Methods of isolating neoantigen-specific t cell receptor sequences

Abstract

To provide methods of producing a drug for treating or preventing cancer in mammals.SOLUTION: The methods include: isolating paired T cell receptor (TCR) alpha and beta chain sequences, or an antigen-binding portion thereof, and introducing a nucleotide encoding sequences of the isolated paired TCR alpha and beta chain sequences or antigen-binding portion thereof into host cells to obtain a population of cells that express the paired TCR alpha and beta chain sequences or antigen-binding portion thereof; and formulating the obtained population of cells into a drug for treating or preventing cancer in mammals.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application is a continuation of U.S. Provisional Patent Application No. 62 / 479, filed March 31, 2017. 398, which is incorporated by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under Project No. ZIABC010985 awarded by the National Institutes of Health, National Cancer Institute. The Government holds certain rights in this invention. has.

[0003] Incorporation by Reference of Electronically Submitted Materials Incorporated herein by reference in its entirety is a computer readable nucleotide / amino acid sequence listing identified below in one 5,530 byte ASCII (text) file entitled "737921 SeqListing_ST25.txt", dated March 22, 2018, which was submitted concurrently herewith. [Background technology]

[0004] 2. Background of the Invention Adoptive cell therapy (ACT) using cells that have been genetically engineered to express cancer antigen (e.g., neoantigen)-specific T cell receptors (TCRs) can produce positive clinical responses in some cancer patients. Nevertheless, obstacles remain to successfully use TCR-engineered cells to treat a wide range of cancers and other diseases. For example, TCRs that specifically recognize cancer antigens (e.g., neoantigens) can be difficult to identify and / or isolate from patients. Thus, there is a need for improved methods to obtain cancer-reactive (e.g., neoantigen-reactive) TCRs. Summary of the Invention

[0005] One aspect of the invention provides a method for isolating paired T cell receptor (TCR) alpha and beta chain sequences, or antigen-binding portions thereof, comprising the steps of: (a) isolating from a biological sample T cells having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation; (b) co-culturing the isolated T cells with an antigen-presenting cell (APC) that presents the mutated amino acid sequence, whereby the T cells express one or more T cell activation markers; (c) sorting the co-cultured T cells to separate single T cell antigens; (d) isolating mRNA from each separate single T cell sample; (e) sequencing the mRNA from each separate single T cell sample, wherein the sequencing comprises the steps of: (i) generating cDNA from the mRNA and amplifying the cDNA; (ii) generating multiple fragments of the amplified cDNA and tagging the multiple fragments; (iii) amplifying the tagged multiple fragments of the cDNA; and (iv) sequencing the amplified, tagged multiple fragments of the cDNA; wherein the sequencing identifies the sequence of each of the multiple fragments of the cDNA; (f) (g) aligning the sequence of each of the diverse fragments of the cDNA against known sequences of one or more T cell activation markers to identify which single T cell samples contain single T cells expressing one or more T cell activation markers; (g) aligning the sequence of each of the diverse fragments of the cDNA against a reference TCR sequence database to identify TCR alpha chain variable (V) segment sequences and TCR beta chain V segment sequences of the diverse fragments of the cDNA of each isolated single T cell sample identified in (f) as expressing one or more T cell activation markers; (h) aligning the TCR alpha chain variable (V) segment sequences and TCR beta chain V segment sequences of the diverse fragments of the cDNA of each isolated single T cell sample identified in (g). identifying TCR complementarity determining region 3 (CDR3) sequences within the cDNA variant fragments containing the TCR beta chain V segment sequence identified in (g); (i) counting the number of cDNA variant fragments sharing the same alpha chain CDR3 amino acid sequence and the number of cDNA variant fragments sharing the same beta chain CDR3 amino acid sequence; (j) counting the most abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence, the most abundant cDNA variant fragments encoding the same beta chain CDR3 sequence, and optionally the second most abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence. (k) identifying the TCR alpha and beta chain CDR3 sequences of the most abundant cDNA diverse fragment obtained in (j), the TCR beta chain V segment sequence of the most abundant cDNA diverse fragment obtained in (j), and optionally the TCR alpha chain V segment sequence of the second abundant cDNA diverse fragment obtained in (j), and identifying the TCR alpha and beta chain V segment sequences;and (l) assembling one or more nucleotide sequences encoding a TCR alpha chain comprising the V segment sequence of the TCR alpha chain identified in (k) and the CDR3 sequence of the TCR alpha chain taken in (j), and a TCR beta chain comprising the V segment sequence of the TCR beta chain identified in (k) and the CDR3 sequence of the TCR beta chain taken in (j), and optionally assembling one or more second nucleotide sequences encoding a second TCR alpha chain comprising the V segment sequence of the TCR alpha chain of the second most abundant cDNA diverse fragment identified in (k) and the CDR3 sequence of the TCR alpha chain of the second most abundant cDNA diverse fragment taken in (j), and a TCR beta chain comprising the V segment sequence of the TCR beta chain identified in (k) and the CDR3 sequence of the TCR beta chain taken in (j), to produce isolated and paired TCR alpha and beta chain sequences, or antigen-binding portions thereof;

[0006] Another aspect of the invention provides a method for automatically identifying T cell receptor (TCR) alpha and beta chain V segment sequences and TCR CDR3 sequences having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: (a) receiving, at a user's computer device, sequences of a polymorphic fragment of cDNA, where the cDNA is encoded by mRNA produced by a single T cell following co-culture of the T cell with an antigen presenting cell (APC) that presents the mutated amino acid sequence, whereby the T cell expresses one or more T cell activation markers; (b) computerized alignment of the sequence of each of the polymorphic fragments of cDNA against a reference TCR sequence database to identify variable (V) segment sequences of TCR alpha chain and V segment sequences of TCR beta chain of the polymorphic fragment of cDNA; (c) identifying a variable (V) segment sequence of TCR alpha chain and a V segment sequence of TCR beta chain of the polymorphic fragment of cDNA comprising the V segment sequence of TCR alpha chain identified in (b) and a V segment sequence of TCR beta chain identified in (b). (d) performing a computerized identification of TCR complementarity determining region 3 (CDR3) sequences within the cDNA polymorphic fragments that contain a CDR3 amino acid sequence of the TCR alpha and beta chain; (e) performing a computerized measurement of the number of cDNA polymorphic fragments that share the same alpha chain CDR3 amino acid sequence and the number of cDNA polymorphic fragments that share the same beta chain CDR3 amino acid sequence; (e) performing a computerized collection of the most abundant cDNA polymorphic fragments that encode the same alpha chain CDR3 sequence, the most abundant cDNA polymorphic fragments that encode the same beta chain CDR3 sequence, and optionally the second abundant cDNA polymorphic fragments that encode the same alpha chain CDR3 sequence, to identify TCR alpha and beta chain CDR3 sequences, wherein the alpha chain CDR3 sequence encoded by the second abundant cDNA polymorphic fragment is different from the alpha chain CDR3 sequence encoded by the most abundant cDNA polymorphic fragment; and (f) performing a computerized measurement of the number of TCR alpha chain V segment CDR3 sequences within the most abundant cDNA polymorphic fragments collected in (e). and performing computerized identification of the TCR alpha and beta chain V segment sequences of the most abundant cDNA diverse fragment obtained in (e), the TCR beta chain V segment sequence of the most abundant cDNA diverse fragment obtained in (e), and optionally, the TCR alpha chain V segment sequence of the second most abundant cDNA diverse fragment obtained in (e), to identify the TCR alpha and beta chain V segment sequences.

[0007] Another aspect of the invention provides a method for preparing a cell population expressing paired TCR alpha and beta chain sequences, or antigen binding portions thereof, comprising isolating the paired TCR alpha and beta chain sequences, or antigen binding portions thereof, by any of the methods of the invention described herein, and introducing a nucleotide sequence encoding the isolated paired TCR alpha and beta chain sequences, or antigen binding portions thereof, into a host cell to obtain cells expressing the paired TCR alpha and beta chain sequences, or antigen binding portions thereof.

[0008] A further aspect of the present invention provides a pair of TCR alpha and beta chain sequences, or antigen-binding portions thereof, isolated by any of the methods of the invention described herein.

[0009] Yet another aspect of the present invention provides an isolated cell population prepared by any of the methods of the present invention described herein.

[0010] Further aspects of the invention provide related pharmaceutical compositions and methods for the treatment or prevention of cancer. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram of the method for identifying neoantigen-reactive TILs. [Diagram 2] FIG. 2 is a schematic diagram of a method for identifying neoantigen-specific TCRs. [Figure 3A]Figures 3A and 3B are graphs showing the percentage of IFN-γ (A) and IL-2 (B) reads within total R1 reads measured in 4090F7 T cells cocultured with TMG-5-pulsed autologous dendritic cells (DCs) for 4 hours and then subjected to single cell RNA-seq analysis. [Figure 3B] Figures 3A and 3B are graphs showing the percentage of IFN-γ (A) and IL-2 (B) reads within total R1 reads measured in 4090F7 T cells cocultured with TMG-5-pulsed autologous dendritic cells (DCs) for 4 hours and then subjected to single-cell RNA-seq analysis. [Figure 3C] Figure 3C is a graph showing the amount of IFN-γ (pg / mL) secreted by donor T cells, untransduced (unshaded bars) or transduced with 4090TCR (shaded bars), upon co-culture with dendritic cells (DCs) pulsed with TMG-5 or TMG-6. DCs pulsed without TMG ("w / o") served as a negative control. [Figure 3D] FIG. 3D is a graph showing the amount of IFN-γ (pg / mL) secreted by 4090TCR-transduced T cells upon co-culture with 4090 dendritic cells (DCs) pulsed with a mutated 25mer peptide corresponding to one of the indicated minigenes derived from TMG-5. [Figure 3E] FIG. 3E is a graph showing the amount of IFN-γ (pg / mL) secreted by 4090TCR-transduced T cells upon co-culture with 4090 dendritic cells (DCs) pulsed with purified 25-mer wild-type (WT) (open circles) or mutated (filled circles) USP8 peptides at the indicated concentrations (conc.) (μM). [Figure 4A] Figures 4A and 4B are graphs showing the percentage of IFN-γ (A) and IL-2 (B) reads within total R1 reads measured in 4095F5 T cells cocultured with TMG-1-pulsed autologous dendritic cells (DCs) for 4 hours and then subjected to single-cell RNA-seq analysis. [Figure 4B] Figures 4A and 4B are graphs showing the percentage of IFN-γ (A) and IL-2 (B) reads within total R1 reads measured in 4095F5 T cells cocultured with TMG-1-pulsed autologous dendritic cells (DCs) for 4 hours and then subjected to single-cell RNA-seq analysis. [Figure 4C] FIG. 4C is a graph showing the percentage of IFN-γ and IL-2 leads within the total R1 leads measured in single cells that expressed detectable IL-2 leads in FIG. 4B. [Figure 4D] Figure 4D is a graph showing the amount of IFN-γ (pg / mL) secreted by untransduced (unshaded bars) or 4095TCR-transduced (shaded bars) donor T cells upon co-culture with dendritic cells (DCs) pulsed with full-length wild-type (WT) or mutated KRAS mRNA. Dendritic cells (DCs) pulsed without peptide ("w / o") served as a negative control. [Figure 4E] FIG. 4E is a graph showing the amount of IFN-γ (pg / mL) secreted by 4095TCR-transduced T cells upon co-culture with 4095 dendritic cells (DCs) pulsed with the indicated concentrations (μM) of purified 9-mer wild-type (WT) (open circles) or mutated (filled circles) KRAS peptides. [Figure 5A] Figures 5A and 5B are graphs showing the percentage of IFN-γ (A) and IL-2 (B) reads within total R1 reads measured in 4112F5 T cells cocultured with TMG-9-pulsed autologous dendritic cells (DCs) for 4 hours and then subjected to single-cell RNA-seq analysis. [Figure 5B] Figures 5A and 5B are graphs showing the percentage of IFN-γ (A) and IL-2 (B) reads within total R1 reads measured in 4112F5 T cells cocultured with TMG-9-pulsed autologous dendritic cells (DCs) for 4 hours and then subjected to single-cell RNA-seq analysis. [Figure 5C]FIG. 5C is a graph showing the percentage of IFN-γ and IL-2 leads within the total R1 leads measured in the eight single cells that expressed detectable IL-2 leads in FIG. 5B. [Figure 5D] Figure 5D is a graph showing the amount of IFN-γ (pg / mL) secreted by untransduced (unshaded bars) or 4112TCR-transduced (shaded bars) donor T cells upon co-culture with TMG-9 or TMG-10 pulsed dendritic cells (DCs). Dendritic cells (DCs) pulsed without peptide ("w / o") served as a negative control. [Figure 5E] Figure 5E is a graph showing the amount of IFN-γ (pg / mL) secreted by 4112TCR-transduced T cells upon co-culture with EBV-transformed B cells pulsed with one of the indicated pools of short peptides (SPP-1 to SPP-10). EBV-transformed B cells pulsed without peptide ("w / o") served as a negative control. [Figure 5F] Figure 5F is a graph showing the amount of IFN-γ (pg / mL) secreted by 4112TCR-transduced T cells upon co-culture with EBV-transformed B cells pulsed with SPP-9 or one of the indicated short peptides: VWDALFADGLSLCL (SEQ ID NO: 18); WRRVAWSYDSTLL (SEQ ID NO: 19); WSYDSTLL (SEQ ID NO: 20); WSYDSTLLA (SEQ ID NO: 21); WSYDSTLLAY (SEQ ID NO: 22); YLALVDKNIIGY (SEQ ID NO: 23); or YSEPDVSGK (SEQ ID NO: 24). EBV-transformed B cells pulsed without peptide ("w / o") served as a negative control. [Figure 5G] FIG. 5G is a graph showing the amount of IFN-γ (pg / mL) secreted by 4112TCR-transduced T cells upon co-culture with EBV-transformed B cells pulsed with purified mutated (closed circles) NBAS peptide WSYDSTLLAY(C>S) (SEQ ID NO:4) or its wild-type (WT) (open circles) counterpart. [Figure 6]FIG. 6 is a block diagram illustrating a system according to some embodiments of the present invention. [Figure 7] FIG. 7 is a block diagram illustrating components of a computing device according to one embodiment of the present invention. [Figure 8] FIG. 8 is a flow diagram of method steps for automatically identifying T cell receptor (TCR) alpha and beta chain V segment sequences and TCR CDR3 sequences that have antigen specificity for mutated amino acid sequences encoded by cancer-specific mutations, according to one embodiment of the present invention. [Figure 9A] Figure 9A is a graph showing the number of IFN-γ positive spots detected after screening TIL4171F6 T cells against a library of 25mer long peptide pools (PP) encoding mutations in an ELISPOT assay. T cells treated with OKT3 antibody served as a positive control. T cells cultured without peptide pools (w / o) served as a negative control. [Figure 9B] Figures 9B and 9C are graphs showing expression of IFN-γ (FPKM (fragments per thousand bases of transcript per million mapped reads)) (Figure 9B) and IL-2 (Figure 9C) by TIL4171 F6 T cells upon co-culture with autologous dendritic cells (DCs) pulsed with PP-3. [Figure 9C] Figures 9B and 9C are graphs showing expression of IFN-γ (FPKM (fragments per thousand bases of transcript per million mapped reads)) (Figure 9B) and IL-2 (Figure 9C) by TIL4171 F6 T cells upon co-culture with autologous dendritic cells (DCs) pulsed with PP-3. [Figure 9D] FIG. 9D is a two-dimensional scatter plot combining the data from FIGS. 9B and 9C showing the relationship between IFN-γ and IL-2 expression in each single cell (each point represents a single cell). [Figure 9E]Figure 9E is a graph showing the amount of IFN-γ (pg / mL) produced following co-culture of untransduced (unshaded bars) or 4171TCR-transduced (shaded bars) cells with PP-pulsed dendritic cells (DCs). T cells cultured without peptide pool (w / o) served as a negative control. [Figure 9F] Figure 9F is a graph showing the amount of IFN-γ produced (pg / mL) following co-culture of 4171TCR-transduced T cells with dendritic cells (DCs) pulsed with the indicated peptides. T cells cultured without peptide pools (w / o) served as a negative control. [Figure 9G] Figure 9G is a graph showing the amount of IFN-γ produced (pg / mL) following co-culture of 4171TCR-transduced T cells with dendritic cells (DCs) pulsed with the indicated concentrations (μM) of wild-type (WT) (open circles) or mutated (filled circles) SIN3A peptides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention In one aspect, the invention provides a method for isolating paired TCR alpha and beta chain sequences, or antigen-binding portions thereof.

[0013] The methods of the invention may address any of a variety of different challenges for the identification and isolation of functional TCRs with the desired antigenic specificity. These challenges may include, for example, the large degree of diversity in TCR sequences, the requirement for the TCR α and β chains to be correctly paired to provide the desired antigenic specificity, and the requirement that up to about one-third of mature T cells may express two functional TCR α chains, only one of which may have the desired specificity.

[0014] The methods of the invention may provide any of a variety of advantages. For example, the methods of the invention may significantly reduce the time and / or cost required to isolate and identify TCR sequences having antigen specificity for a cancer antigen (e.g., neoantigen) following removal of a biological sample (e.g., a tumor sample) from a patient. Following isolation and identification of the TCR sequences, host cells (e.g., autologous T cells) may be transduced with the TCR sequences, the transduced cells expanded in number, and the expanded transduced cells administered to the patient for the treatment and / or prevention of cancer. The methods of the invention may include (i) ) identify both the cancer antigen and the sequence of the TCR that recognizes that cancer antigen, and / or (ii) facilitate highly personalized TCR therapy targeting cancer antigens (e.g., neoantigens). Further advantageously, the method of the present invention can be less time-consuming, less laborious, and have a higher success rate than the method of isolating paired TCR alpha / beta sequences using limiting dilution T cell cloning. The method of the present invention can also efficiently identify the correct pair of TCR alpha and beta chains in those T cells that have more than one functional TCR alpha gene. The method of the present invention can also identify and isolate paired TCR alpha and beta chain sequences (with the desired antigen specificity) from a highly diverse T cell population.

[0015] The αβTCR is a heterodimer consisting of an α and a β protein chain. Each chain contains two extracellular domains, a variable (V) region and a constant (C) region, followed by a transmembrane domain region and a short cytoplasmic tail. The variable domains of each TCR α and β chain contain three "complementarity determining regions" (CDR1, CDR2, and CDR3) that contact and recognize peptide-MHC complexes. In particular, CDR3 of α and β are responsible for the recognition of processed antigens. There is a very high degree of polymorphism in the amino acid sequences of CDR3α and CDR3β among T cells. This level of polymorphism is necessary for T cells to recognize the broad range of antigens that the immune system faces. Polymorphism in the amino acid sequences of CDR3α and CDR3β results from DNA rearrangements within the TCRα and β genes that occur during T cell maturation.

[0016] The gene encoding the TCR is composed of cassettes of coding sequences called "V segments" and "J segments" in the TCR α gene and "V segments", "D segments" and "J segments" in the TCR β chain. Stochastic rearrangements in genomic DNA result in the juxtaposition of these DNA segments, which have functions. These rearrangements may be imprecise, and the Vα-Jα and Vβ-Dc-Jβ segment junctions may be highly variable. The CDR3 of the alpha chain is encoded by part of the V segment and all of the J segment. The CDR3 of the beta chain is encoded by part of the V segment, all of the J segment, and all of the D segment.

[0017] The method can include isolating T cells having antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation from the biological sample. Any suitable biological sample can be used. In one embodiment of the present invention, the biological sample is a tumor sample or a peripheral blood sample. Examples of biological samples that can be used in the present invention include, but are not limited to, tissue from a primary tumor, tissue from a site of a metastatic tumor, exudate, transudate, ascites, fractionated peripheral blood cells, bone marrow, peripheral blood buffy coat, and cerebrospinal fluid. Thus, the biological sample can be obtained by any suitable means, including, but not limited to, aspiration, biopsy, resection, venipuncture, arterial puncture, lumbar puncture, shunt, catheterization, or drain replacement.

[0018] The T cells isolated from the biological sample have antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. As used herein, the phrase "antigen specificity" means that the TCR or an antigen-binding portion thereof can specifically bind to and immunologically recognize the mutated amino acid sequence encoded by the cancer-specific mutation. The cancer-specific mutation may be any mutation in any gene that encodes the mutated amino acid sequence (also called a "non-silent mutation") and is expressed in cancer cells but not in normal, non-cancerous cells. Methods for isolating T cells having antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation are described, for example, in International Publication No. 2016 / 053338 and International Publication No. 2016 / 053339. For example, a method for isolating T cells having antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation may be described, for example, in International Publication No. 2016 / 053338 and International Publication No. 2016 / 053339. Isolating a T cell having the mutated amino acid sequence may include the steps of: identifying one or more genes in nucleic acid of the patient's cancer cells, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing the patient's autologous antigen presenting cells (APCs) to present the mutated amino acid sequence; co-culturing the patient's autologous T cells with autologous APCs that present the mutated amino acid sequence; and selecting autologous T cells that (a) are co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigen specificity for the mutated amino acid sequence presented in the context of a major histocompatibility complex (MHC) molecule expressed by the patient to provide isolated T cells having antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

[0019] Once T cells with antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation are isolated, the method of the present invention further comprises co-culturing the isolated T cells with APCs presenting the mutated amino acid sequence, whereby the T cells express one or more T cell activation markers. APCs may comprise any cell that presents a peptide fragment of a protein in association with a major histocompatibility complex (MHC) molecule on their cell surface. APCs may comprise, for example, any one or more of macrophages, dendritic cells (DCs), Langerhans cells, B lymphocytes, and T cells. Preferably, the APCs are dendritic cells (DCs). Any one or more of a variety of T cell activation markers may be used to identify those T cells with antigen specificity for the mutated amino acid sequence. Examples of T cell activation markers include, but are not limited to, any one or more of programmed cell death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, OX40, CD107a, granzyme B, interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor alpha (TNF-α), granulocyte / monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

[0020] This method involves sorting the co-cultured T cells to separate them into a single T cell sample. and isolating mRNA from each isolated single T cell sample. The steps of sorting into isolated single T cell samples and isolating mRNA may be automated. For example, the steps of sorting into isolated single T cell samples and isolating mRNA can be performed using the FLUIDIGM C1 Automated Single Cell Isolation and Preparation System (available from Fluidigm, South San Francisco, Calif.). The method of the present invention can advantageously provide any number of isolated single cell mRNA samples (e.g., about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). In one embodiment of the present invention, the method comprises the preparation of about 96 isolated single cell mRNA samples.

[0021] In one embodiment of the invention, the method may further comprise labeling the mRNA from each isolated single T cell sample with a different tag (e.g., barcode) for each isolated single T cell sample. For example, the mRNA from each isolated single T cell sample may be labeled using an ILLUMINA NEXTERA XT DNA Library Preparation Kit (available from Illumina, San Diego, Calif.).

[0022] The method of the present invention further comprises the step of sequencing the mRNA from each isolated single T cell sample. The sequencing can be carried out in any suitable manner known in the art. A suitable example of a sequencing technique that may be useful in the method of the present invention is next generation sequencing. "NGS" (also called "massively parallel sequencing technology" or "deep sequencing") or third generation sequencing. NGS refers to high-performance DNA sequencing technology that is not based on the Sanger method. With NGS, millions or billions of DNA strands can be sequenced in parallel, resulting in substantially higher performance and minimizing the need for fragment cloning methods that are often used in Sanger sequencing of genomes. In NGS, nucleic acid templates are generated in parallel across the entire genome by breaking the entire genome down into small pieces. The plates can be read randomly. NGS can advantageously provide the nucleic acid sequence information from each isolated single T cell mRNA sample within a very short period of time, for example, within about 1 week to about 2 weeks, preferably within about 1 day to about 7 days, or most preferably within about 24 hours. A variety of NGS platforms that are commercially available or described in the literature, such as those described in Zhang et al. J. Genet. Genomics, 38(3): 95-109 (2011) and Voelkerding et al. Clinical Chemistry, 55: 641-658 (2009), can be used in connection with the method of the present invention.

[0023] Non-limiting examples of NGS technologies and platforms include sequencing by synthesis (also known as "pyrosequencing") (e.g., the GS-FLX454 Genomic Sequencing System, 454 Life Sciences, Branford, Connecticut, the ILLUMINA SOLEXA Genomic Analysis System (Illumina, Inc., San Diego, California), the ILLUMINA sequencing-by-ligation (e.g., using the SOLID platform (Life Technologies Corporation, Carlsbad, CA) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)); single-molecule sequencing (e.g., using the PACBIO platform); These include the RS system (performed using the Pacific Biosciences (Menlo Park, CA) or the HELISCOPE platform (Helicos Biosciences (Cambridge, MA)), nanotechnologies for single molecule sequencing (e.g., performed using the GRIDON platform from Oxford Nanopore Technologies (Oxford, UK), the Hybridization Assisted Nanopore Sequencing (HANS) platform developed by Navsys (Providence, RI), and a ligase-based DNA sequencing platform with DNA nanoball (DNB) technology called probe-anchor ligation (cPAL)), electron microscopy-based techniques for single molecule sequencing, and ion semiconductor sequencing.

[0024] In this regard, sequencing the mRNA from each isolated single T cell sample can include generating cDNA from the mRNA, amplifying the cDNA, generating multiple fragments of the amplified cDNA and tagging the multiple fragments, amplifying the tagged multiple fragments of the cDNA, and sequencing the amplified, tagged multiple fragments of the cDNA. Tagging can include adding nucleotide sequences to each multiple fragment, allowing the multiple fragments to be distinguished from one another. Sequencing identifies the sequence of each of the multiple fragments of the cDNA. The sequence of each of the multiple fragments of the cDNA is also referred to as a "read." Sequencing of mRNA can generate any number of reads. For example, mRNA sequencing may generate about 1,000,000 reads, about 900,000 reads, about 800,000 reads, about 700,000 reads, about 600,000 reads, about 500,000 reads, about 400,000 reads, about 300,000 reads, about 200,000 reads, about 100,000 reads or more, or a range defined by any two of the foregoing values, for each single T cell sample. There may be two reading directions in the S platform: one forward reading (also called "read 1" or "R1") and the other reverse reading (also called "read 2" or "R2"). For a cDNA fragment, R1 and R2 may be complementary to each other. In one embodiment of the invention, the method comprises measuring only the R1 read, only the R2 read, or both the R1 and R2 reads. R1 may have a higher sequencing quality than R2. Preferably, the method comprises measuring only the R1 read.

[0025] The method further includes aligning the sequence of each of the diverse fragments of the cDNA to known sequences of one or more T cell activation markers to identify which single T cell samples contain single T cells expressing the one or more T cell activation markers. Following co-culture with APCs presenting the mutated amino acid sequence encoded by the cancer-specific mutation, the one or more single T cell(s) expressing the one or more single T cell activation markers are identified as expressing a TCR with antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

[0026] The method further includes aligning the sequence of each of the diverse cDNA fragments against a reference TCR sequence database to identify TCR alpha chain variable (V) region sequences and TCR beta chain V region sequences of the diverse cDNA fragments of each isolated single T cell sample identified as expressing one or more T cell activation markers. In this regard, the sequence of each of the diverse cDNA fragments is aligned against known TCR variable segment sequences to identify which cDNA fragments contain all or a portion of a variable segment sequence and to determine the approximate location of the 3' end of the variable segment sequence of the cDNA fragment(s). The 3' end of the variable segment sequence indicates the approximate location of the CDR3.

[0027] The reference TCR sequence database may be any suitable reference TCR sequence database. An example of a reference TCR sequence database is the International IMMUNOGENETICS Information System (IMGT) database described in Lefranc et al. Nucleic Acids Res., 43: D413-422 (2015). The sequences of the various fragments of the cDNA may include sequences obtained from the reference TCR sequence database (http: / / www.imgt.org). Alignment of the sequences of each of the various fragments of the cDNA to the reference TCR sequence database may be performed using the Burrows-Wheeler Aligner (BWA) software package (http: / / bio-bwa.sourceforge.net / ), described, for example, in Li et al., Bioinformatics, 25: 1754-60 (2009) and Li et al., Bioinformatics, 26(5): 589-95 (2010).

[0028] The method further comprises identifying TCR complementarity determining region 3 (CDR3) sequences within the various fragments of cDNA that contain the identified TCR alpha chain V segment sequences and within the various fragments of cDNA that contain the identified TCR beta chain V segment sequences. The CDR3 region sequences can be identified in any suitable manner. In one embodiment of the invention, the TCR CDR3 sequences are identified by identifying cDNA sequences that encode conserved amino acid residues located near the C-terminus of the amino acid sequences encoded by the alpha and beta chain V segments. For example, the TCR CDR3 sequences can be identified by YX1CX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 (SEQ ID NO:5) The method can be carried out by identifying any cDNA sequence(s) encoding the amino acid sequence motif of 10 From X 21 each of which is no amino acid or any naturally occurring amino acid, and 22is phenylalanine or tryptophan. The amino acid sequence motif of SEQ ID NO:5 is a conserved amino acid sequence motif located near the C-terminus of the amino acid sequence encoded by the V segment.

[0029] In one embodiment of the invention, the method further comprises the steps of: The method includes identifying a constant (C) region sequence of a TCR alpha chain of a fragment of a diverse cDNA of the most abundant cDNA collected, and a C region sequence of a TCR beta chain of a fragment of a diverse cDNA of the most abundant cDNA collected. Optionally, the method further includes identifying a C region sequence of a TCR alpha chain of a fragment of a diverse cDNA of the second most abundant cDNA collected. The TCR alpha chain has one candidate constant region amino acid sequence. The TCR beta chain has one of two candidate constant region amino acid sequences.

[0030] The method further includes the step of determining the number of different fragments of cDNA that share the same alpha chain CDR3 amino acid sequence and the number of different fragments of cDNA that share the same beta chain CDR3 amino acid sequence.

[0031] The method further includes taking the most abundant cDNA diverse fragments encoding the same alpha chain CDR3 sequence, the most abundant cDNA diverse fragments encoding the same beta chain CDR3 sequence, and optionally the second abundant cDNA diverse fragments encoding the same alpha chain CDR3 sequence, and identifying TCR alpha and beta chain CDR3 sequences. The alpha chain CDR3 sequence encoded by the second abundant cDNA diverse fragment is different from the alpha chain CDR3 sequence encoded by the most abundant cDNA diverse fragment. The identified CDR3 sequences may include the beta chain CDR3 sequence and the alpha chain CDR3 sequence of a TCR with antigen specificity for the mutated amino acid sequence encoded by the cancer specific mutation, and optionally an additional alpha chain CDR3 sequence expressed by the T cell that does not pair with the beta chain CDR3 sequence to form a TCR with antigen specificity for the mutated amino acid sequence encoded by the cancer specific mutation. It is estimated that about one-third of mature T cells can express two TCR alpha chains. Only one of the expressed alpha chains will pair with an expressed TCR beta chain to provide a TCR with antigen specificity for the amino acid sequence encoded by the cancer-specific mutation.

[0032] The method further includes identifying the TCR alpha chain V segment sequence of the most abundant cDNA diverse fragment collected, the TCR beta chain V segment sequence of the most abundant cDNA diverse fragment collected, and optionally the TCR alpha chain V segment sequence of the second most abundant cDNA diverse fragment collected, and identifying the TCR alpha and beta chain V segment sequences. The number of cDNA diverse fragments encoding the CDR3 sequence of the dominant TCR expressed by a single, activated T cell will exceed the number of cDNA fragments encoding the CDR3 sequence of any other TCR that may be present due to contamination by about 10 to about 100 times. The source of contamination may be a nearby single cell sample or may be of unknown origin. The dominant TCR expressed by a single T cell that expresses one or more T cell activation markers in response to co-culture with an APC presenting the mutated amino acid sequence is a TCR with antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

[0033] The method further includes assembling one or more nucleotide sequences encoding a TCR alpha chain comprising the identified TCR alpha chain V segment sequence and the collected TCR alpha chain CDR3 sequence, and a TCR beta chain comprising the identified TCR beta chain V segment sequence and the collected TCR beta chain CDR3 sequence. The various fragments of different cDNAs encoding the same CDR3 sequence may be of different lengths and may overlap with each other. By aligning the various fragments of different cDNAs encoding the same alpha chain CDR3 sequence of different lengths with each other, it is possible to determine the sequence of the entire V segment, J segment, and optionally the constant region of the predominant TCR alpha chain. By aligning the various fragments of different cDNAs encoding the same beta chain CDR3 sequence of different lengths with each other, it is possible to determine the sequence of the entire V segment, J segment, D segment, and optionally the constant region of the predominant TCR beta chain. Nucleotide sequences encoding the entire V segment, J segment, and optionally the constant region of the predominant TCR alpha chain, and the entire V segment, J segment, D segment, and optionally the constant region of the predominant TCR beta chain can be assembled using routine techniques. Isolated and paired TCR alpha and beta chain sequences, or antigen-binding portions thereof, can be produced.

[0034] In one embodiment of the present invention, assembling one or more nucleotide sequences includes assembling a TCR alpha chain comprising a V segment sequence of a TCR alpha chain identified in a sample, a C region sequence of a TCR alpha chain identified in a sample, and a CDR3 sequence of a TCR alpha chain obtained, and assembling a TCR beta chain comprising a V segment sequence of a TCR beta chain identified in a sample, a C region sequence of a TCR beta chain identified in a sample, and a CDR3 sequence of a TCR beta chain obtained. In this regard, the assembled nucleotide sequence may comprise an endogenous C region sequence.

[0035] In one embodiment of the invention, assembling one or more nucleotide sequences includes assembling a TCR alpha chain comprising a V-segment sequence of a TCR alpha chain identified in the sample, a C-region sequence of an exogenous TCR alpha chain, and a CDR3 sequence of a harvested TCR alpha chain, and assembling a TCR beta chain comprising a V-segment sequence of a TCR beta chain identified in the sample, a C-region sequence of an exogenous TCR beta chain, and a CDR3 sequence of a harvested TCR beta chain. An exogenous C-region sequence is a C-region sequence that is not native (not naturally occurring) to the T cell. In this regard, the isolated paired TCR alpha and beta chain sequences, or antigen-binding portions thereof, produced by the method may be chimeric or hybrid TCRs, consisting of amino acid sequences obtained from TCRs derived from two different mammalian species. For example, the TCR may comprise a variable region derived from a human TCR and a constant region of a mouse TCR, such that the TCR is "murinized". Methods for generating chimeric or hybrid TCRs are described, for example, in Cohen et al. Cancer Res., 66: 8878-8886 (2006); Cohen et al. Cancer Res., 67: 3898-3903 (2007); and Haga-Friedman et al. J. Immunol., 188: 5538-5546 (2012).

[0036] A single T cell typically expresses one TCR beta chain and one or two TCR alpha chains. The presence of more than one TCR beta chain in a single sample may be the result of imperfect sorting of T cells into separate T cell samples. Complete sorting may inadvertently result in the inclusion of more than one T cell in a sample. If a single sample is found to express more than one TCR beta chain, that sample may be excluded from subsequent analysis.

[0037] As discussed above, it is estimated that about one third of mature T cells may express two TCR alpha chains. Only one of the expressed alpha chains will pair with the expressed TCR beta chain to provide a TCR with antigen specificity for the amino acid sequence encoded by the cancer-specific mutation. To determine which TCR alpha chain will pair with the TCR beta chain to provide the desired specificity, the method may include assembling a first nucleotide sequence encoding a first TCR alpha chain comprising a first TCR alpha chain V segment sequence of a diverse fragment of the most abundant cDNA identified as described herein and a first TCR alpha chain CDR3 sequence taken as described herein, and a TCR beta chain comprising a TCR beta chain V segment sequence identified as described herein and a TCR beta chain CDR3 sequence taken as described herein. The method optionally further comprises a V segment sequence of the TCR alpha chain of the identified second most abundant cDNA polymorphic fragment, and a CDR3 sequence of the TCR alpha chain of the identified second most abundant cDNA polymorphic fragment. The method may include assembling one or more second nucleotide sequences encoding a second TCR alpha chain and a TCR beta chain comprising the V segment sequence of the identified TCR beta chain and the CDR3 sequence of the harvested TCR beta chain.

[0038] The method may further include independently introducing the first and second nucleotide sequences into a first and second population of host cells, respectively, and independently co-culturing the first and second populations of host cells with APCs that present the mutated amino acid sequence encoded by the cancer-specific mutation. The method may further include selecting a population of host cells that (a) are co-cultured with the APCs that present the mutated amino acid sequence, and (b) have antigenic specificity for the mutated amino acid sequence. The co-cultured population of host cells that have antigenic specificity for the mutated amino acid sequence will express a TCR alpha chain that provides the desired specificity along with a TCR beta chain.

[0039] Cells having antigen specificity for the mutated amino acid sequence may be identified by any suitable means known in the art. For example, cells having antigen specificity for the mutated amino acid sequence may be identified based on expression of one or more T cell activation markers and / or one or more cytokines, e.g., as described in WO 2016 / 053338 and WO 2016 / 053339. T cell activation markers may be as described herein with respect to other aspects of the invention. Cytokines may include any cytokine whose secretion by a T cell is characteristic of T cell activation (e.g., a TCR expressed by a T cell that specifically binds and immunologically recognizes the mutated amino acid sequence). Non-limiting examples of cytokines whose secretion is characteristic of T cell activation include IFN-γ, IL-2, granzyme B, and tumor necrosis factor alpha (TNF-α), granulocyte / monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

[0040] In some embodiments, one or more steps of the methods of the invention are performed using a software system. In this regard, one embodiment of the invention provides a method for automatically identifying TCR alpha and beta chain V segment sequences and TCR CDR3 sequences that have antigen specificity for mutated amino acid sequences encoded by cancer-specific mutations.

[0041] FIG. 6 is a block diagram of a system 100 according to an embodiment of the present invention. The system 100 includes one or more sequencer computer device(s) 10. The system may include a user computing device 103 and a network connection 102 between the user computing device 103 and the sequencer computing device 101. The sequencer computing device 101 may be any system capable of sequencing the mRNA from each isolated single T cell sample. Examples of sequencer computing devices 101 may include any of the NGS technologies and platforms described herein with respect to other aspects of the invention.

[0042] The user computing device 101 can be any type of communication device that supports network communications, including a personal computer, a laptop computer, or a personal digital assistant (PDA), etc. In some embodiments, the user computing device 101 can support multiple types of networks. For example, the user computing device 101 may have wired or wireless network connectivity using IP (Internet Protocol), or may have mobile network connectivity allowing cellular and data networks.

[0043] As described in more detail herein, the user computing device 103 is used to capture the sequence of each of the various fragments of cDNA provided by the sequencer computing device 101. The sequences may be transmitted over a network connection 102. An example of type 2 is shared disk space.

[0044] FIG. 7 is a block diagram of basic functional elements of a computing device 103 in accordance with some aspects of the present invention. In the embodiment depicted in FIG. 7, the computing device 103 includes one or more processors 202, memory 204, network interface 206, storage 208, power source 210, one or more output devices 212, one or more input devices 214, and software modules-operation system 216 and sequencing application 218 embedded within memory 204. Although the software modules are provided as being included within memory 204, in some embodiments the software modules are included within storage 208 or a combination of memory 204 and storage 208. Each of the elements including the processor 202, memory 204, network interface 206, storage 208, power source 210, output devices 212, input devices 214, operation system 216, and sequencing application 218 are physically, communicatively, and / or operatively interconnected for communication between the components.

[0045] As depicted, the processor 202 is configured to execute instructions of functions and / or processes for execution within the client device 103. For example, the processor 202 executes instructions stored in the memory 204 or instructions stored in the storage device 208. The memory 204 may be a non-transitory, computer-readable storage medium configured to store information within the client device 103 during operation. In some embodiments, the memory 204 includes a temporary memory, which is a storage area for information that should not be maintained when the client device 103 is turned off. Examples of such temporary memory include volatile memory, such as random access memory (RAM), dynamic random access memory (DRAM), and static random access memory (SRAM). The memory 204 may be a non-transitory, computer-readable storage medium configured to store information within the client device 103 during operation. In some embodiments, the memory 204 includes a non-transitory memory, which is a storage area for information that should not be maintained when the client device 103 is turned off. Examples of such temporary memory include volatile memory, such as random access memory (RAM), dynamic random access memory (DRAM), and static random access memory (SRAM). The memory 204 may be a , and also hold program instructions for execution by processor 202.

[0046] Storage 208 also includes one or more non-transitory computer readable storage media. Storage 208 is generally configured to store a larger amount of information than memory 204. Storage 208 may also be configured for long-term storage of information. In some embodiments, storage 208 includes a non-volatile storage element. Non-limiting examples of non-volatile storage elements include a magnetic hard disk, an optical disk, a floppy disk, flash memory, or an electrically programmable memory (EPROM) or an electrically erasable and programmable (EEPROM) memory.

[0047] The user computing device 103 may use a network interface 206 for communicating with external sequencer computing device 101 via one or more networks 102 (see FIG. 6) and other types of networks through which communications with the user computing device 103 may be established. The network interface 206 may be a network interface card, such as an Ethernet card, an optical transceiver, a radio frequency transceiver, or any other type of transceiver capable of sending and receiving information. Other non-limiting examples of network interfaces include Bluetooth, 3G and Wi-Fi radios, and Universal Serial Bus (USB) in the client computing device.

[0048] The user computing device 103 includes one or more power sources 210 to power the device. Non-limiting examples of power sources 210 include single-use power sources, rechargeable power sources, and / or power sources developed from nickel-cadmium, lithium ion, or other suitable materials.

[0049] One or more output devices 212 are also included within the user computing device 103. The output devices 212 are configured to provide output to the user using tactile, auditory, and / or visual stimuli. The output devices 212 may include a display screen (part of a presence-responsive screen), a sound card, a video graphics adapter card, or any other type of device for converting signals into an appropriate form that is human or machine understandable. Additional examples of output devices 212 include speakers, such as headphones, a cathode ray tube (CRT) monitor, a liquid crystal display (LCD), or any other type of device capable of generating output that is understandable to a user.

[0050] The user computing device includes one or more input devices 214. The input devices 214 are configured to receive input from the user or from the user's surrounding environment via tactile, auditory, and / or visual feedback. Non-limiting examples of input devices 214 include photo and video cameras, presence sensitive screens, mice, keyboards, voice responsive systems, and the like. The presence sensitive screen may include a touch sensitive screen, a display, a keyboard, a microphone, or any other type of input device.

[0051] The client device 103 includes an operating system 216. The operating system 216 controls the operation of the components of the client device 103. For example, the operating system 216 facilitates the interaction of the processor(s) 202, memory 204, network interface 206, storage device(s) 208, input devices 214, output devices 212, and power source 210.

[0052] As described in more detail herein, the user computing device uses the sequencing application 218 to capture sequences of various fragments of the cDNA of the single identified T cell(s) identified as expressing one or more T cell activation markers following co-culture with APCs presenting the mutated amino acid sequence. In some embodiments, the sequencing application 218 may interface with and receive input from a sequencer computing device. In some embodiments, the user may download the sequences of various fragments of the cDNA of the single identified T cell from the sequencer computing device 101 to a removable disk, such as, for example, a USB flash drive. The user computing device may obtain the sequences of various fragments of the cDNA of the single identified T cell from the removable disk.

[0053] The user computing device 103 may include software stored in memory and executed by the processor to identify TCR alpha and beta chain V segment sequences and TCR CDR3 sequences that have antigen specificity for the mutated amino acid sequences encoded by the cancer-specific mutations as described herein in connection with other aspects of the present invention.

[0054] 8 is a flow diagram of steps of a method for automatically identifying TCR alpha and beta chain V segment sequences and TCR CDR3 sequences that have antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. As shown, the method 400 begins at step 402, where a user computing device 103 receives a sequence of multiple fragments of cDNA of a single T cell(s) identified as expressing one or more T cell activation markers following co-culture with an APC that displays the mutated amino acid sequence encoded by the cancer-specific mutation. The method may include receiving the sequence of multiple fragments of cDNA at the computing device by electronic network or removable disk (e.g., USB drive).

[0055] In step 403, the user computing device 103 performs a computerized alignment of the sequences of each of the various fragments of the cDNA against a reference TCR sequence database to identify the sequences of the various fragments of the cDNA that are to be co-cultured with APCs that display the mutated amino acid sequences encoded by the cancer-specific mutations. The TCR alpha chain V segment sequences and TCR beta chain V segment sequences of multiple fragments of the cDNA of single T cells identified as expressing one or more T cell activation markers are then identified.

[0056] In step 404, the user computing device 103 performs a computerized identification of TCR CDR3 sequences within various fragments of cDNA that contain the identified TCR alpha chain V segment sequences and within various fragments of cDNA that contain the identified TCR beta chain V segment sequences.

[0057] In step 405, the user computing device 103 performs a computerized measurement of the number of diverse fragments of cDNA that share the same alpha chain CDR3 amino acid sequence and the number of diverse fragments of cDNA that share the same beta chain CDR3 amino acid sequence.

[0058] In step 406, the user computing device performs a computerized collection of the most abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence, the most abundant cDNA variant fragments encoding the same beta chain CDR3 sequence, and optionally the second abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence, and identifies TCR alpha and beta chain CDR3 sequences, where the alpha chain CDR3 sequence encoded by the second abundant cDNA variant fragment is different from the alpha chain CDR3 sequence encoded by the most abundant cDNA variant fragment.

[0059] In step 407, the user computing device 103 performs a computerized identification of the TCR alpha chain V segment sequence of the most abundant cDNA diverse fragment collected, the TCR beta chain V segment sequence of the most abundant cDNA diverse fragment collected, and optionally, the TCR alpha chain V segment sequence of the second most abundant cDNA diverse fragment collected, to identify the TCR alpha and beta chain V segment sequences.

[0060] Another aspect of the invention provides a paired TCR alpha and beta chain sequence, or an antigen-binding portion thereof, isolated according to any of the methods described herein in relation to other aspects of the invention. One aspect of the invention provides an isolated TCR comprising two polypeptides (i.e., polypeptide chains), such as a TCR alpha (α) chain and a TCR beta (β) chain. The polypeptides, or antigen-binding portions thereof, of the isolated paired TCR alpha and beta chain sequences of the invention (also referred to herein as the "TCR(s) of the invention") can comprise any amino acid sequence, provided that the TCR or antigen-binding portion thereof has antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

[0061] As used herein, an "antigen-binding portion" of an isolated paired TCR alpha and beta chain sequence refers to any portion comprising consecutive amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to a mutated amino acid sequence encoded by a cancer-specific mutation as described herein in connection with other aspects of the invention. The term "antigen-binding portion" refers to any portion or fragment of a TCR isolated by the methods of the invention, which portion or fragment maintains the biological activity of the TCR of which it is a part (the parent TCR). An antigen-binding portion encompasses, for example, a portion of a TCR that specifically binds to a mutated amino acid sequence or maintains the ability to detect, treat, or prevent cancer to a similar, equal, or greater extent compared to the parent TCR. An antigen-binding portion with reference to a parent TCR can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent TCR.

[0062] The antigen-binding portion may be an antigen-binding portion of either or both of the α and β chains of a TCR isolated by the methods of the invention, e.g., an antigen-binding portion of the α and / or β chain of a TCR isolated by the methods of the invention. The antigen-binding portion may comprise a portion comprising one or more of the complementarity determining regions (CDR) 1, CDR2, and CDR3 of the variable region(s). In one embodiment of the invention, the antigen-binding portion may comprise the amino acid sequence of the CDR1 of the alpha chain (CDR1α), the CDR2 of the alpha chain (CDR2α), the CDR3 of the alpha chain (CDR3α), the CDR1 of the beta chain (CDR1β), the CDR2 of the beta chain (CDR2β), the CDR3 of the beta chain (CDR3β), or any combination thereof. Preferably, the antigen-binding portion comprises the amino acid sequences of CDR1α, CDR2α, and CDR3α; the amino acid sequences of CDR1β, CDR2β, and CDR3β; or all of the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the TCR isolated by the method of the invention.

[0063] In one embodiment of the invention, the antigen binding portion may comprise a variable region of a TCR isolated by the methods of the invention, for example comprising a combination of the CDR regions set out above (e.g. all six CDR regions set out above). In this regard, the antigen binding portion may comprise the amino acid sequence of the variable region of the α chain (Vα), the amino acid sequence of the variable region of the β chain (Vβ), or the amino acid sequence of both the Vα and Vβ of a TCR isolated by the methods of the invention.

[0064] In one embodiment of the invention, the antigen binding portion may comprise a combination of variable and constant regions. In this context, the antigen binding portion may comprise the full-length α or β chain, or both the α and β chains, of a TCR isolated by the method of the invention.

[0065] The isolated paired TCR alpha and beta chain sequences, or antigen binding portions thereof, isolated by the methods of the invention may be useful for the preparation of cells for adoptive cell therapy. In this regard, another aspect of the invention provides a method for preparing a cell population expressing paired TCR alpha and beta chain sequences, or antigen binding portions thereof. The method may comprise isolating the paired TCR alpha and beta chain sequences, or antigen binding portions thereof, according to any of the methods described herein in relation to other aspects of the invention.

[0066] The method may further comprise the step of introducing nucleotide sequences encoding the isolated paired TCR alpha and beta chain sequences, or antigen binding portions thereof, into a host cell to obtain a cell expressing the paired TCR alpha and beta chain sequences, or antigen binding portions thereof. In this regard, the method may comprise introducing nucleotide sequences encoding the isolated paired TCR alpha and beta chain sequences, or antigen binding portions thereof, into a recombinant expression vector, such as those described in Green et al. (eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory For purposes of this specification, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and allows expression of the mRNA, protein, polypeptide, or peptide by a host cell when the construct is contacted with a cell under conditions sufficient to allow expression of the mRNA, protein, polypeptide, or peptide by the cell. The vectors of the present invention are not naturally occurring as a whole. However, it is possible for portions of the vector to be naturally occurring. Recombinant expression vectors can contain any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or derived in part from natural sources, and can contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0067] The recombinant expression vector of the present invention can be any suitable recombinant expression vector. The vectors can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression, or both, such as plasmids and viruses. The vectors can be selected from the group consisting of transposon / transposase, pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector.

[0068] Introduction of nucleotide sequences (e.g., recombinant expression vectors) encoding isolated paired TCR alpha and beta chain sequences, or antigen-binding portions thereof, into a host cell can be accomplished by a variety of different techniques known in the art, such as those described in Green et al., supra. Non-limiting examples of techniques useful for introducing a nucleotide sequence into a host cell include transformation, transduction, transfection, and electroporation.

[0069] In one embodiment of the invention, the method comprises introducing nucleotide sequences encoding the isolated and paired TCR alpha and beta chain sequences, or antigen-binding portions thereof, into host cells that are autologous to the patient providing the biological sample. In this regard, the TCRs or antigen-binding portions thereof identified and isolated by the method of the invention may be personalized for each patient. In another embodiment, however, the method of the invention may identify and isolate TCRs or antigen-binding portions thereof that have antigen specificity for mutated amino acid sequences encoded by recurrent (also called "hotspot") cancer-specific mutations. In this regard, the method may comprise introducing nucleotide sequences encoding the isolated and paired TCR alpha and beta chain sequences, or antigen-binding fragments thereof, into host cells that are allogeneic to the patient. For example, the method may comprise introducing nucleotide sequences encoding the isolated and paired TCR alpha and beta chain sequences, or antigen-binding portions thereof, into host cells of another patient whose tumor expresses the same mutation in the context of the same MHC molecule as the patient who originally expressed the TCR.

[0070] In one embodiment of the invention, the host cells are peripheral blood mononuclear cells (PBMCs). The PBMCs may include T cells. The T cells may be obtained from a number of sources in a patient, including, but not limited to, tumors, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cells may include any type of T cell and may be at any stage of development, 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., tumor infiltrating lymphocytes (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.

[0071] Without being bound to a particular theory or mechanism, it is believed that less differentiated "younger" T cells may be associated with any one or more of greater in vivo persistence, proliferation, and anti-tumor activity compared to more differentiated "older" T cells. Therefore, the method of the present invention is advantageously directed to the production of antibodies against mutated amino acid sequences. A paired TCR alpha and beta chain sequence or antigen-binding portion thereof with the original specificity may be identified and isolated, and the paired TCR alpha and beta chain sequence or antigen-binding portion thereof may be introduced into a "younger" T cell, which may provide any one or more of greater in vivo persistence, proliferation, and anti-tumor activity compared to an "older" T cell (e.g., an effector cell within a patient's tumor) from which the TCR or antigen-binding portion may have been isolated.

[0072] In one embodiment of the invention, the method further comprises expanding the number of host cells into which the TCR or antigen-binding portion thereof has been introduced. Expanding the number of cells can be accomplished by, for example, expanding the number of host cells into which the TCR or antigen-binding portion thereof has been introduced, as described in U.S. Patent No. 8,034,334; U.S. Patent No. 8,383,099; U.S. Patent Application Publication No. 2012 / 024442; Expansion of cell numbers can be accomplished by any of a number of methods known in the art, including those described in, for example, Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods,128:189-201 (1990). In one embodiment, expansion of cell numbers is performed by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0073] Another aspect of the invention provides an isolated cell population prepared according to any of the methods described herein with respect to other aspects of the invention. The cell population can be a heterogeneous population, comprising host cells expressing the isolated TCR or antigen binding portion thereof, as well as host cells (e.g. PBMCs) that do not express the isolated TCR or antigen binding portion thereof, or at least one other cell other than a T cell, such as a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc. Alternatively, the cell population can be a substantially homogenous population, comprising primarily (e.g. consisting essentially of) host cells expressing the isolated TCR or antigen binding portion thereof therein. The population can also be a clonal cell population, in which all cells in the population are clones of a single PBMC expressing the isolated TCR or antigen binding portion thereof, such that all cells of the population express the isolated TCR or antigen binding portion thereof. In one embodiment of the invention, the cell population is a clonal population comprising host cells expressing the isolated TCR or antigen binding portion thereof as described herein. By introducing a nucleotide sequence encoding the isolated TCR or an antigen-binding portion thereof into a host cell, the method of the present invention may advantageously provide a cell population that contains a high percentage of host cells that express the isolated TCR and have antigen specificity for the mutated amino acid sequence. In one embodiment of the present invention, about 1% to about 100%, for example about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, or a range defined by any two of the aforementioned values, of the cell population contains host cells that express the isolated TCR and have antigen specificity for the mutated amino acid sequence.Without being bound to a particular theory or mechanism, it is believed that a cell population that contains a high percentage of host cells that express the isolated TCR and have antigen specificity for the mutated amino acid sequence contains a lower percentage of unrelated cells that may interfere with the function of the host cells, e.g., the ability of the host cells to target and destroy cancer cells and / or treat or prevent cancer.

[0074] The TCRs or antigen binding portions thereof, and cell populations of the invention may be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention provides pharmaceutical compositions comprising either the TCRs or antigen binding portions thereof, or cell populations of the invention, and a pharma- ceutical carrier. Pharmaceutical compositions of the invention may comprise a TCR or antigen binding portion thereof, or cell population of the invention, in combination with other pharma- ceutical active agent(s) or drug(s), such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, bilirubin, ribosomal proteinase (RIP), ... It may be included in combination with chemotherapy drugs such as ramblastine, vincristine, etc.

[0075] Preferably, the carrier is a pharma- ceutically acceptable carrier. In the context of pharmaceutical compositions, the carrier may be any of those commonly used for the particular TCR of the invention, or antigen-binding portion thereof, or cell population under consideration. Such pharma- ceutical acceptable carriers are well known to those skilled in the art and are readily available commercially. Preferably, the pharma- ceutical acceptable carrier is one that has no adverse side effects or toxicity under the conditions contemplated for use.

[0076] The choice of carrier will be determined in part by the particular TCR, antigen-binding portion thereof, or cell population of the invention, as well as the particular method used to administer the TCR, antigen-binding portion thereof, or cell population of the invention. Thus, there are a variety of formulations suitable for the pharmaceutical compositions of the invention. Suitable formulations may include any for oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or intraperitoneal administration. More than one route may be used to administer the TCR or cell population of the invention, and in some cases, a particular route may provide a more rapid and more effective response than another route.

[0077] Preferably, the TCR, antigen-binding fragment thereof, or cell population of the present invention is administered by injection, for example, intravenous injection. When the cell population of the present invention is administered, the pharma- ceutically acceptable carrier for the cells for injection may include any isotonic carrier, such as normal saline (about 0.90% w / v sodium chloride in water, about 300 mOsm / L sodium chloride in water, or about 9.0 g sodium chloride per liter of water), NORMOSOL® electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, or lactated Ringer's solution, etc. In one embodiment, the pharma- ceutical acceptable carrier is supplemented with human serum albumin.

[0078] It is believed that the TCRs, antigen-binding portions thereof, cell populations, and pharmaceutical compositions of the present invention can be used in methods of treating or preventing cancer. Without being bound to a particular theory or mechanism, it is believed that the TCRs or antigen-binding portions thereof of the present invention, when expressed by a cell, specifically bind to the mutated amino acid sequence encoded by the cancer-specific mutation such that the TCRs or antigen-binding portions thereof are capable of mediating an immune response against target cells expressing the mutated amino acid sequence. In this regard, one aspect of the present invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, isolated TCR alpha and beta chain sequence pairs, antigen-binding portions thereof, or cell populations of the present invention described herein in an amount effective to treat or prevent cancer in the mammal.

[0079] Another aspect of the invention provides any of the TCRs or antigen-binding portions thereof, cell populations, or pharmaceutical compositions of the invention as described herein in relation to other aspects of the invention for use in the treatment or prevention of cancer in a mammal.

[0080] As used herein, the terms "treatment" and "prevention," and words derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art recognizes as having potential benefits or therapeutic effects. In this regard, the methods of the invention can provide treatment or prevention of cancer in a mammal at any level and in any amount. Furthermore, the treatment or prevention provided by the methods of the invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, the treatment or prevention can include promoting tumor regression. For purposes of this specification, "prevention" can also include delaying the onset of cancer or a symptom or condition thereof.

[0081] For purposes of the present invention, the amount or dose of the TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the present invention administered (e.g., the number of cells when the cell population of the present invention is administered) should be sufficient to produce an effect (e.g., a therapeutic or prophylactic response) in the mammal over a reasonable time frame. For example, the dose of the TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the present invention should be sufficient to bind to the mutated amino acid sequence encoded by the cancer-specific mutation and to detect, treat, or prevent cancer for a period of about 2 hours or more, e.g., 12-24 hours or more, from the time of administration. In some embodiments, the period may even be longer. The dose will be determined by the potency of the particular TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the present invention administered and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) being treated.

[0082] Many assays for measuring the administered dose are known in the art. For purposes of the present invention, an assay that involves comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a TCR of the present invention, or an antigen-binding portion thereof, when a particular dose of such T cells is administered to the mammal, between a set of mammals each receiving different doses of T cells, can be used to determine a starting dose to administer to the mammal. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a dose can be assayed by methods known in the art.

[0083] The dosage of the TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the invention will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the invention. Typically, the attending physician will determine the dosage of the TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the invention with which to treat each individual patient, taking into account a variety of factors, such as age, body weight, general health, diet, sex, the TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the invention to be administered, the route of administration, and the severity of the condition to be treated.

[0084] In one embodiment in which the cell population of the invention is administered, the number of cells administered by injection can vary, for example, from 1 million to 100 billion cells; however, amounts below or above this exemplary range are also within the scope of the invention. For example, the daily dose of the host cells of the present invention is about 1 million to about 150 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 60 billion cells, about 80 billion cells, about 100 billion cells, about 120 billion cells, about 130 billion cells, about 150 billion cells, or a range defined by any two of the aforementioned values), preferably about 10 million to about 130 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, or a range defined by any two of the aforementioned values). cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells, or a range defined by any two of the aforementioned values), more preferably about 100 million cells to about 130 billion cells (for example, about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells, or a range defined by any two of the aforementioned values).

[0085] For purposes of the methods of the invention in which a cell population is administered, the cells can be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0086] Another aspect of the invention is the use of the compounds described herein for the treatment or prevention of cancer in a mammal. The present invention provides either a TCR, an antigen-binding portion thereof, an isolated cell population, or a pharmaceutical composition of the present invention for use in

[0087] The cancer is preferably selected from acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, bile duct cancer, chronic lymphocytic leukemia, chronic bone marrow cancer, colon cancer, cancer of the esophagus, cancer of the cervix, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, cancer of the peritoneum, omentum, and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, cancer), skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer The cancer may be any cancer, including cancer of the ureter, bladder, solid tumor, and liquid tumor. Preferably, the cancer is epithelial cancer. In one embodiment, the cancer is any cancer, including bile duct cancer, melanoma, colon cancer, or rectal cancer.

[0088] The mammal referred to in the methods of the present invention may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. Suitably, the mammal is from the order Carnivora, including Feline (cat) and Canine (dog). The mammal is preferably from the order Bovine, including Bovine (cow) and Swine (pig), or from the order Equine. The mammal is preferably from the order Equine, including Equine (horse). The preferred mammal is a mammal of the order Acanthoptidae, Ceboidoides, or Simoides (monkeys), or a mammal of the order Anthropoides (humans and apes). A more preferred mammal is a human. In a particularly preferred embodiment, the mammal is a patient expressing a cancer-specific mutation.

[0089] The present invention is further illustrated in the following examples which, of course, should not be construed as in any way limiting its scope.

[0090] Examples 1 to 5 For the experiments described in Examples 1-5, the following materials and methods were employed.

[0091] Screening for neoantigen-reactive TILs All patient material was obtained from a National Cancer Institute Review Board-approved clinical trial (trial registration ID: NCT01174121). Methods for identifying neoantigens and neoantigen-reactive TIL populations are described in International Publication No. 2016 / 053338. Specifically, tumor fragments were excised and cultured in medium containing IL-2 (6000 IU / mL) for 3 to 6 weeks (Dudley et al., J. Immunother., 26: 332-342 (2003)). The expanded TIL cultures were screened for neoantigen recognition. To screen the expanded TIL cultures for neoantigen recognition, nonsynonymous mutations in the tumor were identified by whole exome and RNA sequencing (RNA-seq). A tandem minigene (TMG) library encompassing nonsynonymous mutations was synthesized. Autologous dendritic cells (DCs) expressing TMG were screened to identify the neoantigen(s) recognized by the TILs (Lu et al., Clin. Cancer Res., 20: 3401-3410 (2014)) (see Figure 1).

[0092] Identification of neoantigen-specific TCR sequences from single-cell RNA sequencing data Neoantigens from single cell RNA sequencing (RNA-seq) data The method for identifying neoantigen-specific TCR sequences is outlined in the schematic diagram shown in Figure 2. After identifying neoantigen-reactive TIL cultures, 1 × 10 6 1×10 TILs 6 T's MG were co-cultured with pulsed dendritic cells (DCs) for 4 h. After co-culture, T cells were resuspended and washed extensively. T cells were then subjected to single-cell sorting according to the manufacturer's instructions (Fludig, San Francisco, CA) and Clontech, Mountain View, CA) to prepare RNA sequencing samples. All 96 single-cell RNA sequencing samples were barcoded using a NEXTERA XT DNA Library Preparation Kit (Illumina, San Diego, CA) and then sequenced using the ILLUMINA MISEQ system using Reagent Kit V3 (2 × 250 base pairs (bp)).

[0093] Single-cell RNA sequencing data were analyzed using TCR sequences derived from the International Immunogenetics Information System (IMGT) database (Lefranc et al., Nucleic Acids Res., 43: D413-422 (2015)). The α / β variable (V) region sequences were used for alignment by the Burrows-Wheeler aligner (BWA) (Li et al., Bioinformatics, 25: 1754-60 (2009); Li et al., Bioinformatics, 26(5): 589-95 (2010)). The sequences of the CDR3 regions were identified based on the conserved amino acid residues near the C-terminus of the V regions (C...F / W), and were analyzed and reported by the software. Some TCRs with non-productive (out-of-frame) sequences were removed from the analysis. In addition, some samples may contain more than one T cell due to the imperfect sorting mechanism of the FLUIDIGM C1 single-cell mRNA sequencing system. As a result, samples with one or more TCRβ were excluded from subsequent analysis. Separately, RNA sequencing data were aligned against sequences of IFN-γ, IL-2 or other potential T cell activation markers. Aligned TCR fragments associated with single cells with high IFN-γ were extracted and TCR V, CDR3 and constant (C) regions were identified. To assemble the paired full-length TCR sequences, the incomplete 5'V region sequences were assembled with identified human full-length TCR V region sequences from the IMGT database. To promote pairing and avoid TCRα / β mispairing, the 3'C region sequence was replaced with a modified mouse constant region sequence (Cohen et al., Cancer Res., 66: 8878-8886 (2006); Cohen et al., Cancer Res., 67: 3898-3903 (2007); Haga-Friedman et al., J. Immunol., 188: 5538-5546 (2012)) (Figure 2).

[0094] Validation of neoantigen-specific TCR A detailed protocol is described in Morgan et al., Science, 314: 126-129 (2006), with some minor modifications as described herein. The full-length TCRα and TCRβ sequences linked by NPGP (SEQ ID NO: 1) were synthesized and The MSGV8-TCR plasmid (1.5 μg) and 0.75 μg of VSV-G (RD114) plasmid were cloned into the SGV8 retroviral expression vector (Wargo et al., Cancer Immunol. Immunother., 58: 383-394 (2009)). 1 × 10 6 The retroviral supernatants were harvested after 48 h and spun at 3000 revolutions per minute (rpm) for 10 min to remove debris. The retroviral supernatants were loaded onto 6-well plates coated with RETRONECTIN Reagent (Takara, Otsu, Japan) by centrifugation at 2000 g for 2 h.

[0095] Separately, 1 × 10 6 Peripheral blood mononuclear cells (PBMCs) at 2 × 10 cells / mL were stimulated with 50 ng / mL anti-CD3 monoclonal antibody OKT3 and 300 IU / mL IL-2 in AIM V medium containing 5% human serum. After 2 days, stimulated cells were harvested and resuspended in the same medium without OKT3. Stimulated PBMCs were cultured at 2 × 10 cells / mL. 6 100 cells / well were added to each retrovirus-loaded well and spun at 1000 g for 10 minutes. The plates were incubated overnight at 37°C. The following day, PBMCs were transferred to new retrovirus-loaded wells and the transduction procedure was repeated. The cells were incubated at 300 I for another 5 days before the experiment. TCR-transduced T cells were continuously cultured in AIM V medium with U / mL IL-2 and 5% human serum.

[0096] To test the specificity of TCR-transduced T cells, autologous dendritic cells (DCs) or EBV-transformed B cells were pulsed with TMG RNA, full-length mRNA, or peptide for 24 h. 1 × 10 5 T cells were then cocultured overnight with 1 × 105 dendritic cells (DCs) or EBV-transformed B cells in 96-well U-bottom plates. Supernatants were collected and IFN-γ secretion from T cells was measured by enzyme-linked immunosorbent assay (ELISA) (Thermo Fisher Scientific).

[0097] Example 1 This example demonstrates how to isolate neoantigen-specific TCR paired alpha and beta chain sequences from TIL4090 cultures.

[0098] TIL4090 cultures were grown from metastatic lung lesions resected from patients with colorectal cancer. One of the cultures, TIL4090F7, recognized TMG-5 based on the results of TMG library screening. To isolate neoantigen-specific TCRs, TIL4090F7 cells were co-cultured with autologous dendritic cells (DCs) pulsed with TMG-5 for 4 h and subjected to single-cell RNA sequencing analysis. Single cell RNA Among all sequence reads in the A-sequencing data, two single cells expressed a high percentage of IFN-γ reads (6.42% and 12.25% of total R1 reads) (Figure 3A). The remaining single cells expressed only 0–0.16% of IFN-γ reads (Figure 3A). No single cells expressed detectable IL-2 using this approach (Figure 3B). These data suggest that these two T cells react specifically with neoantigens presented by dendritic cells (DCs). Next, TCRα / β variable regions and CDR3 sequences were identified from the single-cell RNA sequencing data of these two T cells, and the TCR sequences derived from both T cells were identical (Table 1).

[0099] [Table 1]

[0100] Example 2 This example shows that T cells transduced with the TCR alpha and beta chain sequences isolated in Example 1 specifically recognize neoantigens expressed by the cancer of the patient in Example 1.

[0101] To validate the TCR isolated from TIL4090F7, full-length TCRα and TCRβ sequences with modified mouse constant regions joined by a furin SGSGP2A linker were synthesized and cloned into the MSGV8 retroviral expression vector. Peripheral blood T cells were transduced with 4090TCR and co-cultured overnight with TMG-5 pulsed 4090 dendritic cells (DCs). Based on the secretion of IFN-γ by T cells, 4090TCR-transduced DCs were shown to be highly potent TCR-specific. The T cells recognized dendritic cells (DCs) pulsed with TMG-5, but not with irrelevant TMG (Fig. 3C).

[0102] Additional experiments were performed to test the specificity of 4090TCR. TMG-5 contained a total of 12 minigenes. A mutated 25-mer long peptide corresponding to each minigene was synthesized and pulsed onto 4090 dendritic cells (DCs) for 24 hours. After washing, the peptide-pulsed DCs were co-cultured overnight with 4090TCR-transduced T cells. 4090TCR-transduced T cells expressed a mutated USP8 (ubiquitin-specific peptidase 8) peptide, WAKFLDPITGTF HThe 4090TCR only reacted with dendritic cells (DCs) pulsed with YYHSPTNTVHMY(R>H) (SEQ ID NO:2), suggesting that the 4090TCR recognizes the mutated USP8 (Figure 3D). Finally, the mutated USP8 long peptide and its wild-type (WT) equivalent, purified by high performance liquid chromatography (HPLC), were pulsed with 4090 dendritic cells (DCs) for 24 h. The peptide-pulsed DCs were then co-cultured overnight with 4090TCR-transduced T cells. The 4090TCR-transduced T cells reacted with the mutated USP8 peptide at a minimum concentration of 0.01 μM, but did not show any significant recognition of the wild-type (WT) USP8 peptide (Figure 3E).

[0103] Example 3 This example demonstrates how to isolate neoantigen-specific TCR paired alpha and beta chain sequences from TIL4095 cultures.

[0104] TIL4095 cultures were grown from metastatic lung lesions resected from patients with colorectal cancer. Based on the results of the TMG library screening, TIL4095F5 recognized TMG-1. To isolate neoantigen-specific TCRs, TIL4095F5 cells were co-cultured with autologous dendritic cells (DCs) pulsed with TMG-1 for 4 h and subjected to single-cell RNA sequencing analysis. All single cells with high levels of IFN-γ reads (0.79%-3.74%) were identified (Figure 4A). All of these T cells contained identical TCRα / β variable and CDR3 sequences (Table 2). Only one single cell expressed detectable IL-2 reads (0.03%) (Figure 4B), and this single cell co-expressed high levels of IFN-γ (1.07%) (Figure 4C).

[0105] [Table 2]

[0106] Example 4 This example shows that T cells transduced with the TCR alpha and beta chain sequences isolated in Example 3 specifically recognize neoantigens expressed by the cancer of the patient in Example 3.

[0107] To validate the TCR isolated from TIL4095F5 in Example 3, full-length TCRα and TCRβ sequences with modified mouse constant regions were synthesized and expressed using the MSGV8 retrovirus. The antibodies were cloned into a KRAS expression vector and then transduced into donor T cells. Previous studies have identified a TCR that recognizes the mutated KRAS(G12D) peptide in an HLA-C0802-restricted manner (Tran et al., Science, 350: 1387-1390 (2015)). Because 4095 was found to be positive for HLA-C0802 and KRAS(G12D), and TMG-1 encodes KRAS(G12D), we tested whether the 4095 TCR also recognizes HLA-C0802-restricted KRAS(G12D). As shown in Figure 4D, 4095 TCR-transduced T cells were co-cultured overnight with autologous dendritic cells (DCs) pulsed with full-length wild-type (WT) KRAS or G12D mRNA. 4095 TCR-transduced T cells recognized KRAS(G12D)-pulsed dendritic cells (DCs), but not wild-type (WT) KRAS-pulsed dendritic cells (DCs). Finally, autologous dendritic cells (DCs) were transfected with the minimal epitope GA of the HLA-C0802-restricted KRAS(G12D) antigen. D GVGKSA (SEQ ID NO: 3) was pulsed for 2 hours. 4095TCR-transduced T cells recognized the KRAS(G12D) epitope at a minimum concentration of 0.01 μM, but not the wild-type (WT) counterpart (FIG. 4E).

[0108] Example 5 This example demonstrates how to isolate neoantigen-specific TCR paired alpha and beta chain sequences from TIL4112 cultures.

[0109] TIL4112 cultures were grown from metastatic liver lesions resected from a patient with cholangiocarcinoma. One of the cultures, TIL4112F5, was found to recognize TMG-9 based on the results of TMG library screening. To identify the neoantigen-specific TCR, TIL4112F5 cells were co-cultured with autologous dendritic cells (DCs) pulsed with TMG-9 for 4 h and subjected to single-cell RNA sequencing analysis. Twenty-two single cells with high levels of IFN-γ reads (>2%) contained the exact same TCR sequence (Figure 5A, Figure 5C, and Table 3). However, 13 of the 22 single cells did not contain detectable TCRα due to the low level of TCRα expression in this clonotype. Eight single cells expressed detectable IL-2 reads ranging from 0.01% to 0.1% (Figure 5B and Figure 5C). Among them, six single cells expressed the same TCRα / β sequence. One single cell expressed the same TCRβ sequence, but no detectable TCRα, and one single cell did not express any detectable TCRα / β sequence (Figures 5A-5C).

[0110] [Table 3]

[0111] To validate the TCR identified from TIL4112F5, full-length TCRα and TCRβ sequences with modified mouse constant regions were synthesized and subsequently transduced into donor T cells. T cells transduced with the 4112TCR recognized dendritic cells (DCs) pulsed with TMG-9, but not with unrelated TMG (Figure 5D). The amino acid sequence of TMG-9 was then submitted to the Immune Epitope Database (IEDB) and Analysis Resources website (iedb.org) and the Center for Biological Sequence Analysis (CBS) Analysis NetMHC website (cbs.dtu.dk / services / NetMHC / ) and identified as TCR-specific TCR in patient 4. Peptides with high affinity were predicted for 112 of the 6 HLAs. A total of 67 predicted high affinity peptides from IEDB (rank <1%) and NetMHC (rank <2%) were synthesized and mixed into 10 pools. 4112TCR-transduced T cells recognized short peptide pool (SPP)-9 pulsed with autologous EBV-transformed B cells (Figure 5E). Subsequent experiments identified a mutated NBAS (neuroblastoma amplified sequence) peptide, WSYDSTLLAY(C>S) (SEQ ID NO: 4), as the minimal epitope recognized by 4112TCR-transduced T cells (Figures 5F, 5G).

[0112] Example 6 This example demonstrates how to isolate neoantigen-specific TCR paired alpha and beta chain sequences from TIL4171 cultures.

[0113] TIL4171 cultures were grown from metastatic lung lesions resected from patients with colorectal cancer. 128 long peptides (25mers) were synthesized, each peptide containing a nonsynonymous mutation flanked on either side by 12 normal amino acids. TIL4171 cultures were screened against a peptide library, and one of the cultures, TIL4171F6, recognized peptide pool 3 (PP-3) (Figure 9A). TIL4171F6 cells were then co-cultured with autologous dendritic cells (DCs) pulsed with PP-3 for 4 h and subjected to single-cell RNA sequencing analysis. Expression of IFN-γ and IL-2 was measured (Figure 9B-9D). Nine samples contained high levels of IFN-γ mRNA (FPKM (fragments per thousand bases of transcript per million mapped reads) ranging from 2209 to 24845) (Figure 9B). Between them, six samples had the same TCRβ CDR3 sequence. Two samples did not contain any detectable TCRβ, and one sample contained two different TCRβ CDR3 sequences, likely resulting from contamination with other T cells. However, none of these samples contained any detectable TCRα chain sequence. Similarly, four samples contained detectable IL-2 mRNA (331.2-1497 FPKM). Although all of these samples contained the same TCRβ CDR3 sequence, none of the samples had any detectable TCRα chain sequence.

[0114] The single-cell RNA sequencing data obtained in this experiment were further examined in an attempt to discover the missing TCR α chain. + of single cells and 2 IL-2 +We found that single cells expressed unique TCR chains, including the V gene segment DV3, the J gene segment AJ56, and the C gene segment AC. Several V gene segments were shared between the TCRα and TCRδ chains, including AV14 / DV4, AV23 / DV6, AV29 / DV5, AV36 / DV7, and AV38-2 / DV8 (Lefranc, Current Protocols in Immunology, John Wiley & Sons, Inc., pp. A.1O.1-A.1O.23 (2001)). These V gene segments were recombined with the AJ joining gene segments of TCRα. The DD diversity gene segments and the DJ junction genes of TCR delta are rearranged. In particular, the transcriptional direction of DV3 is reversed. It has not been previously reported that the TCR α chain can utilize the DV3 gene segment.

[0115] To test the function of this unique TCR chain, it was combined with the identified TCR β chain and then cloned into a retroviral vector. T cells transduced with 4171TCR had strong reactivity to PP-3 (Figure 9E). This peptide pool PP-3 contained 14 mutated 25mer peptides.

[0116] Autologous dendritic cells (DCs) were then pulsed with individual peptides from the peptide pool PP-3 for 24 hours. Peptide-pulsed DCs were transduced with 4171 TCR. When co-cultured with transduced T cells, the 4171 TCR recognized dendritic cells (DCs) pulsed with the mutated peptide SIN3A (SIN3 transcriptional regulatory family member A) (Figure 9F).

[0117] Finally, purified 25-mer wild-type (WT) or mutated SIN3A peptide (LGKFPELFNWFKIFLGYKESVHLET (SEQ ID NO: 25), N>I) was pulsed onto autologous dendritic cells (DCs) for 24 hours. Peptide-pulsed DCs were co-cultured with transduced T cells. IFN-γ secretion from T cells was measured by ELISA. 4171TCR-transduced T cells were shown to specifically recognize the mutated SIN3A peptide but not the wild-type counterpart (Figure 9G).

[0118] Thus, this unique TCR was functional and could specifically recognize the mutated SIN3A. Similar to other V gene segments, these data suggested that the DV3 gene segment may be shared between the TCRα and TCRδ chains.

[0119] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was fully set forth herein.

[0120] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in connection with the description of the invention (particularly in connection with the claims which follow) should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are used interchangeably herein. " " is an open-ended term (i.e., "including but not limited to"), unless otherwise noted. Reference to numerical ranges herein is intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely to serve as a means to better illustrate the invention. It is intended to be illustrative, and not to pose a limitation on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0121] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that such variations will be adopted by those of skill in the art as appropriate, and the inventors intend the invention to be practiced otherwise than as expressly described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, unless otherwise indicated herein or otherwise clearly contradicted by context, any combination of the above-described elements in all possible variations thereof is encompassed by the invention.

Claims

1. 1. A method for isolating paired T cell receptor (TCR) alpha and beta chain sequences, or antigen binding portions thereof, comprising: (a) isolating from the biological sample T cells having antigen specificity for a mutated amino acid sequence encoded by the cancer-specific mutation; (b) co-culturing the isolated T cells with antigen-presenting cells (APCs) that present the mutated amino acid sequence, whereby the T cells express one or more T cell activation markers; (c) sorting the co-cultured T cells into a separated single T cell sample; (d) isolating mRNA from each separated single T cell sample; (e) sequencing mRNA from each separated single T cell sample, wherein the sequencing comprises: (i) preparing cDNA from the mRNA and amplifying the cDNA; (ii) generating multiple fragments of the amplified cDNA and tagging the multiple fragments; (iii) amplifying various fragments of the tagged cDNA; and (iv) sequencing the amplified, tagged, multiple fragments of the cDNA; wherein the sequencing identifies the sequence of each of the multiple fragments of the cDNA; (f) aligning the sequence of each of the diverse fragments of cDNA to known sequences of one or more T cell activation markers to identify which single T cell samples contain single T cells that express one or more T cell activation markers; (g) aligning the sequence of each of the various cDNA fragments against a reference TCR sequence database to identify the variable (V) TCR alpha chain and V TCR beta chain segment sequences of the various cDNA fragments of each separated single T cell sample identified in (f) as expressing one or more T cell activation markers; (h) identifying TCR complementarity determining region 3 (CDR3) sequences within the various fragments of cDNA containing the TCR alpha chain V segment sequences identified in (g) and within the various fragments of cDNA containing the TCR beta chain V segment sequences identified in (g); (i) counting the number of different fragments of cDNA that share the same alpha chain CDR3 amino acid sequence and the number of different fragments of cDNA that share the same beta chain CDR3 amino acid sequence; (j) taking the most abundant cDNA variant fragment encoding the same alpha chain CDR3 sequence, the most abundant cDNA variant fragment encoding the same beta chain CDR3 sequence, and optionally the second abundant cDNA variant fragment encoding the same alpha chain CDR3 sequence, and identifying TCR alpha and beta chain CDR3 sequences, wherein the alpha chain CDR3 sequence encoded by the second abundant cDNA variant fragment is different from the alpha chain CDR3 sequence encoded by the most abundant cDNA variant fragment; (k) identifying the V segment sequence of the TCR alpha chain of the most abundant cDNA variegated fragment collected in (j), the V segment sequence of the TCR beta chain of the most abundant cDNA variegated fragment collected in (j), and optionally the V segment sequence of the TCR alpha chain of the second most abundant cDNA variegated fragment collected in (j), and identifying the V segment sequences of the TCR alpha and beta chains; and (l) a TCR alpha chain comprising the TCR alpha chain V segment sequence identified in (k) and the TCR alpha chain CDR3 sequence taken in (j); and A TCR beta chain comprising the V segment sequence of the TCR beta chain identified in (k) and the CDR3 sequence of the TCR beta chain taken in (j); Assembling one or more nucleotide sequences encoding Optionally, a TCR antigen of a variant fragment of the second most abundant cDNA identified in (k). a second TCR alpha chain comprising the V segment sequence of a lupus chain and the CDR3 sequence of the TCR alpha chain of the second most abundant cDNA variant fragment taken in (j); and A TCR beta chain comprising the V segment sequence of the TCR beta chain identified in (k) and the CDR3 sequence of the TCR beta chain taken in (j); and assembling one or more second nucleotide sequences encoding Producing isolated and paired TCR alpha and beta chain sequences, or antigen-binding portions thereof; The method includes:

2. 2. The method of claim 1, wherein the one or more T cell activation markers comprise one or more of interferon (IFN)-gamma, interleukin (IL)-2, tumor necrosis factor alpha (TNF-α), programmed cell death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, OX40, CD107a, granzyme B, granulocyte / monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

3. 3. The method of claim 1 or 2, further comprising labeling the mRNA from each separated single T cell sample with a different tag for each separated single T cell sample.

4. 4. The method of any one of claims 1 to 3, wherein (h) comprises identifying TCR CDR3 sequences by identifying cDNA sequences encoding conserved amino acid residues located near the C-terminus of amino acid sequences encoded by the V segments of the alpha and beta chains.

5. The method of any one of claims 1 to 4, wherein (k) further comprises identifying the constant (C) region sequence of the TCR alpha chain of the most abundant cDNA diverse fragment collected in (j) and the constant (C) region sequence of the TCR beta chain of the most abundant cDNA diverse fragment collected in (j).

6. The method of claim 5, wherein (l) comprises the steps of assembling a TCR alpha chain comprising the V segment sequence of the TCR alpha chain identified in (k), the C region sequence of the TCR alpha chain identified in (k), and the CDR3 sequence of the TCR alpha chain taken in (j), and the steps of assembling a TCR beta chain comprising the V segment sequence of the TCR beta chain identified in (k), the C region sequence of the TCR beta chain identified in (k), and the CDR3 sequence of the TCR beta chain taken in (j).

7. The method of any one of claims 1 to 4, wherein (l) comprises the steps of assembling a TCR alpha chain comprising the V segment sequence of the TCR alpha chain identified in (k), the C region sequence of an exogenous TCR alpha chain, and the CDR3 sequence of the TCR alpha chain taken in (j), and assembling a TCR beta chain comprising the V segment sequence of the TCR beta chain identified in (k), the C region sequence of the exogenous TCR beta chain, and the CDR3 sequence of the TCR beta chain taken in (j).

8. 8. The method of any one of claims 1 to 7, further comprising the step of receiving, on a user computer device, a sequence of a diverse fragment of the cDNA of a single T cell identified in (f); wherein (g) is a computerized alignment of the sequence of each of the various fragments of the cDNA against a reference TCR sequence database to identify the variable (V) segment sequence of the TCR alpha chain and the variable (V) segment sequence of the TCR vector of the various fragments of the cDNA of the single T cell identified in (f). identifying a V segment sequence of the data chain; wherein (h) comprises computerized identification of TCR CDR3 sequences within the various fragments of cDNA containing the TCR alpha chain V segment sequence identified in (g) and within the various fragments of cDNA containing the TCR beta chain V segment sequence identified in (g); wherein (i) comprises the steps of performing a computerized measurement of the number of diverse fragments of cDNA that share the same alpha chain CDR3 amino acid sequence and the number of diverse fragments of cDNA that share the same beta chain CDR3 amino acid sequence; wherein (j) comprises performing a computerized collection of the most abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence, the most abundant cDNA variant fragments encoding the same beta chain CDR3 sequence, and optionally the second abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence, to identify TCR alpha and beta chain CDR3 sequences, wherein the alpha chain CDR3 sequence encoded by the second abundant cDNA variant fragment is different from the alpha chain CDR3 sequence encoded by the most abundant cDNA variant fragment; and wherein (k) comprises a step of computerized identification of the TCR alpha chain V segment sequence of the most abundant cDNA diverse fragment collected in (j), the TCR beta chain V segment sequence of the most abundant cDNA diverse fragment collected in (j), and optionally the TCR alpha chain V segment sequence of the second most abundant cDNA diverse fragment collected in (j), to identify the TCR alpha and beta chain V segment sequences; method.

9. 1. A method for preparing a cell population expressing paired TCR alpha and beta chain sequences, or antigen binding portions thereof, the method comprising: Isolating paired TCR alpha and beta chain sequences or antigen-binding portions thereof by the method of any one of claims 1 to 8; introducing nucleotide sequences encoding the isolated paired TCR alpha and beta chain sequences or antigen-binding portions thereof into a host cell to obtain a cell expressing the paired TCR alpha and beta chain sequences or antigen-binding portions thereof; The method includes:

10. 10. The method of claim 9, further comprising the step of expanding the number of host cells expressing paired TCR alpha and beta chain sequences or antigen-binding portions thereof.

11. A paired TCR alpha and beta chain sequence or an antigen-binding portion thereof isolated by the method of any one of claims 1 to 8.

12. 11. An isolated cell population prepared according to claim 9 or 10.

13. 13. A pharmaceutical composition comprising: (a) the TCR alpha and beta chain pair of claim 11 or an antigen-binding portion thereof; or (b) the isolated T cell population of claim 12, and a pharma- ceutical acceptable carrier.

14. 14. The pair of TCR alpha and beta chain sequences or antigen-binding portions thereof of claim 11, the isolated cell population of claim 12, or the pharmaceutical composition of claim 13 for use in the treatment or prevention of cancer in a mammal.

15. 15. The isolated cell population, or a pharmaceutical composition comprising the isolated cell population, for use according to claim 14, wherein the cell population is autologous to the mammal.

16. 1. A method for automatically identifying T cell receptor (TCR) alpha and beta chain V segment sequences and TCR CDR3 sequences having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: (a) receiving, on a user's computer device, sequences of multiple fragments of a cDNA, wherein the cDNA is encoded by mRNA produced by a single T cell following co-culture of the T cell with an antigen presenting cell (APC) that presents a mutated amino acid sequence, whereby the T cell expresses one or more T cell activation markers; (b) performing a computerized alignment of the sequence of each of the various cDNA fragments against a reference TCR sequence database to identify the variable (V) segment sequences of the TCR alpha chain and the V segment sequences of the TCR beta chain of the various cDNA fragments; (c) performing a computerized identification of TCR complementarity determining region 3 (CDR3) sequences within the various fragments of cDNA that contain the TCR alpha chain V segment sequences identified in (b) and within the various fragments of cDNA that contain the TCR beta chain V segment sequences identified in (b); (d) performing a computerized measurement of the number of diverse fragments of cDNA that share the same alpha chain CDR3 amino acid sequence and the number of diverse fragments of cDNA that share the same beta chain CDR3 amino acid sequence; (e) performing a computerized collection of the most abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence, the most abundant cDNA variant fragments encoding the same beta chain CDR3 sequence, and optionally the second abundant cDNA variant fragments encoding the same alpha chain CDR3 sequence, to identify TCR alpha and beta chain CDR3 sequences, wherein the alpha chain CDR3 sequence encoded by the second abundant cDNA variant fragment is different from the alpha chain CDR3 sequence encoded by the most abundant cDNA variant fragment; and (f) performing a computerized identification of the TCR alpha chain V segment sequence of the most abundant cDNA diverse fragment collected in (e), the TCR beta chain V segment sequence of the most abundant cDNA diverse fragment collected in (e), and optionally the TCR alpha chain V segment sequence of the second most abundant cDNA diverse fragment collected in (e) to identify TCR alpha and beta chain V segment sequences; The method includes:

17. 17. The method of claim 16, wherein (c) comprises identifying the CD3 sequence of the TCR by identifying cDNA sequences encoding conserved amino acid residues located near the C-terminus of the amino acid sequences encoded by the V segments of the alpha and beta chains.

18. Conserved amino acid residues YX １ CX ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ X １１ X １２ X １３ X １４ X １５ X １６ X １７ X １８ X １９ X ２０ X ２１ X ２２ (Array number 5) comprising the amino acid sequence Where: X １ -X ９ each of which is any naturally occurring amino acid; X １０ -X ２１ each of which is no amino acid or any naturally occurring amino acid; and X ２２ is phenylalanine or tryptophan; 20. The method of claim 17.

19. The method of any one of claims 16 to 18, wherein (f) further comprises a step of performing a computerized identification of the constant (C) region sequences of the TCR alpha chain of the diverse fragments of the most abundant cDNA collected in (e) and the C region sequences of the TCR beta chain of the diverse fragments of the most abundant cDNA collected in (e).

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