Method for isolating t cell receptors with antigen specificity for cancer-specific mutations

Abstract

Disclosed is a method of isolating a TCR having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in a nucleic acid of a cancer cell of a patient, each gene containing a cancer-specific mutation encoding a mutated amino acid sequence; inducing the patient's autologous APC to present the mutated amino acid sequence; co-culturing the autologous T cell of the patient with the autologous APC presenting the mutated amino acid sequence; selecting autologous T cells; and isolating a nucleotide sequence encoding a TCR from the selected autologous T cell, wherein the TCR has antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. Related methods of making a cell population, cell populations, TCRs, pharmaceutical compositions, and methods of treating or preventing cancer are also disclosed.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201480082932.3. The application date of Chinese Patent Application No. 201480082932.3 is October 2, 2014, and all the contents are incorporated herein.

[0002] Incorporation by Reference of Electronic Submissions

[0003] Incorporated herein by reference in its entirety is a computer readable nucleotide / amino acid sequence listing filed concurrently with the present application and identified as follows: 29,577 bytes ASCII (text) with the file name "718291ST25.TXT", dated September 15, 2014. Background Art

[0004] Adoptive cell therapy (ACT) using cells genetically engineered to express T cell receptors (TCRs) against cancer antigens can produce positive clinical responses in some cancer patients. However, barriers to the successful application of TCR-engineered cells for the broad treatment of cancer and other diseases remain. For example, TCRs that specifically recognize cancer antigens can be difficult to identify and / or isolate from patients. Therefore, improved methods for obtaining cancer-reactive TCRs are needed. SUMMARY OF THE INVENTION

[0005] One embodiment of the present invention provides a method for isolating a TCR or its antigen binding portion that has antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: 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 the autologous APCs presenting the mutated amino acid sequence; selecting autologous T cells that (a) are co-cultured with the autologous APCs presenting the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a major histocompatibility complex (MHC) molecule expressed by the patient; and isolating a nucleotide sequence encoding the TCR or its antigen binding portion from the selected autologous T cells, wherein the TCR or its antigen binding portion has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

[0006] Another embodiment of the present invention provides a method for preparing a cell population expressing a TCR or its antigen binding portion that has antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: 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 APCs to present the mutated amino acid sequence; co-culturing the patient's autologous T cells with the autologous APCs presenting the mutated amino acid sequence; selecting autologous T cells that (a) are co-cultured with the autologous APCs presenting the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of MHC molecules expressed by the patient; isolating a nucleotide sequence encoding a TCR or its antigen binding portion from the selected autologous T cells, wherein the TCR or its antigen binding portion has antigenic specificity for the mutated amino acid sequence encoded by the cancer-specific mutation; and introducing the nucleotide sequence encoding the isolated TCR or its antigen binding portion into peripheral blood mononuclear cells (PBMCs) to obtain cells expressing the TCR or its antigen binding portion.

[0007] Other embodiments of the invention provide related cell populations, TCRs or antigen-binding portions thereof, pharmaceutical compositions, and methods for treating or preventing cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 A shows the expression of IFN-γ per 1×10 TILs measured by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay after DCs transfected with OKT3 or with green fluorescent protein (GFP) RNA or the indicated tandem minigene (TMG) constructs were co-cultured with 3737-TILs for 20 h. 3 (1e3) The number of spots in each cell. ">" indicates that every 1×10 3 Mock transfected cells were treated with transfection reagent only without the addition of nucleic acid.

[0009] Figure 1 B shows a graph of the percentage of CD4+3737-TILs that are OX40+ after co-culture with OKT3 or dendritic cells (DCs) transfected with GFP RNA, TMG-1, or the indicated wild-type (wt) genes ALK, CD93, ERBB2IP, FCER1A, GRXCR1, KIF9, NAGS, NLRP2, or RAC3. Mock-transfected cells were treated with transfection reagents only without the addition of nucleic acids.

[0010] Figure 2A-2C shows the expression of 1×10 T cells per 1×10 T cells measured by IFN-γ ELISPOT assay at 20 h for 3737-TIL (A), DMF5 T cells (B), or T4 T cells (C) co-cultured with DCs transfected with TMG-1 (A) or 624-CIITA cells (B) and (C) without preincubation with anything, or the indicated HLA-blocking antibodies (against MHC-I, MHC-II, HLA-DP, HLA-DQ, or HLA-DR). 3 (AC) Graphs of the number of spots for (1e3) cells.

[0011] Figure 2 D shows the expression of 1×10 ALK per 1×10 T cells measured by IFN-γ ELISPOT assay at 20 h for 3737-TILs co-cultured with autologous DQ-0301 / -0601 B cells (grey bars) or allogeneic EBV-B cells partially matched at the HLA-DQ 05 / 0601 locus (black bars) or HLA-DQ-0201 / 0301 locus (unshaded bars) pulsed overnight with DMSO, mutant (mut) ALK, or mutant (mut) ERBB2IP 25-AA long peptide. 3 (1e3) Graph of the number of spots in cells. ETGHLENGNKYPNLE (SEQ ID NO: 53);

[0012] Figure 2E shows the expression of mut ERBB2IP peptides FLSINSKEETGHLENGNKYPNLE (SEQ ID NO:30), SINSKEETGHLENGNKYPNLE (SEQ ID NO:31), NSKEETGHLENGNKYPNLE (SEQ ID NO:32), KEETGHLENGNKYPNLE (SEQ ID NO:33), ETGHLENGNKYPNLE (SEQ ID NO:53), TSFLSINSKEETGHL (SEQ ID NO:34), TSFLSINSKEETGHLEN (SEQ ID NO:35), TSFLSINSKEETGHLENGNKYPNLE (SEQ ID NO:36), TSFLSINSKEETGHLENGNKYPNLE (SEQ ID NO:37), or TSFLSINSKEETGHLENGNKYPNLE (SEQ ID NO:38). NO:38) 3737-TIL co-cultured with autologous B cells pulsed overnight, measured by IFN-γ ELISPOT assay at 20 hours per 1×10 3 (1e3) Plot of the number of spots for each cell.

[0013] Figure 3 A shows a graph of the percentage of various TCR Vβ clonotypes in 3737-TILs measured by flow cytometry gating on live CD4+ (shaded) or CD8+ (non-shaded) T cells.

[0014] Figure 3 B shows a graph of IFN-γ levels (pg / ml) detected in patient 3737 serum samples measured before and at the indicated days after adoptive cell transfer of 3737-TIL on day 0 (indicated by arrows). Error bars are standard error of the mean (SEM).

[0015] Figure 3 C shows a graph of total tumor burden (circles) (measured as a percentage of pre-treatment baseline) or tumor burden in the lung (triangles) or liver (squares) at the indicated months relative to cell metastasis at day 0 (indicated by arrows).

[0016] Figure 3 D shows a graph of the percentages of various TCR Vβ clonotypes in CD4+Vβ22-OX40+3737- TILs measured by flow cytometry.

[0017] Figure 4 A and 4B show the frequencies of two ERBB2IP-mutation-specific TCRβ-CDR3 clonotypes Vβ22+ (A) and Vβ5.2+ (B) in the blood of patient 3737 (circles) at different times before and after adoptive cell transfer with 3737-TIL, in tumors before cell transfer (diamonds), and in various tumors after cell transfer (Tu-1-Post (squares), Tu-2-Post (▲), and Tu-3-Post (▼)). The shaded bars indicate the frequencies of two ERBB2IP-mutation-specific TCRβ-CDR3 clonotypes Vβ22+ (A) and Vβ5.2+ (B) in transferred cells (3737-TIL). "X" indicates "not detected".

[0018] Figure 4 C shows a graph of the expression of ERBB2IP relative to ACTB in various tumors before (TU-Pre) and after (Tu-1-post, Tu-2-post, and Tu-3-post) 3737-TIL (T cells) and adoptive cell transfer.

[0019] Figure 4 D shows total tumor burden (circles) (measured as a percentage of pre-treatment baseline) or tumor burden in the lung (triangles) or liver (squares) at the indicated months relative to cell metastasis (indicated by arrows).

[0020] Figure 5A is a schematic diagram of an example of a tandem minigene (TMG) construct encoding a polypeptide flanked by the six identified mutant amino acid residues (12 amino acids on either side) at its N- and C-termini. The mutant KIF2C sequence is DSSLQARLFPGLTIKIQRSNGLIHS (SEQ ID NO: 57).

[0021] Figure 5B shows a graph of IFN-γ levels (pg / mL) secreted by TIL 2359T cells co-cultured overnight with autologous melanocytes or with COS-7 cells co-transfected with HLA-A*0205 and TMG constructs RJ-1 (structure as shown in Figure 5A), RJ-2, RJ-3, RJ-4, RJ-5, RJ-6, RJ-7, RJ-8, RJ-9, RJ-10, RJ-11, RJ-12 or empty vector.

[0022] Figure 5C shows a graph of IFN-γ levels (pg / mL) secreted by TIL 2359 co-cultured with COS-7 cells transfected with HLA-A*0205 and RJ-1 variants (where the genes indicated as "wt" in the table were converted back to WT sequences). The KIF2C WT sequence is DSSLQARLFPGLAIKIQRSNGLIHS (SEQ ID NO: 65).

[0023] Figure 5D shows a graph of IFN-γ levels (pg / mL) secreted by TIL 2359 co-cultured with COS-7 cells transfected with an empty vector, cDNA constructs of KIF2C WT or mutated KIF2C and cDNA constructs of HLA (identify each shaded bar from left to right: HLA-A*0101 (non-shaded bar), HLA-A*0201 (grey bar), or HLA-A*0205 (black bar)).

[0024] FIG5E shows the expression of KIF2C in the presence of various concentrations (μM). 10-19 WT (RLFPGLAIKI; SEQ ID NO: 58) (bottom line in the figure) or mutant KIF2C 10-19 Graph of IFN-γ levels (pg / mL) secreted by TIL 2359 T cells co-cultured overnight with HEK293 cells stably expressing HLA-A*0205 pulsed with (RLFPGLTIKI; SEQ ID NO: 59) (top line in the figure).

[0025] FIG6A shows a graph of IFN-γ levels (pg / mL) secreted by TIL 2591 T cells co-cultured with autologous melanocytes or with HEK293 cells stably expressing HLA-C*0701 transfected with an empty vector or a TMG construct selected from DW-1 to DW-37.

[0026] Figure 6B shows a schematic diagram of the structure of TMG construct DW-6. The mutated POLA2 sequence is TIIEGTRSSGSHFVFVPSLRDVHHE (SEQ ID NO: 64).

[0027] Figure 6C shows a graph of IFN-γ levels (pg / mL) secreted by TIL 2591 co-cultured with COS-7 cells transfected with HLA-C*0701 and DW-6 variants (where the genes indicated as "wt" in the table were converted back to WT sequences). The POLA2 WT sequence is TIIEGTRSSGSHLVFVPSLRDVHHE (SEQ ID NO: 66).

[0028] Figure 6D shows a graph of IFN-γ levels (pg / mL) secreted by TIL 2591 co-cultured with COS-7 cells transfected with cDNA constructs of empty vector, POLA2 WT, or mutated POLA2 and cDNA constructs of HLA (identify the bars from left to right: HLA-C*0401 (non-shaded bars), HLA-C*0701 (grey bars), or HLA-C*0702 (black bars)).

[0029] FIG6E shows the results of the experiment with various concentrations (μM) of POLA2. 413-422 WT (TRSSGSHLVF; SEQ ID NO: 67) (bottom line in the figure) or mutant POLA2 413-422 A graph showing the level of IFN-γ secreted (pg / mL) by TIL 2591 T cells co-cultured overnight with HEK293 cells stably expressing HLA-C*0701 (TRSSGSHFVF; SEQ ID NO: 68) (top line in the figure).

[0030] Figure 7 A-7F are computed tomography (CT) scans of the lungs of patient 3737 obtained before (AC) and six months after (DF) the second administration of mutation-reactive cells. Arrows point to cancerous lesions. DETAILED DESCRIPTION OF THE INVENTION

[0031] One embodiment of the present invention provides a method for separating a TCR or its antigen binding portion having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. The present invention provides many advantages. For example, the method of the present invention can quickly evaluate a large number of mutations limited by all MHC molecules of a patient at one time, which can identify the full composition of the patient's mutation-reactive T cells. In addition, by distinguishing immunogenic cancer mutations from (a) silent cancer-specific mutations (which do not encode mutated amino acid sequences) and (b) cancer-specific mutations encoding non-immunogenic amino acid sequences, the method of the present invention can identify one or more cancer-specific mutated amino acid sequences that can be targeted by TCR or its antigen binding portion. In addition, the present invention can provide a TCR and its antigen binding portion having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation unique to a patient, thereby providing a "personalized" TCR and its antigen binding portion that can be used to treat or prevent cancer in a patient. The method of the present invention can also avoid the technical bias inherent in traditional methods for identifying cancer antigens, such as those using cDNA libraries, and can also be more time-saving and labor-saving than those methods. For example, the method of the present invention can select mutation-reactive T cells without co-culturing the T cells with tumor cell lines (especially, for example, epithelial cancers) that may be difficult to produce. Without being limited by a particular theory or mechanism, it is believed that the method of the present invention can identify and separate such TCRs or their antigen binding portions, which target and destroy cancer cells while minimizing or eliminating the destruction of normal non-cancerous cells, thereby reducing or eliminating toxicity. Therefore, the present invention can also provide such TCRs or their antigen binding portions, which successfully treat or prevent cancer, such as cancers that are unresponsive to other types of treatments (such as chemotherapy alone, surgery or radiation).

[0032] The method may include identifying one or more genes in a nucleic acid of a patient's cancer cell, each gene containing a cancer-specific mutation of an amino acid sequence encoding a mutation. Cancer cells may be obtained from any body sample derived from a patient, wherein the patient contains or is expected to contain a tumor or cancer cell. The body sample may be any tissue sample, such as blood, a tissue sample obtained from a primary tumor or from a tumor metastasis, or any other sample containing a tumor or cancer cell. The nucleic acid of the cancer cell may be DNA or RNA.

[0033] To identify cancer-specific mutations, the method can further include sequencing nucleic acids (such as DNA or RNA) of normal non-cancerous cells and comparing the sequences of cancer cells with the sequences of normal non-cancerous cells.Normal non-cancerous cells can be obtained from a patient or a different individual.

[0034] Cancer-specific mutations can be any mutation in any gene that encodes an amino acid sequence that is mutated (also referred to as a "non-silent mutation") and is expressed in cancer cells but not in normal non-cancer cells. Non-limiting examples of cancer-specific mutations that can be identified in the methods of the present invention include missense, nonsense, insertion, deletion, duplication, frameshift, and repeat expansion mutations. In one embodiment of the invention, the method includes identifying at least one gene that contains a cancer-specific mutation of an amino acid sequence that encodes a mutation. However, the number of genes containing such cancer-specific mutations that can be identified using the methods of the invention is not limited, and may include more than one gene (e.g., about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). Likewise, in one embodiment of the invention, the method includes identifying cancer-specific mutations of at least one amino acid sequence encoding a mutation. However, the number of such cancer-specific mutations that can be identified using the methods of the invention is not limited, and may include more than one cancer-specific mutation (e.g., about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the foregoing values). In embodiments where more than one cancer-specific mutation is identified, the cancer-specific mutations may be located in the same gene or in different genes.

[0035] In one embodiment, one or more genes in the nucleic acid of the cancer cell are identified, including sequencing the entire exome, whole genome or whole transcriptome of the cancer cell. Sequencing can be performed in any suitable manner known in the art. Examples of sequencing techniques that can be used for the method of the present invention include next generation sequencing (NGS) (also referred to as "massively parallel sequencing technology") or third generation sequencing. NGS refers to a high-throughput DNA sequencing technology based on non-Sanger methods. Using NGS, millions or billions of DNA chains can be sequenced in parallel, resulting in significantly more flux and minimizing the need for fragment cloning methods commonly used in Sanger method sequencing of genomes. In NGS, nucleic acid templates can be randomly read in parallel along the entire genome by breaking the entire genome into small pieces. NGS can advantageously provide nucleic acid sequence information of the whole genome, exome or transcriptome in a very short time, such as within about 1 to about 2 weeks, preferably within about 1 to about 7 days, or most preferably within a time of less than about 24 hours. A variety of NGS platforms that are commercially available or described in the literature can be used in the methods of the present invention, for example, those described in Zhang et al., J. Genet. Genomics, 38(3):95-109 (2011) and Voelkerding et al., Clinical Chemistry, 55:641-658 (2009).

[0036] Non-limiting examples of NGS techniques and platforms include: sequencing by synthesis (also known as "pyrosequencing") (as performed, for example, using a GS-FLX 454 Genome Sequencer, 454 Life Sciences (Branford, CT), an ILLUMINASOLEXA Genome Analyzer (Illumina Inc., San Diego, CA), or an ILLUMINA HISEQ 2000 Genome Analyzer (Illumina), or as described, for example, in Ronaghi et al., Science, 281(5375):363-365 (1998)), sequencing by ligation (as performed, for example, using the SOLID platform (Life Technologies, Carlsbad, CA) or the POLONATOR G.007 platform (Dover System, Salem, NH)), single molecule sequencing (as performed, for example, using the PACBIO RS system (Pacific Biosciences (Menlo Park, CA) or the HELISCOPE platform (Helicos Biosciences (Cambridge, MA)), nanotechnology for single-molecule sequencing (as implemented, for example, using the GRIDON platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nanopore sequencing (HANS) platform developed by Nabsys (Providence, RI), and a ligase-based DNA sequencing platform with DNA nanoball (DNB) technology (called probe-anchor ligation (cPAL)), electron microscopy-based technologies for single-molecule sequencing, and ion semiconductor sequencing.

[0037] The method may include inducing the patient's own antigen presenting cells (APCs) to present the mutated amino acid sequence. The APCs may include any cell presenting a peptide fragment of a protein bound to a major histocompatibility complex (MHC) molecule on its cell surface. The APCs may include, for example, any one or more of macrophages, DCs, Langerhans cells, B lymphocytes, and T cells. Preferably, the APCs are DCs. By using autologous APCs from patients, the method of the present invention can advantageously identify TCRs and their antigen binding portions that have antigen-specific amino acid sequences encoded by cancer-specific mutations presented in the context of MHC molecules expressed by patients. MHC molecules may be any MHC molecules expressed by patients, including but not limited to: class I MHC, class II MHC, HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules. The methods of the present invention can advantageously identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient, without using, for example, an epitope prediction algorithm to identify MHC molecules or mutated amino acid sequences, which can only be used to select a few class I MHC alleles and may be limited by the limited availability of reagents for selecting mutation-reactive T cells (e.g., an incomplete set of MHC tetramers). Thus, in one embodiment of the present invention, the methods of the present invention advantageously identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient, and are not limited to any particular MHC molecule. Preferably, the autologous APCs are antigen-negative autologous APCs.

[0038] Any suitable method known in the art can be used to induce the patient's autologous APC to present the mutated amino acid sequence. In one embodiment of the present invention, the patient's autologous APC is induced to present the mutated amino acid sequence, including pulsing the autologous APC with a peptide or peptide library containing the mutated amino acid sequence, each peptide in the peptide library containing a different mutated amino acid sequence. Each mutated amino acid sequence in the peptide library can be encoded by a gene containing a cancer-specific mutation. In this regard, the autologous APC can be cultured with a peptide or peptide library containing a mutated amino acid sequence so that the APC internalizes the peptide and displays the mutated amino acid sequence bound to the MHC molecule on the cell membrane. In one embodiment of identifying more than one gene, each gene containing a cancer-specific mutation encoding a mutated amino acid sequence, the method can include pulsing the autologous APC with a peptide library, each peptide in the peptide library containing a different mutated amino acid sequence. The method of pulsing APC is known in the art and is described in, for example, Solheim (Ed.), Antigen Processing and Presentation Protocols (Methods in Molecular Biology), Human Press, (2010). The peptide used to pulse the APC may include a mutated amino acid encoded by a cancer-specific mutation. The peptide further includes any suitable number of consecutive amino acids from an endogenous protein encoded by an identified gene on each carboxyl side and amino side of the mutated amino acid. The number of consecutive amino acids from endogenous proteins flanking each side of the mutation is not limited and may be, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or a range defined by any two of the foregoing values. Preferably, the peptide includes about 12 consecutive amino acids from endogenous proteins on each side of the mutated amino acid.

[0039] In one embodiment of the present invention, inducing the patient's autologous APC to present a mutated amino acid sequence comprises introducing a nucleotide sequence encoding the mutated amino acid sequence into the APC. The nucleotide sequence is introduced into the APC so that the APC expresses and displays the mutated amino acid sequence bound to the MHC molecule on the cell membrane. The nucleotide sequence encoding the mutated amino acid can be RNA or DNA. The nucleotide sequence is introduced into the APC, and any of a variety of different ways known in the art can be performed, which are described in, for example, Solheim et al. (supra). Non-limiting examples of techniques that can be used to introduce nucleotide sequences into APCs include transformation, transduction, transfection, and electroporation. In embodiments where more than one gene is identified, the method may include preparing more than one nucleotide sequence, each encoding a mutated amino acid sequence encoded by a different gene, and introducing each nucleotide sequence into different autologous APC populations. In this regard, multiple autologous APC populations can be obtained, each population expressing and displaying different mutated amino acid sequences.

[0040] In embodiments where more than one gene is identified, each gene containing a cancer-specific mutation encoding a mutated amino acid sequence, the method may include introducing a nucleotide sequence encoding more than one gene. In this regard, in one embodiment of the invention, the nucleotide sequence introduced into the autologous APC is a TMG construct, each minigene comprising a different gene, each gene comprising a cancer-specific mutation encoding a mutated amino acid sequence. As described herein with respect to other aspects of the invention, each minigene may encode a mutation identified by the method of the invention, with any suitable number of consecutive amino acids from an endogenous protein encoded by the identified gene flanking each side of the mutation. The number of minigenes in the construct is not limited, and may include, for example, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, or more, or a range defined by any two of the foregoing values. The APC expresses the mutated amino acid sequence encoded by the TMG construct on the cell membrane and displays the mutated amino acid sequence (which binds to the MHC molecule). In one embodiment, the method may include preparing more than one TMG construct, each construct encoding a different set of mutated amino acid sequences encoded by different genes, and introducing each TMG construct into a different population of autologous APCs. In this regard, multiple populations of autologous APCs may be obtained, each population expressing and displaying mutated amino acid sequences encoded by a different TMG construct.

[0041] The method may include culturing the patient's autologous T cells with autologous APCs presenting the mutated amino acid sequence. T cells can be obtained from many sources in 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 cells and may be at any stage of development, including but not limited to: CD4+ / CD8+ double positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+T cells (e.g., cytotoxic T cells), tumor infiltrating cells (e.g., tumor infiltrating lymphocytes (TIL)), peripheral blood T cells, memory T cells, initial T cells, etc. T cells may be CD8+T cells, CD4+T cells, or both CD4+T cells and CD8+T cells. The method may include co-culturing autologous T cells and autologous APCs so that T cells encounter the mutated amino acid sequence presented by APCs in such a manner that the autologous T cells specifically bind and immunorecognize the mutated amino acid sequence presented by APCs. In one embodiment of the invention, autologous T cells are co-cultured in direct contact with autologous APCs.

[0042] The method may include selecting such autologous T cells, which: (a) are co-cultured with autologous APCs presenting the mutated amino acid sequence, and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of the MHC molecules expressed by the patient. As used herein, the phrase "antigen specificity" means that the TCR or its antigen binding portion expressed by the autologous T cells can specifically bind to and immunologically recognize the mutated amino acid sequence encoded by the cancer-specific mutation. The selection may include identifying T cells with antigenic specificity for the mutated amino acid sequence and separating them from T cells that do not have antigenic specificity for the mutated amino acid sequence. Autologous T cells with antigenic specificity for the mutated amino acid sequence can be selected in any suitable manner. In one embodiment of the invention, the method includes amplifying the number of autologous T cells, for example, by co-culturing with T cell growth factors such as interleukin (IL) -2 or IL-15 before selecting the autologous T cells, or as described herein in relation to other aspects of the invention. In one embodiment of the present invention, the method does not include expanding the number of autologous T cells with T cell growth factors such as IL-2 or IL-15 before selecting the autologous T cells.

[0043] For example, when co-culturing autologous T cells with APCs presenting mutated amino acid sequences, T cells with antigen-specificity for the mutated amino acid sequences may express any one or more of various T cell activation markers, which may be used to identify T cells with antigen-specificity for the mutated amino acid sequences. Such T cell activation markers may include, but are not limited to, programmed cell death factor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, OX40, and CD107a. Therefore, in one embodiment of the present invention, autologous T cells with antigen-specificity for the mutated amino acid sequences are selected, including selecting T cells expressing any one or more of the following substances: PD-1, LAG-3, TIM-3, 4-1BB, OX40, and CD107a. Cells expressing one or more T cell activation markers can be sorted based on the expression of the marker using any of a variety of techniques known in the art, such as fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS), which are described in, for example, Turcotte et al., Clin. Cancer Res., 20(2):331-43 (2013) and Gros et al., J. Clin. Invest., 124(5):2246-59 (2014).

[0044] In another embodiment of the present invention, autologous T cells with antigenic specificity for the mutated amino acid sequence are selected, including selecting such T cells: (i) secreting more one or more cytokines when co-cultured with APCs presenting the mutated amino acid sequence than the amount of one or more cytokines secreted by the negative control, or (ii) compared to the number of negative control T cells that secrete one or more cytokines, wherein at least twice the number of T cells secrete one or more cytokines when co-cultured with APCs presenting the mutated amino acid sequence. The one or more cytokines may include any cytokine, the secretion of which by T cells is a characteristic of T cell activation (e.g., a TCR expressed by T cells that specifically binds to and immunorecognizes the mutated amino acid sequence). Non-limiting examples of cytokines (cytokine secretion is a characteristic of T cell activation) include: IFN-γ, IL-2 and tumor necrosis factor α (TNF-α), granulocyte / monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17 and IL-22.

[0045] For example, a TCR or antigen-binding portion thereof, or a T cell expressing the TCR or antigen-binding portion thereof, can be considered to be "antigen-specific" for a mutated amino acid sequence if the T cell, or T cell expressing the TCR or antigen-binding portion thereof, secretes at least twice the amount of IFN-γ secreted by a negative control when co-cultured with: (a) an antigen-negative APC pulsed with a concentration of a peptide comprising a mutated amino acid sequence (e.g., about 0.05 ng / mL to about 10 μg / mL, e.g., 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 100 ng / mL, 1 μg / mL, 5 μg / mL, or 10 μg / mL), or (b) an APC into which a nucleotide sequence encoding the mutated amino acid sequence has been introduced. A negative control can be, for example, (i) T cells expressing a TCR or an antigen-binding portion thereof co-cultured with: (a) antigen-negative APCs pulsed with the same concentration of an irrelevant peptide (e.g., a wild-type amino acid sequence, or some other peptide having a sequence different from the mutated amino acid sequence), or (b) APCs into which a nucleotide sequence encoding an irrelevant peptide sequence has been introduced, or (ii) untransduced T cells (e.g., from PBMCs that do not express a TCR or an antigen-binding portion thereof) co-cultured with: (a) antigen-negative APCs pulsed with the same concentration of a peptide comprising the mutated amino acid sequence, or (b) APCs into which a nucleotide sequence encoding the mutated amino acid sequence has been introduced. A TCR or its antigen binding portion, or a T cell expressing said TCR or its antigen binding portion, may also be "antigen-specific" for a mutated amino acid sequence if, compared to a negative control (e.g., any of the negative controls described above), a T cell or a T cell expressing a TCR or its antigen binding portion secretes greater amounts of IFN-γ when co-cultured with an antigen-negative APC pulsed with a higher concentration of a peptide comprising the mutated amino acid sequence. IFN-γ secretion can be measured by methods known in the art, such as an enzyme-linked immunosorbent assay (ELISA).

[0046] Alternatively or additionally, a TCR or its antigen binding portion, or a T cell expressing said TCR or its antigen binding portion, can be considered to have "antigen specificity" for a mutated amino acid sequence if at least twice the number of T cells or T cells expressing a TCR or its antigen binding portion secrete IFN-γ when co-cultured with the following substances: (a) an antigen-negative APC pulsed with a certain concentration of a peptide comprising a mutated amino acid sequence, or (b) an APC into which a nucleotide sequence encoding a mutated amino acid sequence is introduced, compared to the number of negative control T cells secreting IFN-γ. The concentration of the peptide and the negative control can be as described herein for other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art (e.g., ELISPOT).

[0047] T cells having antigenic specificity for the mutated amino acid sequence can both (1) express any one or more T cell activation markers described herein and (2) secrete greater amounts of one or more cytokines as described herein. In an embodiment of the present invention, T cells having antigenic specificity for the mutated amino acid sequence can express any one or more T cell activation markers without secreting greater amounts of one or more cytokines, or can secrete greater amounts of one or more cytokines without expressing any one or more T cell activation markers.

[0048] In another embodiment of the present invention, autologous T cells with antigenic specificity for the mutated amino acid sequence are selected, including selectively cultivating autologous T cells with antigenic specificity for the mutated amino acid sequence. In this regard, the method can include co-culturing autologous T cells with autologous APCs in such a way that the growth of T cells with antigenic specificity for the mutated amino acid sequence is favored compared to T cells that do not have antigenic specificity for the mutated amino acid sequence. Therefore, such a T cell population is provided, wherein the proportion of T cells with antigenic specificity for the mutated amino acid sequence is higher than that of T cells that do not have antigenic specificity for the mutated amino acid sequence.

[0049] In one embodiment of the present invention, the method further includes obtaining multiple fragments from the patient's tumor, separately co-culturing autologous T cells from each of the multiple fragments with autologous APCs presenting the mutated amino acid sequence described herein for other aspects of the present invention, and separately evaluating T cells from each of the multiple fragments that have antigen specificity for the mutated amino acid sequence (as described herein for other aspects of the present invention).

[0050] In one embodiment of the invention, wherein T cells are co-cultured with autologous APCs expressing multiple mutated amino acid sequences (e.g., multiple mutated amino acid sequences encoded by a TMG construct or multiple mutated amino acid sequences in a peptide library pulsed to an autologous APC), selecting the autologous T cells further includes separately evaluating autologous T cells with antigen-specificity for each of the multiple mutated amino acid sequences. For example, the method of the present invention further includes separately inducing the patient's autologous APCs to present each mutated amino acid sequence encoded by the construct (or included in the peptide library) (as described herein for other aspects of the present invention) (e.g., by providing separate APC groups, each of which presents a different mutated amino acid sequence encoded by the construct (or included in the peptide library)). The method further includes separately co-culturing the patient's autologous T cells with different groups of autologous APCs presenting each mutated amino acid sequence (as described herein for other aspects of the present invention). The method further comprises selecting such autologous T cells, respectively, which: (a) are co-cultured with autologous APCs presenting the mutated amino acid sequence, and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of a major histocompatibility complex (MHC) molecule expressed by the patient, as described herein for other aspects of the invention. In this regard, the method can include determining that the mutated amino acid sequence encoded by the TMG construct encoding a plurality of mutated amino acid sequences (or included in the peptide library) is immunologically recognized by the autologous T cells (e.g., by elimination treatment).

[0051] The method may further include isolating a nucleotide sequence encoding a TCR or an antigen binding portion thereof from a selected autologous T cell, wherein the TCR or its antigen binding portion has antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. In one embodiment of the invention, before isolating the nucleotide sequence encoding the TCR or its antigen binding portion, the number of selected autologous T cells having antigenic specificity for the mutated amino acid sequence can be amplified. The number of amplified T cells can be achieved by any of the methods known in the art, which are described in, for example, U.S. Patent No. 8,034,334; U.S. Patent No. 8,383,099; U.S. Patent Application Publication No. 2012 / 0244133; Dudley et al., J. Immunother., 26: 332-42 (2003); and Riddell et al., J. Immunol. Methods, 128: 189-201 (1990). In one embodiment, the number of T cells is expanded by culturing T cells with OKT3 antibody, IL-2 and feeder PBMC (e.g., irradiated allogeneic PBMC). In another embodiment of the invention, the number of autologous T cells selected that have antigen-specificity for the mutated amino acid sequence is not expanded before isolating the nucleotide sequence encoding the TCR or its antigen-binding portion.

[0052] As used herein, the "antigen binding portion" of a TCR refers to any portion of the continuous amino acids comprising a TCR (which is a part of a TCR), as long as the antigen binding portion specifically binds to the amino acid sequence of the mutation encoded by the gene identified as described in other aspects of the present invention. The term "antigen binding portion" refers to any portion or fragment of the TCR of the present invention, which retains the biological activity of the TCR (maternal TCR) (which is a part of the TCR). Antigen binding portions include, for example, those of the TCR, when compared to the parent TCR, retain the amino acid sequence of the specific binding mutation or the ability to detect, treat or prevent cancer to a similar degree, the same degree or a higher degree. With reference to the parent TCR, the functional portion can include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent TCR.

[0053] The antigen binding portion may include an antigen binding portion of either or both of the α chain and β chain of the TCR of the present invention, such as a portion comprising one or more of the complementary determining regions (CDR) 1, CDR2, and CDR3 of the variable region of the α chain and / or β chain of the TCR of the present invention. In one embodiment of the present invention, the antigen binding portion may include an amino acid sequence of CDR1 (CDR1α) of the α chain, CDR2 (CDR2α) of the α chain, CDR3 (CDR3α) of the α chain, CDR1 (CDR1β) of the β chain, CDR2 (CDR2β) of the β chain, CDR3 (CDR3β) of the β chain, or any combination thereof. Preferably, the antigen binding portion comprises an amino acid sequence of CDR1α, CDR2α, and CDR3α; an amino acid sequence of CDR1β, CDR2β, and CDR3β; or an amino acid sequence of all CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of the TCR of the present invention.

[0054] In one embodiment of the invention, the antigen binding portion may include, for example, a variable region of a TCR of the invention comprising a combination of CDR regions as listed above. In this regard, the antigen binding portion may include an amino acid sequence of the variable region (Vα) of the α chain of the TCR of the invention, an amino acid sequence of the variable region (Vβ) of the β chain, or an amino acid sequence of both Vα and Vβ.

[0055] In one embodiment of the invention, the antigen binding portion may include a combination of a variable region and a constant region. In this regard, the antigen binding portion may include the full length of the α chain or β chain, or both α chain and β chain of the TCR of the invention.

[0056] Nucleotide sequences encoding TCRs or antigen binding portions thereof can be isolated from selected autologous T cells in any suitable manner known in the art. For example, methods can include using established molecular cloning techniques and reagents (e.g., 5' rapid amplification (RACE) polymerase chain reaction (PCR) of cDNA ends using constant primers for TCR-α and -β chains), isolating RNA from autologous T cells, and sequencing the TCRs or antigen binding portions thereof.

[0057] In one embodiment of the present invention, the method may include cloning a nucleotide sequence encoding a TCR or its antigen binding portion into a recombinant expression vector using established molecular cloning techniques as described in Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012). For the purposes of this article, the term "recombinant expression vector" refers to an oligonucleotide or polynucleotide construct that allows a host cell to express a genetically modified mRNA, protein, polypeptide or peptide when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide or peptide, and the vector is in contact with the cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector of the present invention, as a whole, is not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vector may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from a natural source, and it may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring, non-naturally occurring nucleotide internucleotide connections, or these two types of connections. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not prevent transcription or replication of the vector.

[0058] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, and can be used for transforming or transfecting any suitable host cell. Suitable vectors include those designed for breeding and amplification or for expression or for both vectors, such as plasmids and viruses. The vector can be selected from: transposon / transposase, pUC series (Fermentas LifeSciences), pBluescript series (Stratagene, LaJolla, 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, λZap II (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.

[0059] The TCR or its antigen binding portion separated by the method of the present invention can be used to prepare cells for adoptive cell therapy. In this regard, one embodiment of the present invention provides a method for preparing a cell population expressing a TCR or its antigen binding portion that has antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising separating TCR or its antigen binding portion as described herein for other aspects of the present invention, and introducing a nucleotide sequence encoding the separated TCR or its antigen binding portion into PBMC to obtain cells expressing the TCR or its antigen binding portion.

[0060] Nucleotide sequences encoding isolated TCRs or antigen binding portions thereof (e.g., recombinant expression vectors) can be introduced into PBMCs in any of a variety of different ways known in the art, such as described in, e.g., Green et al. (supra). Non-limiting examples of techniques that can be used to introduce nucleotide sequences into PBMCs include transformation, transduction, transfection, and electroporation.

[0061] In one embodiment of the invention, the method includes introducing the nucleotide sequence of the TCR or its antigen binding portion thereof encoded for separation into the PBMC of the patient's autologous body. In this regard, the TCR or its antigen binding portion thereof identified and separated by the inventive method can be personalized for each patient. However, in another embodiment, the inventive method can identify and separate the amino acid sequence of the mutation encoded by the recurrent (also referred to as "hot spot") cancer-specific mutation with antigen-specific TCR or its antigen binding portion thereof. In this regard, the method may include introducing the nucleotide sequence of the TCR or its antigen binding portion thereof encoded for separation into the PBMC that is allogeneic for the patient. For example, the method may include introducing the nucleotide sequence of the TCR or its antigen binding portion thereof encoded for separation into the PBMC of another patient, and the tumor of the other patient expresses the same mutation in the same MHC molecule environment.

[0062] In one embodiment of the present invention, PBMC includes T cells.T cells can be any type of T cells, for example, any T cells in those T cells as described herein for other aspects of the present invention.Not bound to a specific theory or mechanism, it is believed that, compared to more differentiated "older" T cells, less differentiated "younger" T cells may be associated with any one or more of greater in vivo persistence, proliferation and anti-tumor activity.Therefore, the method of the present invention can advantageously identify and separate TCR or its antigen binding portion having antigen-specificity to the amino acid sequence of mutation, and TCR or its antigen binding portion is introduced into "younger" T cells, wherein compared to "older" T cells (for example, effector cells in patient tumors) that can separate TCR or its antigen binding portion, "younger" T cells can provide any one or more of greater in vivo persistence, proliferation and anti-tumor activity.

[0063] In one embodiment of the invention, the method further comprises the number of PBMCs expressing TCR or its antigen binding portion thereof by amplification. For example, as described herein for other aspects of the invention, the number of PBMCs can be amplified. In this respect, the method of the present invention can advantageously produce a large number of T cells that have antigen-specificity to the amino acid sequence of mutation.

[0064] Another embodiment of the present invention provides a TCR or its antigen binding portion separated by any of the methods described herein for other aspects of the present invention. One embodiment of the present invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha chain of a TCR, a beta chain of a TCR, a gamma chain of a TCR, a delta chain of a TCR, or a combination thereof. Another embodiment of the present invention provides an antigen binding portion of a TCR comprising one or more CDR regions, one or more variable regions, or one or both of the alpha and beta chains of a TCR as described herein for other aspects of the present invention. The polypeptide of the TCR or its antigen binding portion of the present invention may include any amino acid sequence, as long as the TCR or its antigen binding portion has antigen specificity for the mutated amino acid sequence encoded by a cancer-specific mutation.

[0065] Another embodiment of the present invention provides a cell group prepared according to any one of the methods described herein for other aspects of the present invention. The cell group can be a heterogeneous group, which includes the PBMC expressing the TCR or its antigen binding portion thereof, and at least one other cell that does not express the TCR or its antigen binding portion thereof separated, such as a host cell (such as PBMC), or a cell other than a T cell, such as a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a myocyte, a brain cell, etc. Alternatively, the cell group can be a substantially homogeneous cell group, wherein the cell group mainly includes the PBMC (for example, substantially composed of PBMC) expressing the TCR or its antigen binding portion thereof separated. The cell group can also be a cloned cell group, wherein all cells of the cell group are clones of the PBMC of a single expression of the TCR or its antigen binding portion thereof separated, so that all cells of the cell group express the TCR or its antigen binding portion thereof separated. In one embodiment of the present invention, the cell group is a cloned cell group comprising the PBMC of the TCR or its antigen binding portion thereof separated as described herein. By introducing the nucleotide sequence encoding the isolated TCR or its antigen binding portion into the PBMC, the method of the present invention can advantageously provide a cell population containing a high proportion of PBMC cells expressing the isolated TCR and having antigen specificity for the mutated amino acid sequence. In one embodiment of the present invention, about 1% to about 100%, such as about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, or a cell population of the range defined by any two of the foregoing values, contains PBMC cells expressing the isolated TCR and having antigen specificity for the mutated amino acid sequence. Without being bound by a particular theory or mechanism, it is believed that a cell population comprising a high proportion of PBMC cells expressing an isolated TCR having antigenic specificity for a mutated amino acid sequence has a lower proportion of irrelevant cells that may hinder the function of the PBMC cells (e.g., the ability of the PBMC cells to target and destroy cancer cells and / or treat or prevent cancer).

[0066] The TCR of the present invention or its antigen binding portion thereof and cell group can be formulated into a composition, such as a pharmaceutical composition. In this regard, the invention provides a pharmaceutical composition comprising any one of the TCR of the present invention or its antigen binding portion thereof or cell group and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may include a TCR of the present invention or its antigen binding portion thereof or cell group combined with another pharmaceutically active agent or drug (such as a chemotherapeutic agent, for example, asparaginase, busulfan (busulfan), carboplatin, cisplatin, daunorubicin (daunorubicin), doxorubicin (doxorubicin), fluorouracil, gemcitabine (gemcitabine), hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.) or its antigen binding portion thereof or cell group.

[0067] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any of those conventionally considered for use in a particular TCR or antigen binding portion thereof or cell population of the present invention. Such pharmaceutically acceptable carriers are well known to those skilled in the art and are readily available to the public. Preferably, the pharmaceutically acceptable carrier is a carrier that has no harmful side effects or toxicity under the conditions of use.

[0068] The selection of carrier will be determined in part by a specific TCR of the present invention, its antigen binding portion thereof, or cell group and a specific method for administering the TCR of the present invention, its antigen binding portion thereof, or cell group. Therefore, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include any of those for oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal or intraperitoneal administration. More than one approach can be used to administer the TCR or cell group of the present invention, and in some cases, a specific approach can provide a more direct and more effective response than another approach.

[0069] Preferably, the TCR, antigen binding portion thereof, or cell population of the present invention is administered by injection, e.g., intravenously. When the cell population of the present invention is to be administered, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier, e.g., saline (about 0.90% w / v NaCl aqueous solution, about 300 mOsm / L NaCl aqueous solution, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose aqueous solution, or lactated Ringer's solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0070] It is contemplated that the TCR, its antigen binding portion thereof, cell group and pharmaceutical composition of the present invention can be used for treating or preventing a method for cancer. Not subject to the constraints of a particular theory or mechanism, the TCR of the present invention or its antigen binding portion thereof is considered to specifically bind to the amino acid sequence of the mutation encoded by the cancer-specific mutation, so that the TCR or its antigen binding portion thereof (when it is expressed by a cell) can mediate an immune response of a target cell for expressing the amino acid sequence of the mutation. In this regard, the present invention provides a method for treating or preventing a cancer in a mammal, comprising administering to a mammal any of a pharmaceutical composition as described herein, a TCR, its antigen binding portion thereof or a cell group in an amount that can effectively treat or prevent a cancer in a mammal.

[0071] As used herein, the terms "treatment" and "prevention" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. On the contrary, there are different degrees of treatment or prevention that are recognized by those of ordinary skill in the art as having potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide any amount of treatment or prevention of any level of cancer in a mammal. In addition, the treatment or prevention provided by the methods of the present invention may include the treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention may include promoting the regression of a tumor. In addition, for the purposes of this article, "prevention" may include delaying the onset of cancer or its symptoms or its condition.

[0072] For the purposes of the present invention, the quantity or dosage of the TCR, its antigen binding portion, cell group or pharmaceutical composition of the present invention administered (e.g., the number of cells when the cell group of the present invention is administered) should be sufficient to work in a mammal within a reasonable time frame (e.g., therapeutic or preventive response). For example, the dosage of the TCR, its antigen binding portion, cell group or pharmaceutical composition of the present invention should be sufficient to bind to the mutated amino acid sequence encoded by the cancer-specific mutation, or detect, treat or prevent cancer during about 2 hours or longer (e.g., 12 to 24 hours or longer) from the time of administration. In certain embodiments, the time period can be even longer. The dosage is determined by the efficacy of the specific TCR, its antigen binding portion, cell group or pharmaceutical composition of the present invention administered and the condition of a mammal (e.g., a human), as well as the weight of the mammal (e.g., a human) to be treated.

[0073] Many assay methods for determining the dosage of administration are known in the art. For the purposes of the present invention, an assay method, which includes when a given dose of such T cells is administered to a mammal in a mammal group (each of which is administered with different doses of T cells), the target cells are compared to be dissolved or the T cells expressing the TCR of the present invention or its antigen binding portion secrete IFN-γ, and this assay method can be used to determine the starting dose administered to a mammal. When a certain dose is administered, the target cells are dissolved or the IFN-γ secreted by the T cells can be determined by methods known in the art.

[0074] The dosage of the TCR, antigen binding portion thereof, cell group or pharmaceutical composition of the invention can also be determined by the presence, nature and extent of any adverse side effects that may accompany the administration of a particular TCR, antigen binding portion thereof, cell group or pharmaceutical composition of the invention. Typically, the attending physician will consider various factors such as age, weight, health status, diet, sex, the TCR, antigen binding portion thereof, cell group or pharmaceutical composition of the invention to be administered, the route of administration, and the severity of the condition being treated to determine the dosage of the TCR, antigen binding portion thereof, cell group or pharmaceutical composition of the invention for treating each individual patient.

[0075] In one embodiment, where a cell population of the invention is to be administered, the number of cells administered per infusion may vary, for example, from 1 million to 100 billion cells; however, amounts below or above this exemplary range are within the scope of the invention. For example, a daily dose of host cells of the present invention can be 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 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 120 billion cells, about 130 billion cells, about 150 billion cells, or a range defined by any two of the foregoing values). The present invention relates to a method for producing a cell-free human plasma membrane with a plurality of cell-free cells, and a range of about 100 billion cells to about 130 billion cells (e.g., about 120 million cells, about 250 million cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells, or a range defined by any two of the foregoing values), more preferably about 100 million cells to about 130 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells, or a range defined by any two of the foregoing values).

[0076] For the purposes of the methods of the present invention, where a population of cells is administered, the cells may be allogeneic cells or autologous cells to the mammal. Preferably, the cells are autologous cells to the mammal.

[0077] Another embodiment of the invention provides any of the TCRs, antigen binding portions thereof, isolated cell populations or pharmaceutical compositions described herein for use in treating or preventing cancer in a mammal.

[0078] The cancer may advantageously be any cancer, including any of the following: acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vaginal cancer, vulvar cancer, bile duct cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer. Preferably, the cancer is an epithelial cancer. In one embodiment, the cancer is a cholangiocarcinoma, a melanoma, a colon cancer, or a rectal cancer.

[0079] The mammal mentioned in the method of the present invention can be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to: rodent mammals such as mice and hamsters, and lagomorpha mammals such as rabbits. Preferred mammals are from the order Carnivora, including Felidae (cats) and Canidae (dogs). Preferably, the mammal is from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs), or Perissodactyla, including Equine (horses). Preferably, the mammal is a primate, ape (Ceboids) or simian (Simoids) (monkeys), or anthropoid (human and apes). A more preferred mammal is a human. In a particularly preferred embodiment, the mammal is a patient expressing a cancer-specific mutation.

[0080] The following examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way. Example

[0081] The materials and methods for Examples 1-7 are given below.

[0082] Whole Exome Sequencing

[0083] Whole exome sequencing of cryopreserved tumor tissue (embedded in OCT) and normal peripheral blood cells was performed by Personal Genome Diagnostics (PGDx, Baltimore, MD) as described in Jones et al., Science 330:228-231 (2010). The average number of clear high-quality sequence reads at each base was 155 and 160 for tumor and normal (PBMC) DNA, respectively.

[0084] Patient treatment and generation of tumor-infiltrating lymphocytes (TILs) for adoptive cell therapy

[0085] Patient 3737 was recruited from the institutional review board (IRB)-approved protocol: "A Phase II Study of Autologous Tumor-Infiltrating Lymphocytes in Short-Term Culture after a Lymphoid-Depletion Regimen in Metastatic Gastrointestinal Cancer" (Trial Registration ID: NCT01174121), which was designed to evaluate the safety and efficacy of adoptive transfer of autologous ex vivo expanded tumor-infiltrating lymphocytes (TILs) in patients with gastrointestinal cancer.

[0086] TILs for the first treatment of patients were generated as described by Jin et al., J. Immunother., 35: 283-292 (2012). Briefly, the excised tumor was finely minced into pieces of approximately 1-2 mm, and individual pieces were placed in wells of a 24-well plate containing 2 ml of complete culture medium (CM) containing high-dose IL-2 (6000 IU / ml, Chiron, Emeryville, CA). CM consisted of RPMI supplemented with 10% in-house human serum, 2 mM L-glutamine, 25 mM HEPES, and 10 μg / ml gentamicin. In addition, the mixed tumor digest was cultured in CM containing high-dose IL-2. After T cells began to grow (between 2-3 weeks), 5×10 6 T cells were rapidly expanded in a gas permeable G-Rex100 bottle using irradiated allogeneic PBMCs at a ratio of 1 to 100 in 400 ml of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / ml of IL-2 and 30 ng / ml of OKT3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). The 50 / 50 medium consists of a 1:1 mixture of CM and AIM-V. All cells were cultured at 37°C and 5% CO2. Cell numbers were rapidly expanded two weeks before infusion. Patient 3737 underwent a non-myeloablative lymphodepletion regimen consisting of cyclophosphamide and fludarabine before receiving 42.4 billion total T cells and 4 doses of high-dose IL-2.

[0087] In a similar manner to the first treatment, TILs for the second treatment of the patient were produced with the following changes. The first treatment product (patient 3737-TIL) was composed of 5 separate TIL cultures. The CD4 and Vβ22 expression of these 5 cultures and the reactivity to the mutant ERBB2IP were evaluated separately, and it was found that Vβ22+ERBB2IP mutation-reactive CD4+T cells were highly enriched in one culture. As described above, this TIL culture (with high-dose IL-2 after initial growth) was then rapidly expanded. Before receiving 126 billion total T cells and 4 doses of high-dose IL-2, patient 3737 underwent the same non-myeloablative lymphoid depletion regimen as the first treatment.

[0088] Generation of TMG constructs

[0089] In brief, for each non-synonymous substitution mutation identified by full exome sequencing, a "minigene" construct encoding corresponding amino acid changes and 12 amino acids flanked by wild-type protein sequences was made. Multiple minigenes were fused together to produce TMG constructs. These minigene constructs were codon optimized and synthesized into DNA string constructs (Life Technologies, Carlsbad CA). TMG was then cloned into pcDNA3.1 vectors using In-Fusion technology (Clontech, Mountain View, CA). Site-directed mutagenesis was used to produce 9 TMG-1 constructs (Gene Oracle, Mountain View, CA) of "wild-type reversal". The nucleotide sequences of all TMGs were verified by standard Sanger sequencing (Macrogen and Gene Oracle).

[0090] Generation of autologous APCs

[0091] Monocyte-derived immature DCs were generated using the plastic adherence method. Briefly, autologous samples were thawed, washed, and cultured in pure AIM-V medium (Life Technologies) at a concentration of 5–10 × 10 6 cells / ml, and then about 1×10 6 cells / cm 2Incubate in tissue culture flasks of appropriate size and incubate at 37°C, 5% CO2. After 90 minutes (min), collect non-adherent cells, and wash the flask vigorously with AIM-V culture medium, and then incubate for 60min with AIM-V culture medium. Then wash the flask vigorously again with AIM-V culture medium, and then incubate the adherent cells with DC culture medium. DC culture medium contains RPMI containing 5% human serum (collected and processed by yourself), 100U / ml penicillin and 100μg / ml streptomycin, 2mM L-glutamine, 800IU / ml GM-CSF and 800U / ml IL-4 (culture medium supplement is from LifeTechnologies, cytokines are from Peprotech). On the 3rd day, fresh DC culture medium is added to the culture. 5-7 days after initial stimulation, use fresh or frozen / thawed DC in the experiment. In all experiments, flow cytometry was used to genotype cells expressing CD11c, CD14, CD80, CD86, and HLA-DR (all from BD Bioscience) to ensure that the cells were mainly immature DCs (CD11c+, CD14-, CD80 低 , CD86+ and HLA-DR+; data not shown).

[0092] Antigen presenting B cells were generated by CD40L and IL-4 stimulation. In brief, human CD19 microbeads (Miltenyi Biotec) were used to positively select B cells from autologous extracted samples. CD19+ cells were then cultured in B cell culture medium with irradiated (6000 rads) 3T3 cells (3T3-CD40L) that stably expressed CD40L at a ratio of approximately 1:1. B cell culture medium included IMDM culture medium (Life Technologies) supplemented with 7.5-10% human serum (self-owned), 100U / ml penicillin and 100μg / ml streptomycin (Life Technologies), 10μg / mL gentamicin (CellGro, Manassas, VA), 2mM L-glutamine (Life Technologies) and 200U / ml IL-4 (Peprotech). Fresh B cell culture medium was added starting on the 3rd day, and culture medium was added or replaced every 2-3 days thereafter. Additional irradiated 3T3-CD40L feeder cells were also added as needed. Antigen presenting B cells were used in experiments 2-3 weeks after initial stimulation.

[0093] Generating in vitro transcribed (IVT) RNA

[0094] The plasmid encoding the tandem minigene was linearized with the restriction endonuclease Sac II. The control pcDNA3.1 / V5-His-TOPO vector encoding GFP was linearized with Not I. The restriction digest was terminated with EDTA, sodium acetate, and ethanol precipitation. The intact plasmid digest was verified by standard agarose gel electrophoresis. Approximately 1 μg of the linearized plasmid was used to generate IVT RNA using the Message Machine T7 Ultra kit (Life Technologies) according to the manufacturer's instructions. The RNA was precipitated using the lithium chloride method, and the purity and concentration of the RNA were assessed using a NanoDrop spectrophotometer. The RNA was then aliquoted into microtubes and stored at -80°C until use.

[0095] RNA transfection

[0096] APCs (DCs or B cells) were harvested, washed once with PBS, and then cultured at 10-30 × 10 6 Cells / ml were resuspended in Opti-MEM (Life Technologies). IVT RNA (4 μg or 8 μg) was aliquoted to the bottom of the electroporation tank with a 2 mm gap, and 50 μl or 100 μl of APC was added directly to the electroporation tank. Therefore, the final RNA concentration used in the electroporation was 80 μg / mL. Electroporation was performed using a BTX-830 square wave electroporator. DCs were electroporated with 150V, 10ms, 1 pulse, and B cells were electroporated with 150V, 20ms, 1 pulse. As evaluated with GFP RNA (data not shown), the transfection efficiency using these settings is typically 70%-90%. All steps were performed at room temperature. After electroporation, the cells were immediately transferred to a polypropylene tube containing DC cells or B cell culture medium supplemented with appropriate cytokines. The transfected cells were incubated overnight (12-14h) at 37°C, 5% CO2. Cells were washed once with PBS before use in co-culture assays.

[0097] Peptide pulse

[0098] Autologous B cells were harvested, washed, and then cultured at 1 × 10 6Cells / ml were resuspended in B cell culture medium supplemented with IL-4 and then incubated with 1 μg / ml of 25-mer peptide overnight (12-14 h) at 37°C, 5% CO2. After the overnight pulse, the B cells were then washed twice with PBS and then resuspended in T cell culture medium and immediately used in the co-culture assay. The peptides used were: mutant ERBB2IP (TSFLSINSKEETGHLENGNKYPNLE (SEQ ID NO: 73)); wild-type ERBB2IP (TSFLSINSKEETEHLENGNKYPNLE (SEQ ID NO: 45)); and as a negative control, mutant ALK (RVLKGGSVRKLRHAKQLVLELGEEA (SEQ ID NO: 46)). Mutant ERBB2IP peptides were purchased from three different sources (GenScript, Piscataway, NJ, Peptide 2.0, Chantilly, VA, and SelleckChem, Houston TX) and all produced the same in vitro results, while wild-type ERBB2IP and mutant ALK peptides were purchased from Peptide 2.0. To culture allogeneic EBV-B cells, RPMI medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin (Life Technologies), 10 μg / ml gentamicin (CellGro), and 2 mM L-glutamine was used instead of B cell culture medium.

[0099] T cell sorting, expansion and cloning

[0100] BD FACSAria IIu and BD FACSJazz were used in all experiments requiring cell sorting. In the specified experiment, allogeneic feeder cells (white blood cell removal samples from three different donors) that were over-irradiated (4000 rads) were used to expand the sorted T cells in a 50 / 50 culture medium containing 30ng / ml anti-CD3 antibody (OKT3) and 3000IU / ml of IL-2. Limited dilution cloning was performed in 96-well round-bottom plates with the above-mentioned stimulation conditions of 5e4 feeder cells per well and 1-2 T cells per well. The culture medium was exchanged starting about 1 week after stimulation, and then every other day or as needed. About 2-3 weeks after the initial stimulation, the cells are usually used for determination or further expansion.

[0101] Co-culture assays: IFN-γ ELISPOT and ELISA, flow cytometry for cell surface activation markers, and intracellular cytokine staining (ICS)

[0102] When DCs are used as APCs, use approximately 3.5 × 10 4 Up to 7×10 4 When B cells are used as APCs, approximately 2 × 10 DCs are used per well of a 96-well round-bottom plate. 5 In the ELISPOT assay, 1 × 10 cells were used per well. 3 Up to 1×10 4 Effector T cells were used in each well for flow cytometry assays. 5 Effector T cells. T cells were typically thawed and recovered in 50 / 50 medium containing IL-2 (3000 IU / ml of IL-2) for two days and then washed (3×) with PBS prior to co-culture assays. All co-cultures were performed without exogenously added cytokines. For all assays, plate-bound OKT3 (0.1 μg / ml or 1 μg / ml) was used as a positive control.

[0103] In experiments involving HLA blocking antibodies, the following antibodies were used: Pan-class II (clone: ​​IVA12), Pan-class I (clone: ​​W6 / 32), HLA-DR (clone: ​​HB55), HLA-DP (clone: ​​B7 / 21) and HLA-DQ (clone: ​​SPV-L3). Cells were blocked with 20-50 μg / ml of the indicated antibodies for 1-2 hours at 37°C, 5% CO2 prior to co-culture with T cells. T4 are T cells transduced with an HLA-DR4-restricted TCR reactive to a tyrosinase epitope. DMF5 is an HLA-A2-restricted T cell line reactive to MART-1. 624-CIITA is an HLA-A2 and HLA-DR4-positive melanoma cell line that stably expresses MHC-II and is positive for MART-1 and tyrosinase expression due to ectopic expression of CIITA (class II, MHC, transactivator).

[0104] For IFN-γ ELISPOT assay, briefly, each well of ELIIP plate (Millipore, MAIPSWU) was pretreated with 50 μl of 70% ethanol for 2 minutes, washed 3 times with PBS, and then coated with 50 μl of 10 μg / ml IFN-γ capture antibody (Mabtech, clone: ​​1-D1K), and incubated overnight in a refrigerator. For OKT3 control, a mixture of IFN-γ capture antibody (10 μg / ml) and OKT3 (1 μg / ml) was coated on the wells. Before co-culture, the plate was washed 3 times with PBS and then blocked with 50 / 50 culture medium at room temperature (RT) for at least 1 hour. After 20-24 hours of co-culture, the cells were flicked off the plate, washed 6 times with PBS + 0.05% Tween-20 (PBS-T), and then incubated at room temperature for 2 hours with 100 μl / well of 1 μg / mL biotinylated anti-human IFN-γ detection antibody solution (MABTECH, clone: ​​7-B6-1) filtered through 0.22 μm. The plate was then washed 3 times with PBS-T, followed by incubation for 1 hour with 100 μl / well of streptavidin-ALP (Mabtech, Cincinatti, OH, 1:3000 dilution). The plate was then washed 6 times with PBS, followed by color development with 100 μl / well of 0.45 μm filtered BCIP / NBT substrate solution (KPL, Inc.). The reaction was stopped by rinsing thoroughly with cold tap water. The ELISPOT plates were scanned and counted using an immunospot plate reader and associated software (Cellular Technologies, Ltd, Shaker Heights, OH).

[0105] At approximately t = 22-26 hours after stimulation, the expression of T cell activation markers OX40 and 4-1BB was assessed by flow cytometry. Briefly, the cells were pelleted, washed with FACS buffer (1X PBS supplemented with 1% FBS and 2mM EDTA), and then stained with appropriate antibodies for approximately 30 minutes at 4°C in the dark. Before collection from a BD FACSCanto II flow cytometer, the cells were washed at least once with FACS buffer. All data were gated for live (PI-negative) single cells.

[0106] Cytokine production was assessed using intracellular cytokine staining (ICS) and flow cytometry. In brief, after target cells and effector cells were merged in the wells of a 96-well plate, both GolgiStop and GolgiPlug were added to the culture (BD Biosciences). GolgiStop and GolgiPlug were used at 1 / 2 of the manufacturer's recommended concentration. At t = 6 hours after stimulation, cells were treated with a Cytofix / Cytoperm kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. In brief, the cells were pelleted, washed with FACS buffer, and then cell surface markers were stained (as described above). The cells were then washed twice with FACS buffer before fixation and permeabilization. The cells were then washed with Perm / Wash buffer and stained with antibodies against cytokines for 30 minutes at 4 ° C in the dark. Before collection from a FACSCantoII flow cytometer, the cells were washed twice with Perm / Wash buffer and resuspended in FACS buffer. All flow cytometry data were analyzed using FLOWJO software (TreeStar Inc).

[0107] IFN-γ in serum samples was detected using a human IFN-γ ELISA kit according to the manufacturer's instructions (Thermo Scientific, Waltham, MA).

[0108] Antibodies for flow cytometry

[0109] The following titrated anti-human antibodies were used for cell surface staining: CCR7-FITC (clone: ​​150503), CD45RO-PE-Cy7 (clone: ​​UCHL1), CD62L-APC (clone: ​​DREG-56), CD27-APC-H7 (clone: ​​M-T271), CD4-efluor 605NC (clone: ​​OKT4), CD57-FITC (clone: ​​NK-1), CD28-PE-Cy7 (clone: ​​CD28.2), CD127-APC (clone: ​​eBioRDR5), CD3-AF700 (clone: ​​UCHT1), CD4-FITC, PE-Cy7, APC-H7 (clone: ​​SK3), CD8-PE-Cy7 (clone: ​​SK1), Vβ22-PE (clone: ​​IMMU 546), Vβ5.2-PE (clone: ​​36213), OX40-PE-Cy7 or FITC (clone: ​​Ber-ACT35), 4-1BB-APC (clone: ​​4B4-1) and CD107a-APC-H7 (clone: ​​H4A3). All antibodies except CD4-efluor605NC (eBioscience), Vβ22-PE and Vβ5.2-PE (Beckman Coulter), and 4-1BB-APC and OX40-PE-Cy7 (BioLegend) were from BD Biosciences. The following optimally titrated anti-human antibodies were used for intracellular cytokine staining: IFN-γ-FITC (clone: ​​4S.B3), IL-2-APC (clone: ​​MQ1-17H12), TNF-PerCPCy5.5 or APC (clone: ​​MAb11), IL-17-PE (clone: ​​eBio64DEC17) and IL-4-PE-Cy7 (clone: ​​8D4-8). Except for IL-4-PE-Cy7 (BD Bioscience), all ICS antibodies are from eBioscience. IOMark Β Mark TCR V kit is used to evaluate all TCR-Vβ (Beckman Coulter).

[0110] Sequencing of ERBB2IP mutations

[0111] Sanger sequencing was used to verify the ERBB2IP mutations found by whole exome sequencing. Total RNA was extracted from snap-frozen T cells or tumor tissue (OCT blocks) using the RNeasy Mini kit (Qiagen). Total RNA was then reverse transcribed into cDNA using ThermoScript reverse transcriptase and oligo-dT primers (Life Technologies). Normal cDNA and tumor cDNA were then used as templates in PCR, and the following ERBB2IP primers were flanked on the mutation: ERBB2IP Seq forward: 5'-TGTTGA CTC AAC AGC CAC AG-3' (SEQ ID NO: 47); and ERBB2IP Seq reverse: 5'-CTG GAC CACTTT TCT GAG GG-3' (SEQ ID NO: 48). Phusion DNA polymerase (Thermo Scientific) and the recommended three-step method (annealing temperature of 58°C (15 seconds) and extension at 72°C (30 seconds)) were used. PCR products were separated by standard agarose gel electrophoresis and gel extraction (Clontech). The products were directly sequenced using the same PCR primers (Macrogen).

[0112] Quantitative PCR

[0113] Total RNA was extracted from snap-frozen T cells or tumor tissue (OCT blocks) using the RNeasy Mini kit (Qiagen, Venlo, Netherlands). Total RNA was then reverse transcribed into cDNA using qScript cDNA Supermix (Quanta Biosciences, Gaithersburg, MD). Gene-specific Taqman primers and probe sets for human β-actin (Catalog #: 401846) and ERBB2IP (Catalog #: 4331182) were purchased from Life Technologies. Quantitative PCR was performed using a 7500 fast real-time PCR instrument with TAQMAN Fast Advanced Master Mix (both from Applied Biosystems). The specificity of the amplified products was verified by standard agarose gel electrophoresis. All calculated threshold cycle numbers (Ct) were 30 or less.

[0114] TCR-Vβ deep sequencing

[0115] TCR-Vβ deep sequencing was performed by immunoSEQ (Adaptive Biotechnologies, Seattle, WA) on genomic DNA isolated from peripheral blood cells, T cells, and frozen tumor tissue using the DNeasy blood and tissue kit (Qiagen). The number of reads of total productive TCRs for each sample ranged from 279,482 to 934,672. Only productive TCR rearrangements were used to calculate TCR frequencies.

[0116] TCR sequencing and construction of ERBB2IP-mutation-responsive TCRs

[0117] T cells were pelleted and total RNA was isolated (RNeasy Mini kit, Qiagen). Then, according to the manufacturer's instructions (SMARTer RACE cDNA amplification kit, Clontech), total RNA was subjected to 5'RACE using TCR-α and -β chain constant primers. Protocol 1 of the kit was used for PCR, where the extension time was adjusted (2min, instead of 3min). The sequences of the α and β chain constant primers were: TCR-α, 5'-GCC ACA GCA CTG TGC TCT TGA AGT CC-3' (SEQ ID NO: 49); TCR-β, 5'-CAG GCA GTA TCT GGA GTC ATT GAG-3 (SEQ ID NO: 50). The PCR products of TCR were then separated by standard agarose gel electrophoresis and gel extraction (Clontech). The products were then sequenced directly, or TOPO-TA cloned and then single colony sequenced (Macrogen). For sequencing of known Vβ22+T cell clones, cDNA was generated from RNA using qScript cDNA supermix (Quanta Biosciences). These cDNAs were then used as templates in PCR, and TCR-β constant primers (described above) and Vβ22 specific primers: 5'-CAC CAT GGA TAC CTG GCT CGT ATG C-3' (SEQ ID NO: 51). PCR products were separated by standard agarose gel electrophoresis and gel extraction (Clontech). The products were sequenced directly (Macrogen) using nested TCR-β chain constant primers: 5'-ATT CAC CCA CCA GCT CAG-3' (SEQ ID NO: 52).

[0118] By fusing the Vβ22+TCR-αV-DJ region with the mouse TCR-α constant chain and fusing the Vβ22+TCR-β-VDJ region with the mouse TCR-β constant chain, the construction of the Vβ22+ERBB2IP mutant TCR is completed. The α and β chains are separated by furin SGSG P2A linkers. The use of mouse TCR constant regions promotes the pairing of the introduced TCRs and is also conducive to the use of antibodies specific to the mouse TCR-β chain (eBioscience) to identify positively transduced T cells by flow cytometry. The TCR construct was synthesized and cloned into the MSGV1 retroviral vector (Gene Oracle).

[0119] TCR transduction of peripheral blood T cells

[0120] The autologous samples were thawed and set up at 2 × 10 6 10 cells / ml in a 50 / 50 mixture of RPMI medium and AIM-V medium supplemented with 5% free human serum, 10 μg / ml gentamicin (CellGro), 100 U / ml penicillin and 100 μg / ml streptomycin, 1.25 μg / ml amphotericin B (Fungizone), and 2 mM L-glutamine (all from Life Technologies). Prior to retroviral transduction, 2×10 cells / ml were stimulated with 50 ng / ml soluble OKT3 (Miltenyi Biotec) and 300 IU / ml recombinant human (rhu) IL-2 (Chiron) in 24-well plates. 6 To generate transient retroviral supernatants, the retroviral vector MSGV1 encoding Vβ22-positive, ERBB2IP mutation-specific TCR (1.5 μg / well) and the envelope encoding plasmid RD114 (0.75 μg / well) were co-transfected into the retroviral packaging cell line 293GP (6-well poly-D-lysine-coated plates, 1×10 cells / well) using Lipofectamine 2000 (Life Technologies). 6 42-48 h after transfection, the retroviral supernatant was collected, diluted 1:1 with DMEM medium, and then centrifuged at 2,000 g for 2 h at 32°C into retronectin-coated (10 μg / mL, Takara), non-tissue culture treated 6-well plates. Then, activated T cells (2 × 10 cells per well) were plated with 6 cells, 0.5 × 10 in T cell culture medium containing IL-2 6Cells / ml) were centrifuged at 300 g for 10 minutes onto retroviral plates. Activated T cells were transduced overnight, removed from the plates and further cultured in T cell culture medium containing IL-2. GFP and mock transduction controls were included in the transduction experiments. Cells were usually analyzed 10-14 days after retroviral transduction.

[0121] Example 1

[0122] This example demonstrates a method for 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.

[0123] A 43-year-old female (patient (Pt.) 3737) with extensive metastatic cholangiocarcinoma who had undergone multiple chemotherapy regimens was enrolled in a TIL-based adoptive cell therapy (ACT) program for patients with gastrointestinal (GI) cancer. The clinical characteristics of patient 3737 are shown in Table 1.

[0124] Table 1

[0125]

[0126] *Collected sites for TIL generation and whole exome sequencing

[0127] + Functional status: ECOG, Eastern Cooperative Oncology Group

[0128] The lung metastases were resected and used as a source for whole exome sequencing and generation of therapeutic T cells. Table 2 shows the somatic mutations identified by whole exome sequencing of metastatic lung nodules from patient 3737. Pathological analysis of hematoxylin and eosin (H&E) stained sections estimated that the tumor nodules were approximately 70% tumor. Whole exome sequencing revealed 26 non-synonymous mutations

[0129] (Table 2).

[0130]

[0131]

[0132]

[0133] Example 2

[0134] This example demonstrates the following method: inducing a patient's autologous APCs to present a mutated amino acid sequence; co-culturing the patient's autologous T cell population with the autologous APCs presenting the mutated amino acid sequence; and selecting the following autologous T cells, which: (a) are co-cultured with the autologous APCs presenting the mutated amino acid sequence, and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of MHC molecules expressed by the patient.

[0135] For each mutation identified in Example 1, a minigene construct was designed that encodes a mutated amino acid flanked by 12 amino acids from an endogenous protein on each side. Multiple minigenes were synthesized in tandem to generate a tandem minigene (TMG) construct (Table 3). In Table 3, the underline indicates the mutated amino acid and the new sequence encoded by a point mutation or nucleotide insertion or deletion. For splice site donor mutations (HLA-DOA and LONRF3), based on the assumption that these mutations prevent splicing at this site, mutant minigene transcripts were designed that continued to the downstream intron until the next stop codon. The splice site acceptor mutation of DIP2C was not evaluated.

[0136] Table 3

[0137]

[0138] The TMG constructs were then used as templates to generate in vitro transcribed (IVT) RNAs. Each of these IVT TMG RNAs was then individually transfected into autologous APCs (DCs) and subsequently co-cultured with TILs to determine if there was any processing and that the presented mutated antigens were recognized by the TILs. It was observed that 3737-TILs were reactive to the mutated antigens presented in TMG-1 but not TMG-2 or TMG-3 ( Figure 1 A). In addition, the reactivity is in the majority in the CD4+T cell population, as confirmed by the upregulation of activation markers OX40 and 4-1BB (Table 4A and Table 4B). Table 4A and Table 4B show the percentage of 3737-TIL detected by flow cytometry, after co-culturing with DC cultured with nonspecific stimulator OKT3 or DC transfected with green fluorescent protein (GFP) RNA, it has a specified phenotype, or has a specified tandem minigene (TMG) construct encoding various mutations identified by whole exome sequencing. The cells of the simulated transfection were treated with transfection reagent only without adding nucleic acid. The data set gates for live CD3+ cells.

[0139] Table 4A

[0140] 4-1BB- / CD4- 4-1BB+ / CD4- 4-1BB- / CD4+ 4-1BB+ / CD4+ simulation 49 0 51 0 GFP 49 0 51 0 TMG-1 47 4 38 11 TMG-2 47 0 53 0 TMG-3 48 0 52 0 OKT3 4 41 23 32

[0141] Table 4B

[0142] OX40- / CD4- OX40+ / CD4- OX40- / CD4+ OX40+ / CD4+ simulation 49 0 51 0 GFP 48 0 51 1 TMG-1 49 2 16 33 TMG-2 47 0 53 0 TMG-3 48 0 52 0 OKT3 38 6 11 45

[0143] Although some 4-1BB upregulation was observed in the CD4-negative (CD8+) T cell population, these cells were sorted and no reactivity against TMG was found. To determine which of the nine mutations in TMG-1 were recognized by 3737-TIL, nine additional TMG-1 constructs were synthesized, each containing a reversion of one of the mutations back to the wild-type sequence. The reactivity of 3737-TIL against TMG-1 was abolished only when the ERBB2 interacting protein (ERBB2IP) mutation was reverted to the wild-type sequence, indicating that TIL specifically recognizes ERBB2IP E805G mutation( Figure 1 B).

[0144] Molecular characterization of ERBB2IP-mutation reactive T cell responses. IFN-γ ELISPOT assays were performed and results were determined at 20 hours. Patient 3737-TILs were co-cultured with DCs transfected with TMG-1 or designated HLA-blocking antibodies (anti-MHC-I, MHC-II, HLA-DP, HLA-DQ, or HLA-DR) without preincubation ( Figure 2 A). As a control for antibody blocking, HLA-A2-restricted MART-reactive T cells DMF5 ( Figure 2 B) and HLA-DR restricted tyrosinase-reactive T cells T4 ( Figure 2 C), all co-cultured with MART and pre-incubated with nothing added to the tyrosinase-positive 624-CIITA melanoma cell line or the indicated HLA-blocking antibodies. The response of 3737-TIL was blocked by anti-HLA-DQ antibody ( Figure 2 A).

[0145] Additional IFN-γ ELISPOT assays were performed and the results were determined at 20 hours. Patient 3737-TILs were treated with DMSO, mutant (mut) ALK, or mut ERBB2IP 25-AA long peptide ( Figure 2 D) Co-culture of autologous B cells or allogeneic EBV-B cells partially matched at the HLA-DQ locus pulsed overnight.

[0146] Additional IFN-γ ELISPOT assays were performed and results were determined at 20 hours. Patient 3737-TILs were co-cultured with mutERBB2IP 25-AA peptide or the indicated truncated mut ERBB2IP peptides ( Figure 2 E) Co-culture of autologous B cells pulsed overnight.

[0147] like Figure 2As shown in A-2E, the 3737-TIL response is restricted by the HLA-DQB1*0601 allele, and the minimal epitope is located within the 13 amino acid sequence NSKEETGHLENGN (SEQ ID NO: 29).

[0148] Example 3

[0149] This example demonstrates that autologous open repertoire peripheral blood T cells genetically engineered with the TCR-Vβ22 chain (matching its α chain) of the ERBB2IP-specific CD4+ T cells identified in Example 2 are endowed with specific reactivity against mutant ERBB2IP peptides.

[0150] Using OX40 as an activation marker, the clonality of the mutated ERBB2IP-specific CD4+T cells identified in Example 2 was characterized by sorting them after antigen-specific activation. These cells were then expanded as a whole and cloned by limiting dilution. Measurement of the entire TCR-Vβ based on flow cytometry showed that the overall amplified population was greater than 95% Vβ22+, and 10 / 11 clones evaluated were pure Vβ22+. TCR sequence analysis revealed the same TCRβV-DJ sequence in 6 / 6 of the Vβ22+ clones tested (Table 5), indicating that most ERBB2IP-mutation reactive T cells contained a major Vβ22+T cell clone.

[0151] Table 5

[0152]

[0153] When stimulated with mutated ERBB2IP peptide, the T cell clone expressing the Vβ22TCR specifically produces cytokine IFN-γ (Table 6). CD4+Vβ22+ clones were co-cultured with OKT3 or autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutated (mut) ALK or mutERBB2IP 25-AA long peptide for 6 hours. Table 6 shows the percentage of CD4+Vβ22+ and Vβ22- clones that produce intracellular IFN-γ (IFN-γ+) or do not produce intracellular IFN-γ (IFN-γ-) after co-culture measured by flow cytometry. The data represent 2 clones sharing the same TCR-Vβ sequence.

[0154] Table 6

[0155] IFN-γ- / Vβ22- IFN-γ+ / Vβ22- IFN-γ- / Vβ22+ IFN-γ+ / Vβ22+ mutALK 1 0 98 1 wtERBB2IP 1 0 99 0 mutERBB2IP 1 0 19 80 OKT3 3 4 59 34

[0156] Furthermore, autologous open whole peripheral blood T cells genetically modified with this TCR-Vβ22 chain (matching its α chain) (Table 7) were endowed with specific reactivity against mutant ERBB2IP peptides (Tables 8A and 8B), indicating that this TCR specifically recognizes ERBB2IP E805G Mutation. Autologous open whole peripheral blood T cells were transduced (Td) with TCRs from Vβ22+ clones (Table 8A), or treated with transduction reagents only without adding nucleic acids (mock) (Table 8B), and then the reactivity as described in Table 6 was evaluated. The constant region of endogenous Vβ22+TCR was exchanged with the mouse constant region, which allows the use of antibodies against mouse TCRβ constant region (mTCRβ) to detect the introduced TCR. Plate-bound OKT3 was used as a control in all assays. Tables 8A and 8B show the percentage of mTCRβ+ and mTCRβ- cells that produce intracellular IFN-γ (IFN-γ+) or do not produce intracellular IFN-γ (IFN-γ-) measured by flow cytometry.

[0157] Table 7

[0158]

[0159] Table 8A

[0160] IFN-γ- / mTCRβ- IFN-γ+ / mTCRβ- IFN-γ- / mTCRβ+ IFN-γ+ / mTCRβ+ mutALK 16 0 84 0 wtERBB2IP 15 0 85 0 mutERBB2IP 19 0 69 12 OKT3 14 2 74 10

[0161] Table 8B

[0162] IFN-γ- / mTCRβ- IFN-γ+ / mTCRβ- IFN-γ- / mTCRβ+ IFN-γ+ / mTCRβ+ mutALK 99 1 0 0 wtERBB2IP 100 0 0 0 mutERBB2IP 100 0 0 0 OKT3 83 17 0 0

[0163] Example 4

[0164] This example demonstrates a method of using the autologous cells identified in Example 2 to treat cancer.

[0165] Patient 3737 received adoptive transfer of 42.4 billion CD4+ERBB2IP mutation-reactive T cells containing TILs, followed by 4 doses of IL-2 to enhance T cell proliferation and function. For treatment, patient 3737 underwent resection of the lung lesion. The tumor was then minced into small fragments and incubated with high-dose IL-2 to expand tumor-infiltrating lymphocytes (TILs). After the cell number was initially expanded in IL-2, the number of selected TIL cultures was further expanded for 2 weeks using a rapid expansion protocol (REP) (including irradiated allogeneic peripheral blood feeder cells, OKT3 and IL-2). Prior to cell infusion, the patient was preconditioned with cyclophosphamide (CTX: 60 mg / kg, once a day for two days), followed by fludarabine (Flu: 25 mg / m 2, for 5 days) preconditioning. Patient 3737-TIL contained 42.4 billion TILs containing more than 10 billion (25%) ERBB2IP mutation-reactive T cells and was administered on day 0, followed by IL-2 (aldesleukin, 7.2e5 IU / kg) every 8 hours. The patient received a total of 4 doses of IL-2.

[0166] 3737-TILs were co-cultured with DCs transfected with TMG-1 or TMG-1 encoding wild-type (wt) ERBB2IP reversed, and flow cytometry was used to assess OX40 and Vβ22 expression on CD4+ T cells 24 hours after stimulation. Plate-bound OKT3 stimulation was used as a positive control. Flow cytometric analysis showed that approximately 25% of the entire 3737-TIL product administered contained Vβ22+, mutation-reactive T cells ( Figure 3 A, Table 9), equivalent to infusing over 10 billion ERBB2IP mutation-specific CD4+ T cells. Table 9 shows the percentage of Vβ22+ and Vβ22- cells expressing OX40 (OX40+) or not expressing OX40 (OX40-) as measured by flow cytometry.

[0167] Serum samples from patient 3737 were subjected to IFN-γ ELISA assays before and after adoptive cell transfer of 3737-TIL. The results are shown in Figure 3 B. If Figure 3 As shown in B, elevated IFN-γ levels were detected in the patient's serum during the first five days after cell infusion.

[0168] Although the patient had clear evidence of progressive disease prior to cell infusion, tumor regression was observed at two months of follow-up, and all target lung and liver lesions continued to regress, reaching a maximum reduction of 30% 7 months after treatment ( Figure 3 C). The patient experienced stable disease for approximately 13 months following cell infusion, after which disease progression was observed only in the lungs (not the liver).

[0169] Table 9

[0170]

[0171] Example 5

[0172] This example demonstrates the phenotype and function of the cells of Example 4 in vitro.

[0173] To determine whether there is evidence that CD4+ERBB2IP mutation-reactive T cells play a role in disease stabilization, the phenotype and function of the cells in vitro were evaluated. 3737-TIL was co-cultured with autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK or mut ERBB2IP 25-AA long peptide for 6 hours. In the CD4+ group, flow cytometry was used to evaluate the expression of Vβ22 and detect IFN-γ (Table 10A), tumor necrosis factor (TNF) (Table 10B) and IL-2 (Table 10C) produced in the cells. The percentage of cells with the specified phenotype is shown in Tables 10A-10C. Table 10D shows the percentage of Vβ22+ cells expressing the specified number of cytokines. It was found that Vβ22+ERBB2IP-mutation reactive CD4+ T cells were multifunctional Th1 cells that induced robust co-expression of IFN-γ, TNF, and IL-2 (Tables 10A-10C), but little or no IL-4 or IL-17 when stimulated with mutant ERBB2IP peptide.

[0174] Table 10A

[0175] Vβ22- / IFN-γ- Vβ22- / IFN-γ+ Vβ22+ / IFN-γ- Vβ22+ / IFN-γ+ mutALK 45 0 55 0 wtERBB2IP 44 0 56 0 mutERBB2IP 40 8 6 47 OKT3 29 33 24 14

[0176] Table 10B

[0177] Vβ22- / TNF- Vβ22- / TNF+ Vβ22+ / TNF- Vβ22+ / TNF+ mutALK 45 0 55 0 wtERBB2IP 43 1 56 0 mutERBB2IP 37 10 3 50 OKT3 10 52 6 32

[0178] Table 10C

[0179] Vβ22- / IL-2- Vβ22- / IL-2+ Vβ22+ / IL-2- Vβ22+ / IL-2+ mutALK 45 0 55 0 wtERBB2IP 43 1 56 0 mutERBB2IP 38 10 5 47 OKT3 27 36 23 14

[0180] Table 10D

[0181]

[0182] Further phenotypic characteristics show that these cells are mainly effector memory CD4+T cells with cytolytic potential (Tables 11 and 12). The expression of Vβ22 (representing ERBB2IP-mutation reactive T cells) and T cell differentiation markers CD28, CD45RO, CD57, CCR7, CD127, CD62L and CD27 of patient 3737-TIL was evaluated by flow cytometry. The data were gated for live CD3+CD4+ cells. Positive and negative quadrant gates were set with homotypic staining or unstained cells. The percentage of cells with a specified phenotype is shown in Table 11. Human peripheral blood cells (including T cells at all stages of differentiation) were included in the experiment to ensure that the antibody worked.

[0183] Table 11

[0184] Vβ22- / CD28- Vβ22- / CD28+ Vβ22+ / CD28- Vβ22+ / CD28+ 1 56 1 42 Vβ22- / CD45RO- Vβ22- / CD45RO+ Vβ22+ / CD45RO- Vβ22+ / CD45RO+ 0 57 0 43 Vβ22- / CD57- Vβ22- / CD57+ Vβ22+ / CD57- Vβ22+ / CD57+ 48 9 42 1 Vβ22- / CCR7- Vβ22- / CCR7+ Vβ22+ / CCR7- Vβ22+ / CCR7+ 57 0 43 0 Vβ22- / CD127- Vβ22- / CD127+ Vβ22+ / CD127- Vβ22+ / CD127+ 25 32 2l 22 Vβ22- / CD62L- Vβ22- / CD62L+ Vβ22+ / CD62L- Vβ22+ / CD62L+ 49 8 42 1 Vβ22- / CD27- Vβ22- / CD27+ Vβ22+ / CD27- Vβ22+ / CD27+ 57 0 43 0

[0185] Patient 3737-TIL was co-cultured with OKT3 or autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK or mut ERBB2IP25-AA long peptide for 6 hours. At the beginning of co-culture, antibodies specific to the degranulation marker CD107a were added. Flow cytometry was used to evaluate the expression of Vβ22 and detect the cell surface movement of CD107a. The data were gated for the CD4+ group. The percentages of cells with the specified phenotype are shown in Table 12.

[0186] Table 12

[0187] Vβ22- / CD107a- Vβ22- / CD107a+ Vβ22+ / CD107a- Vβ22+ / CD107a+ mutALK 51 0 48 1 wtERBB2IP 51 1 48 0 mutERBB2IP 53 6 19 22 OKT3 42 19 26 13

[0188] It can be seen that there are small groups of multifunctional Vβ22-negative, ERBB2IP-mutation reactive CD4+T cells in 3737-TIL (Tables 9 and 10). These Vβ22-negative cells were sorted by FACS, followed by a 2-day rest in a medium containing IL-2, and then co-cultured with autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK or mut ERBB2IP 25-AA long peptides. In the CD4+ group, 6 hours (h) after stimulation, flow cytometry was used to evaluate the expression of Vβ22, and IL-2 (Table 13C), TNF (Table 13B) and IFN-γ (Table 13A) produced in the cells were detected. The percentage of cells with a specified phenotype is shown in Tables 13A-13C.

[0189] Table 13A

[0190] Vβ22- / IFN-γ- Vβ22- / IFN-γ+ Vβ22+ / IFN-γ- Vβ22+ / IFN-γ+ mutALK 99 0 1 0 wtERBB2IP 99 0 1 0 mutERBB2IP 85 14 0 1 OKT3 50 49 0 1

[0191] Table 13B

[0192] Vβ22- / TNF- Vβ22- / TNF+ Vβ22+ / TNF- Vβ22+ / TNF+ mutALK 99 0 1 0 wtERBB2IP 97 2 1 0 mutERBB2IP 78 21 0 1 OKT3 9 90 0 1

[0193] Table 13C

[0194] Vβ22- / IL-2- Vβ22- / IL-2+ Vβ22+ / IL-2- Vβ22+ / IL-2+ mutALK 99 0 1 0 wtERBB2IP 97 2 1 0 mutERBB2IP 78 21 0 1 OKT3 36 63 0 1

[0195] 24 hours after stimulation, flow cytometry was used to assess the expression of OX40 and Vβ22 in the CD4+ population. Cells that upregulated OX40 were sorted, the number of cells was expanded, and the total TCR-Vβ was analyzed by flow cytometry. The results are shown in Figure 3 D. Sorting of Vβ22-negative cells and subsequent activation of these cells revealed the presence of one or more additional clonotypes reactive to this epitope in 3737-TIL (Tables 13A-13C), the most predominant of which was Vβ5.2 ( Figure 3 D).

[0196] Will be like Figure 3The sorted cells shown in D were co-cultured for 6 hours with autologous B cells pulsed overnight with wt ERBB2IP, mut ALK or mut ERBB2IP 25-AA long peptides. In the CD4+ population, flow cytometry was used to assess the expression of Vβ5.2 and to detect intracellular production of IL-2 (Table 14C), TNF (Table 14B) and IFN-γ (Table 14A). Table 15 shows the percentage of Vβ5.2+ cells expressing the specified number of cytokines.

[0197] Table 14A

[0198] Vβ5.2- / IFN-γ- Vβ5.2- / IFN-γ+ Vβ5.2+ / IFN-γ- Vβ5.2+ / IFN-γ+ mutALK 51 0 49 0 wtERBB2IP 54 0 46 0 mutERBB2IP 42 13 20 25 OKT3 28 23 25 24

[0199] Table 14B

[0200] Vβ5.2- / TNF- Vβ5.2- / TNF+ Vβ5.2+ / TNF- Vβ5.2+ / TNF+ mutALK 50 2 48 0 wtERBB2IP 52 2 46 0 mutERBB2IP 33 21 3 43 OKT3 5 46 3 46

[0201] Table 14C

[0202] Vβ5.2- / IL-2- Vβ5.2- / IL-2+ Vβ5.2+ / IL-2- Vβ5.2+ / IL-2+ mutALK 51 1 48 0 wtERBB2IP 54 1 45 0 mutERBB2IP 38 17 14 31 OKT3 31 21 27 21

[0203] Table 15

[0204]

[0205] Vβ22-negative cells that upregulate OX40 when stimulated with mutated ERBB2IP are sorted and the number of the cells is amplified. Then, RNA from these cells is isolated and rapid amplification of 5' complementary DNA ends (5'RACE) is performed with TCR-β constant chain primers to identify the expressed TCR-Vβ sequence. TOPO-TA cloning is performed on polymerase chain reaction (PCR) products, and then individual clones are sequenced. Flow cytometry confirmed that 40-50% of these T cells are Vβ5.2 (TRBV5-6). By Sanger sequencing, 3 / 7 TOPO-TA clones are Vβ5.2 (TRBV5-6) with sequences shown in Table 16. Table 16 shows the most frequent TCRβV-DJ sequences of Vβ22-negative ERBB2IP mutation-reactive T cells.

[0206] Table 16

[0207]

[0208] Most Vβ5.2+ cells produce a variety of cytokines in an antigen-specific manner (Tables 14A-14C, Tables 15 and 16). It can be seen that there are small populations of Vβ5.2-negative (and Vβ22-negative) CD4+T cells that recognize mutated ERBB2IP (Tables 14A-14C and Table 15). Therefore, the TIL used to treat patient 3737 contains at least three different multifunctional CD4+T cell clones that recognize the same mutation in ERBB2IP, indicating that the mutation is highly immunogenic.

[0209] Example 6

[0210] This example demonstrates the persistence of the cells of Example 4 in vivo.

[0211] To determine whether there is evidence that CD4+ERBB2IP-mutation reactive T cells contribute to disease stabilization, the in vivo persistence of these cells was evaluated. TCR-Vβ deep sequencing revealed that these clonotypes were rare or undetectable in peripheral blood before ACT ( Figure 4 A and Figure 4 B) Ten days after ACT, both clones accounted for more than 2% of total T cells in peripheral blood, but decreased to less than 0.3% 34 days after cell infusion ( Figure 4 A and Figure 4 B). Three lung metastases resected nearly one and a half years after ACT were infiltrated by ERBB2IP mutation-reactive T cells ( Figure 4 A and 4B), indicating that these cells promote cancer regression and disease stabilization. The Vβ22+ERBB2IP mutation-reactive clone was the most frequent clone detected in Tumor Nodule-3 (Tu-3-Post) and represented nearly 8% of the total T cells in the tumor ( Figure 4 A and Figure 4 B), while in tumor nodules-1 and -2, this clone was the 2nd and 12th most common, respectively. The Vβ5.2+ERBB2IP-mutation reactive clone was also enriched compared to its frequency in the blood of all three tumor nodules ( Figure 4 A and 4B). Thus, after receiving more than 10 billion ERBB2IP-mutation-specific multifunctional Th1 cells that infiltrated and persisted in metastatic lesions, patient 3737 experienced tumor regression and stable disease for more than one year.

[0212] Reverse transcriptase quantitative PCR (RT-qPCR) analysis was performed for ERBB2IP expression in patient 3737-TIL (T cells) and tumors before (Tu-Pre) and after adoptive cell transfer. Three separate metastatic lung lesions (Tu-1, -2, -3-Post) were resected approximately 17 months after cell infusion. Figure 4C, and is relative to β-actin (ACTB). Figure 4 A 350 base pair (bp) fragment of the mutated ERBB2IP gene was amplified by PCR from the cDNA sample described in C and subjected to Sanger sequencing. The mutation was located at nucleotide position 2414 of the coding sequence, corresponding to a change at position 805 of the amino acid sequence. As determined by quantitative RT-PCR, relatively high levels of ERBB2IP expression were observed in primary and recurrent lung lesions ( Figure 4 C), and Sanger sequencing confirmed the presence of ERBB2IP mutations in all tumor lesions.

[0213] Immunohistochemical analysis was performed for T cell infiltration and MHC expression before and after ACT. Tumors after ACT were harvested approximately 17 months after the first ACT. All staining included a positive control (tonsil). T cell infiltration and MHC in situ expression of tumors are summarized in Tables 17 and 18, respectively.

[0214] Table 17

[0215]

[0216] 0, no infiltration

[0217] 1. Very little to very little

[0218] 2. Medium density

[0219] 3. Very dense

[0220] Table 18

[0221] Tumor nodule HLA-I HLA-II (HLA-DR) Pre-1A 1-2,>50％ 0 Pre-2A 1-2,>50％ 0 Pre-3A 1,>50％ 0 Pre-3B 2,>50％ 0 Post-1A 2-3,>50％ 0 Post-1B 3,>50％ 0 Post-2A 2,>50％ 0

[0222] >50% means that more than 50% of the tumor cells are positive.

[0223] 0, negative

[0224] 1. Weak positive

[0225] 2. Moderately positive

[0226] 3. Strong positive

[0227] Example 7

[0228] This example demonstrates the contribution of mutation-reactive Th1 cells to the anti-tumor response of Example 4.

[0229] To specifically evaluate the contribution of mutation-reactive Th1 cells to the in vivo anti-tumor response, a TIL product containing greater than 95% Vβ22+ERBB2IP-mutation-reactive Th1 cells (approximately 120 billion mutation-reactive cells) was generated and adoptively transferred to patient 3737.

[0230] The TIL products used for retreatment were analyzed by flow cytometry. Table 19 shows that after gating on CD3, 97% were CD4+ / CD8-, and among these, after further gating on CD4+ cells, 98% were Vβ22+ (Table 20). The retreatment TILs were co-cultured for 6 hours with autologous B cells pulsed overnight with wild-type (wt) or mutant (mut) ERBB2IP 25-AA long peptides. The production of intracellular TNF in the CD4+ population was detected by flow cytometry (Table 20).

[0231] Table 19

[0232] CD8- / CD4- CD8- / CD4+ CD8+ / CD4- CD8+ / CD4+ 0 97 3 0

[0233] Table 20

[0234] Vβ22- / TNF- Vβ22- / TNF+ Vβ22+ / TNF- Vβ22+ / TNF+ wtERBB2IP 2 0 98 0 mutERBB2IP 1 3 3 93

[0235] Again, the patient experienced a reduction in target lesions, but unlike the first treatment, tumor regression was observed even at the first month of follow-up and continued at 4 months after the second treatment ( Figure 4 D) Tumor regression continued at follow-up 8 months after the second treatment.

[0236] Six months after the second administration of mutation-reactive cells, computed tomography (CT) scans of patient 3737's lungs were performed, and the images obtained were as follows: Figure 7 AC. These images are compared with images obtained before the second administration of mutation-responsive cells ( Figure 7 D-7F). Figure 7 As shown in A-7F, approximately 36% reduction in cancerous lesions was observed, which provided a partial response (PR) by the criteria of Response Evaluation Criteria in Solid Tumors (RECIST).

[0237] Eight months after the second administration of mutation-reactive cells, positron emission tomography (PET) scans were performed on the liver and lungs of patient 3737. It was observed that the target lesions continued to shrink. A radiolabeled glucose analog (FDG (fluorodeoxyglucose)) was administered to assess the uptake of glucose by the tumor to measure the metabolic activity of the tumor. The PET scans showed no glucose uptake in both liver lesions and only some uptake in the lung lesions.

[0238] Examples 8-10

[0239] The materials and methods for Examples 8-10 are given below.

[0240] Patient material and cell lines

[0241] All patient materials were obtained during the course of a clinical trial approved by the Institutional Review Board of the National Cancer Institute. Dudley et al., J. Clin. Oncol., 26: 5233-9 (2008) described that patients 2359 and 2591 were enrolled in a clinical trial (respectively, trial registration ID: NCT00096382 and ID: NCT00335127). TIL cell lines and tumor cell lines were established from patients who underwent resection. TILs used for this study were generated by the method described by Dudley et al., J. Immunother., 26: 332-42 (2003). In brief, tumor fragments were excised and cultured in a medium containing IL-2. The TIL cultures that were expanded in number were screened to identify autologous or HLA-matched tumors, and the number of reactive TILs was expanded to large numbers for patient infusion using a rapid expansion protocol (REP) with IL-2, anti-CD3 antibodies, and irradiated feeder cells (Riddell et al., Science, 257:238-41 (1992)). A small portion of the TILs underwent a second REP. For co-culture assays, T cells and tumor cells were cultured at a 1:1 ratio for 16 hours (hr) on a 96-well plate with 200 μL of culture medium (AIM-V medium supplemented with 5% human serum).

[0242] In order to assess the antigenic reactivity of clinically active TIL, two patients with metastatic melanoma who experienced a lasting complete response to adoptive TIL treatment were studied. Patient 2359 suffered from primary cutaneous melanoma in the right knee, which was transferred to thigh, iliac and inguinal lymph nodes. This individual experienced a complete regression of all metastatic lesions transferred in response to autologous TIL, which lasted for more than 8 years after treatment. Patient 2591 suffered from primary back melanoma, which was transferred to abdominal wall, mesenteric lymph nodes, right colon and supraclavicular lymph nodes. This individual experienced a complete regression of all metastatic lesions transferred in response to autologous TIL and remained disease-free for 9 years after treatment.

[0243] Whole Exome Sequencing

[0244] The method is described in Robbins et al., Nat. Med., 19: 747-52 (2013). Genomic DNA purification, library construction, exome capture of approximately 20,000 coding genes, and next-generation sequencing of tumor and normal samples were performed at Personal Genome Diagnostics (Baltimore, MD). In brief, genomic DNA from tumor and normal samples was fragmented and used for Illumina TRUSEQ library construction (Illumina, San Diego, CA). Exon regions were captured in solution using the Agilent SURESELECT 50Mb kit (version 3) according to the manufacturer's instructions (Agilent, Santa Clara, CA). Double-end sequencing (generating 100 bases from each end of each fragment) was performed using the HISEQ 2000 genome analyzer (Illumina). Sequence data was matched to the reference human genome sequence, and sequence changes were determined by comparing more than 50 million bases of tumor and normal DNA. For each sample, more than 8 billion bases of sequence data were obtained, and a high fraction of bases came from captured coding regions. More than 43 million bases of target DNA were analyzed in tumor and normal samples, and an average of 42-51 reads were obtained at each base in normal and tumor DNA samples.

[0245] Bioinformatics analysis is carried out by the genomics and medical pathology service department of personal genome diagnosis and genome technology access center, Washington University School of Medicine.Use the ELAND algorithm of CASAVA 1.6 software (Illumina), label is compared with human genome reference sequence (hg18).Use the Chastity filter of Illumina's BASECALL software to read with the sequence selected for subsequent analysis.Then the ELANDv2 algorithm of CASAVA 1.6 software is applied to identify point mutations and small insertions and deletions.Remove the known polymorphism recorded in dbSNP from analysis.As Jones et al., Science, 330:228-31 (2010) described in, filter and visually estimate potential somatic mutations.

[0246] Construction of tandem mini-gene library

[0247] Identification of nonsynonymous mutations in melanoma samples from whole exome sequencing data. A tandem minigene construct encoding a polypeptide containing the six identified mutant amino acid residues (12 amino acids on either side) flanked at its N- and C-termini was synthesized (Integrated DNA Technologies, Coralville, Iowa) and then cloned into the pcDNA3.1 expression vector using the IN-FUSION Advantage PCR Cloning Kit (Clontech) according to the manufacturer's instructions.

[0248] IFN-γ ELISPOT assay

[0249] The responses to tumor cell lines and peptide-pulsed target cells were quantified in an IFN-γ ELISPOT assay using 96-well PVDF membrane filter plates (EMD Millipore, Billerica, MA) coated with 15 μg / mL of the monoclonal anti-IFN-γ antibody 1D1K (Mabtech, Inc., Cincinnati, OH). Bound cytokines were detected using 1 μg / ml of the biotinylated anti-IFN-γ antibody 7-B6-1 (Mabtech). HEK293 cells expressing HLA-A*0201, HLA-A*0205, or HLA-C*0701 were pulsed with peptides for 2 hours at 37°C. The following peptides were used: MART-1: AAGIGILTV (SEQ ID NO: 54), mutated KIF2C: RLFPGLTIKI (SEQ ID NO: 55), mutated POLA2: TRSSGSHFVF (SEQ ID NO: 56). T cells were co-cultured with target cells or medium containing 50 ng / ml PMA plus 1 μM ionomycin (PMA / I) overnight. 5 The number of T cells.

[0250] Example 8

[0251] This example demonstrates that TIL 2359 recognizes mutant antigens evaluated by minigene library screening.

[0252] The responsiveness of TIL 2359 was evaluated using TMG constructs generated based on nonsynonymous mutations identified by exome analysis of tumor and normal DNA. Each TMG construct encodes up to 6 separate minigene fragments corresponding to mutant codons flanked on both sides by 12 additional codons present in the normal gene product. An example is shown in Figure 5A.

[0253] Based on the exome DNA sequence containing non-synonymous point mutations identified from Mel 2359, COS-7 cells were transiently transfected individually with one of the 12 tandem minigenes encoding 71 minigenes. These COS-7 cells were also co-transfected with HLA-A*0205, the major HLA restriction element for autologous tumor cells recognized by this TIL. These transfectants were co-cultured with TIL 2359, resulting in the recognition of one of the 12 TMG constructs (RJ-1) (Figure 5B). As shown in Figure 5A, RJ-1 encodes mutant fragments of EPHB2, KIF2C, SLC44A5, ABCA4, DENND4B and EPRS genes. Subsequently, 6 RJ-1 variant constructs were formed, each of which encodes WT instead of the mutant residue present in one of the 6 minigenes (Figure 5C). TIL 2359 recognized COS-7 cells co-transfected with HLA-A*0205 plus 5 of the 6 individually transfected RJ-1 variants, but not the variant encoding the WT KIF2C sequence, indicating that the minigene encodes a mutant epitope recognized by TIL 2359 (Figure 5C). To further test this observation, COS-7 cells were co-transfected with WT or mutant full-length KIF2C cDNA transcripts amplified from Mel 2359, together with HLA-A*0101, HLA-A*0201, or HLA-A*0205 cDNAs. Co-culture experiments showed that TIL 2359 T cells recognized COS-7 cells co-transfected with mutant but not wild (WT) KIF2C gene products in an HLA-A*0205-restricted manner (Figure 5D).

[0254] The mutated KIF2C coding region contains a single C to A transversion at nucleotide 46, which results in the replacement of alanine with threonine at position 16 in the native protein KIF2C. Exome sequencing results showed that the DNA from Mel 2359 corresponded only to the mutant residue at position 46 instead of the normal residue, which was confirmed by direct Sanger sequencing of Mel 2359 DNA, indicating loss of heterozygosity at this locus. To identify the mutant KIF2C epitope recognized by TIL 2359, a peptide containing the KIF2C mutation that was predicted to bind to HLA-A*0205 with high affinity was synthesized (Hoof et al., Immunogenetics, 61: 1-13 (2009)) and pulsed to HEK293 cells stably expressing HLA-A*0205 (Table 21). HEK293-A*0205 cells pulsed with decamers corresponding to residues 10-19 stimulated high levels of IFN-γ release from TIL 2359T cells, and the peptide was recognized at a minimum concentration of 0.1 nM. In contrast, the corresponding WT peptide did not induce significant IFN-γ release at concentrations up to 10 μM (Figure 5E).

[0255] Table 21

[0256]

[0257] Example 9

[0258] This example demonstrates that TIL 2591 recognizes mutant antigens identified by mini-library screening.

[0259] Based on the exome DNA sequence containing non-synonymous point mutations identified from Mel 2591, mutated T cell antigens recognized by TIL 2591 were identified by synthesizing 37 TMG constructs encoding 217 minigenes. TIL 2591 recognizes autologous tumor cells in the context of multiple HLA restriction elements. Therefore, HEK293 cell lines stably expressing each of the six MHC class I HLA molecules isolated from Mel 2591 were transiently transfected individually with the 37 TMG constructs and subsequently co-cultured with TIL 2591 overnight. Preliminary results show that TIL 2591 recognizes HLA-C*0701 transiently transfected with the minigene DW-6 + HEK293 cells (HEK293-C*0701) were resistant to WT POLA2 but failed to respond significantly to other minigene constructs (Figure 6A). Each of the six individual mutant minigenes in the DW-6 tandem construct (Figure 6B) was then individually reverted to the WT sequence (Figure 6C). Evaluation of the response to the WT variants showed that TIL 2591 recognized COS-7 cells transfected with each of the DW-6 variants in addition to the construct encoding the WT POLA2 fragment (Figure 6C). To test these findings, COS-7 cells were transfected with cDNA constructs of WT or mutated full-length POLA2, together with cDNAs of HLA-C*0401, HLA-C*0701, or HLA-C*0702. TIL 2591 T cells only recognized target cells transfected with HLA-C*0701 plus mutant POLA2 constructs, but not the corresponding WT transcripts (Figure 6D). A single C to T transition at nucleotide 1258 in the POLA2 coding region results in a replacement of phenylalanine with leucine at position 420 of the WT POLA2 protein. Sanger sequencing showed that both genomic DNA and cDNA from Mel 2591 RNA contained both the WT and the mutant nucleotide at position 1258, and thus the genomic DNA isolated from PBMCs of patient 2591 corresponded to the WT sequence, indicating that this represented a heterozygous somatic mutation in Mel 2591 cells.

[0260] The HLA-C*0701 binding algorithm was then used to identify candidate POLA2 peptides that overlapped with the mutated leucine residue at position 420 (Table 22). The co-culture results showed that HLA-C*0701 pulsed with decamers corresponding to residues 413-422 of mutant POLA2 + HEK293 cells stimulated IFN-γ release from TIL 2591T cells at a minimum concentration of 10 nM. In contrast, the corresponding WT peptide did not induce significant IF-γ release at concentrations up to 10 μM ( FIG. 6E ).

[0261] Table 22

[0262]

[0263] The proportion of T cells in TIL2359 that recognize mutant KIF2C and TIL2591 that recognize mutant POLA2 was then estimated using an IFN-γ enzyme-linked immunospot (ELISPOT) assay. + When TIL 2591 was used to target the HLA-A2 restricted MART-1 epitope, approximately 2,000 spots were generated (per 100,000 T cells), which is similar to that observed in response to autologous melanoma (Table 23). TIL 2591 generated more than 7,000 spots in response to the HLA-A2 restricted MART-1 epitope, while only a portion of T cells responded to the HLA-C*0701 restricted mutated POLA2 epitope (Table 23).

[0264] Table 23

[0265]

[0266] Example 10

[0267] This example demonstrates methods for identifying T cells reactive to mutant antigens present in gastrointestinal (GI) cancers identified by minigene library screening.

[0268] Whole exome sequencing is performed on metastatic lesions from GI cancer patients to determine mutations. Next, a small gene construct encoding each mutation is generated and transfected into autologous APCs to allow processing and presentation of all mutations expressed by the tumor. These APCs are then co-cultured with tumor infiltrating lymphocytes (TILs), and T cell reactivity to mutations is determined by IFN-γ ELISPOT and upregulation of 4-1BB and OX40 by flow cytometry.

[0269] In a patient with colon cancer, the mutational reactivity of 119 mutations was evaluated. It was found that several (but not all) TIL cultures contained highly variable proportions of CD8+ T cells that specifically recognized the mutation (67F→V) in CASP8. When further expanded in vitro, these mutation-reactive CD8+ T cells significantly outnumbered other cells in the culture. The patient was administered 40.3×10 9 TILs (which were estimated to contain approximately 0.31% (approximately 127 million) mutation-reactive cells) did not result in a clinical response at the first follow-up approximately 6 weeks after administration of the cells. The patient died approximately 6 weeks later. Without being bound by a particular theory or mechanism, it is believed that any one or more of the very late stage of the disease prior to treatment, the poor overall condition of the patient, and the patient's poor tolerance to lymphodepleting chemotherapy prior to administration of adoptive cell therapy may be factors that contributed to the patient's death. TCRs reactive to mutant CASP8 are isolated from TILs, and T cells transduced to express the TCRs are reactive to DCs pulsed with mutant CASP8.

[0270] In another patient with rectal cancer, mutation reactivity of 155 mutations was assessed. At least 3 different mutation reactivities were found, 2 of which included CD8+ T cell responses and 1 CD4+ response. Administration of mutation-reactive TILs to the patient initially resulted in mixed responses at approximately 1.5 months after treatment, but the patient subsequently developed progressive disease approximately 3.5 months after treatment. Potential mutation-reactive TCRs were isolated from CD4+ TILs and CD8+ TILs.

[0271] In the third patient (cholangiocarcinoma), no T cell reactivity was detected for the 38 mutations tested. For this patient, the "mutation call" threshold was lowered, and another 125 putative mutations were evaluated. The "mutation call" is an arbitrary threshold value set when the sequence is identified as a mutation using bioinformatics. In this case, as the first pass, the threshold is relatively high (e.g., providing a high level of confidence that the identified mutation is a true mutation). The threshold is then reduced, thereby providing a lower level of confidence that the identified mutation is a true mutation, however, the possibility that the identified mutation is a true mutation still exists.

[0272] These data suggest that the ability of the human immune system to mount a T-cell response against somatic mutations may not be a rare event in metastatic gastrointestinal cancer. The study is ongoing.

[0273] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0274] Unless otherwise specified herein or clearly contradicted by context, the terms "a / an" and "the" and "at least one" and similar expressions used in the context of describing the present invention (especially in the context of the appended claims) will be interpreted as covering both the singular and the plural. Unless otherwise specified herein or clearly contradicted by 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 as referring to one item (A or B) selected from the listed items, or any combination of two or more listed items (A and B). Unless otherwise specified, the terms "comprising", "having", "including" and "containing" will be interpreted as open-ended terms (i.e., meaning "including, but not limited to"). Unless otherwise specified herein, the numerical ranges mentioned herein are intended only to be used as a shorthand method of referring individually to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually listed herein. Unless otherwise specified herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. No language in the specification should be construed as indicating any non-claimed element necessary to implement the present invention.

[0275] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. It will be apparent to those of ordinary skill in the art, upon reading the above description, that various modifications of these preferred embodiments will be apparent. The inventors anticipate that those skilled in the art will adopt these modifications where appropriate, and the inventors expect that the present invention will be implemented in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradictory to the context, any combination of the above elements in all possible variations is included in the present invention.

Claims

1. A method for preparing a pharmaceutical composition suitable for parenteral administration, the pharmaceutical composition comprising (a) a pharmaceutically acceptable carrier and (b) an isolated population of T cells expressing a T cell receptor (TCR) or an antigen binding portion thereof having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in nucleic acid from cancer cells of a patient, wherein the cancer is selected from the group consisting of colon cancer and rectal cancer, 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; Identifying T cells with antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation presented in the context of a major histocompatibility complex (MHC) class I molecule expressed by the patient; Selecting autologous T cells that (a) are co-cultured with autologous APCs presenting the mutated amino acid sequence and (b) have antigenic specificity for the mutated amino acid sequence presented in the context of MHC molecules expressed by the patient; isolating a nucleotide sequence encoding a TCR or an antigen binding portion thereof from a selected autologous T cell, wherein the TCR or the antigen binding portion thereof has antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation; introducing a nucleotide sequence encoding the isolated TCR or antigen binding portion thereof into peripheral blood mononuclear cells (PBMCs) to obtain cells expressing the TCR or antigen binding portion thereof, wherein the obtained cells provide any one or more of greater in vivo persistence, proliferation, and anti-tumor activity compared to effector cells of the isolated TCR or antigen binding portion thereof in a colon or rectal tumor of a patient; and The obtained T cell population is formulated with a pharmaceutically acceptable carrier for parenteral administration.

2. The method of claim 1, wherein inducing the patient's autologous APCs to present the mutated amino acid sequence comprises pulsing the APCs with a peptide or a peptide library comprising the mutated amino acid sequence, each peptide in the library comprising a different mutated amino acid sequence.

3. The method of claim 1, wherein inducing the patient's autologous APCs to present the mutated amino acid sequence comprises introducing a nucleotide sequence encoding the mutated amino acid sequence into the APCs.

4. The method of claim 3, wherein the nucleotide sequence introduced into the autologous APC is a tandem minigene (TMG) construct, each minigene comprising a different gene, each gene comprising a cancer-specific mutation encoding a mutated amino acid sequence.

5. The method of any one of claims 1 to 4, further comprising obtaining multiple fragments of a colon or rectal tumor from the patient, co-culturing autologous T cells from each of the multiple fragments with autologous APCs presenting the mutated amino acid sequence, and evaluating the antigen specificity of T cells from each of the multiple fragments to the mutated amino acid sequence.

6. The method of any one of claims 1 to 4, wherein selecting autologous T cells having antigen specificity for the mutated amino acid sequence comprises selectively cultivating autologous T cells having antigen specificity for the mutated amino acid sequence.

7. The method of any one of claims 1-4, wherein selecting autologous T cells having antigenic specificity for the mutated amino acid sequence comprises selecting T cells expressing any one or more of the following: programmed cell death factor 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-3 (TIM-3), 4-1BB, OX40, and CD107a.

8. The method of any one of claims 1-4, wherein selecting autologous T cells having antigenic specificity for a mutated amino acid sequence comprises selecting T cells that: (i) secrete a greater amount of one or more cytokines when co-cultured with APCs presenting the mutated amino acid sequence than the amount of one or more cytokines secreted by a negative control, or (ii) wherein at least twice as many T cells secrete the one or more cytokines when co-cultured with APCs presenting the mutated amino acid sequence than the number of negative control T cells secreting the one or more cytokines.

9. The method of claim 8, wherein the one or more cytokines comprise: 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.

10. The method of any one of claims 1-4, wherein identifying one or more genes in the nucleic acid of a cancer cell comprises sequencing the whole exome, whole genome, or whole transcriptome of the cancer cell.

11. The method of any one of claims 1-4, further comprising expanding the number of PBMCs expressing the TCR or antigen binding portion thereof.

12. Use of the pharmaceutical composition prepared according to the method according to any one of claims 1 to 11 in the preparation of a medicament for treating or preventing cancer in a mammal, wherein the cancer is selected from colon cancer and rectal cancer.

13. The use according to claim 12, wherein the PBMCs are autologous to the patient.

14. The use of claim 12, wherein the PBMCs are allogeneic to the patient.

15. The method of any one of claims 1-4, further comprising identifying T cells having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation presented in the context of an MHC class II molecule expressed by the patient.

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