Methods of isolating t cells with antigenic specificity for cancer-specific mutations

Abstract

Disclosed are methods of isolating T cells having antigenic 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 patient cancer cell, each gene containing a cancer-specific mutation encoding a mutated amino acid sequence; inducing autologous APCs of the patient to present the mutated amino acid sequence; co-culturing autologous T cells of the patient with the autologous APCs presenting the mutated amino acid sequence; and selecting the autologous T cells. Also disclosed are related methods of making a cell population, a plurality of cell populations, a pharmaceutical composition, and a method of treating or preventing cancer.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201480082922.X, the entire contents of which are incorporated herein by reference.

[0002] By incorporating electronic submission materials

[0003] Incorporated herein by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing filed concurrently with this application and identified as follows: a 29,568-byte ASCII (text) file named “718292ST25.TXT” dated September 30, 2014. Background Technology

[0004] Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) can produce positive clinical responses in some cancer patients. However, obstacles remain to the successful application of ACT in the broad treatment of cancer and other diseases. For example, T cells that specifically recognize cancer antigens may be difficult to identify and / or isolate from patients. Therefore, improved methods for obtaining cancer-reactive T cells are needed. Invention Overview

[0005] One embodiment of the present invention provides a method for isolating T cells that are antigen-specific to a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in the nucleic acids 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; and selecting (a) autologous T cells that are antigen-specific to the mutated amino acid sequence presented in the presence of major histocompatibility complex (MHC) molecules expressed in the patient, to provide isolated T cells that are antigen-specific to the mutated amino acid sequence encoded by the cancer-specific mutation.

[0006] Another embodiment of the present invention provides a method for preparing a population of T cells with antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying genes of 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 (a) autologous T cells co-cultured with the autologous APCs presenting the mutated amino acid sequence and (b) autologous T cells that are antigen specific to the mutated amino acid sequence presented in the presence of MHC molecules expressed by the patient; and expanding the number of the selected autologous T cells to obtain a population of T cells with antigen specificity to the mutated amino acid sequence encoded by the cancer-specific mutation.

[0007] Other embodiments of the present invention provide related cell populations, pharmaceutical compositions, and methods for treating or preventing cancer. Attached Figure Description

[0008] Figure 1 A shows the results of co-culturing dendritic cells (DCs) transfected with OKT3 or with green fluorescent protein (GFP) RNA or a designated tandem small gene (TMG) construct with 3737-TIL for 20 hours, measured by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay. 3 A curve showing the number of spots per (1e3) cells. ">" indicates the number of spots per 1×10⁻⁶ cells. 3 Each cell has more than 500 spots. Mock transfected cells are treated with only the transfection reagent, without the addition of nucleic acids.

[0009] Figure 1 B shows the gene associated with OKT3 or with GFP RNA, TMG-1, or the specified wild-type (wt) gene. ALK, CD93 ERBB2IP, FCER1A, GRXCR1, KIF9, NAGS, NLRP2 or RAC3 A graph showing the percentage of OX40+ CD4+ 3737-TILs after co-culturing transfected dendritic cells (DCs). 。 The cells that were simulated for transfection were treated with transfection reagents only, without the addition of nucleic acids.

[0010] Figure 2A-2C shows the percentage of 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) (which are not pre-incubated with anything) or designated HLA-blocking antibodies (targeting MHC-I, MHC-II, HLA-DP, HLA-DQ, or HLA-DR) as measured by IFN-γ ELISPOT assay at 20 hours. 3 A curve (AC) showing the number of spots in (1e3) cells.

[0011] Figure 2 D shows the percentage of 3737-TILs co-cultured with autologous DQ-0301 / -0601 B cells (gray bars) pulsed overnight with DMSO, mutated ALK, or mutated ERBB2IP 25-AA long peptide (black bars) or partially matched allogeneic EBV-B cells at the HLA-DQ 05 / 0601 locus (unshaded bars) at the HLA-DQ-0201 / 0301 locus (unshaded bars) at 20 hours, measured by IFN-γ ELISPOT assay. 3 A curve showing the number of spots in (1e3) cells. ETGHLENGNKYPNLE (SEQ ID NO: 53); Figure 2E shows the results for the use of the mut ERBB2IP 25-AA peptide TSFLSINSKEETGHLENGNKYPNLE (SEQ ID NO: 73) or the specified truncated 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), TSFLSINSKEETGHLENGN (SEQ ID NO: 36), TSFLSINSKEETGHLENGNKY (SEQ ID NO: 37) or TSFLSINSKEETGHLENGNKYPN (SEQ ID NO: 38). 38) 3737-TILs co-cultured with autologous B cells pulsed overnight, measured at 20 hours using the IFN-γ ELISPOT assay at a concentration of 1 × 10⁻⁶ cells / mL. 3 A curve showing the number of spots in (1e3) cells.

[0012] Figure 3 A plot shows the percentage of various TCR Vβ clones in 3737-TILs as measured by flow cytometry with gates on live CD4+ (shaded) or CD8+ (unshaded) T cells.

[0013] Figure 3 B shows a graph of IFN-γ levels (pg / ml) detected in patient 3737 serum samples measured before and after adoptive cell transfer of 3737-TIL on day 0 (indicated by the arrow). Error bars are the standard deviations of the mean (SEM).

[0014] Figure 3 C shows a graph of total tumor burden (circle) (measured as a percentage of pre-treatment baseline) or tumor burden in the lungs (triangle) or liver (square) at a specified number of months relative to cell metastasis on day 0 (indicated by arrows).

[0015] Figure 3 D shows a graph illustrating the percentage of various TCR Vβ clones in CD4+ Vβ22- OX40+ 3737-TIL as measured by flow cytometry.

[0016] Figure 4 A and 4B show the patient's blood (rounds), tumors before cell transfer (diamonds), and various tumors after cell transfer (Tu-1-Post (squares), Tu-2-Post (squares), and Tu-3737-TIL) at different time points before and after adoptive cell transfer. ) and Tu-3-Post( In the figure, the frequencies of the two ERBB2IP-mutation-specific TCRβ-CDR3 clonal types Vβ22+ (A) and Vβ5.2+ (B) are shown. The shaded bars indicate the frequencies of the two ERBB2IP-mutation-specific TCRβ-CDR3 clonal types Vβ22+ (A) and Vβ5.2+ (B) in the metastatic cells (3737-TIL). “X” indicates “not detected”.

[0017] Figure 4 C illustrates various tumors before (TU-Pre), after (Tu-1-post, Tu-2-post, and Tu-3-post) 3737-TIL (T cells) and adoptive cell metastasis. ERBB2IP Compared to ACTB The graph represents the expression of the value.

[0018] Figure 4 D shows the total tumor burden (circle) (measured as a percentage of pre-treatment baseline) or the tumor burden in the lungs (triangle) or liver (square) at a specified number of months relative to cell metastasis (indicated by arrows).

[0019] Figure 5A is a schematic diagram of an example of a tandem small gene (TMG) construct encoding a polypeptide with six identified mutant amino acid residues flanking its N-terminus and C-terminus (12 amino acids on each side). The mutant KIF2C sequence is DSSLQARLFPGLTIKIQRSNGLIHS (SEQ ID NO: 57).

[0020] Figure 5B shows the results of autologous melanocytes or HLA-A therapy. A graph showing the IFN-γ levels (pg / mL) secreted by TIL 2359T cells co-cultured overnight with COS-7 cells co-transfected with 0205 and TMG constructs RJ-1 (structure 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.

[0021] Figure 5C shows the comparison with HLA-A. A graph showing the IFN-γ levels (pg / mL) secreted by TIL 2359 cells co-cultured with COS-7 cells transfected with the 0205 and RJ-1 variants (where the gene indicated as "wt" in the table was converted back to the WT sequence). KIF2C The WT sequence is DSSLQARLFPGLAIKIQRSNGLIHS (SEQ ID NO: 65).

[0022] Figure 5D shows the use of an empty carrier. KIF2C WT or mutation KIF2C cDNA constructs of HLA and HLA (identifying each shaded bar from left to right: HLA-A) 0101 (non-shaded bar), HLA-A 0201 (gray bar) or HLA-A A graph showing the IFN-γ level (pg / mL) secreted by TIL 2359 cells co-cultured with COS-7 cells transfected with 0205 (black bar).

[0023] Figure 5E shows the results of using KIF2C at various concentrations (μM). 10-19 WT (RLFPGLAIKI; SEQ ID NO: 58) (bottom line in the figure) or mutated KIF2C 10-19 (RLFPGLTIKI; SEQ ID NO: 59) (Top line in figure) Stable expression of HLA-A pulses A graph showing the IFN-γ level (pg / mL) secreted by TIL 2359 T cells co-cultured overnight with HEK293 cells from cell line 0205.

[0024] Figure 6A shows stable HLA-C expression in autologous melanocytes or in TMG constructs transfected with an empty vector or selected from DW-1 to DW-37. A graph showing the IFN-γ level (pg / mL) secreted by TIL 2591 T cells co-cultured with HEK293 cells from cell line 0701.

[0025] Figure 6B shows a schematic diagram of the structure of the TMG construct DW-6. The mutated POLA2 sequence is TIIEGTRSSGSHFVFVPSLRDVHHE (SEQ ID NO: 64).

[0026] Figure 6C shows the comparison with HLA-C. A graph showing the IFN-γ levels (pg / mL) secreted by TIL 2591 cells co-cultured with COS-7 cells transfected with 0701 and DW-6 variants (where the gene indicated as "wt" in the table was converted back to the WT sequence). POLA2 The WT sequence is TIIEGTRSSGSHLVFVPSLRDVHHE (SEQ ID NO: 66).

[0027] Figure 6D shows the use of an empty carrier. POLA2 WT or mutation POLA2 cDNA constructs of HLA and HLA cDNA constructs (identifying individual bars from left to right: HLA-C) 0401 (non-shaded bar), HLA-C 0701 (gray bar) or HLA-C A graph showing the IFN-γ level (pg / mL) secreted by TIL 2591 cells co-cultured with COS-7 cells transfected with 0702 (black bar).

[0028] Figure 6E shows the results of using POLA2 at various concentrations (μM). 413-422 WT (TRSSGSHLVF; SEQ ID NO: 67) (bottom line in the figure) or mutated POLA2 413-422 (TRSSGSHFVF; SEQ ID NO: 68) (Top line in figure) Stable expression of HLA-C pulse A graph showing the IFN-γ level (pg / mL) secreted by TIL 2591 T cells co-cultured overnight with HEK293 cells from cell 0701.

[0029] Figure 7 A-7F consists of computed tomography (CT) scans of the lungs of patient 3737 obtained before the second administration of mutant reactive cells (AC) and six months later (DF). Arrows point to cancerous lesions. Invention Details

[0030] One embodiment of the present invention provides a method for isolating T cells that are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations. The present invention offers numerous advantages. For example, the method of the present invention can rapidly assess a large number of mutations restricted by all MHC molecules of a patient at once, which can identify the full composition of the patient's mutation-responsive T cells. Furthermore, by distinguishing between immunogenic cancer mutations and (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 T cells. In addition, the present invention can provide T cells that are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations unique to the patient, thereby providing a “personalized” T cell population that can be used to prepare cells for adoptive cell therapies (such as for treating or preventing cancer in patients). The method of the present invention also avoids the technical biases inherent in conventional methods for identifying cancer antigens, such as those using cDNA libraries, and can be more time- and labor-saving than those methods. For example, the method of the present invention can select mutant reactive T cells without co-culturing said T cells with tumor cell lines that may be difficult to generate (especially, for example, epithelial carcinoma). Not limited to any particular theory or mechanism, the method of the present invention is believed to identify and isolate such T cells that target and destroy cancer cells while minimizing or eliminating destruction of normal non-cancer cells, thereby reducing or eliminating toxicity. Therefore, the present invention can also provide such T cells that successfully treat or prevent cancers, such as cancers that do not respond to other types of treatment (e.g., chemotherapy, surgery, or radiation alone).

[0031] The method may include identifying one or more genes in the nucleic acids of a patient's cancer cells, each gene containing a cancer-specific mutation encoding a mutated amino acid sequence. Cancer cells can be obtained from any body sample derived from a patient who has or is expected to have a tumor or cancer cells. The body sample can be any tissue sample, such as blood, tissue samples obtained from a primary tumor or tumor metastases, or any other sample containing a tumor or cancer cells. The nucleic acids of the cancer cells can be DNA or RNA.

[0032] To identify cancer-specific mutations, the method may further include sequencing the nucleic acids (such as DNA or RNA) of normal non-cancerous cells and comparing the sequences of cancer cells with those of normal non-cancerous cells. Normal non-cancerous cells can be obtained from patients or different individuals.

[0033] Cancer-specific mutations can be any mutation in any gene that encodes the mutated amino acid sequence (also known 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 this invention include missense, nonsense, insertion, deletion, duplication, frameshift, and amplification mutations. In one embodiment of the invention, the method includes identifying at least one gene containing a cancer-specific mutation encoding the mutated amino acid sequence. However, the number of genes containing such cancer-specific mutations that can be identified using the method of the present 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). Similarly, in one embodiment of the invention, the method includes identifying at least one cancer-specific mutation encoding an amino acid sequence that has undergone a mutation. However, the number of such cancer-specific mutations that can be identified using the methods of the present 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 has been identified, the cancer-specific mutation may be located in the same gene or different genes.

[0034] In one embodiment, identifying one or more genes in the nucleic acids of cancer cells includes sequencing the entire exome, whole genome, or entire transcriptome of the cancer cells. Sequencing can be performed in any suitable manner known in the art. Examples of sequencing technologies that can be used in the methods of the present invention include next-generation sequencing (NGS) (also known as “mass parallel sequencing”) or third-generation sequencing. NGS refers to high-throughput DNA sequencing technology based on non-Sanger methods. Using NGS, millions or billions of DNA strands can be sequenced in parallel, resulting in significantly greater throughput and minimizing the need for fragment cloning methods commonly used for genome sequencing using Sanger methods. In NGS, nucleic acid templates can be read randomly 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, for example, in about 1 to 2 weeks, preferably in about 1 to 7 days, or most preferably in less than about 24 hours. Several commercially available or described in the literature NGS platforms can be used in the methods of the present invention, for example those described in the following literature: Zhang et al., J. Genet. Genomics , 38(3): 95-109 (2011) and Voelkerding et al., Clinical Chemistry , 55:641-658 (2009).

[0035] Non-limiting examples of NGS technologies and platforms include: sequencing-by-synthesis (also known as “pyrosequencing”) (as implemented, for example, using the GS-FLX 454 genome sequencer, 454 Life Sciences (Branford, CT), the ILLUMINA SOLEXA genome analyzer (Illumina Inc., San Diego, CA), or the ILLUMINA HISEQ 2000 genome analyzer (Illumina), or as described, for example, Ronaghi et al. Science281(5375):363-365(1998)), sequencing-while-ligating (as implemented, e.g., using the SOLID platform (Life Technologies, Carlsbad, CA) or the POLONATOR G.007 platform (Dover System, Salem, NH)), single-molecule sequencing (as implemented, e.g., 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, e.g., using the GRIDON platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nanopore sequencing (HANS) platform developed by Nabsys (Providence, RI), and ligase-based DNA sequencing platforms with DNA nanosphere (DNB) technology (called probe-anchor ligation (cPAL)), electron microscopy-based techniques for single-molecule sequencing, and ion semiconductor sequencing.

[0036] The method may include inducing the patient's autologous antigen-presenting cells (APCs) to present a mutated amino acid sequence. The APC may include any cell that presents a peptide fragment of a protein that binds to a major histocompatibility complex (MHC) molecule on its cell surface. The APC may include any one or more of, for example, macrophages, dendritic cells (DCs), Langerhans cells, B lymphocytes, and T cells. Preferably, the APC is a dendritic cell (DC). By using autologous APCs from the patient, the method of the present invention can advantageously identify T cells that have antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation presented in the context of a patient-expressed MHC molecule. The MHC molecule may be any MHC molecule expressed by the patient, 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 method of the present invention can advantageously identify mutated amino acid sequences presented in the context of any MHC molecule expressed by a patient, without using, for example, epitope prediction algorithms 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 mutant reactive T cells (e.g., incomplete MHC tetramer groups). Therefore, in one embodiment of the invention, the method of the present invention advantageously identifies mutated amino acid sequences presented in the context of any MHC molecule expressed by a patient, and is not limited to any specific MHC molecule. Preferably, the autologous APC is an antigen-negative autologous APC.

[0037] Any suitable method known in the art can be used to induce a patient's autologous APCs to present a mutated amino acid sequence. In one embodiment of the invention, inducing a patient's autologous APCs to present a mutated amino acid sequence includes pulsating the autologous APCs with a peptide or peptide library containing the mutated amino acid sequence, wherein each peptide in the peptide library contains a different mutated amino acid sequence. Each mutated amino acid sequence in the peptide library may be encoded by a gene containing a cancer-specific mutation. In this regard, the autologous APCs can be cultured with a peptide or peptide library containing the mutated amino acid sequence in such a manner that the APCs internalize the peptides and display the mutated amino acid sequence bound to an MHC molecule on the cell membrane. In one embodiment, in identifying more than one gene, each containing a cancer-specific mutation encoding the mutated amino acid sequence, the method may include pulsating the autologous APCs with a peptide library, wherein each peptide in the peptide library contains a different mutated amino acid sequence. Methods for pulsating APCs are known in the art and described, for example, in Solheim (Ed.). Antigen Processing and Presentation Protocols (Methods in Molecular Biology)Human Press, (2010). The peptide used to pulse APC may include mutated amino acids encoded by cancer-specific mutations. The peptide further includes any suitable number of consecutive amino acids from an endogenous protein encoded by an identified gene on each carboxyl and amino side of the mutated amino acid. The number of consecutive amino acids from the endogenous protein 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 the endogenous protein on each side of the mutated amino acid.

[0038] In one embodiment of the invention, inducing a patient's autologous APCs to present a mutated amino acid sequence includes introducing a nucleotide sequence encoding the mutated amino acid sequence into the APC. Introducing the nucleotide sequence into the APC causes the APC to express and display the mutated amino acid sequence bound to an MHC molecule on the cell membrane. The nucleotide sequence encoding the mutated amino acid can be RNA or DNA. The introduction of the nucleotide sequence into the APC can be performed in any of a variety of different manners known in the art, as described, for example, by Solheim et al. (ibid.). Non-limiting examples of techniques that can be used to introduce the nucleotide sequence into the APC include transformation, transduction, transfection, and electroporation. In embodiments identifying more than one gene, 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 groups of autologous APCs. In this respect, multiple groups of autologous APCs can be obtained, each group expressing and displaying a different mutated amino acid sequence.

[0039] In embodiments for identifying more than one gene, each 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 including 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, each flanking the mutation being linked with any suitable number of consecutive amino acids from an endogenous protein encoded by the identified gene. 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 an MHC molecule). In one embodiment, the method may include preparing more than one TMG construct, each construct encoding a different group of mutated amino acid sequences encoded by different genes, and introducing each TMG construct into a different autologous APC population. In this respect, multiple autologous APC populations can be obtained, each population expressing and displaying the mutated amino acid sequences encoded by different TMG constructs.

[0040] The method may include culturing the patient's autologous T cells with autologous APCs presenting a 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 cell and may be at any developmental stage, including but not limited to: CD4+ / CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating cells (e.g., tumor-infiltrating lymphocytes (TILs)), peripheral blood T cells, memory T cells, naive 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 such that the T cells encounter a mutated amino acid sequence presented by the APC in such a manner that the autologous T cells specifically bind to and immunely recognize the mutated amino acid sequence presented by the APC. In one embodiment of the invention, autologous T cells are co-cultured in direct contact with autologous APCs.

[0041] The method may include selecting autologous T cells that: (a) are co-cultured with autologous APCs presenting a mutated amino acid sequence, and (b) are antigen-specific to the mutated amino acid sequence presented in the presence of MHC molecules expressed in the patient. As used herein, the phrase "antigen-specific" means that the autologous T cells can specifically bind to and immunely recognize a mutated amino acid sequence encoded by a cancer-specific mutation. The selection may include identifying T cells that are antigen-specific to the mutated amino acid sequence and separating them from T cells that are not antigen-specific to the mutated amino acid sequence. Autologous T cells that are antigen-specific to the mutated amino acid sequence may be selected in any suitable manner. In one embodiment of the invention, the method includes expanding the number of autologous T cells, for example, by co-culturing with T cell growth factors such as interleukin (IL)-2 or IL-15 prior to selection of autologous T cells, or as described herein with respect to other aspects of the invention. In one embodiment of the 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 prior to selection of autologous T cells.

[0042] For example, when autologous T cells are co-cultured with APCs presenting mutated amino acid sequences, T cells that are antigen-specific to the mutated amino acid sequence may express any one or more of various T cell activation markers, which can be used to identify T cells that are antigen-specific to the mutated amino acid sequence. 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 invention, selecting autologous T cells that are antigen-specific to the mutated amino acid sequence includes selecting T cells that express 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 marker expression using any of a variety of techniques known in the art, such as fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS), as described, for example, by Turcotte et al. Clin. Cancer Res. , 20(2): 331-43 (2013) and Gros et al., J. Clin. Invest ., 124(5):2246-59 (2014).

[0043] In another embodiment of the invention, selecting autologous T cells that are antigen-specific to the mutated amino acid sequence includes 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) secrete at least twice as many of the number of negative control T cells that secrete one or more cytokines when co-cultured with APCs presenting the mutated amino acid sequence. The one or more cytokines may include any cytokines whose secretion by T cells is a characteristic of T cell activation (e.g., T cell receptors (TCRs) expressed by T cells that specifically bind to and immunely recognize the mutated amino acid sequence). Non-limiting examples of cytokines (where 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.

[0044] For example, if T cells secrete at least twice the amount of IFN-γ compared to the amount secreted by a negative control when co-cultured with: (a) an antigen-negative APC pulsed with a concentration of a peptide containing 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 in which a nucleotide sequence encoding the mutated amino acid sequence has been introduced, the autologous T cells can be considered to have “antigen specificity” to the mutated amino acid sequence. Negative controls can be, for example, autologous T cells (e.g., derived from peripheral blood mononuclear cells (PBMCs)) co-cultured with: (a) an antigen-negative APC pulsed with the same concentration of an unrelated 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) an APC in which a nucleotide sequence encoding the unrelated peptide sequence has been introduced. If, compared to a negative control (e.g., the negative control described above), the T cells secrete a greater amount of IFN-γ when co-cultured with an antigen-negative APC pulsed with a higher concentration of a peptide containing the mutated amino acid sequence, the autologous T cells may also exhibit “antigen specificity” for the mutated amino acid sequence. IFN-γ secretion can be measured by methods known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0045] Optionally or additionally, autologous T cells may be considered to have “antigen specificity” to the mutated amino acid sequence if, when co-cultured with at least twice the number of negative control T cells secreting IFN-γ compared to the number of negative control T cells secreting IFN-γ: (a) antigen-negative APCs pulsed with a peptide containing a mutated amino acid sequence at a certain concentration, or (b) APCs wherein a nucleotide sequence encoding the mutated amino acid sequence has been introduced. The peptide concentration and negative control may be as described herein with respect to other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art (e.g., ELISPOT).

[0046] T cells that are antigen-specific to the mutated amino acid sequence can (1) express any one or more T cell activation markers described herein and (2) secrete more of one or more cytokines as described herein. In embodiments of the present invention, T cells that are antigen-specific to the mutated amino acid sequence can express any one or more T cell activation markers without secreting more of one or more cytokines, or can secrete more of one or more cytokines without expressing any one or more T cell activation markers.

[0047] In another embodiment of the invention, selecting autologous T cells that are antigen-specific to the mutated amino acid sequence includes selectively culturing autologous T cells that are antigen-specific to the mutated amino acid sequence. In this regard, the method may include co-culturing autologous T cells with autologous APCs in a manner that favors the growth of T cells that are antigen-specific to the mutated amino acid sequence compared to T cells that are not antigen-specific to the mutated amino acid sequence. Therefore, a population of T cells is provided in which the proportion of T cells that are antigen-specific to the mutated amino acid sequence is higher than that of T cells that are not antigen-specific to the mutated amino acid sequence.

[0048] In one embodiment of the invention, the method further includes obtaining a plurality of fragments from a patient's tumor, co-culturing autologous T cells from each of the plurality of fragments with autologous APCs presenting mutated amino acid sequences as described herein with respect to other aspects of the invention, and evaluating T cells from each of the plurality of fragments that are antigen-specific to the mutated amino acid sequences (as described herein with respect to other aspects of the invention).

[0049] In one embodiment of the invention, 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 autologous APCs). Selecting the autologous T cells further includes evaluating autologous T cells that are antigen-specific to each of the multiple mutated amino acid sequences. For example, the method of the invention further includes 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 with respect to other aspects of the invention) (e.g., by providing separate populations of APCs, each presenting a different mutated amino acid sequence encoded by the construct (or included in the peptide library)). The method further includes co-culturing the patient's autologous T cells with different populations of autologous APCs presenting each mutated amino acid sequence (as described herein with respect to other aspects of the invention). The method further includes selecting autologous T cells that: (a) are co-cultured with autologous APCs presenting mutated amino acid sequences, and (b) are antigen-specific to mutated amino acid sequences presented in the presence of major histocompatibility complex (MHC) molecules expressed in a patient, as described herein with respect to other aspects of the invention. In this regard, the method may include determining that mutated amino acid sequences encoded by a TMG construct (or included in the peptide library) are recognized by autologous T cell immunity (e.g., by elimination treatment).

[0050] In one embodiment of the invention, the method further includes amplifying the number of selected autologous T cells to obtain a population of T cells that are antigen-specific to a mutated amino acid sequence encoded by a cancer-specific mutation. The amplification of the number of selected cells can be achieved by any of a number of methods known in the art, as described, for example, in 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 fed PBMCs (e.g., irradiated allogeneic PBMCs). In this respect, the method of the present invention can advantageously generate large numbers of T cells with antigen specificity to mutated amino acid sequences.

[0051] T cells isolated using the method of the present invention can be used to prepare cells for use in adoptive cell therapy. In this regard, one embodiment of the invention provides a method for preparing a population of T cells with antigen specificity to mutated amino acid sequences encoded by cancer-specific mutations, the method comprising isolating T cells (as described herein with respect to other aspects of the invention) and amplifying a selected number of autologous T cells to obtain a population of T cells with antigen specificity to mutated amino acid sequences encoded by cancer-specific mutations. As described herein with respect to other aspects of the invention, the number of selected cells can be amplified.

[0052] Another embodiment of the invention provides an isolated cell population prepared according to any of the methods described herein with respect to other aspects of the invention. The cell population may be a heterogeneous population comprising T cells having antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation, and at least one other cell type, such as PBMCs or cells other than T cells that do not have antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell population may be a substantially homogeneous cell population, wherein the cell population primarily comprises T cells having antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation (e.g., substantially composed of said T cells). The cell population may also be a clonal cell population, wherein all cells in the cell population are clones of single T cells, such that all cells in the cell population have antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation. In one embodiment of the invention, as described herein, the cell population is a clonal cell population comprising T cells having antigen specificity to a mutated amino acid sequence encoded by a cancer-specific mutation. In one embodiment of the invention, a cell population comprising from 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 any two of the ranges defined in the foregoing values, contains T cells that are antigen-specific to the mutated amino acid sequence. Not bound by any particular theory or mechanism, a cell population believed to contain a high proportion of T cells that are antigen-specific to the mutated amino acid sequence may advantageously have a low proportion of unrelated cells that may impair T cell function (e.g., the ability of T cells to target and destroy cancer cells and / or treat or prevent cancer).

[0053] The cell populations of the present invention can be formulated into compositions, such as pharmaceutical compositions. In this regard, the present invention provides pharmaceutical compositions comprising any of the cell populations of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may comprise the cell populations of the present invention in combination with other pharmaceutically active agents or drugs (such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, etc.).

[0054] Preferably, the carrier is a pharmaceutically acceptable carrier. For a pharmaceutical composition, the carrier can be any of those conventionally used for a particular cell population of the 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 one that does not cause harmful side effects or toxicity under the conditions of use.

[0055] The choice of carrier will be determined in part by the specific cell population of the invention and the specific method of administering the cell population of the invention. Therefore, there are various suitable formulations of the pharmaceutical compositions of the invention. Suitable formulations may include those for oral, parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, intrathecal, or intraperitoneal administration. More than one route may be used to administer the cell population of the invention, and in some cases, a particular route may provide a more direct and effective response than another.

[0056] Preferably, the cell population of the present invention is administered by injection, for example, intravenous administration. When administering the cell population of the present invention, the pharmaceutically acceptable carrier for the injected cells may include any isotonic carrier, such as physiological saline (about 0.90% w / v aqueous NaCl solution, about 300 mOsm / L aqueous NaCl solution, or about 9.0 g of NaCl per liter of water), NORMOSOL R electrolyte (Abbott, Chicago, IL), PLASMALYTE A (Baxter, Deerfield, IL), about 5% dextran aqueous solution, or lactated Ringer's solution. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0057] It is anticipated that the cell populations and pharmaceutical compositions of the present invention can be used in methods for treating or preventing cancer. Unbound by any particular theory or mechanism, the T cells of the present invention are thought to specifically bind to mutated amino acid sequences encoded by cancer-specific mutations, such that the TCRs expressed by said cells can mediate an immune response against target cells expressing the mutated amino acid sequences. In this regard, the present invention provides a method for treating or preventing cancer in mammals, comprising administering to a mammal any of the cell populations or pharmaceutical compositions described herein in an amount effective in treating or preventing cancer in mammals.

[0058] As used herein, the terms “treatment” and “prevention,” and those derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, there exist different degrees of treatment or prevention that are recognized by those skilled in the art as having potential benefit or therapeutic effect. In this respect, the methods of the present invention can provide any amount of treatment or prevention for any level of cancer in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention may include 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 tumor regression. Moreover, for the purposes of this document, “prevention” may include delaying the onset of cancer or its symptoms or conditions.

[0059] For the purposes of this invention, the quantity or dose of the cell population or pharmaceutical composition of the invention applied (e.g., the number of cells when the cell population of the invention is applied) should be sufficient to act in mammals (e.g., a therapeutic or preventative response) within a reasonable timeframe. For example, the dose of the cell population or pharmaceutical composition of the invention should be sufficient to bind to a mutated amino acid sequence encoded by a cancer-specific mutation, or to detect, treat, or prevent cancer for a period of about 2 hours or longer (e.g., 12 to 24 hours or longer) from the time of application. In some embodiments, the time period may be even longer. The dose is determined by the efficacy of the specific cell population or pharmaceutical composition of the invention applied and the condition of the mammal (e.g., a human), as well as the weight of the mammal (e.g., a human) to be treated.

[0060] Many assays for determining the administered dose are known in the art. For the purposes of this invention, one assay includes comparing the IFN-γ levels of target cells lysed or secreted by T cells when a given dose of such T cells is administered to mammals in a mammalian group (each given different doses of T cells). This assay method can be used to determine the initial dose administered to mammals. When a certain dose is administered, target cells are lysed or T cells secrete IFN-γ. The degree of [something] can be determined by methods known in the art.

[0061] The dosage of the cell population or pharmaceutical composition of the present invention can also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a specific cell population or pharmaceutical composition of the present invention. Typically, the attending physician will consider various factors such as age, weight, health status, diet, sex, the cell population or pharmaceutical composition of the present invention to be administered, route of administration, and severity of the condition being treated to determine the dosage of the cell population or pharmaceutical composition of the present invention for treating each individual patient.

[0062] In one embodiment, the number of cells to be administered and 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, the daily dose of host cells in this invention can be from about 1 million to about 150 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 60 billion cells, about 80 billion cells, about 100 billion cells, about 120 billion cells, about 130 billion cells, about 150 billion cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 130 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about...). 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, approximately 100 billion cells, approximately 110 billion cells, approximately 120 billion cells, approximately 130 billion cells, or a range defined by any two of the foregoing values), more preferably approximately 100 million cells to approximately 130 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, approximately 100 billion cells, approximately 110 billion cells, approximately 120 billion cells, approximately 130 billion cells, or a range defined by any two of the foregoing values).

[0063] For the purposes of the method of the present invention, when the cell population is applied, the cells may be allogeneic cells or autologous cells for mammals. Preferably, the cells are autologous cells for mammals.

[0064] Another embodiment of the invention provides any of the isolated cell populations or pharmaceutical compositions described herein for the treatment or prevention of cancers in mammals.

[0065] Cancer can be any type of cancer, including any of the following: acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectal region, 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 carcinoma, colon cancer, esophageal cancer, cervical cancer, and gastrointestinal cancer. Benign tumors, gliomas, Hodgkin's lymphomas, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, bladder cancer, solid tumors, and fluid-filled tumors. Preferably, the cancer is epithelial cancer. In one embodiment, the cancer is bile duct cancer, melanoma, colon cancer, or rectal cancer.

[0066] The mammals mentioned in the method of this invention can be any mammal. As used herein, the term "mammal" means any mammal, including but not limited to: rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. Preferred mammals are from the order Carnivora, including Felidae (cats) and Canidae (dogs). Preferably, the mammals are from the order Artiodactyla, including Bovidae (cattle) and Suaeda (pigs), or Perissodactylus, including Equidae (horses). Preferably, the mammals are from the orders Primates, Cebooids, or Simoids (monkeys), or Great Apes (humans and great apes). More preferably, the mammal is a human. In a particularly preferred embodiment, the mammal is a patient expressing a cancer-specific mutation.

[0067] The following embodiments further illustrate the invention, but should not be construed as limiting its scope in any way.

[0068] Example

[0069] The materials and methods of Examples 1-7 are given below.

[0070] Whole exome sequencing

[0071] Whole-exome sequencing of cryopreserved tumor tissue (embedded in OCT) and normal peripheral blood cells using Personal Genome Diagnostics (PGDx, Baltimore, MD) was described by Jones et al. Science 330: 228-231 (2010). The average number of distinctly high-quality sequence reads at each base for tumor and normal (PBMC) DNA were 155 and 160, respectively.

[0072] The generation of tumor-infiltrating lymphocytes (TILs) for patient treatment and adoptive cell therapy

[0073] Patient 3737 was recruited under an IRB-approved protocol: “A Phase II study of short-cultured autologous tumor-infiltrating lymphocytes following a lympholytic depletion protocol in metastatic gastrointestinal cancer” (Trial Registration ID: NCT01174121), designed to evaluate the safety and efficacy of adoptive transfer of autologous, ex vivo expanded tumor-infiltrating lymphocytes (TILs) in patients with gastrointestinal cancer.

[0074] Such as Jin et al. J. Immunother As described in ., 35: 283-292 (2012), TILs were produced for the first-line treatment of patients. In short, the excised tumor was finely diced into fragments of approximately 1-2 mm, and individual fragments were placed in the wells of a 24-well plate containing 2 ml of complete culture medium (CM) containing a high dose of IL-2 (6000 IU / ml, Chiron, Emeryville, CA). The CM consisted of RPMI supplemented with 10% in-house human serum, 2 mM L-glutamine, 25 mM HEPES, and 10 μg / ml gentamicin. Additionally, the mixed tumor digestion products were cultured in the CM containing a high dose of IL-2. After T cells began to grow (between 2-3 weeks), 5 × 10⁶ cells from the selected culture were... 6T cells were rapidly expanded in 400 ml of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / ml IL-2, and 30 ng / ml OKT3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) in a gas-permeable G-Rex100 flask using irradiated allogeneic PBMCs at a 1:100 ratio. The 50 / 50 medium consisted 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 prior to infusion. Patient 3737 underwent a non-myeloablative lympholytic regimen consisting of cyclophosphamide and fludarabine prior to receiving 42.4 billion total T cells and four high-dose IL-2.

[0075] The TIL for the second treatment was generated in a manner similar to the first treatment, with the following modifications. The first treatment product (patient 3737-TIL) consisted of five separate TIL cultures. The CD4 and Vβ22 expression and responsiveness to the mutant ERBB2IP were individually assessed in these five cultures, and it was found that Vβ22+ ERBB2IP mutant-responsive CD4+ T cells were highly enriched in one culture. As described above, this TIL culture (with a high dose of IL-2 after initial growth) was then rapidly expanded. Patient 3737 underwent the same non-myeloablative lympholytic depletion protocol as the first treatment prior to receiving 126 billion total T cells and four doses of the high-dose IL-2.

[0076] Generating TMG Constructs

[0077] In short, for each nonsynonymous substitution mutation identified by whole-exome sequencing, 12-amino acid "small gene" constructs encoding the corresponding amino acid changes and flanked by wild-type protein sequences were created. Multiple small genes were fused together to generate TMG constructs. These small gene constructs were codon-optimized and synthesized into DNA string constructs (Life Technologies, Carlsbad, CA). The TMGs were then cloned into the pcDNA3.1 vector using in-fusion technology (Clontech, MountainView, CA). Site-directed mutagenesis was used to generate nine "wild-type reversed" TMG-1 constructs (Gene Oracle, Mountain View, CA). The nucleotide sequences of all TMGs were validated using standard Sanger sequencing (Macrogen and Gene Oracle).

[0078] Generation of autologous APC

[0079] Immature DCs derived from mononuclear cells were generated using a plastic adhesion method. In short, autologous samples were thawed, washed, and cultured in pure AIM-V medium (Life Technologies) at a concentration of 5-10 × 10⁻⁶. 6 cells / ml, then at approximately 1×10 6 cells / cm 2 Incubate in appropriately sized tissue culture flasks at 37°C and 5% CO2. After 90 minutes, collect non-adherent cells and vigorously wash the flasks with AIM-V medium, then incubate again with AIM-V medium for 60 minutes. Wash the flasks again vigorously with AIM-V medium, then incubate adherent cells with DC medium. The DC medium contains 5% human serum (collected and processed by the patient), 100 U / ml penicillin and 100 μg / ml streptomycin, 2 mM L-glutamine, 800 IU / ml GM-CSF, and 800 U / ml IL-4 (medium supplement from Life Technologies, cytokines from Peprotech) in RPMI. On day 3, add fresh DC medium to the culture. Use fresh or frozen / thawed DCs in experiments 5–7 days after initial stimulation. 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 primarily immature dendritic cells (DCs) expressing CD11c+, CD14-, CD80-, or CD11c-. 低 CD86+ and HLA-DR+; data not shown).

[0080] Antigen-presenting B cells were generated using a CD40L and IL-4 stimulation method. In short, human CD19 microbeads (Miltenyi Biotec) were used to positively select B cells from autologous samples. CD19+ cells were then cultured at approximately a 1:1 ratio with irradiated (6000 rads) 3T3 cells stably expressing CD40L (3T3-CD40L) in B cell culture medium. The B cell culture medium contained IMDM medium (Life Technologies) supplemented with 7.5–10% human serum (homesourced), 100 U / ml penicillin and 100 μg / ml streptomycin (Life Technologies), 10 μg / ml gentamicin (CellGro, Manassas, VA), 2 mM L-glutamine (Life Technologies), and 200 U / ml IL-4 (Peprotech). Fresh B cell culture medium was added starting on day 3, and subsequently added or replaced every 2–3 days. Additional irradiated 3T3-CD40L feeder cells were added as needed. Antigen-presenting B cells were used in experiments 2-3 weeks after initial stimulation.

[0081] Generates in vitro transcribed RNA (IVT)

[0082] The plasmid encoding the tandem small gene was linearized using the restriction endonuclease Sac II. The control pcDNA3.1 / V5-His-TOPO vector encoding GFP was linearized using Not I. Restriction digestion was terminated by precipitation with EDTA, sodium acetate, and ethanol. 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. RNA was precipitated using the lithium chloride method, and the purity and concentration of RNA were assessed using a NanoDrop spectrophotometer. The RNA was then aliquoted into microtubes and stored at -80°C until use.

[0083] RNA transfection

[0084] Harvest APCs (DCs or B cells), wash once with PBS, and then... 6Cells were resuspended at 100 cells / mL in Opti-MEM (Life Technologies). IVT RNA (4 µg or 8 µg) was aliquoted into the bottom of the 2 mm gap electroporation wells, and 50 μl or 100 μl of APC was added directly to the well. Therefore, the final RNA concentration used in electroporation was 80 μg / mL. Electroporation was performed using a BTX-830 square wave electroporator. DCs were electroporated at 150 V, 10 ms, 1 pulse, and B cells were electroporated at 150 V, 20 ms, 1 pulse. Transfection efficiency using these settings was typically 70%–90%, as assessed using GFP RNA (data not shown). All steps were performed at room temperature. Immediately after electroporation, cells were transferred to polypropylene tubes containing DC or B cell culture medium supplemented with appropriate cytokines. Transfected cells were incubated overnight (12–14 h) at 37°C and 5% CO2. Cells were washed once with PBS before being used for co-culture assays.

[0085] peptide pulse

[0086] Harvest autologous B cells, wash, and then use 1×10 6B cells / ml were resuspended in IL-4-supplemented B cell culture medium and then incubated overnight (12-14 h) at 37°C, 5% CO2 with 1 μg / ml of 25-mer peptide. After pulse incubation overnight, B cells were washed twice with PBS, resuspended in T cell culture medium, and immediately used in co-culture assays. The peptides used were: mutant ERBB2IP (TSFLSINSKEETEHLENGNKYPNLE (SEQ ID NO: 73)); wild-type ERBB2IP (TSFLSINSKEETEHLENGNKYPNLE (SEQ ID NO: 45)); and mutant ALK (RVLKGGSVRKLRHAKQLVLELGEEA (SEQ ID NO: 46)) as a negative control. Mutant ERBB2IP peptides were purchased from three different sources (GenScript, Piscataway, NJ; Peptide 2.0, Chantilly, VA; and SelleckChem, HoustonTX) and all produced identical in vitro results. 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 medium.

[0087] T cell sorting, expansion, and cloning

[0088] BD FACSAria IIu and BD FACSJazz were used in all experiments requiring cell sorting. In the designated experiments, sorted T cells were expanded using allogeneic feeder cells (from three different donor leukocyte-cleared samples) subjected to excessive irradiation (4000 rads) in 50 / 50 medium containing 30 ng / ml anti-CD3 antibody (OKT3) and 3000 IU / ml IL-2. Limiting dilution clones were produced in 96-well round-bottom plates under the above stimulation conditions of 5e4 feeder cells per well and 1-2 T cells per well. Medium exchange was initiated approximately one week after stimulation, and then every other day or as needed. Cells were typically used for assays or further expansion approximately 2-3 weeks after initial stimulation.

[0089] Co-culture assays: IFN-γ ELISPOT and ELISA, flow cytometry targeting cell surface activation markers, and Intracellular cytokine staining (ICS)

[0090] When DC is used as APC, each hole of the 96-hole flat or round-bottom plate uses approximately 3.5 × 10.4 Up to 7×10 4 10 DCs. When B cells are used as APCs, each well of the 96-well round-bottom plate uses approximately 2 × 10⁻⁶ cells. 5 Cells. In the ELISPOT assay, 1 × 10⁶ cells were used per well. 3 Up to 1×10 4 10 effector T cells, and in the flow cytometry assay, 1 × 10⁶ cells were used per well. 5 Effector T cells. T cells were typically thawed and thawed for two days in 50 / 50 medium containing IL-2 (3000 IU / ml IL-2), then washed with PBS (3×) before co-culture assays. All co-cultures were performed without the addition of exogenous cytokines. For all assays, plate-bound OKT3 (0.1 μg / ml or 1 μg / ml) was used as a positive control.

[0091] In experiments involving HLA-blocking antibodies, the following antibodies were used: pan-II (clone: ​​IVA12), pan-I (clone: ​​W6 / 32), HLA-DR (clone: ​​HB55), HLA-DP (clone: ​​B7 / 21), and HLA-DQ (clone: ​​SPV-L3). Cells were blocked with the specified antibody at 20-50 μg / ml for 1-2 hours at 37°C and 5% CO2 before co-culturing with T cells. T4 is a T cell line transduced with HLA-DR4-restricted TCRs that are responsive to tyrosinase epitopes. DMF5 is an HLA-A2-restricted T cell line responsive to MART-1. 624-CIITA is an HLA-A2 and HLA-DR4 positive melanoma cell line that stably expresses MHC-II due to ectopic expression of CIITA (class II, MHC, transactivator), and is positive for MART-1 and tyrosinase expression.

[0092] For IFN- In short, the ELISPOT assay involves pretreating each well of an ELIIP plate (Millipore, MAIPSWU) with 50 μl of 70% ethanol for 2 minutes, washing three times with PBS, and then pretreating with 50 μl of 10 μg / ml IFN-γ. The capture antibody (Mabtech, clone: ​​1-D1K) was coated and incubated overnight in a refrigerator. For the OKT3 control, IFN-γ was used. The wells were coated with a mixture of capture antibody (10 µg / ml) and OKT3 (1 μg / ml). Prior to co-culturing, the plates were washed three times with PBS, followed by blocking with 50 / 50 medium at room temperature (RT) for at least 1 hour. After co-culturing for 20–24 hours, the cells were gently ejected from the plates, washed six 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 plates were then washed three times with PBS-T and incubated for 1 hour with 100 μl / well of streptavidin-ALP (Mabtech, Cincinatti, OH, 1:3000 dilution). The plate was then washed six 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 thoroughly rinsing with cold tap water. The ELISPOT plates were scanned and counted using an immunospot plate reader and related software (Cellular Technologies, Ltd, Shaker Heights, OH).

[0093] At approximately t = 22–26 hours post-stimulation, the expression of T cell activation markers OX40 and 4-1BB was assessed by flow cytometry. Briefly, cells were pelleted, washed with FACS buffer (1X PBS supplemented with 1% FBS and 2 mM EDTA), and then stained with appropriate antibodies in the dark at 4°C for approximately 30 minutes. Cells were washed at least once with FACS buffer before collection from the BD FACSCanto II flow cytometer. All data were gated to live (PI-negative) single cells.

[0094] Cytokine production was assessed using intracellular cytokine staining (ICS) and flow cytometry. Briefly, after target and effector cells were pooled 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 half the manufacturer's recommended concentration. At t = 6 h post-stimulation, cells were treated using the Cytofix / Cytoperm kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. Briefly, cells were pelleted, washed with FACS buffer, and then stained with cell surface markers (as described above). Cells were then washed twice with FACS buffer before fixation and permeabilization. Cells were then washed with Perm / Wash buffer and stained with antibodies against cytokines at 4°C in the dark for 30 min. Cells were washed twice with Perm / Wash buffer and resuspended in FACS buffer before collection from the FACSCantoII flow cytometer. All flow cytometry data were analyzed using FLOWJO software (TreeStar Inc).

[0095] Following the manufacturer's (Thermo Scientific, Waltham, MA) instructions, use human IFN- ELISA kit for detecting IFN-γ in serum samples .

[0096] Antibodies from flow cytometry

[0097] 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 were derived from BD Biosciences, except for CD4-efluor605NC (eBioscience), Vβ22-PE and Vβ5.2-PE (Beckman Coulter), and 4-1BB-APC and OX40-PE-Cy7 (BioLegend). 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). All ICS antibodies except IL-4-PE-Cy7 (BD Bioscience) were from eBioscience. All TCR-Vβ antibodies were evaluated using the IOMark B Mark TCR V kit (Beckman Coulter).

[0098] Sequencing of ERBB2IP mutations

[0099] Sanger sequencing was used to validate findings discovered through whole-exome sequencing. ERBB2IPMutation. Total RNA was extracted from frozen T cells or tumor tissue (OCT blocks) using the RNeasy Mini kit (Qiagen). The total RNA was then reverse transcribed into cDNA using ThermoScript reverse transcriptase with oligo-dT primers (Life Technologies). Normal cDNA and tumor cDNA were then used as templates for PCR, with the following ERBB2IP primers side-linked to 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 at 58°C for 15 seconds and extension at 72°C for 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).

[0100] Quantitative PCR

[0101] Total RNA was extracted from frozen T cells or tumor tissue (OCT blocks) using the RNeasy Mini kit (Qiagen, Venlo, Netherlands). The total RNA was then reverse transcribed into cDNA using qScript cDNA ultramix (Quanta Biosciences, Gaithersburg, MD). Gene-specific TaqMan primers and probes for human β-actin (catalog number #: 401846) and ERBB2IP (catalog number #: 4331182) were purchased from Life Technologies. Quantitative PCR was performed using a 7500 Rapid 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 cycle number thresholds (Ct) were 30 or lower.

[0102] TCR-Vβ deep sequencing

[0103] Genomic DNA isolated from peripheral blood cells, T cells, and frozen tumor tissue using the DNeasy Blood and Tissue Kit (Qiagen) was subjected to TCR-Vβ deep sequencing using immunoSEQ (Adaptive Biotechnologies, Seattle, WA). The total number of productive TCR reads for each sample ranged from 279,482 to 934,672. Only productive TCR rearrangements were used to calculate TCR frequencies.

[0104] TCR sequencing and construction of ERBB2IP-mutation-responsive TCRs

[0105] T cells were pelleted and total RNA was isolated (RNeasy Mini Kit, Qiagen). Then, following the manufacturer's instructions (SMARTer RACE cDNA Amplification Kit, Clontech), the total RNA underwent 5' RACE using constant primers for the TCR-α and -β chains. Protocol 1 of the kit was used for PCR, with adjustments made to the extension time (2 min instead of 3 min). The sequences of the constant primers for the α and β chains 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 isolated by standard agarose gel electrophoresis and gel extraction (Clontech). The products were then sequenced directly, or by TOPO-TA cloning followed by single-colony sequencing (Macrogen). For sequencing of known Vβ22+ T cell clones, cDNA was generated from RNA using qScript cDNA ultramixing solution (Quanta Biosciences). These cDNAs were then used as templates in PCR, with constant TCR-β primers (as described above) and Vβ22-specific primers: 5'-CAC CAT GGA TAC CTGGCT CGT ATG C-3' (SEQ ID NO: 51). PCR products were separated by standard agarose gel electrophoresis and gel extraction (Clontech). The products were directly sequenced (Macrogen) using nested TCR-β chain constant primers: 5'-ATT CAC CCA CCAGCT CAG-3' (SEQ ID NO: 52).

[0106] The Vβ22+ ERBB2IP mutant TCR was constructed by fusing the Vβ22+ TCR-α VDJ region to the mouse TCR-α constant chain and the Vβ22+ TCR-β-VDJ region to the mouse TCR-β constant chain. The α and β chains were separated by a furin SGSG P2A linker. The use of the mouse TCR constant region facilitated the pairing of the introduced TCR and also facilitated the identification of positively transduced T cells by flow cytometry using antibodies specific to the mouse TCR-β chain (eBioscience). The TCR construct was synthesized and cloned into the MSGV1 retroviral vector (Gene Oracle).

[0107] TCR transduction of peripheral blood T cells

[0108] The autologous extracted sample was thawed and placed in T cell culture medium at 2×10⁻⁶. 6 The T cell culture medium consisted of a 50 / 50 mixture of RPMI medium and AIM-V medium supplemented with 5% autologous 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, cells were stimulated 2 × 10⁶ cells / ml in 24-well plates with 50 ng / ml soluble OKT3 (Miltenyi Biotec) and 300 IU / ml recombinant human (rhu) IL-2 (Chiron). 6 1 cell (1 ml) for 2 days. To generate transient retroviral supernatant, the retroviral vector MSGV1 (1.5 µg / well) encoding a Vβ22-positive, ERBB2IP mutation-specific TCR 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 × 10⁶ cells / well) using a Lipofectamine 2000 (Life Technologies). 6 Cells were seeded one day prior to transfection. 42–48 h post-transfection, retroviral supernatant was collected, diluted 1:1 with DMEM, and centrifuged at 2,000 g for 2 hours at 32°C into 6-well plates coated with retrotronectin (10 μg / mL, Takara), without tissue culture treatment. Then, activated T cells (2 × 10⁶ cells per well) were added. 6 0.5 × 10⁶ cells in IL-2-containing T cell culture medium. 6Cells were centrifuged at 300 g for 10 minutes (cells / ml) into a retroviral plate. Activated T cells were transduced overnight, removed from the plate, and further cultured in T cell medium containing IL-2. GFP and a simulated transduction control were included in the transduction assay. Cells were typically analyzed 10–14 days after retroviral transduction.

[0109] Example 1

[0110] This embodiment illustrates a method for identifying one or more genes in the nucleic acids of a patient's cancer cells, each gene containing a cancer-specific mutation encoding a mutated amino acid sequence.

[0111] A 43-year-old woman (patient (Pt.) 3737) with extensively metastatic cholangiocarcinoma who had undergone multiple chemotherapy regimens was enrolled in a TIL-based adoptive cell therapy (ACT) regimen for patients with gastrointestinal (GI) cancer. The clinical characteristics of patient 3737 are shown in Table 1.

[0112] Table 1 Acquisition sites for generating TILs and whole exome sequencing + Functional Status: ECOG, Eastern Cooperative Oncology Group Lung metastases were resected and used as a source for whole-exome sequencing and the 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 approximately 70% of the tumor nodules were tumors. Whole-exome sequencing revealed 26 nonsynonymous mutations (Table 2).

[0113] Table 2

[0114] Example 2

[0115] This embodiment illustrates the following method: inducing a patient's autologous APCs to present a mutated amino acid sequence; co-culturing a patient's autologous T cell population with autologous APCs presenting the mutated amino acid sequence; and selecting autologous T cells that: (a) are co-cultured with autologous APCs presenting the mutated amino acid sequence, and (b) have antigen specificity for the mutated amino acid sequence presented in the context of MHC molecules expressed by the patient.

[0116] For each mutation identified in Example 1, small gene constructs were designed encoding mutated amino acids with 12 amino acids from endogenous proteins flanked on each side. Multiple small genes were tandemly synthesized to produce tandem small gene (TMG) constructs (Table 3). In Table 3, underlined lines indicate mutated amino acids and new sequences encoded by point mutations or nucleotide insertions or deletions. For splice site donor mutations ( HLA-DOA and LONRF3 Based on the hypothesis that these mutations prevent splicing at this site, mutant small gene transcripts were designed that persist downstream introns until the next stop codon. No evaluation was conducted. DIP2C Receptor mutation at the splice site.

[0117] Table 3

[0118] Then, the TMG construct was used as a template to generate in vitro transcribed (IVT) RNA. Each of these IVT TMG RNAs was then individually transfected into autologous APCs (DCs) and subsequently co-cultured with TIL to determine if any processing had occurred and whether the presented mutated antigen was recognized by the TIL. It was observed that 3737-TIL was reactive to the mutated antigen presented in TMG-1, but not in TMG-2 or TMG-3. Figure 1 A). Furthermore, the reactivity was predominant in the CD4+ T cell population, as evidenced by the upregulation of activation markers OX40 and 4-1BB (Tables 4A and 4B). Tables 4A and 4B show the percentage of 3737-TILs detected by flow cytometry, after co-culturing DCs with the non-specific stimulant OKT3, or DCs transfected with green fluorescent protein (GFP) RNA, exhibiting a specified phenotype, or having specified tandem small gene (TMG) constructs encoding various mutations identified by whole-exome sequencing. Simulated transfection cells were treated with the transfection reagent only without the addition of nucleic acids. Data are gated for live CD3+ cells.

[0119] Table 4A

[0120] Table 4B

[0121] Although some 4-1BB upregulation was observed in CD4-negative (CD8+) T cell populations, sorting of these cells revealed no responsiveness to TMG. To determine which of the nine TMG-1 mutations was recognized by 3737-TIL, nine additional TMG-1 constructs were synthesized, each containing a reverse sequence of one of the mutations reverting to the wild-type sequence. 3737-TIL's responsiveness to TMG-1 was only observed when the ERBB2 interacting protein (… ERBB2IP The mutation was eliminated when reverted to the wild-type sequence, indicating that TIL specifically recognizes ERBB2IP. E805G mutation( Figure 1 B).

[0122] Molecular characterization of ERBB2IP-mutant reactive T cell responses. IFN-γ ELISPOT assay was performed, with results measured at 20 hours. Patient 3737-TILs were co-cultured with DCs transfected with TMG-1 (pre-incubated without any additives) or designated HLA-blocking antibodies (anti-MHC-I, MHC-II, HLA-DP, HLA-DQ, or HLA-DR). 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 cell T4 ( Figure 2 C), all were co-cultured with MART and tyrosinase-positive 624-CIITA melanoma cell lines that were pre-incubated without any additives, or with a specified HLA-blocking antibody. The response of 3737-TIL was blocked by anti-HLA-DQ antibody. Figure 2 A).

[0123] Perform another IFN- ELISPOT assay was performed, with results measured 20 hours later. Patients with 3737-TIL were compared with those who had received DMSO, mutated (mut)ALK, or mutated ERBB2IP 25-AA long peptide (…). Figure 2 D) Pulsed overnight co-culture of autologous B cells or allogeneic EBV-B cells partially matched at the HLA-DQ locus.

[0124] Perform another IFN- ELISPOT assay, with results measured 20 hours later. The patient's 3737-TIL was compared with that of mut ERBB2IP 25-AA peptide, or the specified truncated mut ERBB2IP peptide (…). Figure 2 E) Co-culture of autologous B cells pulsed overnight.

[0125] like Figure 2 As shown in A-2E, the 3737-TIL response is provided by HLA-DQB1. The 0601 allele is restricted, and the smallest epitope is located within the 13-amino acid sequence NSKEETGHLENGN (SEQ ID NO: 29).

[0126] Example 3

[0127] This example demonstrates that autologous open repertoire peripheral blood T cells, genetically modified with the TCR-Vβ22 chain (matching its α chain) of ERBB2IP-specific CD4+ T cells identified in Example 2, are endowed with specific responsiveness to the mutant ERBB2IP peptide.

[0128] Using OX40 as an activation marker, the clonality of the mutant ERBB2IP-specific CD4+ T cells identified in Example 2 was characterized by sorting them after antigen-specific activation. These cells were then expanded whole and cloned using limiting dilutions. Flow cytometry measurements of the total TCR-Vβ population showed that the total expanded population was greater than 95% Vβ22+, and the evaluated 10 / 11 clones were pure Vβ22+. TCR sequence analysis revealed identical TCRβ VDJ sequences in 6 / 6 of the tested Vβ22+ clones (Table 5), indicating that the majority of ERBB2IP-mutant reactive T cells contain a dominant Vβ22+ T cell clone.

[0129] Table 5

[0130] When stimulated with the mutated ERBB2IP peptide, T cell clones expressing this Vβ22 TCR specifically produce the cytokine IFN-γ. (Table 6). CD4+ Vβ22+ clones were co-cultured for 6 hours with OKT3 or autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK, or mutERBB2IP 25-AA long peptide. Table 6 shows the percentage of CD4+ Vβ22+ and Vβ22- clones that produced intracellular IFN-γ (IFN-γ+) or did not produce intracellular IFN-γ (IFN-γ-) after co-culture, as measured by flow cytometry. Data represent two clones sharing the same TCR-Vβ sequence.

[0131] Table 6

[0132] Furthermore, autologous open peripheral blood T cells genetically modified with this TCR-Vβ22 chain (matching its α chain) (Table 7) were conferred a specific reactivity against the mutant ERBB2IP peptide (Tables 8A and 8B), indicating that this TCR specifically recognizes ERBB2IP. E805GMutation. Autologous open peripheral blood T cells were transduced (Td) from the Vβ22+ clone (Table 8A), or treated with the transduction reagent alone without the addition of nucleic acid (simulated) (Table 8B), and reactivity was then assessed as described in Table 6. The constant region of the endogenous Vβ22+ TCR was exchanged with the mouse constant region, which allowed the introduction of TCR to be detected using antibodies against the mouse TCRβ constant region (mTCRβ). 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-) as measured by flow cytometry.

[0133] Table 7

[0134] Table 8A

[0135] Table 8B

[0136] Example 4

[0137] This embodiment illustrates a method for treating cancer using the autologous cells identified in Example 2.

[0138] Patient 3737 received adoptive transfer of 42.4 billion CD4+ ERBB2IP-mutant reactive T cells containing TILs, followed by four doses of IL-2 to enhance T cell proliferation and function. For treatment, patient 3737 underwent resection of a lung lesion. The tumor was then fragmented into small pieces and incubated with high-dose IL-2 to expand tumor-infiltrating lymphocytes (TILs). After initial expansion of cell numbers in IL-2, the selected TIL cultures were further expanded for two 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 daily for two days), followed by fludarabine (Flu: 25 mg / m²). 2 Pre-conditioning (5-day period). Patient 3737-TIL contained 42.4 billion TILs with more than 10 billion (25%) ERBB2IP mutant reactive T cells and was administered on day 0, followed by IL-2 (aldeleukin, 7.2e5 IU / kg) every 8 hours. Patients received a total of 4 doses of IL-2.

[0139] 3737-TIL was co-cultured with DCs transfected with TMG-1 or TMG-1 encoding wild-type (wt) ERBB2IP reversed, and the expression of OX40 and Vβ22 on CD4+ T cells was assessed by flow cytometry 24 hours post-stimulation. Plate-bound OKT3 stimulation was used as a positive control. Flow cytometry analysis showed that approximately 25% of the total 3737-TIL product administered contained Vβ22+, mutant reactive T cells (…). Figure 3 A (Table 9) is equivalent to the infusion of 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.

[0140] Serum samples from patients with 3737-TIL were subjected to IFN-γ assays before and after adoptive cell transfer. ELISA assay. Results are shown in... Figure 3 B. For example Figure 3 As shown in Figure B, elevated IFN-γ levels were detected in the patient's serum during the first five days after cell infusion. level.

[0141] Although the patient had clear evidence of progressive disease prior to cell infusion, tumor regression was observed at two months of follow-up, as well as continued regression of all targeted lung and liver lesions, with a maximum reduction of 30% achieved at 7 months post-treatment. Figure 3 C). Following cell infusion, the patient experienced approximately 13 months of disease stabilization, after which disease progression was observed only in the lungs (not the liver).

[0142] Table 9

[0143] Example 5

[0144] This example demonstrates the phenotype and function of the cells from Example 4 in vitro.

[0145] To determine whether there is evidence that CD4+ ERBB2IP mutant reactive T cells play a role in disease stabilization, the phenotype and function of the cells were evaluated in vitro. 3737-TILs were co-cultured for 6 hours with autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK, or mut ERBB2IP 25-AA long peptide. In the CD4+ population, Vβ22 expression was assessed using flow cytometry, and intracellular IFN-γ production was detected. (Table 10A), tumor necrosis factor (TNF) (Table 10B), and IL-2 (Table 10C). 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 cytokine numbers. Vβ22+ ERBB2IP-mutant reactive CD4+ T cells were found to be multifunctional Th1 cells that, when stimulated with the mutant ERBB2IP peptide, induced robust co-expression of IFN-γ, TNF, and IL-2 (Tables 10A-10C), but with little or no IL-4 or IL-17.

[0146] Table 10A

[0147] Table 10B

[0148] Table 10C

[0149] Table 10D

[0150] Further phenotypic characterization revealed that these cells were primarily effector memory CD4+ T cells with cytolytic potential (Tables 11 and 12). Vβ22 expression (representing ERBB2IP-mutant reactive T cells) and the expression of T cell differentiation markers CD28, CD45RO, CD57, CCR7, CD127, CD62L, and CD27 in patient 3737-TILs were assessed by flow cytometry. Data were gated to live CD3+CD4+ cells. Positive and negative quadrant gates were established using isotype-stained or unstained cells. The percentage of cells exhibiting the specified phenotype is shown in Table 11. Human peripheral blood cells (containing T cells at all stages of differentiation) were included in the experiments to ensure antibody efficacy.

[0151] Table 11

[0152] Patient 3737-TILs were co-cultured for 6 hours with OKT3 or autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK, or mut ERBB2IP25-AA long peptide. At the start of co-culture, an antibody specific for the degranulation marker CD107a was added. Flow cytometry was used to assess Vβ22 expression and to detect cell surface migration of CD107a. Data were gated to the CD4+ population. The percentage of cells with the specified phenotype is shown in Table 12.

[0153] Table 12

[0154] A small population of multifunctional Vβ22-negative, ERBB2IP-mutant reactive CD4+ T cells was observed in 3737-TIL (Tables 9 and 10). These Vβ22-negative cells were sorted by FACS, rested for 2 days in IL-2-containing medium, and then co-cultured with autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK, or mutant ERBB2IP 25-AA long peptide. At 6 hours (h) post-stimulation, Vβ22 expression in the CD4+ population was assessed by flow cytometry, and intracellular production of IL-2 (Table 13C), TNF (Table 13B), and IFN-γ (Table 13A) was detected. The percentage of cells with the specified phenotype is shown in Tables 13A-13C.

[0155] Table 13A

[0156] Table 13B

[0157] Table 13C

[0158] Twenty-four hours post-stimulation, the expression of OX40 and Vβ22 in the CD4+ population was assessed by flow cytometry. Cells with upregulated OX40 were sorted, and their numbers were expanded. Overall TCR-Vβ levels were analyzed by flow cytometry. Results are shown in... Figure 3 D. Sorting Vβ22-negative cells and subsequently activating them revealed the presence of one or more additional clonal types responsive to this epitope in 3737-TIL (Tables 13A-13C), with the most predominant clonal type being Vβ5.2. Figure 3 D).

[0159] Will as Figure 3 Cells sorted as 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 peptide. Vβ5.2 expression in the CD4+ population was assessed by flow cytometry, and intracellular production of IL-2 (Table 14C), TNF (Table 14B), and IFN-γ was detected. (Table 14A). Table 15 shows the percentage of Vβ5.2+ cells expressing specified cytokine numbers.

[0160] Table 14A

[0161] Table 14B

[0162] Table 14C

[0163] Table 15

[0164] Vβ22-negative cells with upregulated OX40 upon stimulation with mutant ERBB2IP were sorted and their numbers were expanded. RNA from these cells was then isolated and rapidly amplified at the 5' complementary DNA ends (5' RACE) using TCR-β constant-strand primers to identify expressed TCR-Vβ sequences. TOPO-TA cloning was performed on polymerase chain reaction (PCR) products, and individual clones were sequenced. Flow cytometry confirmed that 40–50% of these T cells were Vβ5.2 (TRBV5-6). By Sanger sequencing, 3 / 7 of the TOPO-TA clones were Vβ5.2 (TRBV5-6) with sequences as shown in Table 16. Table 16 shows the most frequent TCRβ VDJ sequences in Vβ22-negative ERBB2IP mutant reactive T cells.

[0165] Table 16

[0166] Most Vβ5.2+ cells produce a variety of cytokines in an antigen-specific manner (Tables 14A-14C, 15, and 16). Vβ5.2-negative (and Vβ22-negative) CD4+ T cell populations recognizing the mutated ERBB2IP are observed (Tables 14A-14C and 15). Therefore, the TIL used to treat patient 3737 contains at least three distinct polyfunctional CD4+ T cell clones recognizing the same mutation in ERBB2IP, indicating that this mutation is highly immunogenic.

[0167] Example 6

[0168] This example demonstrates the persistence of the cells from Example 4 in vivo.

[0169] To determine whether there is evidence that CD4+ ERBB2IP-mutant reactive T cells play a role in disease stabilization, the in vivo persistence of these cells was evaluated. Deep TCR-Vβ sequencing revealed that these clones are rare or undetectable in peripheral blood prior to ACT (Acute Coronary Intervention). Figure 4 A and Figure 4 B). Ten days after ACT, two clones were greater 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 a year and a half after ACT were infiltrated by ERBB2IP-mutated reactive T cells. Figure 4 (A and 4B), indicating that these cells promote cancer regression and disease stabilization. This Vβ22+ ERBB2IP mutant reactive clone was the most frequently detected clone in tumor nodules-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- mutant reactive clone was also enriched in the blood compared to its frequency in all three tumor nodules. Figure 4 (A and 4B). Therefore, after receiving more than 10 billion ERBB2IP-mutation-specific multifunctional Th1 cells that were infiltrating and persistent in metastatic lesions, patient 3737 experienced tumor regression and disease stabilization for more than a year.

[0170] For patients with 3737-TIL (T cells) and adoptive cell metastasis before (Tu-Pre) and after tumors ERBB2IP Expression was analyzed by reverse transcriptase quantitative PCR (RT-qPCR). Three independent metastatic lung lesions (Tu-1, -2, -3-Post) were resected approximately 17 months after cell infusion. Results are as follows: Figure 4 As shown in C, and relative to β-actin ( ACTB From such Figure 4 The cDNA sample described in C was amplified by PCR into a 350-base-pair (bp) fragment containing the mutated ERBB2IP gene, and then sequenced using Sanger sequencing. The mutation was located at nucleotide position 2414 of the coding sequence, corresponding to a change at amino acid position 805. As determined by quantitative RT-PCR, relatively high levels were observed in both primary and recurrent lung lesions. ERBB2IP Express( Figure 4 C), and Sanger sequencing confirmed the presence of ERBB2IP mutations in all tumor lesions.

[0171] Immunohistochemical analyses of T cell infiltration and MHC expression were performed before and after ACT. Tumors were harvested approximately 17 months after the first ACT. All staining included a positive control (tonsils). T cell infiltration and in situ MHC expression in tumors are summarized in Tables 17 and 18, respectively.

[0172] Table 17

[0173] 0, no wetting

[0174] 1. Very few to very few

[0175] 2. Medium density

[0176] 3. Very dense

[0177] Table 18

[0178] >50% means that more than 50% of the tumor cells are positive.

[0179] 0, negative

[0180] 1. Weak positive

[0181] 2. Moderately positive

[0182] 3. Strongly positive

[0183] Example 7

[0184] This example demonstrates the contribution of mutant reactive Th1 cells to the antitumor response of Example 4.

[0185] To specifically evaluate the contribution of mutant reactive Th1 cells to the in vivo antitumor response, a TIL product containing more than 95% Vβ22+ ERBB2IP- mutant reactive Th1 cells (approximately 120 billion mutant reactive cells) was generated and adopted into patient 3737.

[0186] Flow cytometry analysis was performed on TIL products used for retreatment. Table 19 shows that after CD3 gating, 97% were CD4+ / CD8-, and among these, 98% were Vβ22+ after further gating to CD4+ cells (Table 20). Retreated TILs were co-cultured for 6 hours with autologous B cells pulsed overnight with wild-type (wt) or mutant (mut) ERBB2IP 25-AA long peptide. Flow cytometry was used to detect intracellular TNF production in the CD4+ population (Table 20).

[0187] Table 19

[0188] Table 20

[0189] Furthermore, the patient experienced a reduction in the target lesion, but unlike the first treatment, even though tumor regression was observed at the first month of follow-up, the tumor continued to regress at the 4-month follow-up after the second treatment. Figure 4 D). The tumor continued to regress during the follow-up at 8 months after the second treatment.

[0190] Six months after the second administration of the mutant reactive cells, computed tomography (CT) scans were performed on the lungs of patient 3737. The images obtained are as follows: Figure 7 As shown in AC. These images are compared with images obtained before the second application of mutant reactive cells. Figure 7 (D-7F). For example... Figure 7 As shown in A-7F, a reduction of approximately 36% was observed in cancerous lesions, providing a partial response (PR) according to the Response Evaluation Criteria in Solid Tumors (RECIST).

[0191] Eight months after the second administration of mutant reactive cells, positron emission tomography (PET) scans were performed on the liver and lungs of patient 3737. The target lesions were observed to continue shrinking. Radiolabeled glucose analogue (FDG) was administered to assess tumor glucose uptake, thus measuring tumor metabolic activity. PET scans showed no glucose uptake in either of the two liver lesions, while only some uptake was observed in the lung lesion.

[0192] Examples 8-10

[0193] The materials and methods of Examples 8-10 are given below.

[0194] Patient materials and cell lines

[0195] All patient materials were obtained during the process of obtaining approval for clinical trials from the Institutional Review Board of the National Cancer Institute. Dudley et al. J. Clin. Oncol Journal of Tumor Imaging Research (2008) described patients 2359 and 2591 enrolled in a clinical trial (Trial Enrollment ID: NCT00096382 and NCT00335127, respectively). TIL cell lines and tumor cell lines were established from patients who underwent resection. (By Dudley et al.) J. Immunother The method described in Riddell et al., 26: 332-42 (2003) produces TILs for this study. In short, tumor fragments are excised and cultured in a medium containing IL-2. Cultures of amplified TILs are screened to identify autologous or HLA-matched tumors, and reactive TILs are amplified to large quantities for patient infusion using a rapid amplification protocol (REP) with IL-2, anti-CD3 antibody, and irradiated feeder cells (Riddell et al.). Science , 257: 238-41 (1992)). A small fraction of 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) in 96-well plates with 200 μL of medium (AIM-V medium supplemented with 5% human serum).

[0196] To evaluate the antigenic reactivity of clinically active tumor-associated lymphoid tissue (TILs), two patients with metastatic melanoma who experienced durable complete responses to adoptive TIL therapy were studied. Patient 2359 had primary cutaneous melanoma in the right knee that metastasized to the thigh, iliac, and groin lymph nodes. This individual experienced complete regression of all metastatic lesions in response to autologous TIL therapy, which persisted for more than 8 years post-treatment. Patient 2591 had primary back melanoma that metastasized to the abdominal wall, mesenteric lymph nodes, right colon, and supraclavicular lymph nodes. This individual experienced complete regression of all metastatic lesions in response to autologous TIL therapy and remained disease-free for 9 years post-treatment.

[0197] Whole exome sequencing

[0198] The method is described by Robbins et al. Nat. Med ., 19: 747-52 (2013). Genomic DNA purification, library construction, exon 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 short, 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 50 Mb kit (version 3) following the manufacturer's instructions (Agilent, Santa Clara, CA). Paired-end sequencing (100 bases from each end of each fragment) was performed using an Illumina HISEQ 2000 genome analyzer. Sequence data were matched against a reference human genome sequence, and sequence alterations were identified by comparing more than 50 million bases in tumor and normal DNA. For each sample, over 8 billion bases of sequence data were obtained, with a high base fraction derived from the captured coding regions. Over 43 million bases of target DNA were analyzed in tumor and normal samples, yielding an average of 42–51 reads per base in both normal and tumor DNA samples.

[0199] Bioinformatics analysis was performed through the Personal Genome Diagnostics and Genome Technology Access Center and the Genomics and Medical Pathology Service at the University of Washington School of Medicine. Tags were aligned to the human genome reference sequence (hg18) using the ELAND algorithm in CASAVA 1.6 software (Illumina). The Chastity filter in Illumina's BASECALL software was used to select sequence reads for subsequent analysis. The ELANDv2 algorithm in CASAVA 1.6 software was then applied to identify point mutations, small insertions, and deletions. Known polymorphisms already recorded in dbSNPs were removed from the analysis. (As Jones et al.) Science As described in , 330: 228-31 (2010), potential somatic mutations are filtered and visually detected.

[0200] Construction of tandem small gene banks

[0201] Nonsynonymous mutations in melanoma samples were identified from whole-exome sequencing data. A tandem small gene construct encoding a polypeptide containing six identified mutant amino acid residues flanking its N- and C-termini (12 amino acids in total) 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.

[0202] IFN-γ ELISPOT assay

[0203] Responses to tumor cell lines and target cells of peptide-pulse assays were quantified using a 96-well PVDF membrane filter plate (EMD Millipore, Billerica, MA) coated with 15 μg / mL monoclonal anti-IFN-γ antibody 1D1K (Mabtech, Inc., Cincinnati, OH). Binding cytokines were detected using 1 μg / mL biotinylated anti-IFN-γ antibody 7-B6-1 (Mabtech). HLA-A expression cells were also included. 0201, HLA-A 0205 or HLA-C HEK293 cells (0701) were pulsed with peptides at 37°C for 2 hours. The following peptides were used: MART-1: AAGIGILTV (SEQ ID NO: 54), mutant KIF2C: RLFPGLTIKI (SEQ ID NO: 55), and mutant POLA2: TRSSGSHFVF (SEQ ID NO: 56). T cells were co-cultured overnight with target cells or in medium containing 50 ng / ml PMA plus 1 μM iomycin (PMA / I). Calculations were performed every 10... 5 The number of points per T cell.

[0204] Example 8

[0205] This example demonstrates that TIL 2359 recognizes mutant antigens assessed through small gene library screening.

[0206] The reactivity of TIL 2359 was assessed using TMG constructs generated from nonsynonymous mutations identified by exome analysis of tumor and normal DNA. Each TMG construct encodes up to six separate small gene segments corresponding to mutant codons flanked by 12 additional codons present in the normal gene product. An example is shown in Figure 5A.

[0207] Based on the exon DNA sequence containing a nonsynonymous point mutation identified from Mel 2359, COS-7 cells were transiently transfected alone with one of 12 tandem small genes encoding 71 small genes. HLA-A was also used. 0205. These COS-7 cells were co-transfected with 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… EPHB2, KIF2C, SLC44A5 ABCA4、DENND4B and EPRS The mutated fragment of the gene. Subsequently, six RJ-1 variant constructs were formed, each encoding WT instead of the mutated residue present in one of the six small genes (Figure 5C). TIL 2359 recognition is associated with HLA-A. COS-7 cells co-transfected with 5 of the 6 individually transfected RJ-1 variants (0205) but unable to recognize the encoding WT KIF2C The sequence variants indicate that this small gene encodes a mutant epitope recognized by TIL 2359 (Figure 5C). To further test this observation, the full-length mutation from Mel 2359 was amplified using WT or other methods. KIF2C cDNA transcripts, with HLA-A 0101, HLA-A 0201 or HLA-A 0205 cDNA was co-transfected into COS-7 cells. Co-culture experiments showed that TIL 2359 T cells used HLA-A... 0205 - Restricted identification using mutants instead of wild-type (WT) KIF2C COS-7 cells co-transfected with gene products (Fig. 5D).

[0208] Mutant KIF2C The coding region contains a single C-to-A transversion at nucleotide 46, resulting in a threonine substitution for an alanine at position 16 of the native protein KIF2C. Exome sequencing results showed that the DNA from Mel 2359 corresponded only to the mutated residue at position 46, not the normal residue, a result confirmed by direct Sanger sequencing of Mel 2359 DNA, indicating loss of heterozygosity at this locus. To identify the mutated KIF2C epitope recognized by TIL 2359, a protein predicted to bind to HLA-A with high affinity was synthesized. 0205 contains a peptide with a KIF2C mutation (Hoof et al., Immunogenetics ,61: 1-13 (2009)), and stable expression of HLA-A HEK293 cells of 0205 were pulsed (Table 21). HEK293-A was pulsed with the decamer corresponding to residues 10-19. 0205 cells were stimulated to release high levels of IFN-γ from TIL 2359 T 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 (Fig. 5E).

[0209] Table 21

[0210] Example 9

[0211] This example demonstrates that TIL 2591 recognizes mutant antigens identified through screening from a small gene pool.

[0212] Based on exon DNA sequences containing nonsynonymous point mutations identified from Mel 2591, mutated T-cell antigens recognized by TIL 2591 were identified by synthesizing 37 TMG constructs encoding 217 small genes. TIL 2591 recognizes autologous tumor cells in the presence 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 individually transiently transfected with each of the 37 TMG constructs and subsequently co-cultured overnight with TIL 2591. Preliminary results indicate that TIL 2591 recognizes HLA-C cells transiently transfected with the small gene DW-6. 0701 + HEK293 cells (HEK293-C) 0701), but did not respond significantly to other small gene constructs (Fig. 6A). Then, each of the six individual mutant small genes in the DW-6 tandem construct (Fig. 6B) was individually reverted to the WT sequence (Fig. 6C). Evaluation of the response to the WT variants showed that, in addition to encoding WT, POLA2 Outside of the construct, TIL 2591 recognizes each COS-7 cell transfected with the DW-6 variant (Fig. 6C). To test these findings, full-length or mutated fragments were used. POLA2 The cDNA construct, with HLA-C 0401, HLA-C 0701 or HLA-C 0702 cDNA was co-transfected into COS-7 cells. TIL 2591 T cells only recognized HLA-C. 0701 plus mutation POLA2 The target cells were transfected with the construct rather than the corresponding WT transcript (Figure 6D). POLA2 A single C-to-T conversion at nucleotide 1258 in the coding region resulted in a leucine substitution for a phenylalanine at position 420 of the WT POLA2 protein. Sanger sequencing revealed that both the genomic DNA and cDNA from Mel 2591 RNA contained the WT sequence and the mutated nucleotide at position 1258, thus the genomic DNA isolated from the PBMCs of patient 2591 corresponded to the WT sequence, indicating a heterozygous cell mutation in Mel 2591 cells.

[0213] Then, use HLA-C The 0701 algorithm was used to identify candidate POLA2 peptides overlapping with the mutated leucine residue at position 420 (Table 22). Co-culture results showed that HLA-C pulses corresponding to residues 413-422 of the mutated POLA2 were effective. 0701 +HEK293 cells were stimulated to release IFN-γ from TIL 2591 T 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).

[0214] Table 22

[0215] Then, the proportion of T cells in TIL 2359, which recognizes the mutant KIF2C, and TIL 2591, which recognizes the mutant POLA2, was estimated using an IFN-γ enzyme-linked immunospot (ELISPOT) assay. TIL 2359 responded to HLA-A pulses generated by the mutant KIF2C epitope. 0205 + When T cells respond to HLA-A2-restricted MART-1 epitopes, approximately 2,000 spots are generated (per 100,000 T cells), similar to what is observed in response to autologous melanoma (Table 23). TIL 2591 generates more than 7,000 spots in response to the HLA-A2-restricted MART-1 epitope, while only a subset of T cells respond to HLA-C... The POLA2 epitope with the 0701 restriction was responsive (Table 23).

[0216] Table 23

[0217] Example 10

[0218] This embodiment demonstrates a method for identifying T cells that are responsive to mutated antigens present in gastrointestinal (GI) cancers identified by screening from a small gene library.

[0219] Whole-exome sequencing was performed on metastatic lesions from GI cancer patients to identify mutations. Small gene constructs encoding each mutation were then generated and transfected into autologous APCs to allow for the processing and presentation of all mutations expressed by the tumor. These APCs were then co-cultured with tumor-infiltrating lymphocytes (TILs), and T cell responsiveness to the mutations was determined by IFN-γ ELISPOT and by upregulation of 4-1BB and OX40 via flow cytometry.

[0220] Mutation reactivity to 119 mutations was evaluated in a colorectal cancer patient. Several (but not all) TIL cultures were found to contain a highly variable proportion of specific recognition of mutations in CASP8 (67 F). CD8+ T cells (V). When further expanded in vitro, these mutant reactive CD8+ T cells significantly outperformed other cell types in culture. The patient was administered 40.3 × 10⁻⁶ cells. 9The TIL (which is estimated to contain approximately 0.31% (about 127 million) of mutant reactive cells) did not result in a clinical response at the first follow-up approximately 6 weeks after cell administration. The patient died approximately 6 weeks later. Without being bound to a specific theory or mechanism, it is believed that one or more of the following factors may have contributed to the patient's death: a very late stage of disease prior to treatment, poor overall patient condition, and poor tolerance to lymphocyte-reducing chemotherapy prior to adoptive cell therapy. The TCRs responsive to mutant CASP8 were isolated from the TILs, and T cells transduced to express TCRs were responsive to DCs pulsed with mutant CASP8.

[0221] In another rectal cancer patient, mutation reactivity was evaluated for 155 mutations. At least three distinct mutation reactivity profiles were identified, two of which included CD8+ T cell responses and one CD4+ response. Administration of a mutation-reactive TIL initially resulted in a mixed response at approximately 1.5 months post-treatment, but the patient subsequently developed progressive disease at approximately 3.5 months post-treatment. Potential mutation-reactive TCRs were isolated from both CD4+ and CD8+ TILs.

[0222] In the third patient (cholangiocarcinoma), no T-cell responsiveness was detected to the 38 mutations tested. For this patient, the "mutation call" threshold was lowered, and an additional 125 putative mutations were evaluated. The "mutation call" is an arbitrarily set threshold used to identify a sequence as a mutation using bioinformatics. In this case, as a 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 lowered, thus 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.

[0223] These data suggest that the ability of the human immune system to load T-cell responses against somatic mutations may not be uncommon in metastatic gastrointestinal cancers. The study is ongoing.

[0224] All references cited herein (including publications, patent applications and patents) are incorporated herein by reference to the extent that each reference is individually and specifically indicated as incorporated herein by reference, and are set forth in their entirety herein.

[0225] Unless otherwise stated herein or obviously contradicted by the context, the terms “a / an,” “the,” and “at least one,” and similar expressions used in the context of describing the invention (especially in the context of the appended claims) shall be construed as covering both the singular and the plural. Unless otherwise stated herein or obviously contradicted by the context, the use of the term “at least one,” followed by a list of one or more items (e.g., “at least one of A and B”), shall be construed as referring to one item selected from the list (A or B), or any combination of two or more of the list items (A and B). Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” shall be construed as open-ended terms (i.e., meaning “including, but not limited to”). Unless otherwise stated herein, the numerical ranges mentioned herein are intended only as a concise way of expressing that each individual value falls within that range, and each individual value is incorporated into the specification as if it were individually listed herein. Unless otherwise stated herein or obviously contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise required, the use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in the specification should be construed as representing any unclaimed element necessary for carrying out the invention.

[0226] This document describes preferred embodiments of the invention, including the best modes known to the inventors for carrying out the invention. Various variations of these preferred embodiments will be apparent to those skilled in the art upon reading the above description. The inventors anticipate that those skilled in the art will employ these variations where appropriate, and the inventors expect the invention to be practiced in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, any combination of the foregoing elements is included in the invention in all possible variations.

Claims

1. A method for isolating T cells that are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations, the method comprising: Identify one or more genes in the nucleic acids of a patient's cancer cells, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; Inducing the patient's autoantigen-presenting cells (APCs) to present mutated amino acid sequences; The patient's autologous T cells were co-cultured with autologous APCs presenting mutated amino acid sequences; and Select (a) autologous APCs that present mutated amino acid sequences and (b) autologous T cells that are antigen-specific to mutated amino acid sequences presented in the context of major histocompatibility complex (MHC) molecules expressed in patients, to provide isolated T cells that are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations.

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

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

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

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

6. The method of any one of claims 1-4, wherein selecting autologous T cells that are antigen-specific to the mutated amino acid sequence includes selectively culturing autologous T cells that are antigen-specific to the mutated amino acid sequence.

7. A method for preparing a population of T cells with antigen specificity to mutated amino acid sequences encoded by cancer-specific mutations, the method comprising: The method for isolating T cells according to any one of claims 1-6, and The number of selected autologous T cells is expanded to obtain a population of T cells that are antigen-specific to mutated amino acid sequences encoded by cancer-specific mutations.

8. The isolated cell population prepared by the method according to claim 7.

9. A pharmaceutical composition comprising the isolated cell population of claim 8 and a pharmaceutically acceptable carrier.

10. Use of the isolated cell population of claim 8 or the pharmaceutical composition of claim 9 in the preparation of a medicament for the treatment or prevention of cancer in mammals.

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