WO2017048614A1

WO2017048614A1 - Methods of isolating tumor-reactive t cell receptors from tumor or peripheral blood

Info

Publication number: WO2017048614A1
Application number: PCT/US2016/051227
Authority: WO
Inventors: Anna PASETTO; Alena Gros; Steven A. Rosenberg
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2015-09-15
Filing date: 2016-09-12
Publication date: 2017-03-23

Abstract

The invention provides methods for generating or obtaining tumor-reactive T cells. Briefly, a high frequency CDR3β clonotype is used to select a full length β chain. The appropriate TCR α chain for pairing with this β chain is identified and polynucleotides encoding both chains of the TCR chains are constructed. These polynucleotides are introduced into PBMCs. The antitumor activity of TCR expressing transformants is determined and highly active transformants are considered for use in treating patients.

Description

METHODS OF ISOLATING TUMOR-REACTIVE T CELL RECEPTORS FROM

TUMOR OR PERIPHERAL BLOOD

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/218,763, filed September 15, 2015, which is incorporated by reference in its entirety herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 13,893 Byte ASCII (Text) file named "726292_ST25.txt," dated

September 12, 2016.

BACKGROUND OF THE INVENTION

[0003] Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) or cells that have been genetically engineered to express a tumor reactive T cell receptor (TCR) can produce positive clinical responses in some cancer patients. Nevertheless, obstacles to the successful use of ACT for the widespread treatment of cancer and other diseases remain. For example, T cells and TCRs that are tumor-reactive may be difficult to identify and/or isolate from a patient. Accordingly, there is a need for improved methods of obtaining tumor-reactive T cells and TCRs.

BRIEF SUMMARY OF THE INVENTION

[0004] An embodiment of the invention provides a method of obtaining tumor- reactive T cells comprising: (a) obtaining a bulk population of T cell clonotypes from a biological sample from a patient, wherein each T cell clonotype comprises a T cell receptor (TCR) comprising a beta chain comprising a complementarity determining region 1 beta ("CDRi p"), a CDR2 , and a CDR3P and an alpha chain comprising a CDR1 alpha (CDRl a), a CDR2a, and a CDR3a; (b) sequencing the CDR3a and the CDR3 of one or more T cell clonotypes obtained in (a) to provide a CDR3a amino acid sequence and a CDR3P amino acid sequence; (c) pairing a CDR3a amino acid sequence of (b) with a CDR3P amino acid sequence of (b); (d) sequencing the CDRla and the CDR2a of the alpha chain comprising the CDR3a amino acid sequence of (c) and the CDRlp and the CDR2P of the beta chain comprising the CDR3P amino acid sequence of (c); (e) preparing one or more polynucleotide(s) encoding a TCR comprising the CDRla, CDR2a, CDR3a, CDRl p, CDR2p, and CDR3P of (d); (f) introducing the one or more polynucleotide(s) of (e) into peripheral blood

mononuclear cells (PBMCs) and expressing the TCR; (g) screening the PBMCs expressing the TCR for tumor reactivity; and (h) selecting the PBMCs of (g) that are tumor-reactive.

[0005] Another embodiment of the invention provides a pharmaceutical composition comprising the tumor-reactive T cells obtained by the inventive methods.

[0006] Still another embodiment of the invention provides a method of treating or preventing a tumor in a mammal, comprising administering the inventive

pharmaceutical composition to the mammal in an amount effective to treat or prevent the tumor in the mammal.

BRIEF DESCRIPTION OF THE FIGURES

[0007] Figure 1 is a schematic presentation of the germline and rearranged TCRa and TCRP genes and mature TCRafic complex.

[0008] Figure 2 is a bar graph showing the frequency (%) of the indicated T cell populations in bulk preparations obtained from the tumors of 22 patients with metastatic melanoma.

[0009] Figure 3 A is a graph showing the mean number of T cell β clonotype sequences (log 10) in the indicated T cell populations for 22 patients with metastatic melanoma. (Counting total productive sequences.)

[0010] Figure 3B is a graph showing the mean number of T cell beta clonotype sequences (log 10) in the indicated T cell populations for 22 patients with metastatic melanoma. (Counting productive unique sequences.)

[0011] Figure 4A is a graph showing the estimated clonality of the indicated T cell populations.

[0012] Figure 4B is a graph showing the maximum frequency (%) of TCRp clonotypes in the indicated T cell populations. [0013] Figures 4C-4D are graphs showing the frequency of TCR beta clonotypes clonotypes in CD8+ PD-1 + (C) and CD8+ PD-1- (D) T cells.

[0014] Figures 5A-5D are bar graphs showing the νβ clonotype frequency (%) (unshaded portions of bars) of bulk T cells, CD8+ population, CD8+PD1- population, and the CD8+PD1+ population from Patient 3713 (A), Patient 3922 (B), Patient 3926 (C), and Patient 3998 (D) with metastatic melanoma.

[0015] Figure 6A is a graph showing the frequency of a and β clonotypes (per loglO of TCR beta clonotype sequences).

[0016] Figure 6B is a graph showing the level of clonality of CD8+ PD-1+ a and β clonotypes.

[0017] Figure 7 is a bar graph showing the percentage (%) of TIL from an ovarian carcinoma having the indicated phenotypes.

[0018] Figure 8 is a bar graph showing the clonotype frequency (%) of the variable region of the beta chain (white portions of bars) in the materials for administration to a patient.

[0019] Figures 9 is a graph showing the clonality of PBMCs after various periods in patients with a cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The αβ T cell receptor is a heterodimer composed of a and β protein chains (Figure 1). Each chain includes two extracellular domains, the variable (V) region and the constant (C) region, followed by a transmembrane region and a short cytoplasmic tail. The variable domains of both the TCR a-chain and β-chain have three "complementarity determining regions" (CDR1 , CDR2 and CDR3) which contact and recognize a peptide-MHC complexes. In particular, the a and β CDR3s are responsible for recognizing processed antigen. From T cell to T cell, there is an extremely high degree of polymorphism in the amino acid sequences of the CDR3a and CDR3β. This level of polymorphism is necessary for T cells to recognize the wide scope of antigens that confront the immune system. The polymorphism in the amino acid sequences of the CDR3a and CDR3β result from DNA rearrangements within the TCR a and β genes that occur during the maturation of a T cell.

[0021] The genes that encode the TCR are made up cassettes of coding sequence referred to as "V" and "J" segments in the TCR a-gene and "V", "D" and "J "in the TCR β-chain. Stochastic rearrangement in the genomic DNA results in the juxtaposition of these DNA segments resulting in a functional TCR gene. However, these rearrangements may be imprecise and junctions of the Va-Ja and νβ-Dc-jp regions may be highly variable. In addition, the DNA-repair process may add new palindromic sequences and random nucleotides. This process may occur at the part of the TCR gene encoding the CDR3 regions and may account for the great diversity of T-cell receptors. It is estimated that this process can result in about 5.2 x l O¹⁵ CDR3 encoding sequences.

[0022] An embodiment of the invention provides methods for generating or obtaining tumor-reactive T cells. Briefly, a high frequency CDR3P clonotype is used to select a full length β chain. The appropriate TCR a chain for pairing with this β chain is identified and polynucleotides encoding both chains of the TCR chains are constructed. In an embodiment of the invention, a high frequency CDR3a clonotype is used to select a full length a chain. The appropriate TCR β chain for pairing with this a chain is identified and polynucleotides encoding both chains of the TCR chains are constructed. These polynucleotides may be introduced into PBMCs. The antitumor activity of TCR-expressing transformants may be determined, and highly active transformants may be selected for use in treating patients.

[0023] An embodiment of the invention provides methods for generating or obtaining tumor-reactive T cells based on clonotypic frequencies. Without desiring to be bound by any particular theories, an increased frequency of a clonotype may reflect a strong interaction between the TCR expressing the clonotype and an antigen, resulting a rapid expansion T cells of that clonotype. An embodiment of the invention provides methods for the preparation TCRs, or functional variants thereof, based on the identification of high frequency of T cells clonotypes. The rationale is that if this T cell reacts with an antigen, the number T cells expressing that clonotype should be increased. Therefore, in a tumor, the high frequency clonotypes are likely to be among the most active against the tumor.

[0024] In an embodiment of the invention, the method comprises obtaining a bulk population of T cell clonotypes from a biological sample from a patient, wherein each T cell clonotype comprises a T cell receptor (TCR) comprising a β chain comprising a "complementarity determining region 1 β ("CDR^"), a CDR2(3, and a CDR3|3 and an a chain comprising a CDR1 a (CDRla), a CDR2a, and a CDR3a. [0025] As used herein, the term "clonotype" refers to any TCR amino acid sequence that can be used to differentiate one TCR sequence from all other TCR sequences. This TCR sequence can serve as a marker of the lineage of T cells. The frequency of each clonotype in a population of T cells can serve as measurement of the immune response against a tumor antigen.

[0026] Any suitable biological sample can be used in accordance with the invention. In an embodiment of the invention, the biological sample is a tumor sample or a sample of peripheral blood. Examples of biological samples that may be used in accordance with invention include, without limitation, normal tissue, tissue from a primary tumors, tissue from the site of metastatic tumors, exudates, effusions, ascites, fractionated peripheral blood cells, bone marrow, peripheral blood buffy coat, cerebrospinal fluid, urine, feces, saliva, and cerebrospinal fluid. As such, the biological sample may be obtained by any suitable means, including, without limitation, aspiration, biopsy, resection, venous puncture, arterial puncture, lumbar spinal puncture, shunts, catheterization, or the placement of a drain. Preferred sources of the biological sample include tissue from melanomas and blood from patients with melanoma. Suitable patients include patients with primary or metastatic tumors, preferred embodiments of the invention include patients with melanoma. In an embodiment of the invention, the tumor is a melanoma, leukemia, astrocytoma, glioblastoma, retinoblastoma, lymphoma, glioma, Hodgkin's lymphoma, chronic lymphocytic leukemia or a tumor of the pancreas, breast, thyroid, ovary, uterus, testis, prostate, pituitary gland, adrenal gland, kidney, stomach, esophagus, rectum, small intestine, colon, liver, gall bladder, bile ducts, head and neck, tongue, mouth, eye and orbit, bone, joints, brain, nervous system, skin, blood, nasopharyngeal tissue, lung, larynx, urinary tract, cervix, vagina, exocrine glands, and endocrine glands. In another embodiment of the invention, the tumor is a bile duct carcinoma or an ovarian carcinoma.

[0027] The size of the bulk T cell population in the biological sample will vary depending upon the amount, source, and age of the biological sample.

[0028] Suitable patients include patients with primary or metastatic tumors, preferred embodiments of the invention include patients with melanoma.

[0029] The method may comprise obtaining a bulk population of T cells from a biological sample by any suitable method known in the art. For example, a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which specific cell populations can be selected. Suitable methods of obtaining a bulk population of T cells may include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting) the tumor, and aspiration (e.g., as with a needle).

[0030] The bulk population of T cells obtained from a biological sample may comprise any suitable type of T cell. Preferably, the bulk population of T cells obtained from a tumor sample comprises tumor infiltrating lymphocytes (TILs). The biological sample may be obtained from any mammal. Unless stated otherwise, as used herein, the term "mammal" refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits; the order Carnivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and Swines (pigs); or of the order Perssodactyla, including Equines (horses). The mammal may be a non-human primate, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some embodiments, the mammal may be a mammal of the order Rodentia, such as mice and hamsters. Preferably, the mammal is a non-human primate or a human. An especially preferred mammal is the human.

[0031] A preferred embodiment of the invention isolates bulk T cells from the site of metastatic melanoma. Another preferred embodiment of the invention isolates bulk T cells from the peripheral blood of a patient with a tumor. A preferred tumor is a melanoma.

[0032] In an embodiment of the invention, the method further comprises sorting the bulk population of T cell clonotypes for tumor-reactive T cells. In an embodiment of the invention, the method comprises (a) obtaining a bulk population of T cells from a biological sample; (b) specifically selecting CD8⁺ T cells that express any one or more of TIM-3, LAG-3, 4- I BB, and PD-1 from the bulk population; and (c) separating the cells selected in (b) from unselected cells to obtain a cell population enriched for tumor-reactive T cells. The separating may be carried out in any suitable manner such as, for example, by using fluorescence activated cell sorting ("FACS"), magnetic separation, or affinity chromatography. [0033] The method may comprise specifically selecting T cells expressing one or more of PD-1 , LAG-3, TIM-1 , 4-1BB, and CD 8 and separating the selected T cells from the bulk population. In a preferred embodiment, the method comprises selecting cells that also express CD3. The method may comprise specifically selecting the cells in any suitable manner, including but not limited to panning, affinity chromatography, or flow cytometry based on cell size (forward light scatter) or cell cytoplasmic complexity (side light scatter). Preferably, the selecting is carried out using antibodies and separation using fluoresce activated cell sorting (FACS) with a flow cytometer. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. For example, the specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3, anti-4-lBB, or anti- PD-1 antibodies, respectively. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome.

[0034] In an embodiment of the invention, selecting may comprise specifically selecting T cells that co-express PD-1 and any one or more of CD3, CD4, CD8, ΤΓΜ- 3, and CD27 and separating the selected T cells from the bulk population. In this regard, specifically selecting may comprise specifically selecting T cells that are single positive for expression of any one of TIM-3, LAG-3, 4-1BB, and PD-1 or specifically selecting T cells that are double, triple, or quadruple positive for simultaneous co-expression of any two, three or four of TIM-3, LAG-3, 4-1 BB, and PD-1. In an embodiment of the invention, the method comprises specifically selecting CD8⁺ T cells that express TIM-3 from the bulk population. In another embodiment, the method comprises specifically selecting CD8⁺ T cells that express LAG-3 from the bulk population. In still another embodiment, the method comprises specifically selecting CD8⁺ T cells that express 4- IBB from the bulk population. In still another embodiment of the invention, the method comprises specifically selecting CD8 T cells that express PD-1 from the bulk population. An additional embodiment of the invention provides a method comprising specifically selecting CD8⁺ T cells that are (i) 4-lBB⁺/PD-l⁺, (ii) 4-lBB7PD-l⁺, and/or (iii) 4-lBB⁺/PD-L from the bulk population. Another embodiment of the invention provides a method comprising specifically selecting CD8⁺ T cells that are (i) LAG-3⁺/PD-l⁺, (ii) LAG-37PD-1⁺, and/or (iii) LAG-3⁺/PD-l ^" from the bulk population. Still another embodiment of the invention provides a method comprising specifically selecting CD8⁺ T cells that are

(i) TIM-3⁺/PD-l⁺, (ii) TIM-37PD-1⁺, or (iii) TIM-3⁺/PD-l^" from the bulk population. Still another embodiment of the invention provides a method comprising specifically selecting CD8⁺ T cells that are (i) TIM-3⁺/LAG-3⁺, (ii) TIM-37LAG-3⁺, or (iii) ΤΓΜ- 3⁺/LAG-3^" from the bulk population. Another embodiment of the invention provides a method comprising specifically selecting CD8⁺ T cells that are (i) 4-l BB⁺/LAG-3⁺,

(ii) 4-lBB7LAG-3⁺, or (iii) 4-lBB⁺/LAG-3^" from the bulk population. Still another embodiment of the invention provides a method comprising specifically selecting CD8⁺ T cells that are (i) 4-lBB⁺/TIM-3⁺, (ii) 4-lBB7TIM-3⁺, or (iii) 4-l BB⁺/TIM-3^" from the bulk population. In another embodiment of the invention, any of the methods described herein may further comprise selecting cells that also express CD3⁺.

[0035] In an embodiment of the invention, specifically selecting may comprise specifically selecting combinations of CD8⁺ cells expressing any of the markers described herein. In this regard, the method may produce a cell population that is enriched for tumor-reactive cells that comprises a mixture of cells expressing any two, three, four, or more of the biomarkers described herein. In an embodiment of the invention, specifically selecting comprises specifically selecting any of the following combinations of cells: (a) PD-1⁺ cells and 4-l BB⁺ cells, (b) PD-1⁺ cells and LAG-3⁺ cells, (c) PD-1⁺ cells and TIM-3⁺ cells, (d) 4-l BB⁺ cells and LAG-3⁺ cells, (e) 4-lBB⁺ cells and TIM-3⁺ cells, (f) LAG-3⁺ cells and TIM-3⁺ cells, (g) PD-1⁺ cells, 4-lBB⁺ cells, and LAG-3⁺ cells, (h) PD-1⁺ cells, 4-lBB⁺ cells, and TIM-3⁺ cells, (i) PD-1 ⁺ cells, LAG-3⁺ cells, and TIM-3⁺ cells, 0^') 4-lBB⁺ cells, LAG-3⁺ cells, and TIM-3⁺ cells, and/or (k) PD-1⁺ cells, 4-l BB⁺ cells, LAG-3⁺ cells, and TIM-3⁺ cells. In another embodiment of the invention, any of the methods described herein may further comprise selecting cells that also express CD8 and/or CD3 .

[0036] An embodiment of the invention may comprise separating the selected cells from unselected cells to obtain a cell population enriched for tumor-reactive T cells. In this regard, the selected cells may be physically separated from the unselected cells. The selected cells may be separated from unselected cells by any suitable method such as, for example, sorting. Separating the selected cells from the unselected cells preferably produces a cell population that is enriched for tumor- reactive T cells.

[0037] The method may comprise selecting T cells that express PD-1 from the bulk population. In an embodiment of the invention, the T cells that express PD-1 may be PD-1 hi cells. In a preferred embodiment, selecting T cells that express PD-1 from the bulk population comprises selecting T cells that co-express (a) PD-1 and (b) any one or more of CD3, CD4, CD8, T cell immunoglobulin and mucin domain 3 (TIM-3), and CD27. In an embodiment of the invention, the cells that express CD3, CD4, CD8, TIM-3, or CD27 may be CD3hi, CD4hi, CD8hi, TIM-3hi, or CD27hi cells, respectively. The method may comprise specifically selecting the cells in any suitable manner. Preferably, the selecting is carried out using flow cytometry. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. For example, the specific selection of PD-1 , CD3, CD4, CD8, TIM-3, or CD27 may be carried out using anti-PD-1 , anti-CD3, anti-CD4, anti-CD8, anti-TIM-3, or anti-CD27 antibodies, respectively. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry is fluorescence-activated cell sorting (FACS).

[0038] In an embodiment of the invention, selecting may comprise specifically selecting PD-1+ T cells that are also positive for expression of (i) any one of CD4, CD8, TIM-3, and CD27; (ii) both of CD8 and TIM-3; (iii) both of CD8 and CD27; (iv) both of TIM-3 and CD27; (v) all three of CD8, TIM-3, and CD27; (vi) both of CD4 and TIM-3; (vii) both of CD4 and CD27; or (viii) all three of CD4, TIM-3, and CD27. In another embodiment of the invention, any one or more of the populations of (i)-(viii) may also co-express CD3.

[0039] In an embodiment of the invention, selecting T cells that express PD-1 from the bulk population comprises selecting any one or more of (a) CD8+PD-1+; (b) PD-1+TIM-3+; (c) PD-1 +CD27+; (d) CD8+PD-lhi; (e) CD8+PD-1+TIM-3+; (f) CD8+PD-l+CD27hi; (g) CD8+PD-1+CD27+; (h) CD8+PD-1+TIM-3-; (i) CD8+PD- 1+CD27-; 0^') CD4+PD-1 +; (k) CD4+PD-lhi; (1) CD4+PD-1+TIM-3+; (m) CD4+PD- l+CD27hi; (n) CD4+PD-1+CD27+; (o) CD4+PD-1+TIM-3-; and (p) CD4+PD- 1+CD27- T cells. In another embodiment of the invention, any one or more of the populations of (a)-(p) may also co-express CD3.

[0040] As used herein, the term "positive" (which may be abbreviated as "+"), with reference to expression of the indicated cell marker, means that the cell expresses the indicated cell marker at any detectable level, which may include, for example, expression at a low (but detectable) level as well as expression at a high (hi) level. The term "negative" (which may be abbreviated as "-"), as used herein with reference to expression of the indicated cell marker, means that the cell does not express the indicated cell marker at a detectable level. The term "high" (which may be abbreviated as "hi"), as used herein with reference to expression of the indicated cell marker, refers to a population of cells that are positive for expression of the indicated cell marker which stain more brightly for the indicated cell marker using

immunohistochemical methods (e.g., FACS, flow cytometry, immunofluorescence assays and microscopy) than other cells that are positive for expression of the indicated cell marker. For example, cells with a "high" level of expression of the indicated cell marker may stain more brightly than about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%, or a range of any two of the foregoing values, of the other cells that are positive for expression of the indicated cell marker.

[0041] In an embodiment of the invention, selecting T cells that express PD-1 may comprise selecting combinations of PD-1 ⁺ cells, each PD-1+ cell co-expressing any one, two, or more different markers as described herein. In this regard, the method may produce a cell population that is enriched for tumor-reactive cells that comprises a mixture of PD-1 + cells, each PD-1+ cell co-expressing any one, two, or more different markers described herein. In an embodiment of the invention, selecting T cells that express PD-1 comprises selecting a combination of (i) both PD- 1+CD8+ cells and PD-1+TIM-3+ cells; (ii) both PD-1+CD8+ cells and PD-1+CD27+ cells; (iii) both PD-1+TIM-3+ cells and PD-1 +CD27+ cells; (iv) all of PD-1+CD8+ cells, PD-1+TIM-3+ cells, and PD-1+CD27+ cells; (v) both PD-1 +CD4+ cells and PD-1+TIM-3+ cells; (vi) both PD-1+CD4+ cells and PD-1+CD27+ cells; (vii) all of PD-1+CD4+ cells, PD-1+TIM-3+ cells, and PD-1+CD27+ cells, or (viii) a combination of any of the populations of (i)-(vii). In another embodiment of the invention, any one or more of the populations of (i)-(vii) may also co-express CD3. In another embodiment of the invention, selecting T cells that express PD-1 comprises selecting a combination of any two or more of (a) CD8+PD-1+; (b) PD-1+TIM-3+; (c) PD-1+CD27+; (d) CD8+PD-lhi; (e) CD8+PD-1+TIM-3+; (f) CD8+PD- l+CD27hi; (g) CD8+PD-1+CD27+; (h) CD8+PD-1+TIM-3-; (i) CD8+PD-1+CD27-; 0) CD4+PD-1+; (k) CD4+PD-lhi; (1) CD4+PD-1+TIM-3+; (m) CD4+PD-l+CD27hi; (n) CD4+PD-1+CD27+; (o) CD4+PD-1+TIM-3-; and (p) CD4+PD-1+CD27- cells. In another embodiment of the invention, any one or more of the populations of (a)-(p) may also co-express CD3.

[0042] The method may comprise separating the T cells that express PD-1 from cells that do not express PD-1 to obtain a T cell population enriched for T cells that express PD-1. In this regard, the selected cells may be physically separated from unselected cells, i.e., the cells that do not express PD-1. The selected cells may be separated from unselected cells by any suitable method such as, for example, sorting.

[0043] The method may further comprise sequencing the CDR3a and the CDR3P of one or more T cell clonotypes to provide a CDR3a amino acid sequence and a CDR3P amino acid sequence which can be used to identify full length TCR gene clones. In an embodiment of the invention, each clonotype in the bulk population occurs at a frequency in the population, the frequency of each clonotype ranging from most frequent in the population to least frequent in the population. In an embodiment of the invention, the method may comprise sequencing the CDR3P of at least the 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, or 190 most frequent clonotypes in the population.

[0044] Sequencing the CDR3a and the CDR3P of one or more T cell clonotypes may be carried out in any suitable manner. In an embodiment of the invention, the sequencing may be carried out by deep sequencing the CDR3a and the CDR3p of coding sequences from a plurality of T cells obtained from the bulk culture to provide a CDR3a amino acid sequence and a CDR3P amino acid sequence. Sequencing may be carried out in any suitable manner known in the art. Examples of suitable sequencing techniques that may be useful in the inventive methods include the so- called "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 technologies. With NGS, millions or billions of DNA strands may be sequenced in parallel, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing of genomes. In NGS, nucleic acid templates may be randomly read in parallel along the entire genome by breaking the entire genome into small pieces. NGS may, advantageously, provide nucleic acid sequence information of a whole genome, exome, or transcriptome in very short time periods, e.g., within about 1 to about 2 weeks, preferably within about 1 to about 7 days, or most preferably, within less than about 24 hours. Multiple NGS platforms which are commercially available or which are described in the literature can be used in the context of the inventive methods, e.g., those described in Zhang et al., J. Genet. Genomics, 38(3): 95-109 (201 1) and Voelkerding et al., Clinical Chemistry, 55: 641-658 (2009).

[0045] Non-limiting examples of NGS technologies and platforms include sequencing-by-synthesis (also known as "pyrosequencing") (as implemented, e.g., using the GS-FLX 454 Genome Sequencer, 454 Life Sciences (Branford, CT), Illumina Solexa Genome Analyzer (Illumina Inc., San Diego, CA), or the Illumina HiSeq 2000 Genome Analyzer (Illumina), or as described in, e.g., Ronaghi et al., Science, 281 (5375): 363-365 (1998)), sequencing-by-ligation (as implemented, e.g., using the SOLid platform (Life Technologies Corporation, Carlsbad, CA) or the Polonator G.007 platform (Dover Systems, Salem, NH)), single-molecule sequencing (as implemented, e.g., using the PacBio RS system (Pacific Biosciences (Menlo Park, CA) or the HeliScope platform (Helicos Biosciences (Cambridge, MA)), nano- technology for single-molecule sequencing (as implemented, e.g., using the GridON platform of Oxford Nanopore Technologies (Oxford, UK), the hybridization-assisted nano-pore sequencing (HANS) platforms developed by Nabsys (Providence, RI), and the ligase-based DNA sequencing platform with DNA nanoball (DNB) technology referred to as probe-anchor ligation (cPAL)), electron microscopy-based technology for single-molecule sequencing, and ion semiconductor sequencing.

[0046] In an embodiment of the invention, the bulk T cell population is oligoclonal and deep sequencing allows for ranking of the CDR3a and CDR3P clonotypes based on their frequencies in the oligoclonal T cell population.

[0047] An embodiment of the invention provides methods for generating or obtaining tumor-reactive T cells based on clonotypic frequencies. In preferred embodiments, the invention provides methods for preparing T cell Receptors (TCRs), or functional variants thereof, based on the relative frequencies of the recurrent amino acid sequences of TCR CDRs (colonotypes). [0048] The method may further comprise pairing a CDR3a amino acid sequence with a CDR3P amino acid sequence. Pairing a CDR3a amino acid sequence with a CDR3P amino acid sequence may be carried out in any of a variety of different ways. In an embodiment of the invention, pairing may be carried out by isolating a single T cell from the population and sequencing the CDR3a and the CDR3P of the TCR of that single T cell. In this regard, the TCRa and β CDR3 coding regions of the single cell may be amplified using, e.g., PCR, and sequenced. Since the amplification products are from a single T cell, the α/β pairing that this technique identifies is likely to provide a functional TCR.

[0049] Alternatively or additionally, the method may comprise obtaining a monoclonal T cell population by limiting dilution cloning and sequencing the CDR3a and the CDR3P of the TCR of the monoclonal T cell population. Limiting dilution may generate single cell cultures, the numbers of which are then allowed to expand. PCR may then be used to identify the correct TCRa that pairs with the TCRp.

[0050] In another embodiment of the invention, wherein the population is oligoclonal, the pairing of a CDR3a amino acid sequence with a CDR3p amino acid sequence may be carried out by determining (i) frequency ranks of the CDR3a amino acid sequences in the oligoclonal T cell population and (ii) frequency ranks of the CDR3P amino acid sequences in the oligoclonal T cell population; and pairing a CDR3a amino acid sequence at a frequency rank with a CDR3 amino acid sequence with the same or different frequency rank. This technique may employ the sequences identified by deep sequencing to pair high-frequency TCR β clonotypes with high- frequency TCRa clonotypes based on their having identical or nearly identical frequencies.

[0051] In another embodiment of the invention, the pairing of a CDR3a amino acid sequence with a CDR3 amino acid sequence may be carried out by high throughput pairing of the CDR3a with a CDR3 . High throughput pairing of the CDR3a with a CDR3p may be carried out, for example, as described in Howie et al., Sci. Transl. Med., 7: 301ral 31 (2015). High throughput pairing of the CDR3a with a CDR3P may also be carried out by commercial entities such as, for example, using the PAIRSEQ assay from Adaptive Biotechnologies (Seattle, WA). High-throughput pairing may take advantage of the frequency of α/β coamplifications. Pools of cells may be distributed into a 96 well plate and sequenced in bulk for TCRa and TCRp. Pairs of TCRa and TCRP may be identified based on statistical calculations. PCR barcodes may be employed to identify the well from which each sequence is obtained.

[0052] The method may further comprise sequencing the CDRl a and the CDR2a of the a chain comprising the CDR3a amino acid sequence and the CDRi and the CDR2P of the β chain comprising the CDR3P amino acid sequence. The sequencing may be carried out as described herein with respect to other aspects of the invention.

[0053] The method may further comprise preparing one or more polynucleotide(s) encoding a TCR comprising the CDRla, CDR2a, CDR3a, CDRl p, CDR2p, and CDR3p; and introducing the one or more polynucleotide(s) into peripheral blood mononuclear cells (PBMCs) and expressing the TCR.

[0054] The polynucleotide(s) may encode an antigen binding portion of the TCR. The "the antigen-binding portion" of the TCR, as used herein, refers to any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to the mutated amino acid sequence encoded by the gene identified as described herein with respect to other aspects of the invention. The term "antigen-binding portion" refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Antigen-binding portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to the mutated amino acid sequence, or detect, treat, or prevent tumor, to a similar extent, the same extent, or to a higher extent, as compared to the parent TCR. In reference to the parent TCR, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent TCR.

[0055] The TCR antigen-binding portions can comprise an antigen-binding portion of either or both of the a and β chains of the TCR of the invention, such as a portion comprising one or more of the complementarity determining region (CDR)l , CDR2, and CDR3 of the variable region(s) of the a chain and/or β chain of the TCR of the invention. In an embodiment of the invention, the antigen-binding portion can comprise the amino acid sequence of the CDRl of the a chain (CDRla), the CDR2 of the a chain (CDR2a), the CDR3 of the a chain (CDR3a), the CDRl of the β chain (CDRl p), the CDR2 of the β chain (CDR2β), the CDR3 of the β chain (CDR3P), or any combination thereof. Preferably, the antigen-binding portion comprises the amino acid sequences of CDRla, CDR2a, and CDR3a; the amino acid sequences of CDRi p, CDR2p, and CDR3P; or the amino acid sequences of all of CDRl a, CDR2a, CDR3a, CDRl p, CDR2p, and CDR3p of the inventive TCR.

[0056] In an embodiment of the invention, the antigen-binding portion can comprise, for instance, the variable region of the inventive TCR comprising a combination of the CDR regions set forth above. In this regard, the antigen-binding portion can comprise the amino acid sequence of the variable region of the a chain (Va), the amino acid sequence of the variable region of the β chain (νβ), or the amino acid sequences of both of the Va and νβ of the inventive TCR.

[0057] In an embodiment of the invention, the antigen-binding portion may comprise a combination of a variable region and a constant region. In this regard, the antigen-binding portion can comprise the entire length of the a or β chain, or both of the a and β chains, of the inventive TCR.

[0058] Isolating the nucleotide sequence that encodes the TCR, or the antigen- binding portion thereof, from the selected T cells may be carried out in any suitable manner known in the art. For example, the method may comprise isolating RNA from the selected T cells and sequencing the TCR, or the antigen-binding portion thereof, using established molecular cloning techniques and reagents such as, for example, 5' Rapid Amplification of cDNA Ends (RACE) polymerase chain reaction (PCR) using TCR-a and -β chain constant primers.

[0059] In an embodiment of the invention, the method may comprise cloning the nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, into a recombinant expression vector using established molecular cloning techniques as described in, e.g., Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012). For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally- occurring. The recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA (e.g., complementary DNA (cDNA)) and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non-naturally- occurring internucleotide linkages, or both types of linkages. Preferably, the non- naturally occurring or altered nucleotides or internucleotide linkages does not hinder the transcription or replication of the vector.

[0060] The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of transposon/transposase, the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λβΤΙ Ο, XGTl 1 , ZapII (Stratagene), EMBL4, and λΝΜΙ 149, also can be used. Examples of plant expression vectors include pBIOl , pBI101.2, pBI101 .3, pBI121 and pBIN19

(Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, e.g., a retroviral vector.

[0061] The TCR, or the antigen-binding portion thereof, isolated by the inventive methods may be useful for preparing cells for adoptive cell therapies. In this regard, an embodiment of the invention provides a method of preparing a population of cells that express a TCR, or an antigen-binding portion thereof, which are shown to be tumor-reactive. This tumor-reactivity may be from having antigenic specificity for a known mutated amino acid sequence encoded by a tumor-specific mutation, the method comprising isolating a TCR, or an antigen-binding portion thereof, as described herein with respect to other aspects of the invention, and introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into host cells to obtain cells that express the TCR, or the antigen-binding portion thereof.

[0062] Introducing the nucleotide sequence (e.g., a recombinant expression vector) encoding the isolated TCR, or the antigen-binding portion thereof, into host cells may be carried out in any of a variety of different ways known in the art as described in, e.g., Green et al. supra. The method may comprise introducing a polynucleotide or polynucleotides encoding complete or functioning portions of the TCR a and β chains into PBMCs. The polynucleotide or polynucleotides may be introduced into PBMCs by any suitable means as described in, for example, Greenet al. supra. The polynucleotide(s) may be introduced by, for example, transduction. The transduction may employ any one or more of an adenoviral, retroviral, lentiviral, poxviral, or adeno-associated viral vector. Alternatively or additionally, the polynucleotide or polynucleotides can be introduced by transfection using

electroporation, salt precipitation, DEAE dextran, liposomes, or dendrimers. In a preferred embodiment, the polynucleotide is introduced into PBMCs by transduction with a bicistrontic lentivirus vetor.

[0063] The host cell into which the nucleotide sequence encoding the TCR, or antigen binding portion thereof, is introduced may be any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a cell. For purposes of producing the TCR, or antigen binding portion thereof, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a PBMC. More preferably, the host cell is a T cell.

[0064] Without being bound to a particular theory or mechanism, it is believed that less differentiated, "younger" T cells may be associated with any one or more of greater in vivo persistence, proliferation, and antitumor activity as compared to more differentiated, "older" T cells. Accordingly, the inventive methods may,

advantageously, identify and isolate a TCR, or an antigen-binding portion thereof, that has antigenic specificity for the mutated amino acid sequence and introduce the TCR, or an antigen-binding portion thereof, into "younger" T cells that may provide any one or more of greater in vivo persistence, proliferation, and antitumor activity as compared to "older" T cells (e.g., effector cells in a patient's tumor).

[0065] In an embodiment of the invention, the host cells are autologous to the patient. In this regard, the TCRs, or the antigen-binding portions thereof, identified and isolated by the inventive methods may be personalized to each patient. However, in another embodiment, the inventive methods may identify and isolate TCRs, or the antigen-binding portions thereof, that have antigenic specificity against a mutated amino acid sequence that is encoded by a recurrent (also referred to as "hot-spot") tumor-specific mutation. In this regard, the method may comprise introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into host cells that are allogeneic to the patient. For example, the method may comprise introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into the host cells from another patient whose tumors express the same mutation in the context of the same MHC molecule.

[0066] The method may further comprise screening the PBMCs expressing the TCR for tumor reactivity; and selecting the PBMCs that are tumor-reactive.

Screening the PBMCs expressing the TCR for tumor reactivity may be carried out in a variety of different ways. In an embodiment of the invention, screening for tumor reactivity may be carried out by identifying one or more genes in the nucleic acid of a tumor cell of the patient, each gene containing a tumor-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; and co-culturing the PBMC with the autologous APCs that present the mutated amino acid sequence to screen the PBMCs expressing the TCR for tumor reactivity as described in, for example, WO 2016/053338; WO 2016/053339; and Tran et al., Science, 344: 641- 645 (2014).

[0067] The method may comprise inducing autologous APCs of the patient to present the mutated amino acid sequence. The APCs may include any cells which present peptide fragments of proteins in association with MHC molecules on their cell surface. The APCs may include, for example, any one or more of macrophages, dendritic cells (DCs), langerhans cells, B-lymphocytes, and T-cells. Preferably, the APCs are DCs. By using autologous APCs from the patient, the inventive methods may, advantageously, identify T cells, TCRs, and antigen-binding portions thereof, that have antigenic specificity for a mutated amino acid sequence encoded by a tumor-specific mutation that is presented in the context of an MHC molecule expressed by the patient. The MHC molecule can 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, H LA-DP, HLA-DQ, and HLA-DR molecules. The inventive methods may, advantageously, identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient without using, for example, epitope prediction algorithms to identify MHC molecules or mutated amino acid sequences, which may be useful only for a select few MHC class I alleles and may be constrained by the limited availability of reagents to select mutation- reactive T cells (e.g., an incomplete set of MHC tetramers). Accordingly, in an embodiment of the invention, the inventive methods advantageously identify mutated amino acid sequences presented in the context of any MHC molecule expressed by the patient and are not limited to any particular MHC molecule. Preferably, the autologous APCs are antigen-negative autologous APCs.

[0068] Inducing autologous APCs of the patient to present the mutated amino acid sequence may be carried out using any suitable method known in the art. In an embodiment of the invention, inducing autologous APCs of the patient to present the mutated amino acid sequence comprises pulsing the autologous APCs with peptides comprising the mutated amino acid sequence or a pool of peptides, each peptide in the pool comprising a different mutated amino acid sequence. Each of the mutated amino acid sequences in the pool may be encoded by a gene containing a tumor specific mutation. In this regard, the autologous APCs may be cultured with a peptide or a pool of peptides comprising the mutated amino acid sequence in a manner such that the APCs internalize the peptide(s) and display the mutated amino acid sequence(s), bound to an MHC molecule, on the cell membrane. In an embodiment in which more than one gene is identified, each gene containing a tumor-specific mutation that encodes a mutated amino acid sequence, the method may comprise pulsing the autologous APCs with a pool of peptides, each peptide in the pool comprising a different mutated amino acid sequence. Methods of pulsing APCs are known in the art and are described in, e.g., Solheim (Ed.), Antigen Processing and Presentation Protocols (Methods in Molecular Biology), Human Press, (2010). The peptide(s) used to pulse the APCs may include the mutated amino acid(s) encoded by the tumor- specific mutation. The peptide(s) may further comprise any suitable number of contiguous amino acids from the endogenous protein encoded by the identified gene on each of the carboxyl side and the amino side of the mutated amino acid(s). The number of contiguous amino acids from the endogenous protein flanking each side of the mutation is not limited and may be, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or a range defined by any two of the foregoing values. Preferably, the peptide(s) comprise(s) about 12 contiguous amino acids from the endogenous protein on each side of the mutated amino acid(s).

[0069] In an embodiment of the invention, inducing autologous APCs of the patient to present the mutated amino acid sequence comprises introducing a nucleotide sequence encoding the mutated amino acid sequence into the APCs. The nucleotide sequence is introduced into the APCs so that the APCs express and display the mutated amino acid sequence, bound to an MHC molecule, on the cell membrane. The nucleotide sequence encoding the mutated amino acid may be RNA or DNA. Introducing a nucleotide sequence into APCs may be carried out in any of a variety of different ways known in the art as described in, e.g., Solheim et al. supra. Non- limiting examples of techniques that are useful for introducing a nucleotide sequence into APCs include transfom ation, transduction, transfection, and electroporation. In an embodiment in which more than one gene is identified, the method may comprise preparing more than one nucleotide sequence, each encoding a mutated 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, each population expressing and displaying a different mutated amino acid sequence, may be obtained.

[0070] In an embodiment in which more than one gene is identified, each gene containing a tumor-specific mutation that encodes a mutated amino acid sequence, the method may comprise introducing a nucleotide sequence encoding the more than one gene. In this regard, in an embodiment of the invention, the nucleotide sequence introduced into the autologous APCs is a tandem minigene (TMG) construct, each minigene comprising a different gene, each gene including a tumor-specific mutation that encodes a mutated amino acid sequence. Each minigene may encode one mutation identified by the inventive methods flanked on each side of the mutation 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 invention. The number of minigenes in the construct is not limited and may include for example, about 5, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 20, about 25, or more, or a range defined by any two of the foregoing values. The APCs express the mutated amino acid sequences encoded by the TMG construct and display the mutated amino acid sequences, bound to an MHC molecule, on the cell membranes. In an embodiment, the method may comprise preparing more than one TMG construct, each construct encoding a different set of mutated amino acid sequences encoded by different genes, and introducing each TMG construct into a different population of autologous APCs. In this regard, multiple populations of autologous APCs, each population expressing and displaying mutated amino acid sequences encoded by different TMG constructs, may be obtained.

[0071] The method may comprise co-culturing PBMCs expressing the TCR with the autologous APCs that present the mutated amino acid sequence. The method may comprise co-culturing the PBMCs expressing the TCR and autologous APCs so that the T cells encounter the mutated amino acid sequence presented by the APCs in such a manner that the T cells specifically bind to and immunologically recognize a mutated amino acid sequence.

[0072] The method may further comprise selecting the PBMCs that are tumor- reactive. Selecting the PBMCs that are tumor-reactive may be carried out in any suitable manner. In an embodiment of the invention, the method comprises selecting the PBMCs that express 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- 1 BB, OX40, and CD 107a upon co-culture with the autologous APCs.

[0073] In another embodiment of the invention, selecting the PBMCs that are tumor-reactive may be carried out by selecting the PBMCs (i) that secrete a greater amount of one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the amount of the one or more cytokines secreted by a negative control or (ii) in which at least twice as many of the numbers of PBMCs secrete one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the numbers of negative control T cells that secrete the one or more cytokines. Non-limiting examples of cytokines, the secretion of which is characteristic of T cell activation, include IFN-γ, IL-2, and tumor necrosis factor alpha (TNF-a), granulocyte/monocyte colony stimulating factor (GM-CSF), IL- 4, IL-5, IL-9, IL-10, IL-17, and IL-22.

[0074] In another embodiment of the invention, selecting the PBMCs that are tumor-reactive may be carried out by selecting the PBMCs that express 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 upon co- culture with autologous tumor cells.

[0075] In another embodiment of the invention, selecting the PBMCs that are tumor-reactive may be carried out by selecting the PBMCs (i) that secrete a greater amount of one or more cytokines upon co-culture with autologous tumor cells as compared to the amount of the one or more cytokines secreted by a negative control or (ii) in which at least twice as many of the numbers of PBMCs secrete one or more cytokines upon co-culture with autologous tumor cells as compared to the numbers of negative control T cells that secrete the one or more cytokines. The cytokine may be as described herein with respect to other aspects of the invention.

[0076] In an embodiment of the invention, the method further comprises expanding the numbers of PBMCs that are tumor reactive in vitro. The numbers of T cells may be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold), more preferably at least about 100-fold, more preferably at least about 1 ,000 fold, or most preferably at least about 100,000-fold. The numbers of T cells may be expanded using any suitable method known in the art. Exemplary methods of expanding the numbers of cells are described in U.S. Patent 8,034,334 and U.S. Patent Application Publication No. 2012/0244133, each of which is incorporated herein by reference.

[0077] In an embodiment of the invention, the method further comprises culturing the PBMCs that are tumor reactive obtained by the inventive methods in the presence of any one or more of TWSl 19, interleukin (IL)-21 , IL-12, IL-15, IL-7, transforming growth factor (TGF) beta, and AKT inhibitor (AKTi). Without being bound to a particular theory, it is believed that culturing the enriched cell population in the presence of TWSl 19, IL-21 , and/or IL-12 may, advantageously, enhance the antitumor reactivity of the enriched cell population by preventing or retarding the differentiation of the enriched cell population. [0078] In an embodiment of the invention, the method further comprises transducing or transfecting the PBMCs that are tumor reactive obtained by any of the inventive methods described herein with a nucleotide sequence encoding any one or more of IL-12, IL-7, IL-15, IL-2, IL-21 , mirl 55, and anti-PD-1 siRNA.

[0079] The term "tumor antigen" as used herein refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) solely or predominantly expressed or over- expressed by a tumor cell, such that the antigen is associated with the tumor. The cancer antigen can additionally be expressed by normal or non-tumor cells. However, in such cases, the expression of the tumor antigen by normal or non-tumor cells is not as robust as the expression by tumor cells. In this regard, the tumor cells can over- express the antigen or express the antigen at a significantly higher level, as compared to the expression of the antigen by normal or non-tumor cells. Also, the tumor antigen can additionally be expressed by cells of a different state of development or maturation. For instance, the tumor antigen can be additionally expressed by cells of the embryonic or fetal stage, which cells are not normally found in an adult host. Alternatively, the tumor antigen can be additionally expressed by stem cells or precursor cells, which cells are not normally found in an adult host.

[0080] Another embodiment of the invention provides a TCR, or an antigen- binding portion thereof, isolated by any of the methods described herein with respect to other aspects of the invention. An embodiment of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (a) chain of a TCR, a beta (β) chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of a TCR, or a combination thereof. Another embodiment of the invention provides an antigen-binding portion of the TCR comprising one or more CDR regions, one or more variable regions, or one or both of the a and β chains of the TCR, as described herein with respect to other aspects of the invention. The polypeptides of the inventive TCR, or the antigen-binding portion thereof, can comprise any amino acid sequence, provided that the TCR, or the antigen-binding portion thereof, has tumor- reactivity. Such tumor-reactivity includes, but is not limited to, antigenic specificity for the mutated amino acid sequence encoded by a tumor-specific mutation.

[0081] Another embodiment of the invention provides an isolated population of cells prepared according to any of the methods described herein with respect to other aspects of the invention. The population of cells can be a heterogeneous population comprising the host cells expressing the isolated TCR, or the antigen-binding portion thereof, in addition to at least one other cell, e.g., a host cell (e.g., a PBMC), which does not express the isolated TCR, or the antigen-binding portion thereof, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) expressing the isolated TCR, or the antigen-binding portion thereof. The population also can be a clonal or oligoclonal population of cells, in which all cells of the population are clones of a single host cell expressing the isolated TCR, or the antigen- binding portion thereof, such that all cells of the population express the isolated TCR, or the antigen-binding portion thereof. In one embodiment of the invention, the population of cells is a clonal population comprising host cells expressing the isolated TCR, or the antigen-binding portion thereof, as described herein. By introducing the nucleotide sequence encoding the isolated TCR, or the antigen binding portion thereof, into host cells, the inventive methods may, advantageously, provide a population of cells that comprises a high proportion of host cells that express the isolated TCR and have tumor-reactivity, which may result from antigenic specificity for the mutated amino acid sequence. In an embodiment of the invention, about 1 % to about 100%, for example, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%o, 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 foregoing values, of the population of cells comprises host cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence. Without being bound to a particular theory or mechanism, it is believed that populations of cells that comprise a high proportion of host cells that express the isolated TCR and have antigenic specificity for the mutated amino acid sequence have a lower proportion of irrelevant cells that may hinder the function of the host cell, e.g., the ability of the host cell to target the destruction of tumor cells and/or treat or prevent tumor.

[0082] The inventive tumor-reactive TCRs, or the antigen-binding portions thereof, and populations of cells can be formulated into a composition, such as a pharmaceutical composition. In this regard, an embodiment of the invention provides a pharmaceutical composition comprising any of the inventive TCRs, or the antigen- binding portions thereof, or populations of cells and a pharmaceutically acceptable carrier. The inventive pharmaceutical composition can comprise an inventive TCR, or an antigen-binding portion thereof, or population of cells in combination with another pharmaceutically active agent(s) or drug(s), such as a chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0083] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the particular inventive TCR, or the antigen-binding portion thereof, or population of cells under consideration. Such pharmaceutically acceptable carriers are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.

[0084] The choice of carrier will be determined in part by the particular inventive TCR, the antigen-binding portion thereof, or population of cells, as well as by the particular method used to administer the inventive TCR, the antigen-binding portion thereof, or population of cells. Accordingly, there are a variety of suitable

formulations of the pharmaceutical composition of the invention. Suitable

formulations may include any of those for oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, or interperitoneal administration. More than one route can be used to administer the inventive TCR or population of cells, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0085] Preferably, the inventive TCR, the antigen-binding portion thereof, or population of cells is administered by injection, e.g., intravenously. When the inventive population of cells is to be administered, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaCl in water, 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 Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0086] It is contemplated that the inventive tumor-reactive TCRs, or the antigen- binding portions thereof, populations of cells, and pharmaceutical compositions can be used in methods of treating or preventing tumor. Without being bound to a particular theory or mechanism, the inventive TCRs, or the antigen-binding portions thereof, are believed to tumor-reactive or, preferably, bind specifically to a mutated amino acid sequence encoded by a tumor-specific mutation, such that the TCR, or the antigen-binding portion thereof, when expressed by a cell, is able to mediate an immune response against a target cell expressing the mutated amino acid sequence. In this regard, an embodiment of the invention provides a method of treating or preventing tumor in a patient, comprising administering to the patient any of the pharmaceutical compositions, TCRs, antigen-binding portions thereof, or populations of cells described herein, in an amount effective to treat or prevent tumor in the patient.

[0087] The terms treat," and "prevent", as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of tumor in a patient. Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the tumor being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumor. Also, for purposes herein, "prevention" can encompass delaying the onset of the tumor or its recurrence, or a symptom or condition thereof.

[0088] For purposes of the invention, the amount or dose of the inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition administered (e.g., numbers of cells when the inventive population of cells is administered) should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the patient over a reasonable time frame. For example, the dose of the inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition should be sufficient to demonstrate tumor-reactivity, including but not limited to, to bind to a mutated amino acid sequence encoded by a tumor-specific mutation, or detect, treat or prevent tumor in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition administered and the condition of the patient, as well as the body weight of the patient to be treated.

[0089] Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing the inventive TCR, or the antigen-binding portion thereof, or the inventive populations of cells, upon administration of a given dose of such T cells to a mammal among a set of mammals of which is each given a different dose of the cells, could be used to determine a starting dose to be administered to a patient. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a certain dose can be assayed by methods known in the art.

[0090] The dose of the inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition. Typically, the attending physician will decide the dosage of the inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive TCR, the antigen-binding portion thereof, population of cells, or pharmaceutical composition to be administered, route of administration, and the severity of the condition being treated.

[0091] In an embodiment in which the inventive population of cells is to be administered, the number of cells administered per infusion may vary, for example, in the range of one million to 200 billion cells; however, amounts below or above this exemplary range are within the scope of the invention. For example, the daily dose of inventive host cells can be about 1 million to about 200 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, about 160 billion cells, about 170 billion cells, about 180 billion cells, about 190 billion cells, about 200 billion cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 200 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 1 10 billion cells, about 120 billion cells, about 130 billion cells, about 140 billion cells, about 150 billion cells, about 160 billion cells, about 170 billion cells, about 180 billion cells, about 190 billion cells, about 200 billion cells, or a range defined by any two of the foregoing values), more preferably about 100 million cells to about 200 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 1 10 billion cells, about 120 billion cells, about 130 billion cells, about 140 billion cells, about 150 billion cells, about 160 billion cells, about 170 billion cells, about 180 billion cells, about 190 billion cells, about 200 billion cells, or a range defined by any two of the foregoing values).

[0092] For purposes of the inventive methods, wherein populations of cells are administered, the cells can be cells that are allogeneic or autologous to the patient. Preferably, the cells are autologous to the patient.

[0093] Another embodiment of the invention provides any of the TCRs, the antigen-binding portions thereof, isolated population of cells, or pharmaceutical compositions described herein for use in treating or preventing tumor in a patient.

[0094] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0095] This example demonstrates that PD 1 + expression is significantly higher in the CD8+ population. [0096] Table 1 A is a list of the patients under study in these Examples.

TABLE 1A

PR=partial response

CR=complete response

NR= no response

N/A= not applicable

[0097] PBMCs were obtained by either leukapheresis or venipuncture, prepared over Ficoll-Hypaque gradient (LSM; ICN Biomedicals Inc.), and cryopreserved until analysis. Briefly, tumor specimens were minced under sterile conditions, followed by enzymatic digestion (RPMI-1640 with 1-glutamine [Lonza],l mg/ml collagenase IV [Sigma- Aldrich], 30 U/ml DNAse [Genentech], and antibiotics) overnight at room temperature or for several hours at 37 °C and intermittent mechanical tissue separation using gentle MACS (Miltenyi Biotech). Tumor single-cell suspensions were cryopreserved until further analysis.

[0098] Fluorescently conjugated antibodies were obtained from BD Biosciences (UCHT1 , 1.6:100, CD3 PE-CF594; RPTA-T4, 3.4: 100, CD4 V500; NK-1 , 3:100, CD57 FITC, J 168-540, 1.2: 100, BTLA PE), eBioscience (MIH-4, 1.6:100, PD-1 APC), Biolegend (0323, 2: 100, CD27 BV605; BD96, 1.4:100, CD25 BV650;

EH12.2H7, 0.7:100, PD-1 BV421 ; c398.4A, 1 :100, ICOS Pacific Blue), R&D

(344823, 2.6: 100, TIM-3 PE and APC), Enzo Life Sciences (17B4, 1 : 100, LAG-3 FITC), and Miltenyi (4B4-1, 2.6: 100, 4-lBB PE). Cell sorting by flow cytometry was perfomied on a modified FORTESA instrument (BD Biosciences), equipped to detect 18 fluorescent parameters. Data were compensated and analyzed with FLOWJO software (Treestar). T cell populations were separated using a modified FACSARIA instrument (BD Biosciences). Antibodies and tetramers were titrated for optimal staining.

[0099] The phenotypic characterization of CD8+ T cells and cell sorting were done using flow cytometry. PBMCs and tumor samples were thawed in the presence of 3 U/ml DNAse (Genentech Inc.) and rested for 16-24 hours in a 1 :1 mix of RPMI- 1640 with 1 glutamine (Lonza) and AIMV (Gibco) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 12.5 mM HEPES, and 5% human serum

(noncommercial, prepared from healthy donors) without cytokines (T cell media) at a concentration of 2 x 10⁶ live cells (determined by trypan blue exclusion) per well in a 24-well plate at 37 °C in 5% C0₂. This overnight resting period was carefully assessed and was believed to facilitate the recovery of expressed cell surface receptors affected by cryopreservation or subject to cleavage during the enzymatic digest. Cells were harvested, counted, and blocked with Fc block for human cells (Miltenyi). Live cells (1 10⁶) were stained for 30 minutes in a 50 μΐ volume of F ACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) with the antibodies. Propidium iodide (PI) was added to exclude dead cells.

[0100] The Mann- Whitney test, Dunn test for multiple comparisons, and

Wilcoxon signed-rank tests were used to determine the statistical significance of the data. To determine the statistical difference in marker expression between CD8+ T cells in PBLs and TILs, P were values for the number of comparisons. P values of 0.05 or less were considered significant. The significant P values are shown in Figure 2.

[0101] As shown in Figure 2, PD1+ expression was significantly higher in the CD8+ population. As shown in Figure 2, no preferential infiltration of CD8+ cells compared to CD8- cells was observed. The difference in frequency between

CD8+PD-1+ and CD8+PD-1- populations was statistically significant (P=0.0001).

EXAMPLE 2

[0102] This example demonstrates the use of deep sequencing to identify oligoclonal T cell populations in melanoma TILs.

[0103] Cells of interest were sorted into a 1 :1 mix of PBS and FBS (Hyclone Defined, Logan), and the cell pellet was resuspended in 200 μΐ RNA later

(Invitrogen). mRNA was extracted and the TRB gene products of sorted CD8+ T cell populations were amplified using a template-switch anchored RT-PCR, as previously described (Gros A, et al., Clin Cancer Res. 2012;18(19):5212-5223), followed by ILLUMINA sequencing. TCRP annotation was performed by combining a custom Java program and NCBI BLAST+. Briefly, BLAST+ was used to identify the V and J germline genes of a TCRP read. The sequence of interest was determined by finding the conserved cysteine at the 5' end of the CDR3 and the conserved phenylalanine at the 3' end of the CDR3. Unique TCRP species, which is defined as a unique TRBV- CDR3 (nucleotide)-TRBJ combination, were then collapsed to determine the count for each species. Similar cell numbers were sorted from each population from the fresh tumor to ensure comparable coverage (total number of TCRP sequence counts from a population/initial cell input of population) to enable comparison of clonotypic diversity among different populations. In addition to determining the frequency of the clonotypes, data from deep sequencing were used to determine the "clonality" of different T cell populations. "Clonality" refers to a measure equal to the inverse of the normalized Shannon entropy of all productive clones in a sample. Values for clonality range from 0 to 1. Values near 1 represent samples with one or a few predominant clones (monoclonal or oligoclonal samples) dominating the observed repertoire. Clonality values near 0 represent more polyclonal samples. [0104] As shown in Figures 3A and 3B, the number of total productive sequences is comparable between the different samples analyzed, including blood pre-treatment, bulk TIL population, and the sorted TIL populations (CD8-, CD8+, CD8+PD1-, CD8+PD1+), indicating no bias in the depth of sequencing. The number of unique sequences in the samples decreased in the CD8+ population as compared to the CD8- population and in the CD8+PD1+ population compared to the CD8+PD1- population, indicating more oligoclonality in these populations. The same number of cells were sorted for each of these populations. In addition, the bulk TIL population from fresh tumor appeared to be more oligoclonal than the blood pre-treatment sample.

[0105] As shown in Figure 4A, there is a high degree of "clonality"

(oligoclonality) in CD8+PD1+ T cells. The CD8+ population is significantly more clonal than the CD8+PD1- population. In addition, Figure 4B shows that the

CD8+PD1+ population contains TCRB clonotypes at a higher frequency. Taken together, these data suggest that clonal expansion due to antigen recognition has occurred. As shown in Figure 4C, the most frequent clonotype in the CD8+PD1+ T cell population is not present or is present at a low frequency in the CD8+PD1- population, indicating that there is a different composition of TCRB clonotypes in these two populations.

[0106] Figures 5A-5D shows the size of the oligoclonal subpopulations in the bulk T cells, CD8+ population, CD8+PD1- population, and CD8+PD1+ T cell population from four patients with metastatic melanoma (white portion of bars in Figures 5A-5D).

[0107] Table I B lists the clonotype CDR3 beta amino acid sequences and frequencies in the boxed portion of Figure 5A (Patient 3713).

TABLE I B

[0108] Table 1C lists the clonotype CDR3 beta amino acid sequences and frequencies in the boxed portion of Figure 5B (Patient 3922).

TABLE 1 C

[0109] Table ID lists the clonotype CDR3 beta amino acid sequences and frequencies in the boxed portion of Figure 5C (Patient 3926). TABLE ID

[0110] Table IE lists the clonotype CDR3 beta amino acid sequences and frequencies in the boxed portion of Figure 5D (Patient 3998).

TABLE IE

EXAMPLE 3A

[0111] This example demonstrates pairing alpha and beta chains to provide a functional TCR.

[0112] After high frequency clonotypes of the TCR CDR3 beta locus were identified in Example 2, the task became to pair each β chain containing each CDR3 beta locus with an appropriate TCRa chain to provide a functional TCR.

[0113] The TCR beta (TCRB) and TCR alpha (TCRA) sequences were obtained from the same sorted CD8+PD1 + population for each sample and the frequency of each TCR chain in the CD8+PD1+ population was analyzed. The most frequent TCR is paired with the most frequent TCRA from the same sorted CD8+PD1+ population. As shown in Figures 6A and 6B, the clonality values of the TCR alpha chain and the TCR beta chain were comparable. Accordingly, the most frequent TCRB was paired with the most frequent TCRA from this oligoclonal subpopulation.

[0114] In a separate experiment, the frequency of the most dominant TCRB and the most dominant TCRA in the same CD8+PD1+ population were compared in each of six patients. The results are shown in Table IF. As shown in Table IF, the maximum frequency TCR clonotype was identified for each of the six patients.

TABLE IF

[0115] A retrospective analysis of pairing alpha and beta chains based

frequencies was carried out. The results are shown in Table 1G.

TABLE 1G

[0116] As shown in Table 1 G, the most frequent TCRB was paired with the most frequent TCRA from the same sample. Without being bound to a particular theory or mechanism, it is believed that pairing the most frequent TCRB with the most frequent TCRA will be most effective with respect to oligoclonal samples. EXAMPLE 3B

[0117] This example demonstrates that PBMCs transduced with a nucleotide sequence encoding the TCR alpha and beta chain pair identified in Example 3A are tumor-reactive.

[0118] As a test of the function of the TCR alpha and beta chain pair identified in Example 3 A, a polynucleotide was encoding the TCR alpha and beta chain was constructed. The polynucleotide was transduced into PBMCs.

[0119] 4- IBB upregulation was used to measure recognition of target cells by the transduced cells.

[0120] 4- IBB is upregulated transiently in response to TCR stimulation, regardless of the effector cytokines produced or the differentiation state of the cell, and its expression on stimulated CD8+ cells peaks at 24-36 hours. After 15 days of in vitro expansion of the numbers of transduced PBMCs prior to co-culture with tumor cell targets, basal expression of 4- IBB was consistently negative.

[0121] For assessment of tumor cell recognition by the transformed PBMCs, the transduced effector cells were thawed 2 days prior to co-culture with target tumor cells. The transduced effector cells were cultured at 1 10⁶ cells/ml in 3,000 IU/ml IL-2 at 37 °C in 5% C0₂. Two days later, effector cells were washed and cultured, either alone or with target cells (1 x 105:1 x 105) or plate-bound anti-CD3 (O T3, Miltenyi), in 96-well plates in T cell media without IL-2.

[0122] In antibody-blocking experiments, melanoma cells were preincubated with 0.05 mg/ml HLA-I or HLA-II blocking antibodies (clones W6/32 and IVA12, respectively) for 3 hours, followed by coculture with T cells. 24 hours later, supematants were harvested (duplicates) and analyzed, and cells were stained in 40 μΐ PBS containing 0.5% BSA and 2 mM EDTA with anti-CD3, anti-CD8, and anti- 4- 1 BB antibodies and acquired on Canto II (BD Biosciences). Data were analyzed with FlowJo software (Treestar).

[0123] The results showed that expression of the TCR β was accompanied by increased 4-1 BB expression after 24 hours of co-culture with an autologous tumor- cell line. As a positive control, autologous B cells, loaded with mutant peptides, elicited an increased expression of 4- IBB when TCRs for the mutant peptides were used. EXAMPLE 4

[0124] This example demonstrates that tumor-reactive TCRs may be among top ten most frequent TCRs in the population. As shown in Table 2, the most frequent TCR clonotypes were often tumor reactive.

TABLE 2

*Not all TCRs tested because of difficulty in matching a and β chains

*N/A not available

EXAMPLE 5

[0125] This example demonstrates the clonality of TILs from ovarian tumors and gastrointestinal tumors.

[0126] TIL were obtained from an ovarian tumor. The TIL were sorted into the following populations: bulk lymphocytes, CD3+ cells, CD8+ cells, CD8- cells, CD8+PD1+ cells, and CD8-PD1+ cells. As shown in Figure 7, a preferential infiltration of CD8- cells was observed in the ovarian tumor TIL. The PD1 expression of the ovarian tumor TIL was also lower than that observed in the melanoma TIL (Figure 7).

[0127] The CDR3 region of the beta chain of TILs from the ovarian tumor of three patients were sequenced, and the frequency of each CDR3 beta sequence in the population was ranked. The results are shown in Tables 3-5. The TIL from ovarian tumors appeared to show oligoclonality (Tables 3-5). [0128] Preliminary data from gastrointestinal (GI) carcinomas from three patients show that the TIL in gastrointestinal carcinoma also appears to be oligoclonal. From this limited cohort of three GI carcinoma patients, one mutation-reactive TCR was identified for each patient from within the top 10 most frquent clonotypes in the bulk TIL population.

TABLE 3

N/A = below detection limit.

TABLE 4

N/A = below detection limit.

TABLE 5

N/A = below detection limit.

EXAMPLE 6

[0129] This example demonstrates that cells in the infusion bag (made up of cells selected for use in treatment and expanded in vitro) are significantly more oligoclonal than TIL in the corresponding fresh tumor.

[0130] The CDR3 beta regions of CD8+PD1+ TIL in the infusion bag were sequenced, and the frequency of each CDR3 beta sequence in the population was ranked. The results are shown in Figure 8.

TABLE 6

N/A = below detection limit.

[0131] As shown in Figure 8, the infusion bag (made up of cells selected for use in treatment and expanded in vitro) were significantly more oligoclonal than the TIL in the corresponding fresh tumor (FrTu). On average, only a few clonotypes accounted for 50% of the infusion product. It is believed that the specificity of those clonotypes may be relevant for the clinical outcome.

[0132] In the 14 melanoma patients examined, a maximum of three of the top 10 CD8+PD1+ clonotypes in the fresh tumor appeared to be within the top 10 clonotypes in the infusion bag, as shown in Table 6. [0133] In a separate experiment, the clonality of TILs in various samples (blood pre-treatment, fresh tumor bulk, infusion bag, blood 1 month post-treatment, and blood 1 year post treatment) was measured. The results are shown in Figure 9. As shown in Figure 9, the clonality in the infusion bag was higher as compared to the clonality in the fresh tumor, and that clonality appears to last at least one month.

[0134] It was also observed that mutation-reactive clonotypes persist and can be found at a high percentage 1 year after treatment.

EXAMPLE 7

[0135] This example demonstrates a method of obtaining tumor-reactive TCRs from peripheral blood.

[0136] Peripheral blood T cells were harvested from the PBMCs which had been obtained by either leukapheresis or venipuncture, prepared over Ficoll-Hypaque gradient (LSM; ICN). Similar cell numbers were sorted from each population from the fresh tumor to ensure comparable coverage (total number of TCRP sequence counts from a population/initial cell input of population) to enable comparison of clonotypic diversity among different populations. Expansion of CD8+ T cells and establishment of T cell clones (Biomedicals Inc.) was carried out. The cells were cryopreserved until immunophenotypic analysis or deep sequencing.

[0137] Cells of interest were sorted into a 1 :1 mix of PBS and FBS (Hyclone Defined, Logan), and the cell pellet was resuspended in 200 μΐ RNAlater (Invitrogen). mRNA was extracted and TRB gene products of sorted CD8+ T cell populations were amplified using a template-switch anchored RT-PCR, followed by ILLUMINA deep sequencing of the DNA. TCRP annotation was performed by combining cultures. CD25 depletion was carried out followed by CD4-positive selection using the BD IMag cell separation system (BD Biosciences).

[0138] For Patient #3998, clonotype beta chains were paired with alpha chains and construction of TCR Nos. 1 ,2, 3 A and 3B, TCR4, TCR5A and TCR5B, TCR6, TCR7, TCR9, TCR 10, TCR1 1 , TCR 12, TCR 13, TCR 14, TCR 16, TCR 19, TCR20 and TCR21 in the circulating CD8+PD-1+ cells led to the isolation of 6 TCRs recognizing tumor antigen NY-ESO-1 , and 3 TCRs recognizing a cancer specific neo- antigen MAGE-A6 (see Table 7). The results were encouraging; out of 19 TCRs constructed, 1 1 TCRs were capable of recognizing the autologous tumor cell line 3998mel. In addition, TCRl , which was NY-ESO-1 specific and recognized tumor, was the most frequent TCR clonotype in the peripheral blood CD8+ population before sorting. These results demonstrated that, for this patient, selection of CD8+PD-1 + cells and construction of the T-cell receptors of the most dominant clonotypes in this population led to the rapid isolation of tumor-reactive T-cell receptors that can be used for treatment.

TABLE 7

EXAMPLE 8

[0139] This example demonstrates the identification of productive TCR pairs using single cell RT-PCR on CD8+ TIL or using the pairSEQ approach on single cell suspensions from unsorted fresh tumors.

[0140] Without being bound to a particular theory or mechanism, it is believed that discordance of pairing based on frequency of the TCRA is likely due to the presence of more than 1 a-chain in some cells as well as the variable efficiency of primers used in the TCRA sequencing. Thus, it was decided to identify the productive TCR pairs with 2 different approaches. After using deep sequencing to identify the most frequent TCRBs in the CD8+PD-1+ population, the corresponding TCRA was identified using single cell RT-PCR on CD8+PD-1+ FACS sorted TIL or CD8+ TIL expanded in vitro. Alternatively, the pairSEQ approach (Howie et al., Sci. Transl. Med., 7: 301ral31 (2015)) was used on single cell suspensions from unsorted fresh tumors. The efficiency of the single cell RT-PCR was between 26% and 90% depending on the sample. Using this method, a median value of 29 (range 9-43) unique TCRA-TCRB pairs were identified in each of the CD8+ or CD8+PD-1 + samples. Using pairSEQ on unsorted fresh tumors, a median value of 217 (range 1 1- 883) unique pairs were identified for each sample. A total of 93 (median value 6, range 0-21) TCRA-TCRB pairs were identified using both methods (congruent pairs in Table 8). 83 of these pairs ranked within the top 10 CD8+PD-1+ clonotypes, so the evaluation of their anti-tumor activity was tested.

[0141] Expression vector constructs encoding the appropriate TCRA-TCRB pairs, linked with murine constant chain sequences that improve stability and avoid mismatches with endogenous human TCRs (Cohen et al., Cancer Res., 66: 8878-86 (2006)), were then generated and used to genetically engineer fresh PBL. The frequency of T-cells that expressed the recombinant TCRs following either retroviral transduction or transfection with a Sleeping Beauty transposon construct ranged between 24.4 and 97.6%.

TABLE 8

Sample Number of Number of Number of Number of

ID Unique TCR Unique TCR Unique reconstructed TCR pairs identified pairs identified congruent pairs evaluated within by Single cell by pairSEQ* TCR pairs the top 10 CD8+PD-1 +

RT-PCR# clonotypes

1913 29 136 3 8

2650 30 21 3 7

3678 32 1 1 0 4 Sample Number of Number of Number of Number of

RT-PCRff clonotypes

3713 21 829 21 4

3759 34 133 15 7

3784 15 883 7 9

3903 14 156 5 10

3922 9 351 3 5

3926 33 737 6 8

3977 29 21 2 8

3992 20 278 9 5

3998 43 349 19 8

ft Single cell PCR was performed on sorted CD8+PD-1 + TIL and for 1913, 2650, 3713, 3784 on sorted CD8+ expanded TIL due to limited availability of tumor samples.

* PairSEQ was performed on bulk TIL.

EXAMPLE 9

[0142] This example demonstrates that high frequency CD8+PD-1+ clonotypes display tumor and mutation reactivity.

[0143] The anti-tumor activity of T-cells engineered to individually express each of the 83 (median value 8 per patient; range 4-10) TCR pairs that belonged to the 10 highest frequency TCRB in the CD8+PD-1+ TIL from 12 metastatic melanoma samples (Table 8) was then evaluated. The TCRs obtained from 10 of the 12 patients were evaluated for reactivity against candidate neo-epitopes identified by whole exome sequencing of autologous tumor (TCRs from samples 2650 and 3977 were only evaluated against the tumor cell (TC) line). All of the TCR pairs were also tested against autologous or HLA -matched antigen presenting cells transfected with full-length RNA encoding the melanoma/melanocyte shared differentiation antigens MART-1 , gpl OO and tyrosinase (TYR) and cancer-germline antigens NY-ESO-1 , MAGEA3 and SSX2.

[0144] Evaluation of the response against the corresponding autologous TC and/or autologous antigen presenting cells that had either been pulsed with mutated tumor-specific neoantigen minimal epitopes or transfected with tandem minigene (TMG) (Robbins et al., Nat. Med., 19: 747-52 (2013); Lu et al., Clin. Cancer Res., 20:3401-10 (2014); Lu et al, J. Immunol, 190: 6034-42 (2013)) constructs provided evidence for tumor antigen reactivity in 1 1 of the 12 patients that were evaluated.

[0145] For example, for patient 3998, the reactivity of some of the top 8 most frequent TCR pairs based on the frequency of TCRB (3998-1 , 3998-2, 3998-3A1 , 3998-3A2, 3998-4, 3998-6, 3998-7 and 3998-8) against the autologous TC was initially evaluated. Six of the TCR pairs tested (3998-1 , 3998-2, 3998-4, 3998-6, 3998-7 and 3998-8) showed MHC restricted recognition of the autologous tumor (as measured by CD 137 up-regulation greater than 1 %, 3 times the background and inhibited at least 50% by pan MHC-I blocking antibody (clone W6-32)). The TCRB clonotype ranking 3^ld in frequency in the CD8+PD-1+ TIL was associated with 2 productive TCRA chains but none of the 2 combinations (3998-3A1 and 3998-3A2) were tumor reactive. For the tumor sample 3998, 345 non-synonymous mutations were identified. The reactivity of the 6 TCR pairs (3998-1, 3998-2, 3998-4, 3998-6, 3998-7 and 3998-8) against 1 15 mutated antigens encoded by 7 TMGs and 6 shared melanoma/melanocyte differentiation antigens and cancer-germline antigens (MART- 1 , gpl OO, SSX2, TYR, NY-ESO-1 , MAGE A3) was next evaluated. The 115 mutated antigens were selected for screening among the 345 non-synonymous mutations found based on RNAseq data of their expression level. Two TCR pairs (3998-7 and 3998-8) were reactive to TMG-1. Further testing identified MAGEA6^E168K as the specific mutation recognized within the antigens encoded by TMG-1. Reactivity against 1 shared antigen (NY-ESO-1) was found for TCR pair 3998-5 (Table 9).

[0146] A summary of the findings of all 12 samples is presented in Table 3. All of the TCR pairs that were considered positive fit the following criteria: 3 times the background, CD137 expression >1 % in response to the autologous tumor and, inhibition > 50% by pan MHC-I blocking antibody. For example, in sample 1913, the TCRB ranking 2, 3 and 4 were specific for the autologous TC line, and the clonotypes ranking 2 and 4 also recognized a mutation in the HLA-1 1 gene (Table 9 (N/A = below detection limit)). Moreover, the most frequent TCR clonotype was found to be tumor reactive for 7 samples (2650, 3759, 3903, 3922, 3926, 3977 and 3998). For all but patient 3992, up to 5 tumor-reactive TCRs were found among the 5 most frequently expressed TCRs in the CD8+PD-1+ TIL. Reactivity against autologous neoantigens was found in 5 of the 10 patients whose TCRs were screened against putative autologous mutations. In summary, it was found that 36 TCR pairs were reactive against autologous tumor and 11 were directed against mutated tumor- specific neoantigens. This indicates that it is possible to identify tumor-reactive TCR pairs in the majority of melanoma samples simply based on their frequency in the CD8+PD-1+ TIL compartment.

[0147] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0148] The use of the temis "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to 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 indicated herein or clearly contradicted by context. The terms "comprising," "having,"

"including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0149] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to 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 otherwise clearly contradicted by context.

Claims

CLAIMS:

1. A method of obtaining tumor-reactive T cells comprising:

(a) obtaining a bulk population of T cell clonotypes from a biological sample from a patient, wherein each T cell clonotype comprises a T cell receptor (TCR) comprising a beta chain comprising a complementarity determining region 1 beta ("CDRl p"), a CDR2p, and a CDR3p and an alpha chain comprising a CDR1 alpha (CDRla), a CDR2a, and a CDR3a;

(b) sequencing the CDR3a and the CDR3P of one or more T cell clonotypes obtained in (a) to provide a CDR3a amino acid sequence and a CDR3P amino acid sequence;

(c) pairing a CDR3a amino acid sequence of (b) with a CDR3p amino acid sequence of (b);

(d) sequencing the CDRla and the CDR2a of the alpha chain comprising the CDR3a amino acid sequence of (c) and the CDRl p and the CDR2P of the beta chain comprising the CDR3P amino acid sequence of (c);

(e) preparing one or more polynucleotide(s) encoding a TCR comprising the CDRla, CDR2a, CDR3a, CDRi p, CDR2p, and CDR3p of (d);

(f) introducing the one or more polynucleotide(s) of (e) into peripheral blood mononuclear cells (PBMCs) and expressing the TCR;

(g) screening the PBMCs expressing the TCR for tumor reactivity; and

(h) selecting the PBMCs of (g) that are tumor-reactive.

2. The method of claim 1 , wherein (a) further comprises selecting T cells expressing one or more of PD-1 , LAG-3, TIM-1, 4- IBB, and CD8 and separating the selected T cells from the bulk population.

3. The method of claim 2, wherein (a) comprises selecting T cells that co- express PD-1 and any one or more of CD3, CD4, CD8, TIM-3, and CD27 and separating the selected T cells from the bulk population.

4. The method of claim 2, wherein (a) comprises selecting T cells that co- express CD8 and PD-1 and separating the selected cells from the bulk population.

5. The method of claim 2, wherein (a) comprises selecting T cells that are

(i) CD8+PD-1+;

(ii) PD-1+TIM-3+;

(iii) PD-1+CD27+;

(iv) CD8+PD-lhi;

(v) CD8+PD-1+TIM-3+;

(vi) CD8+PD-1+CD27W;

(vii) CD8+PD-1+CD27+;

(viii) CD8+PD-1+TIM-3-;

(ix) CD8+PD-1+CD27-

(x) CD4+PD-1+;

(xi) CD4+PD-lhi;

(xii) CD4+PD-1+TIM-3+;

(xiii) CD4+PD-l+CD27hi;

(xiv) CD4+PD-1+CD27+;

(xv) CD4+PD-1+TIM-3-; or

(xvi) CD4+PD-1+CD27-,

and separating the selected T cells from the bulk population.

6. The method of any one of claims 1-5, wherein the separating is carried out using fluorescence activated cell sorting ("FACS"), magnetic separation, or affinity chromatography.

7. The method of any one of claims 1-6, wherein the sequencing of (b) is carried out by deep sequencing.

8. The method of any one of claims 1 -7, wherein (c) is carried out by isolating a single T cell from the population and sequencing the CDR3a and the CDR3p of the TCR of the single T cell.

9. The method of any one of claims 1-7, wherein (c) is earned out by obtaining a monoclonal T cell population by limiting dilution cloning and sequencing the CDR3a and the CDR3P of the TCR of the monoclonal T cell population.

10. The method of any one of claims 1-7, wherein the population is oligoclonal and (c) is carried out by

determining (i) frequency ranks of the CDR3a amino acid sequences in the oligoclonal T cell population and (ii) frequency ranks of the CDR3P amino acid sequences in the oligoclonal T cell population; and

pairing a CDR3a amino acid sequence at a frequency rank with a CDR3p amino acid sequence at the same or different frequency rank.

11. The method of any one of claims 1 -7, wherein (c) is carried out using high throughput pairing of the CDR3a of (b) with a CDR3P of (b).

12. The method of any one of claims 1 -1 1 , wherein the polynucleotide is introduced by transduction.

13. The method of claim 12, wherein the transduction employs an adenoviral, retroviral, lentiviral, poxviral, or adeno-associated viral vector.

14. The method of any one of claims 1-13, wherein the polynucleotide is introduced by transfection.

15. The method of claim 14, wherein transfection is carried out using electroporation, salt precipitation, DEAE dextran, liposomes, or dendrimers.

16. The method of any one of claims 1 -15, wherein the polynucleotide is introduced into PBMCs by transduction with a bicistrontic lentivirus vetor.

17. The method of any one of claims 1 -16, wherein (g) is earned out by identifying one or more genes in the nucleic acid of a tumor cell of the patient, each gene containing a tumor-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; and

co-culturing the PBMC with the autologous APCs that present the mutated amino acid sequence to screen the PBMCs expressing the TCR for tumor reactivity.

18. The method of claim 17, wherein (h) comprises selecting the PBMCs that express 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- IBB, OX40, and CD 107a upon co-culture with the autologous APCs.

19. The method of claim 17, wherein (h) comprises selecting the PBMCs (i) that secrete a greater amount of one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the amount of the one or more cytokines secreted by a negative control or (ii) in which at least twice as many of the numbers of PBMCs secrete one or more cytokines upon co-culture with APCs that present the mutated amino acid sequence as compared to the numbers of negative control T cells that secrete the one or more cytokines.

20. The method of any of claims 1-16, wherein (h) comprises selecting the PBMCs that express 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 CD 107a upon co-culture with autologous tumor cells.

21. The method of any one of claims 1 -16, wherein (h) comprises selecting the PBMCs (i) that secrete a greater amount of one or more cytokines upon co-culture with autologous tumor cells as compared to the amount of the one or more cytokines secreted by a negative control or (ii) in which at least twice as many of the numbers of PBMCs secrete one or more cytokines upon co-culture with autologous tumor cells as compared to the numbers of negative control T cells that secrete the one or more cytokines.

22. The method of any one of claims 1-21, further comprising introducing one or more polynucleotides encoding any one or more of IL-12, IL-7, IL-15, IL-2, IL-21 , mirl 55, and anti-PD-1 siRNA into the PBMCs.

23. The method of claim 22, wherein the biological sample is a tumor sample.

24. The method of claim 22, wherein the biological sample is a peripheral blood sample.

25. The method of any one of claims 1-24, wherein the tumor is a melanoma, leukemia, astrocytoma, glioblastoma, retinoblastoma, lymphoma, glioma, Hodgkin's lymphoma, chronic lymphocytic leukemia or a tumor of the pancreas, breast, thyroid, ovary, uterus, testis, prostate, pituitary gland, adrenal gland, kidney, stomach, esophagus, rectum, small intestine, colon, liver, gall bladder, bile ducts, head and neck, tongue, mouth, eye and orbit, bone, joints, brain, nervous system, skin, blood, nasopharyngeal tissue, lung, larynx, urinary tract, cervix, vagina, exocrine glands, and endocrine glands.

26. The method of claim 25, wherein the tumor is a melanoma.

27. The method of claim 25, wherein the tumor is a bile duct carcinoma.

28. The method of claim 25, wherein the tumor is an ovarian carcinoma.

29. A pharmaceutical composition comprising the tumor-reactive T cells obtained by the method of any one of claims 1 -28 and pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29 for use in the treatment or prevention of a tumor in a mammal.