JP7340144B2 - Method for isolating T cells with antigenic specificity for cancer-specific mutations - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Presented at the American Association for Cancer Research (AACR) meeting held on April 7, 2014

Application of Article 30, Paragraph 2 of the Patent Act May 8, 2014 https://www. nytimes. com/2014/05/09/health/doctors-use-patients-immune-cells-to-shrink-cancer-tumors. Announcement through html

Application of Article 30, Paragraph 2 of the Patent Act May 8, 2014 https://www. wsj. com/articles/patients-immune-system-harnessed-to-attack-cancer-1399579965? KEYWORDS=Patient%27s%20Cells%20Battle%20Tumors&mg=reno64-wsj&tesla=y

Application of Article 30, Paragraph 2 of the Patent Act May 8, 2014 https://www. cancer. Published through gov/newscenter/newsfromnci/2014/ACTepithelial

Application of Article 30, Paragraph 2 of the Patent Act May 8, 2014 https://www. nbcnews. Published through com/health/cancer/new-immune-therapy-approach-tackles-womens-rare-cancer-n100811

Application of Article 30, Paragraph 2 of the Patent Act May 9, 2014 https://science. sciencemag. org/content/344/6184/641. Announced through full

Application of Article 30, Paragraph 2 of the Patent Act July 1, 2014 https://clincancerres. aacrjournals. org/content/20/13/3401. full, https://clincancerres. aacrjournals. org/content/20/13/3401. full-text. Announcement through pdf

Application of Article 30, Paragraph 2 of the Patent Act July 19, 2014 https://www. impactjournals. com/oncotarget/index. php? Published through journal=oncotarget&page=article&op=view&path%5B%5D=2234

Incorporation by Reference of Electronically Submitted Material The computer-readable nucleotide/amino acid sequence listing filed contemporaneously with this specification and identified as follows is incorporated herein by reference in its entirety: One ASCII (text) file dated September 30, 2014, file name "718292ST25.TXT", 29,568 bytes.

BACKGROUND OF THE INVENTION Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) can produce beneficial clinical responses in some cancer patients. Nevertheless, obstacles remain to the successful and widespread treatment of cancer and other diseases using ACT. For example, T cells that specifically recognize cancer antigens can be difficult to identify and/or isolate from a patient. Therefore, there is a need for improved methods of obtaining cancer-reactive T cells.

SUMMARY OF THE INVENTION One embodiment of the invention is a method of isolating T cells having antigenic specificity for a mutant amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in a nucleic acid of a patient's cancer cells that contain a cancer-specific mutation encoding an amino acid sequence; co-culturing the patient's autologous T cells with autologous APCs presenting the mutant amino acid sequence; and (a) co-culturing the autologous APCs presenting the mutant amino acid sequence, and ( b) Select autologous T cells that have antigenic specificity for the mutated amino acid sequence presented in the context of major histocompatibility complex (MHC) molecules expressed by the patient and encoded by cancer-specific mutations. providing an isolated T cell having antigenic specificity for a mutant amino acid sequence in which the mutant amino acid sequence is a mutated amino acid sequence.

Another embodiment of the invention is a method of preparing a T cell population having antigenic specificity for a mutant amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in the nucleic acid of the patient's cancer cells that contain a cancer-specific mutation encoding the patient; inducing the patient's autologous APC to present the mutant amino acid sequence; co-culturing autologous T cells of the patient with autologous APCs presenting the mutant amino acid sequence; (a) co-cultivating the autologous APCs presenting the mutant amino acid sequence; selecting autologous T cells that have antigenic specificity for a mutant amino acid sequence presented in a molecular context; and expanding the number of selected autologous T cells encoded by a cancer-specific mutation; A method is provided comprising obtaining a population of T cells having antigenic specificity for a mutant amino acid sequence.

Further embodiments of the invention provide related cell populations, pharmaceutical compositions, and methods of treating or preventing cancer.

Figure 1A shows that after 20 hours of co-culture of 3737-TIL with dendritic cells (DCs) transfected with OKT3 or green fluorescent protein (GFP) RNA or the indicated tandem minigene (TMG) constructs, interferon (IFN)-γ Enzyme-linked immunosorbent spot (ELISPOT) assay is a graph showing the number of spots per 1×10 ³ (1e3) cells. “>” indicates more than 500 spots per 1×10 ³ cells. Mock transfected cells were treated with transfection reagent alone without addition of nucleic acid. Figure 1B shows the results after co-culture with DCs transfected with OKT3 or GFP RNA, TMG-1, or the indicated wild-type (wt) genes ALK, CD93, ERBB2IP, FCER1A, GRXCR1, KIF9, NAGS, NLRP2, or RAC3. Figure 3 is a graph showing the percentage of CD4+ 3737-TILs that are OX40+. Mock transfected cells were treated with transfection reagent alone without addition of nucleic acid. Figures 2A-2C show TMG-1 without or preincubated with the indicated HLA-blocking antibodies (MHC-1, MHC-II, HLA-DP, HLA-DQ, or HLA-DR). 20 hours for 3737-TIL (A), DMF5T cells (B), or T4T cells (C) co-cultured with DCs transfected with (A) or 624-CIITA cells ((B) and (C)). (A-C) shows the number of spots per 1×10 ³ (1e3) cells measured by IFN-γ ELISPOT assay in FIG. Figure 2D shows autologous DQ-0301/-0601B cells (gray bars) or HLA-DQ05/0601 loci pulsed overnight with DMSO, 25 amino acid (-AA) long peptides of mut ALK or mutERBB2IP. IFN-γ ELISPOT assay at 20 hours for 3737-TIL co-cultured with partially matched allogeneic EBV-B cells at (black bars) or HLA-DQ-0201/0301 loci (bars without hatching) FIG. 2 is a graph showing the number of spots per 1×10 ³ (1e3) cells, as measured by . ETGHLENGNKYPNLE (SEQ ID NO: 53); FIG. 2E shows the 25 amino acid peptide of mutERBB2IP TSFLSINSKEETGHLENGNKYPNLE (SEQ ID NO: 73) or the indicated truncated mutERBB2IP peptide FLSINSKEETGHLENGNKYPNLE (SEQ ID NO: 30), S INSKEETGHLENGNKYPNLE (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), TS FLSINSKEETGHLENGNKY (SEQ ID NO: 37) or TSFLSINSKEETGHLENGNKYPN (SEQ ID NO: 38) Figure 3 is a graph showing the number of spots per 1 x ¹⁰³ (1e3) cells measured by IFN-gamma ELISPOT assay at 20 hours for 3737-TIL co-cultured with autologous B cells pulsed overnight with 3737-TIL. FIG. 3A is a graph showing the percentage of various TCRVβ clonotypes in 3737-TILs as determined by flow cytometry gated on live, CD4+ (shaded) or CD8+ (unshaded) T cells. . Figure 3B is a graph showing the amount of IFN-γ detected in serum samples of patient 3737 (pg/ml) measured on the indicated days before and after day 0 (indicated by arrows) of adoptive cell transfer of 3737-TIL. It is. Error bars are standard error of the mean (SEM). Figure 3C shows total tumor burden (circles) (measured as % of pretreatment reference) or tumor burden in the lungs (triangles) or liver (squares) at the indicated months relative to day 0 of cell transfer (indicated by arrows). This is a graph showing. FIG. 3D is a graph showing the percentage of various TCRVβ clonotypes in CD4+Vβ22-OX40+ 3737-TILs as determined by flow cytometry. Figures 4A and 4B show various tumors before and after adoptive cell transfer using 3737-TIL (tumor before cell transfer (diamond) and after cell transfer (Tu-1-Post (square), Tu-2-Post (▲ ), and Tu-3-Post (▼))), two ERBB2IP mutation-specific TCRβ-CDR3 clonotypes, Vβ22+ (A) and Vβ5.2+, in the blood (circles) of patient 3737. It is a graph showing the frequency of (B). Shaded bars indicate the frequency of two ERBB2IP-mutant-specific TCRβ-CDR3 clonotypes, Vβ22+ (A) and Vβ5.2+ (B), in transfected cells (3737-TIL). "X" indicates "no detection". Figure 4C shows ACTB in 3737-TIL (T cells) and tumors before (Tu-Pre) and after (Tu-1-post, Tu-2-post and Tu-3-post) different adoptive cell transfers. FIG. 3 is a graph showing the comparative expression of ERBB2IP. FIG. 4D is a graph showing total tumor burden (circles) (measured as % of pretreatment baseline) or tumor burden in the lungs (triangles) or liver (squares) at the indicated months versus cell transfer (indicated by arrows). be. Figure 5A shows an example of a tandem minigene (TMG) construct encoding a polypeptide containing the identified six mutated amino acid residues flanked by 12 amino acids on either side at its N- and C-termini. It is a schematic diagram. The mutant KIF2C sequence is DSSLQARLFPGLTIKIQRSNGLIHS (SEQ ID NO: 57). Figure 5B shows autologous melanocytes or HLA-A ^* 0205 and TMG constructs RJ-1 (structure shown in Figure 9A), RJ-2, RJ-3, RJ-4, RJ-5, RJ-6, RJ-7. Amount of IFN-γ secreted by TIL2359T cells co-cultured overnight with COS-7 cells co-transfected with , RJ-8, RJ-9, RJ-10, RJ-11, RJ-12, or empty vector. (pg/mL). FIG. 5C is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2359 co-cultured with COS-7 cells transfected with HLA-A ^* 0205 and the RJ-1 mutant (in the table, Genes labeled "wt" were reconverted to WT sequences). The KIF2C WT sequence is DSSLQARLFPGLAIKIQRSNGLIHS (SEQ ID NO: 65). Figure 5D shows HLA cDNA constructs (identified in each bar from left to right): HLA-A ^* 0101 (unshaded bars), HLA-A ^* 0201 (gray bars), or HLA-A ^* 0205 ( Graph showing the amount of IFN-γ (pg/mL) secreted by TIL2359 co-cultured with COS-7 cells transfected with empty vector, KIF2C WT, or mutant KIF2C cDNA constructs (black bars). be. Figure 5E shows various concentrations (μM) of KIF2C _10-19 WT (RLFPGLAIKI; SEQ ID NO: 58) (lower line in the graph) or mutant KIF2C _10-19 (RLFPGLTIKI; SEQ ID NO: 59) (upper line in the graph). Figure 2 is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2359T cells co-cultured overnight with HEK293 cells stably expressing HLA-A ^* 0205 and pulsed with (line). FIG. 6A shows autologous melanocytes or co-cultured with HEK293 cells stably expressing HLA-C ^* 0701 transfected with empty vector or a TMG construct selected from the group consisting of DW-1 to DW-37. It is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2591T cells. FIG. 6B is a schematic diagram showing the structure of TMG construct DW-6. The mutant POLA2 sequence is TIIEGTRSSGSHFVFVPSLRDVHHE (SEQ ID NO: 64). FIG. 6C is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2591 co-cultured with COS-7 cells transfected with HLA-C ^* 0701 and the DW-6 mutant (in the table, Genes labeled "wt" were reconverted to WT sequences). The POLA2 WT sequence is TIIEGTRSSGSHLVFVPSLRDVHHE (SEQ ID NO: 66). Figure 6D shows HLA cDNA constructs (identified by each bar from left to right): HLA-C ^* 0401 (unshaded bars), HLA-C ^* 0701 (gray bars), or HLA-C ^* 0702 ( Graph showing the amount of IFN-γ (pg/mL) secreted by TIL2591 co-cultured with COS-7 cells transfected with empty vector, POLA2 WT, or mutant POLA2 cDNA constructs (black bars). be. Figure 6E shows various concentrations (μM) of POLA2 _413-422 WT (TRSSGSHLVF; SEQ ID NO: 67) (lower line in the graph) or mutant POLA2 _413-422 (TRSSGSHFVF; SEQ ID NO: 68) (upper line in the graph). FIG. 2 is a graph showing the amount of IFN-γ (pg/mL) secreted by HEK293 cells stably expressing HLA-C ^* 0701 and TIL2591T cells co-cultured overnight, pulsed with (line). Figures 7A-7F are computed tomography (CT) scans of patient 3737's lungs taken before (A-C) and 6 months after (D-F) the second dose of mutation-responsive cells. . Arrow points to cancerous lesion.

DETAILED DESCRIPTION OF THE INVENTION One embodiment of the invention provides a method of isolating T cells with antigenic specificity for mutant amino acid sequences encoded by cancer-specific mutations. The invention provides many advantages. For example, the methods of the invention can rapidly assess a patient's entire MHC molecule-restricted large number of mutations at once and identify the patient's entire repertoire of mutation-reactive T cells. Additionally, the methods of the present invention can be used to identify immunogenic cancer mutations that include (a) cancer-specific silent mutations (that do not encode a mutant amino acid sequence) and (b) non-immunogenic amino acid sequences that encode By distinguishing cancer-specific mutations, one or more cancer-specific mutant amino acid sequences can be identified that can be targeted by T cells. Furthermore, the present invention can provide T cells with antigenic specificity for mutant amino acid sequences encoded by cancer-specific mutations specific to a patient, thereby, for example, treating or treating the patient's cancer. Provided is an isolated, "personalized" population of T cells that may be useful in preparing cells for preventive adoptive cell therapy. The methods of the invention may also avoid the technical biases inherent in traditional methods of identifying cancer antigens, such as those using cDNA libraries, and may be less time-consuming and labor-intensive than those methods. . For example, the methods of the invention can select mutation-responsive T cells without co-cultivating T cells with tumor cell lines, which can be difficult to generate, particularly for epithelial cancers (eg, epithelial cancers). Without being bound to any particular theory or mechanism, the methods of the present invention allow for the destruction of cancerous cells while minimizing or avoiding the destruction of normal, non-cancerous cells, thereby reducing or eliminating toxicity. It is believed that T cells of interest can be identified and isolated. Thus, the present invention may also provide T cells that successfully treat or prevent cancers, such as cancers that do not respond to other types of treatments, such as chemotherapy alone, surgery or radiation, for example.

The method can include identifying one or more genes in the nucleic acid of the patient's cancer cells, each gene containing a cancer-specific mutation encoding a mutant amino acid sequence. Cancer cells can be obtained from any body sample from a patient that contains or is suspected of containing tumor cells or cancer cells. The body sample can be any tissue sample, such as blood, a tissue sample obtained from a primary tumor or from a metastatic tumor, or any other sample containing tumor or cancer cells. Cancer cell nucleic acids may be DNA or RNA.

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

A cancer-specific mutation is a mutation in any gene that encodes a mutated amino acid sequence (also referred to as a "non-silent mutation") and is expressed in cancer cells but not in normal, non-cancerous cells. It can be a mutation. Non-limiting examples of cancer-specific mutations that can be identified in the methods of the invention include missense, nonsense, insertions, deletions, duplications, frameshifts and repeat expansion mutations. In one embodiment of the invention, the method comprises identifying at least one gene containing a cancer-specific mutation encoding a mutant amino acid sequence. However, the number of genes containing such cancer-specific mutations that can be identified using the methods of the invention is not limited and can be greater than 1 (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 above values). Similarly, in one embodiment of the invention, the method comprises identifying at least one cancer-specific mutation encoding a mutant amino acid sequence. However, the number of such cancer-specific mutations that may be identified using the methods of the invention is not limited, and may be greater than 1 (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 above values). In embodiments where more than one cancer-specific mutation is identified, the cancer-specific mutations may be located in the same gene or in different genes.

In one embodiment, identifying one or more genes in the nucleic acid of the cancer cell comprises sequencing the whole exome, whole genome, or whole transcriptome of the cancer cell. Sequencing may be performed in any suitable manner known in the art. Examples of sequencing techniques that may be useful in the methods of the invention include next generation sequencing (NGS) (also referred to as "massively parallel sequencing technology") or third generation sequencing. It will be done. NGS refers to non-Sanger-based high-throughput DNA sequencing technology. With NGS, millions or billions of DNA strands can be sequenced in parallel, yielding significantly higher throughput and minimizing the need for fragment cloning methods, commonly used for Sanger sequencing of genomes. . In NGS, nucleic acid templates can be read randomly across the genome in parallel by dividing the entire genome into small fragments. NGS advantageously produces genomes, exomes, or It can provide nucleic acid sequence information for the entire transcriptome. A number of NGS platforms that are commercially available or described in the literature can be used in connection with the methods of the invention (e.g., Zhang et al., J. Genet. Genomics, 38(3):95- 109 (2011) and Voelkerding et al., Clinical Chemistry, 55:641-658 (2009)).

Non-limiting examples of NGS technologies and platforms include (GS-FLX 454
Genome sequencer, 454 Life Sciences (Branford, CT), ILLUMINA SOLEXA genome analyzer (Illumina Inc., San Diego, CA), or ILLUMINA HISEQ 2000 genome analyzer (Illumina). (example), or Ronaghi et al. , Sequencing-by-synthesis (also known as "pyrosequencing"), as performed as described in , Science, 281(5375):363-365 (1998) (Examples); Sequencing-by-ligation (eg, as performed using the SOLID platform (Life Technologies Corporation, Carlsbad, Calif.) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)) ing-by-ligation) , (PACBIO RS system (Pacific Biosciences (Menlo Park, CA)) or HELISCOPE platform (Helicos Bioscie
single molecule sequencing (e.g., as performed using the Nanopore Technologies (Cambridge, MA)), the GRIDON platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization assay developed by Nabsys (Providence, RI); Single molecule sequencing (e.g. as performed using Stead Nanopore Sequencing (HANS) platform, ligase-based DNA sequencing platform by DNA nanoball (DNB) technology called probe-anchor ligation (cPAL)) technologies, electron microscopy-based techniques for single molecule sequencing, and ionic semiconductor sequencing.

The method may include inducing autologous antigen presenting cells (APCs) of the patient to present the mutant amino acid sequence. APCs can include any cells that present peptide fragments of proteins in association with major histocompatibility complex (MHC) molecules on their cell surface. APCs can include, for example, any one or more of macrophages, DCs, Langerhans cells, B lymphocytes, and T cells. Preferably the APC is a DC. By using autologous APC from a patient, the methods of the present invention advantageously provide antigens against mutant amino acid sequences encoded by cancer-specific mutations that are presented in the context of MHC molecules expressed in the patient. T cells with specificity can be identified. Examples of MHC molecules include MHC class I, MHC class II, HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules. It can be any MHC molecule expressed by the patient, including but not limited to. The methods of the invention may be useful, for example, only for selecting a few MHC class I alleles, and may be useful only for selecting a few MHC class I alleles, using reagents for selecting mutation-reactive T cells, e.g. , an incomplete set of MHC tetramers) expressed by a patient, without the use of epitope prediction algorithms to identify MHC molecules or mutated amino acid sequences. Mutant amino acid sequences presented in molecular relationships can be conveniently identified. Accordingly, in one embodiment of the invention, the method of the invention advantageously identifies mutant amino acid sequences that are presented in the context of any MHC molecule expressed by a patient and Not restricted. Preferably, the autologous APC is an antigen-negative autologous APC.

Inducing a patient's autologous APC to present the mutant amino acid sequence may be performed using any suitable method known in the art. In one embodiment of the invention, inducing the patient's autologous APCs to present the mutant amino acid sequence comprises pulsing the autologous APCs with a peptide or peptide pool comprising the mutant amino acid sequence; Each peptide of contains a different mutant amino acid sequence. Each of the mutant amino acid sequences in the pool can be encoded by a gene containing a cancer-specific mutation. In this regard, autologous APCs internalize the peptide(s) containing the mutant amino acid sequence(s) in such a way that the APC internalizes the peptide(s) and presents the mutant amino acid sequence(s) on the cell membrane bound to MHC molecules. or can be incubated with a pool of peptides. In embodiments in which more than one gene is identified and each gene contains a cancer-specific mutation encoding a mutant amino acid sequence, the method provides a peptide in which each peptide in the pool comprises a different mutant amino acid sequence. It may include pulsing the self APC in the pool. Methods of pulsing APC are known in the art and are described in Solheim (Ed.), Antigen Processing and Presentation Protocols (Methods in Molecular Biology), Human Press, (2003). 010) (Example). The peptide(s) used to pulse APCs may include mutant amino acid(s) encoded by cancer-specific mutations. The peptide(s) further comprises, on each of the carboxyl and amino sides of the mutant amino acid(s), any suitable number of consecutive amino acids from the endogenous protein encoded by the identified gene. may be included. The number of contiguous amino acids from the endogenous protein flanking each side of the mutation is not limited, e.g., about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, It can be 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 above values. Preferably, the peptide(s) comprises about 12 contiguous amino acids from the endogenous protein on each side of the mutant amino acid(s).

In one embodiment of the invention, inducing the patient's autologous APC to present the mutant amino acid sequence comprises introducing into the APC a nucleotide sequence encoding the mutant amino acid sequence. The nucleotide sequence is introduced into the APC, which expresses the mutant amino acid sequence, binds it to MHC molecules, and displays it on the cell membrane. The nucleotide sequence encoding the mutant amino acid may be RNA or DNA. Introduction of nucleotide sequences into APC is described by Solheim et al. This can be done in any of a variety of different ways known in the art, as described in Examples (above). Non-limiting examples of techniques useful for introducing nucleotide sequences into APCs include transformation, transduction, transfection, and electroporation. In embodiments where more than one gene is identified, the method includes preparing more than one nucleotide sequence, each encoding a mutant amino acid sequence encoded by a different gene, and The method may include introducing different populations of APCs. In this regard, multiple populations of autologous APCs may be obtained, each population expressing and displaying a different mutant amino acid sequence.

In embodiments where more than one gene is identified and each gene contains a cancer-specific mutation encoding a mutant amino acid sequence, the method includes introducing nucleotide sequences encoding the more than one gene. may be included. In this regard, in one embodiment of the invention, the nucleotide sequences introduced into autologous APCs are TMG constructs, each minigene comprising a different gene, each gene encoding a mutant amino acid sequence. Contains mutations. Each minigene contains one mutation (on each side of the mutation, an endogenous gene encoded by the identified gene) identified by the methods of the invention, as described herein with respect to other aspects of the invention. any suitable number of contiguous amino acids from a natural protein). The number of minigenes in the construct is not limited, e.g., about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25 or more, or any of the above values. A range defined by any two of the following may be mentioned. APC expresses the mutant amino acid sequence encoded by the TMG construct and displays the mutant amino acid sequence on the cell membrane bound to MHC molecules. In one embodiment, the method comprises preparing more than one TMG construct, each construct encoding a different set of mutant amino acid sequences encoded by a different gene, and combining each TMG construct with a different set of self-APCs. Including introduction into a population. In this regard, multiple populations of autologous APCs may be obtained, each population expressing and displaying mutant amino acid sequences encoded by different TMG constructs.

The method may include culturing the patient's autologous T cells and autologous APCs displaying the mutant amino acid sequence. T cells can be obtained from many sources within a patient including, but not limited to, tumor, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cells include CD4+/CD8+ double-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), and tumor-infiltrating cells (e.g., tumor-infiltrating lymphocytes). (TIL)), peripheral blood T cells, memory T cells, naive T cells, etc., may be included and may be at any stage of development. The T cells can be CD8+ T cells, CD4+ T cells, or both CD4+ and CD8+ T cells. The method involves a T cell encountering a mutant amino acid sequence presented by an APC in such a manner that the autologous T cell specifically binds to and immunologically recognizes the mutant amino acid sequence presented by the APC. The method may include co-culturing autologous T cells and autologous APCs to do so. In one embodiment of the invention, autologous T cells are co-cultured in direct contact with autologous APCs.

The method comprises: (a) co-cultured with autologous APC presenting the mutant amino acid sequence; and (b) having antigenic specificity for the mutant amino acid sequence presented in the context of an MHC molecule expressed by the patient. , may include selecting autologous T cells. The term "antigen-specific" as used herein refers to the ability of an autologous T cell to specifically bind to and immunologically recognize a mutant amino acid sequence encoded by a cancer-specific mutation. means. Selecting may include identifying T cells that have antigenic specificity for the mutant amino acid sequence and separating them from T cells that do not have antigenic specificity for the mutant amino acid sequence. Selecting autologous T cells with antigenic specificity for the mutant amino acid sequence may be performed in any suitable manner. In one embodiment of the invention, the method comprises, for example, prior to selection of autologous T cells, by co-culturing with T cell growth factors such as interleukin (IL)-2 or IL-15; As described herein with respect to other aspects of the invention, including expanding the number of autologous T cells. In one embodiment of the invention, the method does not include expanding the number of autologous T cells with a T cell growth factor, such as IL-2 or IL-15, prior to selection of autologous T cells.

For example, during co-culture of autologous T cells and APCs presenting a mutant amino acid sequence, T cells with antigen specificity for the mutant amino acid sequence T cells may express any one or more of a variety of T cell activation markers that can be used to identify T cells. Such T cell activation markers include programmed cell death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3) 4-1BB, OX40, and CD107a, but are not limited to these. Accordingly, in one embodiment of the invention, selecting autologous T cells with antigenic specificity for mutant amino acid sequences includes PD-1, LAG-3, TIM-3, 4-1BB, OX40, and and selecting T cells that express any one or more of CD107a. Cells expressing one or more T cell activation markers can be determined based on the expression of the markers, for example, as described by Turcotte et al. , Clin. Cancer Res. , 20(2):331-43 (2013) and Gros et al. , J. Clin. Invest. , 124(5): 2246-59 (2014) (Example), using various methods known in the art, such as fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS). Can be screened using any of the following techniques:

In another embodiment of the invention, selecting autologous T cells with antigenic specificity for the mutant amino acid sequence comprises: (i) by means of a negative control upon co-culture with APCs presenting the mutant amino acid sequence; (ii) upon co-culture with APCs that display a mutant amino acid sequence; selecting T cells in which at least twice as many T cells secrete one or more cytokines as compared to the number of negative control T cells secreting one or more cytokines. One or more cytokines whose secretion by a T cell activates the T cell (e.g., a T cell receptor (TCR) expressed on the T cell that specifically binds and immunologically recognizes a mutant amino acid sequence) may include any cytokine characteristic of. Non-limiting examples of cytokines whose secretion is characteristic of T cell activation include IFN-γ, IL-2, and tumor necrosis factor alpha (TNF-α), granulocyte/monocyte colony stimulating factor (GM -CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22.

For example, autologous T cells may (a) have a concentration of a peptide comprising a mutant 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, (b) antigen-negative APCs pulsed with 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) encoding a mutant amino acid sequence. A mutation is identified if the T cells secrete at least twice as much IFN-γ when co-cultured with APCs into which the nucleotide sequence has been introduced, compared to the amount of IFN-γ secreted by the negative control. It can be considered to have "antigen specificity" for an amino acid sequence. Negative controls can be, for example, (a) antigen-negative APCs pulsed with the same concentration of an unrelated peptide (e.g., the wild-type amino acid sequence, or some other peptide with a sequence that differs from the mutant amino acid sequence); or (b) ) Autologous T cells (eg, derived from peripheral blood mononuclear cells (PBMCs)) may be co-cultured with APCs into which a nucleotide sequence encoding an unrelated peptide sequence has been introduced. Autologous T cells also showed higher amounts of T cells when cocultured with antigen-negative APCs pulsed with higher concentrations of peptides containing mutant amino acids compared to negative controls (e.g., the negative control described above). of IFN-γ, it may have "antigenic specificity" for the mutant amino acid sequence. IFN-γ secretion can be measured by methods known in the art, such as, for example, enzyme-linked immunosorbent assay (ELISA).

Alternatively, or additionally, the autologous T cells are (a) antigen-negative APCs pulsed with a concentration of a peptide containing the mutant amino acid sequence, or (b) a number of negative control T cells secreting IFN-γ. If at least twice as many T cells secrete IFN-γ upon co-culture with APCs into which a nucleotide sequence encoding the mutant amino acid sequence has been introduced compared to the mutant amino acid sequence It can be considered to have "antigenic specificity" for a sequence. Concentrations of peptides and negative controls can be as described herein with respect to other aspects of the invention. The number of cells secreting IFN-γ can be determined by methods known in the art, such as, for example, ELISPOT.

In one embodiment of the invention, T cells having antigenic specificity for a mutant amino acid sequence are characterized by: (1) expression of any one or more T cell activation markers described herein; T cells with antigenic specificity for the mutant amino acid sequence may both secrete greater amounts of one or more cytokines as described herein, but T cells with antigenic specificity for the mutant amino acid sequence may secrete greater amounts of one or more cytokines as described herein. of any one or more T cell activation markers without secreting cytokines, or a greater amount of one or more T cell activation markers without secreting any one or more T cell activation markers. May secrete cytokines.

In another embodiment of the invention, selecting autologous T cells that have antigenic specificity for the mutant amino acid sequence selectively expands autologous T cells that have antigenic specificity for the mutant amino acid sequence. Including. In this regard, the method provides for autologous cell proliferation in a manner that favors the proliferation of T cells that have antigenic specificity for the mutant amino acid sequence over T cells that do not have antigenic specificity for the mutant amino acid sequence. It may include co-culturing T cells and autologous APCs. Thus, a population of T cells is provided that has a high proportion of T cells that have antigen specificity for the mutant amino acid sequence compared to T cells that do not have antigen specificity for the mutant amino acid sequence.

In one embodiment of the invention, the method comprises obtaining a plurality of tumor fragments from a patient, each of the autologous T cells from the plurality of fragments separately as described herein with respect to other aspects of the invention. T cells from each of the plurality of fragments are co-cultured with autologous APCs displaying the mutant amino acid sequence, as described herein with respect to other aspects of the invention. Further comprising separately assessing antigen specificity for the sequences.

the T cell expresses multiple mutant amino acid sequences (e.g., multiple mutant amino acid sequences encoded by a TMG construct or multiple mutant amino acid sequences in a peptide pool pulsed onto the self APC); In embodiments of the invention where the autologous T cells are co-cultured, selecting the autologous T cells may further include evaluating the autologous T cells separately for antigen specificity for each of the plurality of mutant amino acid sequences. For example, the methods of the invention can be used as described herein with respect to other aspects of the invention (e.g., each presenting a different mutant amino acid sequence encoded by (or included in a pool) a construct). , by providing separate populations of APCs) may further include separately inducing the patient's autologous APCs to present each mutant amino acid sequence encoded by the construct (or included in the pool). . The method comprises separately co-culturing the patient's autologous T cells and different autologous APC populations displaying each mutant amino acid sequence, as described herein with respect to other aspects of the invention. may further include. The method comprises: (a) co-cultured with autologous APC displaying the mutant amino acid sequence; and (b) MHC molecules expressed by the patient, as described herein with respect to other aspects of the invention. It may further include separately selecting autologous T cells that have antigenic specificity for the mutant amino acid sequence presented in the context. In this regard, the method determines which mutant amino acid sequences encoded by (or contained in a pool of) a TMG construct encoding a plurality of mutant amino acid sequences can be immunologically (e.g., removed through a removal process) by an autologous T cell. (by).

In one embodiment of the invention, the method comprises expanding the number of selected autologous T cells to create a T cell population with antigenic specificity for a mutant amino acid sequence encoded by a cancer-specific mutation. It further includes obtaining. Amplification of the number of selected cells is described, for example, in U.S. Patent No. 8,034,334; U.S. Patent No. 8,383,099; , J. Immunother. , 26:332-42 (2003); and Riddell et al. , J. Immunol. Methods, 128:189-201 (1990), which may be accomplished by any of a number of methods known in the art. In one embodiment, expansion of T cell numbers is performed by culturing T cells with OKT3 antibody, IL-2, and feeder PBMC (eg, irradiated allogeneic PBMC). In this regard, the methods of the invention advantageously allow the generation of large numbers of T cells with antigenic specificity for mutant amino acid sequences.

T cells isolated by the methods of the invention may be useful for preparing cells for adoptive cell therapy. In this regard, one embodiment of the invention provides a method of preparing a population of T cells with antigenic specificity for a mutant amino acid sequence encoded by a cancer-specific mutation, the method comprising: Isolating T cells as described herein with respect to other aspects of the invention to obtain a population of T cells with antigenic specificity for the mutant amino acid sequence encoded by the tumor-specific mutation. and expanding the number of selected autologous T cells. Expanding the number of selected cells can be performed as described herein with respect to other aspects of the invention.

Another embodiment of the invention provides an isolated cell population prepared according to any method described herein with respect to other aspects of the invention. The cell population includes at least one other cell (e.g., PBMC that does not have antigen specificity for the mutant amino acid sequence encoded by the cancer-specific mutation) or a cell other than T cells (e.g., B cell, macrophages, neutrophils, red blood cells, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc.), as well as T cells with antigenic specificity for mutant amino acid sequences encoded by cancer-specific mutations. It can be a heterogeneous population of cells. Alternatively, the cell population may be substantially homogeneous, such that the population primarily comprises (e.g., consists essentially of) T cells that have antigenic specificity for the mutant amino acid sequence encoded by the cancer-specific mutation. It can be a group of people. The population is also such that all cells of the population are clones of a single T cell, such that all cells of the population have antigenic specificity for the mutant amino acid sequence encoded by the cancer-specific mutation. It can be a clonal population of cells. In one embodiment of the invention, the cell population is a clonal population comprising T cells with antigenic specificity for a mutant amino acid sequence encoded by a cancer-specific mutation, as described herein. . In one embodiment of the invention, about 1% to about 100% of the cell population, 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 The range defined by 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, or any two of the above values, provides antigen specificity for the mutant amino acid sequence. Contains T cells with Without being bound by any particular theory or mechanism, cell populations that contain a high proportion of T cells with antigenic specificity for the mutant amino acid sequence advantageously have significant T cell function (e.g. It is believed that there may be a low proportion of inappropriate cells that may interfere with the ability of the patient to target the destruction of cancer cells, and/or the ability to treat or prevent cancer.

Cell populations of the invention can be formulated into compositions such as pharmaceutical compositions. In this regard, the invention provides a pharmaceutical composition comprising any of the cell populations of the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention combine the cell populations of the invention with another pharmaceutically active substance(s) or drug(s), such as a chemotherapeutic agent (e.g., asparaginase, busulfan). , carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea yurea), methotrexate, paclitaxel, rituximab ), vinblastine, vincristine, etc.).

Preferably the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any of those conventionally used for the particular inventive cell population under consideration. 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 have harmful side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particular cell population of the invention and by the particular method used to administer the cell population of the invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the present invention. Suitable formulations may include any formulation for oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and intraperitoneal administration. More than one route may be used to administer the cell populations of the invention, and in certain instances, certain routes may provide a more rapid and effective response than others.

Preferably, the cell populations of the invention are administered intravenously (eg, by injection). When the cell populations of the invention are administered, pharmaceutically acceptable carriers for cells for injection include any isotonic carrier, such as normal saline (about 0.90% w in water). /v NaCl, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), Examples include about 5% dextrose in water, or lactated Ringer's solution. In one embodiment, human serum albumin is added to the pharmaceutically acceptable carrier.

It is contemplated that the cell populations and pharmaceutical compositions of the invention may be used in methods of treating or preventing cancer. Without being bound to any particular theory or mechanism, the T cells of the present invention are cancer-specific, such that the TCR expressed on the cells can mediate an immune response against target cells expressing the mutant amino acid sequence. It is believed that the amino acid sequence specifically binds to the mutant amino acid sequence encoded by the target mutation. In this regard, the present invention provides a method of treating or preventing cancer in a mammal, comprising an effective amount of a pharmaceutical composition or cell population as described herein for treating or preventing cancer in a mammal. A method comprising administering to a mammal any of the following.

The terms "treat" and "prevent" and their derivatives, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that those skilled in the art would recognize as having potential benefit or therapeutic effect. In this regard, the methods of the invention may provide any level and amount of treatment or prevention of cancer in a mammal. Additionally, treatment or prevention provided by the methods of the invention may include treatment or prevention of one or more conditions or symptoms of cancer that are being treated or prevented. For example, treatment or prevention can include promoting tumor regression. Also, for purposes herein, "prevention" may include delaying the onset of cancer, or a symptom or condition thereof.

For purposes of the present invention, the amount or dose of the cell population or pharmaceutical composition of the present invention to be administered (e.g., the number of cells if the cell population of the present invention is administered) is determined, e.g., in a mammal, as appropriate. should be sufficient to produce a therapeutic or prophylactic response over a period of time. For example, a dose of a cell population or pharmaceutical composition of the invention may be administered to bind to a mutant amino acid sequence encoded by a cancer-specific mutation, or for about 2 hours or more from the time of administration, The period of time or more must be sufficient to detect, treat or prevent cancer. In some embodiments, the period may be longer. The dosage will be determined by the efficacy of the particular cell population or pharmaceutical composition of the invention being administered and the condition of the animal (eg, human) and the weight of the animal (eg, human) being treated.

Many assays for determining dosage are known in the art. For purposes of the present invention, the extent to which target cells are lysed upon administration of a given dose of such T cells to a mammal of a group of mammals each receiving a different dose of T cells. Alternatively, an assay involving comparing the extent to which IFN-γ is secreted by T cells can be used to determine the starting dose to be administered to the mammal. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a fixed dose can be assayed by methods known in the art.

The dosage of a cell population or pharmaceutical composition of the invention will also be determined by the existence, nature, and extent of any untoward side effects that may accompany administration of a particular cell population or composition of the invention. Typically, the attending physician will assess various factors such as age, weight, general health, diet, sex, the cell population or pharmaceutical composition of the invention being administered, the route of administration, and the severity of the condition being treated. The dosage of the cell population or pharmaceutical composition of the invention to treat each individual patient is determined by considering the following.

In embodiments in which cell populations of the invention are administered, the number of cells per injection administered can vary, for example, in the range of 1 million to 100 billion cells; however, below this exemplary range, or greater amounts are within the scope of this invention. For example, a daily dose of host cells of the invention may range 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 any of the above values 2), 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) Cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells cells or the range defined by any two of the above values), more preferably from about 100 million cells to about 130 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells) 450 million cells, 650 million cells, 800 million cells, 900 million cells, 3 billion cells, 30 billion cells, 45 billion cells, 50 billion cells, 75 billion cells about 90 billion cells, about 100 billion cells, about 110 billion cells, about 120 billion cells, about 130 billion cells (or a range defined by any two of the above values).

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

Another embodiment of the invention provides any of the isolated cell populations or pharmaceutical compositions described herein for use in treating or preventing cancer in a mammal.

The cancer is advantageously acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, ocular cancer. cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva. Cancer, bile duct cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer. Cancer of the peritoneum, omentum and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer It can be any tumor, including cancer, uterine cancer, ureteral cancer, bladder cancer, solid tumors and liquid tumors. Preferably the cancer is an epithelial cancer. In one embodiment, the cancer is bile duct cancer, melanoma, colon cancer, or rectal cancer.

The mammal referred to in the method of the invention may be any mammal. The term "mammal" as used herein refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. Preferably, the mammal is from the order Felidae, which includes felines (cats) and canines (dogs). Preferably, the mammal is from the order Bovidae, which includes animals of the family Bovidae (cows) and animals of the family Boaridae (pigs), or from the order Perssodactyla, which includes animals of the family Equinidae (horses). Preferably, the mammal is of the order Primates, Ceboids or Simoids (monkeys) or of the order Anthropoids (humans and apes). A more preferred mammal is a human. In particularly preferred embodiments, the mammal is a patient who expresses a cancer-specific mutation.

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

For Examples 1-7, materials and methods are shown below.

Whole exome sequencing Jones et al. Whole exome sequencing of cryopreserved tumor tissue (embedded in OCT) and normal peripheral blood cells was performed using Personal Genome Diagnostics (PGDx, B
altimore, MD). The average number of unambiguous, high-quality reads at each base of the sequence was 155 and 160 for tumor and normal (PBMC) DNA, respectively.

Patient Treatment and Generation of Tumor-Infiltrating Lymphocytes (TILs) for Adoptive Cell Therapy Patient 3737 was treated with an Institutional Review Board (IRB) approved protocol: The patient was enrolled in a Phase II study using intercultured autologous tumor-infiltrating lymphocytes (Clinical trial registration ID: NCT01174121). The protocol was designed to evaluate the safety and efficacy of adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) in gastrointestinal cancer patients.

The TIL used for initial treatment of patients was described by Jin et al. , J. Immunother. , 35:283-292 (2012). Briefly, excised tumors were minced into approximately 1-2 mm pieces and individual pieces were placed in 2 ml of complete medium (CM) containing high dose IL-2 (6000 IU/ml, Chiron, Emeryville, CA). ) into the wells of a 24-well plate. CM consisted of RPMI supplemented with in-house 10% human serum, 2mM L-glutamine, 25mM HEPES, and 10μg/ml gentamicin. Additionally, mixed tumor digests were also cultured in CM with high doses of IL-2. After initial expansion of T cells (between 2-3 weeks), 5 x ¹⁰ T cells from the selective culture were cultured using irradiated allogeneic PBMC in gas permeable G-Rex 100 flasks. , rapidly at a ratio of 1:100 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). Amplified. The 50/50 medium consisted of a 1:1 mixture of CM and AIM-V medium. All cells were cultured at 37°C, 5% _CO2 . Prior to injection, cell numbers were rapidly expanded for 2 weeks. Patient 3737 received non-myeloablative lymphodepleting therapy consisting of cyclophosphamide and fludarabine prior to receiving a total of 42.4 billion T cells in conjunction with four doses of high-dose IL-2. Regimen).

The TILs used for the second patient treatment were made in a similar manner to the first treatment with the following modifications. The first therapeutic product (patient 3737-TIL) was composed of a combination of 5 individual TIL cultures. These 5 cultures were individually evaluated for CD4 and Vβ22 expression and reactivity to the mutant ERBB2IP, and one culture was found to be highly enriched in Vβ22+ERBB2IP mutation-reactive CD4+ T cells. This one TIL culture (after initial expansion with high dose IL-2) was then rapidly expanded as described above. The patient received the same non-myeloablative lymphodepletion therapy as the initial treatment, prior to receiving a total of 126 billion T cells, along with four doses of high-dose IL-2.

Generation of TMG Constructs Briefly, for each non-synonymous substitution mutation identified by whole exome sequencing, a "minigene" encoding the corresponding amino acid mutation (flanking by 12 amino acids of the wild-type protein sequence) is generated. The construct was created. Multiple minigenes were genetically fused to create TMG constructs. These minigene constructs were codon-optimized and synthesized as DNA String constructs (Life Technologies, Carlsbad CA). TMG was then cloned into the pcDNA3.1 vector using In-Fusion technology (Clontech, Mountain View, Calif.). Nine "wild type reversion" TMG-1 constructs (Gene Oracle, Mountain View, CA) were generated using site-directed mutagenesis. Standard Sanger sequencing (Macro
The nucleotide sequence of all TMG was confirmed by Gene and Gene Oracle).

Generation of autologous APCs Monocyte-derived immature DCs were generated using the plastic attachment method. Briefly, autopheresis samples were thawed, washed, set to 5-10 x ¹⁰ cells/ml using pure AIM-V medium (Life Technologies), and then placed in appropriately sized tissue culture flasks. The medium was incubated at 37° C., 5% CO ₂ at approximately 1×10 ⁶ cells/cm ² . After 90 minutes, non-adherent cells were harvested and the flasks were thoroughly washed with AIM-V medium and then incubated with AIM-V medium for an additional 60 minutes. The flasks were then thoroughly washed again with AIM-V medium, and the adherent cells were then incubated with DC medium. DC medium consisted of RPMI containing 5% human serum (collected and processed in-house), 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine, 800 IU/ml GM-CSF, and 800 U/ml IL-4. (media supplements from Life Technologies, cytokines from Peprotech). On the third day, fresh DC medium was added to the culture. Fresh or frozen/thawed DCs were used for experiments 5-7 days after initial stimulation. In all experiments, cells were phenotyped for expression of CD11c, CD14, CD80, CD86, and HLA-DR (all from BD Bioscience) using flow cytometry, indicating that cells were primarily immature DCs (CD11c+, CD14-, CD80 ^low , CD86+, and HLA-DR+; data not shown).

Antigen-presenting B cells were generated using CD40L and IL-4 stimulation methods. Briefly, positive B cells were selected from autopheresis samples using human CD19 microbeads (Miltenyi Biotec). CD19+ cells were then cultured in B cell medium at an approximately 1:1 ratio with irradiated (6000 rad) 3T3 cells stably expressing CD40L (3T3-CD40L). B cell culture medium contained 7.5-10% human serum (our laboratory), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life
IMDM medium (Life Technologies) supplemented with 10 μg/ml gentamicin (CellGro, Manassas, VA), 2 mM L-glutamine (Life Technologies), and 200 U/ml IL-4 (Peprotech). Gies). Fresh B cell medium was added starting on day 3, and medium was added or replaced every 2-3 days thereafter. Irradiated 3T3-CD40L feeder cells were also added as needed. Antigen-presenting B cells were typically used in experiments 2-3 weeks after initial stimulation.

Generation of in vitro transcribed RNA (IVT) RNA The plasmid encoding the tandem minigene was linearized with the restriction enzyme SacII. A control pcDNA3.1/V5-His-TOPO vector encoding GFP was linearized with NotI. Restriction digests were terminated with EDTA, sodium acetate and ethanol precipitation. Complete digestion of the plasmid was confirmed by standard agarose gel electrophoresis. Approximately 1 μg of linearized plasmid was used to generate IVT RNA using the message machine T7 Ultra kit (Life Technologies) as per the manufacturer's instructions. RNA was precipitated using the _LiCl2 method and RNA purity and concentration was assessed using a NanoDrop spectrophotometer. The RNA was then aliquoted into microtubes and stored at -80°C until use.

RNA Transfection APCs (DCs or B cells) were collected, washed once with PBS, and then resuspended in Opti-MEM (Life Technologies) at 10-30×10 ⁶ cells/ml.
IVT RNA (4 μg or 8 μg) was aliquoted to the bottom of a 2 mm gap electroporation cuvette and 50 μl or 100 μl of APC was added directly to the cuvette. Therefore, the final concentration of RNA used in electroporation was 80 μg/ml. Electroporation was performed using a BTX-830 square wave electroporator. DCs were electroporated with 150V, 10 ms, 1 pulse, and B cells were electroporated with 150V, 20 ms, 1 pulse. Transfection efficiency using these settings was consistently between 70 and 90% as assessed with GFP RNA (data not shown). All steps were performed at room temperature. Immediately after electroporation, cells were transferred to polypropylene tubes containing DC medium or B cell medium supplemented with appropriate cytokines. Transfected cells were incubated overnight (12-14 hours) at 37°C, 5% _CO2 . Cells were washed once with PBS prior to use in co-culture assays.

Peptide Pulse Autologous B cells were harvested, washed, and then resuspended at 1 x ¹⁰ cells/ml in B cell medium supplemented with IL-4 and then treated with 1 μg/ml 25-mer peptide overnight ( 12-14 hours) at 37°C and 5% _CO2 . After overnight pulsing, B cells were then washed twice with PBS, then resuspended in T cell medium and used immediately for co-culture assays. The peptides used were: mutant ERBB2IP (

(SEQ ID NO: 73)); wild type ERBB2IP (

(SEQ ID NO: 45)); mutant ALK (RVLKGGSVRKLRHAKQLVLELGEEA (SEQ ID NO: 46)) was used as a negative control. Mutant ERBB2IP peptides were purchased from three different sources (GenScript, Piscataway, NJ, Peptide 2.0, Chantilly, VA and SelleckChem, Houston TX), all yielding the same in vitro results. Meanwhile, wild type ERBB2IP peptide and mutant ALK peptide were purchased from Peptide 2.0. To culture allogeneic EBV-B cells, B cell medium was replaced with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies), 10 μg/ml gentamicin (CellGro) and 2 mM L- RPMI medium containing glutamine was used.

T Cell Sorting, Expansion, and Cloning A BD FACSAria IIu and BD FACSJazz were used in all experiments requiring cell sorting. In the experiments shown, sorted T cells were cultured with over-irradiated (4000 rad) allogeneic feeder cells (donor A pool of three leukocyte-depleted transfusion samples with different values was used for amplification. Limiting dilution cloning was carried out in 96-well round bottom plates with 5e4 feeder cells per well and 1 to 100 cells per well.
2T cells using the stimulation conditions described above. The medium was changed starting approximately one week after stimulation and then every other day or as needed. Cells are typically used in assays or further expanded approximately 2-3 weeks after initial stimulation.

Co-culture assays: IFN-γ ELISPOT and ELISA, flow cytometry for cell surface activation markers, and intracellular cytokine staining (ICS)
When using DCs as APCs, approximately 3.5×10 ⁴ to 7×10 ⁴ DCs were used per well of a 96-well flat bottom or round bottom plate. When using B cells as APCs, approximately 2×10 ⁵ cells were used per well of a 96-well round bottom plate. In ELISPOT assays, 1×10 ³ to 1×10 ⁴ effector T cells were used per well, and in flow cytometry assays, 1×10 ⁵ effector T cells were used per well. T cells are typically thawed and rested for 2 days in 50/50 medium containing IL-2 (3000 IU/ml IL-2) and then incubated (three times) with PBS prior to co-culture assays. ) Washed. All co-cultures were performed in the absence of externally added cytokines. For all assays, plate-bound OKT3 (0.1 μg/ml or 1 μg/ml) was used as a positive control.

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

For the IFN-γ ELISPOT assay, briefly, ELIIP plates (Millipore, MAIPSWU) were pretreated with 50 μl of 70% ethanol per well for 2 min, washed 3 times with PBS, and then treated with 50 μl of 10 μg/ml IFN. -γ capture antibody (Mabtech, clone: 1-D1K) and incubated overnight in the refrigerator. For OKT3 controls, wells were coated with a mixture of IFN-γ capture antibody (10 μg/ml) and OKT3 (1 μg/ml). Prior to co-cultivation, plates were washed three times with PBS and subsequently blocked with 50/50 medium for at least 1 hour at room temperature (RT). After 20 to 24 hours of co-culture, cells were flicked out of the plate, washed six times with PBS (with 0.05% Tween-20 (PBS-T)), and then incubated at room temperature. for 2 hours with 100 μl/well of 0.22 μm filtered 1 μg/ml biotinylated anti-human IFN-γ detection antibody solution (Mabtech, clone: 7-B6-1). Plates were then washed three times with PBS-T followed by incubation for 1 hour with 100 μl/well streptavidin-ALP (Mabtech, Cincinatti, OH, diluted 1:3000). Plates were then washed six times with PBS followed by development with 100 μl/well of 0.45 μm filtered BCIP/NBT substrate solution (KPL, Inc.). The reaction was stopped by rinsing thoroughly with cold tap water. ELISPOT plates were scanned and counted using an ImmunoSpot plate reader and associated software (Cellular Technologies, Ltd, Shaker Heights, OH).

Expression of T cell activation markers OX40 and 4-1BB was assessed by flow cytometry at approximately t=22-26 hours post-stimulation. Briefly, cells were pelleted, washed with FACS buffer (1x PBS supplemented with 1% FBS and 2mM EDTA), and then stained with appropriate antibodies for approximately 30 min at 4°C in the dark. Cells were washed at least once with FACS buffer prior to acquisition on a BD FACSCanto II flow cytometer. All data were gated on live single cells (PI negative).

Cytokine production was assessed using intracellular cytokine staining (ICS) and flow cytometry. Briefly, target cells and effector cells were mixed in the wells of a 96-well plate before both GolgiStop and GolgiPlug were added to the culture (BD Biosciences). GolgiStop and GolgiPlug were used at 1/2 the manufacturer's recommended concentration. After t=6 hours of 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 for cell surface markers (described above). Cells were then washed twice with FACS buffer prior to fixation and permeabilization. Cells were then washed with Perm/Wash buffer and stained with antibodies against cytokines for 30 minutes at 4°C in the dark. Cells were washed twice with Perm/Wash buffer and resuspended in FACS buffer prior to acquisition on a FACS Canto II flow cytometer. All flow cytometry data were generated using FLOWJO software (TreeStar
Inc.).

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

Flow Cytometry Antibodies The following titered anti-human antibodies were used for cell surface staining: CCR7-FITC (clone: 150503), CD45RO-PE-Cy7 (clone: UCHL1), CD62L-APC (clone: DREG). -56), CD27-APC-H7 (clone: M-T271), CD4-efluor 605NC (clone: OKT4), CD57-FITC (clone: NK-1), CD28-PE-Cy7 (clone: CD28.2) , CD127-APC (clone: eBioRDR5), CD3-AF700 (clone: UCHT1), CD4-FITC, PE-Cy7, APC-H7 (clone: SK3), CD8-PE-Cy7 (clone: SK1), Vβ22-PE (clone: IMMU 546), Vβ5.2-PE (clone: 36213), OX40-PE-Cy7 or FITC (clone: Ber-ACT35), 4-1BB-APC (clone: 4B4-1), and CD107a-APC -H7 (clone: H4A3). All antibodies except CD4 -EFLUOR605NC (EBIOSCIENCE), Vβ22 -PE and Vβ5.2 -PE (Vβ5.2 -PE (VECKMAN COULTER)), 4-1 BB -APC and OX40 -PE -CY7 (BioleND). It was from BD BIOSCIENCES. For intracellular cytokine staining, the following were appropriately titrated: 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) anti-human antibodies were used. All ICS antibodies except IL-4-PE-Cy7 (BD Bioscience) were from eBioscience. The TCR-Vβ repertoire was evaluated using the IO Mark Beta Mark TCR V kit (Beckman Coulter).

Sequencing of ERBB2IP mutations Sanger sequencing was used to verify the ERBB2IP mutations found by whole exome sequencing. Total RNA was extracted from snap-frozen T cells or tumor tissue (OCT blocks) using the RNeasy Mini kit (Qiagen). Total RNA was then reverse transcribed into cDNA using ThermoScript reverse transcriptase with oligo dT primer (Life Technologies). The normal cDNA and tumor cDNA were then combined with the following ERBB2IP primers flanking the mutation: ERBB2IP Seq Forward: 5'-TGT TGA CTC AAC AGC CAC AG-3' (SEQ ID NO: 47) and ERBB2IP Seq Reverse: 5'-CTG It was used as a template in PCR using GAC CAC TTT TCT GAG GG-3' (SEQ ID NO: 48). Phusion DNA polymerase (Thermo Scientific) was used with the recommended 3-step protocol with an annealing temperature of 58°C (15 seconds) and extension at 72°C (30 seconds). PCR products were isolated by standard agarose gel electrophoresis and gel extraction (Clontech). Products were directly sequenced using the same PCR primers (Macrogen).

quantitative PCR
Total RNA was extracted from snap-frozen T cells or tumor tissue (OCT blocks) using the RNeasy Mini kit (Qiagen, Venlo, Netherlands). Total RNA was then reverse transcribed into cDNA using qScript cDNA Supermix (Quanta Biosciences, Gaithersburg, MD). Gene-specific Taqman primers and probe sets for human β-actin (catalog number: 401846) and ERBB2IP (catalog number: 4331182) were purchased from Life Technologies. Quantitative PCR was performed using a 7500 Fast Real Time PCR machine using TAQMAN Fast
It was performed using Advanced Master Mix (all from Applied Biosystems). Specificity of the amplified products was confirmed by standard agarose gel electrophoresis. All calculated threshold cycles (Ct) were 30 or less.

TCR-Vβ deep sequencing Genomic DNA isolated from peripheral blood, T cells, and frozen tumor tissue was subjected to TC sequencing by immunoSEQ, Adaptive Biotechnologies (Seattle, WA) using the DNeasy blood and tissue kit (Qiagen). R -Vβ deep sequencing was performed. The total number of productive TCR reads per sample ranged from 279,482 to 934,672. Only useful rearrangements of TCRs were used in calculating TCR frequencies.

TCR Sequencing and Construction of ERBB2IP Mutation-Responsive TCR T cells were pelleted and total RNA was isolated (RNeasy Mini kit, Qiagen). 5′ RACE was then performed on total RNA using TCR α and β chain constant primers as instructed by the manufacturer (SMARTer RACE cDNA amplification kit, Clontech). Program 1 of the kit was used for PCR with a modified extension time (2 minutes instead of 3 minutes). The sequences of the α chain and β chain constant primers are: 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 TCR PCR product was then isolated by standard agarose gel electrophoresis and gel extraction (Clontech). The products were then sequenced directly or TOPO-TA cloned followed by sequencing of individual colonies (Macrogen). For sequencing of known Vβ22+ T cell clones, qScript cDNA Supermix (
cDNA was generated from RNA using Quanta Biosciences). These cDNAs were then used as templates in a PCR using the TCR-β constant primer (described above) and the Vβ22-specific primer: 5'-CAC CAT GGA TAC CTG GCT CGT ATG C-3' (SEQ ID NO: 51). PCR products were isolated by standard agarose gel electrophoresis and gel extraction (Clontech). The product was directly sequenced (Macrogen) using the TCR-β constant chain nested primer: 5'-ATT CAC CCA CCA GCT CAG-3' (SEQ ID NO: 52).

Construction of the Vβ22+ERBB2IP-mutant TCR involves fusing the VDJ region of Vβ22+TCR-α to the mouse TCR-α constant chain and the Vβ22+TCR-β-VDJ region to the mouse TCR-β constant chain. This was done by letting The α and β chains were separated by a furin SGSG P2A linker. The use of murine TCR constant regions facilitates pairing of the introduced TCR and identification of positively transduced T cells by flow cytometry using an antibody specific for the murine TCR-β chain (eBioscience). becomes easier. The TCR construct was synthesized and cloned into the MSGV1 retroviral vector (Gene Oracle).

TCR transduction of peripheral blood T cells Autopheresis samples were thawed and treated with 5% in-house human serum, 10 μg/ml gentamicin (CellGro), 100 U/ml penicillin and 100 μg/ml streptomycin, 1.25 μg/ml amphotericin B (Fungizone). and 2×10 ⁶ cells/ml in T cell medium consisting of a 50/50 mixture of RPMI and AIM-V medium supplemented with 2 mM L-glutamine (both from Life Technologies). 2× ¹⁰ cells (1 ml) were stimulated in 24-well plates containing 50 ng/ml soluble OKT3 (Miltenyi Biotec) and 300 IU/ml rhu IL-2 (Chiron) for 2 days before retroviral transduction. . To generate transient retroviral supernatants, Lipofectamine 2000 (Life Technologies) was used to generate retroviral vector MSGV1 (1.5 μg/well) encoding a Vβ22-positive ERBB2IP mutation-specific TCR and an envelope encoding Plasmid RD114 (0.75 μg/well) was co-transfected into the retroviral packaging cell line 293GP (1×10 cells per well of a 6-well poly-D ^- lysine coated plate, plated the day before transfection). did. At 42-48 hours post-transfection, retroviral supernatants were collected, diluted 1:1 in DMEM medium, and transferred into retronectin-coated (10 μg/ml, Takara) non-tissue culture treated 6-well plates for 32 hours. Centrifuged at 2,000 g for 2 hours at °C. Activated T cells (2×10 ⁶ per well, 0.5×10 ⁶ cells/ml in T cell medium containing IL-2) were then spun into retroviral plates at 300 g for 10 minutes. Activated T cells were transduced overnight, removed from plates, and further cultured in T cell medium containing IL-2. GFP and mock transduction controls were included in the transduction experiments. Cells were typically assayed 10-14 days after retroviral transduction.

Example 1
This example illustrates a method for identifying one or more genes in the nucleic acid of a patient's cancer cells, each gene containing a cancer-specific mutation encoding a mutant amino acid sequence.

TIL-based adoptive cell therapy for patients with gastrointestinal (GI) cancer treated a 43-year-old woman (Patient (Pt.) 3737) with widely metastatic cholangiocarcinoma that had progressed through multiple chemotherapy regimens. (ACT) protocol. The clinical characteristics of patient 3737 are shown in Table 1.

Lung metastases were excised and used as a source for whole exome sequencing and generation of therapeutic T cells. Table 2 shows somatic mutations identified by whole exome sequencing of metastatic lung nodules from patient 3737. Approximately 70% of the tumor nodules were estimated to be tumors based on pathological analysis of hematoxylin and eosin (H&E) stained sections. Whole exome sequencing revealed 26 nonsynonymous mutations (Table 2).

Example 2
This example describes a method for inducing a patient's autologous APC to present a mutant amino acid sequence; a method for co-culturing a patient's autologous T cell population with autologous APC presenting a mutant amino acid sequence; and (a (b) selecting autologous T cells that have antigenic specificity for the mutant amino acid sequence presented in the context of MHC molecules expressed by the patient; The method will be illustrated with an example.

For each mutation identified in Example 1, a minigene construct encoding the mutant amino acid was designed flanked on each side by 12 amino acids from the endogenous protein. Multiple minigenes were synthesized in tandem to create tandem minigene (TMG) constructs (Table 3). In Table 3, underlining indicates mutant amino acids and novel sequences encoded by point mutations or nucleotide insertions or deletions. For splice-site donor mutations (HLA-DOA and LONRF3), an abrupt mutation that enters the downstream intron and continues until the next stop codon is based on the assumption that the mutation prevents splicing at that site. Mutant minigene transcripts were designed. Splice-site acceptor mutations in DIP2C were not evaluated.

The TMG construct was then used as a template for the generation of in vitro transcribed (IVT) RNA. Each of these IVT TMG RNAs was then individually transfected into autologous APCs (DCs) followed by co-culture with TILs to determine which of the processed and presented mutant antigens were recognized by TILs. It was determined. 3737-TIL was observed to be reactive against a mutant antigen present in TMG-1 but not in TMG-2 or TMG-3 (Fig. 1A). Furthermore, reactivity was predominant in the CD4+ T cell population, as demonstrated by upregulation of activation markers OX40 and 4-1BB (Tables 4A and 4B). Tables 4A and 4B display the indicated tandem minigenes (TMG ) Shows the percentage of 3737-TILs with the indicated phenotypes as detected by flow cytometry after co-culture with DCs transfected with the constructs. Mock transfected cells were treated with transfection reagent alone without addition of nucleic acid. Data were gated on live CD3+ cells.

Although some 4-1BB upregulation was observed in the CD4 negative (CD8+) T cell population, these cells were sorted but no reactivity to TMG was found. To determine which of the nine mutations in TMG-1 was recognized by 3737-TIL, TMG-1, each containing one of the mutations reverted to the wild-type sequence, Nine additional constructs were synthesized. Only when the ERBB2-interacting protein (ERBB2IP) mutation was reverted to the wild-type sequence, the reactivity of 3737-TIL to TMG-1 was abolished, indicating that TIL could specifically recognize the ERBB2IP ^E805G mutation. (Fig. 1B).

ERBB2IP mutation-reactive T cell responses were molecularly characterized. IFN-γ ELISPOT assay was performed and results were measured at 20 hours. Patient 3737-TIL was preincubated with nothing or with the indicated HLA blocking antibodies (against MHC-I, MHC-II, HLA-DP, HLA-DQ, or HLA-DR). DCs transfected with TMG-1 were co-cultured (FIG. 2A). As controls for antibody blocking, HLA-A2-restricted MART-reactive T cells DMF5 (Fig. 2B) and HLA-DR-restricted tyrosinase-reactive T cells T4 (Fig. 2C) were tested without or indicated. MART and tyrosinase positive 624-CIITA melanoma cell line were co-cultured with HLA blocking antibody and pre-incubated. The 3737-TIL response was blocked by anti-HLA-DQ antibody (Figure 2A).

Another IFN-γ ELISPOT assay was performed and results were measured at 20 hours. Patient 3737-TIL and autologous B cells or allogeneic EBV-B with partially matched HLA-DQ loci pulsed overnight with DMSO, 25 amino acid long peptides of mutated ALK or mutated ERBB2IP. cells were co-cultured (Fig. 2D).

Another IFN-γ ELISPOT assay was performed and results were measured at 20 hours. Patient 3737-TILs were co-cultured with autologous B cells pulsed overnight with the mutERBB2IP 25 amino acid peptide or the indicated truncated mutERBB2IP peptides (Figure 2E).

As shown in Figures 2A-2E, the 3737-TIL response was HLA-DQB1 ^* 0601 allele-restricted and the minimal epitope was located within the 13 amino acid sequence, NSKEETGHLENGN (SEQ ID NO: 29).

Example 3
This example describes the use of autologous open repertoire peripheral blood T cells genetically modified with the TCR-Vβ22 chain (corresponding to its α chain) of the ERBB2IP-specific CD4+ T cells identified in Example 2.
blood T cells) conferred specific reactivity towards the mutant ERBB2IP peptide.

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 amplified in bulk and cloned by limiting dilution. Flow cytometry-based investigation of the TCR-Vβ repertoire demonstrated that >95% of the bulk amplified population was Vβ22+ and 10/11 of the clones evaluated were purely Vβ22+. TCR sequence analysis revealed that the TCRβV-DJ sequences were identical in 6/6 of the Vβ22+ clones tested (Table 5), indicating that the majority of ERBB2IP mutation-reactive T cells were the dominant Vβ22+ T cells. It was shown to be composed of clones.

T cell clones expressing this Vβ22 TCR specifically produced the cytokine IFN-γ upon stimulation with the mutant ERBB2IP peptide (Table 6). CD4+Vβ22+ clones were co-cultured for 6 hours with autologous B cells pulsed overnight with OKT3 or a 25 amino acid long peptide of wild type (wt) ERBB2IP, mutant (mut) ALK, or mutERBB2IP. Table 6 shows Vβ22+ and Vβ22 in CD4+ that produce intracellular IFN-γ (IFN-γ+) or do not produce intracellular IFN-γ (IFN-γ−) after co-culture, as measured by flow cytometry. - indicates the percentage of clones. Data are representative of two clones that both share identical TCR-Vβ sequences.

Furthermore, autologous open repertoire peripheral blood T cells genetically modified by matching this TCR-Vβ22 chain with its α chain (Table 7) showed specific reactivity against the mutant ERBB2IP peptide (Tables 8A and 8B). granted. This demonstrated that this TCR specifically recognizes the ERBB2IP ^E805G mutation. Autologous open repertoire peripheral blood T cells were transduced (Td) with TCR from Vβ22+ clones (Table 8A) or treated with transduction reagent alone without the addition of nucleic acid (Mock) (Table 8B) and then reacted with The properties were evaluated as shown in Table 6. The endogenous Vβ22+ TCR constant region was replaced with a mouse constant region, allowing detection of the introduced TCR using an antibody against mouse TCRβ constant region (mTCRβ). Plate-bound OKT3 was used as a control in all assays. Tables 8A and 8B show that mTCRβ+ and mTCRβ−, producing intracellular IFN-γ (IFN-γ+) or not producing intracellular IFN-γ (IFN-γ−), as measured by flow cytometry. Percentages of cells are shown.

Example 4
This example illustrates a method of treating cancer using the autologous cells identified in Example 2.

Patient 3737 received 4 doses of IL-2 to enhance T cell proliferation and function after adoptive transfer of 42.4 billion TILs containing CD4+ERBB2IP mutation-reactive T cells. Patient 3737 underwent resection of the pulmonary lesion for treatment. Tumors were then minced into small pieces and incubated with high doses of IL-2 to amplify tumor-infiltrating lymphocytes (TILs). After initial cell number expansion in IL-2, the number of selected TIL cultures was further expanded using a rapid amplification protocol (REP) containing irradiated allogeneic peripheral blood feeder cells, OKT3 and IL-2. Amplified for 2 weeks. Prior to cell infusion, patients were preconditioned with cyclophosphamide (CTX: 60 mg/kg once daily for 2 days) followed by fludarabine (Flu: 25 mg/m ² for 5 days). Patient 3737-TIL contains 42.4 billion TIL containing >10 billion (25%) ERBB2IP mutation-reactive T cells and was administered on day 0 followed by IL-2 (aldesleukin, 7.2e5 IU /kg) every 8 hours. The patient received a total of 4 doses of IL-2.

3737-TILs were co-cultured with DCs transfected with TMG-1 or TMG-1 encoding wild-type (wt) reverted ERBB2IP, and flow cytometry was used to detect the presence of CD4+ T cells at 24 hours post-stimulation. Expression of OX40 and Vβ22 was evaluated. Stimulation of plate-bound OKT3 was used as a positive control. Flow cytometry analysis showed that approximately 25% of the total administered 3737-TIL product was composed of Vβ22+ mutation-reactive T cells (Figure 3A, Table 9), indicating that infusion of >10 billion ERBB2IP mutation-specific CD4+ T cells It has been proven that it is equal to Table 9 shows the percentage of Vβ22+ and Vβ22− cells that express OX40 (OX40+) or do not express OX40 (OX40−) as determined by flow cytometry.

IFN-γ ELISA assays were performed on serum samples from patient 3737 before and after adoptive cell transfer of 3737-TIL. The results are shown in Figure 3B. As shown in Figure 3B, increased levels of IFN-γ were detected in patient serum during the first 5 days after cell injection.

Although the patient had clear signs of progressive disease prior to cell injection, tumor regression was observed at 2-month follow-up, and target lesions in the lungs and liver all continued to regress, and after treatment A maximum of 30% decline was reached after a month (Figure 3C). The patient experienced disease stabilization for approximately 13 months after cell infusion, after which disease progression was observed only in the lungs and not in the liver.

Example 5
This example demonstrates the in vitro phenotype and function of the cells of Example 4.

To determine whether there is evidence that CD4+ERBB2IP mutation-reactive T cells play a role in disease stabilization, the in vitro phenotype and function of the cells were evaluated. 3737-TIL and autologous B cells pulsed overnight with a 25 amino acid long peptide of wild type (wt) ERBB2IP, mutant (mut) ALK or mut ERBB2IP were co-cultured for 6 hours. Flow cytometry was used to assess the expression of Vβ22 and detect intracellular production of IFN-γ (Table 10A), tumor necrosis factor (TNF) (Table 10B), and IL-2 (Table 10C) in the CD4+ population. did. The percentages of cells with the indicated phenotypes are shown in Tables 10A-10C. Table 10D shows the percentage of Vβ22+ cells that expressed the indicated number of cytokines. Stimulation with the mutant ERBB2IP peptide induced robust co-expression of IFN-γ, TNF and IL-2 (Tables 10A-10C), but little or no IL-4 or IL-17; Vβ22+ERBB2IP mutation-reactive CD4+ T cells were found to be multifunctional Th1 cells.

Further phenotypic characterization revealed that these cells were primarily effector memory CD4+ T cells with cytolytic potential (Tables 11 and 12). Patient 3737-TIL was evaluated by flow cytometry for expression of Vβ22 (corresponding to ERBB2IP mutation-reactive T cells) and T cell differentiation markers CD28, CD45RO, CD57, CCR7, CD127, CD62L and CD27. Data were gated on live CD3+CD4+ cells. Positive and negative quadrant gates were set using isotype stained or unstained cells. The percentages of cells with the indicated phenotypes are shown in Table 11. Human peripheral blood cells (containing T cells at all stages of differentiation) were included in the experiment to confirm that the antibodies were working.

Patient 3737-TIL and autologous B cells pulsed overnight with OKT3 or a 25 amino acid long peptide of wild type (wt) ERBB2IP, mutant (mut) ALK or mutERBB2IP were co-cultured for 6 hours. At the start of co-culture, an antibody specific for the degranulation marker CD107a was added. Flow cytometry was used to assess the expression of Vβ22 and detect cell surface mobilization of CD107a. Data were gated on the CD4+ population. The percentages of cells with the indicated phenotypes are shown in Table 12.

There also appeared to be a small population of polyfunctional, Vβ22-negative, ERBB2IP mutation-reactive CD4+ T cells present in 3737-TILs (Tables 9 and 10). These Vβ22-negative cells were sorted by FACS and then incubated with IL before co-culturing with autologous B cells pulsed overnight with wild type (wt) ERBB2IP, mutant (mut) ALK or mutERBB2IP 25 amino acid long peptides. -2-containing medium for 2 days (rest). Flow cytometry was used to assess the expression of Vβ22, IL-2 (Table 13C), TNF (Table 13B), and IFN-γ (Table 13A) cells in the CD4+ population 6 hours (h) after stimulation. Endogenous production was detected. The percentages of cells with the indicated phenotypes are shown in Tables 13A-13C.

Expression OX40 and Vβ22 in the CD4+ population was assessed 24 hours post-stimulation using flow cytometry. Cells that upregulated OX40 were sorted, cell numbers expanded, and TCR-Vβ repertoires analyzed by flow cytometry. The results are shown in Figure 3D. Upon selection of Vβ22-negative cells and subsequent activation of these cells, there is one or more additional clonotypes among 3737-TILs that are reactive to this epitope (Tables 13A-13C), of which the most predominant The predominant clonotype was revealed to be Vβ5.2 (Fig. 3D).

The sorted cells described in FIG. 3D and autologous B cells pulsed overnight with a 25 amino acid long peptide of wtERBB2IP, mut ALK, or mutERBB2IP were co-cultured for 6 hours. Flow cytometry was used to assess the expression of Vβ5.2 and detect intracellular production of IL-2 (Table 14C), TNF (Table 14B), and IFN-γ (Table 14A) in the CD4+ population. Table 15 shows the percentage of Vβ5.2+ cells that expressed the indicated number of cytokines.

Upon stimulation with mutant ERBB2IP, Vβ22-negative cells that upregulated OX40 were selected and cell numbers expanded. RNA from these cells was then isolated and rapid amplification of 5' complementary DNA ends (5'RACE) was performed using TCR-β constant chain primers to identify the expressed TCR-Vβ sequences. TOPO-TA cloning was performed on polymerase chain reaction (PCR) products and then sequenced on individual colonies. Flow cytometry demonstrated that 40-50% of these T cells were Vβ5.2 (TRBV5-6). According to Sanger sequencing, 3/7 of the TOPO-TA colonies were Vβ5.2 (TRBV5-6) with the sequence shown in Table 16. Table 16 shows the most frequent TCRβVDJ sequences of Vβ22-negative ERBB2IP mutation-reactive T cells.

Most Vβ5.2+ cells produced multiple cytokines in an antigen-specific manner (Tables 14A-14C, 15 and 16). There also appeared to be a small population of Vβ5.2 negative (and Vβ22 negative) CD4+ T cells that recognized mutant ERBB2IP (Tables 14A-14C and 15). Therefore, the TILs used to treat patient 3737 contain at least three clones of different multifunctional CD4+ T cells that recognize the same mutation in ERBB2IP, and this mutation is highly immunogenic. showed that.

Example 6
This example demonstrates the in vivo persistence of the cells of Example 4.

To determine whether there is evidence that CD4+ERBB2IP mutation-reactive T cells play a role in disease stabilization, the in vivo persistence of the cells was assessed. TCR-Vβ deep sequencing revealed that these clonotypes were rare or undetectable in peripheral blood before ACT (FIGS. 4A and 4B). Ten days after ACT, both clones represented >2% of the total T cells in the peripheral blood, but this decreased to <0.3% by day 34 after cell injection (FIGS. 4A and 4B). The three lung metastases resected approximately 1.5 years after ACT were infiltrated by ERBB2IP mutation-reactive T cells (Figures 4A and 4B), and these cells were responsible for cancer regression and disease stabilization. was shown to contribute to The Vβ22+ERBB2IP mutation-reactive clone was the most frequent clone detected in tumor node 3 (Tu-3-Post), representing approximately 8% of the total T cells in the tumor (Figures 4A and 4B). However, this clone was the 2nd and 12th most frequent among tumor nodules 1 and 2, respectively. Vβ5.2+ERBB2IP mutation-reactive clones were also enriched in all three tumor nodules compared to their frequency in blood (FIGS. 4A and 4B). Thus, patient 3737 underwent tumor regression with disease stabilization for over 1 year after receiving >10 billion ERBB2IP mutation-specific multifunctional Th1 cells that infiltrated and persisted in metastatic lesions.

Quantitative reverse transcriptase PCR (RT-qPCR) analysis of ERBB2IP expression in patient 3737-TIL (T cells) and tumors before adoptive cell transfer (Tu-Pre) was performed. Approximately 17 months after cell injection, three separate metastatic lesions in the lung (Tu-1, -2, -3-Post) were excised. The results are shown in Figure 4C relative to β-actin (ACTB). A 350 base pair (bp) segment of the ERBB2IP gene containing the mutation was PCR amplified and Sanger sequenced from the cDNA sample described in Figure 4C. The location of the mutation was nucleotide position 2414 of the coding sequence, corresponding to a change at position 805 of the amino acid sequence. Relatively high levels of ERBB2IP expression were observed in both the original and recurrent lung lesions as determined by quantitative RT-PCR (Fig. 4C), and in all tumor lesions by Sanger sequencing. The existence of a mutation was confirmed.

Immunohistochemical analysis of T cell infiltration and MHC expression before and after ACT was performed. Post-ACT tumors were harvested approximately 17 months after the initial ACT. Positive controls (tonsils) were included for all stainings. In situ tumor T cell infiltration and MHC expression are summarized in Tables 17 and 18, respectively.

Example 7
This example demonstrates the contribution of mutation-reactive Th1 cells to the anti-tumor response of Example 4.

To specifically assess the contribution of mutation-responsive Th1 cells to antitumor responses in vivo, >95% of Vβ22+ERBB2IP mutation-responsive Th1 cells (approximately 120 billion mutation-responsive cells) A constructed TIL product was generated and adoptively transferred to patient 3737.

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

The patient again underwent target lesion reduction, but unlike the first treatment, tumor regression was observed at the first 1-month follow-up and continued at the 4-month follow-up after the second treatment. (Figure 4D). At follow-up 8 months after the second treatment, tumor regression continued.

Six months after the second dose of mutation-reactive cells, a computed tomography (CT) scan of the lungs of patient 3737 was taken. The obtained images are shown in FIGS. 7A-C. These images were compared to those taken prior to the second administration of mutation-responsive cells (Figures 7D-7F). As shown in FIGS. 7A-7F, approximately 36% reduction in cancerous lesions was observed, resulting in a partial response (PR) based on RECIST criteria.

Eight months after the second dose of mutation-reactive cells, positron emission tomography (PET) scans of the liver and lungs of patient 3737 were taken. Continuous shrinkage of the target lesion was observed. To measure the metabolic activity of the tumor, a radiolabeled glucose analog, FDG (fluorodeoxyglucose), was administered to assess glucose uptake by the tumor. PET scans showed no glucose uptake in the two liver lesions and only partial uptake in the lung lesions.

Examples 8-10
Materials and methods for Examples 8-10 are shown below.

Patient Materials and Cell Lines All patient materials were obtained during the course of clinical trials approved by the National Cancer Institute's Institutional Review Board. Patients 2359 and 2591 were treated as described by Dudley et al. , J. Clin. Oncol. , 26:5233-9 (2008) (study registration ID: NCT00096382 and ID: NCT00335127, respectively). The patient underwent resection and both TIL and tumor cell lines were established. The TIL used in this study was developed by Dudley et al. , J. Immunother. , 26:332-42 (2003). Briefly, tumor fragments were excised and cultured in IL-2 containing medium. The number of amplified TIL cultures was screened for recognition of autologous or HLA-matched tumors and the number of reactive TIL was determined using the rapid amplification protocol (REP) (Riddell et al., Science, 257:238-41 (1992)). were amplified in large quantities for patient injection with IL-2, anti-CD3 antibodies and irradiated feeder cells (Riddell et al., Science, 257:238-41 (1992)). A small number of TILs underwent a second REP. T cells and tumor cells were cultured for 16 hours (hr) at a 1:1 ratio in 96-well plates containing 200 μL of medium (AIM-V medium supplemented with 5% human serum) for co-culture assays.

To assess the antigen reactivity of clinically active TILs, we investigated two metastatic melanoma patients who underwent sustained complete remission with adoptive TIL therapy. Patient 2359 had a primary cutaneous melanoma in his right knee that metastasized to the femoral, iliac, and inguinal lymph nodes. This individual underwent complete regression of all metastatic lesions in response to autologous TIL transfer, which was ongoing for over 8 years after treatment. Patient 2591 had a primary back melanoma that metastasized to the abdominal wall, mesenteric lymph nodes, right colon, and supraclavicular lymph nodes. This individual underwent complete regression of all metastatic lesions in response to autologous TIL transfer and remained disease-free for 9 years post-treatment.

Whole Exome Sequencing The method was described by Robbins et al. , Nat. Med. , 19:747-52 (2013). Genomic DNA purification, library construction, exome capture of approximately 20,000 coding genes, and next generation sequencing of tumor and normal samples were performed at Personal Genome Diagnostics (Baltimore, MD). Briefly, genomic DNA from tumor and normal samples was fragmented and used in Illumina TRUSEQ library construction (Illumina, San Diego, CA). Exon regions were captured in solution using the Agilent SURESELECT 50 Mb kit (version 3) according to the manufacturer's instructions (Agilent, Santa Clara, CA). Paired-end sequencing was performed using a HISEQ 2000 Genome Analyzer (Illumina) to obtain 100 bases from each end of each fragment. Sequence data was mapped to the reference human genome sequence, and sequence alterations were determined by comparison of over 50 million bases of tumor and normal DNA. More than 8 billion bases of sequence data were obtained for each sample, many of which were derived from the captured coding region. Over 43 million bases of target DNA were analyzed in tumor and normal samples, with an average of 42-51 reads for each base in normal and tumor DNA samples.

Bioinformatics analyzes were performed by the Center for Personal Genomic Diagnosis and Genomic Technology Access, Division of Genomics and Pathology, University of Washington School of Medicine. The tags were aligned with the human genome reference sequence (hg18) using the ELAND algorithm in CASAVA 1.6 software (Illumina). Sequence reads for subsequent analysis were selected using the purity filters of Illumina's BASECALL software. The ELANDv2 algorithm of CASAVA 1.6 software was then applied to identify point mutations and small insertions and deletions. Known polymorphisms recorded in dbSNP were excluded from the analysis. Possible somatic mutations were described by Jones et al. , Science, 330:228-31 (2010) and visually verified.

Construction of tandem minigene libraries Nonsynonymous mutations from melanoma samples were identified from whole exome sequencing data. A tandem minigene construct encoding a polypeptide containing the identified six mutated amino acid residues (flanked on either side of its N- and C-terminus by 12 amino acids on each side) was synthesized (Integrated DNA Technologies, Coralville, USA). 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.

IFN-γ ELISPOT Assay Responses to tumor cell lines and peptide-pulsed target cells were measured using 15 μg/ml anti-I
Quantification was performed in an IFN-γ ELISPOT assay using 96-well PVDF membrane filter plates (EMD Millipore, Billerica, MA) coated with the FN-γ monoclonal antibody 1D1K (Mabtech, Inc., Cincinnati, OH). Bound cytokines were detected using 1 μg/ml biotinylated anti-IFN-γ antibody 7-B6-1 (Mabtech). HEK293 cells expressing HLA-A ^* 0201, HLA-A ^* 0205 or HLA-C ^* 0701 were pulsed with peptide for 2 hours at 37°C. The following peptides were used: MART-1: AAGIGILTV (SEQ ID NO: 54), mutant KIF2C: RLFPGLTIKI (SEQ ID NO: 55), mutant POLA2: TRSSGSHFVF (SEQ ID NO: 56). T cells were co-cultured overnight with target cells or medium containing 50 ng/ml PMA and 1 μM ionomycin (PMA/I). The number of spots per 10 ⁵ T cells was calculated.

Example 8
This example demonstrates that TIL2359 recognizes mutant antigens as assessed by minigene library screening.

The reactivity of TIL2359 was evaluated using TMG constructs generated based on nonsynonymous mutations identified by exome analysis of tumor and normal DNA. Each TMG construct encoded up to six individual minigene fragments, corresponding to the mutant codons flanked on either side by an additional 12 codons present in the normal gene product. An example is shown in FIG. 5A.

Each one of the 12 minigenes in tandem, encoding 71 minigenes based on exome DNA sequences containing nonsynonymous point mutations identified from Mel2359, was transiently introduced into COS-7 cells separately. was transfected. These COS-7 cells were also transfected with HLA-A ^* 0205, the predominant HLA-restricted element used for autologous tumor cell recognition by this TIL. Co-culture of these transfectants with TIL2359 resulted in recognition of RJ-1, one of the 12 constructs of TMG (Figure 5B). As shown in Figure 5A, RJ-1 encoded mutant fragments of EPHB2, KIF2C, SLC44A5, ABCA4, DENND4B, and EPRS genes. Six constructs of RJ-1 mutants, each encoding a WT in place of the existing mutated residue in one of the six minigenes, were subsequently generated (FIG. 5C). TIL2359 recognized COS-7 cells cotransfected with HLA-A ^* 0205 and five of the six individually transfected mutants of RJ-1, but not the mutant encoding the WT KIF2C sequence. I couldn't recognize it. This indicated that this minigene encodes the mutant epitope recognized by TIL2359 (Figure 5C). To further verify this observation, COS-7 cells were incubated with WT or mutant full-length KIF2C amplified from Mel2359 along with either HLA-A ^* 0101, HLA-A ^* 0201 or HLA-A ^* 0205 cDNA. Either of the cDNA transcripts were cotransfected. Co-culture experiments showed that TIL2359T cells recognized COS-7 cells co-transfected with the mutant KIF2C gene product in an HLA-A ^* 0205-restricted manner, but not with the WT KIF2C gene product. (Figure 5D).

The mutant KIF2C coding region contained a single C to A transversion at nucleotide 46, resulting in the substitution of a threonine for alanine at position 16 in the native KIF2C protein. Exome sequencing results showed that Mel2359-derived DNA matched exclusively the mutated residue at position 46, but not the normal residue, and direct Sanger sequencing of Mel2359 DNA showed that the results It was confirmed. This indicated loss of heterozygosity at this locus. In an attempt to identify the mutant KIF2C epitope recognized by TIL2359, we synthesized a peptide encompassing the KIF2C mutation that was predicted to bind to HLA-A ^* 0205 with high affinity (Hoof et al., Immunogenetics). , 61:1-13 (2009)) into HEK293 cells stably expressing HLA-A ^* 0205 (Table 21). HEK293-A ^* 0205 cells pulsed with decamer corresponding to residues 10-19 promoted the release of large amounts of IFN-γ from TIL2359 T cells, and the peptide was recognized at the lowest concentration of 0.1 nM. In contrast, the corresponding WT peptide did not induce significant IFN-γ release at 10 μM (Fig. 5E).

Example 9
This example demonstrates that TIL2591 recognizes mutant antigens identified by minigene library screening.

Mutant T cell antigens recognized by TIL2591 were identified by synthesizing 37 TMG constructs encoding 217 minigenes based on exome DNA sequences containing nonsynonymous point mutations identified from Mel2591. . TIL2591 recognized autologous tumor cells in association with multiple HLA-restricted elements. Therefore, HEK293 cell lines stably expressing each of the MHC class I HLA6 molecules isolated from Mel2591 were transiently transfected individually with 37 TMG constructs, followed by overnight co-incubation with TIL2591. Culture was performed. Initial results showed ^{that TIL2591} was able to transform HLA- ^{C *} 0701 ⁺ HEK293 cells (HEK293-C*) transiently transfected with the minigene DW-6 ^. 0701) cells), but did not significantly respond to other minigene constructs (Figure 6A). Each of the individual mutant 6 minigenes in the DW-6 tandem construct (Fig. 6B) was then separately reverted to the WT sequence (Fig. 6C). Evaluation of the response to the WT mutants showed that TIL2591 recognized COS-7 cells transfected with each of the DW-6 mutants (except for the construct encoding the WT POLA2 fragment) (Fig. 6C) . To verify these findings, COS-7 cells were transfected with either WT or mutant full-length POLA2 cDNA constructs along with HLA-C ^* 0401, HLA-C ^* 0701, or HLA-C ^* 0702 cDNA. did. TIL2591 T cells only recognized target cells transfected with HLA-C ^* 0701 and the mutant POLA2 construct, but not the corresponding WT transcript (Figure 6D). A single C to T change at nucleotide 1258 of the POLA2 coding region resulted in the substitution of leucine for phenylalanine at position 420 of the WT POLA2 protein. Genomic DNA and Mel259 by Sanger sequencing
1 RNA-derived cDNA contained both WT and mutant nucleotides at position 1258, whereas genomic DNA isolated from patient 2591's PBMC was shown to correspond to the WT sequence; this showed that this represents a heterozygous somatic mutation in Mel2591 cells.

The HLA-C ^* 0701 binding algorithm was then used to identify potential POLA2 peptides that overlapped with the mutant leucine residue at position 420 (Table 22). Co-culture results show that HLA-C ^* 0701 ⁺ HEK293 cells pulsed with decamer corresponding to residues 413-422 of mutant POLA2 stimulate the release of IFN-γ from TIL2591T cells at a lowest concentration of 10 nM. It has been shown. In contrast, the corresponding WT peptide did not induce significant IFN-γ release at a high concentration of 10 μM (Fig. 6E).

The percentage of T cells recognizing mutated KIF2C and POLA2, respectively, in TIL2359 and 2591 was then estimated using an IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay. TIL2359 generated approximately 2,000 spots per 100,000 T cells in response to HLA-A ^* 0205 ⁺ cells pulsed with the mutant KIF2C epitope, similar to that observed in response to autologous melanoma ( Table 23). TIL2591 generated >7000 spots in response to the HLA-A2-restricted MART-1 epitope, whereas only a few T cells responded to the HLA-C ^* 0701-restricted mutant POLA2 epitope ( Table 23).

Example 10
This example demonstrates a method for identifying T cells reactive against mutant antigens present in gastrointestinal (GI) cancers identified by minigene library screening.

Whole exome sequencing was performed to identify mutations in metastatic lesions from GI cancer patients. Minigene constructs encoding each mutation were then generated and transfected into autologous APCs to allow processing and presentation of all mutations expressed in the tumor. These APCs were then co-cultured with tumor-infiltrating lymphocytes (TILs) and T cell reactivity to mutations was measured by 4-1BB and OX40 upregulation based on IFN-γ ELISPOT and flow cytometry.

In one colon cancer patient, 119 mutations were evaluated for mutational reactivity. Some, but not all, TIL cultures were found to contain highly variable proportions of CD8+ T cells that specifically recognized the mutation in CASP8 (67F→V). Upon further amplification in vitro, other cells in the culture proliferated significantly more than these mutation-reactive CD8+ T cells. Initial follow-up approximately 6 weeks after administration of cells, depending on patient administration of 40.3x10 ⁹ TILs estimated to contain approximately 0.31% (approximately 127 million) mutation-reactive cells. No clinical response was produced. The patient died approximately 6 weeks later. Without being bound by any particular theory or mechanism, prior to treatment, it is important to note that the disease is in a very late stage, that the patient's overall condition is poor, and that the patient's It is believed that any one or more of the following poor tolerance to the lymphocyte-depleting chemotherapy administered may have contributed to the patient's death. TCRs reactive against mutated CASP8 were isolated from TILs, and T cells transduced to express the TCR were reactive against DCs pulsed with mutant CASP8.

In another rectal cancer patient, 155 mutations were evaluated for mutational reactivity. At least three distinct mutational reactivities were found, two involving CD8+ T cell responses and one CD4+ response. Administration of mutation-responsive TILs to patients initially produced a mixed response approximately 1.5 months after treatment, but the patient subsequently developed progressive disease approximately 3.5 months after treatment. . Candidate mutation-responsive TCRs were isolated from CD4+ and CD8+ TILs.

In the third patient (cholangiocarcinoma), no reactive T cells were detected for the 38 mutations tested. For this patient, the threshold for "mutant calling" has been lowered, so an additional 125 putative mutations
mutations) are evaluated. A "mutation call" is an arbitrarily set threshold at which a sequence is identified as a mutation using bioinformatics. In this case, for the first pass, the threshold was relatively high (eg, provided a high level of confidence that the identified mutation was a true mutation). The threshold was then lowered and the level of confidence that the identified mutation was a true mutation became lower, but the possibility that the identified mutation was a true mutation remained.

These data indicate that the ability of the human immune system to mount a T cell response to somatic mutations in metastatic gastrointestinal cancer may not be an uncommon event. This research is ongoing.

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

With respect to the description of the invention (and particularly with respect to the claims that follow), the use of the terms "a" and "an" and "the" and "at least one" and similar referents herein are It should be construed to cover both the singular and the plural unless otherwise clearly contradicted by the context. The use of the term "at least one" after a listing of one or more items (e.g., "at least one of A and B") does not include the use of the term "at least one" unless otherwise specified herein or clearly contradicted by context. It should be interpreted to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B). The terms “comprising,” “having,” “including,” and “containing” are used, unless specified otherwise, as open-ended terms (i.e., “including but limited to”). should be interpreted as meaning "not done"). The description of ranges of values herein, unless otherwise specified herein, is intended only to serve as shorthand for individually referring to each individual value falling within the range, and each individual value falls within the range. Values are incorporated herein as if individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as") provided herein is only intended to better explain the invention, and the use of No limitations on the scope of the invention are intended unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations on these preferred embodiments may be apparent to those skilled in the art upon reading the above description. The inventors expect those skilled in the art to use such variations as appropriate, and the inventors expect that the invention may be practiced otherwise than as specifically described herein. It is intended that Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the invention, unless stated otherwise herein or clearly contradicted by context.

Claims (20)

A method for producing a medicament for treating or preventing cancer in a patient, the method comprising:
identifying one or more genes in the nucleic acid of the patient's cancer cells, each gene containing a cancer-specific mutation encoding a mutant amino acid sequence;
inducing the patient's autologous antigen presenting cells (APCs) to present the mutant amino acid sequence;
co-culturing the patient's autologous T cells with autologous APCs presenting the mutant amino acid sequence;
(a) co-cultured with autologous APCs presenting the mutant amino acid sequence; and (b) antigen directed against the mutant amino acid sequence presented in the context of major histocompatibility complex (MHC) molecules expressed by the patient. selecting autologous T cells with specificity and providing isolated T cells with antigenic specificity for a mutant amino acid sequence encoded by a cancer-specific mutation;
expanding the number of selected autologous T cells to obtain a T cell population having antigenic specificity for the mutant amino acid sequence encoded by the cancer-specific mutation; and subjecting the T cell population to the medicament. wherein the medicament comprises 1 million to 150 billion T cells per injection. 2. The method of claim 1, wherein expanding the number of cells comprises culturing the selected cells with feeder PBMC, interleukin (IL)-2, and OKT3 antibody. 3. The method of claim 1 or 2, wherein the cancer is epithelial cancer. 3. The method of claim 1 or 2, wherein the cancer is bile duct cancer, melanoma, colon cancer or rectal cancer. 5. The method of any one of claims 1-4, wherein the T cell population is autologous to the patient. Inducing the patient's autologous APC to present the mutant amino acid sequence includes pulsing the APC with a peptide or peptide pool containing the mutant amino acid sequence, each peptide in the pool presenting a different mutant amino acid sequence. A method according to any one of claims 1 to 5, comprising: 6. The method of any one of claims 1-5, wherein inducing the patient's autologous APC to present the mutant amino acid sequence comprises introducing into the APC a nucleotide sequence encoding the mutant amino acid sequence. 7. The nucleotide sequence introduced into the autologous APC is a tandem minigene (TMG) construct, each minigene comprising a different gene, each gene comprising a cancer-specific mutation encoding a mutant amino acid sequence. the method of. 9. The method of any one of claims 1-8, wherein the medicament comprises 10 million to 130 billion T cells per injection. 9. The method of any one of claims 1-8, wherein the medicament comprises 100 million to 130 billion T cells per injection. 9. The method of any one of claims 1-8, wherein the medicament comprises between 5 million and 120 billion T cells per injection. 9. The method of any one of claims 1 to 8, wherein the medicament comprises 25 million to 100 billion T cells per injection. 9. The method of any one of claims 1 to 8, wherein the medicament comprises 500 million to 80 billion T cells per injection. 9. The method of any one of claims 1 to 8, wherein the medicament comprises between 20 million and 120 billion T cells per injection. 9. The method of any one of claims 1-8, wherein the medicament comprises 30 million to 110 billion T cells per injection. 9. The method of any one of claims 1 to 8, wherein the medicament comprises 40 million to 100 billion T cells per injection. 9. The method of any one of claims 1-8, wherein the medicament comprises 120 million to 110 billion T cells per injection. 9. The method of any one of claims 1-8, wherein the medicament comprises between 250 million and 100 billion T cells per injection. 9. The method of any one of claims 1 to 8, wherein the medicament comprises 350 million to 90 billion T cells per injection. 9. The method of any one of claims 1-8, wherein the medicament comprises 450 million to 75 billion T cells per injection.