JP2024056826A - Method for isolating T cell receptors with antigen specificity for cancer specific mutations - Google Patents

Abstract

[Problem] To provide a method for isolating a TCR having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. [Solution] A method is disclosed that includes identifying one or more genes in nucleic acid of a patient's cancer cells, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing the patient's autologous APCs to present the mutated amino acid sequence; co-culturing the patient's autologous T cells with the autologous APCs presenting the mutated amino acid sequence; selecting the autologous T cells; and isolating a nucleotide sequence encoding a TCR from the selected autologous T cells, wherein the TCR has antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. Also disclosed are related methods of preparing cell populations, cell populations, TCRs, pharmaceutical compositions, and methods of treating or preventing cancer. [Selected Figures] None

Description

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

Application for application of Article 30, Paragraph 2 of the Patent Law submitted May 8, 2014 (Heisei 26) Published via http://www.nytimes.com/2014/05/09/health/doctors-use-patients-immune-cells-to-shrink-cancer-tumors.html

Application for application of Article 30, Paragraph 2 of the Patent Law filed May 8, 2014 (Heisei 26) Announced via 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 for application of Article 30, Paragraph 2 of the Patent Act submitted May 8, 2014 (Heisei 26) Announced via http://www.cancer.gov/newscenter/newsfromncci/2014/ACTepithelial

Application for application of Article 30, Paragraph 2 of the Patent Law submitted May 8, 2014 (Heisei 26) Published via http://www.nbcnews.com/health/cancer/new-immune-therapy-approach-tackles-womens-rare-cancer-n100811

Application for application of Article 30, Paragraph 2 of the Patent Law filed May 9, 2014 (Heisei 26) Science (2014) Vol. 344, pp. 641-645 Published via <http://science.sciencemag.org/content/344/6184/641.full>

Application for application of Article 30, Paragraph 2 of the Patent Law filed July 1, 2014 Clinical Cancer Research (2014) Vol. 20, pp. 3401-3410 <http://clincancerres. aacrjournals. org/content/20/13/3401. full http://clincancerres. aacrjournals. org/content/20/13/3401. full-text. pdf>

Application for application of Article 30, Paragraph 2 of the Patent Law filed July 19, 2014 Oncotarget (2014) Vol. 5, No. 13, pp. 4579-4580 <http://www.impactjournals.com/oncotarget/index.php?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, which was submitted contemporaneously herewith and is identified as follows, is incorporated herein by reference in its entirety: One 29,577 byte ASCII (text) file dated September 15, 2014, filed as "718291ST25.TXT."

2. Background of the Invention Adoptive cell therapy (ACT) using cells genetically engineered to express anti-cancer antigen T cell receptors (TCRs) can result in beneficial clinical responses in some cancer patients. Nevertheless, obstacles remain to successfully and broadly treat cancer and other diseases using TCR-engineered cells. For example, TCRs that specifically recognize cancer antigens can be difficult to identify and/or isolate from patients. Thus, there is a need for improved methods of obtaining cancer-reactive TCRs.

SUMMARY OF THE DISCLOSURE One embodiment of the present invention provides a method of isolating a TCR or antigen-binding portion thereof having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in nucleic acid of a patient's cancer cells, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence; inducing autologous antigen-presenting cells (APCs) of the patient to present the mutated amino acid sequence; co-culturing autologous T cells of the patient with autologous APCs presenting the mutated amino acid sequence; selecting autologous T cells that are (a) co-cultured with autologous APCs presenting the mutated amino acid sequence and (b) have antigen specificity for the mutated amino acid sequence presented in the context of a major histocompatibility complex (MHC) molecule expressed by the patient; and isolating from the selected autologous T cells a nucleotide sequence encoding the TCR or antigen-binding portion thereof, wherein the TCR or antigen-binding portion thereof has antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

Another embodiment of the present invention provides a method for preparing a cell population expressing a TCR or antigen-binding portion thereof with antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising: identifying one or more genes in the nucleic acid of a patient's cancer cells, each of which contains a cancer-specific mutation encoding a mutated amino acid sequence; inducing the patient's autologous APCs to present the mutated amino acid sequence; co-culturing the patient's autologous T cells with autologous APCs presenting the mutated amino acid sequence; selecting autologous T cells that (a) are co-cultured with the autologous APCs presenting the mutated amino acid sequence and (b) have antigen specificity for the mutated amino acid sequence presented in the context of an MHC molecule expressed by the patient; isolating a nucleotide sequence encoding the TCR or antigen-binding portion thereof from the selected autologous T cells, the TCR or antigen-binding portion thereof having antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation; and introducing the nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into peripheral blood mononuclear cells (PBMCs) to obtain cells expressing the TCR or antigen-binding portion thereof.

Further embodiments of the invention provide related cell populations, TCRs or antigen-binding portions thereof, pharmaceutical compositions, and methods for treating or preventing cancer.

Figure 1A is a graph showing the number of spots per 1x103 (1e3) cells measured by interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISPOT) assay after 20 hours of co-culture of 3737-TIL with OKT3 or dendritic cells (DCs) transfected with green fluorescent protein (GFP) RNA or the indicated tandem minigene (TMG) constructs. ">" indicates >500 spots per ^{1x103 cells} . Mock-transfected cells were treated with transfection reagent alone without added nucleic acid. (B) is a graph showing the percentage of CD4+ 3737-TILs that are OX40+ 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. Mock-transfected cells were treated with transfection reagent alone without the addition of nucleic acid. 2A-2C are graphs showing the number of spots per 1x10 (1e3) cells measured by IFN-γ ELISPOT assay at 20 hours for 3737-TIL (A), DMF5 T cells (B), or T4 T cells (C) cocultured with TMG-1-transfected DC (A) or 624-CIITA cells ((B) and (C)), either precultured without or with the indicated HLA-blocking antibodies (MHC-I, MHC ^- II, HLA-DP, HLA-DQ, or HLA-DR) (A-C). FIG. 2D is a graph showing the number of spots per 1×10 (1e3) cells measured by IFN-γ ELISPOT assay at 20 hours for 3737-TILs cocultured with autologous DQ-0301/-0601 B cells (gray bars) or allogeneic EBV-B cells partially matched at the HLA-DQ05/0601 locus (black bars) or HLA-DQ-0201/0301 locus (open bars) pulsed overnight with DMSO, mutant (mut) ALK, or mut ERBB2IP ²⁵ amino acid (-AA) long peptides. FIG. 2E shows the 25 amino acid peptide of mutERBB2IP, TSFLSINSKEETGHLENGNKYPNLE (SEQ ID NO:73) or the truncated mutERBB2IP peptides FLSINSKEETGHLENGNKYPNLE (SEQ ID NO:30), SINSKEETGHLENGNKYPNLE (SEQ ID NO:31), NSKEETGHLENGNKYPNLE (SEQ ID NO:32), KEETGHLENGNKYPNLE (SEQ ID NO:33), ETGHLE Graph showing number of spots per 1×10 (1e3) cells measured by IFN-γ ELISPOT assay at 20 hours for 3737-TILs co-cultured with autologous B cells pulsed overnight with NGNKYPNLE (SEQ ID NO:53), TSFLSINSKEETGHL (SEQ ID NO:34), TSFLSINSKEETGHLEN (SEQ ID NO:35), TSFLSINSKEETGHLENGN (SEQ ID NO:36), TSFLSINSKEETGHLENGNKY (SEQ ID NO: ³⁷ ), or TSFLSINSKEETGHLENGNKYPN (SEQ ID NO:38). Figure 3A is a graph showing the percentage of various TCRVβ clonotypes in 3737-TILs as measured by flow cytometry gated on live CD4+ (shaded) or CD8+ (unshaded) T cells. Figure 3B is a graph showing the amount of IFN-γ (pg/ml) detected in serum samples from patient 3737 as measured at the indicated days before and after day 0 (indicated by arrows) of adoptive cell transfer of 3737-TILs. Error bars are standard error of the mean (SEM). Figure 3C is a graph showing total tumor burden (circles) (measured as % of pretreatment baseline) or tumor burden in lungs (triangles) or liver (squares) at the indicated months relative to day 0 (indicated by arrows) of cell transfer. Figure 3D is a graph showing the percentage of various TCRVβ clonotypes in CD4+Vβ22-OX40+ 3737-TILs as measured by flow cytometry. 4A and 4B are graphs showing the frequency of two ERBB2IP mutation-specific TCRβ-CDR3 clonotypes, Vβ22+ (A) and Vβ5.2+ (B), in the blood (circles) of patient 3737 at various time points before and after adoptive cell transfer with 3737-TILs (tumors before cell transfer (diamonds) and various tumors after cell transfer (Tu-1-Post (squares), Tu-2-Post (▲), and Tu-3-Post (▼)). Shaded bars indicate the frequency of two ERBB2IP-mutation-specific TCRβ-CDR3 clonotypes, Vβ22+ (A) and Vβ5.2+ (B), in transferred cells (3737-TILs). "X" indicates "not detected." Figure 4C is a graph showing expression of ERBB2IP relative to ACTB in 3737-TIL (T cells) and tumors pre- (Tu-Pre) and post (Tu-1-post, Tu-2-post, and Tu-3-post) various adoptive cell transfers. Figure 4D is a graph showing total tumor burden (circles) (measured as % of pretreatment baseline) or tumor burden in lung (triangles) or liver (squares) at the indicated months versus cell transfer (indicated by arrows). 5A is a schematic diagram of an example of a tandem minigene (TMG) construct encoding a polypeptide containing the identified mutated 6 amino acid residues flanked at its N- and C-termini by 12 amino acids on either side. The mutated KIF2C sequence is DSSLQARLFPGLTIKIQRSNGLIHS (SEQ ID NO:57). FIG. 5B is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2359 T cells cocultured overnight with autologous melanocytes or COS-7 cells cotransfected with HLA-A ^* 0205 and TMG constructs RJ-1 (structure shown in FIG. 9A ), RJ-2, RJ-3, RJ-4, RJ-5, RJ-6, RJ-7, RJ-8, RJ-9, RJ-10, RJ-11, RJ-12, or empty vector. 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 RJ-1 mutants (genes marked with "wt" in the table were reconverted to the WT sequence). The KIF2C WT sequence is DSSLQARLFPGLAIKIQRSNGLIHS (SEQ ID NO:65). FIG. 5D is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2359 cocultured with COS-7 cells transfected with empty vector, KIF2C ^WT , or mutant KIF2C cDNA constructs together with 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 (black bars). FIG. 5E is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2359 T cells cocultured overnight with HEK293 cells stably expressing HLA-A ^* 0205 pulsed with 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). FIG. 6A is a graph showing the amount of IFN-γ (pg/mL) secreted by autologous melanocytes or TIL2591T cells cocultured with HEK293 cells stably expressing HLA-C ^* 0701 transfected with an empty vector or a TMG construct selected from the group consisting of DW-1 to DW-37. 6B is a schematic diagram showing the structure of TMG construct DW-6. The mutated POLA2 sequence is TIIEGTRSSGSHFVFVPSLRDVHHE (SEQ ID NO:64). 6C is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2591 cells co-cultured with COS-7 cells transfected with HLA-C ^* 0701 and DW-6 mutants (genes marked with "wt" in the table were reconverted to the WT sequence). The POLA2 WT sequence is TIIEGTRSSGSHLVFVPSLRDVHHE (SEQ ID NO:66). FIG. 6D is a graph showing the amount of IFN-γ (pg/mL) secreted by ^TIL2591 cells cocultured with COS-7 cells transfected with empty vector, POLA2 WT, or mutant POLA2 cDNA constructs together with 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 (black bars). Figure 6E is a graph showing the amount of IFN-γ (pg/mL) secreted by TIL2591T cells cocultured overnight with HEK293 cells stably expressing HLA-C ^* 0701 pulsed with various concentrations (μM) of POLA2 _413-422 WT (TRSSGSHLVF; sequence number 67) (lower line in the graph) or mutant POLA2 _413-422 (TRSSGSHFVF; sequence number 68) (upper line in the graph). 7A-7F are computed tomography (CT) scans of the lungs of patient 3737 taken before (A-C) and 6 months (D-F) after the second dose of mutation-reactive cells. Arrows point to cancer lesions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention provides a method for isolating a TCR or antigen-binding portion thereof with antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. The present invention provides many advantages. For example, the method of the present invention can rapidly evaluate a large number of mutations in the entire MHC molecule restriction of a patient at once, and identify the entire repertoire of mutation-reactive T cells of the patient. Furthermore, the method of the present invention can identify one or more cancer-specific mutated amino acid sequences that can be targeted by a TCR or antigen-binding portion thereof by distinguishing immunogenic cancer mutations from (a) cancer-specific silent mutations (that do not code for mutated amino acid sequences) and (b) cancer-specific mutations that code for non-immunogenic amino acid sequences. Furthermore, the present invention can provide a TCR or antigen-binding portion thereof with antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation unique to a patient, thereby providing a "personalized" TCR or antigen-binding portion thereof that can be useful for treating or preventing cancer in a patient. The method of the present invention can also avoid the technical biases inherent in traditional methods of identifying cancer antigens, such as those using cDNA libraries, and may require less time and effort than those methods. For example, the methods of the invention may select for mutation-responsive T cells without co-culturing T cells with tumor cell lines, which may be difficult to generate, particularly for epithelial cancers (e.g.). Without being bound to a particular theory or mechanism, it is believed that the methods of the invention may identify and isolate TCRs or antigen-binding portions thereof that target the destruction of cancer cells while minimizing or avoiding the destruction of normal, non-cancerous cells, thereby reducing or eliminating toxicity. Thus, the invention may also provide TCRs or antigen-binding portions thereof that successfully treat or prevent cancers, such as cancers that are unresponsive to other types of treatment, such as chemotherapy alone, surgery, or radiation.

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

To identify cancer-specific mutations, the method may further include sequencing nucleic acid, such as DNA or RNA, of normal, non-cancerous cells and comparing the sequence of the cancer cells to the sequence of the normal, non-cancerous cells. The normal, non-cancerous cells may be obtained from the patient or a different individual.

A cancer-specific mutation can be any 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. Non-limiting examples of cancer-specific mutations that can be identified in the methods of the invention include missense, nonsense, insertion, deletion, duplication, frameshift and repeat expansion mutations. In one embodiment of the invention, the method includes identifying at least one gene that contains a cancer-specific mutation that encodes a mutated amino acid sequence. However, the number of genes containing such cancer-specific mutations that can be identified using the methods of the present invention is not limited and can include more than one gene (e.g., about 2, about 3, about 4, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000 or more, or a range defined by any two of the above values). Similarly, in one embodiment of the present invention, the method includes identifying at least one cancer-specific mutation that encodes a mutant amino acid sequence. However, the number of such cancer-specific mutations that can be identified using the methods of the present invention is not limited and can include more than one (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 in which more than one cancer-specific mutation is identified, the cancer-specific mutations can be located in the same gene or different genes.

In one embodiment, identifying one or more genes in the nucleic acid of the cancer cell includes sequencing the entire exome, genome, or transcriptome of the cancer cell. Sequencing can be performed in any suitable manner known in the art. Examples of sequencing techniques that may be useful in the methods of the present invention include Next Generation Sequencing (NGS) (also referred to as "Massively Parallel Sequencing Technology") or Third Generation Sequencing. NGS refers to non-Sanger-based high-throughput DNA sequencing technology. With NGS, millions or billions of DNA strands can be sequenced in parallel, providing a significantly higher throughput and minimizing the need for fragment cloning methods that are often used for Sanger sequencing of genomes. In NGS, nucleic acid templates can be read randomly in parallel across the entire genome by dividing the entire genome into small fragments. NGS can advantageously provide genome, exome or transcriptome-wide nucleic acid sequence information in a very short period of time, e.g., within about 1 to about 2 weeks, preferably within about 1 to about 7 days, or most preferably within less than 24 hours. Several NGS platforms that are commercially available or described in the literature can be used in conjunction with the methods of the present invention (e.g., those described in 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
For example, using a genome sequencer, 454 Life Sciences (Branford, CT), an ILLUMINA SOLEXA genome analyzer (Illumina Inc., San Diego, CA), or an ILLUMINA HISEQ 2000 genome analyzer (Illumina), or as described by Ronaghi et al. , Science, 281(5375):363-365 (1998) (e.g.), sequencing-by-synthesis (also known as "pyrosequencing") (as performed, e.g., using the SOLID platform (Life Technologies Corporation, Carlsbad, CA) or the POLONATOR G.007 platform (Dover Systems, Salem, NH)), sequencing-by-ligation (as performed, e.g., using the PACBIO RS system (Pacific Biosciences, Menlo Park, CA) or the HELISCOPE platform (Dover Systems, Salem, NH)), sequencing-by-synthesis (as performed, e.g., using the PACBIO RS system (Pacific Biosciences, Menlo Park, CA) or the HELISCOPE platform (Dover Systems, Salem, NH)), sequencing-by-synthesis (as performed, e.g., using the PACBIO RS system (Pacific Biosciences, These include single molecule sequencing (e.g., as performed using the Helicos Biosciences (Cambridge, MA) platform), techniques for single molecule sequencing (e.g., as performed using the GRIDON platform from Oxford Nanopore Technologies (Oxford, UK), the Hybridization-Assisted Nanopore Sequencing (HANS) platform developed by Nabsys (Providence, RI), a ligase-based DNA sequencing platform with DNA nanoball (DNB) technology called probe-anchor ligation (cPAL), electron microscopy-based techniques for single molecule sequencing, and ion semiconductor sequencing.

The method may include inducing the patient's autologous antigen presenting cells (APCs) to present the mutant amino acid sequence. APCs may include any cell that presents a peptide fragment of a protein in the context of a major histocompatibility complex (MHC) molecule on their cell surface. APCs may include, for example, any one or more of macrophages, DCs, Langerhans cells, B lymphocytes, and T cells. Preferably, the APCs are DCs. By using autologous APCs from the patient, the method of the invention may advantageously identify a TCR or antigen-binding portion thereof that has antigen specificity for the mutant amino acid sequence encoded by the cancer-specific mutation that is presented in the context of an MHC molecule expressed by the patient. The MHC molecule may be any MHC molecule expressed by the patient, including, but not limited to, MHC class I, MHC class II, HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules. The method of the invention can advantageously identify mutant amino acid sequences presented in the context of any MHC molecule expressed by the patient without using epitope prediction algorithms to identify MHC molecules or mutant amino acid sequences, which may be useful, for example, to select only a few MHC class I alleles and may be constrained by limited availability of reagents (e.g., an incomplete set of MHC tetramers) for selecting mutant-reactive T cells. Thus, in one embodiment of the invention, the method of the invention advantageously identifies mutant amino acid sequences presented in the context of any MHC molecule expressed by the patient and is not limited to any particular MHC molecule. Preferably, the autologous APC is an antigen-negative autologous APC.

Inducing the patient's autologous APCs to present the mutant amino acid sequence can be performed using any suitable method known in the art. In one embodiment of the present invention, inducing the patient's autologous APCs to present the mutant amino acid sequence comprises pulsing the autologous APCs with a peptide or a pool of peptides comprising the mutant amino acid sequence, where each peptide in the pool comprises a different mutant amino acid sequence. Each of the mutant amino acid sequences in the pool can be encoded by a gene that contains a cancer-specific mutation. In this regard, the autologous APCs can be cultured with the peptide or pool of peptides comprising the mutant amino acid sequence in such a manner that the APCs internalize the peptide(s) and present the mutant amino acid sequence(s) bound to MHC molecules on the cell membrane. In an embodiment in which more than one gene is identified and each gene contains a cancer-specific mutation that encodes a mutant amino acid sequence, the method can comprise pulsing the autologous APCs with a pool of peptides, where each peptide in the pool comprises a different mutant amino acid sequence. Methods for pulsing APCs are known in the art and are described in Solheim (Ed.), Antigen Processing and Presentation Protocols (Methods in Molecular Biology), Human Press, (2010) (for example). The peptide(s) used to pulse APCs may include mutant amino acid(s) encoded by a cancer-specific mutation. The peptide(s) may further include any suitable number of consecutive amino acids from the endogenous protein encoded by the identified gene on each of the carboxyl and amino sides of the mutant amino acid(s). The number of contiguous amino acids from the endogenous protein flanking each side of the mutation is not limited and can be, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or a range defined by any two of the above values. Preferably, the peptide(s) include about 12 contiguous amino acids from the endogenous protein on each side of the mutated amino acid(s).

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

In embodiments in which more than one gene is identified and each gene contains a cancer-specific mutation that encodes a mutant amino acid sequence, the method may include introducing a nucleotide sequence encoding more than one gene. In this regard, in one embodiment of the present invention, the nucleotide sequence introduced into the autologous APC is a TMG construct, each minigene containing a different gene, each gene containing a cancer-specific mutation that encodes a mutant amino acid sequence. Each minigene may encode one mutation identified by the method of the present invention, flanked on each side by any suitable number of contiguous amino acids from the endogenous protein encoded by the identified gene, as described herein with respect to other aspects of the present invention. The number of minigenes in the construct is not limited and may include, for example, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25 or more, or a range defined by any two of the above values. The APC expresses the mutant amino acid sequence encoded by the TMG construct and presents the mutant amino acid sequence bound to an MHC molecule on the cell membrane. In one embodiment, the method includes preparing more than one TMG construct, each construct encoding a different set of mutant amino acid sequences encoded by a different gene, and introducing each TMG construct into a different population of autologous APCs. In this regard, multiple populations of autologous APCs can be obtained, each population expressing and displaying mutant amino acid sequences encoded by a different TMG construct.

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

The method may include selecting autologous T cells that have antigen specificity for the mutated amino acid sequence (a) co-cultured with autologous APCs presenting the mutated amino acid sequence and (b) presented in the context of MHC molecules expressed by the patient. The phrase "antigen specificity" as used herein means that the TCR or antigen-binding portion thereof expressed by the autologous T cells can specifically bind to and immunologically recognize the mutated amino acid sequence encoded by the cancer-specific mutation. Selecting may include identifying T cells that have antigen specificity for the mutated amino acid sequence and separating them from T cells that do not have antigen specificity for the mutated amino acid sequence. Selecting autologous T cells that have antigen specificity for the mutated amino acid sequence may be performed in any suitable manner. In one embodiment of the invention, the method includes expanding the number of autologous T cells, for example, by co-culture with a T cell growth factor such as interleukin (IL)-2 or IL-15, or as described herein with respect to other aspects of the invention, prior to the selection of the autologous T cells. In one embodiment of the present 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 the autologous T cells.

For example, upon co-culture of autologous T cells with APCs presenting the mutated amino acid sequence, T cells having antigen specificity for the mutated amino acid sequence may express any one or more of a variety of T cell activation markers that may be used to identify those T cells having antigen specificity for the mutated amino acid sequence. Such T cell activation markers may include, but are not limited to, 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. Thus, in one embodiment of the present invention, selecting autologous T cells having antigen specificity for the mutated amino acid sequence includes selecting T cells expressing any one or more of PD-1, LAG-3, TIM-3, 4-1BB, OX40, and CD107a. Cells expressing one or more T cell activation markers may be selected based on, for example, expression of the markers, as described in Turcotte et al., Clin. Cancer Res. , 20(2):331-43 (2013) and Gros et al., J. Clin. Invest., 124(5):2246-59 (2014) (e.g.), may be sorted using any of a variety of techniques known in the art, such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

In another embodiment of the invention, selecting autologous T cells having antigen specificity for the mutated amino acid sequence comprises selecting T cells that (i) secrete a greater amount of one or more cytokines upon co-culture with APCs presenting the mutated amino acid sequence compared to the amount of one or more cytokines secreted by a negative control, or (ii) upon co-culture with APCs presenting the mutated amino acid sequence, at least a two-fold greater number of T cells secrete one or more cytokines compared to the number of negative control T cells secreting the one or more cytokines. The one or more cytokines can include any cytokine whose secretion by the T cell is characteristic of T cell activation (e.g., a TCR expressed on a T cell that specifically binds and immunologically recognizes the mutated amino acid sequence). Non-limiting examples of cytokines whose secretion is characteristic of T cell activation include IFN-γ, IL-2, 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, a TCR or an antigen-binding portion thereof, or a T cell expressing a TCR or an antigen-binding portion thereof, may be considered to have "antigen specificity" for a mutant amino acid sequence if, upon co-culture with (a) an antigen-negative APC pulsed with a concentration of a peptide containing the 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, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 100 ng/mL, 1 μg/mL, 5 μg/mL or 10 μg/mL)), or (b) an APC into which a nucleotide sequence encoding the mutant amino acid sequence has been introduced, the T cell or T cell expressing the TCR or an antigen-binding portion thereof secretes at least two-fold more IFN-γ compared to the amount of IFN-γ secreted by a negative control. A negative control can be, for example, (i) a T cell expressing a TCR or an antigen-binding portion thereof co-cultured with (a) an antigen-negative APC pulsed with the same concentration of an irrelevant peptide (e.g., a wild-type amino acid sequence or any other peptide having a sequence different from the mutated amino acid sequence), or (b) an APC into which a nucleotide sequence encoding an irrelevant peptide sequence has been introduced, or (ii) a non-transduced T cell (e.g., derived from a PBMC that does not express a TCR or an antigen-binding portion thereof) co-cultured with (a) an antigen-negative APC pulsed with the same concentration of a peptide comprising a mutated amino acid sequence, or (b) an APC into which a nucleotide sequence encoding a mutated amino acid sequence has been introduced. A TCR or an antigen-binding portion thereof, or a T cell expressing a TCR or an antigen-binding portion thereof, may also have "antigen specificity" for a mutated amino acid sequence if, when the T cell or the T cell expressing a TCR or an antigen-binding portion thereof is co-cultured with an antigen-negative APC pulsed with a higher concentration of a peptide containing the mutated amino acid, the T cell or the T cell expressing a TCR or an antigen-binding portion thereof secretes a greater amount of IFN-γ compared to a negative control (e.g., any of the negative controls described above). IFN-γ secretion may be measured by methods known in the art, such as, for example, enzyme-linked immunosorbent assay (ELISA).

Alternatively, or additionally, the TCR or antigen-binding portion thereof, or T cells expressing the TCR or antigen-binding portion thereof, may be considered to have "antigen specificity" for the mutant amino acid sequence if at least a two-fold greater number of T cells or T cells expressing the TCR or antigen-binding portion thereof secrete IFN-γ upon co-culture with APCs into which a nucleotide sequence encoding the mutant amino acid sequence has been introduced, compared to (a) antigen-negative APCs pulsed with a concentration of a peptide containing the mutant amino acid sequence, or (b) the number of negative control T cells secreting IFN-γ. The concentrations of peptide and negative control may be as described herein for other aspects of the invention. The number of cells secreting IFN-γ may be measured by methods known in the art, such as, for example, ELISPOT.

In one embodiment of the invention, T cells with antigen specificity for a mutated amino acid sequence may both (1) express any one or more T cell activation markers as described herein, and (2) secrete greater amounts of one or more cytokines as described herein, whereas T cells with antigen specificity for a mutated amino acid sequence may express any one or more T cell activation markers without secreting greater amounts of one or more cytokines, or
Or, they may secrete greater amounts of one or more cytokines without expressing any one or more T cell activation markers.

In another embodiment of the invention, selecting autologous T cells having antigen specificity for the mutated amino acid sequence comprises selectively expanding autologous T cells having antigen specificity for the mutated amino acid sequence. In this regard, the method may comprise co-culturing autologous T cells with autologous APCs in a manner that favors expansion of T cells having antigen specificity for the mutated amino acid sequence over T cells that do not have antigen specificity for the mutated amino acid sequence. Thus, a population of T cells is provided that has a higher proportion of T cells having antigen specificity for the mutated amino acid sequence compared to T cells that do not have antigen specificity for the mutated amino acid sequence.

In one embodiment of the invention, the method further comprises obtaining a plurality of tumor fragments from the patient, separately co-culturing each of the plurality of fragments with an autologous APC presenting the mutant amino acid sequence as described herein for other aspects of the invention, and separately evaluating the T cells from each of the plurality of fragments for antigen specificity to the mutant amino acid sequence as described herein for other aspects of the invention.

In embodiments of the invention in which T cells are co-cultured with autologous APCs expressing 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 autologous APCs), selecting autologous T cells may further include evaluating the autologous T cells separately for antigen specificity for each of the multiple mutant amino acid sequences. For example, the method of the invention may further include separately inducing the patient's autologous APCs to present each mutant amino acid sequence encoded by the construct (or contained in the pool) as described herein with respect to other aspects of the invention (e.g., by providing separate APC populations each presenting a different mutant amino acid sequence encoded by the construct (or contained in the pool). The method may further include separately co-culturing the patient's autologous T cells with different autologous APC populations presenting each mutant amino acid sequence, as described herein with respect to other aspects of the invention. The method may further comprise separately selecting autologous T cells that have antigen specificity for the mutated amino acid sequence (a) co-cultured with the autologous APCs presenting the mutated amino acid sequence and (b) presented in the context of MHC molecules expressed by the patient, as described herein with respect to other aspects of the invention. In this regard, the method may comprise determining which mutated amino acid sequences encoded by (or contained in) the TMG construct encoding multiple mutated amino acid sequences are immunologically recognized (e.g., by a depletion process) by the autologous T cells.

The method may further comprise isolating a nucleotide sequence encoding a TCR or an antigen-binding portion thereof from the selected autologous T cells, the TCR or antigen-binding portion thereof having antigen-specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. In one embodiment of the present invention, the number of selected autologous T cells having antigen-specificity for the mutated amino acid sequence may be expanded prior to isolating the nucleotide sequence encoding the TCR or antigen-binding portion thereof. Expanding the number of T cells may be accomplished by any of a number of methods known in the art, e.g., as described in U.S. Pat. No. 8,034,334; U.S. Pat. No. 8,383,099; U.S. Patent Application Publication No. 2012/0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In another embodiment of the invention, the number of selected autologous T cells having antigen specificity for the mutated amino acid sequence is not amplified prior to isolating the nucleotide sequence encoding the TCR or antigen-binding portion thereof.

An "antigen-binding portion" of a TCR, as used herein, refers to any portion, including consecutive amino acids, of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to a mutant amino acid sequence encoded by an identified gene, as described herein with respect to other aspects of the invention. The term "antigen-binding portion" refers to any portion or fragment of a TCR of the invention, which portion or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Antigen-binding portions include, for example, those portions of the TCR that retain the ability to specifically bind to a mutant amino acid sequence or to detect, treat, or prevent cancer to a similar, equal, or greater extent than the parent TCR. With respect to the parent TCR, a functional portion may, for example, comprise about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent TCR.

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

In one embodiment of the invention, the antigen-binding portion may comprise a variable region of a TCR of the invention, e.g., comprising a combination of the CDR regions described above. In this regard, the antigen-binding portion may comprise the amino acid sequence of the variable region of the α chain (Vα), the variable region of the β chain (Vβ), or both the Vα and Vβ of a TCR of the invention.

In one embodiment of the invention, the antigen-binding portion may comprise a combination of variable and constant regions. In this regard, the antigen-binding portion may comprise the full length of the α-chain or β-chain, or both the α-chain and the β-chain, of the TCR of the invention.

Isolation of the nucleotide sequence encoding the TCR or its antigen-binding portion from the selected autologous T cells can be performed in any suitable manner known in the art. For example, the method can include isolating RNA from the autologous T cells and sequencing the TCR or its antigen-binding portion using established molecular cloning techniques and reagents, such as 5' rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) using constant primers for the TCR alpha and TCR beta chains.

In one embodiment of the invention, the method may include cloning a nucleotide sequence encoding the TCR or an antigen-binding portion thereof into a recombinant expression vector using established molecular cloning techniques, such as those described in Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012) (for example). For purposes of this specification, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and that allows for expression of the mRNA, protein, polypeptide, or peptide by a host cell when the construct is contacted with the cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the invention are not naturally occurring in their entirety. However, portions of the vector may be naturally occurring. Recombinant expression vectors may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, some of which may be synthetic or derived from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors may contain naturally occurring internucleotide bonds, non-naturally occurring internucleotide bonds, or both types of bonds. Preferably, the non-naturally occurring or modified nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

The recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and amplification, or for expression, or both, such as plasmids and viruses. Vectors include transposons/transposases, pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia, Inc. ...
The expression vector may be selected from the group consisting of pEX series (Clontech, Palo Alto, Calif.) and pEX series (Stratagene). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector (e.g., a retroviral vector).

The TCR or antigen-binding portion thereof isolated by the methods of the invention may be useful for preparing cells for adoptive cell therapy. In this regard, embodiments of the invention provide a method for preparing a cell population expressing a TCR or antigen-binding portion thereof having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, the method comprising isolating the TCR or antigen-binding portion thereof and introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into PBMCs to obtain cells expressing the TCR or antigen-binding portion thereof, as described herein with respect to other aspects of the invention.

Introduction of a nucleotide sequence (e.g., a recombinant expression vector) encoding an isolated TCR or antigen-binding portion thereof into a PBMC can be accomplished by any of a variety of different methods known in the art, such as those described in Green et al. (supra) (e.g.). Non-limiting examples of techniques useful for introducing a nucleotide sequence into a PBMC include transformation, transduction, transfection, and electroporation.

In one embodiment of the invention, the method comprises introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into PBMCs autologous to the patient. In this regard, the TCR or antigen-binding portion thereof identified and isolated by the method of the invention can be personalized for each patient. However, in another embodiment, the method of the invention can identify and isolate a TCR or antigen-binding portion thereof having antigen specificity for a mutated amino acid sequence encoded by a recurrent (also called "hot spot") cancer-specific mutation. In this regard, the method can comprise introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into PBMCs that are allogeneic to the patient. For example, the method can comprise introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into PBMCs autologous to the patient. In this regard, the method can comprise introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into PBMCs autologous to the patient. In this regard, the method can comprise introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into PBMCs autologous to the patient.
This may include introducing the MC.

In one embodiment of the invention, the PBMCs include T cells. The T cells can be any type of T cell, such as any of those described herein with respect to other aspects of the invention. Without being bound to a particular theory or mechanism, it is believed that less differentiated "younger" T cells may be associated with greater in vivo persistence, proliferation, and/or anti-tumor activity compared to more differentiated "older" T cells. Thus, the methods of the invention can advantageously identify and isolate a TCR or antigen-binding portion thereof having antigen specificity for a mutated amino acid sequence, and introduce the TCR or antigen-binding portion thereof into the "younger" T cells, which may provide greater in vivo persistence, proliferation, and/or anti-tumor activity compared to the "older" T cells from which the TCR or antigen-binding portion may be isolated (e.g., effector cells in a patient's tumor).

In one embodiment of the invention, the method further comprises amplifying the number of PBMCs expressing the TCR or an antigen-binding portion thereof. The number of PBMCs may be amplified, for example, as described herein with respect to other aspects of the invention. In this regard, the method of the invention may advantageously generate large numbers of T cells having antigen specificity for the mutated amino acid sequence.

Another embodiment of the invention provides a TCR or antigen-binding portion thereof isolated by any of the methods described herein with respect to other aspects of the invention. One embodiment of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as a TCR alpha (α) chain, a TCR beta (β) chain, a TCR gamma (γ) chain, a TCR delta (δ) chain, or a combination thereof. Another embodiment of the invention provides an antigen-binding portion of a TCR comprising one or more CDR regions, one or more variable regions, or one or both of the α and β chains of a TCR, as described herein with respect to other aspects of the invention. The polypeptide of the TCR or antigen-binding portion thereof of the invention may comprise any amino acid sequence, provided that the TCR or antigen-binding portion thereof has antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation.

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

The TCRs, or antigen-binding portions thereof, and cell populations of the present invention may be formulated into compositions, such as pharmaceutical compositions. In this regard, the present invention provides pharmaceutical compositions comprising either the TCRs, or antigen-binding portions thereof, or cell populations of the present invention and a pharma- ceutical carrier. The pharmaceutical compositions of the present invention comprise the TCRs, or antigen-binding portions thereof, or cell populations of the present invention in combination with another pharma- ceutical active substance(s) or drug(s), such as a chemotherapeutic agent (e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, or the like). It may be included in combination with bicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

Preferably, the carrier is a pharma- ceutically acceptable carrier. For pharmaceutical compositions, the carrier can be any of those conventionally used for the particular TCR, or antigen-binding portion thereof, or cell population of the invention under consideration. Such pharma-ceutically acceptable carriers are well known to those of skill in the art and are readily available to the public. Preferably, a pharma-ceutically acceptable carrier is one that has no adverse side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particular TCR, antigen-binding portion thereof, or cell population of the invention, and by the particular method used to administer the TCR, antigen-binding portion thereof, or cell population of the invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the 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 TCR or cell population of the invention, and in certain instances, a particular route may provide a more rapid and effective response than another route.

Preferably, the TCR, antigen-binding portion thereof, or cell population of the invention is administered intravenously (for example) by injection. When a cell population of the invention is administered, a pharma- ceutically acceptable carrier for the cells for injection can be any isotonic carrier, such as normal saline (about 0.90% in water).
w/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), about 5% dextrose in water, or lactated Ringer's solution, etc. In one embodiment, the pharma- ceutically acceptable carrier is supplemented with human serum albumin.

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

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

For purposes of the present invention, the amount or dose (e.g., number of cells, if a cell population of the present invention is administered) of the TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the present invention administered should be sufficient to provide a therapeutic or prophylactic response, for example, in a mammal, for an appropriate period of time. For example, the dose of the TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the present invention should be sufficient to bind to a mutated amino acid sequence encoded by a cancer-specific mutation, or to detect, treat, or prevent cancer for a period of about 2 hours or more, e.g., 12-24 hours or more, from the time of administration. In some embodiments, the period can be longer. The dose is determined by the efficacy of the particular TCR, antigen-binding portion thereof, cell population, or pharmaceutical composition of the present invention administered, and the condition of the animal (e.g., human), and the body weight of the animal (e.g., human) to be treated.

Many assays for determining dosages are known in the art. For purposes of the present invention, an assay can be used that involves comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing the TCR or antigen-binding portion thereof upon administration of a given dose of T cells to a mammal in a group of mammals each receiving different doses of such T cells to determine a 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 the TCR, antigen-binding portion thereof, cell population or pharmaceutical composition of the present invention will also be determined by the existence, nature and extent of any adverse side effects that may accompany the administration of a particular TCR, antigen-binding portion thereof, cell population or composition of the present invention. Typically, the attending physician will determine the dosage of the TCR, antigen-binding portion thereof, cell population or pharmaceutical composition of the present invention to treat each individual patient, taking into account a variety of factors such as age, weight, overall physical health, diet, sex, the TCR, antigen-binding portion thereof, cell population or pharmaceutical composition of the present invention to be administered, the route of administration, and the severity of the condition being treated.

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

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

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

The cancer is preferably acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, bile duct cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, The cancer may be any tumor, including lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid 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 present invention may be any mammal. The term "mammal" as used herein refers to any mammal, including, but not limited to, rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). Preferably, the mammal is from the order Bovidae, including bovines (cattle) and scrofa (pigs), or the order Persodactyla, including equines (horses). Preferably, the mammal is from the order Primates, Ceboids, or Simoids (monkeys), or Anthropoids (humans and apes). More preferably, the mammal is a human. In a particularly preferred embodiment, the mammal is a patient expressing a cancer-specific mutation.

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

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

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

Generation of Tumor Infiltrating Lymphocytes (TILs) for Patient Treatment and Adoptive Cell Therapy Patient 3737 was enrolled in an Institutional Review Board (IRB) approved protocol: "Phase II Study of Short-Term Cultured Autologous Tumor Infiltrating Lymphocytes After Lymphodepletion Therapy in Metastatic Gastrointestinal Cancer (Trial Registration ID: NCT01174121)," which was designed to evaluate the safety and efficacy of adoptive transfer of ex vivo expanded autologous tumor infiltrating lymphocytes (TILs) in patients with gastrointestinal cancer.

TILs for initial patient treatment were generated as described in Jin et al., J. Immunother., 35:283-292 (2012). Briefly, resected tumors were minced into approximately 1-2 mm pieces and individual pieces were placed into wells of a 24-well plate containing 2 ml of complete medium (CM) with high dose IL-2 (6000 IU/ml, Chiron, Emeryville, CA). CM consisted of RPMI supplemented with in-house 10% human serum, 2 mM L-glutamine, 25 mM HEPES, and 10 μg/ml gentamicin. Additionally, mixed tumor digests were also cultured in CM with high dose IL-2. After initial expansion of T cells (between 2-3 weeks), ^5x106 T cells from the selection cultures were rapidly expanded with irradiated allogeneic PBMCs at a 1:100 ratio in gas-permeable G-Rex100 flasks 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). The 50/50 medium consisted of a 1:1 mixture of CM and AIM-V medium. All cells were cultured at 37°C with 5% _CO2 . Cell numbers were allowed to rapidly expand for 2 weeks prior to infusion. Patient 3737 received a non-myeloablative lymphodepleting regimen consisting of cyclophosphamide and fludarabine, coupled with four doses of high-dose IL-2, prior to receiving a total of 42.4 billion T cells.

The TILs used in the second patient treatment were generated in a similar manner to the first treatment with the following modifications. The first treatment product (Patient 3737-TIL) consisted of a combination of 5 individual TIL cultures. These 5 cultures were individually assessed for CD4 and Vβ22 expression and reactivity to mutant ERBB2IP, and one culture was found to be highly enriched for Vβ22+ERBB2IP mutant-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 lymphodepleting regimen as the first treatment prior to receiving a total of 126 billion T cells combined with 4 doses of high-dose IL-2.

Generation of TMG constructs Briefly, for each nonsynonymous substitution mutation identified by whole exome sequencing, a "minigene" construct was generated encoding the corresponding amino acid mutation (12 amino acids flanking the wild-type protein sequence). Multiple minigenes were genetically fused to generate the 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, CA). Nine "wild-type revertant" TMG-1 constructs (Gene Oracle, Mountain View, CA) were generated using site-directed mutagenesis. The nucleotide sequences of all TMGs were confirmed by standard Sanger sequencing (Macrogen and Gene Oracle).

Generation of autologous APCs Monocyte-derived immature DCs were generated using the plastic adherence method. Briefly, autopheresis samples were thawed, washed, and set to ^5-10x106 cells/ml with pure AIM-V medium (Life Technologies) and then incubated at approximately ^1x106 cells/ ^cm2 in appropriately sized tissue culture flasks at 37°C, 5% _CO2 . After 90 minutes, non-adherent cells were harvested and the flasks were washed extensively with AIM-V medium and then incubated in AIM-V medium for an additional 60 minutes. The flasks were then washed extensively again with AIM-V medium and the adherent cells were then incubated in 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 (medium supplements from Life Technologies, cytokines from Peprotech). Fresh DC medium was added to the cultures on day 3. Fresh or frozen/thawed DCs were used for experiments 5-7 days after the first stimulation. In all experiments, cells were phenotyped for expression of CD11c, CD14, CD80, CD86, and HLA-DR (all from BD Bioscience) using flow cytometry, confirming that the cells were predominantly 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. Briefly, positive B cells were selected from autopheresis samples using human CD19 microbeads (Miltenyi Biotec). CD19+ cells were then cultured with irradiated (6000 rad) 3T3 cells stably expressing CD40L (3T3-CD40L) at an approximate 1:1 ratio in B cell medium. B cell medium was supplemented with 7.5-10% human serum (Hepatitis C), 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies).
The medium consisted of 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). Fresh B cell medium was added starting on day 3, and medium was added or replaced every 2-3 days thereafter. Additional irradiated 3T3-CD40L feeder cells were added as needed. Antigen-presenting B cells were usually used in experiments 2-3 weeks after initial stimulation.

Generation of in vitro transcribed RNA (IVT) RNA Plasmids encoding the tandem minigenes were linearized with the restriction enzyme SacII. The control pcDNA3.1/V5-His-TOPO vector encoding GFP was linearized with NotI. Restriction digestion was 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 instructed by the manufacturer. RNA was precipitated using the LiCl ₂ method, and RNA purity and concentration were assessed using a NanoDrop spectrophotometer. RNA was then aliquoted into microtubes and stored at −80° C. until use.

RNA transfection APCs (DCs or B cells) were harvested, washed once with PBS, and then resuspended in Opti-MEM (Life Technologies) at ^10-30x106 cells/ml. IVT RNA (4 or 8 μg) was aliquoted to the bottom of a 2 mm gap electroporation cuvette, and 50 or 100 μl of APCs was added directly to the cuvette. Thus, the final RNA concentration used in electroporation was 80 μg/ml. Electroporation was performed using BTX
Transfections were performed using a -830 square wave electroporator. DCs were electroporated with one pulse of 150 V for 10 ms and B cells with one pulse of 150 V for 20 ms. Transfection efficiency using these settings, as assessed with GFP RNA, was routinely between 70-90% (data not shown). All steps were performed at room temperature. Immediately after electroporation, cells were transferred to polypropylene tubes containing DC or B cell medium supplemented with the appropriate cytokines. Transfected cells were incubated overnight (12-14 h) at 37°C, 5% _CO2 . Prior to use in co-culture assays, cells were washed once with PBS.

Peptide pulsing Autologous B cells were harvested, washed, and then resuspended at ^1x106 cells/ml in B cell medium supplemented with IL-4, then incubated overnight (12-14 hours) with 1 μg/ml of 25-mer peptide at 37°C, 5% _CO2 . After overnight pulsing, B cells were then washed twice with PBS, then resuspended in T cell medium, and used immediately in the co-culture assay. The peptides used were: mutant ERBB2IP(

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

(SEQ ID NO: 45); and mutant ALK (RVLKGGSVRKLRHAKQLVLELGEEA (SEQ ID NO: 46)) as a negative control. Mutant ERBB2 IP peptides were purchased from three different sources (GenScript, Piscataway, NJ, Peptide 2.0, Chantilly, VA, and SelleckChem, Houston TX) and all yielded the same in vitro results, whereas wild-type ERBB2 IP peptide and mutant ALK peptide were purchased from Peptide 2.0. To culture allogeneic EBV-B cells, RPMI medium containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies), 10 μg/ml gentamicin (CellGro), and 2 mM L-glutamine was used instead of B cell medium.

T cell sorting, expansion, and cloning. BD FACSAria IIu and BD FACSJazz were used in all experiments requiring cell sorting. In the experiments indicated, sorted T cells were expanded using over-irradiated (4000 rad) allogeneic feeder cells (pool of 3 leukapheresis samples from different donors) in 50/50 medium containing 30 ng/ml anti-CD3 antibody (OKT3) and 3000 IU/ml IL-2. Limiting dilution cloning was performed in 96-well round-bottom plates with 5e4 feeder cells per well and 1-2 T cells per well using the stimulation conditions described above. Medium was changed starting approximately 1 week after stimulation and then every other day or as needed. Cells were usually used in assays or further expanded approximately 2-3 weeks after the first stimulation.

Co-culture assays: IFN-γ ELISPOT and ELISA, flow cytometry for cell surface activation markers, and intracellular cytokine staining (ICS)
When DCs were used as APCs, approximately ^{3.5x104-7x104} DCs were used per well of a 96-well flat- or round-bottom plate. When B cells were used as APCs, approximately ^2x105 cells were used per well of a 96-well round-bottom plate. In the ^ELISPOT assay, ^1x103-1x104 effector T cells were used per well, and in the flow cytometry assay, ^1x105 effector T cells were used per ^well . T cells were usually thawed and rested in 50/50 medium containing IL-2 (3000 IU/ml IL-2) for 2 days, then washed (3x) with PBS prior to the co-culture assay. All co-cultures were performed in the absence of exogenously added cytokines. Plate-bound OKT3 (0.1 or 1 μg/ml) was used as a positive control for all assays.

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% _CO2 . T4 are T cells transduced with an HLA-DR4-restricted TCR reactive to an epitope in tyrosinase. DMF5 is an HLA-A2-restricted T cell line reactive to MART-1. 624-CIITA is an HLA-A2 and HLA-DR4 positive melanoma cell line that stably expresses MHC-II through ectopic expression of CIITA (class II, MHC, transactivator) and is positive for expression of MART-1 and tyrosinase.

For 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 coated with 50 μl of 10 μg/ml IFN-γ capture antibody (Mabtech, clone: 1-D1K) and incubated overnight in the refrigerator. For OKT3 control, wells were coated with a mixture of IFN-γ capture antibody (10 μg/ml) and OKT3 (1 μg/ml). Prior to co-culture, plates were washed 3 times with PBS and subsequently blocked with 50/50 medium for at least 1 h at room temperature (RT). After 20-24 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 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) for 2 hours at room temperature. Plates were then washed three times with PBS-T, followed by incubation with 100 μl/well of streptavidin-ALP (Mabtech, Cincinatti, OH, diluted 1:3000) for 1 hour. 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, Ohio).

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 (1×PBS supplemented with 1% FBS and 2 mM EDTA) and then stained with appropriate antibodies for approximately 30 minutes at 4° C. in the dark. Prior to acquisition on a BD FACSCanto II flow cytometer, cells were washed at least once with FACS buffer. All data was gated on live single cells (PI negative).

Cytokine production was assessed using intracellular cytokine staining (ICS) and flow cytometry. Briefly, target and effector cells were mixed in wells of a 96-well plate, after which both GolgiStop and GolgiPlug were added to the culture (BD Biosciences). GolgiStop and GolgiPlug were used at 1/2 the concentration recommended by the manufacturer. At t=6 hours after stimulation, cells were treated with 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 (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 min in the dark at 4° C. Prior to acquisition on a FACSCanto II flow cytometer, cells were washed twice with Perm/Wash buffer and resuspended in FACS buffer. All flow cytometry data were analyzed using FLOWJO software (TreeStar
Inc.) was used for analysis.

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

Flow cytometry antibodies The following anti-human antibodies, whose titers were measured, were used for cell surface staining: CCR7-FITC (clone: 150503), CD45RO-PE-Cy7 (clone: UCHL1), CD62L-APC (clone: DREG-56), CD27-APC-H7 (clone: M-T271), CD4-efluor 605NC (clone: OKT4), CD57-FITC (clone: NK-1), CD28-PE-Cy7 (clone: CD28.2), CD127-APC (clone: eBioRDR5), CD3-AF700 (clone: UCHT1), CD4-FITC, PE-Cy7, APC-H7 (clone: SK3), CD8-PE-Cy7 (clone: SK1), Vβ22-PE (clone: IMMU 546), Vβ5.2-PE (clone: 36213), OX40-PE-Cy7 or FITC (clone: Ber-ACT35), 4-1BB-APC (clone: 4B4-1), and CD107a-APC-H7 (clone: H4A3). All antibodies were from BD Biosciences, except for CD4-efluor605NC (eBioscience), Vβ22-PE and Vβ5.2-PE (Beckman Coulter), and 4-1BB-APC and OX40-PE-Cy7 (BioLegend). For intracellular cytokine staining, the following anti-human antibodies were used, 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). All ICS antibodies were from eBioscience, except for IL-4-PE-Cy7 (BD Bioscience). The IO Mark B Mark TCR V kit was used to assess the TCR-Vβ repertoire (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 tissues (OCT blocks) using the RNeasy Mini kit (Qiagen). Total RNA was then probed with an oligo-dT primer (Life Technologies) for Th
The cDNA was reverse transcribed using ermoScript reverse transcriptase. Normal and tumor cDNA were then used as templates in PCR 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 GAC CAC TTT TCT GAG GG-3' (SEQ ID NO:48). Phusion DNA polymerase (Thermo Scientific) was used with the recommended three-step protocol with an annealing temperature of 58°C (15 sec) and extension at 72°C (30 sec). 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 tissues (OCT blocks) using 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 with TAQMAN Fast PCR.
The amplifications were performed using the Advanced Master Mix (both from Applied Biosystems). The specificity of the amplified products was confirmed by standard agarose gel electrophoresis. The calculated threshold cycles (Ct) were all below 30.

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

TCR Sequencing and Construction of ERBB2 IP Mutation-Reactive TCRs T cells were pelleted and total RNA was isolated (RNeasy Mini kit, Qiagen). Total RNA was then subjected to 5' RACE using constant primers for the α and β chains of the TCR as directed by the manufacturer (SMARTer RACE cDNA amplification kit, Clontech). Program 1 of the kit was used for PCR with modified extension time (2 min instead of 3 min). The sequences of the α and β chain constant primers were: TCR-α, 5'-GCC ACA GCA CTG TGC TCT TGA AGT CC-3' (SEQ ID NO:49); TCR-β, 5'-CAG GCA GTA TCT GGA GTC ATT GAG-3 (SEQ ID NO:50). TCR PCR products were then isolated by standard agarose gel electrophoresis and gel extraction (Clontech). Products were then either sequenced directly or sequenced of individual colonies after TOPO-TA cloning (Macrogen). For sequencing of known Vβ22+ T cell clones, cDNA was generated from RNA using qScript cDNA Supermix (Quanta Biosciences). These cDNAs were then used as templates in PCR using the TCR-β constant primers (above) and a 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). Products were directly sequenced (Macrogen) using a nested primer for the TCR-β constant chain: 5'-ATT CAC CCA CCA GCT CAG-3' (SEQ ID NO:52).

The Vβ22+ERBB2IP-mutant TCR was constructed by fusing the V-D-J region of Vβ22+TCR-α to the mouse TCR-α constant chain and the Vβ22+TCR-β-V-D-J region to the mouse TCR-β constant chain. The α and β chains were separated by a furin SGSG P2A linker. The use of the mouse TCR constant region promotes pairing of the transduced TCR and facilitates the identification of truly transduced T cells by flow cytometry using an antibody specific for the mouse TCR-β chain (eBioscience). 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 set up at 2x106 cells/ml in T cell medium consisting of a 50/50 mixture of RPMI and AIM-V medium supplemented with 5% autologous human serum, 10 μg/ml gentamicin (CellGro), 100 U/ml penicillin and 100 μg/ml streptomycin, 1.25 μg/ml amphotericin B (Fungizone) and 2 mM L-glutamine (both from Life Technologies). ^2x106 cells (1 ^ml ) were stimulated in 24-well plates with 50 ng/ml soluble OKT3 (Miltenyi Biotec) and 300 IU/ml rhu IL-2 (Chiron) for 2 days prior to retroviral transduction. To generate transient retroviral supernatants, retroviral vector MSGV1 (1.5 μg/well) encoding the Vβ22-positive ERBB2IP mutant-specific TCR and the envelope-encoding plasmid RD114 (0.75 μg/well) were co-transfected into the retroviral packaging cell line 293GP (1×10 ⁶ cells per well of a 6-well poly-D-lysine-coated plate, plated the day before transfection) using Lipofectamine 2000 (Life Technologies). 42-48 hours after transfection, retroviral supernatants were harvested, diluted 1:1 with DMEM medium, and centrifuged at 2,000 g for 2 hours at 32° C. onto Retronectin-coated (10 μg/ml, Takara) non-tissue culture-treated 6-well plates. Activated T cells ( ^2x10 per well, ^0.5x10 cells/ml in T cell medium containing IL-2) were then spun at 300g for 10 minutes into the retroviral plates. Activated T cells were transduced overnight, removed from the plates and further cultured in T cell medium containing IL-2. GFP and mock transduced controls were included in the transduction experiments. Cells were usually assayed 10-14 days after retroviral transduction.

Example 1
This example demonstrates a method to identify one or more genes in the nucleic acid of a patient's cancer cells, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence.

A 43-year-old woman (Pt. 3737) with widespread metastatic cholangiocarcinoma that had progressed through multiple chemotherapy regimens was enrolled in a TIL-based adoptive cellular therapy (ACT) protocol for patients with gastrointestinal (GI) cancer. Clinical characteristics of Patient 3737 are shown in Table 1.

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

Example 2
This example illustrates methods of inducing a patient's autologous APCs to present a mutated amino acid sequence; co-culturing a population of the patient's autologous T cells with autologous APCs presenting the mutated amino acid sequence; and selecting autologous T cells that (a) have been co-cultured with autologous APCs presenting the mutated amino acid sequence and (b) have antigen specificity for the mutated amino acid sequence presented in the context of MHC molecules expressed by the patient.

For each mutation identified in Example 1, a minigene construct was designed that encoded the mutated amino acid flanked on each side by 12 amino acids from the endogenous protein. Multiple minigenes were synthesized in tandem to generate tandem minigene (TMG) constructs (Table 3). In Table 3, underlines indicate mutated amino acids and novel sequences encoded by point mutations or nucleotide insertions or deletions. For splice-site donor mutations (HLA-DOA and LONRF3), mutated minigene transcripts were designed that continued into the downstream intron to the next stop codon, based on the assumption that the mutations would prevent splicing at that site. Splice-site acceptor mutations in DIP2C were not evaluated.

The TMG constructs were then used as templates 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 the TILs. 3737-TILs were observed to be reactive to the mutant antigens present in TMG-1 but not in TMG-2 or TMG-3 (Figure 1A). Furthermore, the activation markers OX40 and OX41 were not recognized by TILs.
Reactivity was predominant in the CD4+ T cell population, as demonstrated by upregulation of -1BB (Tables 4A and 4B). Tables 4A and 4B show the percentage of 3737-TILs that had the indicated phenotypes, as detected by flow cytometry, after co-culture with DCs cultured with the non-specific stimulator OKT3 or with DCs transfected with the indicated tandem minigene (TMG) constructs encoding green fluorescent protein (GFP) RNA or various mutations identified by whole exome sequencing. Mock-transfected cells were treated with transfection reagent alone, without the addition of nucleic acid. Data were gated on live CD3+ cells.

Some 4-1BB upregulation was observed in CD4 negative (CD8+) T cell populations, but these cells were sorted and found to be unreactive to TMG. To determine which of the nine mutations in TMG-1 were recognized by 3737-TIL, nine additional constructs of TMG-1 were synthesized, each containing one of the mutations reverted to the wild-type sequence. Only when the ERBB2 interacting protein (ERBB2IP) mutation was reverted to the wild-type sequence was the reactivity of 3737-TIL to TMG-1 abolished, indicating that the TIL specifically recognizes the ERBB2IP ^E805G mutation (Figure 1B).

ERBB2IP mutation-reactive T cell responses were molecularly characterized. IFN-γ ELISPOT assays were performed and results were measured at 20 hours. Patient 3737-TILs were co-cultured with TMG-1 transfected DCs pre-incubated with either no additions or the indicated HLA-blocking antibodies (against MHC-I, MHC-II, HLA-DP, HLA-DQ, or HLA-DR) (Figure 2A). As controls for antibody blocking, HLA-A2-restricted MART-reactive T cells DMF5 (Figure 2B) and HLA-DR-restricted tyrosinase-reactive T cells T4 (Figure 2C) were co-cultured with MART and tyrosinase-positive 624-CIITA melanoma cell lines pre-incubated with either no additions or the indicated HLA-blocking antibodies. The 3737-TIL response was blocked by anti-HLA-DQ antibodies (Figure 2A).

A separate IFN-γ ELISPOT assay was performed and results were measured at 20 hours. Patient 3
737-TILs were cocultured with autologous B cells or allogeneic EBV-B cells partially matched at the HLA-DQ locus that had been pulsed overnight with DMSO, mutant (mut)ALK, or a 25-mer peptide of mutant ERBB2IP (FIG. 2D).

A separate 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 25 amino acid peptide of mutERBB2IP or the indicated truncated mutERBB2IP peptides (Figure 2E).

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

Example 3
This example describes the generation of autologous open repertoire peripheral blood T cells genetically modified with the TCR-Vβ22 chain (together with its α chain) of the ERBB2IP-specific CD4+ T cells identified in Example 2.
These results show that ERBB2-associated mutated blood T cells conferred specific reactivity to mutant ERBB2 IP peptides.

The clonality of mutant ERBB2IP-specific CD4+ T cells identified in Example 2 was characterized by sorting them after antigen-specific activation using OX40 as an activation marker. These cells were then expanded in bulk and cloned by limiting dilution. Flow cytometry-based survey of the TCR-Vβ repertoire demonstrated that >95% of the bulk amplified population was Vβ22+, with 10/11 of the clones evaluated being purely Vβ22+. TCR sequence analysis revealed identical TCRβ V-D-J sequences in 6/6 of the Vβ22+ clones tested (Table 5), indicating that the majority of ERBB2IP mutation-reactive T cells are composed of predominantly Vβ22+ T cell clones.

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

Furthermore, autologous open repertoire peripheral blood T cells genetically modified with this TCR-Vβ22 chain corresponding to its α chain (Table 7) conferred specific reactivity to mutant ERBB2IP peptides (Tables 8A and 8B), demonstrating that this TCR specifically recognizes the ERBB2IP ^E805G mutation. Autologous open repertoire peripheral blood T cells were transduced (Td) with TCR from a Vβ22+ clone (Table 8A) or treated with transduction reagent alone without added nucleic acid (Mock) (Table 8B) and then assessed for reactivity as described in Table 6. The endogenous Vβ22+ TCR constant region was replaced with a mouse constant region, allowing detection of the transduced TCR with an antibody against the mouse TCRβ constant region (mTCRβ). In all assays, plate-bound OKT3 was used as a control. Tables 8A and 8B show the percentages of mTCRβ+ and mTCRβ− cells that either produce intracellular IFN-γ (IFN-γ+) or do not produce intracellular IFN-γ (IFN-γ−) as measured by flow cytometry.

Example 4
This example demonstrates a method of using the autologous cells identified in Example 2 to treat cancer.

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 lung lesions for treatment. The tumor was then minced into small pieces and incubated with high doses of IL-2 to expand the tumor infiltrating lymphocytes (TILs). After initial cell number expansion in IL-2, the number of selected TIL cultures was expanded for an additional 2 weeks using a rapid expansion protocol (REP) that included irradiated allogeneic peripheral blood feeder cells, OKT3, and IL-2. Prior to cell infusion, the patient was preconditioned with cyclophosphamide (CTX: 60 mg/kg, once daily for 2 days) followed by fludarabine (Flu: 25 mg/ ^m2 , for 5 days). Patient 3737-TIL contained 42.4 billion TIL containing over 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 OX40 and Vβ22 expression on CD4+ T cells was assessed 24 hours after stimulation using flow cytometry. Stimulation with plate-bound OKT3 was used as a positive control. Flow cytometric analysis demonstrated that approximately 25% of the total administered 3737-TIL product was composed of Vβ22+ mutation-reactive T cells (Figure 3A, Table 9), equivalent to an infusion of over 10 billion ERBB2IP mutation-specific CD4+ T cells. Table 9 shows the percentage of Vβ22+ and Vβ22- cells expressing OX40 (OX40+) or not expressing OX40 (OX40-) as measured by flow cytometry.

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

The patient had clear signs of progressive disease prior to cell infusion, but tumor regression was observed at 2-month follow-up, and target lesions in the lungs and liver all continued to regress, reaching a maximum of 30% regression after 7 months of treatment (Figure 3C). The patient experienced disease stabilization for approximately 13 months after cell infusion, after which disease progression was noted 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 mutant-reactive T cells play a role in stabilizing disease, the cells were evaluated for in vitro phenotype and function. 3737-TILs were co-cultured for 6 hours with autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK, or mut ERBB2IP 25 amino acid peptides. Flow cytometry was used to assess Vβ22 expression and detect intracellular production of IFN-γ (Table 10A), tumor necrosis factor (TNF) (Table 10B), and IL-2 (Table 10C) in the CD4+ population. The percentage of cells with the indicated phenotypes is shown in Tables 10A-10C. Table 10D shows the percentage of Vβ22+ cells that expressed the indicated number of cytokines. Stimulation with mutant ERBB2IP peptides induced robust co-expression of IFN-γ, TNF, and IL-2 (Tables 10A-10C), but little or no IL-4 or IL-17, suggesting that Vβ22+ERBB2IP mutant-responsive CD4+ T cells are polyfunctional 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-TILs were assessed 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 percentage of cells with the indicated phenotypes is 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-TILs were co-cultured for 6 hours with autologous B cells pulsed overnight with OKT3 or 25 amino acid peptides of wild-type (wt) ERBB2IP, mutant (mut) ALK, or mutERBB2IP. Antibodies specific for the degranulation marker CD107a were added at the initiation of co-culture. Flow cytometry was used to assess Vβ22 expression 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 before co-culture with autologous B cells pulsed overnight with wild-type (wt) ERBB2IP, mutant (mut) ALK, or mutERBB2IP 25-mer peptides, then rested for 2 days in IL-2-containing medium. Flow cytometry was used to assess Vβ22 expression and detect intracellular production of IL-2 (Table 13C), TNF (Table 13B), and IFN-γ (Table 13A) in the CD4+ population 6 hours (h) after stimulation. The percentages of cells with the indicated phenotypes are shown in Tables 13A-13C.

Expression of OX40 and Vβ22 in the CD4+ population was assessed 24 hours after stimulation using flow cytometry. Cells that upregulated OX40 were selected, expanded, and the TCR-Vβ repertoire analyzed by flow cytometry. Results are shown in Figure 3D. Sorting of Vβ22-negative cells and subsequent activation of these cells revealed the presence of one or more additional clonotypes reactive to this epitope in 3737-TILs (Tables 13A-13C), of which the most predominant clonotype was Vβ5.2 (Figure 3D).

The sorted cells shown in Figure 3D were co-cultured for 6 hours with autologous B cells pulsed overnight with 25 amino acid peptides of wtERBB2IP, mut ALK, or mutERBB2IP. Flow cytometry was used to assess Vβ5.2 expression 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 numbers of cytokines.

Upon stimulation with mutant ERBB2IP, Vβ22-negative cells that upregulated OX40 were selected and the cell numbers were amplified. RNA from these cells was then isolated and rapid amplification of 5' complementary DNA ends (5'RACE) was performed using TCR-β constant strand primers to identify expressed TCR-Vβ sequences. Polymerase chain reaction (PCR) products were subjected to TOPO-TA cloning and then sequenced in individual colonies. Flow cytometry demonstrated that 40-50% of these T cells were Vβ5.2 (TRBV5-6). By Sanger sequencing, 3/7 of the TOPO-TA colonies were Vβ5.2 (TRBV5-6) with the sequences shown in Table 16. Table 16 shows the most frequent TCRβ V-D-J sequences of Vβ22-negative ERBB2IP mutant-responsive 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 the mutant ERBB2IP (Tables 14A-14C, and 15). Thus, the TILs used to treat patient 3737 contained at least three clones of distinct polyfunctional CD4+ T cells that recognized the same mutation in ERBB2IP, indicating that this mutation is highly immunogenic.

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

To determine whether there was evidence that CD4+ERBB2IP mutation-reactive T cells played a role in disease stabilization, the in vivo persistence of the cells was evaluated. TCR-Vβ deep sequencing revealed that these clonotypes were rare or undetectable in peripheral blood prior to ACT (Figures 4A and 4B). Ten days after ACT, both clones represented more than 2% of total T cells in peripheral blood, but declined to less than 0.3% by day 34 after cell infusion (Figures 4A and 4B). Three lung metastases resected approximately 1.5 years after ACT were infiltrated by ERBB2IP mutation-reactive T cells (Figures 4A and 4B), indicating that these cells contribute to cancer regression and disease stabilization. The Vβ22+ERBB2IP mutation-responsive clone was the most frequent clone detected in tumor nodule 3 (Tu-3-Post), representing approximately 8% of all T cells in the tumor (Figures 4A and 4B), whereas this clone was the second and 12th most frequent in tumor nodule 1 and 2, respectively. The Vβ5.2+ERBB2IP mutation-responsive clone was also enriched in all three tumor nodules compared to its circulating frequency (Figures 4A and 4B). Thus, patient 3737 underwent tumor regression with disease stabilization for more than one year after receiving over 10 billion ERBB2IP mutation-specific polyfunctional Th1 cells that infiltrated and persisted in metastatic lesions.

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

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 first ACT. Positive controls (tonsils) were included for all staining. 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 mutant-responsive Th1 cells to the anti-tumor response of Example 4.

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

Flow cytometric analysis of the retreatment TIL product 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). Retreatment TILs were co-cultured for 6 hours with autologous B cells pulsed overnight with a 25-mer peptide of wild-type (wt) or mutant (mut) ERBB2IP. Intracellular TNF production in the CD4+ population was detected using flow cytometry (Table 20).

The patient again experienced a decrease in target lesions, but unlike the first treatment, tumor regression was observed at the first month of follow-up and continued at the 4-month follow-up after the second treatment (Figure 4D). Tumor regression continued at the 8-month follow-up after the second treatment.

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

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

Examples 8 to 10
Materials and methods for Examples 8-10 are provided below.

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

To evaluate the antigen reactivity of TILs with clinical activity, two metastatic melanoma patients who underwent sustained complete remission with adoptive TIL therapy were studied. Patient 2359 had a primary cutaneous melanoma in the right knee that metastasized to femoral, iliac, and inguinal lymph nodes. This individual underwent complete regression of all metastatic disease in response to transfer of autologous TILs, which had been 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 disease in response to transfer of autologous TILs, and has been disease-free for 9 years after treatment.

Whole-exome sequencing The method is described in Robbins et al., Nat. Med., 19:747-52 (2013). Genomic DNA purification, library construction, exome capture of approximately 20,000 coding genes, and next-generation sequencing of tumor and normal samples were performed at Personal Genome Diagnostics (Baltimore, MD). Briefly, genomic DNA from tumor and normal samples was fragmented and used for Illumina TRUSEQ library construction (Illumina, San Diego, CA). Exonic regions were captured in solution using an Agilent SURESELECT 50 Mb kit (version 3) following 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 a reference human genome sequence, and sequence alterations were determined by comparison of over 50 million bases of tumor and normal DNA. Over 8 billion bases of sequence data were obtained for each sample, with the majority of the bases coming from the captured coding regions. Over 43 million bases of target DNA were analyzed in tumor and normal samples, with an average of 42-51 reads obtained at each base in normal and tumor DNA samples.

Bioinformatics analysis was performed by the Department of Genomics and Pathology, Center for Personal Genomic Diagnostics and Genomic Technology Access, Washington University School of Medicine. Tags were aligned to 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 filter in Illumina's BASECALL software. Point mutations and small insertions and deletions were then identified by applying the ELANDv2 algorithm in CASAVA 1.6 software. Known polymorphisms recorded in dbSNP were excluded from the analysis. Potential somatic mutations were filtered and visually verified as described in Jones et al., Science, 330:228-31 (2010).

Construction of tandem minigene libraries Nonsynonymous mutations from melanoma samples were identified from whole-exome sequencing data. Tandem minigene constructs encoding polypeptides containing the identified mutated 6 amino acid residues flanked by 12 amino acids on either side at their N- and C-termini were synthesized (Integrated DNA Technologies, Coralville, Iowa) and then cloned into the pcDNA3.1 expression vector using the IN-FUSION Advantage PCR Cloning Kit (Clontech) according to the manufacturer's instructions.

IFN-γ ELISPOT Assay Responses to tumor cell lines and peptide-pulsed target cells were quantified with IFN-γ ELISPOT assays using 96-well PVDF membrane filter plates (EMD Millipore, Billerica, MA) coated with 15 μg/ml of anti-IFN-γ monoclonal antibody 1D1K (Mabtech, Inc., Cincinnati, OH). Bound cytokines were detected with 1 μg/ml of biotinylated anti-IFN-γ antibody 7-B6-1 (Mabtech). HEK293 cells expressing HLA-A ^* 0201, HLA-A ^* 0205 or HLA-C ^* 0701 were pulsed with peptides for 2 hours at 37°C. The following peptides were used: MART-1: AAGIGILTV (SEQ ID NO: 54), 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 a mutant antigen 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 codon flanked on either side by 12 additional codons present in the normal gene product. An example is shown in Figure 5A.

COS-7 cells were transiently transfected separately with each individual one of the tandem 12 minigenes encoding 71 minigenes based on the exome DNA sequence containing the nonsynonymous point mutations identified from Mel2359. These COS-7 cells were also transfected with HLA-A ^* 0205, the dominant HLA restriction element used for autologous tumor cell recognition by this TIL. Co-culture of these transfectants with TIL2359 led to the recognition of one of the 12 constructs of TMG, RJ-1 (Figure 5B). As shown in Figure 5A, RJ-1 encoded mutant fragments of EPHB2, KIF2C, SLC44A5, ABCA4, DENND4B, and EPRS genes. Subsequently, six constructs of RJ-1 mutants, each encoding WT in place of the mutated residues present in one of the six minigenes, were generated (Figure 5C). TIL2359 recognized COS-7 cells cotransfected with HLA-A ^* 0205 and five of the six mutants of RJ-1 transfected individually, but not with the mutant encoding the WT KIF2C sequence, indicating that the minigene encodes the mutant epitope recognized by TIL2359 (Figure 5C). To further validate this observation, COS-7 cells were cotransfected with either WT or mutant full-length KIF2C cDNA transcripts amplified from Mel2359 together with either HLA-A ^* 0101, HLA-A ^* 0201, or HLA-A ^* 0205 cDNAs. Coculture experiments demonstrated that TIL2359T cells recognized COS-7 cells cotransfected with mutant, but not WT, KIF2C gene products in an HLA-A ^* 0205-restricted manner (FIG. 5D).

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

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

The mutant T cell antigens recognized by TIL2591 were identified by synthesizing 37 TMG constructs encoding 217 minigenes based on the exome DNA sequence containing the nonsynonymous point mutations identified from Mel2591. TIL2591 recognized autologous tumor cells in the context of multiple HLA-restriction elements. Therefore, the 37 TMG constructs were individually and transiently transfected into HEK293 cell lines stably expressing each of the MHC class I HLA6 molecules isolated from Mel2591, followed by overnight co-culture with TIL2591. Initial results showed that TIL2591 recognized HLA-C ^* 0701 ⁺ HEK293 cells (HEK293-C ^* 0701) transiently transfected with the minigene DW-6 (HLA-C ^* 0701 ⁺ HEK293 cells (HEK293-C ^* 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 (Figure 6B) was then separately reverted to the WT sequence (Figure 6C). Evaluation of the response to the WT mutants revealed that:
We 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 validate these findings, COS-7 cells were transfected with either the WT or mutant full-length POLA2 cDNA constructs together with HLA-C ^* 0401, HLA-C ^* 0701, or HLA-C ^* 0702 cDNA. TIL2591 T cells recognized only target cells transfected with HLA-C ^* 0701 and mutant POLA2 constructs, but not the corresponding WT transcripts (Fig. 6D). A single C to T change at nucleotide 1258 of the POLA2 coding region results in a phenylalanine to leucine substitution at position 420 of the WT POLA2 protein. Sanger sequencing showed that both genomic DNA and cDNA derived from Mel2591 RNA contained both the WT and mutant nucleotides at position 1258, while genomic DNA isolated from PBMCs of patient 2591 corresponded to the WT sequence, indicating that this represents a heterozygous somatic mutation in Mel2591 cells.

We then used an HLA-C ^* 0701 binding algorithm to identify candidate POLA2 peptides that overlap with the mutant leucine residue at position 420 (Table 22). Coculture results showed that HLA-C ^* 0701 ⁺ HEK293 cells pulsed with a decamer corresponding to residues 413-422 of mutant POLA2 stimulated IFN-γ release from TIL2591T cells at concentrations as low as 10 nM. In contrast, the corresponding WT peptide did not induce significant IFN-γ release at concentrations as high as 10 μM (Figure 6E).

The percentage of T cells recognizing mutated KIF2C and POLA2, respectively, in TIL2359 and 2591 was then estimated using 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 mutated KIF2C epitopes, similar to what was observed in response to autologous melanoma (Table 23). TIL2591 generated more than 7000 spots in response to HLA-A2-restricted MART-1 epitopes, whereas only a few T cells reacted against HLA-C ^* 0701-restricted mutated POLA2 epitopes (Table 23).

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

Whole exome sequencing was performed on metastatic lesions from patients with GI cancer to identify mutations. 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 the mutations was measured by IFN-γ ELISPOT and flow cytometry-based upregulation of 4-1BB and OX40.

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 expansion in vitro, other cells in the culture significantly out-expanded these mutation-reactive CD8+ T cells. Administration of 40.3×10 ⁹ TILs, estimated to contain approximately 0.31% (approximately 127 million) of mutation-reactive cells, to the patient did not result in a clinical response at initial follow-up approximately 6 weeks after administration of the cells. The patient died approximately 6 weeks later. Without being bound to a particular theory or mechanism, it is believed that one or more of the very late stage of the disease prior to treatment, the patient's poor overall condition, and the patient's poor tolerance to lymphodepleting chemotherapy administered prior to adoptive cell therapy 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 to DCs pulsed with mutated CASP8.

In another patient with rectal cancer, 155 mutations were evaluated for mutation reactivity. At least three distinct mutation reactivity patterns were found, including two CD8+ T cell responses and one CD4+ response. Administration of mutation-reactive TILs to the patient initially resulted in a mixed response approximately 1.5 months after treatment, but then the patient developed progressive disease approximately 3.5 months after treatment. Candidate mutation-reactive 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 "mutation calling" was lowered, so an additional 125 putative mutations were identified.
A "mutation call" is an arbitrarily set threshold at which a sequence is identified as a mutation using bioinformatics. In this case, for a first pass, the threshold was relatively high (e.g., provided a high level of confidence that the identified mutation was a true mutation). The threshold was then lowered, providing a lower level of confidence that the identified mutation was a true mutation, although the possibility remained that the identified mutation was a true mutation.

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 a rare event. This study is ongoing.

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

With respect to the description of the present invention (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 should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The use of the term "at least one" after a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified. The recitation of ranges of values herein is intended only to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise stated herein, and each separate value is incorporated herein as if it were individually stated herein. All methods described herein can be performed in any suitable order unless otherwise stated herein or clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as") provided herein is intended only to better illustrate the invention and does not impose limitations on the scope of the invention unless specifically claimed. No phrase in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

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

Claims (20)

1. A method for isolating a T cell receptor (TCR), or an antigen-binding portion thereof, having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, comprising:
identifying one or more genes in the nucleic acid of the patient's cancer cells, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence;
inducing the patient's autologous antigen presenting cells (APCs) to present the mutated amino acid sequence;
1. A method comprising: co-culturing the patient's autologous T cells with autologous APCs that present a mutated amino acid sequence; selecting autologous T cells that are (a) co-cultured with the autologous APCs that present the mutated amino acid sequence and (b) have antigen specificity for the mutated amino acid sequence presented in the context of a major histocompatibility complex (MHC) molecule expressed by the patient; and isolating from the selected autologous T cells a nucleotide sequence encoding a TCR or an antigen-binding portion thereof, wherein the TCR or antigen-binding portion thereof has antigen specificity for the mutated amino acid sequence encoded by the cancer-specific mutation. The method of claim 1, wherein inducing the patient's autologous APCs to present the mutant amino acid sequence comprises pulsing the APCs with a peptide or pool of peptides that contain the mutant amino acid sequence, each peptide in the pool containing a different mutant amino acid sequence. The method of claim 1, wherein inducing the patient's autologous APCs to present the mutant amino acid sequence comprises introducing a nucleotide sequence encoding the mutant amino acid sequence into the APCs. The method of claim 3, wherein the nucleotide sequence introduced into the autologous APC is a tandem minigene (TMG) construct, each minigene containing a different gene, each gene containing a cancer-specific mutation that encodes a mutated amino acid sequence. The method of any one of claims 1 to 4, further comprising obtaining multiple tumor fragments from a patient, separately co-culturing each of the multiple fragments with autologous APCs presenting the mutant amino acid sequence, and separately evaluating the T cells from each of the multiple fragments for antigen specificity to the mutant amino acid sequence. The method of any one of claims 1 to 5, wherein selecting autologous T cells having antigen specificity for the mutated amino acid sequence comprises selectively expanding autologous T cells having antigen specificity for the mutated amino acid sequence. The method of any one of claims 1 to 6, wherein selecting autologous T cells having antigen specificity for the mutant amino acid sequence comprises selecting T cells expressing one or more of programmed cell death 1 (PD-1), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain 3 (TIM-3), 4-1BB, OX40, and CD107a. The method of any one of claims 1 to 7, wherein selecting autologous T cells having antigen specificity for the mutant amino acid sequence comprises selecting T cells that (i) secrete a greater amount of one or more cytokines when co-cultured with an APC presenting the mutant amino acid sequence compared to the amount of one or more cytokines secreted by a negative control, or (ii) select T cells in which at least a two-fold greater number of T cells secrete one or more cytokines when co-cultured with an APC presenting the mutant amino acid sequence compared to the number of negative control T cells secreting one or more cytokines. The method of claim 8, wherein the one or more cytokines include interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor alpha (TNF-α), granulocyte/monocyte colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, IL-10, IL-17, and IL-22. The method of any one of claims 1 to 9, wherein identifying one or more genes in nucleic acid of the cancer cell comprises sequencing the whole exome, whole genome, or whole transcriptome of the cancer cell. 1. A method for preparing a cell population expressing a TCR or an antigen-binding portion thereof having antigen specificity for a mutated amino acid sequence encoded by a cancer-specific mutation, comprising:
11. A method comprising isolating a TCR or an antigen-binding portion thereof by the method of any one of claims 1 to 10, and introducing a nucleotide sequence encoding the isolated TCR or antigen-binding portion thereof into peripheral blood mononuclear cells (PBMCs) to obtain a cell expressing the TCR or antigen-binding portion thereof. The method of claim 11, further comprising amplifying the number of PBMCs expressing the TCR or an antigen-binding portion thereof. A TCR or an antigen-binding portion thereof isolated by the method of any one of claims 1 to 10. An isolated cell population prepared according to claim 11 or 12. 15. A pharmaceutical composition comprising: (a) the TCR or antigen-binding portion thereof of claim 13; or (b) the isolated population of T cells of claim 14; and a pharma- ceutical acceptable carrier. The TCR or antigen-binding portion thereof of claim 13, the isolated cell population of claim 14, or the pharmaceutical composition of claim 15, for use in treating or preventing cancer in a mammal. The TCR or antigen-binding portion thereof, isolated cell population, or pharmaceutical composition for use according to claim 16, wherein the cancer is an epithelial cancer. The TCR or antigen-binding portion thereof, isolated cell population or pharmaceutical composition for use according to claim 16, wherein the cancer is bile duct cancer, melanoma, colon cancer or rectal cancer. An isolated cell population or a pharmaceutical composition comprising the isolated cell population for use according to any one of claims 16 to 18, wherein the PBMCs are autologous to the patient. An isolated cell population or a pharmaceutical composition comprising the isolated cell population for use according to any one of claims 16 to 18, wherein the PBMCs are allogeneic to the patient.