Methods of isolating neoantigen-specific t cell receptor sequences

Abstract

Methods of isolating paired T cell receptor (TCR) alpha and beta chain sequences or antigen binding portions thereof are disclosed. Methods of automatically identifying TCR alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer specific mutation are also disclosed. Methods of making a population of cells expressing paired TCR alpha and beta chain sequences or antigen binding portions thereof are also disclosed. Isolated TCR alpha and beta chain sequence pairs and isolated populations of cells made by the methods are also disclosed.

Description

[0001] Cross-reference to related applications

[0002] This patent application is a divisional application of Chinese Patent Application No. 201880022673.3 and claims the benefit of U.S. Provisional Patent Application No. 62 / 479,398, filed March 31, 2017, which is incorporated by reference.

[0003] Statement on federally funded research and development

[0004] This invention was completed with government support under grant number ZIABC010985 from the National Institutes of Health and the National Cancer Institute. The government holds certain rights to this invention. This information is incorporated herein by reference in materials submitted electronically.

[0005] By reference in its entirety is a computer-readable nucleotide / amino acid sequence listing, which was submitted with this paper and identified as follows: a 5,530-byte ASCII (text) file named “737921SeqListing_ST25.txt”, dated March 22, 2018. Background of the Invention

[0006] 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. However, obstacles remain to the successful use of TCR-engineered cells for the broad treatment of cancer and other diseases. For example, TCRs that specifically recognize cancer antigens (e.g., neoantigens) may be difficult to identify and / or isolate from patients. Therefore, there is a need for improved methods to obtain cancer-reactive (e.g., neoantigen-reactive) TCRs. Invention Summary

[0007] Embodiments of the present invention provide a method for isolating paired T cell receptor (TCR) α and β chain sequences or their antigen-binding portions, the method comprising: (a) isolating T cells from a biological sample that are antigen-specific to a mutated amino acid sequence encoded by a cancer-specific mutation; (b) co-culturing the isolated T cells with antigen-presenting cells (APCs) that present the mutated amino acid sequence, such that the T cells express one or more T cell activation markers; (c) sorting the co-cultured T cells into individual single T cell samples; (d) isolating mRNA from each individual single T cell sample; and (e) analyzing the mRNA from each individual single T cell sample. Sequencing is performed, wherein the sequencing includes: (i) generating cDNA from the mRNA and amplifying the cDNA; (ii) generating multiple fragments of the amplified cDNA and labeling the multiple fragments; (iii) amplifying the multiple labeled fragments of the cDNA; and (iv) sequencing the multiple amplified labeled fragments of the cDNA; wherein the sequencing identifies the sequence of each of the multiple fragments of the cDNA; and (f) aligning the sequence of each of the multiple fragments of the cDNA with known sequences of one or more T cell activation markers to identify which single T cell sample contains a single T cell expressing one or more T cell activation markers. T cells; (g) Aligning the sequence of each of the plurality of fragments of the cDNA with a reference TCR sequence database to identify the TCR α-chain variable (V) segment sequence and the TCR β-chain V segment sequence of the plurality of fragments of cDNA identified in (f) to express one or more T cell activation markers; (h) Identifying the TCR complementarity-determining region 3 (CDR3) sequence in the plurality of fragments of the cDNA containing the TCR α-chain V segment sequence identified in (g) and in the plurality of fragments of the cDNA containing the TCR β-chain V segment sequence identified in (g); (i) For those sharing the same α-chain CDR3 The number of multiple fragments of cDNA with the same amino acid sequence and the number of multiple fragments of cDNA sharing the same β-chain CDR3 amino acid sequence are counted; (j) the highest number of multiple fragments encoding the same α-chain CDR3 sequence, the highest number of multiple fragments encoding the same β-chain CDR3 sequence and optionally, the second highest number of multiple fragments encoding the same α-chain CDR3 sequence, wherein the α-chain CDR3 sequence encoded by the second highest number of multiple fragments of cDNA is different from the α-chain CDR3 sequence encoded by the highest number of multiple fragments of cDNA, to identify the TCRα and β-chain CDR3 sequences;(k) Identify the TCRα chain V segment sequences of the highest number of fragments of cDNA collected in (j), the TCRβ chain V segment sequences of the highest number of fragments of cDNA collected in (j), and optionally, the TCRα chain V segment sequences of the second highest number of fragments of cDNA collected in (j), to identify the TCRα and β chain V segment sequences; and (l) assemble one or more nucleotide sequences encoding the following: a TCRα chain comprising the TCRα chain V segment sequences identified in (k) and the TCRα chain CDR3 sequences collected in (j), and a TCRβ chain comprising the TCRα chain V segment sequences identified in (k). The TCRβ chain V segment sequence identified in (k) and the TCRβ chain CDR3 sequence collected in (j) are optionally assembled to encode one or more second nucleotide sequences: a second TCRα chain comprising the TCRα chain V segment sequence of the second high number of fragments of the cDNA identified in (k) and the TCRα chain CDR3 sequence of the second high number of fragments of the cDNA collected in (j), and a TCRβ chain comprising the TCRβ chain V segment sequence identified in (k) and the TCRβ chain CDR3 sequence collected in (j), to produce separated paired TCRα and β chain sequences or their antigen-binding portions.

[0008] Another embodiment of the present invention provides a method for automatically identifying the TCRα and β chain V region sequences and CDR3 sequence of a T cell receptor (TCR), the TCR having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: (a) receiving sequences of multiple fragments of cDNA at a user computing device, wherein, after co-culturing a single T cell with an antigen-presenting cell (APC) presenting the mutated amino acid sequence, such that the T cell expresses one or more T cell activation markers, the cDNA is obtained by... (a) encoding mRNA produced by T cells; (b) computer-aligning the sequence of each of the plurality of fragments of the cDNA with a reference TCR sequence database to identify the TCRα chain variable (V) segment sequence and the TCRβ chain V segment sequence of the plurality of fragments of the cDNA; (c) computer-aligning the TCR complementarity-determining region 3 (CDR3) sequence of the plurality of fragments of cDNA containing the TCRα chain V segment sequence identified in (b) and the plurality of fragments of cDNA containing the TCRβ chain V segment sequence identified in (b). (d) Computerized counting of the number of multiple fragments of cDNA sharing the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA sharing the same β-chain CDR3 amino acid sequence; (e) Computerized collection of the highest number of multiple fragments encoding the same α-chain CDR3 sequence, the highest number of multiple fragments encoding the same β-chain CDR3 sequence, and optionally, the second highest number of multiple fragments encoding the same α-chain CDR3 sequence, wherein the second highest number of multiple fragments of cDNA encoding the same α-chain CDR3 sequence... The α-chain CDR3 sequence is different from the α-chain CDR3 sequence encoded by the highest number of fragments of cDNA, to identify the TCRα and β-chain CDR3 sequences; and (f) computer-aided identification of the TCRα chain V segment sequences of the highest number of fragments of cDNA collected in (e), the TCRβ chain V segment sequences of the highest number of fragments of cDNA collected in (e), and optionally, the TCRα chain V segment sequences of the second highest number of fragments of cDNA collected in (e), to identify the TCRα and β-chain V segment sequences.

[0009] Another embodiment of the present invention provides a method for preparing a cell population expressing paired TCRα and β chain sequences or their antigen-binding portions, the method comprising: isolating paired TCRα and β chain sequences or their antigen-binding portions according to any one of the methods of the present invention described herein, and introducing a nucleotide sequence encoding the isolated paired TCRα and β chain sequences or their antigen-binding portions into a host cell to obtain cells expressing said paired TCRα and β chain sequences or their antigen-binding portions.

[0010] Another embodiment of the invention provides a pair of TCRα and β chain sequences or their antigen-binding portions isolated according to any of the methods of the invention described herein.

[0011] Another embodiment of the present invention provides a separated cell population prepared according to any one of the methods of the present invention described herein.

[0012] Further embodiments of the present invention provide related pharmaceutical compositions and methods for treating or preventing cancer.

[0013] Brief descriptions of the accompanying figures

[0014] Figure 1 This is a schematic diagram illustrating the method for identifying neoantigen-reactive TILs.

[0015] Figure 2 This is a schematic diagram illustrating the method for identifying neoantigen-specific TCRs.

[0016] Figure 3A and Figure 3B This is a graph showing the percentage of total R1 reads of IFN-γ (A) and IL-2 (B) reads in 4090F7 T cells co-cultured with autologous DCs with TMG-5-pulse for 4 hours and then subjected to single-cell RNA-seq analysis.

[0017] Figure 3C This graph shows the amount (pg / mL) of IFN-γ secreted by donor T cells that were not transduced (no shaded column) or transduced with 4090TCR (shaded column) after co-culturing with DCs pulsed with TMG-5 or TMG-6. DCs not pulsed with TMG (“w / o”) served as a negative control.

[0018] Figure 3D This is a graph showing the amount (pg / mL) of IFN-γ secreted by 4090TCR-transduced T cells after co-culturing with 4090DCs that have been pulsed with a mutated 25-mer peptide (corresponding to one of the designated small genes from TMG-5).

[0019] Figure 3E This is a graph showing the amount (pg / mL) of IFN-γ secreted by 4090TCR-transduced T cells after co-culturing with 4090DCs pulsed with a specified concentration (conc.) (μM) of purified 25-mer WT (hollow circle) or mutant (solid circle) USP8 peptide.

[0020] Figure 4A and Figure 4BThis is a graph showing the percentage of total R1 reads of IFN-γ (A) and IL-2 (B) reads in 4095F5 T cells co-cultured with autologous DCs with TMG-1-pulse for 4 hours and then subjected to single-cell RNA-seq analysis.

[0021] Figure 4C It is displayed Figure 4B A graph showing the percentage of total R1 reads and IFN-γ and IL-2 reads measured in single cells expressing detectable IL-2 reads.

[0022] Figure 4D This is a graph showing the amount (pg / mL) of IFN-γ secreted by donor T cells that were not transduced (no shaded column) or transduced with 4095TCR (shaded column) after co-culturing with DCs pulsed with full-length WT or mutant KRAS mRNA. DCs not pulsed with peptide (“w / o”) served as a negative control.

[0023] Figure 4E This is a graph showing the amount (pg / mL) of IFN-γ secreted by 4095TCR-transduced T cells after co-culturing with 4095DCs pulsed with purified 9-mer WT (hollow circles) or mutant (solid circles) KRAS peptide at a specified concentration (μM).

[0024] Figure 5A and Figure 5B This is a graph showing the percentage of total R1 reads of IFN-γ (A) and IL-2 (B) reads in 4112F5 T cells co-cultured with autologous DCs with TMG-9-pulse for 4 hours and then subjected to single-cell RNA-seq analysis.

[0025] Figure 5C It is displayed Figure 5B A graph showing the percentage of total R1 reads and IFN-γ and IL-2 reads measured in eight single cells expressing detectable IL-2 reads.

[0026] Figure 5D This is a graph showing the amount (pg / mL) of IFN-γ secreted by donor T cells that were not transduced (no shaded column) or transduced with 4112TCR (shaded column) after co-culturing with DCs pulsed with TMG-9 or TMG-10. DCs not pulsed with peptide (“w / o”) were used as a negative control.

[0027] Figure 5EThis is a graph showing the amount (pg / mL) of IFN-γ secreted by 4112TCR-transduced T cells after co-culturing with EBV-transformed B cells pulsed with the specified short peptide sets (SPP-1 to SPP-10). EBV-transformed B cells not pulsed with peptides (“w / o”) served as a negative control.

[0028] Figure 5F This is a graph showing the amount (pg / mL) of IFN-γ secreted by 4112TCR-transduced T cells after co-culturing with EBV-transformed B cells pulsed with one of the following peptides: SPP-9, 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 not pulsed with the peptide (“w / o”) served as a negative control.

[0029] Figure 5G This is a graph showing the amount (pg / mL) of IFN-γ secreted by 4112TCR-transduced T cells after co-culturing with EBV-transformed B cells pulsed with purified mutant (solid circle) NBAS peptide WSYDSTLLAY (C>S) (SEQ ID NO:4) or its WT (hollow circle) counterpart.

[0030] Figure 6 This is a block diagram illustrating some embodiments of the system according to the present invention.

[0031] Figure 7 This is a block diagram illustrating the components of a computing device according to an embodiment of the present invention.

[0032] Figure 8 This is a flowchart of the method steps for automatically identifying the TCRα and β chain V segment sequences and CDR3 sequences of a T cell receptor (TCR) with antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation, according to an embodiment of the present invention.

[0033] Figure 9A This graph shows the number of IFN-γ positive spots detected in TIL4171F6 T cells after screening with a 25-mer long peptide (PP) library encoding the mutation in an ELISPOT assay. T cells treated with OKT3 antibody were used as a positive control. T cells not cultured with the peptide library (w / o) were used as a negative control.

[0034] Figure 9B and 9C This shows the IFN-γ (FPKM (reads per kilobase fragment / million mappings)) of TIL 4171F6 T cells after co-culturing with PP-3-pulsed autologous DCs. Figure 9B ) and IL-2 ( Figure 9C (The diagram is used to express this idea.)

[0035] Figure 9D It is a combination Figure 9B and 9C A two-dimensional scatter plot of the data shows the relationship between IFN-γ and IL-2 expression in each single cell (each point represents a single cell).

[0036] Figure 9E This is a graph showing the amount (pg / mL) of IFN-γ produced after co-transfection of untransduced (no shaded column) or 4171TCR-transduced (shaded column) cells with PP-pulsed DCs. T cells not cultured with peptide aggregates (w / o) were used as a negative control.

[0037] Figure 9F This is a graph showing the amount (pg / mL) of IFN-γ produced by 4171TCR-transduced T cells after co-culturing with DCs pulsed with the specified peptide. T cells (pg / mL) not cultured with the peptide assembly served as a negative control.

[0038] Figure 9G This is a graph showing the amount (pg / mL) of IFN-γ produced by 4171TCR-transduced T cells after co-culturing with DCs pulsed with a specified concentration (μM) of WT (hollow circle) or mutant (solid circle) SIN3A peptide. Invention Details

[0039] Embodiments of the present invention provide a method for separating paired TCRα and β chain sequences or their antigen-binding portions.

[0040] The method of this invention can address any of the various challenges in the identification and isolation of functional TCRs with desired antigen specificity. These challenges may include, for example, the vast diversity of TCR sequences, the need for proper pairing of TCRα and β chains to provide the desired antigen specificity, and the fact that up to about one-third of mature T cells express two functional TCRα chains, of which only one may have the desired specificity.

[0041] The method of the present invention can provide any of a variety of advantages. For example, the method of the present invention can significantly reduce the time and / or cost required to isolate and identify the sequence of a TCR that is antigen-specific to a cancer antigen (e.g., a neoantigen) after the removal of a biological sample (e.g., a tumor sample) from a patient. After the TCR sequence is isolated and identified, host cells (e.g., autologous T cells) can be transduced with the TCR sequence, the number of transduced cells can be expanded, and the expanded number of transduced cells can be administered to the patient for the treatment and / or prevention of cancer. The method of the present invention can (i) identify the sequence of a cancer antigen and a TCR that recognizes the cancer antigen and / or (ii) facilitate highly personalized TCR therapies targeting cancer antigens (e.g., neoantigens). Furthermore, the method of the present invention can advantageously be more time-saving, less labor-intensive, and have a higher success rate compared to methods that isolate paired TCRα / β sequences using T cell clones through limiting dilution. The method of the present invention can also efficiently identify the correct TCRα and β chain pairs in those T cells that have more than one functional TCRα gene. The method of the present invention can also identify and isolate paired TCRα and β chain sequences (with desired antigen specificity) from highly diverse T cell populations.

[0042] The αβTCR is a heterodimer composed of α and β protein chains. Each chain includes two extracellular domains, a variable (V) region, and a constant (C) region, followed by a transmembrane region and a short cytoplasmic tail. The variable domain of each of the TCR α- and β-chains has three complementarity-determining regions (CDR1, CDR2, and CDR3) that contact and recognize peptide-MHC complexes. In particular, α and βCDR3 are responsible for recognizing processed antigens. A very high degree of polymorphism exists in the amino acid sequences of CDR3α and CDR3β from T cell to T cell. This level of polymorphism is essential for T cells to recognize a wide range of antigens faced by the immune system. The polymorphism in the amino acid sequences of CDR3α and CDR3β arises from DNA rearrangements within the TCRα and β genes that occur during T cell maturation.

[0043] The gene encoding the TCR consists of coding sequence boxes called the "V" and "J" regions in the TCRα-gene and the "V", "D", and "J" regions in the TCRβ-chain. Random rearrangements in the genomic DNA cause these DNA regions to align, resulting in a functional TCR gene. These rearrangements can be imprecise, and the connections between the Vα-Jα and Vβ-Dc-Jβ regions can be highly variable. The CDR3 of the α-chain is encoded by a portion of the V region and all of the J regions. The CDR3 of the β-chain is encoded by a portion of the V region, all of the J regions, and all of the D regions.

[0044] This method may include isolating T cells from a biological sample that are antigen-specific to a mutated amino acid sequence encoded by a cancer-specific mutation. Any suitable biological sample may be used. In embodiments of the invention, the biological sample is a tumor sample or a peripheral blood sample. Examples of biological samples that may be used according to the invention include, but are not limited to, tissue from a primary tumor, tissue from a metastatic tumor site, exudate, effusion, ascites, graded peripheral blood cells, bone marrow, peripheral blood erythrocyte sedimentation rate (ESR) layer, and cerebrospinal fluid. Therefore, the biological sample may be obtained by any suitable means, including but not limited to aspiration, biopsy, resection, venipuncture, arterial puncture, lumbar puncture, shunt, catheter insertion, or placement of a drainage tube.

[0045] T cells isolated from biological samples possess antigen specificity for mutated amino acid sequences encoded by cancer-specific mutations. The phrase "antigen specificity," as used herein, means that the TCR or its antigen-binding portion can specifically bind to and immunely recognize mutated amino acid sequences encoded by cancer-specific mutations. Cancer-specific mutations can be any mutation in a gene that encodes a mutated amino acid sequence (also referred to as a "non-silent mutation") and is expressed in cancer cells but not in normal non-cancerous cells. Methods for isolating T cells possessing antigen specificity for mutated amino acid sequences encoded by cancer-specific mutations are described, for example, in WO 2016 / 053338 and WO 2016 / 053339. For example, isolating T cells that are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations may include: identifying one or more genes in the nucleic acid of a patient's cancer cells, each gene containing a cancer-specific mutation encoding 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 presenting the mutated amino acid sequence; and selecting autologous T cells that (a) are co-cultured with autologous APCs presenting the mutated amino acid sequence and (b) are antigen-specific to the mutated amino acid sequence presented in the context of major histocompatibility complex (MHC) molecules expressed by the patient, to provide isolated T cells that are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations.

[0046] Once T cells exhibiting antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation are isolated, the method of the present invention further includes co-culturing those isolated T cells with an APC that presents the mutated amino acid sequence, such that the T cells express one or more T cell activation markers. The APC may comprise any cell that presents a peptide fragment of a protein associated with a major histocompatibility complex (MHC) molecule on its cell surface. The APC may comprise any one or more of, for example, macrophages, dendritic cells (DCs), Langerhans cells, B lymphocytes, and T cells. Preferably, the APC is a DC. Those T cells exhibiting antigen specificity to the mutated amino acid sequence can be identified using any one or more of a variety of T cell activation markers. Examples of T-cell activation markers include, but are not limited to, any one or more of the following: programmed cell death 1 (PD-1), lymphocyte-activating 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-α (TNF-α), granulocyte / monocyte colony-stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

[0047] The method further includes sorting the co-cultured T cells into individual single-T cell samples and isolating mRNA from each individual single-T cell sample. Sorting into individual single-T cell samples and isolating mRNA can be automated. For example, the FLUIDIGM C1 automated single-cell isolation and preparation system (available from Fluidigm, South San Francisco, CA) can be used for sorting into individual single-T cell samples and isolating mRNA. The method of the present invention can advantageously provide any number of individual 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 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 an embodiment of the present invention, the method includes preparing approximately 96 individual single-cell mRNA samples.

[0048] In embodiments of the invention, the method may further include labeling the mRNA from each individual single T cell sample with a different tag (e.g., a barcode) for each individual single T cell sample. For example, the mRNA from each individual single T cell sample can be labeled using the ILLUMINANextera XT DNA library preparation kit (available from Illumina, San Diego, CA).

[0049] The method of the present invention also includes sequencing the mRNA from each individual single T cell sample. Sequencing can be performed in any suitable manner known in the art. Preferred examples of sequencing technologies that can be used in the method of the present invention include next-generation sequencing (NGS) (also known as “massively parallel sequencing” or “deep sequencing”) or third-generation sequencing. NGS refers to a high-throughput DNA sequencing technology that is not based on Sanger. With NGS, millions or billions of DNA strands can be sequenced in parallel, resulting in significantly greater throughput and minimizing the need for the fragment-cloning method often used in Sanger sequencing of the genome. In NGS, nucleic acid templates can be read randomly in parallel along the entire genome by dividing the entire genome into small chunks. NGS can advantageously provide nucleic acid sequence information from each individual single T cell mRNA sample within a very short time period (e.g., in about 1 to 2 weeks, preferably in about 1 to 7 days, or most preferably in less than 24 hours). A number of commercially available or literature-described NGS platforms can be used in the context of the present invention, 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).

[0050] Non-limiting examples of NGS technologies and platforms include sequencing-by-synthesis (also known as “pyrosequencing”) (e.g., using the GS-FLX 454 genome sequencer, 454Life Sciences (Branford, CT), the ILLUMINA SOLEXA genome analyzer (Illumina Inc., San Diego, CA), the ILLUMINA HISEQ 2000 genome analyzer (Illumina), or the ILLUMINA MISEQ system (Illumina), or as described, for example, in Ronaghi et al., Science, 281(5375):363-365(1998)), sequencing-by-ligation (e.g., using the SOLID platform (Life Technologies Corporation, Carlsbad, CA) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)), and single-molecule sequencing (e.g., using the PACBIO RS system (Pacific Biosciences (MenloPark, CA) or the HELISCOPE platform (Helicos)). Biosciences (Cambridge, MA) implements nanotechnology for single-molecule sequencing (such as, for example, the GRIDON platform using Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nanopore sequencing (HANS) platform developed by Nabsys (Providence, RI), and ligase-based DNA sequencing platforms using DNA nanosphere (DNB) technology (referred to as probe-anchor ligation (cPAL) implementation), electron microscopy-based techniques for single-molecule sequencing, and ion semiconductor sequencing.

[0051] In this regard, sequencing mRNA from each individual single T cell sample may include generating cDNA from the mRNA and amplifying the cDNA, generating multiple fragments of the amplified cDNA and labeling the multiple fragments, amplifying the multiple labeled fragments of the cDNA, and sequencing the multiple amplified labeled fragments of the cDNA. Labeling may include adding a nucleotide sequence to each of the multiple fragments so that the multiple fragments can be distinguished from each other. This sequencing identifies the sequence of each of the multiple fragments of cDNA. The sequence of each of the multiple fragments of cDNA is also referred to as a "read". Sequencing of mRNA can produce any number of reads. For example, for each single T-cell sample, mRNA sequencing may produce approximately 1,000,000 reads, approximately 900,000 reads, approximately 800,000 reads, approximately 700,000 reads, approximately 600,000 reads, approximately 500,000 reads, approximately 400,000 reads, approximately 300,000 reads, approximately 200,000 reads, approximately 100,000 reads, or more reads, or a range defined by any two of the foregoing values. In many NGS platforms, there may be two read directions: one is a forward read (also referred to as "read 1" or "R1"), and the other is a reverse read (also referred to as "read 2" or "R2"). For cDNA fragments, R1 and R2 can be complementary to each other. In embodiments of the invention, the method includes measuring only R1 reads, measuring only R2 reads, or measuring both R1 and R2 reads. R1 may have higher sequencing quality than R2. Preferably, the method includes measuring only the R1 reading.

[0052] The method also includes aligning the sequence of each of a plurality of fragments of cDNA with known sequences of one or more T cell activation markers to identify which single T cell sample contains single T cells expressing one or more T cell activation markers. One or more single T cells expressing one or more T cell activation markers after co-culturing with APCs presenting mutated amino acid sequences encoded by cancer-specific mutations are identified as expressing TCRs with antigen specificity to the mutated amino acid sequences encoded by cancer-specific mutations.

[0053] The method also includes aligning the sequence of each of a plurality of cDNA fragments to a reference TCR sequence database to identify the TCRα chain variable (V) region sequence and TCRβ chain V region sequence of the plurality of cDNA fragments from each individual single T cell sample identified as expressing one or more T cell activation markers. In this regard, the sequence of each of the plurality of cDNA fragments is aligned against known TCR variable region sequences to identify which cDNA fragments contain all or part of the variable region sequence and to locate the approximate 3' end of the variable region sequence on the cDNA fragment. The 3' end of the variable region sequence indicates the approximate location of CDR3.

[0054] The reference TCR sequence database can be any suitable reference TCR sequence database. Examples of reference TCR sequence databases may include sequences obtained from the International Immunogenetic Information System (IMGT) database ( / / www.imgt.org) as described in Lefranc et al., Nucleic Acids Res., 43:D413-422 (2015). The sequence of each of the multiple fragments of cDNA can be compared with the reference TCR sequence database, for example, using the Burrows-Wheeler Aligner (BWA) software package ( / / bio-bwa.sourceforge.net / ) as described in Li et al., Bioinformatics, 25:1754-60 (2009) and Li et al., Bioinformatics, 26(5):589-95 (2010).

[0055] The method further includes identifying the TCR complementarity-determining region 3 (CDR3) sequence in multiple fragments of cDNA containing the identified TCR α chain V region sequence and in multiple fragments of cDNA containing the identified TCR β chain V region sequence. The CDR3 region sequence can be identified in any suitable manner. In embodiments of the invention, the TCR CDR3 sequence is identified by identifying a cDNA sequence encoding conserved amino acid residues located near the C-terminus of the amino acid sequence encoded by the V region of the α and β chains. For example, the TCR CDR3 sequence can be identified by identifying a cDNA sequence encoding YX1CX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X17X 18 X 19 X 20 X 21 X 22The amino acid sequence motif of (SEQ ID NO:5) can be used to determine any cDNA sequence, wherein each of X1-X9 is any naturally occurring amino acid, X... 10 -X 21 Each of the elements is not an amino acid or any naturally occurring amino acid, and X 22 It is 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.

[0056] In embodiments of the invention, the method further includes identifying the TCRα chain constant (C) region sequences of the highest number of fragments of the collected cDNA and the TCRβ chain C region sequences of the highest number of fragments of the collected cDNA. Optionally, the method further includes identifying the TCRα chain C region sequences of a second highest number of fragments of the collected cDNA. The TCRα chain has one possible constant region amino acid sequence. The TCRβ chain has one of two possible constant region amino acid sequences.

[0057] The method also includes counting the number of multiple fragments of cDNA that share the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA that share the same β-chain CDR3 amino acid sequence.

[0058] The method further includes collecting a maximum number of fragments of cDNA encoding the same α-chain CDR3 sequence, a maximum number of fragments of cDNA encoding the same β-chain CDR3 sequence, and optionally, a second maximum number of fragments of cDNA encoding the same α-chain CDR3 sequence, to identify TCRα and β-chain CDR3 sequences. The α-chain CDR3 sequence encoded by the second maximum number of fragments of cDNA differs from the α-chain CDR3 sequence encoded by the maximum number of fragments of cDNA. The identified CDR3 sequences may include β-chain CDR3 sequences and α-chain CDR3 sequences of an antigen-specific TCR encoding a mutated amino acid sequence of a cancer-specific mutation, and optionally, additional α-chain CDR3 sequences expressed by T cells but not paired with the β-chain CDR3 sequence to form an antigen-specific TCR encoding a mutated amino acid sequence of a cancer-specific mutation. It is estimated that approximately one-third of mature T cells express both TCRα chains. Only one expressed α chain pairs with the expressed TCR β chain to provide an antigen-specific TCR for amino acid sequences encoded by cancer-specific mutations.

[0059] The method further includes identifying the TCRα chain V segment sequences of the highest number of fragments of the collected cDNA, the TCRβ chain V segment sequences of the highest number of fragments of the collected cDNA, and optionally, the TCRα chain V segment sequences of the second highest number of fragments of the collected cDNA, to identify the TCRα and β chain V segment sequences. The number of fragments of cDNA encoding the CDR3 sequence of the dominant TCR expressed by a single activated T cell will exceed the number of cDNA fragments encoding any other TCR CDR3 sequence (which may be present due to contamination) by approximately 10 to approximately 100 times. The source of contamination may be a nearby single-cell sample or an unknown source. The dominant TCR expressed by a single T cell (which expresses one or more T cell activation markers in response to co-culturing with APCs presenting mutated amino acid sequences) is an antigen-specific TCR encoding a mutated amino acid sequence encoded by a cancer-specific mutation.

[0060] The method further includes assembling one or more nucleotide sequences encoding a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises an identified TCRα chain V region sequence and a collected TCRα chain CDR3 sequence, and the TCRβ chain comprises an identified TCRβ chain V region sequence and a collected TCRβ chain CDR3 sequence. Multiple fragments of cDNA encoding the same CDR3 sequence can have various lengths and can overlap each other. By aligning multiple fragments of cDNA of various lengths encoding the same α chain CDR3 sequence to each other, the sequences of the entire V region, J region, and optionally constant region of the dominant TCRα chain can be determined. By aligning multiple fragments of cDNA of various lengths encoding the same β chain CDR3 sequence to each other, the sequences of the entire V region, J region, D region, and optionally constant region of the dominant TCRβ chain can be determined. Nucleotide sequences encoding the entire V, J, and optionally constant regions of the dominant TCRα chain, as well as the entire V, J, D, and optionally constant regions of the dominant TCRβ chain, can be assembled using conventional techniques. Separated paired TCRα and β chain sequences or their antigen-binding portions can be generated.

[0061] In embodiments of the invention, assembling one or more nucleotide sequences includes assembling a TCRα chain comprising a TCRα chain V region sequence identified in a sample, a TCRα chain C region sequence identified in a sample, and a collected TCRα chain CDR3 sequence, and assembling a TCRβ chain comprising a TCRβ chain V region sequence identified in a sample, a TCRβ chain C region sequence identified in a sample, and a collected TCRβ chain CDR3 sequence. In this respect, the assembled nucleotide sequence may include an endogenous C region sequence.

[0062] In embodiments of the invention, assembling one or more nucleotide sequences includes assembling a TCRα chain comprising a TCRα chain V region sequence identified in a sample, a foreign TCRα chain C region sequence, and a collected TCRα chain CDR3 sequence, and assembling a TCRβ chain comprising a TCRβ chain V region sequence identified in a sample, a foreign TCRβ chain C region sequence, and a collected TCRβ chain CDR3 sequence. The foreign C region sequence is a C region sequence that is not native to T cells (not naturally present on T cells). In this regard, the isolated paired TCRα and β chain sequences or their antigen-binding portions generated by this method can be chimeric or heterozygous TCRs, consisting of amino acid sequences derived from TCRs from two different mammalian species. For example, a TCR can contain a variable region derived from a human TCR and a constant region derived from a mouse TCR, thus "mouse-derived" the TCR. Methods for preparing chimeric or heterozygous 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).

[0063] A single T cell typically expresses one TCRβ chain and one or two TCRα chains. The presence of more than one TCRβ chain in a single sample may be a result of imperfect sorting of T cells into individual T cell samples. Imperfect sorting may result in a sample inadvertently containing two or more T cells. If a single sample is found to express more than one TCRβ chain, this sample can be removed from subsequent analyses.

[0064] As discussed above, it is estimated that approximately one-third of mature T cells express two TCRα chains. Only one expressed α chain pairs with the expressed TCRβ chain to provide an antigen-specific TCR for amino acid sequences encoded by cancer-specific mutations. To determine which TCRα chain pairs with the TCRβ chain to provide the desired specificity, the method may include assembling a first nucleotide sequence encoding a first TCRα chain comprising a first TCRα chain V segment sequence containing the highest number of fragments of cDNA identified as described herein and a TCRα chain CDR3 sequence collected as described herein, and a TCRβ chain comprising a TCRβ chain V segment sequence identified as described herein and a TCRβ chain CDR3 sequence collected as described herein. The method may optionally further include assembling a second one or more nucleotide sequences encoding: a second TCRα chain containing a TCRα chain V segment sequence comprising a second high number of fragments of the identified cDNA and a TCRα chain CDR3 sequence comprising a second high number of fragments of the collected cDNA, and a TCRβ chain containing a TCRβ chain V segment sequence comprising a identified TCRβ chain and a collected TCRβ chain CDR3 sequence.

[0065] The method may further include independently introducing first and second nucleotide sequences into first and second host cell populations, respectively, and independently co-culturing the first and second host cell populations with APCs presenting a mutated amino acid sequence encoded by a cancer-specific mutation. The method may further include selecting host cell populations that (a) co-culture with APCs presenting the mutated amino acid sequence and (b) possess antigen specificity for the mutated amino acid sequence. The co-cultured host cell populations possessing antigen specificity for the mutated amino acid sequence will express a TCRα chain, which, together with the TCRβ chain, provides the desired specificity.

[0066] Cells with antigen-specific mutated amino acid sequences can be identified by any suitable method known in the art. For example, cells with antigen-specific mutated amino acid sequences can be identified based on the expression of one or more T cell activation markers and / or one or more cytokines, as described, for example, in WO 2016 / 053338 and WO2016 / 053339. T cell activation markers can be as described herein with respect to other aspects of the invention. Cytokines can include any cytokine whose secretion by T cells is characteristic of T cell activation (e.g., TCR expressed by T cells that specifically bind to and immunely recognize 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-α (TNF-α), granulocyte / monocyte colony-stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

[0067] In some embodiments, a software system is used to perform one or more steps of the method of the present invention. In this regard, embodiments of the present invention provide a method for automatically identifying the TCRα and β chain V segment sequences and CDR3 sequences of a TCR, wherein the TCR is antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations.

[0068] Figure 6 This is a block diagram of system 100 according to certain embodiments of the present invention. System 100 may include one or more sequencer computing devices 101, user computing devices 103, and a network connection 102 between user computing devices 103 and sequencer computing devices 101. Sequencing computing devices 101 may be any system capable of sequencing mRNA from each individual single T cell sample. Examples of sequencer computing devices 101 may include any of NGS technology and platforms described herein with respect to other aspects of the invention.

[0069] User computing device 101 can be any type of communication device that supports network communication, including personal computers, laptops, or personal digital assistants (PDAs). In some implementations, user computing device 101 can support multiple types of networks. For example, user computing device 101 can have wired or wireless network connections using IP (Internet Protocol) or mobile network connections that allow access via cellular and data networks.

[0070] As described in more detail herein, user computing device 103 is used to capture the sequence of each of a plurality of fragments of cDNA provided by sequencer computing device 101. The sequences can be transmitted via network connection 102. An example of network connection 102 is shared disk space.

[0071] Figure 7 This is a block diagram of the basic functional components for a computing device 103 according to some aspects of the present invention. Figure 7 In the illustrated embodiment, computing device 103 includes one or more processors 202, memory 204, network interface 206, storage device 208, power supply 210, one or more output devices 212, one or more input devices 214, and software modules—operating system 216 and sequence applications 218—stored in memory 204. The software modules are provided to be contained in memory 204, but in some embodiments, the software modules are contained in storage device 208 or a combination of memory 204 and storage device 208. Each component (including processor 202, memory 204, network interface 206, storage device 208, power supply 210, output device 212, input device 214, operating system 216, and sequence applications 218) is physically, communicatively, and / or operatively interconnected for inter-component communication.

[0072] As shown, processor 202 is configured to implement functions and / or processing instructions for execution within client device 103. For example, processor 202 executes instructions stored in memory 204 or on storage device 208. Memory 204, which may be a non-transitory computer-readable storage medium, is configured to store information within client device 103 during operation. In some embodiments, memory 204 includes temporary memory, i.e., an area that does not retain information when 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). Memory 204 also maintains program instructions for execution by processor 202.

[0073] Storage device 208 also includes one or more non-transitory computer-readable storage media. Storage device 208 is typically configured to store a larger amount of information than memory 204. Storage device 208 may also be configured for long-term storage of information. In some embodiments, storage device 208 includes non-volatile storage elements. Non-limiting examples of non-volatile storage elements include magnetic hard disks, optical disks, floppy disks, flash memory, or electrically programmable memory (EPROM) or electrically erasable and programmable (EEPROM) memory.

[0074] User computing device 103 can use network interface 206 to communicate via one or more networks 102 (see...). Figure 6 Network interface 206 can also establish other types of networks for communication with user computing device 103 and external sequencer computing device 101. Network interface 206 can be a network interface card, such as an Ethernet card, optical transceiver, radio frequency transceiver, or any other type of device capable of sending and receiving information. Other non-limiting examples of network interfaces include those in client computing devices. 3G and Wi-Fi radios, as well as Universal Serial Bus (USB).

[0075] User computing device 103 includes one or more power sources 210 to provide power to the device. Non-limiting examples of power sources 210 include disposable power sources, rechargeable power sources, and / or power sources developed from nickel-cadmium, lithium-ion, or other suitable materials.

[0076] One or more output devices 212 are also included in the user computing device 103. Output devices 212 are configured to provide output to the user using tactile, audio, and / or video stimuli. Output devices 212 may include a display screen (part of a sensitive screen), a sound card, a video graphics adapter card, or any other type of device for converting signals into a suitable form understandable to a human or machine. Further examples of output devices 212 include speakers such as headphones, cathode ray tube (CRT) monitors, liquid crystal displays (LCDs), or any other type of device capable of producing understandable output to the user.

[0077] The user computing device includes one or more input devices 214. Input devices 214 are configured to receive input from a user or the user's surrounding environment via haptic, audio, and / or video feedback. Non-limiting examples of input devices 214 include cameras and video cameras, presence-sensitive screens, mice, keyboards, voice response systems, microphones, or any other type of input device. In some instances, presence-sensitive screens include touch-sensitive screens.

[0078] Client device 103 includes operating system 216. Operating system 216 controls the operation of components of client device 103. For example, operating system 216 facilitates the interaction between processor 202, memory 204, network interface 206, storage device 208, input device 214, output device 212, and power supply 210.

[0079] As described in more detail herein, a user computing device may use sequence application 218 to capture sequences of multiple fragments of cDNA from a single T cell identified as expressing one or more T cell activation markers after co-culturing with APCs presenting mutated amino acid sequences. In some embodiments, sequence application 218 may interface with and receive input from a sequencer computing device. In some embodiments, a user may download sequences of multiple fragments of cDNA from a single identified T cell from sequencer computing device 101 to a removable disk, such as, for example, a USB flash drive. The user computing device may obtain sequences of multiple fragments of cDNA from a single identified T cell from the removable disk.

[0080] User computing device 103 may include software stored in memory and executed by a processor to identify the TCR α and β chain V segment sequences and CDR3 sequence of a TCR, the TCR having antigen specificity for mutated amino acid sequences encoded by cancer-specific mutations, as described herein in other aspects of the invention.

[0081] Figure 8This is a flowchart of method steps for automatically identifying TCRα and β chain V segment sequences and CDR3 sequences of TCRs, wherein the TCRs are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations. As shown, method 400 begins at step 402, wherein a user computing device 103 receives sequences of multiple fragments of cDNA from a single T cell identified as expressing one or more T cell activation markers after co-culturing with an APC presenting mutated amino acid sequences encoded by cancer-specific mutations. The method may include receiving sequences of multiple fragments of cDNA at the computing device via an electronic network or via a removable disk (e.g., a USB drive).

[0082] In step 403, the user computing device 103 performs a computerized alignment of the sequence of each of the multiple fragments of cDNA with a reference TCR sequence database to identify the TCRα chain V segment sequence and TCRβ chain V segment sequence of the multiple fragments of cDNA of a single T cell, the single T cell being identified as expressing one or more T cell activation markers after co-culturing with APCs presenting mutated amino acid sequences encoded by cancer-specific mutations.

[0083] In step 404, the user computing device 103 performs computerized identification of the TCR CDR3 sequence in multiple fragments of cDNA containing the identified TCRα chain V region sequence and in multiple fragments of cDNA containing the identified TCRβ chain V region sequence.

[0084] In step 405, the user computing device 103 performs a computerized count of the number of multiple fragments of cDNA sharing the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA sharing the same β-chain CDR3 amino acid sequence.

[0085] In step 406, the user computing device performs computerized collection of the highest number of fragments of cDNA encoding the same α-chain CDR3 sequence, the highest number of fragments of cDNA encoding the same β-chain CDR3 sequence, and optionally, the second highest number of fragments of cDNA encoding the same α-chain CDR3 sequence, wherein the α-chain CDR3 sequence encoded by the second highest number of fragments of cDNA is different from the α-chain CDR3 sequence encoded by the highest number of fragments of cDNA, to identify TCRα and β-chain CDR3 sequences.

[0086] In step 407, the user computing device 103 performs computerized identification of the TCRα chain V segment sequences of the highest number of fragments of the collected cDNA, the TCRβ chain V segment sequences of the highest number of fragments of the collected cDNA, and optionally, the TCRα chain V segment sequences of the second highest number of fragments of the collected cDNA, to identify the TCRα and β chain V segment sequences.

[0087] Another embodiment of the invention provides a pair of TCR α and β chain sequences or their antigen-binding portions isolated according to any method described herein with respect to other aspects of the invention. Embodiments of the invention provide an isolated TCR comprising two polypeptides (i.e., polypeptide chains), such as an α (α) chain and a β (β) chain of a TCR. The polypeptide of the pair of TCR α and β chain sequences isolated by the invention (also referred to herein as “the TCR of the invention”) or their antigen-binding portions may comprise any amino acid sequence, provided that the TCR or its antigen-binding portions are antigen-specific to a mutated amino acid sequence encoded by a cancer-specific mutation.

[0088] As used herein, the “antigen-binding moiety” of a separated pair of TCR α and β chain sequences refers to any portion of a TCR containing, as a part, a continuous amino acid sequence, provided that the antigen-binding moiety specifically binds to a mutated amino acid sequence encoded by a cancer-specific mutation as described herein with respect to other aspects of the invention. The term “antigen-binding moiety” refers to any portion or fragment of a TCR isolated by the methods of the present invention that retains the biological activity of the TCR (parental TCR) as a part of it. The antigen-binding moiety encompasses, for example, those portions of a TCR that retain the ability to specifically bind mutated amino acid sequences, or to detect, treat, or prevent cancer to a similar, equal, or greater degree compared to the parental TCR. With respect to the parental TCR, the antigen-binding moiety may, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or greater of the parental TCR.

[0089] The antigen-binding portion may comprise one or both of the antigen-binding portions of the α and β chains of the TCR isolated by the method of the present invention, such as portions comprising one or more of the complementarity-determining regions (CDRs) 1, CDR 2, and CDR 3 of the variable regions of the α and / or β chains of the TCR isolated by the method of the present invention. In embodiments of the present invention, the antigen-binding portion may comprise the amino acid sequences of: CDR 1 (CDR 1α), CDR 2 (CDR 2α), CDR 3 (CDR 3α) of the α chain, CDR 1 (CDR 1β), CDR 2 (CDR 2β), CDR 3 (CDR 3β) of the β chain, or any combination thereof. Preferably, the antigen-binding portion comprises the amino acid sequences of CDR1α, CDR2α, and CDR3α of the TCR isolated by the method of the present invention; the amino acid sequences of CDR1β, CDR2β, and CDR3β; or the amino acid sequences of all CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β.

[0090] In embodiments of the present invention, the antigen-binding portion may include, for example, a variable region of a TCR isolated by the method of the present invention, which comprises a combination of the aforementioned CDR regions, such as all six CDR regions described above. In this regard, the antigen-binding portion may comprise the amino acid sequence of the α-chain variable region (Vα) of the TCR isolated by the method of the present invention, the amino acid sequence of the β-chain variable region (Vβ), or the amino acid sequences of both Vα and Vβ.

[0091] In embodiments of the invention, the antigen-binding portion may comprise a combination of a variable region and a constant region. In this respect, the antigen-binding portion may comprise the entire length of the α-chain or β-chain, or both α-chain and β-chain, of the TCR isolated by the method of the invention.

[0092] The isolated paired TCRα and β chain sequences or their antigen-binding portions, separated by the method of the present invention, can be used to prepare cells for adoptive cell therapy. In this regard, another embodiment of the method provides a method for preparing a cell population expressing paired TCRα and β chain sequences or their antigen-binding portions. According to any method described herein with respect to other aspects of the invention, the method may include separating paired TCRα and β chain sequences or their antigen-binding portions.

[0093] This method may also include introducing a nucleotide sequence encoding a separated paired TCRα and β chain sequence or its antigen-binding portion into a host cell to obtain cells expressing the paired TCRα and β chain sequence or its antigen-binding portion. In this regard, the method may include cloning a nucleotide sequence encoding a separated paired TCRα and β chain sequence or its antigen-binding portion into a recombinant expression vector using established molecular cloning techniques, such as those described, for example, Green et al. (ed.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th edition (2012). For the purposes of this document, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, allows the host cell to express mRNA, protein, polypeptide, or peptide when the vector is contacted with the cell under conditions sufficient to enable the cell to express the mRNA, protein, polypeptide, or peptide. The vector of the present invention as a whole is not naturally occurring. However, the portions of the vector may be naturally occurring. Recombinant expression vectors may contain any type of nucleotide, including but not limited to DNA and RNA (which may be single-stranded or double-stranded, synthetic or partially derived from natural sources), and may contain natural, non-natural, or altered nucleotides. Recombinant expression vectors may contain naturally occurring, non-natural nucleotide linkages, or both. Preferably, non-natural or altered nucleotides or nucleotide linkages do not impede transcription or replication of the vector.

[0094] The recombinant expression vectors of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. Vectors can be selected from: transposons / transposases, pUC series (Fermentas LifeSciences), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector.

[0095] The introduction of a nucleotide sequence (e.g., a recombinant expression vector) encoding the isolated paired TCRα and β chain sequences or their antigen-binding portions into a host cell can be performed in any of a variety of different methods known in the art, such as those described above by Green et al. Non-limiting examples of techniques that can be used to introduce nucleotide sequences into host cells include transformation, transduction, transfection, and electroporation.

[0096] In embodiments of the invention, the method includes introducing a nucleotide sequence encoding a separated paired TCRα and β chain sequence or its antigen-binding portion into host cells that are autologous to the patient providing the biological sample. In this respect, the TCR or its antigen-binding portion identified and isolated by the method of the invention can be personalized for each patient. However, in another embodiment, the method of the invention can identify and isolate a TCR or its antigen-binding portion that is antigen-specific to a mutated amino acid sequence encoded by a specific mutation in a recurrent (also referred to as a “hotspot”) cancer. In this respect, the method may include introducing a nucleotide sequence encoding a separated paired TCRα and β chain sequence or its antigen-binding portion into host cells that are allogeneic to the patient. For example, the method may include introducing a nucleotide sequence encoding a separated paired TCRα and β chain sequence or its antigen-binding portion into host cells of another patient whose tumor expresses the same mutation in the same MHC molecule background as the patient who originally expressed the TCR.

[0097] In embodiments of the present invention, the host cell is peripheral blood mononuclear cells (PBMCs). PBMCs may include T cells. T cells can be obtained from a variety of sources from the patient, including but not limited to tumors, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may include any type of T cell and may belong to any developmental stage, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells such as 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, native T cells, etc. T cells may be CD8+ T cells, CD4+ T cells, or both CD4+ and CD8+ T cells.

[0098] Unbound by any particular theory or mechanism, it is believed that less differentiated “younger” T cells can be associated with better in vivo persistence, proliferation, and antitumor activity compared to more differentiated “older” T cells. Therefore, the method of the present invention can advantageously identify and isolate paired TCRα and β chain sequences or their antigen-binding portions that are antigen-specific to mutated amino acid sequences, and introduce these paired TCRα and β chain sequences or their antigen-binding portions into “younger” T cells that provide better in vivo persistence, proliferation, and antitumor activity compared to “older” T cells from which TCRs or their antigen-binding portions can be isolated (e.g., effector cells in a patient’s tumor).

[0099] In embodiments of the invention, the method further includes increasing the number of host cells in which a TCR or its antigen-binding portion has been introduced. The increase in cell number can be accomplished, for example, by any of the many methods known in the art, as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012 / 0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In one embodiment, the increase in cell number is performed by culturing T cells with an OKT3 antibody, IL-2, and fed PBMCs (e.g., irradiated allogeneic PBMCs).

[0100] Another embodiment 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 may be a heterogeneous population comprising host cells expressing the isolated TCR or its antigen-binding portion, and at least one other cell type, such as host cells not expressing the isolated TCR or its antigen-binding portion (e.g., PBMCs), or cells other than T cells (e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc.). Alternatively, the cell population may be a substantially homogeneous population, wherein the population primarily comprises (e.g., substantially consists of) host cells expressing the isolated TCR or its antigen-binding portion. The population may also be a clonal cell population, wherein all cells in the population are clones of a single PBMC expressing the isolated TCR or its antigen-binding portion, such that all cells in the population express the isolated TCR or its antigen-binding portion. In one embodiment of the invention, the cell population is a clonal population comprising host cells expressing the isolated TCR or its antigen-binding portion as described herein. By introducing a nucleotide sequence encoding a segregated TCR or its antigen-binding moiety into host cells, the method of the present invention can advantageously provide a cell population comprising a high proportion of host cells expressing a segregated TCR and possessing antigen specificity to the mutated amino acid sequence. In embodiments of the invention, a cell population comprising from 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 the range defined by any two of the foregoing values, comprises host cells expressing a segregated TCR and possessing antigen specificity to the mutated amino acid sequence. Unbound by any particular theory or mechanism, it is believed to include a high proportion of host cells expressing isolated TCRs and possessing antigen specificity for mutated amino acid sequences, and a low proportion of irrelevant cells that may impede the function of host cells (such as the host cells’ ability to target and destroy cancer cells and / or treat or prevent cancer).

[0101] The TCR or its antigen-binding portion and cell population of the present invention can be formulated into compositions, such as pharmaceutical compositions. In this regard, the present invention provides pharmaceutical compositions comprising any one of the TCR or its antigen-binding portion or cell population of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may comprise the TCR or its antigen-binding portion or cell population of the present invention, and another pharmaceutically active agent or drug such as a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, etc.

[0102] Preferably, the carrier is a pharmaceutically acceptable carrier. Regarding the pharmaceutical composition, the carrier can be any of those conventionally used in the specific TCR of the present invention or its antigen-binding moiety or cell population under consideration. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are readily available to the public. A pharmaceutically acceptable carrier is preferably one that does not cause harmful side effects or toxicity under the conditions of use.

[0103] The choice of carrier will be determined in part by the specific TCR of the present invention, its antigen-binding portion, or cell population, and by the specific method of administering the TCR, its antigen-binding portion, or cell population of the present invention. Therefore, a variety of suitable formulations of the pharmaceutical compositions of the present invention exist. Suitable formulations may include any of the following administration methods: oral, parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, or intraperitoneal. More than one route may be used to administer the TCR or cell population of the present invention, and in some cases, a particular route may provide a more direct and effective response than another.

[0104] Preferably, the TCR, its antigen-binding portion, or cell population of the present invention is administered by injection, such as intravenous injection. When administering the cell population of the present invention, a pharmaceutically acceptable carrier for the injectable cells may include any isotonic carrier, such as, for example, physiological saline (about 0.90% w / v aqueous NaCl solution, about 300 mOsm / L aqueous NaCl solution, or about 9.0 g NaCl / L water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), plasma-LYTE A (Baxter, Deerfield, IL), about 5% dextran aqueous solution, or Ringer's lactate. In embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0105] The TCR, its antigen-binding moiety, cell population, and pharmaceutical composition of the present invention are intended to be used in methods of treating or preventing cancer. Not bound by any particular theory or mechanism, the TCR or its antigen-binding moiety of the present invention is believed to specifically bind to a mutated amino acid sequence encoded by a cancer-specific mutation, such that the TCR or its antigen-binding moiety, when expressed by cells, can mediate an immune response against target cells expressing the mutated amino acid sequence. In this regard, embodiments of the present invention provide a method of treating or preventing mammalian cancer, comprising administering to a mammal any one of the pharmaceutical composition of the present invention described herein, an isolated TCR α and β chain sequence pair, its antigen-binding moiety, or a cell population in an amount effective for treating or preventing mammalian cancer.

[0106] Another embodiment of the invention provides any one of the TCR of the present invention or its antigen-binding portion, cell population or pharmaceutical composition for treating or preventing cancer in mammals as described herein with respect to other aspects of the invention.

[0107] The terms “treatment” and “prevention,” and the words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, different degrees of treatment or prevention exist, which those skilled in the art will consider to have potential benefit or therapeutic effect. In this respect, the methods of the present invention can provide any amount or level of cancer treatment or prevention in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention may include treating or preventing one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention may include promoting tumor regression. Moreover, for the purposes of this document, “prevention” may encompass delaying the onset of cancer or its symptoms or conditions.

[0108] For the purposes of this invention, the dosage of the TCR, its antigen-binding portion, cell population, or pharmaceutical composition of this invention (e.g., the number of cells when the cell population of this invention is applied) should be sufficient to achieve a therapeutic or preventative response in mammals within a reasonable timeframe. For example, the dosage of the TCR, its antigen-binding portion, cell population, or pharmaceutical composition of this invention should be sufficient to bind to a mutated amino acid sequence encoded by a cancer-specific mutation, or to detect, treat, or prevent cancer, for a period of about 2 hours or longer (e.g., 12 to 24 hours or more) from the time of application. In some embodiments, the timeframe may be even longer. The dosage will be determined by the efficacy of the specific TCR, its antigen-binding portion, cell population, or pharmaceutical composition of this invention applied and the condition of the mammal (e.g., a human), as well as the weight of the mammal (e.g., a human) to be treated.

[0109] Many assays for determining the administered dose are known in the art. For the purposes of this invention, one assay can be used to determine the initial dose to be administered to a mammal, said assay comprising, after administering a given dose of T cells to a group of mammals (each of which has been given different doses of T cells expressing the TCR or its antigen-binding portion of the invention), comparing the extent to which target cells are lysed or IFN-γ is secreted by such T cells. The extent to which target cells are lysed or IFN-γ is secreted after the administration of a given dose can be determined by methods known in the art.

[0110] The dosage of the TCR, its antigen-binding portion, cell population, or pharmaceutical composition of the present invention will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular TCR, its antigen-binding portion, cell population, or pharmaceutical composition of the present invention. Typically, the attending physician will consider a variety of factors, such as age, weight, general health condition, diet, sex, the TCR, its antigen-binding portion, cell population, or pharmaceutical composition of the present invention to be administered, the route of administration, and the severity of the condition being treated, to determine the dosage of the TCR, its antigen-binding portion, cell population, or pharmaceutical composition of the present invention for treating each individual patient.

[0111] In embodiments in which the cell population of the present invention is administered, the number of cells administered per infusion may vary, for example, in the range of one million to one hundred billion cells; however, amounts below or above this example range are within the scope of the present invention. For example, the daily dose of host cells in this invention can be from 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 foregoing values), preferably from 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, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, approximately 100 billion cells, approximately 110 billion cells, approximately 120 billion cells, approximately 130 billion cells, or a range defined by any two of the foregoing values), more preferably approximately 100 million cells to approximately 130 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, approximately 100 billion cells, approximately 110 billion cells, approximately 120 billion cells, approximately 130 billion cells, or a range defined by any two of the foregoing values).

[0112] For the purposes of the method of the present invention, a cell population is applied, and the cells may be allogeneic or autologous cells of a mammal. Preferably, the cells are autologous to the mammal.

[0113] Another embodiment of the invention provides any one of the TCR of the invention described herein, its antigen-binding portion, isolated cell populations, or pharmaceutical compositions for the treatment or prevention of cancers in mammals.

[0114] Cancer can be any cancer, including any of the following: acute lymphoblastic carcinoma, acute myeloid leukemia, pulmonary alveolar retinoblastoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, bile duct cancer, chronic lymphocytic leukemia, chronic myeloma, colon cancer, esophageal cancer, cervical cancer, stomach cancer. Intestinal carcinoid tumors, gliomas, Hodgkin's lymphomas, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, greater omentum cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, bladder cancer, solid tumors, and liquid tumors. Preferably, the cancer is epithelial cancer. In an embodiment, the cancer is bile duct cancer, melanoma, colon cancer, or rectal cancer.

[0115] The mammals mentioned in the method of this invention can be any mammal. As used herein, the term "mammal" means any mammal, including but not limited to rodents such as mice and hamsters, and lagomorphs such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). Preferably, the mammal is from the order Artiodactyla, including bovids (cattle) and suidae (pigs), or belongs to the order Perissodactyla, including equines (horses). Preferably, the mammal belongs to the order Primates, Cebooids, or Simoids (monkeys) or the order Anthropoids (humans and apes). More preferably, the mammal is a human. In a particularly preferred embodiment, the mammal is a patient expressing a cancer-specific mutation.

[0116] This application also relates to the following implementation schemes:

[0117] Implementation Scheme 1. A method for isolating paired T cell receptor (TCR) α and β chain sequences or their antigen-binding portions, said method comprising:

[0118] (a) Isolation of T cells from biological samples that have antigen specificity to mutated amino acid sequences encoded by cancer-specific mutations;

[0119] (b) Co-culturing isolated T cells with antigen-presenting cells (APCs) that present a mutated amino acid sequence, such that the T cells express one or more T cell activation markers;

[0120] (c) The co-cultured T cells were sorted into individual single T cell samples;

[0121] (d) Isolate mRNA from each individual single T cell sample;

[0122] (e) Sequencing of mRNA from each individual single T cell sample, wherein the sequencing includes:

[0123] (i) Generate cDNA from the mRNA and amplify the cDNA;

[0124] (ii) Generate multiple fragments of amplified cDNA and label the multiple fragments;

[0125] (iii) Amplifying multiple labeled fragments of the cDNA; and

[0126] (iv) Sequencing multiple amplified labeled fragments of the cDNA;

[0127] The sequencing identifies the sequence of each of the plurality of fragments of the cDNA;

[0128] (f) The sequence of each of the plurality of fragments of the cDNA is compared with a known sequence of one or more T cell activation markers to identify which single T cell sample contains a single T cell expressing one or more T cell activation markers;

[0129] (g) The sequence of each of the multiple fragments of the cDNA is compared with a reference TCR sequence database to identify the TCRα chain variable (V) segment sequence and TCRβ chain V segment sequence of the multiple fragments of the cDNA of each individual single T cell sample expressing one or more T cell activation markers identified in (f).

[0130] (h) Identify the TCR complementarity-determining region 3 (CDR3) sequence in multiple fragments of the cDNA containing the TCRα chain V segment sequence identified in (g) and in multiple fragments of the cDNA containing the TCRβ chain V segment sequence identified in (g).

[0131] (i) Count the number of multiple fragments of cDNA that share the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA that share the same β-chain CDR3 amino acid sequence.

[0132] (j) Collect the highest number of fragments of cDNA encoding the same α-chain CDR3 sequence, the highest number of fragments of cDNA encoding the same β-chain CDR3 sequence, and optionally, the second highest number of fragments of cDNA encoding the same α-chain CDR3 sequence, wherein the α-chain CDR3 sequence encoded by the second highest number of fragments of cDNA is different from the α-chain CDR3 sequence encoded by the highest number of fragments of cDNA.

[0133] To identify the CDR3 sequences of the TCRα and β chains;

[0134] (k) Identify the TCRα chain V region sequences of the highest number of fragments of cDNA collected in (j), the TCRβ chain V region sequences of the highest number of fragments of cDNA collected in (j), and optionally, the TCRα chain V region sequences of the second highest number of fragments of cDNA collected in (j).

[0135] To identify the V segment sequences of the TCRα and β chains; and

[0136] (l) Assemble one or more of the following nucleotide sequences:

[0137] The TCRα chain comprises the TCRα chain V segment sequence identified in (k) and the TCRα chain CDR3 sequence collected in (j), and

[0138] The TCRβ chain comprises the TCRβ chain V segment sequence identified in (k) and the TCRβ chain CDR3 sequence collected in (j).

[0139] Optionally assemble one or more of the following second nucleotide sequences:

[0140] The second TCRα chain, comprising the TCRα chain V segment sequence of the second highest number of fragments of cDNA identified in (k) and the TCRα chain CDR3 sequence of the second highest number of fragments of cDNA collected in (j), and

[0141] The TCRβ chain comprises the TCRβ chain V segment sequence identified in (k) and the TCRβ chain CDR3 sequence collected in (j).

[0142] To produce separated paired TCRα and β chain sequences or their antigen-binding portions.

[0143] Implementation Scheme 2. The method according to Implementation Scheme 1, wherein the one or more T cell activation markers include one or more of the following: interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor α (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.

[0144] Implementation Scheme 3. The method according to Implementation Scheme 1 or 2 further includes labeling the mRNA from each individual single T cell sample with a different tag for each individual single T cell sample.

[0145] Implementation Scheme 4. The method according to any one of Implementation Schemes 1-3, wherein (h) includes identifying the TCR CDR3 sequence by identifying a cDNA sequence encoding conserved amino acid residues located near the C-terminus of an amino acid sequence encoded by the V segment of the α and β chains.

[0146] Implementation Scheme 5. The method according to any one of Implementation Schemes 1-4, wherein (k) further comprises identifying the TCRα chain constant (C) region sequence of the highest number of fragments of cDNA collected in (j) and the TCRβ chain C region sequence of the highest number of fragments of cDNA collected in (j).

[0147] Implementation Scheme 6. The method according to Implementation Scheme 5, wherein (l) comprises assembling a TCRα chain containing the TCRα chain V segment sequence identified in (k), the TCRα chain C region sequence identified in (k), and the TCRα chain CDR3 sequence collected in (j), and

[0148] Assemble a TCRβ chain comprising the TCRβ chain V segment sequence identified in (k), the TCRβ chain C segment sequence identified in (k), and the TCRβ chain CDR3 sequence collected in (j).

[0149] Implementation Scheme 7. The method according to any one of Implementation Schemes 1-4, wherein (l) comprises assembling a TCRα chain containing the TCRα chain V region sequence identified in (k), the exogenous TCRα chain C region sequence, and the TCRα chain CDR3 sequence collected in (j), and

[0150] Assemble a TCRβ chain comprising the TCRβ chain V segment sequence identified in (k), the exogenous TCRβ chain C region sequence, and the TCRβ chain CDR3 sequence collected in (j).

[0151] Implementation Scheme 8. The method according to any one of Implementation Schemes 1-7, further comprising receiving at a user computing device the sequence of multiple fragments of cDNA of the single T cell identified in (f);

[0152] (g) includes computer-aligning the sequence of each of the plurality of fragments of the cDNA with a reference TCR sequence database to identify the TCRα chain variable (V) segment sequence and the TCRβ chain V segment sequence of the plurality of fragments of the cDNA of the single T cell identified in (f).

[0153] (h) includes computerized identification of the TCR CDR3 sequence in multiple fragments of cDNA containing the TCRα chain V region sequence identified in (g) and in multiple fragments of cDNA containing the TCRβ chain V region sequence identified in (g).

[0154] (i) includes computerized counting of the number of multiple fragments of cDNA sharing the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA sharing the same β-chain CDR3 amino acid sequence.

[0155] (j) includes the computerized collection of the highest number of fragments encoding the same α-chain CDR3 sequence from cDNA, the highest number of fragments encoding the same β-chain CDR3 sequence from cDNA, and optionally, the second highest number of fragments encoding the same α-chain CDR3 sequence from cDNA, wherein the α-chain CDR3 sequence encoded by the second highest number of fragments of cDNA differs from the α-chain CDR3 sequence encoded by the highest number of fragments of cDNA.

[0156] To identify the CDR3 sequences of the TCRα and β chains; and

[0157] Wherein (k) includes computerized identification of the TCRα chain V region sequences of the highest number of fragments of cDNA collected in (j), the TCRβ chain V region sequences of the highest number of fragments of cDNA collected in (j), and optionally, the TCRα chain V region sequences of the second highest number of fragments of cDNA collected in (j).

[0158] To identify the TCRα and β chain V segment sequences.

[0159] Implementation Scheme 9. A method for preparing a cell population expressing paired TCRα and β chain sequences or their antigen-binding portions, the method comprising:

[0160] The paired TCRα and β chain sequences or their antigen-binding portions are separated according to the method described in any one of embodiments 1-8, and

[0161] Nucleotide sequences encoding isolated paired TCRα and β chain sequences or their antigen-binding portions are introduced into host cells to obtain cells expressing the paired TCRα and β chain sequences or their antigen-binding portions.

[0162] Implementation Scheme 10. The method according to Implementation Scheme 9 further includes increasing the number of host cells expressing the paired TCRα and β chain sequences or their antigen-binding portions.

[0163] Implementation Scheme 11. A pair of TCRα and β chain sequences or their antigen-binding portions, which are isolated according to any one of Implementation Schemes 1-8.

[0164] Implementation Scheme 12. The isolated cell population, prepared according to Implementation Scheme 9 or 10.

[0165] Implementation Scheme 13. A pharmaceutical composition comprising (a) a pair of TCRα and β chain sequences or their antigen-binding portions as described in Implementation Scheme 11, or (b) a population of isolated T cells as described in Implementation Scheme 12, and a pharmaceutically acceptable carrier.

[0166] Implementation Scheme 14. The use of a pair of TCRα and β chain sequences or their antigen-binding portions as described in Implementation Scheme 11, an isolated cell population as described in Implementation Scheme 12, or a pharmaceutical composition as described in Implementation Scheme 13, for the treatment or prevention of cancer in mammals.

[0167] Implementation Scheme 15. An isolated cell population or a pharmaceutical composition comprising the isolated cell population for use according to Implementation Scheme 14, wherein the cell population is autologous to mammals.

[0168] Implementation Scheme 16. A method for automatically identifying the V region sequence and CDR3 sequence of the TCRα and β chains of a T cell receptor (TCR), wherein the TCR has antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising:

[0169] (a) Receiving sequences of multiple fragments of cDNA at a user computing device, wherein the cDNA is encoded by mRNA produced by such T cells after co-culturing a single T cell with an antigen-presenting cell (APC) that presents the mutated amino acid sequence, such that the T cell expresses one or more T cell activation markers;

[0170] (b) The sequence of each of the plurality of fragments of the cDNA is computer-aligned with a reference TCR sequence database to identify the TCRα chain variable (V) segment sequence and the TCRβ chain V segment sequence of the plurality of fragments of the cDNA.

[0171] (c) Computerized identification of the TCR complementarity-determining region 3 (CDR3) sequence in multiple fragments of cDNA containing the TCRα chain V segment sequence identified in (b) and in multiple fragments of cDNA containing the TCRβ chain V segment sequence identified in (b).

[0172] (d) Computerized counting of the number of multiple fragments of cDNA sharing the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA sharing the same β-chain CDR3 amino acid sequence.

[0173] (e) Computerized collection of the highest number of fragments encoding the same α-chain CDR3 sequence, the highest number of fragments encoding the same β-chain CDR3 sequence, and optionally, the second highest number of fragments encoding the same α-chain CDR3 sequence, wherein the α-chain CDR3 sequence encoded by the second highest number of fragments of cDNA differs from the α-chain CDR3 sequence encoded by the highest number of fragments of cDNA.

[0174] To identify the CDR3 sequences of the TCRα and β chains; and

[0175] (f) Computerized identification of the TCRα chain V region sequences of the highest number of cDNA fragments collected in (e), the TCRβ chain V region sequences of the highest number of cDNA fragments collected in (e), and optionally, the TCRα chain V region sequences of the second highest number of cDNA fragments collected in (e).

[0176] To identify the TCRα and β chain V segment sequences.

[0177] Implementation Scheme 17. The method according to Implementation Scheme 16, wherein (c) includes identifying the TCRCDR3 sequence by identifying a cDNA sequence encoding conserved amino acid residues located near the C-terminus of an amino acid sequence encoded by the V segment of the α and β chains.

[0178] Implementation Scheme 18. The method according to Implementation Scheme 17, wherein the conserved amino acid residues comprise the amino acid sequence YX1CX2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22 (SEQ ID NO:5), wherein:

[0179] Each of X1-X9 is any naturally occurring amino acid.

[0180] Each of X10-X21 is not an amino acid or any naturally occurring amino acid, and

[0181] X22 is either phenylalanine or tryptophan.

[0182] Implementation Scheme 19. The method according to any one of Implementation Schemes 16-18, wherein (f) further comprises computer-aided identification of the TCRα chain constant (C) region sequences of the highest number of fragments of cDNA collected in (e) and the TCRβ chain C region sequences of the highest number of fragments of cDNA collected in (e).

[0183] The following examples further illustrate the invention, but should not be construed as limiting its scope in any way.

[0184] Examples 1-5

[0185] The following materials and methods were used in the experiments described in Examples 1-5.

[0186] Screening for neoantigen-reactive TILs

[0187] All patient materials were obtained from clinical trials approved by the National Cancer Institute Institutional Review Board (Trial Registration ID: NCT01174121). Methods for identifying neoantigens and neoantigen-reactive TIL populations were described in WO 2016 / 053338. Briefly, tumor fragments were excised and cultured for 3 to 6 weeks in a medium containing IL-2 (6000 IU / mL) (Dudley et al., J. Immunother., 26:332-342 (2003)). TIL cultures with expanded cell numbers were screened for neoantigen recognition. To screen expanded TIL cultures for neoantigen recognition, nonsynonymous mutations were identified in the tumor by whole-exome sequencing and RNA sequencing (RNA-seq). A tandem small gene (TMG) library covering nonsynonymous mutations was synthesized. Screening autologous dendritic cells (DCs) expressing TMG to identify neoantigens recognized by TILs (Lu et al., Clin. Cancer Res., 20:3401-3410 (2014)) (see...) Figure 1 ).

[0188] Identification of neoantigen-specific TCR sequences from single-cell RNA-seq data

[0189] Methods for identifying neoantigen-specific TCR sequences from single-cell RNA sequencing (RNA-seq) data are summarized in... Figure 2 In the schematic diagram shown, after identifying the neoantigen-reactive TIL culture, 1×10 6 One TIL and 1×10 6T cells were co-cultured with TMG-pulse DCs for four (4) hours (h). After co-culture, T cells were resuspended and thoroughly washed. T cells were then sorted and RNA-seq samples were prepared according to the manufacturer’s instructions (Fluidigm (San Francisco, CA) and Clontech (Mountain View, CA)). All 96 single-cell RNA-seq samples were barcoded using the Nextera XT DNA Library Preparation Kit (Illumina (San Diego, CA)) and then sequenced using the Illumina MiSeq system with Reagent Kit V3 (2 × 250 base pairs (bp)).

[0190] Single-cell RNA-seq data were aligned using Burrows-Wheeler Aligner (BWA) (Li et al., Bioinformatics, 25:1754-60 (2009); Li et al., Bioinformatics, 26(5):589-95 (2010)) with TCRα / β variable (V) region sequences from the International Immunogenetic Information System (IMGT) database (Lefranc et al., Nucleic Acids Res., 43:D413-422 (2015)). CDR3 region sequences were identified based on conserved amino acid residues (C…F / W) near the C-terminus of the V region, analyzed, and reported by software. Some TCRs with non-productive (out-of-frame) sequences were removed from the analysis. Additionally, due to FLUIDIGM… The sorting mechanism of the C1 single-cell mRNA sequencing system is imperfect, and some samples may contain more than one T cell. As a result, samples with more than one TCRβ were eliminated from subsequent analyses. Additionally, RNA-seq data were aligned with sequences of IFN-γ, IL-2, or other potential T cell activation markers. Aligned TCR fragments associated with high IFN-γ single cells were extracted, and TCR V, CDR3, and constant (C) regions were identified. To assemble paired full-length TCR sequences, the incomplete 5'V region sequence was assembled with the identified full-length human TCR V region sequence from the IMGT database. To enhance TCRα / β pairing and avoid 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., 66:8878-8886 (2006)). Res., 67:3898-3903 (2007); Haga-Friedman et al., J. Immunol., 188:5538-5546 (2012) Figure 2 ).

[0191] Validation of neoantigen-specific TCR

[0192] The detailed protocol has been described in Morgan et al., Science, 314:126-129 (2006), and this paper describes some minor modifications. Full-length TCRα and TCRβ sequences with modified mouse constant regions, linked via furin SGSGP2A linker (RAKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO:1), were synthesized and cloned into the MSGV8 retroviral expression vector (Wargo et al., Cancer Immunol. Immunother., 58:383-394 (2009)). Using LIPOFECTAMINE 2000 transfection reagent (Thermo Fisher Scientific), the MSGV8-TCR plasmid (1.5 μg) and 0.75 μg of VSV-G (RD114) plasmid were co-transfected into 1 x 10⁶ wells of each of the 6 wells. 6 293 GP cells were collected. After 48 hours, the supernatant was harvested and rotated at 3000 rpm for 10 min to remove debris. The retrovirus supernatant was then loaded into 6-well plates coated with RETRONECTIN reagent (Takara, Otsu, Japan) by centrifugation at 2000 g for 2 h.

[0193] In addition, in AIM V medium containing 5% human serum, 1×10⁻⁶ cells from healthy donors were stimulated with 50 ng / mL anti-CD3 mAb OKT3 and 300 IU / mL IL-2. 6 / mL PBMC. After 2 days, harvest the stimulated cells and resuspend them in the same medium without OKT3. Stimulated PBMCs were then inoculated at 2×10⁶ mL. 6 Add cells / well to each well loaded with retrovirus and rotate at 1000g for 10 minutes. Incubate the plate overnight at 37°C. The next day, transfer PBMCs to new wells loaded with retrovirus and repeat the transduction procedure. Before the experiment, TCR-transduced T cells were cultured continuously for 5 days in AIM V medium containing 300 IU / mL IL-2 and 5% human serum.

[0194] To test the specificity of TCR-transduced T cells, B cells transformed with autologous DCs or EBV were pulsed with TMG RNA, full-length mRNA, or peptide for 24 hours. Then, 1×10 5 10 T cells and 1×10 5B cells transformed with DCs or EBV were co-cultured overnight in 96-well U-shaped plates. The supernatant was harvested, and IFN-γ secretion from T cells was measured by enzyme-linked immunosorbent assay (ELISA) (Thermo Fisher Scientific).

[0195] Example 1

[0196] This example illustrates a method for isolating the paired α and β strand sequences of a neoantigen-specific TCR from a TIL 4090 culture.

[0197] TIL 4090 cultures were grown from resected metastatic lung lesions in patients with colorectal cancer. Based on TMG library screening, one of the cultures, TIL 4090F7, recognized TMG-5. To isolate the neoantigen-specific TCR, TIL 4090F7 cells were co-cultured with autologous DCs of TMG-5-pulsed cells for 4 hours, and single-cell RNA-seq analysis was performed. Among all sequence reads in the single-cell RNA-seq data, two single cells expressed high percentages of IFN-γ reads (6.42% and 12.25% of total R1 reads, respectively). Figure 3A The remaining single cells expressed only 0–0.16% of IFN-γ. Figure 3A No single cell expressed detectable IL-2 using this method. Figure 3B These data indicate that these two T cells specifically respond to the neoantigens presented by the DCs. Next, the TCRα / β variable region and CDR3 sequence were identified from single-cell RNA-seq data of these two T cells, and the TCR sequences from the two T cells were identical (Table 1).

[0198] Table 1

[0199]

[0200] Example 2

[0201] This example illustrates how T cells transduced with the TCRα and β chain sequences isolated in Example 1 can specifically recognize neoantigens expressed by the cancer of the patient in Example 1.

[0202] To validate the TCR isolated from TIL 4090F7, full-length TCRα and TCRβ sequences with modified mouse constant regions were synthesized via a furin SGSGP2A linker and cloned into the MSGV8 retroviral expression vector. Peripheral blood T cells were transduced with 4090TCRs and co-cultured overnight with TMG-5-pulsed 4090DCs. Based on T cell secretion of IFN-γ, 4090TCR-transduced T cells recognized TMG-5-pulsed DCs but not DCs with unrelated TMG pulses. Figure 3C ).

[0203] Further experiments were conducted to test the specificity of 4090TCR. TMG-5 contains 12 small genes. Long peptides with 25-mer mutations corresponding to each small gene were synthesized and pulsed on 4090 DCs for 24 hours. After washing, the peptide-pulsed DCs were co-cultured overnight with 4090TCR-transduced T cells. 4090TCR-transduced T cells were only cultured with the mutated USP8 (ubiquitous protein-specific peptidase 8) peptide WAKFLDPITGTF. H The DC response to the YYHSPTNTVHMY(R>H)(SEQ ID NO:2) pulse indicates that the 4090TCR recognizes the mutated USP8 ( Figure 3D Finally, the HPLC-purified mutant USP8 long peptide and its wild-type (WT) counterpart were pulsed on a 4090 DC for 24 hours. The peptide-pulsated DC was then co-cultured overnight with 4090TCR-transduced T cells. The 4090TCR-transduced T cells reacted with the mutant USP8 peptide at a minimum concentration of 0.01 μM, but did not show significant recognition of the WTP8 peptide. Figure 3E ).

[0204] Example 3

[0205] This example illustrates a method for isolating the paired α and β strand sequences of a neoantigen-specific TCR from a TIL 4095 culture.

[0206] TIL 4095 cultures were grown from resected metastatic lung lesions in patients with colorectal cancer. Based on TMG library screening results, TIL 4095F5 recognized TMG-1. To isolate neoantigen-specific TCRs, TIL 4095F5 cells were co-cultured with autologous DCs pulsed with TMG-1 for 4 hours, followed by single-cell RNA-seq analysis. All single cells with high levels of IFN-γ readout (0.79%–3.74%) were identified. Figure 4AThese T cells all contained identical TCRα / β variable sequences and CDR3 sequences (Table 2). Only one single cell expressed detectable IL-2 reads (0.03%). Figure 4B The single cell co-expressed IFN-γ at a high level (1.07%). Figure 4C ).

[0207] Table 2

[0208]

[0209] Example 4

[0210] This example illustrates how T cells transduced with the TCRα and β chain sequences isolated in Example 3 can specifically recognize neoantigens expressed by the cancer of the patient in Example 3.

[0211] To validate the TCR isolated from TIL 4095F5 in Example 3, full-length TCRα and TCRβ sequences with modified mouse constant regions were synthesized, cloned into an MSGV8 retroviral expression vector, and then transduced into donor T cells. In previous studies, TCRs recognizing the mutant KRAS(G12D) peptide in an HLA-C0802-restricted manner were identified (Tran et al., Science, 350:1387-1390 (2015)). Because patient 4095 was found to be positive for both HLA-C0802 and KRAS(G12D), and because TMG-1 encodes KRAS(G12D), it was tested whether this 4095 TCR also recognized HLA-C0802-restricted KRAS(G12D). Figure 4D As shown, 4095TCR-transduced T cells were co-cultured overnight with autologous dendritic cells (DCs) pulsed with full-length KRAS WT or G12D mRNA. 4095TCR-transduced T cells recognized KRAS(G12D)-pulsed DCs but not DCs pulsed with WT KRAS. Finally, the minimal epitope GA of the HLA-C0802-restricted KRAS(G12D) antigen was used. D GVGKSA (SEQ ID NO:3) pulsed autologous DCs for 2 hours. 4095TCR-transduced T cells recognized the KRAS (G12D) epitope at a minimum concentration of 0.01 μM and did not recognize the WT counterpart. Figure 4E ).

[0212] Example 5

[0213] This example illustrates a method for isolating paired α and β strand sequences of a neoantigen-specific TCR from a TIL 4112 culture.

[0214] TIL 4112 cultures were grown from metastatic liver lesions resected from patients with cholangiocarcinoma. Based on TMG library screening, one of the cultures, TIL 4112F5, was found to recognize TMG-9. To identify neoantigen-specific TCRs, TIL 4112F5 cells were co-cultured with autologous DCs pulsed with TMG-9 for 4 hours and single-cell RNA-seq analysis was performed. Twenty-two (22) single cells with high levels of IFN-γ reads (>2%) contained identical TCR sequences. Figure 5A , Figure 5C (and Table 3). However, due to low TCRα expression levels in this clone, 13 out of 22 single cells did not contain detectable TCRα. Eight (8) single cells expressed detectable IL-2, ranging from 0.01% to 0.1% (and Table 3). Figure 5B and Figure 5C Of these, six single cells expressed the same TCRα / β sequence. One single cell expressed the same TCRβ sequence, but TCRα was not detected. Furthermore, one single cell did not express any detectable TCRα / β sequence. Figures 5A-5C ).

[0215] Table 3

[0216]

[0217] To validate the TCR identified from TIL 4112F5, full-length TCRα and TCRβ sequences with modified mouse constant regions were synthesized and then transduced into donor T cells. T cells transduced with 4112TCR recognized DCs pulsed with TMG-9, but not those pulsed with unrelated TMG. Figure 5D Next, the amino acid sequence of TMG-9 was submitted to the Immune Epitope Database (IEDB), the Analysis Resource Website (iedb.org), and the Center for Biological Sequences (CBS) for analysis on the NetMHC website (cbs.dtu.dk / services / NetMHC / ) to predict peptides with high affinity for six HLA receptors in patient 4112. A total of 67 predicted high-affinity peptides from IEBDB (Rank <1%) and NetMHC (Rank <2%) were synthesized and grouped into 10 sets. The short peptide set (SPP)-9, which is pulsed on autologous EBV-transduced B cells, was recognized by 4112 TCR-transduced T cells. Figure 5E In subsequent experiments, the mutated NBAS (neuroblastoma amplified sequence) peptide WSYDSTLLAY (C>S) (SEQ ID NO:4) was identified as a minimal epitope recognized by T cells transduced by 4112TCR. Figure 5F 5G).

[0218] Example 6

[0219] This example illustrates a method for isolating the paired α and β strand sequences of a neoantigen-specific TCR from a TIL 4171 culture.

[0220] TIL 4171 cultures were grown from metastatic lung lesions resected from patients with colorectal cancer. 128 long peptides (25-mer) were synthesized, each containing a non-synonymous mutation flanking 12 normal amino acids. TIL 4171 cultures were screened against a peptide library, and one of the cultures, TIL4171F6, recognized peptide set 3 (PP-3). Figure 9A TIL4171F6 cells were then co-cultured with PP-3 pulsed autologous DCs for 4 hours, and single-cell RNA-seq analysis was performed. The expression of IFN-γ and IL-2 was measured. Figure 9B-9D Nine samples contained high levels of IFN-γ mRNA (2209–24845 FPKM (per kilobase transcript fragment per million map reads)). Figure 9B Of these, six samples had the same TCRβCDR3 sequence. Two samples contained no detectable TCRβ, and one sample contained two different TCRβCDR3 sequences, possibly due to another type of T cell contamination. However, none of these samples contained any detectable TCRα chain sequence. Similarly, four samples contained detectable IL-2 mRNA (331.2–1497 FPKM). These samples all contained the same TCRβCDR3 sequence, but none of them contained any detectable TCRα chain sequence.

[0221] To identify the missing TCRα strand, the single-cell RNA-seq data obtained in this experiment were further investigated. Four IFN-γ chains were discovered. + Single cell and 2 IL-2 + Single-cell expression of a unique TCR chain comprises the V gene segment DV3, the J gene segment AJ56, and the C gene segment AC. Several V gene segments are 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 have been found to rearrange into AJ-connecting gene segments for TCRα and into DD-diversity gene segments and DJ-connecting gene segments for TCRδ. Notably, the transcription direction of DV3 is inverted. To date, no reports have indicated that the TCRα chain can utilize the DV3 gene segment.

[0222] To test the function of this unique TCR chain, it was ligated to an identified TCRβ chain and then cloned into a retroviral vector. T cells transduced with 4171TCR showed a strong response to PP-3. Figure 9E This peptide set PP-3 contains 14 mutated 25-mer peptides.

[0223] Next, autologous DCs from each peptide group PP-3 were pulsed for 24 hours. The pulsed DCs were then co-cultured with 4171TCR-transduced T cells. The 4171TCR recognizes the mutant peptide SIN3A (a member of the SIN3 transcription regulator family A)-pulsed DCs. Figure 9F ).

[0224] Finally, purified 25-mer WT or the mutant SIN3A peptide (LGKFPELFNWFKIFLGYKESVHLET (SEQ ID NO: 25), N>I) was pulsed onto autologous dendritic cells (DCs) for 24 hours. The peptide-pulsated DCs were then co-cultured with transduced T cells. IFN-γ secretion from T cells was measured by ELISA. 4171TCR-transduced T cells showed specific recognition of the mutant SIN3A peptide but not of the wild-type counterpart. Figure 9G ).

[0225] Therefore, this unique TCR is functional and can specifically recognize mutated SIN3A. Similar to other V gene segments, these data indicate that the DV3 gene segment can be shared between the TCRα and TCRδ chains.

[0226] All references cited in this article, including publications, patent applications and patents, are incorporated herein by reference to the extent that each reference is individually and specifically indicated by reference and fully elaborated in this article.

[0227] In the context of describing the invention (particularly in the context of the following claims), the terms “a,” “an,” “the,” and “at least one,” and similar indicators, should be interpreted to cover both singular and plural forms, unless otherwise stated herein or obviously contradicted by the context. The use of the term “at least one” followed by a list of one or more items (e.g., “at least one of A and B”) should be interpreted 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 stated herein or obviously contradicted by the context. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise stated herein, references to numerical ranges herein are intended only as a way of abbreviating each individual value falling within that range, and each individual value is incorporated into this specification as if it were individually referenced herein. Unless otherwise stated herein or obviously contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise stated, the use of any and all instances or exemplary language (such as "such as") provided herein is intended only to better illustrate the invention and not to limit its scope. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0228] This document describes preferred embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments will be apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ these variations, and the inventors desire that the invention be carried out in a manner different from that specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the invention covers any combination of the foregoing elements in all possible variations.

Claims

1. A method for preparing a medicament for inducing an immune response against cancer cells expressing a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: (a) Isolation of T cells from biological samples that have antigen specificity to mutated amino acid sequences encoded by cancer-specific mutations; (b) Co-culturing isolated T cells with antigen-presenting cells (APCs) that present mutated amino acid sequences, such that the T cells express one or more T cell activation markers; (c) The co-cultured T cells were sorted into individual single T cell samples; (d) Isolate mRNA from each individual single T cell sample; (e) Sequencing of mRNA from each individual single T cell sample, wherein the sequencing includes: (i) Generate cDNA from the mRNA and amplify the cDNA; (ii) Generate multiple fragments of amplified cDNA and label the multiple fragments; (iii) Amplifying multiple labeled fragments of the cDNA; and (iv) Sequencing multiple amplified labeled fragments of the cDNA; The sequencing identifies the sequence of each of the plurality of fragments of the cDNA; (f) The sequence of each of the plurality of fragments of the cDNA is compared with a known sequence of one or more T cell activation markers to identify which single T cell sample contains a single T cell expressing one or more T cell activation markers; (g) The sequence of each of the multiple fragments of the cDNA is compared with a reference TCR sequence database to identify the TCR α chain variable V segment sequence and TCR β chain V segment sequence of the multiple fragments of cDNA of each individual single T cell sample expressing one or more T cell activation markers identified in (f); (h) Identify the TCR complementarity-determining region 3 (CDR3) sequence in multiple fragments of the cDNA containing the TCR α chain V segment sequence identified in (g) and in multiple fragments of the cDNA containing the TCR β chain V segment sequence identified in (g); (i) Count the number of multiple fragments of cDNA that share the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA that share the same β-chain CDR3 amino acid sequence. (j) Collect the highest number of fragments encoding the same α-chain CDR3 sequence and the highest number of fragments encoding the same β-chain CDR3 sequence. To identify the CDR3 sequences of the TCR α and β chains; (k) Identify the TCR α-strand V region sequences of the highest number of multiple fragments of cDNA collected in (j), and the TCR β-strand V region sequences of the highest number of multiple fragments of cDNA collected in (j). To identify the V segment sequences of the TCR α and β chains; and (l) Assemble one or more of the following nucleotide sequences: The TCR α chain comprises the TCR α chain V segment sequence identified in (k) and the TCR α chain CDR3 sequence collected in (j), and The TCR β chain comprises the TCR β chain V segment sequence identified in (k) and the TCR β chain CDR3 sequence collected in (j). To produce separated, paired TCR α and β chain sequences or their antigen-binding portions, (m) Introducing a nucleotide sequence encoding a separated paired TCR α and β chain sequence or its antigen-binding portion into a host cell to obtain a cell population expressing the paired TCR α and β chain sequence or its antigen-binding portion; and (n) Formulate the cell population obtained in (m) into a drug for inducing an immune response against cancer cells expressing a mutated amino acid sequence encoded by the cancer-specific mutation.

2. The method of claim 1, wherein (j) further comprises collecting a second high number of fragments of cDNA encoding the same α-chain CDR3 sequence, wherein the α-chain CDR3 sequence encoded by the second high number of fragments of cDNA is different from the α-chain CDR3 sequence encoded by the highest number of fragments of cDNA, (k) further comprises identifying the TCR α-chain V segment sequence of the second high number of fragments of cDNA collected in (j), and (l) further comprises assembling a second one or more nucleotide sequences encoding the following: The second TCR α chain, comprising the TCR α chain V segment sequence of the second highest number of fragments of cDNA identified in (k) and the TCR α chain CDR3 sequence of the second highest number of fragments of cDNA collected in (j), and The TCR β chain comprises the TCR β chain V segment sequence identified in (k) and the TCR β chain CDR3 sequence collected in (j).

3. The method according to claim 1 or 2, wherein the one or more T cell activation markers include one or more of the following: interferon IFN-γ, interleukin IL-2, tumor necrosis factor α 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.

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

5. The method according to claim 1 or 2, wherein (h) comprises identifying the TCR CDR3 sequence by identifying a cDNA sequence encoding conserved amino acid residues located near the C-terminus of an amino acid sequence encoded by the V segment of the α and β chains.

6. The method according to claim 1 or 2, wherein (k) further comprises identifying the TCR α-chain constant C region sequence of the highest number of fragments of cDNA collected in (j) and the TCR β-chain C region sequence of the highest number of fragments of cDNA collected in (j).

7. The method of claim 6, wherein (l) comprises assembling a TCR α chain comprising the TCR α chain V segment sequence identified in (k), the TCR α chain C segment sequence identified in (k), and the TCR α chain CDR3 sequence collected in (j), and Assemble a TCR β chain comprising the TCR β chain V segment sequence identified in (k), the TCR β chain C segment sequence identified in (k), and the TCR β chain CDR3 sequence collected in (j).

8. The method according to claim 1 or 2, wherein (l) comprises assembling a TCR α chain comprising the TCR α chain V region sequence identified in (k), the exogenous TCR α chain C region sequence, and the TCR α chain CDR3 sequence collected in (j), and Assemble a TCR β chain comprising the TCR β chain V segment sequence identified in (k), the exogenous TCR β chain C region sequence, and the TCR β chain CDR3 sequence collected in (j).

9. The method of claim 1, further comprising receiving at a user computing device the sequences of multiple fragments of cDNA of the single T cell identified in (f); (g) includes computer-aligning the sequence of each of the plurality of fragments of the cDNA with a reference TCR sequence database to identify the TCR α chain variable V segment sequence and the TCR β chain V segment sequence of the plurality of fragments of the cDNA of the single T cell identified in (f). (h) includes computerized identification of the TCR CDR3 sequence in multiple fragments of cDNA containing the TCR α chain V region sequence identified in (g) and in multiple fragments of cDNA containing the TCR β chain V region sequence identified in (g); (i) includes computerized counting of the number of multiple fragments of cDNA sharing the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA sharing the same β-chain CDR3 amino acid sequence. Where (j) includes the highest number of fragments encoding the same α-chain CDR3 sequence and the highest number of fragments encoding the same β-chain CDR3 sequence. To identify the CDR3 sequences of the TCR α and β chains; and Wherein (k) includes the TCR α-chain V segment sequences of the highest number of fragments of cDNA collected in (j) and the TCR β-chain V segment sequences of the highest number of fragments of cDNA collected in (j). To identify the V segment sequences of the TCR α and β chains.

10. The method of claim 2, further comprising receiving at a user computing device the sequence of a plurality of fragments of cDNA of the single T cell identified in (f); (g) includes computer-aligning the sequence of each of the plurality of fragments of the cDNA with a reference TCR sequence database to identify the TCR α chain variable V segment sequence and the TCR β chain V segment sequence of the plurality of fragments of the cDNA of the single T cell identified in (f). (h) includes computerized identification of the TCR CDR3 sequence in multiple fragments of cDNA containing the TCR α chain V region sequence identified in (g) and in multiple fragments of cDNA containing the TCR β chain V region sequence identified in (g); (i) includes computerized counting of the number of multiple fragments of cDNA sharing the same α-chain CDR3 amino acid sequence and the number of multiple fragments of cDNA sharing the same β-chain CDR3 amino acid sequence. (j) includes the computerized collection of the highest number of fragments encoding the same α-chain CDR3 sequence, the highest number of fragments encoding the same β-chain CDR3 sequence, and the second highest number of fragments encoding the same α-chain CDR3 sequence, wherein the α-chain CDR3 sequence encoded by the second highest number of fragments of cDNA differs from the α-chain CDR3 sequence encoded by the highest number of fragments of cDNA. To identify the CDR3 sequences of the TCR α and β chains; and Wherein (k) includes computerized identification of the TCR α-chain V segment sequences of the highest number of cDNA fragments collected in (j), the TCR β-chain V segment sequences of the highest number of cDNA fragments collected in (j), and the TCR α-chain V segment sequences of the second highest number of cDNA fragments collected in (j). To identify the V segment sequences of the TCR α and β chains.

11. The method of claim 1, further comprising increasing the number of host cells expressing the paired TCR α and β chain sequences or their antigen-binding portions.

12. The method of claim 1, wherein the cell population is the mammalian itself.

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