portion thereof, that are identified or obtained by any of the methods disclosed herein. In one aspect, a TCR sequence comprises one or more of the sequences selected from the group consisting of SEQ ID NOs:1-216 (the sequences provided in Tables 1-18). In another aspect, a TCR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-216 (the sequences provided in Tables 1-18). The present disclosure provides a polynucleotide encoding an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-216 (the sequences provided in Tables 1-18). In one aspect, the present disclosure provides a TCR comprising: (I) an α chain complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: (1+12n), an α chain CDR2 comprising the amino acid sequence of SEQ ID NO: (2+12n), and an α chain CDR3 comprising the amino acid sequence of SEQ ID NO: (3+12n); and (II) a β chain CDR1 comprising the amino acid sequence of SEQ ID NO: (7+12n), a β chain CDR2 comprising the amino acid sequence of SEQ ID NO: (8+12n), and a β chain CDR3 comprising the amino acid sequence of SEQ ID NO: (9+12n), where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In one aspect, the present disclosure provides an isolated or purified TCR. In another aspect, the present disclosure provides an isolated or purified polynucleotide encoding any of the amino acid sequences disclosed herein. The present disclosure also provides a neoantigen / HLA complex, where the neoantigen and the HLA can be any of the neoantigen and HLA sequences disclosed herein or known in the art. In one aspect, Arnold & Porter Ref. P35502WO00 the neoantigen comprises a sequence selected from the group consisting of SEQ ID NOs: 238 to 243 and 310 to 535 and where the HLA comprises a sequence selected from a group consisting of SEQ ID NOs: 301 to 309. The present disclosure also provides recombinant vectors expressing a TCR, or an antigen-binding portion thereof, that are disclosed herein. Production of recombinant vectors is well-known in the art, and a variety of vectors may be utilized, including viral or non-viral vectors. In some aspects, the recombinant vector comprises a polycistronic expression cassette, wherein the polycistronic expression cassette comprises a transcriptional regulatory element operably linked to a polycistronic polynucleotide that comprises: a) a first polynucleotide sequence that encodes a T cell receptor (TCR) alpha chain comprising an alpha chain variable (Vα) region and an alpha chain constant (Cα) region; b) a second polynucleotide sequence that comprises a first 2A element; c) a third polynucleotide sequence that encodes a TCR beta chain comprising a beta chain variable (Vβ) region and a beta chain constant (Cβ) region; d) a fourth polynucleotide sequence that comprises a second 2A element; and e) a fifth polynucleotide sequence that encodes a fusion protein that comprises IL-15, or a functional fragment or functional variant thereof, and IL-15Rα, or a functional fragment or functional variant thereof. In one aspect, the recombinant vector o comprises the first, the second, the third, the fourth, and the fifth polynucleotide sequence in any order from 5’ to 3’. In some aspects, the TCR alpha chain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of TCR alpha chain sequences disclosed in Tables 1-18, and the TCR beta chain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of TCR beta chain sequences disclosed in Tables 1-18. The present disclosure further provides a population of cells that comprise the recombinant vectors disclosed herein. In one aspect, the recombinant vector or the polynucleotide is integrated into the genome of the population of cells. In one aspect, the cells are immune effector cells. In certain aspects, the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, B cells, mast cells, and myeloid-derived phagocytes. The present disclosure provides a pharmaceutical composition comprising a population of cells as disclosed herein. In one aspect, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. The present disclosure further provides a method to treat or to prevent a medical condition, comprising administering a pharmaceutical composition described herein to a patient in need. In one aspect, the medical condition is a cancer. Arnold & Porter Ref. P35502WO00 BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a schematic of the TCR identification and screening platform. FIG.2 is a schematic showing the process for screening of TCRs obtained from TILs. FIG. 3 is a schematic showing the comparison of TCR-based screening and TILs-based screening methods. FIG. 4 presents the results of lentivirus infection of Jurkat NFAT cells to form CD8Lenti cells subsequently infected with CD4 lentivirus, as described in the Examples. Cells are harvested and stained with CD3, CD4, CD8A and CD8B. Jurkat NFAT parental cells are negative for CD8 (99.16% CD8 negative) on CD3+ cells. The results in FIG.2 show that Jurkat LentiCD8 cells line have 43.57% CD8a expression and 43.56% CD8a and CD8b double positive expression. FIG.5 presents the results of single clones with high CD8 and luciferase activity signal to noise ratio. PBMCs from 3 different donors are irradiated and seeded in 96 multiwell plates at 100k / well. Puromycin-selected Jurkat NFAT CD8Lenti stable pool cells are seeded at 0.5 cell / well on top of irradiated PBMCs (96 multiwells) to generate single clones. Single clones are cultured for 1 week with IL-2 (50IU / ml) and PHA (0.25µg / ml). Second week cell medium is replaced with 100 IU / ml of IL-2. Grown back clones are evaluated for CD8a and CD8b expression and luciferase signal / noise ratio (PMA / Ionomycin vs untreated). FIG.5 shows that clones #2, 15, 19, 41 (>95% CD8 expression and >150 signal to noise ratio) are the best clones with higher CD8 expression and higher luciferase activity signal to noise ratio. FIG. 6 presents the results of flowcytometry analysis showing that cells from the Jurkat NFAT_CD8Lenti pool have 46.74% CD8A and CD8B double positive cells. Cells from the #41 clone show 95.74% CD8A and CD8B double positive cells. FIG. 7 presents the results of flow analysis showing that cells from the Jurkat NFAT_CD8Lenti clone #41 infected with pGenLenti-CD4_IRES_Puro lentivirus have a CD4 positive population of 97.8%, compared to cells not infected. FIG.8 presents the results of single clones with high CD8 and luciferase activity signal to noise ratio. Jurkat cells are seeded in RPMI complete medium at 200k cells / well in 96 multiwells. Cells are treated with PMA 50ng / ml and Ionomycin 1µg / ml for 2.5H, 3.5H, 4.5H and 5.5H. Cells are harvested and lysed with passive lysis buffer (Promega) at room temperature for 15 minutes.50μL of cell lysis are mixed with 100μl of luciferase substrate (Promega). Luciferase signal intensities are detected with Luminometer. Luciferase activity folds changes are calculated by dividing PMA / Ionomycin treated condition to vehicle control treated conditions. FIG.8 provides that 4-5 hours is the best time period to harvest cells since signals start to drop from CD8Lenti_CD4Lenti pool. FIG. 9A presents the results of co-culturing Jurkat NFAT CD8Lenti cells with COS-7 cells Arnold & Porter Ref. P35502WO00 transfected with TMG1 or TMG2 with 75ng of HLA A*11:01 and 75ng of HLA A*02:01. FIG.9B presents TCR-mediated reporter activity in Jurkat NFAT cells expressing CD8 co-receptor. FIG. 10 presents Jurkat NFAT cells with or without CD8 co-receptor electroporated with Curie, McClintock cells and stained with CD3, CD4, CD8a, CD8b and mTCR antibodies. Cells are analyzed using flowcytometry to detect the percentage of cells with mTCR expression. Cells are stained with CD3, CD4, CD8a, CD8b and mTCR antibodies. As shown, cells express similar level of mTCR in Jurkat NFAT CD8Lenti cells compared with Jurkat NFAT parental cells. Over 90% of cells are viable in all six cell lines on the next day after electroporation suggesting NEON electroporation system provides highly viable T cells with sufficient percentage of mTCR expression (~20%). This allows coculture experiments to be performed next day without wasting time to recover cells. FIG. 11 presents flowcytometry analysis results for mTCR expression level in 11 TCRs for cells stained with CD3, CD4, CD8a, CD8b antibodies and mTCR antibodies. FIG.11 shows that mTCR expression varied from 8-35% (9 of 11 TCRs expressed above 15%) when cells are gated on CD3+. FIG.12 presents the results of a luciferase activity, indicating the specificity of TMGs matched to 9 of 11 TCRs. FIG. 13 presents the results of an experiment designed to troubleshoot samples with low TCR reactivity of FIG.12. COS-7 cells are transfected with 75 ng of plasmids compared to the COS-7 cells transfected with 25 ng of plasmids in FIG.9. FIG.14 presents results for peptide pulsing with long and short peptides and library TCRs. FIG. 15 presents the results of the development of an anti-TCR positive control using Jurkat cells electroporated with various TCRs and H57 anti-TCR antibody coated multiwell plate. FIG.16 presents a scatter plot showing luciferase activity from anti-TCR positive control (FIG.15, “Anti-TCR (Pos. Ctrl)”) vs. the percent expression of the electroporated TCR as measured by flow cytometry. A trend line (linear regression) is shown as a dotted line. The linear regression model and R2values are shown on the plot. FIG. 17 shows screening Class I TCRs from Patient 0164 against topspot-TMG5. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. Topspot TMG 5 is used for screening since it has EGFR L858R mutation. In addition, this patient has 2 HLA-A, 2 HLA-B, 2 HLA-C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3 (these two are Arnold & Porter Ref. P35502WO00 screened later). The HLA plasmids are separated into 3 groups for class I (HLA A, HLA B and HLA C) to reduce the number of combinations with TMG plasmid. As shown in the figure, TCR 134-1 is specific to the combination of topspot TMG5 and HLA A. FIG. 18 shows identification of HLA-Restriction of 0164-TCR134-1. To further address the HLA allele specificity, COS-7 cells are transfected with individual HLA and topspot TMG5. HLA-A* 11:01 is the specific HLA that TCR134-1 is reactive to. FIG. 19 shows identification of mutation specificity of 0164-TCR134-1. Peptide prediction online tool is used to predict minimal residue of peptide likely to bind with HLA-A *11:01. As shown in the Figure, EGFR L858R 9mer is specific to TCR134-1. No reactivity is found in wide type (WT) peptide. FIG.20 shows screening Class II TCRs from Patient 0198 Against KRAS G12C. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. KRAS G12C 25mer peptide is used for screening. The HLA plasmids are separated into 3 groups for class I (HLA DP, HLA DQ and HLA DR) to reduce the number of combinations with TMG plasmid. 27 TCRs are screened against all HLAs in four replicates. As shown in the figure, TCR 97 is specific to the combination of KRAS G12C and HLA DR. FIG.21 shows identification of HLA-Restriction of 0198-TCR97. HLAs are transfected either alone or together. TCR 97 recognizes DRB1*07:01. TCR97 has reactivity with KRAS G12V suggesting the cross reactivity. FIG. 22 shows expression of engineered TCRs in primary human T cells from four independent donors. The figure shows the percentage expression of transgenic TCRs in primary human T cells. Primary human T cells from four independent donors are gene modified to stably express transgenic TCR 0198-TCR97 using Sleeping Beauty transposon / transposase gene transfer. During ex vivo culture, cells are harvested and mTCR expression is measured. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. FIG.23 shows Mean Fluorescent Intensity (MFI) of engineered TCRs in primary human T cells from four independent donors. The mean fluorescent intensity (MFI) of transgenic TCRs in primary human T cells are shown. Primary human T cells from four independent donors are gene modified to stably express transgenic TCR 0198-TCR97 using Sleeping Beauty transposon / transposase gene transfer. During 28 days of ex vivo culture, cells are harvested and MFI of mTCR expression is measured. The Arnold & Porter Ref. P35502WO00 mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. FIG.24 shows fold expansion of cell growth during ex vivo culture. The figure shows the number of T cells in culture during ex vivo expansion. Primary human T cells from four independent donors are gene modified to stably express transgenic TCR 0198-TCR97 using Sleeping Beauty transposon / transposase gene transfer. At the end of ex vivo culture, cells are harvested and the number of live cells is counted. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. FIG.25 shows 4-1BB expression in TCR-T cells expressing engineered TCRs specific for KRASG12Cand HLA-DRA1*01:01 / HLA-DRB1*07:01. The upregulation of 4-1BB on TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells are shown. TCR-T cells expressing 0198-TCR97 TCR or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG. 26 shows IFN-γ expression from T cells cocultured with HLA-DRA1*01:01 / HLA- DRB1*07:01 DCs pulsed with KRAS WT or KRASG12CMutant peptide. The figure shows the secretion of IFN-γ by TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing 0198-TCR97 TCR or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 4 donors. FIG. 27 shows TCR-T cell mediated cytolysis of tumor cells presenting either wild type KRAS or KRASG12Cantigen in the context of HLA-DRA1*01:01 / HLA-DRB1*07:01. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12X / DRB1*07:01 tumor cells. TCR-T cells expressing 0198-TCR97 TCR or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-DRA1*01:01 / HLA-DRB1*07:01 and are pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. FIG.28 shows screening class I TCRs from Patient 0275 against KRAS G12D. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the Arnold & Porter Ref. P35502WO00 TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. Topspot TMG-1 has KRAS G2D mutation and is used for screening. The HLA plasmids are separated into 3 groups for class I (HLA A, HLA B and HLA C) to reduce the number of combinations with TMG plasmid.27 TCRs are screened against HLA class I in four replicates. As shown in figure, TCR 20-1 is specific to the combination of topspot TMG-1 and HLA A. FIG.29 shows 0275 TCR20-1 parsing confirmed KRAS G12D and A*11:01. HLA A*11:01 or HLA A*01:01 are transfected to test HLA specificity for TCR 20-1. TCR20-1 is specific to A*11:01. FIG.30 shows 0275 TCR20-1 parsing confirmed KRAS G12D and A*11:01. Short peptides against KRAS G12D or G12V are used to test minimal epitope for TCR 20-1. TCR20-1 is specific to KRAS G12D minimal peptide VVVGADGVGK. FIG.31 shows upregulation of 4-1BB by activated KRAS-G12D-A*11:01 TCR-T cells in response to KRASG12Wild Type (WT) or KRASG12Dneoantigen peptides. The upregulation of 4-1BB on TCR- T cells activated with either wild type or mutant peptide-pulsed dendritic cells are shown. TCR-T cells expressing 0275-TCR20-1 TCR, Hubble (NCI licensed TCR S-KRAS-G12D-A*11:01) or non- transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-A*11:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Dmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG. 32 shows secretion of IFN-γ by activated KRAS-G12D-A-1101 TCR-T cells in response of KRASG12(WT) peptide KRASG12Dneoantigen peptides. The figure shows the secretion of IFN-γ by TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing 0275-TCR20-1 TCR or non-transposed (NT) T cells are co-cultured with monocyte- derived dendritic cells generated from an HLA-A*11:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Dmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 2 donors. FIG.33 shows TCR clonotype127 is identified to against Master TMG with HLA DR. Day 1: COS- 7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (50-150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. Master TMG is used for screening. The HLA plasmids are separated into 3 groups for class I (HLA DP, HLA DQ and HLA DR) to reduce the number of combinations with TMG plasmid. As shown in Arnold & Porter Ref. P35502WO00 figure, TCR 127 is specific to the combination of Master TMG and HLA DR. FIG. 34 shows 0166 clonotype127 is reactive to HLA DRB1*07:01. HLA DRB1*03:01, DRB1*07:01, DRB3*02:02 and DRB4*01:03 are transfected individually to test HLA specificity for TCR127. HLA DRB1*07:01 is reactive. FIG.35 shows 0166 clonotype127 is reactive to KRAS G12V. KRAS G12 mutations are all tested for specificity, KRAS G12V has the highest response compared with G12C and G12D. FIG. 36 shows fold expansion of cell growth during ex vivo culture. The figure shows the fold- expansion of T cells modified to express transgenic TCRs. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. At the end of the first phase (PIE) and second phase (PIIE) of ex vivo culture, cells are harvested, counted and the fold expansion is calculated based on the number of live cells at the start of the culture divided into the number of live cells at the end of the culture period. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. Mean and SEM are plotted. FIG. 37 shows expression of KRAS-G12V-DRB1-0701 TCRs on CD3+ T cells during ex vivo generation process. The figure shows the percentage expression of transgenic TCRs in primary human T cells. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. During 27 days of ex vivo culture, cells are harvested and mTCR expression is measured. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. FIG.38 shows flow cytometry contour plots of mTCR expression by CD3. Representative contour plots showing the mTCR expression (y-axis) within the CD3+live cell population of primary human T cells modified to express transgenic TCRs. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. During ex vivo culture, cells are periodically harvested and mTCR expression is measured by flow cytometry. FIG.39 shows frequency of CD4+ and CD8+ cells within the CD3+ T-cell populations in ex vivo TCR-T cell cultures. The figure shows the composition of CD4+and CD8+T cells in ex vivo cultures at harvest. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. At the end of the ex vivo culture, cells are harvested and the frequency of CD4 and CD8 cells within the CD3+ live cell population is determined by flow cytometry. N = 4 donors. Mean and SEM are plotted. Arnold & Porter Ref. P35502WO00 FIG. 40 shows distribution of CD4+ and CD8+ T cells within the mTCR+ population in ex vivo TCR-T cell cultures. The figure shows the distribution of CD4+and CD8+cells within the mTCR+ transgenic cell population at harvest. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. At the end of the ex vivo culture, cells are harvested and the frequency of CD4 and CD8 cells within the CD3+mTCR+ live cell population is determined by flow cytometry. N = 4 donors. Mean and SEM are plotted. FIG.41 shows Mean Fluorescent Intensity of mTCR expression by flow cytometry during ex vivo generation. The figure shows the mean fluorescent intensity (MFI) of transgenic TCRs in primary human T cells. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. During ex vivo culture, cells periodically harvested and MFI of mTCR expression is measured. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. FIG. 42 shows percent recovery of mTCR+ cell following Magnetic-Activated Cell Selection (MACS). The figure shows the recovery efficiency of enrichment for mTCR+ cells from ex vivo cell cultures. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. On Day 14 of the culture, mTCR+ cells are enriched by MACS and the recovery efficiency is calculated. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. FIG. 43 shows upregulation of 4-1BB by activated KRAS-G12V-DRB1-0701 TCR-T cells in response to KRASG12V(MUT) peptide. The figure shows the upregulation of 4-1BB on TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*07:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte- derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG. 44 shows upregulation of 4-1BB by activated KRAS-G12V-DRB1-0701 TCR-T cells in response of KRASG12(WT) peptide. The figure shows the upregulation of 4-1BB on TCR-T cells activated with wild type peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*07:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte- derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with Arnold & Porter Ref. P35502WO00 KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG.45 shows secretion of IFN-γ by activated KRAS-G12V-DRB1-0701 TCR-T cells in response of KRASG12V(MUT) peptide. The figure shows the secretion of IFN-γ by TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*07:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte- derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 4 donors. FIG.46 shows secretion of IFN-γ by activated KRAS-G12V-DRB1-0701 TCR-T cells in response of KRASG12(WT) peptide. The figure shows the secretion of IFN-γ by TCR-T cells activated with wild type peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*07:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte- derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 4 donors. FIG.47 shows KRASG12Vtarget cell cytotoxicity of KRAS-G12V-DRB1-0701-specific TCR-T cells expressing transgenic TCRs. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12V / DRB1*07:01 tumor cells. TCR-T cells expressing S3-KRAS-G12V- DRB1-0701 or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-DRA1*01:01 / HLA-DRBB1*07:01 and are pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG. 48 shows KRASG12Vtarget cell cytotoxicity of 9976-TCR38-2 expressing TCR-T cells. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12V / DRB1*07:01 tumor cells. TCR-T cells expressing 9976-TCR38-2 or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA- DRA1*01:01 / HLA-DRBB1*07:01 and are pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. Arnold & Porter Ref. P35502WO00 FIG.49 shows KRASG12Vtarget cell cytotoxicity of TCR-T cells expressing TCRs identified from Patient 7014. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12V / DRB1*07:01 tumor cells. TCR-T cells expressing 7014-TCR16, 7014-TCR51, 7014- TCR55 or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-DRA1*01:01 / HLA-DRBB1*07:01 and are pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG.50 shows KRASG12Vtarget cell cytotoxicity of 160-TCR70 expressing TCR-T cells. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12V / DRB1*07:01 tumor cells. TCR-T cells expressing 160-TCR70 or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-DRA1*01:01 / HLA-DRBB1*07:01 and are pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptide. After overnight co- culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG. 51 shows KRASG12Vtarget cell cytotoxicity of 166-TCR127 expressing TCR-T cells. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12V / DRB1*07:01 tumor cells. TCR-T cells expressing 166-TCR127 or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA- DRA1*01:01 / HLA-DRBB1*07:01 and are pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG.52 shows 0048 TCR clonotype 82, 485-1 are identified against KRAS G12V with HLA DR. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (50-150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. KRAS G12V25mer peptide is used for screening. The HLA plasmids are separated into 3 groups for class I (HLA DP, HLA DQ and HLA DR) to reduce the number of combinations with TMG plasmid. As shown in figure, TCR 82 and 485-1 are specific to the combination of KRAS G12V and HLA DR. FIG. 53 shows 0048 TCR 82 react to KRAS G12V and DRB1*10:01. HLA DRB1*10:01, DRB1*14:54 and DRB3*02:02 are transfected individually to test HLA specificity for TCR82. HLA DRB1*10:01 is reactive. Arnold & Porter Ref. P35502WO00 FIG.54 shows 0048 TCR 485-1 react to KRAS G12V and DRB1*10:01, cross reactivity is observed against DRB3*02:02. HLA DRB1*10:01, DRB1*14:54 and DRB3*02:02 are transfected individually to test HLA specificity for TCR485-1. HLA DRB1*10:01 and DRB3*02:02 are both reactive with the higher activity observed in DRB1*10:01. FIG. 55 shows upregulation of 4-1BB by activated KRAS-G12V-DRB1-1001 TCR-T cells in response to KRASG12(WT) peptide and KRASG12V(MUT) peptide. The figure shows the upregulation of 4-1BB on TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*10:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA- DRA1*01:01 / HLA-DRB1*10:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4- 1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG. 56 shows upregulation of 4-1BB by activated KRAS-G12V-DRB1-1001 TCR-T cells in response to KRASG12(WT) peptide and KRASG12V(MUT) peptide. The figure shows the upregulation of 4-1BB on TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*10:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA- DRA1*01:01 / HLA-DRB1*10:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4- 1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG. 57 shows upregulation of 4-1BB by activated KRAS-G12V-DRB1-1001 TCR-T cells in response to KRASG12(WT) peptide and KRASG12V(MUT) peptide. The figure shows the upregulation of 4-1BB on TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB3*02:02 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA- DRA1*01:01 / HLA-DRB3*02:02 donor pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4- 1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG.58 shows secretion of IFN-γ by activated KRAS-G12V-DRB1*10:01 TCR-T cells in response to KRASG12Wild Type (WT) or KRASG12Vneoantigen peptides. The figure shows the secretion of IFN-γ by TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*10:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*10:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptides. After overnight Arnold & Porter Ref. P35502WO00 incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 2 donors. FIG.59 shows secretion of IFN-γ by activated KRAS-G12V-DRB1*10:01 TCR-T cells in response to KRASG12Wild Type (WT) or KRASG12Vneoantigen peptides. The figure shows the secretion of IFN-γ by TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*10:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*10:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 2 donors. FIG.60 shows secretion of IFN-γ by activated KRAS-G12V-DRB3*02:02 TCR-T cells in response to KRASG12Wild Type (WT) or KRASG12Vneoantigen peptides. The figure shows the secretion of IFN-γ by TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB3*02:02 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB3*02:02 donor pulsed with KRASG12wildtype (WT) or KRASG12Vmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 2 donors. FIG.61 shows TCR 386 is found to reactive to TMG1 within HLA Group 2. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (100ng each). Day 3: Neon transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co- culture, cells are harvested, and luciferase activity is measured. Topspot TMG-1 has TP53 R175H mutation and is used for screening. As shown in figure, TCR 386 is specific to the combination of topspot TMG-1 and HLA Group2. FIG. 62 shows TCR 386 is found to be reactive to HLA A*02:01. HLA A*02:01, C*02:02 and C*07:27 are transfected individually to test HLA specificity for TCR386. HLA A*02:01 is reactive. FIG.63 shows TCR 386 is found to be reactive to TP53 R175H. Short peptide predicted to against TP53 R175H are used to test minimal epitope for TCR 386. TCR 386 is specific to TP53 R175H minimal peptide. FIG.64 shows expression of TP53-R175H-A-0201 TCRs on CD3+ T cells during ex vivo generation process. The figure shows the percentage expression of transgenic TCRs in primary human T cells. Primary human T cells from four independent donors are gene modified to stably express transgenic Arnold & Porter Ref. P35502WO00 TCRs S3-TP53-R175H-A-0201, 0032-TCR2, 0032-TCR37 or 0032-TCR386 using Sleeping Beauty transposon / transposase gene transfer. During ex vivo culture, cells are harvested and mTCR expression is measured. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. FIG.65 shows Mean Fluorescent Intensity of mTCR expression by flow cytometry during ex vivo generation. The figure shows the mean fluorescent intensity (MFI) of transgenic TCRs in primary human T cells. Primary human T cells from four independent donors are gene modified to stably express transgenic TCR S3-TP53-R175H-A-0201, 0032-TCR2, 0032-TCR37 or 0032-TCR386 using Sleeping Beauty transposon / transposase gene transfer. During 27 days of ex vivo culture, cells are harvested and MFI of mTCR expression is measured. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. Mean and SEM are plotted. FIG. 66 shows fold expansion of cell growth during ex vivo culture. The figure shows the fold- expansion of T cells modified to express transgenic TCRs. Primary human T cells from four independent donors are gene modified to stably express transgenic TCR S3-TP53-R175H-A-0201, 0032-TCR2, 0032-TCR37 or 0032-TCR386 using Sleeping Beauty transposon / transposase gene transfer. At the end of the first phase (PIE) and second phase (PIIE) of ex vivo culture, cells are harvested, counted and the fold expansion is calculated based on the number of live cells at the start of the culture divided into the number of live cells at the end of the culture period. The mean and SEM of each TCR across the four donors is summarized in the plot. N = 4 donors. Mean and SEM are plotted. FIG.67 shows upregulation of 4-1BB by activated TP53-R175H-A-0201 TCR-T cells in response to TP53R175Wild Type (WT) or TP53R175Hneoantigen peptides. The figure shows the upregulation of 4-1BB on TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing S3-TP53-R175H-A-0201, 0032-TCR2, 0032-TCR37, 0032-TCR386 or non- transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-A*02:01 donor pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. Mean and SEM are plotted. FIG. 68 shows secretion of IFN-γ by activated TP53-R175H-A-0201 TCR-T cells in response to TP53R175Wild Type (WT) or TP53R175Hneoantigen peptides. The figure shows the secretion of IFN- γ by TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing S3-TP53-R175H-A-0201, 0032-TCR2, 0032-TCR37, 0032-TCR386 or non- transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-A*02:01 donor pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After Arnold & Porter Ref. P35502WO00 overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 4 donors. Mean and SEM are plotted. FIG. 69 shows TP53R175Htarget cell cytotoxicity of TP53-R175H-A-0201-specific TCR-T cells expressing transgenic TCRs. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting TP53R175H / A*02:01 tumor cells. TCR-T cells expressing S3-TP53-R175H-A-0201 or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-A*02:01 and are pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG. 70 shows TP53R175Htarget cell cytotoxicity of TP53-R175H-A-0201-specific TCR-T cells expressing transgenic TCRs. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting TP53R175H / A*02:01 tumor cells. TCR-T cells expressing 0032-TCR2 or non- transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-A*02:01 and are pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG. 71 shows TP53R175Htarget cell cytotoxicity of TP53-R175H-A-0201-specific TCR-T cells expressing transgenic TCRs. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting TP53R175H / A*02:01 tumor cells. TCR-T cells expressing 0032-TCR37 or non- transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-A*02:01 and are pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG. 72 shows TP53R175Htarget cell cytotoxicity of TP53-R175H-A-0201-specific TCR-T cells expressing transgenic TCRs. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting TP53R175H / A*02:01 tumor cells. TCR-T cells expressing 0032-TCR386 or non- transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-A*02:01 and are pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. Mean and SEM are plotted. FIG. 73 shows CLL000124 TCRs Class I TCRs screening. First round class I TCR screening identifies that TCR Clonotype 10 is reactive to TMGs which contained these two hotspot mutations. The reactivity is observed in HLA B allele. Arnold & Porter Ref. P35502WO00 FIG. 74 shows 0124 second round parsing showing specificity is against TP53 R248W and HLA B*57:01. There are two HLA B in group 2: HLA B* 18:01 and HLA B*57:01. Both topspot-TMG1 (contained KRAS G12D) and topspot-TMG2 (contained TP53 R248W are used for screening. The reactivity is identified in B*57:01 and TMG2, which suggests TP53 R248W. FIG.75 shows top ranked two peptides are specific for TCR10 / HLA B*57:01 interaction. Among all peptides tested, TCR10 recognizes peptide SSCMGGMNW (9mer_1; SEQ ID NO: 238) and NSSCMGGMNW (10mer_1; SEQ ID NO: 239). No reactivity is observed in WT peptide SSCMGGMNR, suggesting TCR 10 is specific against minimal epitope SSCMGGMNW (SEQ ID NO: 238). FIG.76 shows upregulation of 4-1BB by activated TP53-R248W-B*57:01 TCR-T cells in response to TP53R248Wild Type (WT) or TP53R248Wneoantigen peptides. The figure shows the upregulation of 4-1BB on TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to TP53R248W / B*57:01 neoantigen or non-transposed (NT) T cells are co- cultured with monocyte-derived dendritic cells generated from a B*57:01 donor pulsed with TP53R248wildtype (WT) or TP53R248Wmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. FIG.77 shows secretion of IFN-γ by activated TP53-R248W-B*5701 TCR-T cells in response to TP53R248Wild Type (WT) or TP53R248Wneoantigen peptides. The figure shows the secretion of IFN- γ by TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to TP53R248W / B*57:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an B*57:01 donor pulsed with TP53R248wildtype (WT) or TP53R248Wmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 2 donors. FIG.78 shows CLL000105 TCRs Class II TCRs screening. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (50-150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. TP53 R273C 25mer peptide is used for screening. The HLA plasmids are separated into 3 groups for class II (HLA DP, HLA DQ and HLA DR) to reduce the number of combinations with TMG plasmid. As shown in figure, 5 TCRs are specific to the combination of TP53 R273C and HLA DP. Arnold & Porter Ref. P35502WO00 FIG. 79 shows sample 0105 HLA and mutation specificity identification. HLA DPB1*04:02 or DPB1*02:01 are transfected individually to test HLA specificity. All five TCRs are against DPB1*04:02. Two of them are also reactive to DPB1*02:01. FIG. 80 shows CLL000236 TCR screening. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (50-150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. PPP2AR1 R183W 25mer peptide is used for screening. The HLA plasmids are separated into 3 groups for class II (HLA DP, HLA DQ and HLA DR) to reduce the number of combinations. As shown in figure, 2 TCRs are specific to the combination of PPP2AR1 R183W and HLA DR. FIG. 81 shows sample 0236 HLA and mutation specificity identification. HLA DRB1*03:01, DRB3*01:01 or DRB3* 02:02 are transfected individually to test HLA specificity. Two TCRs are either against DRB1*03:01 or DRB3*01:01. FIG.82 shows sample 0075 TIL fragment 2,3 and 12 have reactivity against DRB5*01:01 and TP53 R282W. Transfection is performed to express HLA plasmids in COS-7. 6 groups of HLA are transfected in COS-7. ELISpot are performed in duplicates. HLA DRB5*05:01 are specific to mutant peptide of TP53 R282W in all three TIL fragment. FIG.83 shows sample 0075 TIL coculture scRNA analysis reveals a cluster of activated T cells. TIL fragment are cocultured with COS-7 being transfected with HLA specific for patient 0075 and pulsed peptide for TP53 R282W. T cells are sorted and analyzed at single cells level. A cluster of activated T cells is observed in cluster 24. FIG.84 shows sample 0075 activated clonotypes are picked and packed in plasmids for functional assay. Clonotypes from cluster 24 are picked and ordered plasmids to express in Jurkat cells. FIG.85 shows coculture of reporter cells reveals 3 clonotypes specific for DRB5*01:01 and TP53 R282W. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS- 7 cells are transfected in each well with 450ng of HLAs (50-150ng each). Day 3: Lonza 4D 96 well transfection system is set up the following day and 2 million cells are electroporated in each well with each of the TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. TP53 R282W and TP53 R282 WT peptide are used for screening. As shown in figure, 3 TCRs are specific to the combination of TP53 R282W and HLA DRB5*01:01. Arnold & Porter Ref. P35502WO00 DETAILED DESCRIPTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of 5% to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., up to 5% to 10% below) the value or range remain within the intended meaning of the recited value or range. As used herein, the terms “T cell receptor” and “TCR” are used interchangeably and refer to molecules comprising CDRs or variable regions from α3 T cell receptors. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions of TCRs attached by a flexible linker, TCR chains linked by an engineered disulfide bond, single TCR variable domains, single peptide-HLA-specific TCRs, multi-specific TCRs (including bispecific TCRs), TCR fusions, TCRs comprising co-stimulatory regions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs. In certain aspects, the TCR is a full-length TCR comprising a full-length α chain and a full-length 3 chain. In certain aspects, the TCR is a soluble TCR lacking transmembrane and / or cytoplasmic region(s). In certain aspects, the TCR is a single- chain TCR (scTCR) comprising Vα and V3 linked by a peptide linker, such as a scTCR having a structure as described in PCT Publication No.: WO 2003 / 020763, WO 2004 / 033685, or WO 2011 / 044186, each of which is incorporated by reference herein in its entirety. In certain aspects, the TCR comprises a transmembrane region. In certain aspects, the TCR comprises a co-stimulatory signaling region. As used herein, the term “full-length TCR” refers to a TCR comprising a dimer of a first and a second polypeptide chain, each of which comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In certain aspects, the full- Arnold & Porter Ref. P35502WO00 length TCR comprises one or two unmodified TCR chains, e.g., unmodified α or 3TCR chains. In certain aspects, the full-length TCR comprises one or two altered TCR chains, such as chimeric TCR chains and / or TCR chains comprising one or more amino acid substitutions, insertions, or deletions relative to an unmodified TCR chain. In certain aspects, the full-length TCR comprises a mature, full-length TCR α chain and a mature, full-length TCR β chain. The “antigen-binding portion” of the TCR, as used herein, refers to any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to the target neoantigen as described herein with respect to other aspects of the disclosure. The term “antigen-binding portion” refers to any part or fragment of the TCR of the disclosure, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Antigen-binding portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to the target antigen, or detect, treat, or prevent a condition, to a similar extent, the same extent, or to a higher extent, as compared to the parent TCR. As used herein, the term “TCR variable region” refers to the portion of a mature TCR polypeptide chain (e.g., a TCR α chain or β chain) which is not encoded by the TRAC gene for TCR α chains, either the TRBC1 or TRBC2 genes for TCR β chains, or the TRDC gene for TCR δ chains. In some aspects, the TCR variable region of a TCR α chain encompasses all amino acids of a mature TCR α chain polypeptide which are encoded by a TRAV and / or TRAJ gene, and the TCR variable region of a TCR β chain encompasses all amino acids of a mature TCR β chain polypeptide which are encoded by a TRBV, TRBD, and / or TRBJ gene (see, e.g., Lefranc and Lefranc, (2001) “T cell receptor FactsBook.” Academic Press, ISBN 0-12-441352-8, which is incorporated by reference herein in its entirety). TCR variable regions generally comprise framework regions (FR) 1, 2, 3, and 4 and complementarity determining regions (CDR) 1, 2, and 3. As used herein, the terms “α chain variable region” and “Vα” are used interchangeably and refer to the variable region of a TCR α chain. As used herein, the terms “β chain variable region” and “Vβ” are used interchangeably and refer to the variable region of a TCR β chain. As used herein in the context of a TCR, the term “CDR” or “complementarity determining region” means the noncontiguous antigen combining sites found within the variable regions of a TCR chain (e.g., an α chain or a β chain). These regions have been described in Lefranc, (1999) The Immunologist 7: 132-136; Lefranc et al., (1999) Nucleic Acids Res 27: 209¬212; Lefranc (2001) “T cell receptor FactsBook.” Academic Press, ISBN 0-12-441352-8; Lefranc et al., (2003) Dev Comp Immunol.27(1):55-77; and in Kabat et al., (1991) “Sequences of protein of immunological interest,” each of which is herein incorporated by reference in its entirety. In certain aspects, CDRs are Arnold & Porter Ref. P35502WO00 determined according to the IMGT numbering system described in Lefranc (1999) supra. In certain aspects, CDRs are defined according to the Kabat numbering system described in Kabat supra. In certain aspects, CDRs are defined empirically, e.g., based upon a structural analysis of the interaction of a TCR with a cognate antigen (e.g., a peptide or a peptide-HLA complex). In certain aspects, the α chain and β chain CDRs of a TCR are defined according to different conventions (e.g., according to the Kabat or IMGT numbering systems, or empirically based upon structural analysis). As used herein, the term “constant region” with respect to a TCR refers to the portion of a TCR that is encoded by the TRAC gene (for TCR α chains) or either the TRBC1 or TRBC2 gene (for TCR 3 chains), optionally lacking all or a portion of a transmembrane region and / or all or a portion of a cytoplasmic region. In certain aspects, a TCR constant region lacks a transmembrane region and a cytoplasmic region. A TCR constant region does not include amino acids encoded by a TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV, or TRGJ gene (see, e.g., “T cell receptor Facts Book,” supra). As used herein, the terms “major histocompatibility complex” and “MHC” are used interchangeably and refer to an MHC class I molecule and / or an MHC class II molecule. As used herein, the term “MHC class I” refers to a dimer of an MHC class I α chain and a Beta-2 microglobulin chain and the term “MHC class II” refers to a dimer of an MHC class II α chain and an MHC class II 3 chain. As used herein, the terms “human leukocyte antigen” and “HLA” are used interchangeably and can also refer to the proteins encoded by the MHC genes. HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G refer to major and minor gene products of MHC class I genes. HLA-DP, HLA-DQ, and HLA-DR refer to gene products of MHC class I genes, which are expressed on antigen-presenting cells, B cells, and T cells. As used herein, the term “peptide-HLA complex” refers to an HLA molecule (HLA class I, II or III) with a peptide bound in the art-recognized peptide binding pocket of the HLA. In some aspects, the HLA molecule is a membrane-bound protein expressed on the cell surface. In some aspects, the HLA molecule is a soluble protein lacking transmembrane or cytoplasmic regions. Neoantigens are a class of cancer antigens which arise from cancer-specific mutations in expressed protein. As used herein, the term “neoantigen” relates to a peptide or protein expressed by a cancer cell that includes one or more amino acid modifications compared to the corresponding wild-type (non-mutated) peptide or protein that is expressed by a normal (non-cancerous) cell. A neoantigen may be patient specific. A “cancer-specific mutation” is a somatic mutation that is present in the nucleic acid of a tumor or cancer cell but absent in the nucleic acid of a corresponding normal, i.e., non-tumorous or non-cancerous, cell. Arnold & Porter Ref. P35502WO00 As used herein, the terms “T cell” and “T lymphocyte” are used interchangeably. In one aspect, the T cell is a primary T cell. In another aspect, the T cell is an immortalized T cell line. T cells can be obtained from numerous sources in a patient, including but not limited to tumor, blood, bone marrow, lymph node, the thymus, or other tissues or fluids. The T cells can include any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells (e.g., TILs), peripheral blood T cells, memory T cells, naive T cells, and the like. The T cells may be CD8+ T cells, CD4+ T cells, or both CD4+ and CD8+ T cells. As used herein, the term “reporter T cell” refers to a T cell that comprises a TCR-mediated reporter system. Non-limiting examples of TCR-mediated reporter system include fluorescence-based systems, and those based on luciferase activity or cytokine production. See, e.g., Zong et al., 2020 PLoS ONE, and the references cited therein. A reporter system based on cytokine production may measure the production of one or more cytokines, the secretion of which by a T cell is characteristic of T cell activation (e.g., a TCR expressed by the T cells specifically binding to and immunologically recognizing the mutated amino acid sequence). Non-limiting examples of cytokines, the secretion of which is characteristic of T cell activation, include IFN-γ, IL-2, granzyme B, 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. In certain aspect, a “positive” reporter signal in a reporter T cell is a signal from a reporter gene that is at least 1.5x higher than the average of all of the samples when measured in a 96 well plate having a single TCR, up to 6 TMG sequences in duplicate and five different HLA clusters. In aspects, the reporter signal is luciferase activity. A positive reporter signal is detected when the TCR in the reporter T cell is paired with a matching APC comprising a TMG and matched HLA cluster. The phrase "neoantigen-reactive," as used herein, means that a TCR, or an antigen-binding portion thereof, can bind to and immunologically recognize the mutated amino acid sequence encoded by the cancer-specific mutation. As used herein, the terms “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. In some aspects, the methods of “treatment” employ administration of a TCR or a cell expressing a TCR to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. As used herein, the term “subject” includes any human or non-human animal. In one aspect, the subject is a human or non-human mammal. In one aspect, the subject is a human. As used herein, the Arnold & Porter Ref. P35502WO00 term “polycistronic vector” refers to a polynucleotide vector that comprises a polycistronic expression cassette. As used herein, the term “polycistronic expression cassette” refers to a polynucleotide sequence wherein the expression of three or more transgenes is regulated by common transcriptional regulatory elements (e.g., a common promoter) and can simultaneously express three or more separate proteins from the same mRNA. Exemplary polycistronic vectors, without limitation, include tricistronic vectors (containing three cistrons) and tetracistronic vectors (containing four cistrons). As used herein, the term “polycistronic polynucleotide” refers to a polynucleotide that comprises three or more cistrons. The determination of “percent identity” between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non- limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul S F, (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF, (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., at score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., at score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul S F et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules. Id. When utilizing BLAST, Gapped BLAST, and PSI BLAST programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. Arnold & Porter Ref. P35502WO00 The present disclosure provides a method for identifying a TCR that recognize a target neoantigen, comprising: i) co-culturing a) a reporter T cell comprising a TCR expression cassette, and b) an antigen presenting cell (APC) that expresses a target neoantigen sequence and a matched human leukocyte antigen (HLA) sequence; and ii) evaluating the reporter activity in the reporter T cell to identify a TCR that recognizes the target neoantigen. In one aspect, the methods disclosed herein comprises identifying TCR sequences from tumor infiltrating lymphocytes TILs isolated from a tumor sample. In another aspect, the methods further comprise identifying somatic mutations in the tumor sample and determining the germline HLA typing of the tumor sample. The present disclosure provides a method of identifying a neoantigen-reactive T cell receptor (TCR), comprising: i) obtaining TCR α and β chain sequences from TILs isolated from a tumor sample; ii) obtaining neoantigen sequences comprising somatic mutations present in the tumor sample, and the germline HLA typing of the tumor sample; iii) co-culturing a) a reporter T cell expressing a TCR sequence reconstructed from the TCR α and β chain sequences obtained in step i), and b) an antigen presenting cell (APC) that expresses a neoantigen sequence and a matched human leukocyte antigen (HLA) sequence obtained in step ii); and iv) evaluating the reporter activity in the reporter T cell to identify a neoantigen-reactive TCR. The present disclosure also provides a co-culture reporter system for identifying a T cell receptor (TCR) that recognizes a target neoantigen, comprising: i) a reporter T cell comprising a TCR expression cassette, co-cultured with ii) an antigen presenting cell (APC) that expresses a target neoantigen sequence and a matched human leukocyte antigen (HLA) sequence. The present disclosure provides a TCR identification and screening platform as illustrated in FIG.1. Initially, single-cell gene expression data (e.g., 5’ GEX Analysis) from T cells are utilized to perform unsupervised clustering analysis by employing dimensionality reduction methods such as principal component analysis (PCA), t-distributed Stochastic Neighbor Embedding (tSNE), or Uniform Manifold Approximation and Projection (UMAP) (FIG. 1, STEP 1). Merging the clustered single- cell gene expression analysis with paired, full-length TCR sequences then enables the identification of TCR clonotypes present in each of the distinct clusters. TCR sequences are then selected from the overall single-cell dataset based on frequency, cluster attributes, specific-gene expression signatures, or other criteria employed to increase the likelihood of obtaining TCRs with desired reactivity (i.e., antigen / HLA specificity) (FIG. 1, STEP 2). Selected paired, full-length TCR sequences are then reconstructed in silico, from which expression plasmids encoding the TCR α and β chains are synthesized (FIG. 1, STEP 3). These TCR expression cassettes are then cloned into transposon or other non-viral gene transfer vectors to enable quick translation into process development, manufacturing, and clinical applications. TCR-expression plasmids are then transiently expressed in Arnold & Porter Ref. P35502WO00 a cell line (e.g., Jurkat or SUP-T1) or primary cell (e.g., human ex vivo expanded T cells) that will signal upon TCR recognition of cognate antigen:HLA complexes on the surface of antigen presenting cells (APCs) (FIG. 1, STEP 4). Antigen presenting cells (APCs) are classical professional APCs such as dendritic cells (DCs) or an artificial antigen presenting cell (e.g., COS-7 or 293-HEK). APCs either endogenously express the requisite HLA allele(s) or are transfected with HLA expression plasmids. Antigens are introduced to the APCs either by genetic transfer to antigen encoding plasmids (e.g., Tandem Minigene (TMG) plasmids) or by the pulsing of peptide pools. One aspect of the APC system used is that multiple HLA alleles and antigens are screened within the same set of APCs, thus enable high-throughput assessment of hundreds to thousands of antigen:HLA combinations. Co-culture of the TCR modified cells and APCs is then performed to identify reactive TCRs (FIG.1, STEP 5). Reactive TCRs are those that are found to recognize one of the antigen:HLA conditions tested. These reactive TCRs are then further evaluated in vitro to confirm the findings and deconvolute the multiplexed HLA / antigen. Once all reactive TCRs are identified from a specimen, that binary outcome (reactive vs non-reactive) for each TCR can be mapped back to the initial gene- expression cluster analysis (FIG. 1, STEP 6). By mapping the reactive TCRs back to the gene- expression data, gene signatures or biomarkers which are enriched in the reactive TCR cell population are elucidated and used to further improve and refine the initial selection of TCRs for screening. In one aspect, this process is used to identify TCR sequences and their associated antigen and HLA specificity with a high level of confidence and accuracy from complex starting materials such as tumor tissues or blood samples. In one aspect, the steps of the above-described workflow (FIG.1) comprise the processes as shown in FIG.2 for screening of TCRs obtained from TILs. The process illustrated in FIG.2 correspond to FIG.1 STEPs 1-5. The workflow illustrated in FIG.2 further comprises two parallel processes (indicated with either Alpha [i.e., A, B, C, etc.] or Numeric [i.e., 1, 2, 3, etc.] STEP designators) that diverge from a common starting point (STEP 1 / A) and converge at a common finishing point (STEP 8 / F). STEP 1 / A to STEP 6 illustrate the workflow from TILs isolation to generation of cells expressing TILs-derived TCRs. STEP 1 / A to STEP D illustrate the workflow from patient mutation and HLA calling to the generation of APCs expressing the patient matched HLA and mutation- derived antigens (e.g., neoantigens). In one aspect, a tumor sample is obtained from a cancer patient (FIG. 2, STEP 1 / A). This tumor sample is dissociated into a single-cell suspension and TILs are isolated by fluorescent activated cell sorting (FACS) by staining dissociated tumor samples for lymphocyte, T cell, and live cell markers (FIG. 2, STEP 2). Single-cell transcriptomics is then performed on the sorted TILs to obtain gene expression and TCR V(D)J sequences (FIG. 2, STEP 3). Bioinformatic analysis of the gene- Arnold & Porter Ref. P35502WO00 expression data is used to cluster cells based on transcriptional similarities to aid in the selection of TCR sequences for in vitro evaluation (FIG.2, STEP 4). Once selected, TCRs are reconstructed in silico and synthesized in expression vectors (FIG.2, STEP 5) to enable transgenic expression of the TCRs in cells capable of forming a functional TCR complex with CD3 subunits and CD4 / CD8 co- receptors. These cells are engineered to express any or all necessary protein components of the TCR signaling complex or downstream signaling components. Moreover, these components are modified to further enhance their function in the platform (e.g., CD4 with amino acid substitutions at Q40Y, T45W, P48L, S60R, and / or D63R to enhance affinity to MHC-Class II). Wang et al. 2011 PNAS, 108(38): 15960-15965. TCR expression vectors are transferred into the reporter cells to generate reporter TCR-T cells (FIG.2 STEP 6). In another aspect, in parallel to STEPs 1 – 6 described above, nucleic acids (DNA and RNA) are extracted from the tumor sample (FIG.2, STEP 1 / A). Using Whole Exome Sequencing (WES) and RNA Sequencing (RNAseq) to generate genomic and transcriptional datasets, a bioinformatics pipeline is employed to determine somatic mutations present in the tumor as well as the patient’s germline HLA typing (FIG. 2, STEP B). Somatic mutations are ranked and concatenated so that TMGs and peptide pools can be synthesized (FIG.2, STEP C). These reagents provide the antigen component of the screening assay. Similarly, sequences of the called HLA alleles are synthesized in expression vectors to provide the HLAs necessary for the screening assay. Antigen presenting cells, such as COS-7, are then modified either by stable or transient transfection to express the requisite Class I or Class II HLA alleles either in single-plex or multiplexed within the same cells (FIG. 2, STEP D). Antigen is provided to the APCs either by transfection of relevant TMGs (either as plasmid DNA or in vitro transcribed RNA) and / or peptide pools containing antigens derived from the tumor’s somatic mutations identified. With both the HLA and antigen provided to the APCs, they are able to present peptide:HLA complexes to T cells in vitro. In a further aspect, reporter cells expressing transgenic TCRs (FIG. 2, STEP 6) and antigen / HLA- modified APCs (FIG.2, STEP D) are co-cultured together at a pre-determined ratio of Reporter cells (E) to APCs (T), typically approximately 4:1 to 8:1 (FIG. 2, STEP 7 / E). Positive control wells containing PMA / Ionomycin or coated with H57-597 antibody (anti-transgenic TCR) with the TCR- modified Reporter cells are also set up. Negative control wells of Reporter cells alone or co-cultured with APCs modified with HLA-only, irrelevant antigens, or non-transfected are also set up. All conditions are typically evaluated in duplicate. After the co-culture period, reporter activity (i.e., luciferase activity) is quantified in each co-culture and control well (FIG.2, STEP 8 / F). For a given TCR, the reporter activity is compared across all antigen:HLA conditions evaluated to determine if there is a condition with increased reporter activity which indicates that the transgenic TCR Arnold & Porter Ref. P35502WO00 recognized an antigen:HLA combination present in that well. Because initial screening multiplexes multiple HLA alleles and antigens, when there is specific TCR activity observed, STEP 7 / E and 8 / F are repeated using APCs modified with single HLA and antigens to elucidate the exact specificity of the TCR. Moreover, minimal epitopes can be determined using this co-culture method. Overall, this workflow enables the identification of TCR sequences and the empirical determination of specificity to selected antigens and HLA alleles. The present disclosure provides both a TCR-based screening method (below dotted line) and a TILs- based screening method (above dotted line), as illustrated in FIG. 3. The TCR-based screening method is as described above in the description of FIG. 2 wherein TCR sequences, somatic mutations, and HLA-typing is obtained from primary tumor samples and utilized to screen selected TCRs for reactivity to tumor neoantigens using a co-culture reporter system. Similarly, TILs screening starts with a primary tumor sample obtained from a cancer patient. TILs are expanded from the tumor using standard TILs expansion methods (high-concentration IL-2, feeder cells, muromonab-CD3 (OKT3)). Expanded TILs are then co-cultured in an IFN-γ ELISpot with APCs modified to express the relevant HLA alleles and antigens identified from WES and RNAseq data from the tumor. This is performed in a similar plate layout to TCR screening where multiple HLA alleles and antigens are multiplexed in the same wells, thus increasing the throughput of the assay. Positive controls include PMA / Ionomycin. Negative controls include TILs alone, APCs alone, TILs + APCs without HLA and / or antigen, and no cells. After the overnight co-culture, cells are harvested from the IFN-γ ELISpot and the plate is developed to measure the number of spot-forming colonies (SFCs) of each well. The harvested TILs are also stained and evaluated for upregulation of 4-1BB or other activation molecules (e.g., OX40). TILs from co-culture conditions which produce increased numbers of SFCs and / or activation marker expression are then sorted for either total live T cells or for T cells expressing the activation marker. Single cell gene expression and TCR V(D)J sequencing is then performed on the sorted cells. T cells from a negative control co-culture (typically APCs modified with HLA alone or with HLA and irrelevant antigen) are similarly sorted and analyzed by single-cell transcriptomics. Using the single-cell gene expression data, clusters of activated TILs can be identified. Paired, full-length TCR sequences from these activation clusters are then reconstructed into TCR expression plasmids and screened using the TCR screening methods described in FIG.2. Overall, FIG.3 illustrates parallel workflows with either ex vivo expanded TILs or sorted TILs are utilized to identify tumor-reactive TCRs with potential therapeutic applications in oncology. These general methods are applied to identify therapeutically useful TCRs in other disease indications (e.g., inflammation, auto-immune, etc.) with the appropriate starting material (e.g., a biopsy of inflamed colon from Crohn’s disease patient or a plaque of a patient with psoriasis). Arnold & Porter Ref. P35502WO00 In one aspect, the cells in the methods or systems described herein are mammal cells, such as human cell, mouse cell, or monkey cells. In another aspect, the cells in the methods or systems described herein are non-human primate cells. In one aspect, the reporter T cells and the APCs are from different species. In one aspect, the TCR expression cassette as disclosed herein comprises a TCR sequence reconstructed from TCR α and β chain sequences identified from TILs isolated from a tumor sample, and wherein the target neoantigen sequence and the matched HLA sequence are identified from the same tumor sample. Methods of identifying TCR sequences, antigen or neoantigen sequences, or the HLA sequences from a tumor sample or a normal reference sample are known in the art. Non-limiting examples of some commonly used methods are also disclosed herein. In one aspect, the TCR expression cassette is cloned into a non-viral gene transfer vector. In another aspect, the TCR expression cassette is cloned into a viral gene transfer vector. In a particular aspect, the non-viral gene transfer vector is a transposon. In one aspect, the isolated TILs are first expanded ex vivo and then co-cultured with APCs modified to express relevant HLA alleles and antigens obtained from the tumor sample. In a further aspect, a gene signature for identifying neoantigen reactive TCRs from ex vivo expanded TILs includes one or more gene(s) selected from the group consisting of XCL2, XCL1, IL2, CSF2, IFNG, CCL4, CCL4L2, TNF, CCL3, RGCC, TNFSF9, DUSP2, NFKBID, MIR155HG, NR4A3, EVI2A, CRTAM, ZBED2, FABP5, PIM3, NR4A1, IL10, TNFSF14, NR4A2, LINC00892, ZFP36L1, GZMB, MYC, SPRY1, KDM6B, EGR2, PHLDA1, PPP1R2, VSIR, REL, PRDX1, SLA, CYTOR, DDX21, IER3, PGAM1, NAMPT, HSP90AB1, IL23A, FAM107B, BCL2A1, ZEB2, ZBTB32, BTG2, GADD45B, RILPL2, SEMA7A, TGIF1, SRGN, RAN, CFLAR, MAT2A, SIAH2, PRNP, RNF19A, FASLG, NME1, EVI2B, HSPH1, NOP16, CSRNP1, and TAGAP. In one aspect, the reporter T cell disclosed herein is a primary T cell. In another aspect, the reporter T cell disclosed herein is from an immortalized T cell line. In a certain aspect, the reporter T cell disclosed herein is not a primary T cell. In certain aspects, the immortalized cell is a Jurkat cell or a SUP-T1 cell. In some aspects, the Jurkat cell is Jurkat NFAT. In one aspect, the endogenous T cell receptor of the cells is downregulated or knocked out, such as using routine methods in the art. In one aspect, the reporter T cell disclosed herein expresses any or all protein components of the TCR signaling complex or downstream signaling components. In a certain aspect, the reporter T cell expresses one or more components selected from the group consisting of CD3, CD4, CD8a, and CD8b. In further aspects, these protein components are modified, such as by mutation of one or more amino acids, to enhance their activities. Arnold & Porter Ref. P35502WO00 In one aspect, the antigen presenting cell (APC) disclosed herein is a classical professional APC. In another aspect, the APCs disclosed herein are artificial APCs. In one aspect, the APC described herein does not express an endogenous human HLA. An endogenous human HLA may be knocked out from an APC by methods known in the art, e.g., CRISPR. In a further aspect, the APC comprises the machinery for antigen presentation still and be amenable to modification by transient or stable transgene expression of HLAs. In another aspect, the APC is modified with human beta-2- microglobulin, human CLIP, human TAP1 or TAP2, or any other human-derived molecular components of antigen processing and presentation. In a certain aspect, the APC disclosed herein is not a professional APC. In certain aspects, the APC used in the methods or cell systems disclosed herein is a COS cell. In one aspect, the COS cell is a COS-7 cell. In one aspect, the APC is a 293- HEK cell. In another aspect, the APC is not a 293-HEK cell. In one aspect, the APC endogenously expresses an HLA allele. In another aspect, the APC does not express any endogenous HLA. In one aspect, the APC comprises one or more HLA expression plasmids. In one aspect, the APC expresses multiple HLA alleles in a single cell. In one aspect, the APC expresses a co-stimulatory molecule. Examples of the co-stimulatory molecules include, but not limited to, 4-1BBL, CD40, CD80, CD86, or OX40L. In one aspect, antigen or neoantigen sequences are introduced to the APCs either by genetic transfer to antigen encoding plasmids (e.g., Tandem Minigene (TMG) plasmids) or by the pulsing of peptide pools. A Tandem Minigene is an open reading frame comprising concatenated minigenes which encode about 25 aa each. The minigenes encode the mutated region of the gene as identified from sequencing (typically 12 aa upstream and downstream of the substituted aa residue). These minigenes are flanked at the 5' end with a LAMP1 signal peptide and 3' end DC-LAMP localization signal. One aspect of the APC system used is that multiple HLA alleles and antigens are screened within the same set of APCs, thus enable high-throughput assessment of hundreds to thousands of antigen:HLA combinations. In one aspect, a “matched” HLA sequence of a neoantigen sequence refers to an HLA sequence that is identified from tissue, blood, or tumor samples of the same patient as the TCR sequence and neoantigen sequence. In certain aspect, “matched” HLA sequence may also be used to indicate the HLA sequence of the HLA allele for which a particular TCR is restricted. In some aspects, the reporter T cell disclosed herein comprises a reporter system that is activated by the binding of a TCR to an antigen. Examples of the reporter systems are known in the art and include, but are not limited to, systems based on luciferase activity, fluorescence, or cytokine production. In one aspect of the present disclosure, the reporter T cells and the APCs are co-cultured at a ratio from about 16:1 to about 1:16. In one aspect, the reporter T cells and the APCs are co-cultured at a Arnold & Porter Ref. P35502WO00 ratio of about 4:1. In another aspect, the reporter T cells and the APCs are co-cultured at a ratio of about 8:1. In one aspect, the reporter T cells and the APCs are co-cultured for 1 to 48 hours. In one aspect, the reporter T cells and the APCs are co-cultured for at least one hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, or at least 10 hours. In another aspect, the reporter T cells and the APCs are co-cultured for about one hour, about 2 hours, about 3 hours, about hours, at least 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours. In one aspect, the TCRs disclosed herein interacts with and / or is specific for a peptide from a gene selected from a group comprising KRAS, RHPN2, GFRA2, NUP205, PCSK9, CEP85, HNRNPF, KDM1A, USP9X, LLGL1, ACO2, POLDIP3, EMC8, LCK, RCC1, VARS, LCK, ATP1A1, and CRYBG3. The present disclosure provides TCR sequences, or an antigen-binding portion thereof, that are identified or obtained by any of the methods disclosed herein. In one aspect, a TCR sequence comprises one or more of the sequences selected from the group consisting of SEQ ID NOs: 1-216 (the sequences provided in Tables 1-18). In another aspect, a TCR sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-216 (the sequences provided in Tables 1-18). In one aspect, the present disclosure provides a TCR comprising: (I) an α chain complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: (1+12n), an α chain CDR2 comprising the amino acid sequence of SEQ ID NO: (2+12n), and an α chain CDR3 comprising the amino acid sequence of SEQ ID NO: (3+12n); and (II) a β chain CDR1 comprising the amino acid sequence of SEQ ID NO: (7+12n), a β chain CDR2 comprising the amino acid sequence of SEQ ID NO: (8+12n), and a β chain CDR3 comprising the amino acid sequence of SEQ ID NO: (9+12n), where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In one aspect, the present disclosure provides an isolated or purified TCR. In another aspect, the present disclosure provides an isolated or purified polynucleotide encoding any of the amino acid sequences disclosed herein. In Tables 1 to 18, all of the sequences are fully human except for the “α chain with WT signal peptide and constant Cα” and “β chain with WT signal peptide and constant Cβ.” The sequences in these two sections are chimeric, containing the variable region sequences of the human TCRs combined with the constant region sequences of murine α and β chains. Arnold & Porter Ref. P35502WO00 Table 1. In some aspects, CLL000164-TCR134-1 interacts with and / or is specific for a peptide from gene EGFR. In some aspects, the peptide is from a neoantigen of EGFR and has the amino acid change L858R (in which position 858 of the EGFR protein is mutated from Leu to Arg). In some aspects, CLL000164-TCR134-1 interacts with and / or is specific for the neoantigen in the context of HLA- A*11:01. Arnold & Porter Ref. P35502WO00 Table 2. In some aspects, CLL000198-TCR97 interacts with and / or is specific for a peptide from gene KRAS. In some aspects, the peptide is from a neoantigen of KRAS and has the amino acid change G12C (in which position 12 of the KRAS protein is mutated from Gly to Cys). In some aspects, CLL000198- TCR97 interacts with and / or is specific for the neoantigen in the context of DRB1*07:01. Arnold & Porter Ref. P35502WO00 Table 3. In some aspects, CLL000275-TCR20-1 interacts with and / or is specific for a peptide from gene KRAS. In some aspects, the peptide is from a neoantigen of KRAS and has the amino acid change G12D (in which position 12 of the KRAS protein is mutated from Gly to Asp). In some aspects, CLL000275-TCR20-1 interacts with and / or is specific for the neoantigen in the context of HLA- A*11:01. Arnold & Porter Ref. P35502WO00 Table 4. In some aspects, CLL000166-TCR127 interacts with and / or is specific for a peptide from gene KRAS. In some aspects, the peptide is from a neoantigen of KRAS and has the amino acid change G12V (in which position 12 of the KRAS protein is mutated from Gly to Val). In some aspects, CLL000166-TCR127 interacts with and / or is specific for the neoantigen in the context of DRB1*07:01. Arnold & Porter Ref. P35502WO00 Table 5. In some aspects, In some aspects, CLL000048-TCR82 interacts with and / or is specific for a peptide from gene KRAS. In some aspects, the peptide is from a neoantigen of KRAS and has the amino acid change G12V (in which position 12 of the KRAS protein is mutated from Gly to Val). In some aspects, CLL000048-TCR82 interacts with and / or is specific for the neoantigen in the context of DRB1*10:01. Arnold & Porter Ref. P35502WO00 Table 6. In some aspects, CLL000048-TCR485-1 interacts with and / or is specific for a peptide from gene KRAS. In some aspects, the peptide is from a neoantigen of KRAS and has the amino acid change G12V (in which position 12 of the KRAS protein is mutated from Gly to Val). In some aspects, CLL000048-TCR485-1 interacts with and / or is specific for the neoantigen in the context of DRB1*10:01 or DRB3*02:02. Arnold & Porter Ref. P35502WO00 Table 7. In some aspects, CLL000032-TCR386 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R175H (in which position 175 of the TP53 protein is mutated from Arg to His). In some aspects, CLL000032- TCR386 interacts with and / or is specific for the neoantigen in the context of HLA-A*02:01. Arnold & Porter Ref. P35502WO00 Table 8. In some aspects, CLL000124-TCR10 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R248W (in which position 248 of the TP53 protein is mutated from Arg to Trp). In some aspects, CLL000124- TCR10 interacts with and / or is specific for the neoantigen in the context of HLA-B*57:01. Arnold & Porter Ref. P35502WO00 Table 9. In some aspects, CLL000105-TCR32-2 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R273C (in which position 273 of the TP53 protein is mutated from Arg to Cys). In some aspects, CLL000105- TCR32-2 interacts with and / or is specific for the neoantigen in the context of DPA1*01:03; DPB1*04:02. Arnold & Porter Ref. P35502WO00 Table 10. . In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R273C (in which position 273 of the TP53 protein is mutated from Arg to Cys). In some aspects, CLL000105- TCR67-1 interacts with and / or is specific for the neoantigen in the context of DPA1*01:03; DPB1*04:02. Arnold & Porter Ref. P35502WO00 Table 11. In some aspects, CLL000105-TCR70 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R273C (in which position 273 of the TP53 protein is mutated from Arg to Cys). In some aspects, CLL000105- TCR70 interacts with and / or is specific for the neoantigen in the context of DPA1*01:03; DPB1*04:02 & DPB1*02:01. Arnold & Porter Ref. P35502WO00 Table 12. In some aspects, CLL000105-TCR113 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R273C (in which position 273 of the TP53 protein is mutated from Arg to Cys). In some aspects, CLL000105- TCR113 interacts with and / or is specific for the neoantigen in the context of DPA1*01:03; DPB1*04:02 & DPB1*02:01. Arnold & Porter Ref. P35502WO00 Table 13. In some aspects, CLL000105-TCR304 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R273C (in which position 273 of the TP53 protein is mutated from Arg to Cys). In some aspects, CLL000105- TCR304 interacts with and / or is specific for the neoantigen in the context of DPA1*01:03; DPB1*04:02. Arnold & Porter Ref. P35502WO00 Table 14. PPP2R1A . In some aspects, the peptide is from a neoantigen of PPP2R1A and has the amino acid change R183W (in which position 183 of the PPP2R1A protein is mutated from Arg to Trp). In some aspects, CLL000236-TCR61 interacts with and / or is specific for the neoantigen in the context of DRB1*03:01. Arnold & Porter Ref. P35502WO00 Table 15. In some aspects, CLL000236-TCR78 interacts with and / or is specific for a peptide from gene PPP2R1A. In some aspects, the peptide is from a neoantigen of PPP2R1A and has the amino acid change R183W (in which position 183 of the PPP2R1A protein is mutated from Arg to Trp). In some aspects, CLL000236-TCR78 interacts with and / or is specific for the neoantigen in the context of DRB3*01:01. Arnold & Porter Ref. P35502WO00 Table 16. In some aspects, CLL000075-TCR12 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R282W (in which position 282 of the TP53 protein is mutated from Arg to Trp). In some aspects, CLL000075- TCR12 interacts with and / or is specific for the neoantigen in the context of DRB5*01:01. Arnold & Porter Ref. P35502WO00 Table 17. In some aspects, CLL000075-TCR51 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R282W (in which position 282 of the TP53 protein is mutated from Arg to Trp). In some aspects, CLL000075- TCR51 interacts with and / or is specific for the neoantigen in the context of DRB5*01:01. Arnold & Porter Ref. P35502WO00 Table 18. In some aspects, CLL000075-TCR82 interacts with and / or is specific for a peptide from gene TP53. In some aspects, the peptide is from a neoantigen of TP53 and has the amino acid change R282W (in which position 282 of the TP53 protein is mutated from Arg to Trp). In some aspects, CLL000075- TCR82 interacts with and / or is specific for the neoantigen in the context of DRB5*01:01. The present disclosure provides a polynucleotide encoding an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-216 (the sequences provided in Tables 1-18). Arnold & Porter Ref. P35502WO00 In one aspect, the TCR used herein comprises a sequence selected from the TCR Cα or TCR Cβ provided in Tables 19 and 20. Table 19. Amino acid sequences of TCR Cα regions. Arnold & Porter Ref. P35502WO00 Table 20. Amino acid sequences of TCR Cβ regions. Arnold & Porter Ref. P35502WO00 Non-limiting examples of HLA sequences and neoantigen peptide sequences are provided in Table 21 below. All the sequences are human. Table 21. Non-limiting examples of HLA sequences and neoantigen peptide sequences Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 Arnold & Porter Ref. P35502WO00 The present disclosure also provides a neoantigen / HLA complex, where the neoantigen and the HLA can be any of the neoantigen and HLA sequences disclosed herein or known in the art. In one aspect, the neoantigen comprises a sequence selected from the group consisting of SEQ ID NOs: 238 to 243 and 310 to 535 and where the HLA comprises a sequence selected from a group consisting of SEQ ID NOs: 301 to 309. The present disclosure also provides recombinant vectors expressing a TCR, or an antigen-binding portion thereof, that are disclosed herein. Production of recombinant vectors is well-known in the art, and a variety of vectors may be utilized, including viral or non-viral vectors. The present disclosure also provides recombinant vectors comprising a polycistronic expression cassette comprising a transcriptional regulatory element operably linked to a polycistronic polynucleotide. The present disclosure provides recombinant polycistronic nucleic acid vectors comprising at least three cistrons, wherein the first cistron encodes an α chain of an artificial T-cell receptor (TCR), the second cistron encodes a β chain of an artificial TCR, and the third cistron encodes a fusion protein that comprises IL-15 and IL-15Rα (e.g., mbIL15), or a functional fragment or functional variant thereof. In some aspects, the polycistronic nucleic acid further comprises a fourth cistron that encodes a marker protein (e.g., HER1t). In some aspects, the cistrons are separated by polynucleotide sequence that comprise 2A elements. Any of the TCR alpha or beta chain sequences disclosed herein may be used in the recombinant vectors. Non-limiting examples of the Arnold & Porter Ref. P35502WO00 2A element sequences, the IL-15 sequences, and the sequences are known in the art, e.g., as provided in PCT publication WO 2022 / 183167, which is incorporated by reference herein in its entirety. In some aspects, the recombinant vector comprises a polycistronic expression cassette, where the polycistronic expression cassette comprises a transcriptional regulatory element operably linked to a polycistronic polynucleotide that comprises: a first polynucleotide sequence that encodes a T cell receptor (TCR) alpha chain comprising an alpha chain variable (Vα) region and an alpha chain constant (Cα) region; a second polynucleotide sequence that comprises a first 2A element; a third polynucleotide sequence that encodes a TCR beta chain comprising a beta chain variable (Vβ) region and a beta chain constant (Cβ) region; a fourth polynucleotide sequence that comprises a second 2A element; and a fifth polynucleotide sequence that encodes a fusion protein that comprises IL-15, or a functional fragment or functional variant thereof, and IL-15Rα, or a functional fragment or functional variant thereof. As provided in PCT publication WO 2022 / 183167, the recombinant vector may comprise the five polynucleotide sequence in any order from 5’ to 3’. In some aspects, transgenes of the recombinant vector or any vectors used in the present disclosure are introduced into an immune effector cell via synthetic DNA transposable elements, e.g., a DNA transposon / transposase system, e.g., Sleeping Beauty (SB). SB belongs to the Tc1 / mariner superfamily of DNA transposons. DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner. Transposition is a precise process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome. Exemplary DNA transposon / transposase systems include, but are not limited to, Sleeping Beauty (see, e.g., US6489458, US8227432, the contents of each of which are incorporated by reference in their entirety herein), piggyBac transposon system (see e.g., US9228180, Wilson et al, “PiggyBac Transposon-mediated Gene Transfer in Human Cells,” Molecular Therapy, 15:139-145 (2007), the contents of each of which are incorporated by reference in their entirety herein), piggyBac transposon system (see e.g., Mitra et al., “Functional characterization of piggyBac from the bat Myotis lucifugus unveils an active mammalian DNA transposon,” Proc. Natl. Acad. Sci USA 110:234- 239 (2013), the contents of which are incorporated by reference in their entirety herein), TcBuster (see e.g., Woodard et al. “Comparative Analysis of the Recently Discovered hAT Transposon TcBuster in Human Cells,” PLOS ONE, 7(11): e42666 (Nov. 2012), the contents of which are incorporated by reference in their entirety herein), and the Tol2 transposon system (see e.g., Kawakami, “Tol2: a versatile gene transfer vector in vertebrates,” Genome Biol. 2007; 8(Suppl 1): S7, the contents of each of which are incorporated by reference in their entirety herein). Additional exemplary transposon / transposase systems are provided in US7148203; US8227432; US20110117072; Mates Arnold & Porter Ref. P35502WO00 et al., Nat Genet, 41(6):753- 61 (2009); and Ivies et al., Cell, 91(4):501-10, (1997), the contents of each of which are incorporated by reference in their entirety herein). In some aspects, the transgenes described herein are introduced into an immune effector cell via the SB transposon / transposase system. The SB transposon system comprises a SB a transposase and SB transposon(s). The SB transposon system can comprise a naturally occurring SB transposase or a derivative, variant, and / or fragment that retains activity, and a naturally occurring SB transposon, or a derivative, variant, and / or fragment that retains activity. An exemplary SB system is described in, Hackett et al., “A Transposon and Transposase System for Human Application,” Mol Ther 18:674- 83, (2010), the entire contents of which are incorporated by reference herein. In some aspects, the recombinant vector comprises a Left inverted terminal repeat (ITR), i.e., an ITR that is 5’ to an expression cassette, and a Right ITR, i.e., an ITR that is 3’ to an expression cassette. The Left ITR and Right ITR flank the polycistronic expression cassette of the vector. In some aspects, the Left ITR is in reverse orientation relative to the polycistronic expression cassette, and the Right ITR is in the same orientation relative to the polycistronic expression cassette. In some aspects, the Right ITR is in reverse orientation relative to the polycistronic expression cassette, and the Left ITR is in the same orientation relative to the polycistronic expression cassette. In some aspects, the Left ITR and the Right ITR are ITRs of a DNA transposon selected from the group consisting of a Sleeping Beauty transposon, a piggyBac transposon, TcBuster transposon, and a Tol2 transposon. In some aspects, the Left ITR and the Right ITR are ITRs of the Sleeping Beauty DNA transposon. The present disclosure further provides a population of cells that comprise the recombinant vectors disclosed herein. In one aspect, the recombinant vector or the polynucleotide is integrated into the genome of the population of cells. In one aspect, the cells are immune effector cells. In certain aspects, the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, B cells, mast cells, and myeloid-derived phagocytes. The present disclosure also provides a population of cells comprising a polycistronic expression cassette comprising: a. a first cistron comprising a polynucleotide sequence that encodes a fusion protein that comprises IL-15, or a functional fragment or functional variant thereof, and IL-15Rα, or a functional fragment or functional variant thereof; b. a second cistron comprising a polynucleotide sequence that encodes a TCR beta chain comprising a Vβ region and a Cβ region; and c. a third cistron comprising a polynucleotide sequence that encodes a TCR alpha chain comprising a Vα region and a Cα region. Arnold & Porter Ref. P35502WO00 In some aspects, the recombinant vectors disclosed herein comprise a polynucleotide sequence that encodes an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the TCR alpha or beta chain sequences provided in Tables 1-18 herein. The present disclosure provides a pharmaceutical composition comprising a population of cells as disclosed herein. In one aspect, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. It is contemplated that the TCRs identified by the methods disclosed herein, the antigen-binding portions thereof, populations of cells, and pharmaceutical compositions can be used in methods of treating or preventing medical conditions, such as cancer. Without being bound to a particular theory or mechanism, the TCRs, or the antigen-binding portions thereof, are believed to bind specifically to a mutated amino acid sequence encoded by a cancer-specific mutation, such that the TCR, or the antigen-binding portion thereof, when expressed by a cell, is able to mediate an immune response against a target cell expressing the mutated amino acid sequence. In this regard, an aspect of the disclosure provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, isolated pairs of TCR α and β chain sequences, antigen-binding portions thereof, or populations of cells described herein, in an amount effective to treat or prevent cancer in the mammal. Aspects of the disclosure include a cell or cells encompassed by the disclosure for use in the treatment of a medical condition, such as cancer or a premalignant condition, in a subject. The cells may be used for any type of cancer, including neuroblastoma, breast cancer, cervical cancer, ovary cancer, endometrial cancer, melanoma, bladder cancer, lung cancer, pancreatic cancer, colon cancer, prostate cancer, hematopoietic tumors of lymphoid lineage, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma, multiple myeloma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroid cancer, thyroid follicular cancer, tumors of mesenchymal origin, fibrosarcoma, rhabdomyosarcomas, melanoma, uveal melanoma, teratocarcinoma, neuroblastoma, glioma, glioblastoma, benign tumor of the skin, renal cancer, anaplastic large-cell lymphoma, esophageal squamous cells carcinoma, hepatocellular carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle-invasive cancer, seminal vesicle tumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, small bowel cancer, salivary gland cancer, cancer of uterus, cancer of testicles, cancer of connective tissue, prostatic hypertrophy, myelodysplasia, Waldenstrom's macroglobinaemia, nasopharyngeal, neuroendocrine cancer myelodysplastic syndrome, mesothelioma, angiosarcoma, Kaposi's sarcoma, carcinoid, oesophagogastric, fallopian Arnold & Porter Ref. P35502WO00 tube cancer, peritoneal cancer, papillary serous mullerian cancer, malignant ascites, gastrointestinal stromal tumor (GIST), or a hereditary cancer syndrome selected from Li-Fraumeni syndrome and Von Hippel-Lindau syndrome (VHL). The examples of the present disclosure are offered by way of illustration and explanation, and are not intended to limit the scope of the present disclosure. EXAMPLES EXAMPLE 1: WORKFLOW TO IDENTIFY TUMOR SPECIFIC TCRs FROM PATIENT DERIVED TIL AND DISSOCIATED PRIMARY TUMORS 1.1 TCR Identification and screening platform The fundamental basis for this unbiased TCR identification and screening platform is illustrated in FIG.1. Initially, single-cell gene expression data (e.g., 5’ GEX Analysis) from T cells is utilized to perform unsupervised clustering analysis by employing dimensionality reduction methods such as principal component analysis (PCA), t-distributed Stochastic Neighbor Embedding (tSNE), or Uniform Manifold Approximation and Projection (UMAP) (FIG.1, STEP 1). Merging the clustered single-cell gene expression analysis with paired, full-length TCR sequences then enables the identification of TCR clonotypes present in each of the distinct clusters. TCR sequences are then selected from the overall single-cell dataset based on frequency, cluster attributes, specific-gene expression signatures, or other criteria employed to increase the likelihood of obtaining TCRs with desired reactivity (i.e., antigen / HLA specificity) (FIG.1, STEP 2). Selected paired, full-length TCR sequences are then reconstructed in silico, from which, expression plasmids encoding the TCR α and β chains synthesized (FIG. 1, STEP 3). These TCR expression cassettes are then cloned into transposon or other non-viral gene transfer vector to enable quick translation into process development, manufacturing, and clinical applications. TCR-expression plasmids are then transiently expressed in a cell line (e.g., Jurkat or SUP-T1) or primary cell (e.g., human ex vivo expanded T cells) that will signal upon TCR recognition of cognate antigen:HLA complexes on the surface of antigen presenting cells (APCs) (FIG. 1, STEP 4). Antigen presenting cells (APCs) are classical professional APCs such as dendritic cells (DCs) or an artificial antigen presenting cell (e.g., COS-7 or 293-HEK). APCs either endogenously express the requisite HLA allele(s) or are transfected with HLA expression plasmids. Antigens are introduced to the APCs either by genetic transfer to antigen encoding plasmids (e.g., Tandem Minigene (TMG) plasmids) or by the pulsing of peptide pools. One aspect of the APC system used is that multiple HLA alleles and antigens are screened within the same set of APCs, thus enable high-throughput assessment of hundreds to thousands of antigen:HLA combinations. Co-culture of the TCR modified cells and APCs is then performed to identify reactive TCRs (FIG.1, STEP 5). Reactive TCRs are those that are found to recognize one of the antigen:HLA conditions tested. These reactive TCRs are then further evaluated in vitro to confirm the findings and Arnold & Porter Ref. P35502WO00 deconvolute the multiplexed HLA / antigen. Once all reactive TCRs are identified from a specimen, that binary outcome (reactive vs non-reactive) for each TCR can be mapped back to the initial gene- expression cluster analysis (FIG. 1, STEP 6). By mapping the reactive TCRs back to the gene- expression data, gene signatures or biomarkers which are enriched in the reactive TCR cell population are elucidated and used to further improve and refine the initial selection of TCRs for screening. Overall, this fundamental process is used to identify TCR sequences and their associated antigen and HLA specificity with a high level of confidence and accuracy from complex starting materials such as tumor tissues or blood samples. 1.2 Screening of TCRs from TILs In practice, the steps of the above-described workflow (FIG. 1) can be further broken down into critical processes as shown in FIG. 2 for screening of TCRs obtained from TILs. The process illustrated in FIG.2 correspond to FIG.1 STEPs 1-5. The workflow illustrated in FIG.2 is further broken into two parallel processes (indicated with either Alpha [i.e., A, B, C, etc.] or Numeric [i.e., 1, 2, 3, etc.] STEP designators) that diverge from a common starting point (STEP 1 / A) and converge at a common finishing point (STEP 8 / F). STEP 1 / A to STEP 6 illustrate the workflow from TILs isolation to generation of cells expressing TILs-derived TCRs. STEP 1 / A to STEP D illustrate the workflow from patient mutation and HLA calling to the generation of APCs expressing the patient matched HLA and mutation-derived antigens (e.g., neoantigens). STEP 1 – 6 (TCR): Initially, in STEP 1 / A, a tumor sample is obtained from a cancer patient (FIG. 2). This tumor sample is dissociated into a single-cell suspension and TILs are isolated by fluorescent activated cell sorting (FACS) by staining dissociated tumor samples for lymphocyte, T cell, and live cell markers (FIG.2, STEP 2). Single-cell transcriptomics is then performed on the sorted TILs to obtain gene expression and TCR V(D)J sequences (FIG.2, STEP 3). Bioinformatic analysis of the gene-expression data is used to cluster cells based on transcriptional similarities to aid in the selection of TCR sequences for in vitro evaluation (FIG.2, STEP 4). Once selected, TCRs are reconstructed in silico and synthesized in expression vectors (FIG.2, STEP 5) to enable transgenic expression of the TCRs in cells capable of forming a functional TCR complex with CD3 subunits and CD4 / CD8 co-receptors. These cells are engineered to express any or all necessary protein components of the TCR signaling complex or downstream signaling components. Moreover, these components are modified to further enhance their function in the platform (e.g., CD4 with amino acid substitutions at Q40Y, T45W, P48L, S60R, and / or D63R to enhance affinity to MHC-Class II). Wang et al.2011 PNAS, 108(38): 15960-15965. TCR expression vectors are transferred into the Reporter cells to generate Reporter TCR-T cells (FIG.2 STEP 6). Arnold & Porter Ref. P35502WO00 STEP A – D (Antigen / HLA): In parallel to STEPs 1 – 6, nucleic acids (DNA and RNA) can be extracted from the tumor sample (FIG.2, STEP 1 / A). Using Whole Exome Sequencing (WES) and RNA Sequencing (RNAseq) to generate genomic and transcriptional datasets, a bioinformatics pipeline is employed to determine somatic mutations present in the tumor as well as the patient’s germline HLA typing (FIG. 2, STEP B). Somatic mutations are ranked and concatenated so that TMGs and peptide pools can be synthesized (FIG.2, STEP C). These reagents provide the antigen component of the screening assay. Similarly, sequences of the called HLA alleles are synthesized in expression vectors to provide the HLAs necessary for the screening assay. Antigen presenting cells, such as COS-7, are then modified either by stable or transient transfection to express the requisite Class I or Class II HLA alleles either in single-plex or multiplexed within the same cells (FIG. 2, STEP D). Antigen is provided to the APCs either by transfection of relevant TMGs (either as plasmid DNA or in vitro transcribed RNA) and / or peptide pools containing antigens derived from the tumor’s somatic mutations identified. With both the HLA and antigen provided to the APCs, they are able to present peptide:HLA complexes to T cells in vitro. STEP 7 / E – 8 / F: Reporter cells expressing transgenic TCRs (FIG. 2, STEP 6) and antigen / HLA- modified APCs (FIG.2, STEP D) are co-cultured together at a pre-determined ratio of Reporter cells (E) to APCs (T), typically approximately 4:1 to 8:1 (FIG. 2, STEP 7 / E). Positive control wells containing PMA / Ionomycin or coated with H57-597 antibody (anti-transgenic TCR) with the TCR- modified Reporter cells are also set up. Negative control wells of Reporter cells alone or co-cultured with APCs modified with HLA-only, irrelevant antigens, or non-transfected are also set up. All conditions are typically evaluated in duplicate. After the co-culture period, reporter activity (i.e., luciferase activity) is quantified in each co-culture and control well (FIG.2, STEP 8 / F). For a given TCR, the reporter activity is compared across all antigen:HLA conditions evaluated to determine if there is a condition with increased reporter activity which indicates that the transgenic TCR recognized an antigen:HLA combination present in that well. Because initial screening multiplexes multiple HLA alleles and antigens, when there is specific TCR activity observed, STEP 7 / E and 8 / F are repeated using APCs modified with single HLA and antigens to elucidate the exact specificity of the TCR. Moreover, minimal epitopes can be determined using this co-culture method. Overall, this workflow enables the identification of TCR sequences and the empirical determination of specificity to selected antigens and HLA alleles. 1.3 Relationship between TCR-based and TILs-based screening methods FIG.3 illustrates the relationship between a TCR-based screening method (below dotted line) and TILs-based screening method (above dotted line). The TCR-based screening method is as described above in the description of FIG.2 wherein TCR sequences, somatic mutations, and HLA-typing is Arnold & Porter Ref. P35502WO00 obtained from primary tumor samples and utilized to screen selected TCRs for reactivity to tumor neoantigens using a co-culture reporter system. Similarly, TILs screening starts with a primary tumor sample obtained from a cancer patient. TILs are expanded from the tumor using standard TILs expansion methods (high-concentration IL-2, feeder cells, muromonab-CD3 (OKT3)). Expanded TILs are then co-cultured in an IFN-γ ELISpot with APCs modified to express the relevant HLA alleles and antigens identified from WES and RNAseq data from the tumor. This is performed in a similar plate layout to TCR screening where multiple HLA alleles and antigens are multiplexed in the same wells, thus increasing the throughput of the assay. Positive controls include PMA / Ionomycin. Negative controls include TILs alone, APCs alone, TILs + APCs without HLA and / or antigen, and no cells. After the overnight co-culture, cells are harvested from the IFN-γ ELISpot and the plate is developed to measure the number of spot-forming colonies (SFCs) of each well. The harvested TILs are also stained and evaluated for upregulation of 4-1BB or other activation molecules (e.g., OX40). TILs from co-culture conditions which produce increased numbers of SFCs and / or activation marker expression are then sorted for either total live T cells or for T cells expressing the activation marker. Single cell gene expression and TCR V(D)J sequencing is then performed on the sorted cells. T cells from a negative control co-culture (typically APCs modified with HLA alone or with HLA and irrelevant antigen) are similarly sorted and analyzed by single-cell transcriptomics. Using the single-cell gene expression data, clusters of activated TILs can be identified. Paired, full-length TCR sequences from these activation clusters are then reconstructed into TCR expression plasmids and screened using the TCR screening methods described in FIG.2. Overall, FIG.3 illustrates parallel workflows with either ex vivo expanded TILs or sorted TILs are utilized to identify tumor-reactive TCRs with potential therapeutic applications in oncology. These general methods are applied to identify therapeutically useful TCRs in other disease indications (e.g., inflammation, auto-immune, etc.) with the appropriate starting material (e.g., a biopsy of inflamed colon from Crohn’s disease patient or a plaque of a patient with psoriasis). EXAMPLE 2: DEVELOPMENT OF TCR SCREENING METHODS 2.1 General methods used in the Examples 2.1.1 Nucleic Acid Isolation and Assessment Tumor samples are obtained as either dissociated tumors or frozen tissue. To isolate DNA and RNA from dissociated cells, cells are processed using Qiagen AllPrep DNA / RNA Mini kit per the manufacturer’s protocol. To isolate DNA and RNA from tissue, frozen tissue is disrupted using a mortar and pestle and homogenized using QIAshredder homogenizers. The homogenized tissue is processed through the Qiagen AllPrep DNA / RNA Mini kit according to the manufacturer’s protocol. Arnold & Porter Ref. P35502WO00 Matched normal samples are obtained either as whole blood or as PBMCs. The Qiagen DNeasy Blood & Tissue kit is used to isolate DNA from 200 µL of whole blood per the manufacturer’s protocol and including the optional RNaseA. To isolate DNA and RNA from PBMCs, cells are processed using Qiagen AllPrep DNA / RNA Mini kit per the manufacturer’s protocol. Isolated nucleic acids are quantified by fluorescence spectrometry using the Life Technologies Qubit dsDNA BR Assay kit. Nucleic acids are assessed for fragment size by automated electrophoresis using the Agilent TapeStation 4150. Genomic DNA is assessed using the Agilent Genomic DNA ScreenTape System and RNA is assessed using the Agilent RNA ScreenTape System. 2.1.2 RNAseq To assess gene expression, RNA from tumors are processed through Illumina RNA Prep with Enrichment with an input of 100 ng. Pre-capture libraries are enriched via hybridization with the Illumina Exome Panel. Molarity of final libraries is determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA BR Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 76 on an Illumina NextSeqDx 550 using a 150 cycle High Output kit for a target coverage of 150 M reads. Libraries are subject to on-board demultiplexing to yield FASTQ files. The raw RNA-seq reads are aligned to the hg19 genome using Spliced Transcripts Alignment to a Reference (STAR) with the two-step procedure. Then Cufflinks is applied to the obtained BAM file to calculate the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) value of each gene. The FPKM values are converted to deciles to represent ten gene expression levels. 2.1.3 Single Cell RNAseq To sequence TCRs, dissociated tumor cells are processed through the Chromium Next GEM Single Cell 5’ Reagent Kit v2 from 10x Genomics targeting 10,000 cells when possible. The resulting cDNA is processed through the Chromium Single Cell Human TCR Amplification Kit VDJ per manufacturer’s recommendations. Molarity of final libraries is determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA BR Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 26 + 96 on an Illumina NextSeqDx 550 using a 150 cycle High Output kit for a target coverage of 5000 reads per cell for VDJ and 20,000 reads per gene expression library. Raw bcl files are yielded. 2.1.4 Analysis of the 10x Gene Expression (GEX) and VDJ Sequencing Data Arnold & Porter Ref. P35502WO00 The GEX and VDJ sequencing data are preprocessed using the CellRanger toolkit (version 5.1) provided by 10X Genomics. The BCL files from the Illumina sequencer are converted to raw FASTQ files. The FASTQ files for the GEX and VDJ experiments are processed separately. GEX reads are aligned to the human GRCh38 reference genome. Cell barcodes assignment and UMI counting are then performed to create a single-cell gene expression matrix. Doublets and cells with >10% mitochondria gene counts are filtered out in the study. Then the raw read counts are normalized and scaled using Seurat. About 2,000 highly variable genes are identified using the FindVariableGenes module. Next, principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) are performed for dimension reduction and a shared nearest neighbor (SNN) algorithm is applied to cluster the cells. The raw VDJ reads are assembled into contigs using a graph-based algorithm with the aid of the pre- built reference sequence from the IMGT database. Cells with identical productive V(D)J transcripts are placed into a same clonotype. 2.1.5 Integrating the 10X GEX and VDJ data For each TCR clonotype, the corresponding cells in V(D)J are projected to the identified clusters in the GEX data. The full-length FASTA sequences of both the TRA and TRB chains, as well as the amino acid sequences of the CDR3 regions for each clonotype are also reported. 2.1.6 Whole Exome Sequencing Whole exome sequencing experiments (WES) are performed for the peripheral blood and the tumor tissue of each patient. Somatic single nucleotide variants (SNVs), short insertions and deletions (indels), copy number alterations (CNAs), class I and II HLA types are detected by comparing the tumor versus the normal sequencing data. Each mutant peptide is predicted in silico if it can give rise to a neoantigen. Bulk RNA-Seq is also performed on the tumor tissue to quantify the expression level of each gene. Between 100 and 200 ng of genomic DNA is fragmented enzymatically for 100 bp reads using the Agilent SureSelect Enzymatic Fragmentation Kit. Fragmented DNA is processed through the SureSelect XT HS2 DNA System using v7 probes. Pre-capture libraries the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (BR). A total of 1000 ng of pre-capture library is input into hybridization. Molarity of final libraries is determined using the fragment size between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay Kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 101 on an Illumina NextSeqDx 550 using Arnold & Porter Ref. P35502WO00 a 300 cycle High Output kit for a target coverage of 200x and 100x for tumor and normal libraries, respectively. Libraries are subject to on-board demultiplexing to yield FASTQ files. 2.1.7 Jurkat NFAT cell generation Jurkat NFAT cells are infected with Lentivirus (pGenLenti-CD8A_P2A_CD8B_IRES_Puro) and then selected with puromycin (0.2 μg / mL). Peripheral Blood Mononuclear Cells (PBMCs) from 3 different donors are irradiated and seeded in a 96 multiwell U bottom plate at 100k / well. Puromycin selected stable pools of peptides are seeded at 0.5 cell / well on top of irradiated PBMCs (96 multiwell plates) to generate single clones. Single clones are cultured for 1 week with IL-2 (50 IU / mL) and Phytohaemagglutinin-L (PHA-L) (0.25 μg / mL). Second week cell medium is replaced with 100 IU / mL of IL-2. Grown back clones are evaluated for higher CD3 / CD8 expression and higher luciferase signal / noise ratio (PMA / Ionomycin vs untreated). Clone #41 (having >95% CD8 expression and >150 signal to noise ratio) is selected. In order to better screen class II TCRs, #41 clone is infected with CD4 lentivirus (pGenLenti-CD4_IRES_Puro) to boost CD4 expression. After lentivirus infection CD4 expression is increased to more than 95%. 2.1.8 Mutation Calling, HLA-typing and neoantigen prediction The raw WES reads are aligned to the human hg19 reference genome using Burrows-Wheeler Aligner (BWA) (version 0.7.5a). Duplicate reads are marked using Picard‘s ‘‘MarkDuplicates’’ module. The ‘‘IndelRealigner’’ and ‘‘BaseRecalibrator’’ modules of the Genome Analysis Toolkit are then applied to the obtained BAM files for indel realignment and base quality recalibration. In our workflow, five mutation detection algorithms are applied to the obtained BAM files: Mutect, MuSE, Varscan2, Mutect2 and Strelka, where all of them are used to detect single nucleotide variants (SNVs) and the last three are used to detect short insertions or deletions (indels). Only Mutect2 is used to detect multi-nucleotide variants (MNVs). An SNV is reported if it can be detected by at least three out of the five algorithms. An indel is reported if it can be detected by any of the indel callers. The detected SNVs are annotated with ANNOVA and VEP and compared with public databases such as dbSNP (Sherry et al., 2001), 1,000 genome (http: / / www.1000genomes.org / ) and ESP6500 (http: / / evs.gs.washington.edu / EVS / ). To ensure accuracy, the following criteria is used to filter the SNV and indel list: allele frequency (AF) > 0.05; the coverage is at least 20 reads for the tumor and 10 for the normal; the AF from the normal sample <0.02. Only non-synonymous SNVs, in-frame and frameshift indels are kept for further analysis, as these mutations change the amino acid sequences of the genome and are likely to give rise to neoantigens. For each mutated amino acid that results from a somatic SNV or indel, up to 12 bases are extended to the left and to the right and a peptide sequence of length at most 25 bases (25-mer) is obtained. Since a neoantigen’s length ranges from Arnold & Porter Ref. P35502WO00 8-25 bases, it ensures that any potential neoantigen resulting from the mutation is a subsequence of the 25-mer. The Sequenza algorithm is used to detect the somatic copy number alterations (CNAs) and tumor purity. Optitype and HLA-VBSeq are applied to infer the class I and II HLAs respectively. The 25-mer peptide sequences and the HLA types of each patient are input together to netMHCpan4.1 to predict if the mutant amino acids can lead to a neoantigen. 2.1.9 TCR Plasmid Assembly Approximately 50 T Cell Receptors (TCRs) are selected per patient by a still-developing method according to their abundance in the assessed sample and the association of their corresponding cells with clusters according to gene expression. TCRs are selected considering whether (1) a cluster expresses CD8 or CD4, (2) the function of genes differentially expressed by that cluster, and (3) the abundance of each TCR. Each analysis yields more than 1000 TCR clonotypes, and these are reduced to approximately 50 clonotypes to move on to synthesis. Each cluster is defined by differentially expressed genes. Each cluster is made up of cells, and each cell is associated with a TCR clonotype. The highest abundance clonotypes in every cluster are included such that a total of approximately 50 clonotypes are synthesized across all clusters, giving preference to clonotypes from clusters that are associated with immune response genes. Similarly, if a patient sample has a Class I or Class II HLA allele that is common in the population, preference is given to clusters that more highly express either CD8 or CD4, respectively. 2.1.10 Create beta and α gene sequences in silico The raw beta sequence is curated such that any sequence 5’ of the start of the Variable (V) region is replaced with a NheI site, and the entire constant region is replaced with a BspI site. For the α chain, the sequence 5’ of the start of the V region is replaced with an XmaI site, and the constant region is replaced with a SacII site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used codons for the same amino acid throughout the beta and α open reading frames. NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing codons with other codons encoding the same residues. 2.1.11 Plasmid Assembly Each α and β gene are synthesized and subcloned into pZT2 plasmids using the synthesized restriction sites (NheI and BspEI for beta and XmaI and SacII for α) by GenScript. The final plasmid is prepared in 10 mMTris-HCl, pH 8.0, 1 mM EDTA (TE) with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. Arnold & Porter Ref. P35502WO00 2.1.12 Tandem Minigene Plasmid Assembly When more than 150 non-synonymous mutations are reported for a tumor, the mutations are sorted by gene expression and only the top 150 expressed non-synonymous mutations are included. 2.1.13 TMG Assembly in silico Amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. BamHI, EcoRI, NotI and NheI restriction sites are removed by replacing codons with others encoding the same residues. A set of up to 15 sequences are concatenated together into one open reading frame called a tandem minigene (TMG). The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S)) and has EcoRI site = GAATTC) is added to the 5’ end of the TMG gene, and the nucleotide sequence AAG GAT CCC (codes for K / D / P and has BamHI site = GGATCC) is added to the 3’ end of the TMG gene. 2.1.14 TMG Plasmid Synthesis The TMG, together with the added restriction sites, is synthesized and cloned (GenScript) into masterTMG_pcDNA3.1(+) mammalian expression vector with EcoRI (5’) and BamHI (3’) in frame with existing start and stop codons. The final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 2.1.15 Peptide Design and Synthesis The same amino acid sequences are synthesized up to 25 aa in length with crude quality (GenScript). For peptide sequences longer than 25 residues, multiple peptides of 25 aa in length are synthesized with start sites at 5 aa intervals. For the last window, the last 25 residues are synthesized in place of a peptide shorter than 25 aa. 2.1.16 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each identified allele are downloaded from the IPD-IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. The sequence is then synthesized with BamHI and Kozak sites at the 5’ end and an EcoRI and stop codon on the 3’ end (GenScript). The synthesized sequence is cloned into pcDNA3.1(+) using BamHI and EcoRI. Final plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 2.1.17 Neoantigen specific TCR screening process. On Day 1, COS-7 cells are seeded at 20,000 cells per well (96 multiwell plate) overnight in 37 ℃ incubator. On Day 2, cell medium is replaced with antibiotic-free DMEM medium before transfection. 150 ng of tandem minigene (TMG) and 300 ng HLA plasmids are transfected using lipofectamine 2000. Three to four HLA plasmids (75-100 ng each) are transfected together in one well to enhance screen efficacy. Each condition includes one or two HLA types including A, B, C, Arnold & Porter Ref. P35502WO00 DP, DQ and DR. Twenty-five μL of OptiMEM medium is used to dilute either DNA plasmid (450 ng total) or Lipofectamine (0.6 μL) for each well. DNA tube (A) or lipofectamine tube (B) are mixed well separately and incubated for 5 minutes at room temperature (RT). Tube B is then added to tube A, and the mixture is incubated for 20 minutes at RT. Transfection mix (50 μL) is added to each well and cells are cultured overnight in a 37 ℃ incubator. Jurkat NFAT reporter cells are counted and seeded at 1 million / mL with fresh RPMI1640 complete medium overnight to enhance electroporation efficacy (10% Fetal Bovine Serum (FBS) and 1% Pen / Strep). On Day 3, NeonTMtransfection system is set up in the Biosafety Cabinet (BSC) with program 1,325v, 10 mins, 3 Pulse. 5 mLs of RPMI without Pen / Strep is added into T25 flask and labeled with corresponding murine-TCR (mTCR) number. Flasks are pre-warmed in 37 ℃ incubator while preparing electroporation (EP) Jurkat NFAT cells are spun down at 100g for 10 minutes. Cells are washed with PBS and cell numbers are measured with NC3000.6 million Jurkat NFAT cells are loaded into 15 mL conical tubes and spun down at 100g for 10 minutes. During centrifugation, Buffer R (110 μL each) are prepared in Eppendorf tubes and Electrolytic Buffer E2 (3 mL each) are aliquoted in Neon transfection system tubes. Eleven microliters of mTCR plasmids (2 mg / mL) are added to corresponding Eppendorf tubes containing Buffer R and mixed well. The mixture of DNA and Buffer R is loaded to the Neon tubes using specialty Neon pipette tips. When EP is successful, “COMPLETE” shows on the screen in a few seconds after “START” is clicked. Buffer R / DNA mixture is transferred immediately into a T25 flask containing antibiotic-free RPMI medium. H57- 597 antibody is utilized to coat plate (1 μg / mL, 25 μL / well) overnight to measure EP efficacy next day. For parsing experiment, peptide is prepared at 50 mg / mL and pulsed at 10 μg / mL to identify neoantigen specificity. 2.1.18 Co-culture On Day 4, Jurkat NFAT-mTCR cells are counted and co-cultured (100k / well) on top of transfected COS-7 cells for 4-5 hours. As control, Jurkat NFAT-mTCR cells are also plated on H57 coated plate to perform mTCR functional test. After 4-5 hours incubation, cells from 96 multiwells are transferred to U bottom plates and spined down at 400g for 5 minutes. Cells are then lysed with 1X passive lysis buffer (100 μL / well) for 15 minutes on an orbital shaker at RT. 50 μL cell lysis are loaded onto OPTIPLATE as well as 100 μL of Promega Luciferase substrate. Luciferase activity is measured immediately with BioTek reader. Jurkat NFAT cells mTCR expression is measured with flowcytometry using antibody cocktail CD3, CD4, CD8A, CD8B and H57. HLA expression of COS- 7 cells is measured with flowcytometry using antibody cocktail HLA-A2, HLA-DP, HLA-DQ and HLA-DR. Arnold & Porter Ref. P35502WO00 2.1.19 Neoantigen specific Tumor Infiltrating Leukocytes (TILs) identification process. On Day 1, COS-7 cells are seeded at 20,000 cells per well (96 multiwells) overnight in 37 ℃ incubator. TILs are thawed and recovered with IL-2 at 3000 IU / μL. On Day 2, cell medium is replaced with antibiotic-free DMEM medium before transfection. A Transfection Mix containing 150 ng of tandem minigene (TMG) and 300 ng HLA plasmids are prepared and transfected into the COS-7 cells using lipofectamine 2000. Two HLA plasmids (150 ng each) are transfected together in one well to enhance screening sensitivity. Each condition only includes one HLA type (A, B, C, DP, DQ and DR). 25 μL of OptiMEM medium is used to dilute either DNA plasmids (450 ng total) or Lipofectamine (0.6 μL) for each well. DNA tube (A) and lipofectamine tube (B) are mixed well and incubated separately for 5 minutes at room temperature (RT). Tube B is added to tube A, and the mixture is incubated for 20 minutes at RT. Transfection mix (50μL) is added to each well and cells area cultured overnight in a 37 ℃ incubator. On Day 3, 96 multiwell plates containing COS-7 cells are replaced with fresh medium containing peptide pools. Peptide pools are created by combining the peptides from a given TMG into a pool of equivalent mass ratios of each peptide. Peptides are prepared at 50 mg / mL and pulsed at a final concentration of 10 μg / mL (in well which contains media and COS-7 cells). Peptide pools consist of the synthesized peptides that correspond to the minigenes within a given TMG (i.e., if TMG-1 contains minigenes encoding Peptide 1, Peptide 2, and Peptide 3, a peptide pool containing Peptides 1-3 would be prepared). ELISpot plates are incubated with 70% EtOH (0.22 μm filter, 50 μL / well) for less than 2 mins in the Biosafety Cabinet (BSC) at RT. ELISpot plates are washed 5 times with 200μL / well with sterile PBS. Anti-interferon gamma capture antibody (1-D1K) is mixed with PBS (100 μL / 10 mL / plate) and added 100 μL / well. COS-7 cells are incubated overnight at 4℃. On Day 4, ELISpot plates are washed 5 times with PBS (200 μL / well). Plates are blocked with complete RPMI media (10% FBS), 100μL / well at room temperature for 1 hour. During the one hour, COS-7 cells are harvested from 96 multiwells using trypsin. TILs are counted and resuspended at 400k / mL. Medium is poured out from the ELISPOT plate.50 μL of medium, 100 μL of COS-7 cells, and 100 μL of TILs (40,000 cells) are added sequentially to the ELISpot plates. Plates are transferred to 37℃ incubators with 5% CO2, and incubated for 18-24 hours. On Day 5, the following ELISpot reagents are prepared: 1) IFN-γ biotinylated 7-B6-1 antibody diluted in PBS + 0.5% FBS, then filtered with 0.22 µm filter, and 2) wash buffer (PBS + 0.05% Tween-20). Cells of each well are mixed via pipetting, then 200 μL of cells are carefully transferred from ELISpot plate to a new 96 U-bottom plate. The cells are later stained for phenotyping using cocktail CD3, CD4, CD8 and 41BB with flow cytometry. ELISpot plates are washed 3 times using buffer made by combining PBS with 0.05% tween 20 in the big basin. Anti-IFN-γ antibody (Biotinylated 7-B6-1 biotin) is diluted with PBS and 0.5% FBS then filtered with 0.22μm filter (10 μL / 10 mL / plate, 100 Arnold & Porter Ref. P35502WO00 μL / well). Plates are left at room temperature for 2 hours in the dark covered with aluminum foil. Plates are washed 5 times using PBS with 0.05% tween 20. Streptavidin-ALP is diluted in PBS with 0.05% FBS (10 μL / 10 mL) and added at 100 μL / well at room temperature for 1hr in the dark covered with aluminum foil. Plates are washed 5 times with PBS.5-Bromo-4-chloro-3-indonyl phosphate, X- phosphate, XP, Nitro-blue-tetrazolium chloride, (BCIP / NBT) Alkaline Phosphatase substrate solution is filtered (0.45 μm) and added at 100 μL to every well. Plates are incubated at room temperature for 10-20 mins until distinct spots can be seen. Tap water is used to wash the plates gently but extensively, then the plates are left out until completely dry. Plates are analyzed using the ELISpot reader. HLA expression of COS-7 cells are measured with flowcytometry using antibodies cocktail HLA-A2, HLA-DP, HLA-DQ and HLA-DR. 2.2 Modification of Jurkat Reporter Cells 2.2.1 Adding CD8 and CD4 Lentivirus are prepared using HEK-293Ta cells and Jurkat NFAT cells are transduced. Jurkat NFAT cells are first transduced with CD8 Lentivirus and selected with 0.2 µg / ml puromycin to generate Jurkat NFAT_CD8Lenti cells. Subsequently, Jurkat NFAT_CD8Lenti cells are infected with CD4 Lentivirus and selected with 0.3 µg / ml puromycin. After 4 days selection with 0.3 µg / ml puromycin is adjusted back to 0.2 µg / ml for maintenance. Cells are harvested and stained with CD3, CD4, CD8A and CD8B. Jurkat NFAT parental cells are negative for CD8 (99.16% CD8 negative) within the CD3+ cell population. Results shown in FIG.4 demonstrate that Jurkat LentiCD8 cells have 43.57% CD8A expression and 43.56% CD8A and CD8B double positive expression. Single clones are generated from Jurkat NFAT_CD8Lenti pool. Peripheral Blood Mononuclear Cells (PBMCs) from 3 different donors are irradiated and seeded in 96-multiwell U bottom plates at 100k cells / well. Puromycin selected Jurkat NFAT_CD8Lenti stable pool cells are seeded at 0.5 cell / well on top of irradiated PBMCs to generate single clones. Single clones are cultured for one week with IL-2 (50 IU / ml) and phytohaemagglutinin (PHA; 0.25µg / ml). During the second week cell medium is replaced with 100 IU / ml of IL-2. Grown back clones are evaluated for CD8A and CD8B expression and luciferase signal / noise ratio (PMA / Ionomycin vs untreated). Clones 2, 15, 19, 41 (>95% CD8 expression and >150 signal to noise ratio) are the best clones with higher CD8 expression and higher luciferase activity signal to noise ratio (FIG.5). To better improve screening efficacy, clone #41 is selected from the Jurkat NFAT CD8Lenti pool. Flow analysis is performed to confirm the expression of CD8a and CD8b. Cells are stained with CD3, CD4, CD8A, and CD8B. As shown in FIG. 6, CD8A and CD8B double positive population is increased from 46.74 % in the Jurkat NFAT_CD8Lenti pool to 95.74 % in the #41 clone. This substantial increase of CD8 expression would allow us to capture better neoantigen reactive Class I Arnold & Porter Ref. P35502WO00 TCRs. However, the CD4 expression is still not optimal. To improve the CD4 expression in Jurkat NFAT CD8Lenti #41, the cells are infected with lentivirus (pGenLenti-CD4_IRES_Puro). Flow analysis is then performed to evaluate the expression of CD4 by these cells. Cells are stained with CD3, CD4, CD8a, and CD8b. As shown in FIG. 7, the CD4 positive population is increased from 68 % to 97.8 %. CD8 expression is not changed significantly. Now upgraded #41 clone is both high CD8 and CD4 which improves TCR screening sensitivity. 2.2.2 Reporter activity time course A time course study is performed to determine the best time point to harvest the co-culture. Jurkat cells are seeded in RPMI complete medium at 200k cells / well in 96-multiwell plates. Cells are treated with 50 ng / ml PMA and 1 µg / ml Ionomycin for 2.5, 3.5, 4.5 and 5.5 hrs. Cells are harvested and lysed with passive lysis buffer (Promega) at room temperature for 15 minutes.50 µls of cell lysis is mixed with 100 µl of luciferase substrate (Promega). Luciferase signal intensities are detected with Luminometer. Luciferase activity folds changes are calculated by dividing PMA / Ionomycin treated condition to vehicle control treated conditions. As shown in FIG. 5, 4-5 hours is the best time to harvest cells since luciferase signals start to drop for the CD8Lenti_CD4Lenti pool. Data is shown in FIG.8. 2.3 Optimization of transfection conditions in COS-7 cells Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwell plates. Day 2: COS-7 cells are transfected in each well with 150 ng of TMG1 or TMG2 and 75 ng of HLA A*11:01 and 75 ng of HLA A*02:01. Day 3: NEON transfection system is set up the following day and 5 million cells are electroporated with either TCR002 or TCR010 monkey-TCR (mTCR). Day 4: Jurkat cells are harvested and seeded on top of either transfected COS-7 cells or COS-7 cells stably expressing HLA A*11:01 or HLA A*02:01. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. As shown in FIG.9A, TCR002 TCR electroporated cells specifically recognized TMG2 (contained KRAS G12V mutation). As shown in FIG. 9B TCR010 TCR specifically recognized TMG-1 (contained R175H mutation) transfected COS-7 cells as expected. Transient transfection works better than stable pools in both TCR002 and TCR010 TCRs. In addition, Jurkat NFAT CD8Lenti has higher fold induction compared with Jurkat NFAT parental cells in both TCR co- culture experiments demonstrating the relevance of overexpressing CD8 in Jurkat NFAT cells for Class I TCRs. Jurkat NFAT electroporated with TCR002, TCR010 cells are analyzed using flowcytometry to detect the percentage of cells with mTCR expression. Cells are stained with CD3, CD4, CD8a, CD8b and mTCR antibodies. As shown in FIG. 10, cells express similar level of mTCR in Jurkat NFAT CD8Lenti cells compared with Jurkat NFAT parental cells. Over 90% of cells are viable in all six Arnold & Porter Ref. P35502WO00 cell lines on the next day after electroporation suggesting the NEON electroporation system could provide highly viable T cells with sufficient percentage of mTCR expression (~20%). This would allow co-culture experiments to be performed the next day without wasting time to recover cells. CD8 co-receptor expression did not improve TCR expression therefore suggesting that the addition of CD8 improved the TCR-peptide:MHC interaction to improve the reporter activity. To examinate the reliability of JNR / COS co-culture system, several exemplary TCRs are tested. Flow analyses are performed to evaluate the mTCR expression level in 11 TCRs, and cells are stained with CD3, CD4, CD8a, CD8b and mTCR antibodies. As shown in FIG.11, mTCR expression varied from 8-35% (9 of 11 TCRs expressed above 15%) when cells are gated on CD3+. Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150 ng of TMGs and 150 ng of HLAs (each 25 ng). Day 3: NEON transfection system is set up the following day and 5 million cells are electroporated with each TCR plasmid. Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours of co-culture, cells are harvested, and luciferase activity is measured. Luciferase activity fold change (FC) is calculated based on cells without electroporation using TCR. TCR specific HLAs and matched neoantigens or TMGs are listed on the table below. (FIG.12) Based on the statistical analysis, 9 of 11 TCRs from TCR library are confirmed with specificity against matched TMGs (i.e., a match TMG contained mutations specific to the TCR). No matched TMGs are irrelevant TMGs where no specific mutations are contained in the plasmid to serve as negative control. To troubleshoot the TCRs with low reactivity experiments are designed by transfecting different amounts of HLA plasmids. As you could see from FIG.13, the signal to noise ratio is significantly increased when COS-7 cells are transfected with 75 ng of plasmids compared with 25 ng. This has been observed in all 6 TCRs which show relatively low reactivity based on FIG.12. 2.4 Optimization of peptide pulsing conditions in COS-7 cells Peptide pulsing is tested with certain TCRs. COS-7 cells are pulsed with peptides either overnight or for 2 hours. Long peptides, as well as short peptides are used. 11 TCRs are electroporated for optimization studies. As shown in FIG. 14, three of 7 class I TCR are able to detect long peptide; however, all of the 7 class I TCRs are also able to react to short peptides. In addition, 3 of 4 Class II TCRs are reactive more to long peptides but not short peptides. In conclusion, overnight pulsing of peptide showed stronger signal compared with 2 hours. Class I TCRs recognize short peptide better and Class II TCRs recognize longer peptide better. The COS-7 peptide pulsing worked with most of the TCRs tested which demonstrates that COS-7 cells can be used to identify specific neoantigens in the reactive TMGs. Arnold & Porter Ref. P35502WO00 2.5 Development of assay controls 2.5.1 Anti-TCR Coated Plate Positive Control On the day of electroporation, H57 antibody is coated on the 96 multiwell plate overnight at 4℃ as a positive control. On the next day, Jurkat cells are seeded on the plate for 5 hours. Luciferase activity fold change (FC) is calculated based on cells without electroporation using TCR. Some of the TCRs demonstrated comparable levels of activation as H57 such as TCR002, TCR004, TCR001, TCR007 and TCR008 (FIG.15). Some TCRs including TCR011, TCR009 and TCR006 are not activated as much with matched TMG as they are with H57 coating suggesting that the TCR is successfully electroporated, but not fully activated. This might be due to the sub-optimal formation of HLA- neoantigen-TCR complex. A scatter blot is generated using H57-coated Jurkat NFAT cells luciferase activity and mTCR expression based on the flow analysis. These cells are 12 cell lines shown in FIG. 16. Luciferase activity is positively associated with mTCR expression with R2value of 0.8753 suggesting that luciferase activity from H57 coated plate could serve as optimal control besides flowcytometry for TCR expression and biological function. 2.6 Conclusion The series of data described in this example illustrate the development of a method and cell lines that are used to screen TCRs isolated from primary T cells against various combinations of HLA and antigens. Optimal reporter activity is observed between 4-5 hours after stimulation. It is observed that addition of CD4 and CD8 co-receptors to the reporter cells improved TCR-mediated reporter activity. Isolation of a single CD8-modifed report cell line clone, Clone #41 is achieved which improved the sensitivity of the assay to detect reactive TCRs. Development of an assay positive control, using plate-bound anti-TCR antibody, proved to be a robust control for functional TCR expression and correlated highly with the frequency of TCR expression measured by flow cytometry. Modulation of HLA plasmid amounts in the transfection reaction is found to improve the antigen- presentation and subsequent sensitivity of detecting reactive TCRs in this assay. Overall, the example illustrates the development and optimization of a high-throughput TCR screening platform to enable identification of TCR sequences, antigen-specificity, and HLA-restriction which could be used to identify novel therapeutic TCRs derived from primary tissues. EXAMPLE 3: PATIENT 0164 TCR SCREENING 3.1 Mutation and HLA Calling 3.1.1 Sample Demographics Patient 0164 is a female with stage IV lung cancer. Patient 0164’s tumor specimen is collected when the patient was 69 years old and prior to the start of treatment for the cancer diagnosis. A specimen Arnold & Porter Ref. P35502WO00 of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 3.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 3.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). Arnold & Porter Ref. P35502WO00 3.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 1 hotspot mutations. The mutations are contained on topspot TMGs 5. HLA profiling of the patient reveals HLAs as shown in FIG.17. 3.2 Design and Construction of Synthetic Reagent 3.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 3.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 3.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 3.3.1 Cell preparation Patient 0164 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 80% and a final Arnold & Porter Ref. P35502WO00 concentration of 800 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 10,000 cells. 3.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields 2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves >=Q30 quality score. 3.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 3.4 TCR reconstruction 3.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 423 clonotypes where 281 clonotypes contained exactly one beta chain and one alpha chain. Clonotype frequency ranges from 1 to 28 cells with 48 clonotypes observed in more than one cell and 1 clonotype observed in more than 10 cells. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 27 TCRs for class I. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open Arnold & Porter Ref. P35502WO00 reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 3.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 3.5 Patient 0164 TCR Screening 3.5.1 Experimental design and methods Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 27 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There are 1 TMG used for the relevant mutation, and 27 TCRs are picked from 10x single cell sequencing for this patient 0164. In addition, this patient has 2 HLA-A, 2 HLA-B, 2 HLA-C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmids. Table 22 below summarizes the screening strategy for Patient 0164. Primary screening is performed by separating class I and class II TCRs. Class I TCRs are screened against HLA A, B or C. Class II TCRs are screened against HLA DP, DQ or DR. EGFR L858R is the only hotspot mutation that this patient has. Topspot TMG5 has this mutation and is used for screening. Table 22. Screening strategy for Patient 0164. 3.5.2 Screening Results As shown in FIG.17, TCR 134-1 is specific to the combination of TMG5 and an HLA allele in either locus HLA-A, but not HLA B or HLA C. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and TMG5 and HLA-A*11:01 is found to be the Arnold & Porter Ref. P35502WO00 specific HLA restricting 0164-TCR134-1 (FIG.18). To further define the minimal epitope of 0164- TCR134-1 an online peptide prediction tool is used to predict potential candidates with minimal residue of peptide likely to bind with HLA-A*11:01. To determine which mutation in TMG5 is being recognized by 0164-TCR134-1, EGFR L858R mutant and wildtype peptide are used for coculture. The wildtype form of the EGFR L858R and DMSO control have lower luciferase activity after co- culture, suggesting that EGFR L858R plays critical role in 0164-TCR134-1 reactivity (FIG.19). 3.6 Conclusion The series of data described in this example illustrates the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0164, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA expression vectors encoding eighteen TCRs from Patient 0164 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0164-TCR134-1 is found to be specific for the EGFR L858R neoantigen with minimal epitope KITDFGRAK when presented in the context of HLA-A*11:01. Overall, these data demonstrate a process by which neoantigen-specific TCRs can be identified and functionally validated using a high-throughput TCR screening method. EXAMPLE 4: PATIENT 0198 TCR SCREENING 4.1 Mutation and HLA Calling 4.1.1 Sample Demographics Patient 0198 is an endometrium cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 4.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and Arnold & Porter Ref. P35502WO00 sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 4.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 4.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 1 hotspot mutation. The mutations are contained on topspot TMG 3. HLA profiling of the patient reveals HLAs as shown in FIG.20. Arnold & Porter Ref. P35502WO00 4.2 Design and Construction of Synthetic Reagent 4.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 4.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 4.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 4.3.1 Cell preparation Patient 0198 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 95% and a final concentration of 1200 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 10,000 cells. 4.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields 2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves >=Q30 quality score. 4.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to Arnold & Porter Ref. P35502WO00 belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 4.4 TCR reconstruction 4.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 2325 clonotypes. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 27 TCRs for Class II. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 4.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 4.5 Patient 0198 TCR Screening 4.5.1 Experimental design and methods Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 27 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There is 1 TMG designed for the relevant mutations, and 27 TCRs are picked from 10x single cell sequencing for this patient 0198. In addition, this patient has 2 HLA-A, 2 HLA-B, 2 HLA- C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are Arnold & Porter Ref. P35502WO00 separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmid. Table 23 summarizes the screening strategy for Patient 0198. Primary screening is performed by separating class I and class II TCRs. Class I TCRs are screened against HLA A, B or C. Class II TCRs are screened against HLA DP, DQ or DR. KRAS G12C is the only hotspot mutation this patient has. KRAS G12C 25mer peptide is used for screening. Table 23. Screening strategy for Patient 0198. 4.5.2 Screening Results As shown in FIG.20, TCR 97 is specific to the combination of peptide and an HLA allele in either locus HLA-DR, but not HLA DP or HLA DQ. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and peptide and DRB1*07:01 is found to be the specific HLA restricting 0198-TCR97 (FIG. 21). To further define the minimal epitope of 0198-TCR97 an online peptide prediction tool is used to predict potential candidates with minimal residue of peptide likely to bind with DRB1*07:01. To determine if KRAS G12V has some cross reactivity, KRAS G12V, KRAS G12C and DMSO are used for coculture. Both G12V and G12C has reactivity with TCR97 but not DMSO suggesting that this TCR not only recognize the endogenous mutation from the patient but also has cross activity with G12V (FIG.21). 4.6 Vetting 4.6.1 Generating KRAS-G12C / DRB1*07:01 TCR-T Cells PBMC cells are thawed, spun down, resuspended in electroporation buffer together with one of four TCR transposon plasmids (0198 TCR97) and SB11 Sleeping Beauty transposase encoding plasmid, and electroporated. Following electroporation, cell suspensions are collected, transferred to recovery media (50:50 media), and incubated in a 37°C / 5% CO2incubator overnight. Within 24 hours post- electroporation (Day 1), live cells are transferred to G-REX® culture plates and incubated with a first expansion media (50:50 media containing IL-2 + IL-21 + T Cell TransAct™). Cells are fed regularly with cytokines. After 14 days of first phase expansion, mTCR+ cells are isolated with anti-mTCR antibody. The isolated mTCR+ T cells are transferred to G-REX® culture plates and incubated with a second expansion media (50:50 media containing IL-2 + T Cell TransAct™). Cells are fed regularly Arnold & Porter Ref. P35502WO00 with cytokines. After 13-14 days of second phase expansion, cells are harvested, phenotyped by flow cytometry to determine the mTCR expression, and evaluated in functional assays to determine the TCR specificity and function. FIG. 22 shows expression of engineered TCRs in primary human T cells from four independent donors. FIG.23 shows Mean Fluorescent Intensity (MFI) of engineered TCRs in primary human T cells from four independent donors. FIG. 24 shows fold expansion of cell growth during ex vivo culture. 4.6.2 Generating KRAS-G12C / DRB1*07:01 TCR-T Cells Generation of monocyte-derived dendritic cells The DCs are derived from peripheral blood monocytes by adherence method and differentiation in vitro. Briefly, cryopreserved PBMCs with a known HLA haplotype are thawed, washed, resuspended in AIM-V media (Invitrogen, Carlsbad, CA) and seeded in a T175 flask. The cells are incubated at 37°C, 5% CO2for 2 hours allowing monocytes to adhere to the bottom of the flask. Thereafter, non- adherent PBMCs are removed, and the remaining adherent cells are washed carefully but extensively to ensure maximum removal of non-adherent cells. The remaining adherent cells (monocytes) are cultured in RPMI-1640 media containing 5% human AB serum and supplemented with 800 IU / mL GM-CSF and 200 IU / mL IL-4 to differentiate and maintain the monocyte-derived dendritic cells. Cells are cultured for a total of 6-9 days, with addition of cytokine-supplemented media every 2-3 days. At the end of the culture, both the non-adherent and adherent cell fractions are harvested and phenotyped for DC markers. 4.6.3 Coculture of TCR-T cells with peptide-pulsed dendritic cells For the co-culture assay, DCs are resuspended in 50:50 media and pulsed with KRAS G12C, G12D, G12V peptide or wild type (WT) peptide. The concentration of peptide used is titrated to exponentially decreasing concentrations (10, 1, 0.1, 0.01, and 0.001 μg / mL) to establish a dose- dependent effect of the TCR on its target neoantigen peptide, which also demonstrates the specificity of the TCR. After the incubation, peptide-pulsed DCs are seeded at 2.5 x 104cells / well in a volume of 100 μL of a round-bottom 96 well plate. TCR-T cells are added to the peptide-pulsed DCs at 1 x 105cells / well in a 100 μL volume. DCs pulsed with only DMSO serves as a background control, while DCs alone or T cells alone serves as negative controls. DCs pulsed with WT peptide serves as an additional control to demonstrate the specificity of the TCR target. For positive controls, T cells alone are stimulated with a Cell Activation Cocktail (Biolegend, San Diego, CA) containing an optimized concentration of phorbol 12-myristate-13-acetate (PMA) and ionomycin. The DC-TCR-T cell co-culture is incubated for 18-24 h. Arnold & Porter Ref. P35502WO00 4.6.4 Measurement of T cell activation by 4-1BB expression At the end of the DC / TCR-T cell co-culture, cells are washed and stained with anti-4-1BB antibody to assess for TCR-specific T-cell activation and co-stained with antibodies against mTCRβ (clone H57-597), CD3, CD4 and CD8 for T cell phenotyping. Cells are acquired and analyzed on the flow cytometer (Novocyte Quanteon, Agilent Technologies, Santa Clara, CA), while data analysis is performed using NovoExpress software. FIG.25 shows 4-1BB expression in TCR-T cells expressing engineered TCRs specific for KRASG12Cand HLA-DRA1*01:01 / HLA-DRB1*07:01. The upregulation of 4-1BB on TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells are shown. TCR-T cells expressing 0198-TCR97 TCR or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. 4.6.5 Measurement of T cell activation by IFN-γ secretion At the end of the DC / TCR-T co-culture supernatants are harvested and IFN-γ concentrations are measured using an Enzyme-linked immunosorbent assay (ELISA). Culture supernatants are diluted as necessary to bring the analyte concentrations to within the dynamic range of the assay. FIG. 26 shows IFN-γ expression from T cells cocultured with HLA-DRA1*01:01 / HLA- DRB1*07:01 DCs pulsed with KRAS WT or KRASG12CMutant peptide. The figure shows the secretion of IFN-γ by TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing 0198-TCR97 TCR or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 4 donors. 4.6.6 TCR-T cell cytotoxicity against tumor cells Negative control NT T cells and TCR-T cells are cocultured with HLA-transfected Saos2 tumor cells at an effector:target (E:T) ratio of 4:1 or 1:1 in 96-well white high-binding Optiplates (PerkinElmer, Waltham, MA) for 18-24 hours. Tumor cells are pulsed with 1 μg / mL of TP53R175 WT or TP53R175H mutant peptide for 2 hours at 37°C. All peptides are 25 amino acids in length with 12 amino acids flanking the substituted amino acid. Subsequently, peptides are washed, and T cells are added to initiate the co-culture, which is incubated overnight at 37°C. Arnold & Porter Ref. P35502WO00 After overnight incubation, the viability of adherent tumor cell targets is assessed by the CellTiter- Glo 2.0 Cell Viability Assay (Promega, Madison, WI), an ATP-based assay for detection of viable cells, after removal of cells in suspension (e.g., T cells and detached tumor cells). Luminescence is measured and recorded using a luminescence plate reader, the BioTek Cytation 5 (BioTek, Winooski, VT). Percent target cell lysis is calculated by taking the luminescence from remaining adherent viable tumor cells relative to controls. Peptide-loaded tumor cells and TCR-T cells individually serves as controls for the maximum signal / counts and minimum signal / counts, respectively. FIG. 27 shows TCR-T cell mediated cytolysis of tumor cells presenting either wild type KRAS or KRASG12Cantigen in the context of HLA-DRA1*01:01 / HLA-DRB1*07:01. The figure shows the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12X / DRB1*07:01 tumor cells. TCR-T cells expressing 0198-TCR97 TCR or non-transposed (NT) T cells are co-cultured with Saos-2 tumor cells which have been modified to express HLA-DRA1*01:01 / HLA-DRB1*07:01 and are pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight co-culture, target cell lysis is quantified using the CellTiter Glo assay. N = 4 donors. 4.7 Conclusion The series of data described in this example illustrate the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0198, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA expression vectors encoding eighteen TCRs from Patient 0198 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0198-TCR97 is found to be specific for the KRAS G12C and KRAS G12V neoantigen when presented in the context of DRB1*07:01. However, similar reactivity is observed in wide type peptide suggesting this TCR is not specific. Overall, these data demonstrate a process by which neoantigen-specific TCRs can be identified and functionally validated using a high-throughput TCR screening method. EXAMPLE 5: PATIENT 0275 TCR SCREENING 5.1 Mutation and HLA Calling 5.1.1 Sample Demographics Patient 0275 is a lung cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 5.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis Arnold & Porter Ref. P35502WO00 demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 5.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 5.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 Arnold & Porter Ref. P35502WO00 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 1 hotspot mutation. The mutations are contained on topspot TMG 1. HLA profiling of the patient reveals HLAs as shown in FIG.28. 5.2 Design and Construction of Synthetic Reagent 5.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 5.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 5.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 5.3.1 Cell preparation Patient 0275 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 90% and a final concentration of 240 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 4,000 cells. 5.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina Arnold & Porter Ref. P35502WO00 NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields 2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves >=Q30 quality score. 5.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 5.4 TCR reconstruction 5.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 1148 clonotypes. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 27 TCRs for class I. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 5.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 5.5 Patient 0275 TCR Screening 5.5.1 Experimental design and methods Arnold & Porter Ref. P35502WO00 Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 41 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There is 1 TMG designed for the relevant mutations, and 45 TCRs are picked from 10x single cell sequencing for this patient 0275. In addition, this patient has 2 HLA-A, 2 HLA-B, 2 HLA- C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmid. Table 24 summarizes the screening strategy for Patient 0275. Primary screening is performed by separating class I and class II TCRs. Class I TCRs are screened against HLA A, B or C. Class II TCRs are screened against HLA DP, DQ or DR. This patient has KRAS G12D. Topspot TMG-1 is used for screening. Table 24. Screening strategy for Patient 0275. 5.5.2 Screening Results As shown in FIG.28, TCR 20-1 is specific to the combination of peptide and an HLA allele in either locus HLA-A, but not HLA B or HLA C. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and peptide and A*11:01 is found to be the specific HLA restricting 0275-TCR20-1 (FIG.29). To further define the minimal epitope of 0275- TCR20-1 an online peptide prediction tool is used to predict potential candidates with minimal residue of peptide likely to bind with 0275-TCR20-1. To determine if KRAS G12D has some cross reactivity, KRAS G12V and DMSO are used for coculture. G12D has reactivity with TCR20-1 but not G12V suggesting that this TCR is specific (FIG.30). 5.6 Vetting 5.6.1 Generating KRAS-G12D / A*11:01 TCR-T Cells Arnold & Porter Ref. P35502WO00 PBMC cells are thawed, spun down, resuspended in electroporation buffer together with one of four TCR transposon plasmids (0275 TCR20-1) and SB11 Sleeping Beauty transposase encoding plasmid, and electroporated. Following electroporation, cell suspensions are collected, transferred to recovery media (50:50 media), and incubated in a 37°C / 5% CO2incubator overnight. Within 24 hours post- electroporation (Day 1), live cells are transferred to G-REX® culture plates and incubated with a first expansion media (50:50 media containing IL-2 + IL-21 + T Cell TransAct™). Cells are fed regularly with cytokines. After 14 days of first phase expansion, mTCR+ cells are isolated with anti-mTCR antibody. The isolated mTCR+ T cells are transferred to G-REX® culture plates and incubated with a second expansion media (50:50 media containing IL-2 + T Cell TransAct™). Cells are fed regularly with cytokines. After 13-14 days of second phase expansion, cells are harvested, phenotyped by flow cytometry to determine the mTCR expression, and evaluated in functional assays to determine the TCR specificity and function. 5.6.2 Generating KRAS-G12D / A*11:01 TCR-T Cells Generation of monocyte-derived dendritic cells The DCs are derived from peripheral blood monocytes by adherence method and differentiation in vitro. Briefly, cryopreserved PBMCs with a known HLA haplotype are thawed, washed, resuspended in AIM-V media (Invitrogen, Carlsbad, CA) and seeded in a T175 flask. The cells are incubated at 37°C, 5% CO2for 2 hours allowing monocytes to adhere to the bottom of the flask. Thereafter, non- adherent PBMCs are removed, and the remaining adherent cells are washed carefully but extensively to ensure maximum removal of non-adherent cells. The remaining adherent cells (monocytes) are cultured in RPMI-1640 media containing 5% human AB serum and supplemented with 800 IU / mL GM-CSF and 200 IU / mL IL-4 to differentiate and maintain the monocyte-derived dendritic cells. Cells are cultured for a total of 6-9 days, with addition of cytokine-supplemented media every 2-3 days. At the end of the culture, both the non-adherent and adherent cell fractions are harvested and phenotyped for DC markers. 5.6.3 Coculture of TCR-T cells with peptide-pulsed dendritic cells For the co-culture assay, DCs are resuspended in 50:50 media and pulsed with KRAS G12D peptide or wild type (WT) peptide. The concentration of peptide used is titrated to exponentially decreasing concentrations (10, 1, 0.1, 0.01, and 0.001 μg / mL) to establish a dose-dependent effect of the TCR on its target neoantigen peptide, which also demonstrates the specificity of the TCR. After the incubation, peptide-pulsed DCs are seeded at 2.5 x 104cells / well in a volume of 100 μL of a round- bottom 96 well plate. TCR-T cells are added to the peptide-pulsed DCs at 1 x 105cells / well in a 100 μL volume. DCs pulsed with only DMSO serves as a background control, while DCs alone or T cells alone serves as negative controls. DCs pulsed with WT peptide serves as an additional control to Arnold & Porter Ref. P35502WO00 demonstrate the specificity of the TCR target. For positive controls, T cells alone are stimulated with a Cell Activation Cocktail (Biolegend, San Diego, CA) containing an optimized concentration of phorbol 12-myristate-13-acetate (PMA) and ionomycin. The DC-TCR-T cell co-culture is incubated for 18-24 h. 5.6.4 Measurement of T cell activation by 4-1BB expression At the end of the DC / TCR-T cell co-culture, cells are washed and stained with anti-4-1BB antibody to assess for TCR-specific T-cell activation and co-stained with antibodies against mTCRβ (clone H57-597), CD3, CD4 and CD8 for T cell phenotyping. Cells are acquired and analyzed on the flow cytometer (Novocyte Quanteon, Agilent Technologies, Santa Clara, CA), while data analysis is performed using NovoExpress software. FIG.31 shows upregulation of 4-1BB by activated KRAS-G12D-A*11:01 TCR-T cells in response to KRASG12Wild Type (WT) or KRASG12Dneoantigen peptides. The upregulation of 4-1BB on TCR- T cells activated with either wild type or mutant peptide-pulsed dendritic cells are shown. TCR-T cells expressing 0275-TCR20-1 TCR, Hubble (NCI licensed TCR S-KRAS-G12D-A*11:01) or non- transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-A*11:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Dmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. 5.6.5 Measurement of T cell activation by IFN-γ secretion At the end of the DC / TCR-T co-culture supernatants are harvested and IFN-γ concentrations are measured using an Enzyme-linked immunosorbent assay (ELISA). Culture supernatants are diluted as necessary to bring the analyte concentrations to within the dynamic range of the assay. FIG. 32 shows secretion of IFN-γ by activated KRAS-G12D-A-1101 TCR-T cells in response of KRASG12(WT) peptide KRASG12Dneoantigen peptides. The figure shows the secretion of IFN-γ by TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing 0275-TCR20-1 TCR or non-transposed (NT) T cells are co-cultured with monocyte- derived dendritic cells generated from an HLA-A*11:01 donor pulsed with KRASG12wildtype (WT) or KRASG12Dmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 2 donors. 5.7 Conclusion The series of data described in this example illustrate the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0275, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA Arnold & Porter Ref. P35502WO00 expression vectors encoding 27 TCRs from Patient 0275 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0275-TCR20-1 is found to be specific for the KRAS G12D neoantigen when presented in the context of A*11:01. NCI licensed TCR Hubble is used to compare the specificity and sensitivity, and we have observed that TCR20-1 discovered from hunTR program is comparable with NCI licensed TCR. EXAMPLE 6: PATIENT 0166 TCR SCREENING 6.1 Mutation and HLA Calling 6.1.1 Sample Demographics Patient 0166 is a lung cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 6.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 6.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- Arnold & Porter Ref. P35502WO00 targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 6.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 1 hotspot mutations. HLA profiling of the patient reveals HLAs as shown in FIG.33. 6.2 Design and Construction of Synthetic Reagent 6.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 6.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI Arnold & Porter Ref. P35502WO00 restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 6.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 6.3.1 Cell preparation Patient 0166 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 91% and a final concentration of 1065 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 10,000 cells. 6.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields 2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves >=Q30 quality score. 6.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 6.4 TCR reconstruction 6.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 1957 clonotypes. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 33 TCRs for class II. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start Arnold & Porter Ref. P35502WO00 of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 6.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 6.5 Patient 0166 TCR Screening 6.5.1 Experimental design and methods Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 60 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There is 1 TMG designed for the relevant mutations, and 60 TCRs are picked from 10x single cell sequencing for this patient 0166. In addition, this patient has 2 HLA-A, 2 HLA-B, 2 HLA- C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmid. Table 25 summarizes the screening strategy for Patient 0166. Primary screening is performed by separating class I and class II TCRs. Class I TCRs are screened against HLA A, B or C. Class II TCRs are screened against HLA DP, DQ or DR. Lung patient 0166 has KRAS G12V mutation. Master TMG has mutation KRAS G12V. Table 25. Screening strategy for Patient 0166. Arnold & Porter Ref. P35502WO00 6.5.2 Screening Results As shown in FIG.33, TCR 127 is specific to the combination of G12V and an HLA allele in either locus HLA-DR, but not HLA DP or HLA DQ. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and TMG5 and HLA-DRB1*07:01 is found to be the specific HLA restricting 0166-TCR127 (FIG.34). To further define the minimal epitope of 0166-TCR127 an online peptide prediction tool is used to predict potential candidates with minimal residue of peptide likely to bind with HLA-DRB1*07:01. Other KRAS G12V DRB1*07:01 TCRs found in hunTR as well as NCI licensed TCR are used to compare their sensitivity and specificity (FIG.35). 6.6 Vetting 6.6.1 Generating KRAS-G12V / DRB1*07:01 TCR-T Cells PBMC cells are thawed, spun down, resuspended in electroporation buffer together with one of four TCR transposon plasmids (S3-KRAS-G12V-DRB1-0701-C1, 9976-TCR38-2, 7014-TCR16, 7014- TCR51, 7014-TCR55, 160-TCR70 and 0166 TCR127) and SB11 Sleeping Beauty transposase encoding plasmid, and electroporated. Following electroporation, cell suspensions are collected, transferred to recovery media (50:50 media), and incubated in a 37°C / 5% CO2incubator overnight. Within 24 hours post-electroporation (Day 1), live cells are transferred to G-REX® culture plates and incubated with a first expansion media (50:50 media containing IL-2 + IL-21 + T Cell TransAct™). Cells are fed regularly with cytokines. After 14 days of first phase expansion, mTCR+ cells are isolated with anti-mTCR antibody. The isolated mTCR+ T cells are transferred to G-REX® culture plates and incubated with a second expansion media (50:50 media containing IL-2 + T Cell TransAct™). Cells are fed regularly with cytokines. After 13-14 days of second phase expansion, cells are harvested, phenotyped by flow cytometry to determine the mTCR expression, and evaluated in functional assays to determine the TCR specificity and function. FIG. 36 shows fold expansion of cell growth during ex vivo culture. The figure shows the fold- expansion of T cells modified to express transgenic TCRs. Primary human T cells from four independent donors are gene modified to stably express transgenic TCRs reactive to KRASG12V / DRB1*07:01 neoantigen using Sleeping Beauty transposon / transposase gene transfer. FIG. 37 shows expression of KRAS-G12V-DRB1-0701 TCRs on CD3+ T cells during ex vivo generation process. The figure shows the percentage expression of transgenic TCRs in primary human T cells. FIG.38 shows flow cytometry contour plots of mTCR expression by CD3. Representative contour plots showing the mTCR expression (y-axis) within the CD3+live cell population of primary human Arnold & Porter Ref. P35502WO00 T cells modified to express transgenic TCRs. FIG.39 shows frequency of CD4+ and CD8+ cells within the CD3+ T-cell populations in ex vivo TCR-T cell cultures. FIG. 40 shows distribution of CD4+ and CD8+ T cells within the mTCR+ population in ex vivo TCR-T cell cultures. FIG. 41 shows Mean Fluorescent Intensity (MFI) of mTCR expression by flow cytometry during ex vivo generation. FIG.42 shows percent recovery of mTCR+ cell following Magnetic-Activated Cell Selection (MACS). The figure shows the recovery efficiency of enrichment for mTCR+ cells from ex vivo cell cultures. 6.6.2 Generating KRAS-G12V / DRB1*07:01 TCR-T Cells Generation of monocyte-derived dendritic cells The DCs are derived from peripheral blood monocytes by adherence method and differentiation in vitro. Briefly, cryopreserved PBMCs with a known HLA haplotype are thawed, washed, resuspended in AIM-V media (Invitrogen, Carlsbad, CA) and seeded in a T175 flask. The cells are incubated at 37°C, 5% CO2for 2 hours allowing monocytes to adhere to the bottom of the flask. Thereafter, non- adherent PBMCs are removed, and the remaining adherent cells are washed carefully but extensively to ensure maximum removal of non-adherent cells. The remaining adherent cells (monocytes) are cultured in RPMI-1640 media containing 5% human AB serum and supplemented with 800 IU / mL GM-CSF and 200 IU / mL IL-4 to differentiate and maintain the monocyte-derived dendritic cells. Cells are cultured for a total of 6-9 days, with addition of cytokine-supplemented media every 2-3 days. At the end of the culture, both the non-adherent and adherent cell fractions are harvested and phenotyped for DC markers. 6.6.3 Coculture of TCR-T cells with peptide-pulsed dendritic cells For the co-culture assay, DCs are resuspended in 50:50 media and pulsed with KRAS G12V peptide or wild type (WT) peptide. The concentration of peptide used is titrated to exponentially decreasing concentrations (10, 1, 0.1, 0.01, and 0.001 μg / mL) to establish a dose-dependent effect of the TCR on its target neoantigen peptide, which also demonstrates the specificity of the TCR. After the incubation, peptide-pulsed DCs are seeded at 2.5 x 104cells / well in a volume of 100 μL of a round- bottom 96 well plate. TCR-T cells are added to the peptide-pulsed DCs at 1 x 105cells / well in a 100 μL volume. DCs pulsed with only DMSO serves as a background control, while DCs alone or T cells alone serves as negative controls. DCs pulsed with WT peptide serves as an additional control to demonstrate the specificity of the TCR target. For positive controls, T cells alone are stimulated with a Cell Activation Cocktail (Biolegend, San Diego, CA) containing an optimized concentration of phorbol 12-myristate-13-acetate (PMA) and ionomycin. The DC-TCR-T cell co-culture is incubated for 18-24 h. Arnold & Porter Ref. P35502WO00 6.6.4 Measurement of T cell activation by 4-1BB expression At the end of the DC / TCR-T cell co-culture, cells are washed and stained with anti-4-1BB antibody to assess for TCR-specific T-cell activation and co-stained with antibodies against mTCRβ (clone H57-597), CD3, CD4 and CD8 for T cell phenotyping. Cells are acquired and analyzed on the flow cytometer (Novocyte Quanteon, Agilent Technologies, Santa Clara, CA), while data analysis is performed using NovoExpress software. Upregulation of 4-1BB by activated KRAS-G12V-DRB1-0701 TCR-T cells in response to KRASG12V(MUT) peptide or in response of KRASG12(WT) peptide is shown in FIG. 43 or 44, respectively. The figure shows the upregulation of 4-1BB on TCR-T cells activated with mutant or wildtype peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*07:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte- derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. 6.6.5 Measurement of T cell activation by IFN-γ secretion At the end of the DC / TCR-T co-culture supernatants are harvested and IFN-γ concentrations are measured using an Enzyme-linked immunosorbent assay (ELISA). Culture supernatants are diluted as necessary to bring the analyte concentrations to within the dynamic range of the assay. Secretion of IFN-γ by activated KRAS-G12V-DRB1-0701 TCR-T cells in response of KRASG12V(MUT) peptide or in response of KRASG12(WT) peptide is shown in FIG. 45 or 46, respectively. The figure shows the secretion of IFN-γ by TCR-T cells activated with mutant or wildtype peptide- pulsed dendritic cells. TCR-T cells expressing TCRs reactive to KRASG12V / DRB1*07:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-DRA1*01:01 / HLA-DRB1*07:01 donor pulsed with KRASG12wildtype (WT) or KRASG12C, KRASG12Dor KRASG12Vmutant peptides. 6.6.6 TCR-T cell cytotoxicity against tumor cells Negative control NT T cells and TCR-T cells are cocultured with HLA-transfected Saos2 tumor cells at an effector:target (E:T) ratio of 4:1 or 1:1 in 96-well white high-binding Optiplates (PerkinElmer, Waltham, MA) for 18-24 hours. Tumor cells are pulsed with 1 μg / mL of TP53R175 WT or TP53R175H mutant peptide for 2 hours at 37°C. All peptides are 25 amino acids in length with 12 amino acids flanking the substituted amino acid. Subsequently, peptides are washed, and T cells are added to initiate the co-culture, which is incubated overnight at 37°C. Arnold & Porter Ref. P35502WO00 After overnight incubation, the viability of adherent tumor cell targets is assessed by the CellTiter- Glo 2.0 Cell Viability Assay (Promega, Madison, WI), an ATP-based assay for detection of viable cells, after removal of cells in suspension (e.g., T cells and detached tumor cells). Luminescence is measured and recorded using a luminescence plate reader, the BioTek Cytation 5 (BioTek, Winooski, VT). Percent target cell lysis is calculated by taking the luminescence from remaining adherent viable tumor cells relative to controls. Peptide-loaded tumor cells and TCR-T cells individually serves as controls for the maximum signal / counts and minimum signal / counts, respectively. FIG.47 shows KRASG12Vtarget cell cytotoxicity of KRAS-G12V-DRB1-0701-specific TCR-T cells expressing transgenic TCRs. FIG. 48 shows KRASG12Vtarget cell cytotoxicity of 9976-TCR38-2 expressing TCR-T cells. FIG.49 shows KRASG12Vtarget cell cytotoxicity of TCR-T cells expressing TCRs identified from Patient 7014. FIG.50 shows KRASG12Vtarget cell cytotoxicity of 160-TCR70 expressing TCR-T cells. FIG. 51 shows KRASG12Vtarget cell cytotoxicity of 166-TCR127 expressing TCR-T cells. The figures show the antigen-specific cytotoxicity functions of TCR-T cells targeting KRASG12V / DRB1*07:01 tumor cells. 6.7 Conclusion The series of data described in this example illustrates the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0166, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA expression vectors encoding 33 TCRs from Patient 0166 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0166-TCR127 is found to be specific for the KRAS G12V neoantigen when presented in the context of DRB1*07:01. NCI licensed TCR Hubble is used to compare the specificity and sensitivity, and most of the TCRs from hunTR are comparable to NCI licensed TCR except 7014 TCR16 has some wide type of peptide reactivity. EXAMPLE 7: PATIENT 0048 TCR SCREENING 7.1 Mutation and HLA Calling 7.1.1 Sample Demographics Patient 0048 is a colorectal cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 7.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. Arnold & Porter Ref. P35502WO00 DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 7.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 7.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The Arnold & Porter Ref. P35502WO00 nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 1 hotspot mutation. HLA profiling of the patient reveals HLAs as shown in FIG.52. 7.2 Design and Construction of Synthetic Reagent 7.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 7.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 7.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 7.3.1 Cell preparation Patient 0048 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 92.6% and a final concentration of 1100 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 10,000 cells. 7.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields 2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves >=Q30 quality score. Arnold & Porter Ref. P35502WO00 7.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 7.4 TCR reconstruction 7.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 3273 clonotypes. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 24 TCRs for class II. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 7.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 7.5 Patient 0048 TCR Screening 7.5.1 Experimental design and methods Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 47 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of Arnold & Porter Ref. P35502WO00 transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There is 1 TMG designed for the relevant mutations, and 47 TCRs are picked from 10x single cell sequencing for this patient 0166. In addition, this patient had 2 HLA-A, 2 HLA-B, 2 HLA- C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmid. Table 26 summarizes the screening strategy for Patient 0048. Primary screening is performed by separating class I and class II TCRs. Class I TCRs are screened against HLA A, B or C. Class II TCRs are screened against HLA DP, DQ or DR. This patient has KRAS G12V mutation. Table 26. Screening strategy for Patient 0048. 7.5.2 Screening Results As shown in FIG.52, TCR 82 and 485-1 are specific to the combination of G12V and an HLA allele in either locus HLA-DR, but not HLA DP or HLA DQ. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and KRAS G12V peptide and it is found that HLA-DRB1*10:01 is the specific HLA restricting 0048-TCR82 and TCR485-1 (FIG.53). TCR485-1 is specific also to DRB3*02:02 (FIG.54). 7.6 Vetting 7.6.1 Generating KRAS-G12V / DRB1*10:01 TCR-T Cells PBMC cells are thawed, spun down, resuspended in electroporation buffer together with one of four TCR transposon plasmids (0048 TCR82 and TCR485-1) and SB11 Sleeping Beauty transposase encoding plasmid, and electroporated. Following electroporation, cell suspensions are collected, transferred to recovery media (50:50 media), and incubated in a 37°C / 5% CO2incubator overnight. Within 24 hours post-electroporation (Day 1), live cells are transferred to G-REX® culture plates and incubated with a first expansion media (50:50 media containing IL-2 + IL-21 + T Cell TransAct™). Cells are fed regularly with cytokines. After 14 days of first phase expansion, mTCR+ cells are isolated with anti-mTCR antibody. The isolated mTCR+ T cells are transferred to G-REX® culture plates and incubated with a second expansion media (50:50 media containing IL-2 + T Cell Arnold & Porter Ref. P35502WO00 TransAct™). Cells are fed regularly with cytokines. After 13-14 days of second phase expansion, cells are harvested, phenotyped by flow cytometry to determine the mTCR expression, and evaluated in functional assays to determine the TCR specificity and function. 7.6.2 Generating KRAS-G12V / DRB1*10:01 or DRB3*02:02 TCR-T Cells Generation of monocyte-derived dendritic cells The DCs are derived from peripheral blood monocytes by adherence method and differentiation in vitro. Briefly, cryopreserved PBMCs with a known HLA haplotype are thawed, washed, resuspended in AIM-V media (Invitrogen, Carlsbad, CA) and seeded in a T175 flask. The cells are incubated at 37°C, 5% CO2for 2 hours allowing monocytes to adhere to the bottom of the flask. Thereafter, non- adherent PBMCs are removed, and the remaining adherent cells are washed carefully but extensively to ensure maximum removal of non-adherent cells. The remaining adherent cells (monocytes) are cultured in RPMI-1640 media containing 5% human AB serum and supplemented with 800 IU / mL GM-CSF and 200 IU / mL IL-4 to differentiate and maintain the monocyte-derived dendritic cells. Cells are cultured for a total of 6-9 days, with addition of cytokine-supplemented media every 2-3 days. At the end of the culture, both the non-adherent and adherent cell fractions are harvested and phenotyped for DC markers. 7.6.3 Coculture of TCR-T cells with peptide-pulsed dendritic cells For the co-culture assay, DCs are resuspended in 50:50 media and pulsed with KRAS G12V peptide or wild type (WT) peptide. The concentration of peptide used is titrated to exponentially decreasing concentrations (10, 1, 0.1, 0.01, and 0.001 μg / mL) to establish a dose-dependent effect of the TCR on its target neoantigen peptide, which also demonstrates the specificity of the TCR. After the incubation, peptide-pulsed DCs are seeded at 2.5 x 104cells / well in a volume of 100 μL of a round- bottom 96 well plate. TCR-T cells are added to the peptide-pulsed DCs at 1 x 105cells / well in a 100 μL volume. DCs pulsed with only DMSO serves as a background control, while DCs alone or T cells alone serves as negative controls. DCs pulsed with WT peptide serves as an additional control to demonstrate the specificity of the TCR target. For positive controls, T cells alone are stimulated with a Cell Activation Cocktail (Biolegend, San Diego, CA) containing an optimized concentration of phorbol 12-myristate-13-acetate (PMA) and ionomycin. The DC-TCR-T cell co-culture is incubated for 18-24 h. 7.6.4 Measurement of T cell activation by 4-1BB expression At the end of the DC / TCR-T cell co-culture, cells are washed and stained with anti-4-1BB antibody to assess for TCR-specific T-cell activation and co-stained with antibodies against mTCRβ (clone H57-597), CD3, CD4 and CD8 for T cell phenotyping. Cells are acquired and analyzed on the flow Arnold & Porter Ref. P35502WO00 cytometer (Novocyte Quanteon, Agilent Technologies, Santa Clara, CA), while data analysis is performed using NovoExpress software. FIGs. 55-57 show upregulation of 4-1BB by activated KRAS-G12V-DRB1-1001 TCR-T cells in response to KRASG12(WT) peptide and KRASG12V(MUT) peptide. The figures show the upregulation of 4-1BB on TCR-T cells activated with mutant peptide-pulsed dendritic cells. 7.6.5 Measurement of T cell activation by IFN-γ secretion At the end of the DC / TCR-T co-culture supernatants are harvested and IFN-γ concentrations are measured using an Enzyme-linked immunosorbent assay (ELISA). Culture supernatants are diluted as necessary to bring the analyte concentrations to within the dynamic range of the assay. FIGs.58 and 59 show secretion of IFN-γ by activated KRAS-G12V-DRB1*10:01 TCR-T cells in response to KRASG12Wild Type (WT) or KRASG12Vneoantigen peptides. FIG.60 shows secretion of IFN-γ by activated KRAS-G12V-DRB3*02:02 TCR-T cells in response to KRASG12Wild Type (WT) or KRASG12Vneoantigen peptides. The figures show the secretion of IFN-γ by TCR-T cells activated with mutant peptide-pulsed dendritic cells. 7.7 Conclusion The series of data described in this example illustrates the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0048, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA expression vectors encoding 24 TCRs from Patient 0048 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0048-TCR82 is found to be specific for the KRAS G12V neoantigen when presented in the context of DRB1*10:01. TCR485-1 has some background reactivity with DRB1*10:01. In addition, TCR485-1 is specific to DRB3*02:02 based on the 4-1BB and IFNg release assay. EXAMPLE 8: PATIENT 0032 TCR SCREENING 8.1 Mutation and HLA Calling 8.1.1 Sample Demographics Patient 0032 is a colorectal cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 8.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. Arnold & Porter Ref. P35502WO00 DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 8.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 8.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The Arnold & Porter Ref. P35502WO00 nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 2 hotspot mutations. HLA profiling of the patient reveals HLAs as shown in FIG.61. 8.2 Design and Construction of Synthetic Reagent 8.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 8.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 8.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 8.3.1 Cell preparation Patient 0032 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 99% and a final concentration of 120 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 2,000 cells. 8.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves>=Q30 quality score. Arnold & Porter Ref. P35502WO00 8.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 8.4 TCR reconstruction 8.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 424 clonotypes. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 60 TCRs for both class I and Class II. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 8.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 8.5 Patient 0032 TCR Screening 8.5.1 Experimental design and methods Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 60 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of Arnold & Porter Ref. P35502WO00 transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There is 1 TMG designed for the relevant mutations, and 60 TCRs are picked from 10x single cell sequencing for this patient 0166. In addition, this patient has 2 HLA-A, 2 HLA-B, 2 HLA- C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmid. Table 27 summarizes the screening strategy for Patient 0032. TCRs are tested against all 5 HLA groups. This patient has TP53 R175H. Table 27. Screening strategy for Patient 0032. 8.5.2 Screening Results As shown in FIG.61, TCR 386 is specific to the combination of TP53 R175H and an HLA allele in either locus HLA-A, but not HLA B or HLA C. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and TMG1 and it is found that HLA- A*02:01 is the specific HLA restricting TCR 386 (FIG.62). To further define the minimal epitope of TCR 386 an online peptide prediction tool predicts potential candidates with minimal residue of peptide likely to bind with HLA-A*02:01. Other TP53 R175H HLA-A*02:01 TCRs found in hunTR as well as NCI licensed TCR are used to compare their sensitivity and specificity (FIG.63). 8.6 Vetting 8.6.1 Generating TP53 R175H-A*02:01 TCR-T Cells PBMC cells are thawed, spun down, resuspended in electroporation buffer together with one of four TCR transposon plasmids (S3-TP53-R175H-A-0201-C1, 0032-TCR2, 0032-TCR37 and 0032- TCR485-1) and SB11 Sleeping Beauty transposase encoding plasmid, and electroporated. Following electroporation, cell suspensions are collected, transferred to recovery media (50:50 media), and incubated in a 37°C / 5% CO2incubator overnight. Within 24 hours post-electroporation (Day 1), live cells are transferred to G-REX® culture plates and incubated with a first expansion media (50:50 media containing IL-2 + IL-21 + T Cell TransAct™). Cells are fed regularly with cytokines. After 14 days of first phase expansion, mTCR+ cells are isolated with anti-mTCR antibody. The isolated Arnold & Porter Ref. P35502WO00 mTCR+ T cells are transferred to G-REX® culture plates and incubated with a second expansion media (50:50 media containing IL-2 + T Cell TransAct™). Cells are fed regularly with cytokines. After 13-14 days of second phase expansion, cells are harvested, phenotyped by flow cytometry to determine the mTCR expression, and evaluated in functional assays to determine the TCR specificity and function. FIG.64 shows expression of TP53-R175H-A-0201 TCRs on CD3+ T cells during ex vivo generation process. FIG.65 shows Mean Fluorescent Intensity (MFI) of mTCR expression by flow cytometry during ex vivo generation. FIG.66 shows fold expansion of cell growth during ex vivo culture. 8.6.2 Generating TP53 R175H-A*02:01 TCR-T Cells Generation of monocyte-derived dendritic cells The DCs are derived from peripheral blood monocytes by adherence method and differentiation in vitro. Briefly, cryopreserved PBMCs with a known HLA haplotype are thawed, washed, resuspended in AIM-V media (Invitrogen, Carlsbad, CA) and seeded in a T175 flask. The cells are incubated at 37°C, 5% CO2for 2 hours allowing monocytes to adhere to the bottom of the flask. Thereafter, non- adherent PBMCs are removed, and the remaining adherent cells are washed carefully but extensively to ensure maximum removal of non-adherent cells. The remaining adherent cells (monocytes) are cultured in RPMI-1640 media containing 5% human AB serum and supplemented with 800 IU / mL GM-CSF and 200 IU / mL IL-4 to differentiate and maintain the monocyte-derived dendritic cells. Cells are cultured for a total of 6-9 days, with addition of cytokine-supplemented media every 2-3 days. At the end of the culture, both the non-adherent and adherent cell fractions are harvested and phenotyped for DC markers. 8.6.3 Coculture of TCR-T cells with peptide-pulsed dendritic cells For the co-culture assay, DCs are resuspended in 50:50 media and pulsed with KRAS G12V peptide or wild type (WT) peptide. The concentration of peptide used is titrated to exponentially decreasing concentrations (10, 1, 0.1, 0.01, and 0.001 μg / mL) to establish a dose-dependent effect of the TCR on its target neoantigen peptide, which also demonstrates the specificity of the TCR. After the incubation, peptide-pulsed DCs are seeded at 2.5 x 104cells / well in a volume of 100 μL of a round- bottom 96 well plate. TCR-T cells are added to the peptide-pulsed DCs at 1 x 105cells / well in a 100 μL volume. DCs pulsed with only DMSO serves as a background control, while DCs alone or T cells alone serves as negative controls. DCs pulsed with WT peptide serves as an additional control to demonstrate the specificity of the TCR target. For positive controls, T cells alone are stimulated with a Cell Activation Cocktail (Biolegend, San Diego, CA) containing an optimized concentration of phorbol 12-myristate-13-acetate (PMA) and ionomycin. The DC-TCR-T cell co-culture is incubated for 18-24 h. Arnold & Porter Ref. P35502WO00 8.6.4 Measurement of T cell activation by 4-1BB expression At the end of the DC / TCR-T cell co-culture, cells are washed and stained with anti-4-1BB antibody to assess for TCR-specific T-cell activation and co-stained with antibodies against mTCRβ (clone H57-597), CD3, CD4 and CD8 for T cell phenotyping. Cells are acquired and analyzed on the flow cytometer (Novocyte Quanteon, Agilent Technologies, Santa Clara, CA), while data analysis is performed using NovoExpress software. FIG.67 shows upregulation of 4-1BB by activated TP53-R175H-A-0201 TCR-T cells in response to TP53R175Wild Type (WT) or TP53R175Hneoantigen peptides. The figure shows the upregulation of 4-1BB on TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing S3-TP53-R175H-A-0201, 0032-TCR2, 0032-TCR37, 0032-TCR386 or non- transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-A*02:01 donor pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. 8.6.5 Measurement of T cell activation by IFN-γ secretion At the end of the DC / TCR-T co-culture supernatants are harvested and IFN-γ concentrations are measured using an Enzyme-linked immunosorbent assay (ELISA). Culture supernatants are diluted as necessary to bring the analyte concentrations to within the dynamic range of the assay. FIG. 68 shows secretion of IFN-γ by activated TP53-R175H-A-0201 TCR-T cells in response to TP53R175Wild Type (WT) or TP53R175Hneoantigen peptides. The figure shows the secretion of IFN- γ by TCR-T cells activated with either wild type or mutant peptide-pulsed dendritic cells. TCR-T cells expressing S3-TP53-R175H-A-0201, 0032-TCR2, 0032-TCR37, 0032-TCR386 or non- transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an HLA-A*02:01 donor pulsed with TP53R175wildtype (WT) or TP53R175Hmutant peptide. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 4 donors. Mean and SEM are plotted. 8.6.6 TCR-T cell cytotoxicity against tumor cells Negative control NT T cells and TCR-T cells are cocultured with HLA-transfected Saos2 tumor cells at an effector:target (E:T) ratio of 4:1 or 1:1 in 96-well white high-binding Optiplates (PerkinElmer, Waltham, MA) for 18-24 hours. Tumor cells are pulsed with 1 μg / mL of TP53R175 WT or TP53R175H mutant peptide for 2 hours at 37°C. All peptides are 25 amino acids in length with 12 amino acids flanking the substituted amino acid. Subsequently, peptides are washed, and T cells are added to initiate the co-culture, which is incubated overnight at 37°C. Arnold & Porter Ref. P35502WO00 After overnight incubation, the viability of adherent tumor cell targets is assessed by the CellTiter- Glo 2.0 Cell Viability Assay (Promega, Madison, WI), an ATP-based assay for detection of viable cells, after removal of cells in suspension (e.g., T cells and detached tumor cells). Luminescence is measured and recorded using a luminescence plate reader, the BioTek Cytation 5 (BioTek, Winooski, VT). Percent target cell lysis is calculated by taking the luminescence from remaining adherent viable tumor cells relative to controls. Peptide-loaded tumor cells and TCR-T cells individually serves as controls for the maximum signal / counts and minimum signal / counts, respectively. FIGs.69-72 show TP53R175Htarget cell cytotoxicity of TP53-R175H-A-0201-specific TCR-T cells expressing transgenic TCRs. 8.7 Conclusion The series of data described in this example illustrates the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0032, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA expression vectors encoding 60 TCRs from Patient 0032 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0032-TCR386 is found to be specific for the TP53 R175H neoantigen when presented in the context of A*02:01. NCI licensed TCR TP53 R175H-A*02:01 is used to compare the specificity and sensitivity, and 3 of the TCRs from hunTR are comparable to NCI licensed TCR. EXAMPLE 9: PATIENT 0124 TCR SCREENING 9.1 Mutation and HLA Calling 9.1.1 Sample Demographics Patient 0124 is a colorectal cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 9.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA Arnold & Porter Ref. P35502WO00 HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 9.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 9.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Arnold & Porter Ref. P35502WO00 Mutation panel analysis reveals 2 hotspot mutations. The mutation is contained on topspot TMG 1 and 2. HLA profiling of the patient reveals HLAs as shown in FIG.73. 9.2 Design and Construction of Synthetic Reagent 9.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 9.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 9.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 9.3.1 Cell preparation Patient 0124 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 99% and a final concentration of 1000 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 10,000 cells. 9.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields 2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves >=Q30 quality score. 9.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled Arnold & Porter Ref. P35502WO00 into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 9.4 TCR reconstruction 9.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 1457 clonotypes. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 10 TCRs for class I. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 9.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 9.5 Patient 0124 TCR Screening 9.5.1 Experimental design and methods Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 47 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There are 2 TMGs designed for the relevant mutations, and 42 TCRs are picked from 10x single cell sequencing for this patient 0124. In addition, this patient has 2 HLA-A, 2 HLA-B, 2 HLA- Arnold & Porter Ref. P35502WO00 C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmid. Table 28 summarizes the screening strategy for Patient 0124. Class I TCRs are screened against HLA A, B or C. Class II TCRs are screened against HLA DP, DQ or DR. This patient has TP53- R248W and KRAS-G12D mutations. Table 28. Screening strategy for Patient 0124. 9.5.2 Screening Results As shown in FIG.73, TCR 10 is specific to the combination of TMGs and an HLA allele in locus HLA-B, but not HLA A or HLA C. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and TMG2 and B*57:01 is the specific HLA restricting TCR 10 (FIG.74). Peptide prediction analysis for HLA B*57:01 is performed using web-based tool. Top six ranked peptides are shown in Table 29. The results show that anchor residues serine (pos.2) and tryptophan (pos.9) are critical for TCR binding. Table 29. FIG.75 shows two top ranked peptides are specific for TCR10 / HLA B*57:01 interaction. Among all peptides tested, TCR10 recognizes peptide SSCMGGMNW (9mer_1; SEQ ID NO: 238) and NSSCMGGMNW (10mer_1; SEQ ID NO: 239). No reactivity is observed in WT peptide Arnold & Porter Ref. P35502WO00 SSCMGGMNR (SEQ ID NO: 244), suggesting TCR 10 is specific against minimal epitope SSCMGGMNW (SEQ ID NO: 238). 9.6 Vetting 9.6.1 Generating TP53 R248W / B*57:01 TCR-T Cells PBMC cells are thawed, spun down, resuspended in electroporation buffer together with TCR transposon plasmids (0124 TCR10) and SB11 Sleeping Beauty transposase encoding plasmid, and electroporated. Following electroporation, cell suspensions are collected, transferred to recovery media (50:50 media), and incubated in a 37°C / 5% CO2incubator overnight. Within 24 hours post- electroporation (Day 1), live cells are transferred to G-REX® culture plates and incubated with a first expansion media (50:50 media containing IL-2 + IL-21 + T Cell TransAct™). Cells are fed regularly with cytokines. After 14 days of first phase expansion, mTCR+ cells are isolated with anti-mTCR antibody. The isolated mTCR+ T cells are transferred to G-REX® culture plates and incubated with a second expansion media (50:50 media containing IL-2 + T Cell TransAct™). Cells are fed regularly with cytokines. After 13-14 days of second phase expansion, cells are harvested, phenotyped by flow cytometry to determine the mTCR expression, and evaluated in functional assays to determine the TCR specificity and function. 9.6.2 Generating TP53 R248W / B*57:01 TCR-T Cells Generation of monocyte-derived dendritic cells The DCs are derived from peripheral blood monocytes by adherence method and differentiation in vitro. Briefly, cryopreserved PBMCs with a known HLA haplotype are thawed, washed, resuspended in AIM-V media (Invitrogen, Carlsbad, CA) and seeded in a T175 flask. The cells are incubated at 37°C, 5% CO2for 2 hours allowing monocytes to adhere to the bottom of the flask. Thereafter, non- adherent PBMCs are removed, and the remaining adherent cells are washed carefully but extensively to ensure maximum removal of non-adherent cells. The remaining adherent cells (monocytes) are cultured in RPMI-1640 media containing 5% human AB serum and supplemented with 800 IU / mL GM-CSF and 200 IU / mL IL-4 to differentiate and maintain the monocyte-derived dendritic cells. Cells are cultured for a total of 6-9 days, with addition of cytokine-supplemented media every 2-3 days. At the end of the culture, both the non-adherent and adherent cell fractions are harvested and phenotyped for DC markers. 9.6.3 Coculture of TCR-T cells with peptide-pulsed dendritic cells For the co-culture assay, DCs are resuspended in 50:50 media and pulsed with KRAS G12V peptide or wild type (WT) peptide. The concentration of peptide used is titrated to exponentially decreasing concentrations (10, 1, 0.1, 0.01, and 0.001 μg / mL) to establish a dose-dependent effect of the TCR on its target neoantigen peptide, which also demonstrates the specificity of the TCR. After the Arnold & Porter Ref. P35502WO00 incubation, peptide-pulsed DCs are seeded at 2.5 x 104cells / well in a volume of 100 μL of a round- bottom 96 well plate. TCR-T cells are added to the peptide-pulsed DCs at 1 x 105cells / well in a 100 μL volume. DCs pulsed with only DMSO serves as a background control, while DCs alone or T cells alone serves as negative controls. DCs pulsed with WT peptide serves as an additional control to demonstrate the specificity of the TCR target. For positive controls, T cells alone are stimulated with a Cell Activation Cocktail (Biolegend, San Diego, CA) containing an optimized concentration of phorbol 12-myristate-13-acetate (PMA) and ionomycin. The DC-TCR-T cell co-culture is incubated for 18-24 h. 9.6.4 Measurement of T cell activation by 4-1BB expression At the end of the DC / TCR-T cell co-culture, cells are washed and stained with anti-4-1BB antibody to assess for TCR-specific T-cell activation and co-stained with antibodies against mTCRβ (clone H57-597), CD3, CD4 and CD8 for T cell phenotyping. Cells are acquired and analyzed on the flow cytometer (Novocyte Quanteon, Agilent Technologies, Santa Clara, CA), while data analysis is performed using NovoExpress software. FIG.76 shows upregulation of 4-1BB by activated TP53-R248W-B*57:01 TCR-T cells in response to TP53R248Wild Type (WT) or TP53R248Wneoantigen peptides. The figure shows the upregulation of 4-1BB on TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to TP53R248W / B*57:01 neoantigen or non-transposed (NT) T cells are co- cultured with monocyte-derived dendritic cells generated from a B*57:01 donor pulsed with TP53R248wildtype (WT) or TP53R248Wmutant peptides. After overnight incubation, the cells are harvested and analyzed by flow cytometry for 4-1BB expression within the mTCR+CD3+cell gate. N = 4 donors. 9.6.5 Measurement of T cell activation by IFN-γ secretion At the end of the DC / TCR-T co-culture supernatants are harvested and IFN-γ concentrations are measured using an Enzyme-linked immunosorbent assay (ELISA). Culture supernatants are diluted as necessary to bring the analyte concentrations to within the dynamic range of the assay. FIG. 77 shows secretion of IFN-γ by activated TP53-R248W-B*5701 TCR-T cells in response to TP53R248Wild Type (WT) or TP53R248Wneoantigen peptides. The figure shows the secretion of IFN- γ by TCR-T cells activated with mutant peptide-pulsed dendritic cells. TCR-T cells expressing TCRs reactive to TP53R248W / B*57:01 neoantigen or non-transposed (NT) T cells are co-cultured with monocyte-derived dendritic cells generated from an B*57:01 donor pulsed with TP53R248wildtype (WT) or TP53R248Wmutant peptides. After overnight incubation, the coculture media are harvested and the level of IFN-γ is measured by ELISA. N = 2 donors. Arnold & Porter Ref. P35502WO00 9.7 Conclusion The series of data described in this example illustrates the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0124, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA expression vectors encoding 42 TCRs from Patient 0124 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0124-TCR10 is found to be specific for the TP53 R248W neoantigen when presented in the context of B*57:01. EXAMPLE 10: PATIENT 0105 TCR SCREENING 10.1 Mutation and HLA Calling 10.1.1 Sample Demographics Patient 0105 is a colorectal cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 10.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. Arnold & Porter Ref. P35502WO00 10.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 10.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 1 hotspot mutation TP53 R273C. HLA profiling of the patient reveals HLAs as shown in FIG.78. 10.2 Design and Construction of Synthetic Reagent 10.2.1 TMG Plasmid Synthesis Each TMG, together with the flanking restriction sites, is synthesized, and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 10.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the Arnold & Porter Ref. P35502WO00 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 10.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 10.3.1 Cell preparation Patient 0105 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 99% and a final concentration of 300 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 6,000 cells. 10.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single cell suspensions are processed to distribute single cells into partitions using the 10x Chromium instrument. The resulting single-cell emulsion is processed to yield cDNA. The cDNA library is used as input to prepare a gene expression paired end library (GEX) and a TCR-specific paired end library (VDJ). The final paired end libraries are combined and loaded onto an Illumina NextSeqDx sequencer. Libraries are sequenced at 26 + 10 + 10 + 122 bp read lengths. The sequencing run yields 2 x 637.72 M reads pass filter and 81.91% of non-index bases achieves >=Q30 quality score. 10.3.3 scRNAseq Analysis VDJ sequencing data are preprocessed using the CellRanger toolkit (version 7.0) provided by 10X Genomics. Raw BCL files are converted to FASTQ files. Raw V(D)J sequencing reads are assembled into contigs using a graph-based algorithm with the aid of the pre-built reference sequence from the IMGT (www.imgt.org) database. Cells with identical productive V(D)J transcripts are considered to belong to the same clonotype. The following are reported for each unique clonotype: the amino acid sequence of the CDR3 region, the full-length FASTA sequence of the TRA chain, the full-length FASTA sequence of the TRB chain, and the clonotype frequency, defined as the number of cells in which each clonotype is observed. 10.4 TCR reconstruction 10.4.1 T-Cell Receptor (TCR) Assembly Single-cell RNAseq analysis yields 2623 clonotypes. All clonotypes present in 3 or more cells and containing both an alpha and a beta chain are modified and assembled to create TCRs for a total of 29 TCRs for class II. Each raw beta chain sequence is modified by replacing all sequence 5’ of the start of the V region with an NheI restriction site, and the entire constant region is replaced with a Arnold & Porter Ref. P35502WO00 BspI restriction site. Each alpha chain is modified by the replacement of all sequence 5’ of the start of the V region with an XmaI restriction site, and the constant region is replaced with a SacII restriction site. Rare codons (defined as codons used <10% according to the homo sapiens codon usage table) are replaced with more frequently used synonymous codons throughout the beta and alpha gene open reading frames. Incidental NheI, BspI, XmaI, and SacII restriction sites are eliminated from the open reading frame by replacing bases within the restriction sites with synonymous codons not found within each restriction site. 10.4.2 TCR Plasmid Assembly Each alpha and beta gene is synthesized independently and subcloned into pZT2 using the synthesized restriction sites (NheI and BspEI for the beta gene and XmaI and SacII for the alpha gene). Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 10.5 Patient 0105 TCR Screening 10.5.1 Experimental design and methods Day 1: COS-7 cells are seeded at 20,000 per well overnight in 96 multiwells. Day 2: COS-7 cells are transfected in each well with 150ng of TMGs+300ng of HLAs (75ng each). Day 3: LONZA 4D transfection system is set up the following day and 2 million cells are electroporated with each of the 47 TCR plasmid and negative control (NTC). Day 4: Jurkat cells are harvested and seeded on top of transfected COS-7 cells. After 5 hours co-culture, cells are harvested, and luciferase activity is measured. There are 1 TMG designed for the relevant mutations, and 71 TCRs are picked from 10x single cell sequencing for this patient 0124. In addition, this patient had 2 HLA-A, 2 HLA-B, 2 HLA- C, 2 HLA-DQ-A, 2 HLA-DQ-B, 1 DP-A, 1 DP-B, 2 DRB1 and 2 DRB3. The HLA plasmids are separated into 6 groups (HLA A, B, C, DQ, DP and DR) to reduce the number of combinations with TMG plasmid. Table 30 summarizes the screening strategy for Patient 0105. Class I TCRs are screened against HLA A, B or C. Class II TCRs are screened against HLA DP, DQ or DR. This patient has TP53- R273C mutation. Table 30. Screening strategy for Patient 0105. Arnold & Porter Ref. P35502WO00 10.5.2 Screening Results As shown in FIG.78, TCR 32-2, 67-1, 70, 113, 304 are specific to the combination of peptide and an HLA allele in locus HLA-DP, but not HLA DQ or HLA DR. To further define the HLA allele specificity, COS-7 cells are transfected with individual HLA allele plasmids and DPA1*01:03 DPB1*04:02 is the specific HLA restricting (FIG.79). However, TCR 70, 113 has reactivity against DPB1*02:01. 10.6 Conclusion The series of data described in this example illustrates the application of a high-throughput TCR isolation and screening method in a patient derived tumor specimen. Using a dissociated tumor sample from colorectal cancer Patient 0105, paired TCRα / β sequences are identified from tumor infiltrating T cells. These paired TCR sequences are reconstructed in silico from which DNA expression vectors encoding 29 TCRs from Patient 0105 are generated. Using the TCR screening method, all eighteen TCRs are successfully screened and one TCR, 0105-TCR32-2, 67-1, 70, 113, 304 are found to be specific for the TP53 R273W neoantigen when presented in the context of DPA1*01:03; DPB1*04:02. EXAMPLE 11: PATIENT 0236 TCR SCREENING 11.1 Mutation and HLA Calling 11.1.1 Sample Demographics Patient 0236 is an endometrium cancer patient. A specimen of dissociated tumor cells (DTCs) from this patient is procured through a commercial vendor (Discovery Life Sciences; Huntsville, AL). 11.1.2 Molecular Profiling Sample Processing DNA and RNA are isolated from dissociated tumor sample. Quantification by fluorescence spectrometry indicates that yields are sufficient for downstream applications, and gel electrophoresis demonstrates an absence of degradation in the isolated genomic DNA. RNA is found to be of sufficient quality for paired end library preparation. DNA from tumors are processed through the CareDX AlloSeq TX Version 5.0, Reference AST17.1(24) with an input of 10 ng. Pre-capture libraries are enriched via hybridization with the CareDX AlloSeq Tx Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 5000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D5000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 151bp on an Illumina MiSeqDx using a 300-cycle kit for a target coverage of 10 M reads. Libraries are then subject to on-board demultiplexing to yield FASTQ files. Arnold & Porter Ref. P35502WO00 DNA from tumors are processed through the Illumina DNA Prep with Enrichment protocol with an input of 1000 ng. Pre-captured libraries are enriched via hybridization with the IDT Integrated DNA technologies xGen Lockdown Probe Panel. Molarity of final libraries are determined using the size for fragments between 100 and 1000 bp on the Agilent TapeStation 4150 (Agilent High Sensitivity D1000 ScreenTape assay) and library concentration from Qubit 4 (Life Technologies Qubit dsDNA HS Assay kit). Libraries are pooled with a 1% PhiX spike-in. The library pool is clustered and sequenced at 2 x 149 bp on an Illumina NextSeqDX 550 using a 300-cycle High Output kit for a target coverage of 150 M reads. 11.1.3 Molecular Profiling Analysis For mutation calling, raw sequencing reads are processed using Illumina’s Basespace Enrichment App (3.1.0) into variant call files (VCFs) using the hg19 reference genome. hg19 Xgen-pan-cancer- targets intervals is used as the interval input for the Enrichment App. Basespace Variant Interpreter (2.16.2.1) is then used to annotate the VCFs with protein consequence data and the standard output is used for subsequent analyses. For HLA-typing, raw FASTQ files are imported into the AlloSeq Assign software (CareDx Pty Ltd ; version 1.0.3) for sequence alignment and comparison against the 3.45.1.1 reference consensus sequence. The HLA allele summary is generated using the standard AlloSeq Assign Full Report (protocol Document version 6.0). 11.1.4 Molecular Profiling Results To create peptide fragments containing the patient’s somatic mutations, a total of 192 common mutations in silico strands representing non-synonymous mutations are synthesized as peptides with crude quality. To create vectors containing the same somatic mutations in nucleic acid form, the 75 amino acid sequences are reverse translated in silico and codon optimized for expression in human cells. A total of 16 top spot tandem minigenes (tsTMGs) are designed by concatenating a set of 12 such amino acid sequences into one open reading frame. Incidental BamHI, EcoRI, NotI and NheI sites are removed by replacing codons within the restriction sites with synonymous codons. The nucleotide sequence GAG AAT TCG (codes for Glu (E) / Asn (N) / Ser (S) and contains an EcoRI restriction site) is added to the 5’ end of each TMG gene, and the nucleotide sequence AAG GAT CCC (codes for Lys (K) / Asp (D) / Pro (P) and has a BamHI restriction site) is added to the 3’ end of each TMG gene. Mutation panel analysis reveals 3 hotspot mutation KRAS G12V, PIK3CA E81K PPP2AR1 R183W. HLA profiling of the patient reveals HLAs as shown in FIG.80. 11.2 Design and Construction of Synthetic Reagent 11.2.1 TMG Plasmid Synthesis Arnold & Porter Ref. P35502WO00 Each TMG, together with the flanking restriction sites, is synthesized, and cloned into the masterTMG_pcDNA3.1(+) plasmid in frame with existing start and stop codons using EcoRI (5’) and BamHI (3’) restriction enzymes. Each final plasmid is prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 11.2.2 Human Leukocyte Antigen (HLA) Plasmid Assembly Peptide sequences for each HLA allele found in the patient sample are retrieved from the IPD- IMGT / HLA Database (ebi.ac.uk). Each peptide sequence is reverse translated in silico and codon optimized for expression in human cells. A BamHI restriction site and a Kozak site are added at the 5’ end of the coding sequence and an EcoRI restriction site and stop codon are appended to the 3’ end. The assembled sequence is synthesized and cloned into pcDNA3.1(+) using BamHI and EcoRI restriction enzymes. The resulting plasmids are prepared in TE with 95% ±5% supercoiled plasmid and ≤0.005 EU / μg endotoxin content. 11.3 Single-cell RNA Sequencing (scRNAseq) Analysis of TILs from Dissociated Tumor Sample 11.3.1 Cell preparation Patient 0236 DTCs are thawed, washed, and prepared in a single cell suspension. Cells are counted using an NC3000 automated cell counter (Chemometec). Cell viability is 93% and a final concentration of 500 cells / µL. The single cells suspension is loaded on a Chromium Controller (10x Genomics) with a targeted cell recovery of 10,000 cells. 11.3.2 Single-cell Paired End Library Preparation and Sequencing Prepared single ...

Claims

Arnold & Porter Ref. P35502WO00 CLAIMS:

1. A method for identifying a neoantigen-reactive T cell receptor (TCR), comprising: i) co-culturing a) a reporter T cell comprising a TCR expression cassette, and b) an antigen presenting cell (APC) that expresses a target neoantigen sequence and a matched human leukocyte antigen (HLA) sequence; and ii) identifying a positive reporter signal in the reporter T cell to identify a neoantigen- reactive TCR.

2. A method for identifying a neoantigen-reactive TCR, comprising: i. obtaining single-cell gene expression profiles from a population of tumor infiltrating lymphocytes (TIL) isolated from a patient sample, ii. performing bioinformatics analyses on the single cell gene expression data to identify TCR clonotypes of interests, iii. creating recombinant TCR sequences; iv. preparing a reporter T cell comprising a TCR expression cassette encoding a TCR sequence reconstructed from paired TCR α and β chain sequences identified from the clonotype of interest in step ii, v. preparing a tandem minigene (TMG) expression vector; vi. analyzing the patient sequencing data to identify class I and class II HLA alleles and preparing HLA expression vectors comprising the class I HLA and class II HLA allele sequences; vii. preparing an antigen presenting cell (APC) comprising transfecting the TMG expression vector and one or more HLA expression vectors into a cell wherein each transfection condition comprises a TMG and one or two HLA types; viii. co-culturing the reporter T cell in step iii with the APC of step vii, ix. identifying a positive reporter activity in the reporter T cell to identify a neoantigen- reactive TCR.

3. The method of claim 2, further comprising obtaining whole exome sequence (WES) data from the patient sample and analyzing the WES data to identify class I and class II HLA alleles.

4. The method of claim 2, wherein the performing bioinformatics analysis further comprises clustering the TCR clonotypes and to select a clonotype of interest.

5. The method of claim 2, wherein the creating recombinant TCR sequences comprises designing alpha and beta TCR sequences in silico.Arnold & Porter Ref. P35502WO00 6. The method of claim 2, wherein the TMG comprises nucleic acid sequences for the expression of concatenated amino acid sequences of non-synonymous single nucleotide variants (SNVs) 7. The method of claim 2, wherein the clustering comprises grouping the TCT clonotypes by CD8 or CD4 expression, gene function of differentially expressed genes, and the level of expression of each TCR.

8. The method of claim 2, further comprising culturing the reporter T cell having the identified neoantigen-reactive TCR with one or more peptides representing the non-synonymous single nucleotide variants (SNVs) present in the TMG.

9. The method of claim 1, wherein the APC is a COS-7 cell.

10. The method of claim 1 or 2, wherein the reporter T cell is an immortalized T cell line.

11. The method of claim 10, wherein the immortalized T cell is a Jurkat cell.

12. The method of claim 11, wherein the Jurkat cell is a Jurkat NFAT cell.

13. The method of any one of claims 1 to 12, wherein the TCR expression cassette comprises a TCR sequence reconstructed from TCR α and β chain sequences identified from tumor infiltrating lymphocytes (TILs) isolated from a tumor sample, and wherein the target neoantigen sequence and the matched HLA sequence are identified from the same tumor sample.

14. The method of any one of claims 1 to 13, wherein the method comprises identifying TCR sequences from TILs isolated from a tumor sample.

15. The method of claim 7, wherein the method further comprises identifying somatic mutations in the tumor sample and determining the germline HLA typing of the tumor sample.

16. A method of identifying a neoantigen-reactive T cell receptor (TCR), comprising: i) obtaining TCR α and β chain sequences from tumor infiltrating lymphocytes (TILs) isolated from a patient sample; ii) obtaining neoantigen sequences comprising somatic mutations present in the tumor sample, and the germline HLA typing of the patient sample; iii) co-culturing a) a reporter T cell expressing a TCR sequence reconstructed from the TCR α and β chain sequences obtained in step i), and b) an antigen presenting cell (APC) that expresses a neoantigen sequence and a matched human leukocyte antigen (HLA) sequence obtained in step ii); andArnold & Porter Ref. P35502WO00 iv) evaluating the reporter activity in the reporter T cell to identify a neoantigen-reactive TCR.

17. The method of claim 16, wherein the APC is a COS-7 cell, and the reporter T cell is a Jurkat NFAT cell.

18. The method of claim 13-17, wherein the isolated TILs are first expanded ex vivo and then co-cultured with APCs modified to express relevant HLA alleles and antigens obtained from the tumor sample.

19. The method of any one of claims 1 to 18, wherein the reporter T cell comprises a reporter system that is activated by the binding of the TCR to the neoantigen.

20. The method of any one of claims 1 to 19, wherein the reporter T cell and the APC are co- cultured in a ratio of 1:16, 1:8, 1:4, 1:2, 1:1, 2:1, 4:1, 8:1, or 16:

1.

21. The method of any one of claims 1 to 20, wherein the reporter T cell and the APC are co- cultured for 1-48 hours.

22. A TCR, or an antigen-binding portion thereof, isolated according to the method of any one of claims 1 to 21.

23. A TCR, or an antigen-binding portion thereof, comprising a sequence selected from the group consisting of SEQ ID NOs: 1-216 (the sequences provided in Tables 1-18).

24. A neoantigen / HLA complex, wherein the neoantigen comprises a sequence selected from the group consisting of SEQ ID NOs: 238-243 and 310 to 535 and wherein the HLA comprises a sequence selected from a group consisting of SEQ ID NOs: 301 to 309.

25. The neoantigen / HLA complex of claim 24, wherein the neoantigen is SEQ ID NO: 481 and the HLA sequence is SEQ ID NOs: 303 and 305 or SEQ ID NOs: 304 and 305.

26. The neoantigen / HLA complex of claim 24, wherein the neoantigen is SEQ ID NO: 385 and the HLA sequence is SEQ ID NO:

301.

27. The neoantigen / HLA complex of claim 24, wherein the neoantigen is SEQ ID NO: 394 and the HLA sequence is SEQ ID NOs: 308 and 306.

28. The neoantigen / HLA complex of claim 24, wherein the neoantigen is SEQ ID NO: 405 and the HLA sequence is SEQ ID NO:

302.

29. A recombinant vector expressing the T cell receptor, or an antigen-binding portion thereof, of claim 22 or 23.

30. A polynucleotide encoding an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-216 (the sequences provided in Tables 1-18).Arnold & Porter Ref. P35502WO00 31. A population of cells that comprise the recombinant vector of claim 29, or the polynucleotide of claim 30.

32. The population of cells of claim 31, wherein the recombinant vector or the polynucleotide is integrated into the genome of the population of cells.

33. The population of cells of claim 31 or 32, wherein the cells are immune effector cells.

34. The population of cells of claim 33, wherein the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, B cells, mast cells, and myeloid- derived phagocytes.

35. A pharmaceutical composition comprising the population of cells of any one of claims 31 to 34, and a pharmaceutically acceptable carrier.

36. A method of preparing a medicament for the treatment or prevention of a medical condition, the method comprising preparing the population of cells any one of claims 31 to 34.

37. A method of treating a disease or medical condition, the method comprising administering the pharmaceutical composition of claim 23 to a patient in need.

38. The method of claim 36 or 37, wherein the disease or medical condition is a cancer.

39. A co-culture reporter system for identifying a neoantigen-reactive T cell receptor (TCR), comprising: i) a reporter T cell comprising a TCR expression cassette, co-cultured with ii) an antigen presenting cell (APC) that expresses a target neoantigen sequence and a matched human leukocyte antigen (HLA) sequence.

40. The co-culture reporter system of claim 39, wherein the APC is a COS-7 cell, and the reporter T cell is a Jurkat NFAT cell.

41. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the target neoantigen is expressed in an antigen encoding plasmid.

42. The method or the reporter system of claim 41, wherein the antigen encoding plasmid is a Tandem Minigene (TMG) plasmid.

43. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the target neoantigen is introduced to the APC by the pulsing of peptide pools.

44. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the reporter T cell is a primary T cell.

45. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the reporter cell is from an immortalized T cell line.

46. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the TCR expression cassette comprises a full-length TCR sequence.Arnold & Porter Ref. P35502WO00 47. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the reporter T cell expresses any or all protein components of the TCR signaling complex or downstream signaling components.

48. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the reporter T cell expresses one or more components selected from the group consisting of CD3, CD4, CD8a, and CD8b.

49. The method or the reporter system of claim 48, wherein the reporter T cell is modified to enhance the activity of the one or more protein components.

50. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the TCR expression cassette is cloned into a non-viral gene transfer vector.

51. The method or the reporter system of claim 50, wherein the TCR expression cassette is cloned into a transposon.

52. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the APC is a classical professional APC (such as DC).

53. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the APC is an artificial APC.

54. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the APC endogenously express an HLA allele.

55. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the APC comprises a transgenic HLA expression plasmid.

56. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the APC expresses multiple transgenic HLA alleles in a single cell.

57. The method of claims 1 to 38, or the reporter system of claim 39 or 40, wherein the APC expresses a co-stimulatory molecule.

58. The method or the reporter system of claim 57, wherein the co-stimulatory molecule is one or more selected from the group consisting of 4-1BBL, CD40, CD80, CD86, or OX40L.

59. The method of claim 18, wherein a gene signature for identifying neoantigen reactive TCRs from ex vivo expanded TIL includes one or more gene(s) selected from the group consisting of XCL2, XCL1, IL2, CSF2, IFNG, CCL4, CCL4L2, TNF, CCL3, RGCC, TNFSF9, DUSP2, NFKBID, MIR155HG, NR4A3, EVI2A, CRTAM, ZBED2, FABP5, PIM3, NR4A1, IL10, TNFSF14, NR4A2, LINC00892, ZFP36L1, GZMB, MYC, SPRY1, KDM6B, EGR2, PHLDA1, PPP1R2, VSIR, REL, PRDX1, SLA, CYTOR, DDX21, IER3, PGAM1, NAMPT, HSP90AB1, IL23A, FAM107B, BCL2A1, ZEB2, ZBTB32, BTG2,Arnold & Porter Ref. P35502WO00 GADD45B, RILPL2, SEMA7A, TGIF1, SRGN, RAN, CFLAR, MAT2A, SIAH2, PRNP, RNF19A, FASLG, NME1, EVI2B, HSPH1, NOP16, CSRNP1, and TAGAP.

60. A recombinant vector comprising a polycistronic expression cassette, wherein the polycistronic expression cassette comprises a transcriptional regulatory element operably linked to a polycistronic polynucleotide that comprises: a. a first polynucleotide sequence that encodes a T cell receptor (TCR) alpha chain comprising an alpha chain variable (Vα) region and an alpha chain constant (Cα) region; b. a second polynucleotide sequence that comprises a first 2A element; c. a third polynucleotide sequence that encodes a TCR beta chain comprising a beta chain variable (Vβ) region and a beta chain constant (Cβ) region; d. a fourth polynucleotide sequence that comprises a second 2A element; and e. a fifth polynucleotide sequence that encodes a fusion protein that comprises IL-15, or a functional fragment or functional variant thereof, and IL-15Rα, or a functional fragment or functional variant thereof.

61. The recombinant vector of claim 60, wherein the polycistronic polynucleotide comprises the first, the second, the third, the fourth, and the fifth polynucleotide sequence in any order from 5’ to 3’.

62. The recombinant vector of claim 60 or 61, wherein the TCR alpha chain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of TCR alpha chain sequences disclosed in Tables 1-18, and wherein the TCR beta chain comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of TCR beta chain sequences disclosed in Tables 1-18.

63. A population of cells that comprise the recombinant vector of any one of claims 60-62.

64. A neoantigen-reactive TCR, or an antigen-binding portion thereof, wherein the neoantigen- reactive TCR or the antigen-binding portion thereof can bind to a neoantigen / HLA complex disclosed herein.

Images