Screening of T lymphocytes for cancer-specific antigens

Abstract

Provided herein are methods for identifying cancer-specific antigens that recognize TCRs and TCR engineered T cells with antigen-specific cytotoxic activity. Provided herein are engineered T lymphocytes produced by the methods described herein. Provided herein are methods for treating cancer in a subject, the methods comprising administering engineered T lymphocytes as described herein. Provided herein are antibodies or fragments thereof produced by the methods described herein. Provided herein are methods for treating cancer in a subject, the methods comprising administering antibodies as described herein to the subject. In some embodiments, therapeutic compositions (e.g., engineered lymphocytes, antibodies, etc.) and methods herein are provided as part of a kit or system.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 569,215, filed on October 6, 2017, which is incorporated herein by reference. Technical Field

[0003] Provided herein are methods for identifying cancer-specific antigens that recognize T cell receptors and T cell receptor-engineered T cells having antigen-specific cytotoxic activity. Background Art

[0004] Cancer immunotherapy treats cancer by strengthening the patient's own anti-tumor immune response. Specifically, the success of immune checkpoint inhibitors highlights the importance of anti-cancer immune activity in cancer patients. However, only a few patients show clinical benefits from anti-immune checkpoint therapy, while 70%-80% of cancer patients do not benefit from such treatments or the benefits are minimal. Therefore, it is important and urgent to determine the mechanism of immunotherapy resistance and develop methods to further enhance and improve immune responses (reference 1; incorporated by reference in its entirety). Although cytotoxic T lymphocytes (CTLs) play a key role in cancer immunotherapy, the identification of CTL T cell receptors (TCRs) and their target cancer-specific antigens is difficult and time-consuming. Summary of the invention

[0005] Provided herein are methods for identifying cancer-specific antigens that recognize TCRs and TCR-engineered T cells having antigen-specific cytotoxic activity.

[0006] In some embodiments, provided herein are methods comprising: (a) stimulating target lymphocytes (e.g., CD8 + (b) capturing immunocompetent lymphocytes (e.g., CD8 + The invention relates to a method for capturing immunocompetent T lymphocytes (T-lymphocytes), wherein the capturing comprises contacting the immunocompetent T-lymphocytes with a capture reagent that displays a major histocompatibility complex (MHC) bound to a capture peptide comprising a candidate antigen sequence; and (c) sequencing all or a portion of the TCR of the captured immunocompetent T-lymphocytes.

[0007] In some embodiments, the target lymphocytes are obtained from a healthy donor. In some embodiments, the target lymphocytes are CD8 + Cytotoxic T lymphocytes. In some embodiments, stimulation is performed in vitro (eg, in cell culture).

[0008] In some embodiments, the peptides comprising the candidate antigen sequences are all or fragments of oncogenic antigens and neoantigens. In some embodiments, the candidate antigen sequences are all or fragments of oncogenic antigens and neoantigens.

[0009] In some embodiments, the capture agent is an MHC multimer. In some embodiments, the MHC multimer is an MHC dextramer.

[0010] In some embodiments, sequencing includes next generation sequencing technology. In some embodiments, the portion of the TCR sequenced includes TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced includes one or more complementary determining regions (CDRs) of TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced includes CDR3 of TCR-α and / or TCR-β chains.

[0011] In some embodiments, target lymphocytes (e.g., CD8 + cytotoxic T lymphocytes) are the target lymphocyte population, wherein the stimulatory peptide is one of a population of stimulatory peptides comprising different candidate antigen sequences; and wherein the capturing comprises causing immunocompetent T lymphocytes (e.g., CD8 + A population of cytotoxic T lymphocytes (CTl) is contacted with a capture reagent that displays a major histocompatibility complex (MHC) that binds to a population of capture peptides comprising candidate antigen sequences.

[0012] In some embodiments, provided herein are cancer specific antigens (e.g., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, etc.).

[0013] In some embodiments, provided herein are methods comprising: (a) stimulating target lymphocytes (e.g., CD8 + (b) capturing immunocompetent T lymphocytes (e.g., CD8 + The method comprises: (a) obtaining an engineered T lymphocyte (e.g., CD8 T lymphocyte) by exposing the immunocompetent T lymphocyte to a capture agent that displays a major histocompatibility complex (MHC) bound to a capture peptide comprising a candidate antigen sequence; (b) sequencing all or a portion of the TCR of the captured immunocompetent T lymphocyte; and (c) generating an engineered T lymphocyte (e.g., CD8 T lymphocyte) that displays all or a portion of the TCR of the captured immunocompetent T lymphocyte. + Cytotoxic T lymphocytes), wherein the engineered T lymphocytes recognize antigen presenting cells displaying MHC bound to a peptide comprising a candidate antigen sequence.

[0014] In some embodiments, the engineered T lymphocytes are CD8 + Cytotoxic T lymphocytes.

[0015] In some embodiments, engineered T lymphocytes (e.g., CD8 + The method comprises: (i) cloning a nucleic acid sequence encoding a portion of the TCR of the captured immunocompetent T lymphocyte into a vector; (ii) introducing the vector into a host T lymphocyte (e.g., CD8 + cytotoxic T lymphocytes); and (iii) cultured under conditions where the portion of the TCR of the captured immunocompetent T lymphocytes is expressed and displayed on the engineered T lymphocytes. In some embodiments, the portion of the TCR comprises TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR comprises one or more complementary determining regions (CDRs) of TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced comprises CDR3 of TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45-132. In some embodiments, the engineered T lymphocyte displays a TCR comprising an α and β chain (e.g., CDR3) containing an amino acid sequence pair selected from the group consisting of: SEQ ID NO:45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130 and 131 and 132. In some embodiments, the vector is introduced into a host T lymphocyte from a healthy donor host. In some embodiments, the vector is introduced into a host T lymphocyte from a cancer patient to be treated with engineered T lymphocytes.

[0016] In some embodiments, provided herein are engineered T lymphocytes (e.g., CD8 +In some embodiments, the engineered T lymphocytes are CD8 + Cytotoxic T lymphocytes.

[0017] In some embodiments, provided herein is a method of treating cancer in a subject, the method comprising administering to the subject an engineered T lymphocyte (e.g., CD8 + cytotoxic T lymphocytes).

[0018] In some embodiments, provided herein are methods comprising: (a) stimulating target lymphocytes (e.g., CD8 + (b) capturing immunocompetent T lymphocytes (e.g., CD8 +Cytotoxic T lymphocytes), wherein the capture comprises contacting the immunocompetent T lymphocytes with a capture reagent that displays a major histocompatibility complex (MHC) bound to a capture peptide comprising a candidate antigen sequence; (c) sequencing all or a portion of the TCR of the captured immunocompetent T lymphocytes; and further comprising: (d) producing a therapeutic antibody comprising all or a portion of the sequence of the TCR of the captured immunocompetent T lymphocytes. In some embodiments, the portion of the TCR comprises a TCR-α and / or TCR-β chain. In some embodiments, the portion of the TCR comprises one or more complementary determining regions (CDRs) of the TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR that is sequenced comprises a CDR3 of a TCR-α and / or TCR-β chain. In some embodiments, the portion of the TCR that is sequenced comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45-132. In some embodiments, the therapeutic antibody comprises a CDR3 comprising an amino acid sequence pair selected from the group consisting of: SEQ ID NO:45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130, and 131 and 132. In some embodiments, the antibody is an antibody fragment.

[0019] In some embodiments, provided herein are antibodies produced by the methods described herein. In some embodiments, the antibody is an antibody fragment.

[0020] In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject an antibody described herein.

[0021] The embodiments herein are described as utilizing CD8 + Cytotoxic lymphocytes as target cells and / or for generating engineered CD8 + However, in other embodiments within the scope of the present invention, the target cells and / or engineered lymphocytes described herein may instead contain CD4+ Helper lymphocytes, NK cells, NKT cells, B cells, and dendritic cells are used as target cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A -C. Induction of peptide-specific CTLs derived from FOXM1 and UBE2T and cytotoxic activity of established CTLs. (A) Production of IFN-γ by FOXM1 and UBE2T-specific CTLs was confirmed only when exposed to C1R-A24 cells stimulated with FOXM1 or UBE2T-specific peptides. The R / S ratio indicates the ratio of responding cells (CTLs) / stimulating cells (C1R-A24 cells). (B, C) FOXM1 (B) and UBE2T-specific CTLs (C) exerted significant cell killing effects on HLA-A*24:02-positive SW480 cells, but had no effect on HLA-A*24:02-negative HCC1143 cells or BT549 cells. Both CTLs (2×10 5 cells / well) were all contaminated with cancer cells (2×10 4 cells / well) and incubated for 5 hours.

[0023] Figure 2A -B. Generation of TCR-engineered T cells for FOXM1 and UBE2T. (A) The distribution of TCRA and TCRB CDR3 clonotypes of FOXM1 and UBE2T-specific CTLs is presented in a pie chart with CDR3 sequences. Black indicates the portion of CDR3 clonotypes with a read frequency below 1%. This population contains only one clonotype that is dominant for TCRA and TCRB. (B) Transduction efficiency was checked by staining for CD8 and TCRvβ8 (FOXM1 TCR-engineered T cells) or TCRvβ13 (UBE2T TCR-engineered T cells). Flow cytometry graphs represent FOXM1 or UBE2T-TCR-engineered T cells.

[0024] Figure 3A -H. Cytotoxic activity of TCR-engineered T cells against FOXM1 and UBE2T. (A, B) TCR-engineered T cells against FOXM1 (A) and UBE2T (B) exerted significant cell killing effects on HLA-A*24:02-positive SW480 cells, but had no effect on HLA-A*24:02-negative HCC1143 cells. (C, D) Time course of cancer cell viability co-cultured with FOXM1 (C) and UBE2T TCR-engineered T cells (D). Two sorted TCR-engineered T cells (4×10 5 cells / well) were all contaminated with cancer cells (2×10 4(E, F) Recognition of TCR-engineered T cells against FOXM1 and UBE2T in ELISPOT assays when stimulated with C1R-A24 cells pulsed with or without FOXM1 (E) or UBE2T-specific peptides (F). Sorted TCR-engineered T cells (5×10 4 cells / well) and stimulated cells (2×10 4 Cells / well) were co-incubated in 96-well plates at 37°C for 20 h. (G, H) Protein levels of granzyme B and perforin secreted from primary specific CTLs or TCR-engineered T cells at 0 h, 2.5 h, and 5 h after co-culture with cancer cells.

[0025] Figure 4 . Expression of FOXM1 and UBE2T proteins in cancer cells The expression of endogenous FOXM1 and UBE2T proteins in HLA-A*24:02 positive or negative cancer cell lines was examined by western blot analysis.

[0026] definition

[0027] The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the embodiments described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. However, in the event of a conflict, the present specification including the definitions shall prevail. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0028] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an engineered lymphocyte" is a reference to one or more engineered lymphocytes and equivalents thereof known to those skilled in the art, etc.

[0029] As used herein, the term "comprising" and its language variants indicate the presence of the described features, elements, method steps, etc., without excluding the presence of additional features, elements, method steps, etc. Conversely, the term "consisting of" and its language variants indicate the presence of the described features, elements, method steps, etc., and exclude any undescribed features, elements, method steps, etc., except for commonly associated impurities. The phrase "consisting essentially of" indicates the presence of the described features, elements, method steps, etc. and any additional features, elements, method steps, etc. that do not substantially affect the basic properties of the composition, system or method. Many embodiments herein are described using open "comprising" language. Such embodiments encompass multiple closed "consisting of" and / or "consisting essentially of" embodiments, which may be alternatively claimed or described using such language.

[0030] As used herein, "immune response" refers to cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules (including antibodies, cytokines and complement) produced by any of these cells or the liver to cause selective targeting, binding, destruction, destruction and / or elimination of invading pathogens, cells or tissues infected with pathogens, or cancer cells or other abnormal cells from the subject. Some embodiments herein include generating an immune response in a subject to treat cancer.

[0031] As used herein, the term "immunotherapy" refers to the treatment or prevention of a disease or condition by a method that includes inducing, enhancing, suppressing or otherwise altering an immune response. Some embodiments herein include immunotherapy.

[0032] As used herein, the terms "adoptive immunotherapy" and "adoptive cell transfer" refer to the transfer of immunocompetent cells (e.g., TCR engineered T cells) for the treatment of cancer or infectious diseases (June, CH ed., 2001, Cancer Chemotherapy and Biotherapy: Principles and Practice, Lippincott Williams & Wilkins, Baltimore; Vonderheide et al., 2003, Immun. Research 27: 1-15; incorporated by reference in its entirety). Some embodiments herein include adoptive immunotherapy.

[0033] As used herein, the term "cancer vaccine" refers to a composition (eg, a tumor antigen) that elicits a specific immune response. The response is elicited from the subject's own immune system by administering the cancer vaccine.

[0034] As used herein, the term "natural immune cell" refers to an immune cell naturally present in a subject's immune system. Illustrative examples include, but are not limited to, T cells, NK cells, NKT cells, B cells, and dendritic cells. Some embodiments herein include causing a response to a subject's natural immune cell in a subject.

[0035] As used herein, the term "engineered immune cell" refers to a genetically modified immune cell (eg, T cell, NK cell, NKT cell, B cell, dendritic cell, etc.). Some embodiments herein include generating and / or administering engineered immune cells.

[0036] As used herein, the term "T cell receptor" ("TCR") refers to a molecular complex found on the surface of a T cell (T lymphocyte) that is responsible for recognizing an antigen fragment bound to the major histocompatibility complex (MHC) of an antigen presenting cell. The binding between TCR and an antigenic peptide has a relatively low affinity and is degenerate: that is, many TCRs recognize the same antigenic peptide, and many antigenic peptides are recognized by the same TCR. TCR is a heterodimer consisting of two different protein chains. In 95% of human T cells, TCR is composed of an α (α) chain and a β (β) chain (encoded by TRA and TRB, respectively), while in 5% of T cells, TCR is composed of a γ and δ (γ / δ) chain (encoded by TRG and TRD, respectively). When TCR engages with an antigenic peptide and MHC, T lymphocytes are activated by signal transduction. Some embodiments herein include producing an engineered TCR, preparing cells displaying an engineered TCR, and / or administering cells displaying an engineered TCR to a subject to treat cancer.

[0037] As used herein, the term "human leukocyte antigen" ("HLA") refers to the major histocompatibility complex (MHC) proteins in humans or the gene complex encoding the human MHC proteins.

[0038] As used herein, the term "antibody" refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab' and F(ab')2), which may be polyclonal or monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, etc. As used herein, when an antibody or other entity "specifically recognizes" or "specifically binds" an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules, and the affinity of binding to the antigen or epitope is substantially higher than that to other entities that do not display the antigen or epitope. In this regard, "substantially higher affinity" means that the affinity is high enough to detect the antigen or epitope that is distinguished from the entity using the desired assay or measuring instrument. Typically, it means having at least 10 7 M -1 (e.g. >107 M -1 、>10 8 M -1 、>10 9 M -1 、>10 10 M -1 、>10 11 M -1 、>10 12 M -1 、>10 13 M -1 The binding constant (K a ). In certain such embodiments, the antibody is capable of binding to different antigens, as long as the different antigens contain the specific epitope. In some cases, for example, homologous proteins from different species may contain the same epitope. Some embodiments herein include generating and / or administering antibodies that bind to oncogenic antigens and / or neoantigens.

[0039] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, including at least a portion of the antigen binding region or variable region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, bifunctional antibodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, for example, Hudson et al. (2003) Nat. Med. 9:129-134; incorporated herein by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies produced by recombinant DNA technology (e.g., papain digestion and pepsin digestion of antibodies) or by chemical polypeptide synthesis. For example, a "Fab" fragment contains a light chain, and a C of a heavy chain. H1 The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fab'" fragment contains one light chain and the variable region contained in the C H1 With C H2A heavy chain with an additional constant region extending between the Fab' domains. An interchain disulfide bond can be formed between the two heavy chains of the Fab' fragment, thereby forming a "F(ab')2" molecule. The "Fv" fragment contains variable regions from heavy and light chains, but lacks constant regions. Single-chain Fv (scFv) fragments contain heavy and light chain variable regions connected by a flexible linker, thereby forming a single polypeptide chain with an antigen binding region. Exemplary single-chain antibodies are discussed in detail in WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203; incorporated herein by reference in their entirety. In some cases, a single variable region (e.g., a heavy chain variable region or a light chain variable region) is capable of recognizing and binding to an antigen. A skilled technician will be aware of other antibody fragments. Some embodiments herein include the generation and / or administration of antibody fragments that bind to oncogenic antigens and / or neoantigens.

[0040] As used herein, the term "monoclonal antibody" refers to an antibody that is a member of a substantially homogeneous antibody population that specifically binds to the same epitope. In certain embodiments, the monoclonal antibody is secreted by a hybridoma. In certain such embodiments, the hybridoma is produced according to certain methods known to those skilled in the art. See, for example, Kohler and Milstein (1975) Nature 256:495-499; incorporated herein by reference in its entirety. In certain embodiments, monoclonal antibodies are produced using recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). In certain embodiments, a monoclonal antibody refers to an antibody fragment isolated from a phage display library. See, for example, Clackson et al. (1991) Nature 352:624-628; and Marks et al. (1991) J. Mol. Biol. 222:581-597; incorporated herein by reference in its entirety. The modifier "monoclonal" indicates the antibody properties obtained from a substantially homogeneous antibody population, and does not limit the method of producing the antibody to a specific method. For various other monoclonal antibody production techniques, see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY); incorporated herein by reference in its entirety. Some embodiments herein include generating and / or administering monoclonal antibodies that bind to oncogenic antigens and / or neoantigens.

[0041] The term "antigen binding site" refers to a portion of an antibody that is capable of specifically binding to an antigen. In certain embodiments, the antigen binding site is provided by one or more antibody variable regions.

[0042] The term "epitope" refers to any polypeptide determinant that is capable of specific binding to an immunoglobulin or a T cell or B cell receptor. In certain embodiments, an epitope is a region of an antigen to which an antibody specifically binds. In certain embodiments, an epitope may include a chemically active surface grouping of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group. In certain embodiments, an epitope may have specific three-dimensional structural properties (e.g., a "conformational" epitope) and / or specific charge characteristics.

[0043] If a particular antibody specifically binds to both epitopes, then one epitope is defined as being "identical" to another epitope. In certain embodiments, polypeptides having different primary amino acid sequences may comprise the same epitope. In certain embodiments, the same epitope may have different primary amino acid sequences. If different antibodies compete for specific binding to a particular epitope, then the antibodies are said to bind to the same epitope.

[0044] As used herein, the term "sequence identity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same monomer subunit sequence composition. The term "sequence similarity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are amino acids that share the same biophysical characteristics and can be grouped into families (see above). "Percentage of sequence identity" (or "percentage of sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.); (2) determining the number of positions containing the same (or similar) monomers (e.g., the same amino acid exists in the two sequences, similar amino acids exist in the two sequences), and obtaining the number of matching positions; (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); and (4) multiplying the result by 100 to obtain the percentage of sequence identity or the percentage of sequence similarity. For example, if peptides A and B are both 20 amino acids long and have the same amino acid at all positions except position 1, then peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both are acidic), then peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to the amino acids of a portion of peptide C, then peptides C and D have 70% sequence identity, but the best comparison window of peptide D and peptide C has 93.3% sequence identity. In order to calculate "percentage of sequence identity" (or "percentage of sequence similarity") herein, any gap in the aligned sequence is treated as a mismatch at the position. In some embodiments, the peptide or polypeptide herein comprises the lowest sequence identity with the base sequence.

[0045] The term "effective dose" or "effective amount" refers to the amount of an agent, such as an antibody, that causes a decrease in symptoms or produces a desired biological result in a patient. In certain embodiments, an effective dose or effective amount is sufficient to treat or reduce the symptoms of a disease or disorder.

[0046] As used herein, the terms "administration" and "administering" refer to the action of giving a drug, prodrug or other agent or therapeutic agent to a subject or to cells, tissues and organs in vivo, in vitro or ex vivo. Exemplary human administration routes can be through the space under the arachnoid membrane of the brain, or the spinal cord (intrathecal), eyes (ocular), mouth (oral), skin (superficial or transdermal), nose (nasal), lungs (inhalation), oral mucosa (buccal), ears, rectum, vagina, by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), etc.

[0047] Unless otherwise indicated, the term "treatment" encompasses both therapeutic and prophylactic / preventive measures. Those in need of treatment include, but are not limited to, individuals already suffering from a particular disorder and individuals at risk of developing a particular disorder or condition (e.g., individuals with a genetic or epigenetic predisposition; based on age, sex, lifestyle, etc.). The term "treatment" refers to administering an agent to a subject to achieve therapeutic and / or prophylactic / preventive purposes.

[0048] A "therapeutic agent" refers to an agent that can be administered in vivo to elicit a therapeutic and / or prophylactic / preventive effect.

[0049] "Therapeutic antibody" refers to an antibody that can be administered in vivo to elicit a therapeutic and / or prophylactic / preventive effect.

[0050] As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent / therapy is administered before a second agent / therapy. One skilled in the art will appreciate that the formulation and / or route of administration of the various agents or therapies used may vary. Appropriate doses for co-administration are readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at doses lower than would be appropriate for their individual administration. Thus, co-administration is particularly desirable in embodiments where co-administration of an agent or therapy reduces the necessary dose of a potentially harmful (e.g., toxic) agent and / or when co-administration of two or more agents sensitizes a subject to the beneficial effects of one agent via co-administration of another agent.

[0051] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent (e.g., a binding agent) and an inert or active carrier, such that the composition is particularly suitable for in vitro, in vivo, or ex vivo diagnostic or therapeutic use. As used herein, the term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a composition that does not substantially produce adverse reactions, such as toxic, allergic, or immune responses, when administered to a subject.

[0052] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions) and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference in its entirety.

[0053] As used herein, the term "healthy donor" refers to a mammal, such as a human, who does not suffer from any form of cancer and / or the cells / tissues used in the embodiments herein do not show any signs of cancer (e.g., cancer morphology, cancer biomarkers, etc.). DETAILED DESCRIPTION

[0054] Provided herein are methods for identifying cancer-specific antigens that recognize TCRs and TCR-engineered T cells having antigen-specific cytotoxic activity.

[0055] Cytotoxic T lymphocytes (CTLs) that recognize cancer-specific antigens (oncogenic antigens and new antigens) pre-existing in tumors or blood circulation play a key role in achieving a beneficial clinical response to cancer immunotherapy. For example, a higher number of somatic mutations may increase the probability of producing a larger number of immunogenic new antigens that can be recognized by lymphocytes with high cytolytic activity, which may be further indulged by immune checkpoint inhibitors (references 2-5; incorporated by reference as a whole). In addition, programmed death-ligand 1 (PD-L1) with higher expression levels in cancer cells is upregulated, and programmed death-ligand 1 interacts with programmed death-1 (PD-1) in T cells and is a biomarker for good clinical response (references 4, 6-8; incorporated by reference as a whole). In addition, tumor-infiltrating lymphocytes (TILs) in patients who respond to adoptive TIL transfer therapy include CTLs targeting new antigens and oncogenic antigens (shared antigens) (reference 9; incorporated by reference as a whole).

[0056] In order to enhance CTL-mediated anti-tumor immune responses to further improve the clinical outcomes of cancer immunotherapy, the embodiments herein utilize cancer-specific antigens, oncogenic antigens, and neoantigens as vaccines to activate antigen-specific CTLs in cancer patients. Oncogenic antigens are immunogenic peptides derived from oncogenic proteins that are highly expressed in cancer cells but not expressed in normal organs except testis or fetal organs (reference 10; incorporated by reference in their entirety). It has been expected that immunogenic peptide epitopes derived from oncogenic antigens induce oncogenic antigen-specific CTLs and improve the prognosis of cancer patients (references 11-13; incorporated by reference in their entirety). Neoantigens are immunogenic peptides derived from non-synonymous mutations in cancer cells (reference 10; incorporated by reference in their entirety). Considering some evidence that neoantigen-specific T cells show excellent clinical outcomes (references 14-15; incorporated by reference in their entirety), neoantigen vaccines provide an option to further activate anti-cancer immune responses in patients. However, because the induction of a sufficient number of anti-tumor T cells using vaccine therapy is often very slow and requires several months, this vaccine method does not work for patients with large tumor loads. Therefore, the identification of cancer antigen-specific T cell receptors (TCR), the use of autologous homologous T lymphocytes to produce TCR engineered T cells, and the infusion of such genetically engineered T cells with / without anti-immune checkpoint antibodies provide attractive options for patients with advanced tumors whose host immune system is often significantly suppressed. Preclinical studies and recent clinical trials have shown encouraging results, i.e., onco-antigen / neo-antigen-specific TCR engineered T cells are more effective for large-sized solid tumors (references 16-18; incorporated as a whole by reference). Rapid screening methods for identifying TCR sequences for identifying new antigens and rapid preparation of personalized TCR engineered T cell therapies are provided herein.

[0057] During the development of the embodiments herein, experiments were performed to establish a rapid screening method for detecting oncoantigen / neoantigen specific TCRs. + After T lymphocytes, HLA class I dextran multimers were used to sort CD8 +T cells, and determine the TCR sequences of these cells. Monoclonal or low clonal expansion of unique T cells is achieved by stimulating epitope peptides. TCR cDNA is cloned and TCR engineered T cells are produced. By this method, two antigen-specific CD8+T cell clones are produced; two T cell clones that recognize oncogenic antigens derived from FOXM1 and UBE2T show strong cytotoxic activity against HLA-matched cancer cells expressing target proteins, but do not show cytotoxic activity against HLA-mismatched cancer cells. The method described herein allows for rapid identification of cancer-specific antigens that recognize TCRs after obtaining antigenic peptides. The method allows for the rapid development of personalized T cell immunotherapy for the treatment of cancer. This article provides a channel for identifying cancer-specific antigens that recognize TCRs and establishing TCR-engineered T cells with antigen-specific cytotoxic activity, which integrates in vitro neoantigen stimulation of T cells, right-handed multimer sorting, and TCR sequencing using a next-generation sequencer.

[0058] During the development of the embodiments herein, experiments were conducted to develop a pipeline from screening of putative oncogenic antigen / neoantigen-derived peptides to inducing specific T cells from peripheral blood mononuclear cells (PBMCs) of healthy donors and establishing antigen-specific TCR engineered T cells. Throughout the pipeline, peptides derived from immunogenic oncogenic antigens / neoantigens were identified as being useful in cancer vaccines, and oncogenic antigen / neoantigen-specific TCRs were identified, which led to the establishment of antigen-specific TCR engineered T cells to observe cytotoxic activity against HLA-matched cancer cells.

[0059] As a source of PBMCs, PBMCs from healthy donors allow the detection of candidates for oncogenic antigen / neoantigen-specific CTLs because they have different T cell lineages from cancer patients. T cells obtained from healthy donors broaden the reactivity of neoantigen-specific T cells and are able to target neoantigens that have not yet been recognized by the patient's own immune system (reference 30; incorporated by reference in its entirety). In some embodiments, after identifying cancer-specific antigens that recognize TCRs, TCR-engineered T cells are established from autologous T cells from patients and infused as adoptive cell transfer therapy.

[0060] TCR engineered T cells generated using PBMCs from HLA-A*24:02 positive healthy donors using the methods described herein only recognize HLA-A*24:02 restricted peptides and show significant cytotoxic activity against HLA-A*24:02 matched cancer cells. Considering that TCR engineered T cells targeting HLA-A*02:01 restricted NY-ESO-1-derived peptides using autologous PBMCs show encouraging clinical responses in myeloma patients (reference 21; incorporated by reference in its entirety), it is noteworthy that TCR engineered T cells from healthy donors herein also exert cytotoxic activity against HLA-A matched cancer cells. These results demonstrate the feasibility of preparing TCR engineered T cells from healthy donors, for example, when it is not possible to obtain autologous T cells from patients.

[0061] The channels described herein provide personalized immunotherapy that responds to both oncoantigens and neoantigens. Given that some oncoantigens are often overexpressed in many cancer types, TCR engineered T cell therapy targeting oncoantigens is reasonable because the identified TCRs that recognize specific oncoantigens can be widely used in patients with the same HLA genotype. For example, elevated FOXM1 or UBE2T expression in tumor tissue is associated with poor survival in patients with breast cancer, colon cancer, and prostate cancer (references 31-34; incorporated as a whole by reference). Therefore, in some embodiments, FOXM1 and UBE2T-specific TCR engineered T cells described herein can be used for adoptive transfer therapy. Given that the clinical benefits of current chimeric antigen receptor (CAR) T cell therapy and TCR engineered T cell therapy are limited to hematological malignancies (references 35-36; incorporated as a whole by reference), the channels presented herein for oncoantigen-specific TCR engineered T cells provide another adoptive cell transfer therapy for solid tumors. In contrast, neoantigens are more specific to cancer cells and are considered attractive immune targets, although their presentation depends on somatic mutations of cancer cells. Considering that transfer therapy of neoantigen-specific TILs has shown encouraging clinical results not only for melanoma, but also for solid tumors (references 14-15; incorporated by reference in their entirety), the TCR-engineered T cells against neoantigens described herein provide a therapy for the clinical setting.

[0062] In some embodiments, provided herein are methods for identifying the sequence of an immunologically active TCR, the method comprising stimulating a target lymphocyte (e.g., CD8 +In some embodiments, the stimulatory peptide is a fragment of a protein expressed on cancer and / or tumor cells. In some embodiments, the stimulatory peptide is a fragment of a cancer-specific antigen and / or a tumor-specific antigen. In some embodiments, the target T lymphocytes are obtained from any suitable source (e.g., donor, cell culture, etc.). In some embodiments, the target T lymphocytes are obtained from healthy donors. In some embodiments, the target T lymphocytes are CD8 + Cytotoxic T lymphocytes. In some embodiments, stimulation is performed in vitro (e.g., in cell culture). In some embodiments, the type of cell culture is determined by the type of target T lymphocytes. Conditions and methods suitable for culturing T lymphocytes and stimulating T lymphocytes with stimulatory peptides are known in the art.

[0063] In some embodiments, target lymphocytes (e.g., CD8 + The target T lymphocyte population is a cytotoxic T lymphocyte (e.g., CD8 + A population of cytotoxic T lymphocytes (CTl) is contacted with a capture reagent that displays a major histocompatibility complex (MHC) that binds to a population of capture peptides comprising candidate antigen sequences.

[0064] In some embodiments, the target lymphocytes are CD8 + Cytotoxic lymphocytes, CD4 + Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, etc.

[0065] In some embodiments, the stimulatory peptides comprising candidate antigen sequences are all or fragments of oncogenic antigens and neoantigens. In some embodiments, the candidate antigen sequences are all or fragments of oncogenic antigens and neoantigens. In some embodiments, the stimulatory peptides comprise random amino acid sequences, and the methods herein allow for the identification of peptides capable of eliciting an immune response. In some embodiments, the stimulatory peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44.

[0066] In some embodiments, after stimulating target T lymphocytes with a stimulatory peptide, immunologically active T lymphocytes (e.g., CD8 +Cytotoxic T lymphocytes). In some embodiments, capture includes contacting an immunocompetent T lymphocyte with a capture reagent that displays a major histocompatibility complex (MHC) bound to a capture peptide comprising a candidate antigen sequence. In some embodiments, the capture reagent displays a peptide comprising a sequence of one or more peptides in the stimulating peptide. In some embodiments, the peptide is added to the T lymphocyte in a form bound to the MHC I complex. In some embodiments, the capture reagent is an MHC multimer. In some embodiments, the MHC multimer is an MHC dextrorotatory multimer. For example, the peptide can be presented to a T lymphocyte bound to an MHC dextrorotatory multimer. In some embodiments, the MHC dextrorotatory multimer is a fluorescently labeled MHC multimer bound to a dextran backbone. The use of a multimeric MHC structure has the advantage of presenting multiple copies of a peptide, thereby increasing the likelihood of capture.

[0067] In some embodiments, after capturing immunocompetent T lymphocytes, all or part of the TCR of the captured immunocompetent T lymphocytes are sequenced. In some embodiments, sequencing includes next generation sequencing technology. Next generation sequencing technology is described in more detail below. In some embodiments, the portion of the TCR sequenced includes TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced includes one or more complementary determining regions (CDRs) of TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced includes CDR3 of TCR-α and / or TCR-β chains.

[0068] In some embodiments, provided herein are cancer-specific antigens (e.g., SEQ ID NO: 1, SEQ ID NO: 2, etc.) for identifying TCRs identified by the methods described herein. In some embodiments, cancer-specific antigens identified by the methods described herein are used as therapeutic agents, such as cancer vaccines. Delivery systems for cancer vaccines may include, for example, liposomes, systems made of cholesterol, cholesterol hemisuccinate, or α-tocopherol (e.g., vitamin E), or other amphipathic molecules that can be connected or inserted into modified or synthesized neoantigens. In some embodiments, cancer vaccines include cancer-specific antigens or variants thereof identified by the methods herein. In some embodiments, cancer-specific antigens are provided as fusion peptides. In some embodiments, multiple sequences identified in the methods herein are incorporated. In some embodiments, the peptides used in cancer vaccines are 10-80 amino acids long (e.g., 10, 20, 30, 40, 50, 60, 70, 80, or a range therebetween).

[0069] In some embodiments, provided herein are therapeutic antibodies that bind to cancer-specific antigens that recognize TCRs as described herein. In some embodiments, the therapeutic antibodies herein are antibody fragments. Antibodies and antibody fragments for treating cancer are well known in the art. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are humanized antibodies. In some embodiments, the therapeutic antibodies bind to an antigen comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-44. In some embodiments, the therapeutic antibodies comprise a CDR3 sequence comprising an amino acid sequence pair selected from the group consisting of: SEQ ID NO: NO:45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 1 14, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130 and 131 and 132.

[0070] In some embodiments, provided herein are methods for producing engineered T lymphocytes (e.g., CD8 + In some embodiments, the engineered lymphocytes are CD8 + Cytotoxic lymphocytes, CD4 + Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, etc. In some embodiments, the TCR sequence of an immunocompetent T lymphocyte is used to prepare a nucleic acid and / or vector encoding a TCR that will recognize a target oncogenic antigen or a neoantigen. In some embodiments, such nucleic acids and / or vectors are transformed, transfected, and / or otherwise placed into a T lymphocyte to produce an engineered T lymphocyte. Nucleic acids, vectors, and methods for achieving these purposes are known in the art and are described herein. In some embodiments, the engineered T lymphocyte is a CD8 +In some embodiments, engineered T lymphocytes (e.g., CD8 + The method comprises: (i) cloning a nucleic acid sequence encoding a portion of the TCR of the captured immunocompetent T lymphocyte into a vector; (ii) introducing the vector into a host T lymphocyte (e.g., CD8 + cytotoxic T lymphocytes); and (iii) cultured under conditions where the portion of the TCR of the captured immunocompetent T lymphocytes is expressed and displayed on the engineered T lymphocytes. In some embodiments, the portion of the TCR comprises a TCR-α and / or a TCR-β chain. In some embodiments, the portion of the TCR comprises one or more complementary determining regions (CDRs) of a TCR-α and / or TCR-β chain. In some embodiments, the portion of the TCR sequenced comprises a CDR3 of a TCR-α and / or TCR-β chain. In some embodiments, the portion of the TCR sequenced comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45-132. In some embodiments, the engineered T lymphocyte displays a TCR comprising an α and β chain containing an amino acid sequence pair selected from the group consisting of: SEQ ID NO:45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130 and 131 and 132. In some embodiments, the vector is introduced into a host T lymphocyte from a healthy donor host. In some embodiments, the vector is introduced into a host T lymphocyte from a cancer patient to be treated with engineered T lymphocytes.

[0071] In some embodiments, provided herein are methods for producing engineered T lymphocytes (e.g., CD8 +Cytotoxic T lymphocytes), wherein CAR recognizes an antigen presenting cell displaying MHC bound to a peptide comprising a candidate antigen sequence. In certain embodiments, the antigen binding domain is a single-chain variable fragment (scFv) containing a heavy chain and a light chain variable region (e.g., a variable region identified by the methods herein) that specifically bind to the desired antigen. In some embodiments, CAR also includes a transmembrane domain (e.g., a T cell transmembrane domain (e.g., a CD28 transmembrane domain)) and a signaling domain (e.g., a T cell receptor signaling domain (e.g., a TCR ξ chain) comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, CAR includes one or more costimulatory domains (e.g., a domain that provides a second signal to stimulate T cell activation). The present invention is not limited by the type of costimulatory domain. In some embodiments, the engineered lymphocyte is a CD8 + Cytotoxic lymphocytes, CD4 + Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, etc. In some embodiments, the TCR sequence of an immunocompetent T lymphocyte is used to prepare a CAR that will recognize a target oncogenic antigen or a neoantigen. In some embodiments, nucleic acids and / or vectors encoding such CARs are transformed or transfected into T cells, and / or CARs are otherwise placed into T lymphocytes to produce engineered T lymphocytes. Nucleic acids, vectors, and methods for achieving these purposes are known in the art and are described herein. In some embodiments, the engineered T lymphocyte is a CD8 +Cytotoxic T lymphocytes. In some embodiments, CAR comprises an antigen binding region containing a sequence of a TCR-α and / or TCR-β chain identified in the methods herein. In some embodiments, CAR comprises one or more complementary determining regions (CDRs) of TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced comprises CDR3 of TCR-α and / or TCR-β chains. In some embodiments, the portion of the TCR sequenced comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 45-132. In some embodiments, the engineered T lymphocyte displays a TCR comprising an α and β chain containing an amino acid sequence pair selected from the group consisting of: SEQ ID NO:45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130 and 131 and 132. In some embodiments, the vector is introduced into a host T lymphocyte from a healthy donor host. In some embodiments, the vector is introduced into a host T lymphocyte from a cancer patient to be treated with engineered T lymphocytes.

[0072] In some embodiments, the methods herein are suitable for generating engineered lymphocytes, such as CD4 + Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, etc.

[0073] In some embodiments, nucleic acid (e.g., TCR cDNA) is sequenced. Nucleic acid molecules can be sequenced by a variety of techniques. The analysis can identify the sequence of all or a portion of the nucleic acid. Illustrative non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing and "next generation" sequencing techniques. In some embodiments, RNA is reverse transcribed into cDNA before sequencing.

[0074] Many DNA sequencing techniques are known in the art, including fluorescence-based sequencing methods (see, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, NY; incorporated herein by reference in its entirety). In some embodiments, automated sequencing techniques known in the art are utilized. In some embodiments, systems, devices, and methods employ parallel sequencing of separate amplicons (PCT Publication No: WO2006084132 to Kevin McKernan et al., incorporated herein by reference in its entirety). In some embodiments, DNA sequencing is accomplished by parallel oligonucleotide extension (see, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al. and U.S. Pat. No. 6,306,597 to Macevicz et al., both of which are incorporated herein by reference in their entirety). Additional examples of sequencing techniques include Church polymerase cloning (polony) technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; incorporated herein by reference in their entirety), 454 picotiter pyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380; U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; incorporated herein by reference in their entirety), 20050130173; incorporated herein by reference in its entirety), Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6,373-382; U.S. Patent No. 6,787,308; U.S. Patent No. 6,833,246; incorporated herein by reference in its entirety), Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat. Biotechnol. 18:630-634; U.S. Patent No. 5,695,934; U.S. Patent No. 5,714,330; incorporated herein by reference in its entirety), Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO 00018957; incorporated herein by reference in its entirety), and suitable combinations or alternative methods thereof.

[0075] A group of methods called "next generation sequencing technologies" have emerged as an alternative to Sanger and dye terminator sequencing methods (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; each is incorporated herein by reference in its entirety). Next generation sequencing (NGS) methods share common features of massively parallel high-throughput strategies, with the goal of being lower cost and faster than earlier sequencing methods. NGS methods can be roughly divided into situations where template amplification is required and situations where template amplification is not required.

[0076] Sequencing techniques that can be used in embodiments herein include, for example, Helicos True Single Molecule Sequencing (tSMS) (Harris TD et al. (2008) Science 320:106-109). In tSMS technology, a DNA sample is cleaved into chains of approximately 100 to 200 nucleotides, and a poly A sequence is added to the 3' end of each DNA chain. Each chain is labeled by adding a fluorescently labeled adenosine nucleotide. The DNA chains are then hybridized to a flow cell containing millions of oligo T capture sites fixed to the surface of the flow cell. The template can be at about 100×10 6 Templates / cm 2 The flow cell is then loaded into the sequencer and the laser illuminates the surface of the flow cell to show the position of each template. The CCD camera can locate the position of the template on the surface of the flow cell. The template fluorescent label is then cleaved and washed away. The sequencing reaction begins by introducing DNA polymerase and fluorescently labeled nucleotides. Oligo T nucleic acid is used as a primer. The polymerase incorporates the labeled nucleotides into the primer in a template-guided manner. The polymerase and unincorporated nucleotides are removed. The template that has been guided to incorporate the fluorescently labeled nucleotides is detected by imaging the surface of the flow cell. After imaging, the cleavage step removes the fluorescent label, and the process is repeated with other fluorescently labeled nucleotides until the desired read length is achieved. Sequence information is collected at each nucleotide addition step. Further description of tSMS is shown in, for example, Lapidus et al. (U.S. Patent No. 7,169,560), Lapidus et al. (U.S. Patent Application No. 2009 / 0191565), Quake et al. (U.S. Patent No. 6,818,395), Harris (U.S. Patent No. 7,282,337), Quake et al. (U.S. Patent Application No. 2002 / 0164629), and Braslaysky et al., PNAS (USA), 100:3960-3964 (2003), each of which is incorporated herein by reference in its entirety.

[0077] Another example of a DNA sequencing technology that can be used in the embodiments herein is 454 sequencing (Roche) (Margulies, M et al. 2005, Nature, 437, 376-380; incorporated by reference in its entirety). 454 sequencing involves two steps. In the first step, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt-ended. Oligonucleotide adapters are then connected to the ends of the fragments. The adapters are used as primers for fragment amplification and sequencing. The fragments are connected to DNA capture beads, such as beads coated with streptavidin, using, for example, adapter B containing a 5'-biotin tag. The fragments connected to the beads are PCR amplified in droplets of an oil-in-water emulsion. The result is multiple copies of cloned amplified DNA fragments on each bead. In the second step, the beads are captured in a well (picoliter size). Pyrophosphate sequencing is performed on each DNA fragment simultaneously. Addition of one or more nucleotides generates a light signal that is recorded by a CCD camera in the sequencing instrument. The signal intensity is proportional to the number of nucleotides incorporated. Pyrosequencing utilizes a pyrophosphate group (PPi), which is released upon the addition of a nucleotide. ATP sulfurylase converts PPi to ATP in the presence of 5' adenosine phosphosulfate. Luciferase uses ATP to convert luciferin to oxyluciferin, and this reaction generates light, which is detected and analyzed.

[0078] Another example of DNA sequencing technology that can be used in the embodiments herein is SOLiD technology (Applied Biosystems). In SOLiD sequencing, genomic DNA is sheared into fragments, and adapters are connected to the 5' and 3' ends of the fragments to produce a fragment library. Alternatively, internal adapters can be introduced by: adapters are connected to the 5' and 3' ends of the fragments, the fragments are annularized, the circularized fragments are digested to produce internal adapters, and adapters are connected to the 5' and 3' ends of the resulting fragments to produce an end-pairing library. Then, a cloned bead population is prepared in a microreactor containing beads, primers, templates and PCR components. After PCR, the template is denatured and the beads are enriched to separate the beads with extended templates. The template on the selected beads is subjected to 3' modification, and the modification allows bonding to a slide. The sequence can be determined by continuous hybridization and connection of partially random oligonucleotides with a central determined base (or base pair) identified by a specific fluorophore. After recording the color, the connected oligonucleotides are cracked and removed, and the process is then repeated.

[0079] Another example of a DNA sequencing technology that can be used in the embodiments herein is Ion Torrent sequencing (U.S. Patent Application Nos. 2009 / 0026082, 2009 / 0127589, 2010 / 0035252, 2010 / 0137143, 2010 / 0188073, 2010 / 0197507, 2010 / 0282617, 2010 / 0300559), 2010 / 0300895, 2010 / 0301398, and 2010 / 0304982; incorporated by reference in their entirety). In Ion Torrent sequencing, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt-ended. Oligonucleotide adapters are then attached to the ends of the fragments. The adapters are used as primers for fragment amplification and sequencing. The fragments can be attached to a surface and attached at a resolution that allows the fragments to be individually distinguishable. Addition of one or more nucleotides releases protons (H + ), which is detected and recorded in the sequencing instrument. The signal strength is proportional to the number of incorporated nucleotides.

[0080] Another example of DNA sequencing technology that can be used in the embodiments herein is Illumina sequencing. Illumina sequencing is based on using fold-back PCR and anchor primers to amplify DNA on a solid surface. Genomic DNA is fragmented, and adapters are added to the 5' and 3' ends of the fragments. The DNA fragments connected to the surface of the flow cell channel are extended and bridge amplification is performed. The fragments become double-stranded, and the double-stranded molecules are denatured. Multiple cycles of denaturation after solid phase amplification can establish millions of clusters of approximately 1,000 copies of the same template of single-stranded DNA molecules in each channel of the flow cell. Primers, DNA polymerases, and four fluorophore-labeled reversibly terminated nucleotides are used for continuous sequencing. After the nucleotide is incorporated, a laser is used to excite the fluorophore, and the image is captured and the identity of the first base is recorded. The 3' terminator and fluorophore from each incorporated base are removed and the incorporation, detection, and identification steps are repeated.

[0081] Another example of DNA sequencing technology that can be used in the embodiments herein is the single molecule real-time (SMRT) technology of Pacific Biosciences. In SMRT, each of the four DNA bases is connected to one of four different fluorescent dyes. These dyes are connected through phosphate. The template single-stranded DNA of a single DNA polymerase is fixed with a single molecule at the bottom of a zero-mode waveguide (ZMW). ZMW is a closed structure that can observe the incorporation of a single nucleotide by a DNA polymerase relative to the background of fluorescent nucleotides that diffuse rapidly (in microseconds) outside the ZMW. It takes several milliseconds for the nucleotide to be incorporated into the growing chain. During this period, a fluorescent label is excited, and a fluorescent signal is generated, and the fluorescent label is cleaved. The corresponding fluorescence of the dye is detected to indicate which base is incorporated. Repeat the process.

[0082] Another example of DNA sequencing technology that can be used in the embodiments herein relates to nanopore sequencing (Soni GV and Meller A. (2007) Clin Chem 53: 1996-2001; incorporated by reference in its entirety). A nanopore is a small hole with a diameter of about 1 nanometer. The nanopore is immersed in a conductive fluid and a potential is applied to it, and a slight current is generated due to ion conduction through the nanopore. The amount of current flowing is sensitive to the size of the nanopore. When a DNA molecule passes through the nanopore, each nucleotide on the DNA molecule blocks the nanopore to varying degrees. Therefore, the change in the current passing through the nanopore when the DNA molecule passes through the nanopore represents a read of the DNA sequence.

[0083] Another example of DNA sequencing technology that can be used in the embodiments herein involves sequencing DNA using a chemically sensitive field effect transistor (chemFET) array (e.g., as described in U.S. Patent Application Publication No. 20090026082; incorporated by reference in its entirety). In one example of the technology, a DNA molecule can be placed in a reaction chamber, and the template molecule can be hybridized with a sequencing primer bound to a polymerase. The incorporation of one or more triphosphate groups into a new nucleic acid chain at the 3' end of the sequencing primer can be detected by a chemFET through a change in current. The array can have multiple chemFET sensors. In another example, a single nucleic acid can be attached to a bead, and the nucleic acid can be amplified on the bead, and the individual beads can be transferred to individual reaction chambers on the chemFET array, each of which has a chemFET sensor, and the nucleic acid can be sequenced.

[0084] In some embodiments, other sequencing technologies (eg, NGS technologies) known in the art, or alternative technologies or combinations of the above technologies can be used in the embodiments herein.

[0085] Certain embodiments herein include detecting one or more biomarkers (e.g., detecting cytokines (e.g., IFN-γ) to detect and / or quantify immune responses). In some embodiments of the method, the method further includes isolating one or more biomarkers from a biological sample or in vitro culture (e.g., detecting cytokines (e.g., IFN-γ) to detect and / or quantify immune responses). In some embodiments, a reagent that binds to a biomarker is provided. Such reagents are selected from antibodies, antibody fragments, aptamers, and the like.

[0086] In some embodiments, the detection method includes an enzyme / substrate combination that produces a detectable signal corresponding to the level of the biomarker (e.g., using ELISA, Western blotting, isoelectric focusing techniques). Generally speaking, the enzyme catalyzes the chemical change of a chromogenic substrate, which can be measured using various techniques, including spectrophotometry, fluorescence, and chemiluminescence. Suitable enzymes include, for example, luciferase, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like. In some embodiments, the detection method is a combination of fluorescence, chemiluminescence, radionuclide, or an enzyme / substrate combination that produces a measurement signal. In some embodiments, multimodal signaling has unique and advantageous features in the biomarker assay format.

[0087] In some embodiments, any analytical method is used to detect the presence / level of a biomarker, including singleplex aptamer assays, multiplex aptamer assays, singleplex or multiplex immunoassays, expression profiling, mass spectrometry, histological / cytological methods, etc., as discussed below.

[0088] In some embodiments, suitable immunoassays are used to detect / quantify biomarkers (e.g., detection of cytokines (e.g., IFN-γ) to detect and / or quantify immune responses). Immunoassays are based on the reaction of antibodies to their corresponding targets or analytes, and can detect analytes in samples, depending on the specific assay format. In order to improve the specificity and sensitivity of assays based on immunoreactivity, monoclonal antibodies and fragments thereof are often used because of their specific recognition of epitopes. Polyclonal antibodies have also been successfully used in various immunoassays because these antibodies have increased affinity for targets compared to monoclonal antibodies. Immunoassays for a large number of biological sample matrices have been designed. Immunoassay formats have been designed to provide qualitative, semi-quantitative and quantitative results.

[0089] Many immunoassay formats have been designed. For detecting analytes, ELISA or EIA can be quantitative. This method depends on the attachment of a label to the analyte or antibody, and the label directly or indirectly includes an enzyme. ELISA tests can be formatted for direct, indirect, competitive or sandwich detection of analytes. Other methods depend on, for example, radioisotopes (I 125 ) or fluorescent labeling. Additional techniques include, for example, agglutination, turbidimetry, nephelometry, Western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assays, and the like (see ImmunoAssay: A Practical Guide, Brian Law, ed., Taylor & Francis Ltd., 2005 edition; incorporated herein by reference in its entirety).

[0090] Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescence, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time-resolved-FRET (TR-FRET) immunoassays. Examples of procedures for detecting biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow for size and peptide level differentiation, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.

[0091] The method of detecting and / or quantifying the detectable label or the material producing the signal depends on the nature of the label. The product of the reaction catalyzed by the appropriate enzyme (when the detectable label is an enzyme; see above) may be, but is not limited to, fluorescent, luminescent or radioactive, or it may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, but are not limited to, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, photometers, and densitometers.

[0092] Any method for detection can be in any format that allows any suitable preparation, processing and analysis of the reaction. This can be, for example, in a multi-well assay plate (e.g., 96 or 384 wells) or using any suitable array or microarray. The stock solution of various agents can be made manually or automatically, and all subsequent pipetting, dilution, mixing, distribution, washing, incubation, sample reading, data collection and analysis can be automatically performed using commercially available analysis software, robots and detection instruments capable of detecting detectable labels.

[0093] In some embodiments, the antigenic peptides and sequences thereof used in the embodiments herein are derived from cancer or tumor cell markers. Such markers may be selected from the group including but not limited to: epidermal growth factor receptor (EGFR, EGFR1, ErbB-1, HER1). ErbB-2 (HER2 / neu), ErbB-3 / HER3, ErbB-4 / HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF binding protein (IGFBP), IGFR ligand family (IGF-1R); platelet-derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family: TRK receptor family; epithelial (EPH) receptor family: AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family; discoidin domain receptor (DDR) family; RE T receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; transforming growth factor (TGF-α), TGF-α receptor; transforming growth factor-β (TGF-β), TGF-β receptor; interleukin β receptor α2 chain (IL13Rα2), interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, cytokine receptors, class I (erythropoietin family) and class II (interferon / 1L-10 family) receptors, tumor necrosis factor (TNF) family, TNF-α, tumor necrosis factor (TNF) receptor superfamily (TNTRSF), death receptor family, TRAIL-receptor; cancer-testis (CT) antigen, lineage-specific antigen, differentiation antigen, α-actinin-4, ARTC1, breakpoint cluster region-Abelson (breakpoint clusterregion-Abelson, Bcr-abl) fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, elongation factor 2 (ELF2), Ets variant gene 6 / acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, adhesion protein (FN), GPNMB, low-density lipid receptor / GDP-L fucose: β-D-galactose 2-α-L fucosyltransferase (LDLR / FUT) fusion protein, HLA-A2, MLA-A11, heat shock protein 7 0-2 mutation (HSP70-2M), KIAA0205, MART2, melanoma widespread mutation 1, 2, 3 (MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, myosin class 1, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT12, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, triose phosphate isomerase, BAGE, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2, 3, 4, 5, 6, 7, 8, GnT-V (abnormal N-acetylglucosaminyltransferase V, MGAT5), HERV-KMEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL recognition antigen on melanoma (CAMEL), MAGE-A1 (MAGE-1). MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10. MAGE-All, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5. MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1), MART-1 / melanin-A (Melan-A) (MLANA), gp100, gp100 / Pme117 (S1LV), tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1 / LAGE-2, SAGE, Sp17.SSX-1, 2, 3, 4, TRP2-1NT2, carcinoembryonic antigen (CEA), vasokinetin 4, mammaglobin-A, OA1, prostate-specific antigen (PSA), prostate-specific membrane antigen, TRP-1 / , 75.TRP-2 lipophilin, interferon-induced protein absent in melanoma 2 (AIM-2). BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250 intestinal carboxylesterase (iCE), α-embryoprotein (AFP), M-CSF, mdm-2, MUCI, p53 (TP53), PBF, PRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, survivin (BIRCS), human telomerase reverse transcriptase (hTERT), telomerase, Wilms' tumor gene (Wilms' tumor gene, WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA66I, LDHC, MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHLI7, NXF2TDRD1, TEX 15, FATE, TPTE, immunoglobulin idiotype, Bence-Jones protein, estrogen receptor (ER), androgen receptor (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), β-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96.GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryonic antigen peptide-1 (CAP-1), calcium-activated chloride channel 2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), etc. In some embodiments, the antigenic peptides and their sequences used in the embodiments herein are derived from cell surface markers that are specific to cancer / tumor cells or are mainly displayed on cancer / tumor cells (e.g., recognizable by antibodies and / or immune cells).

[0094] In some embodiments, provided herein are T lymphocytes engineered to express immunocompetent TCRs. In some embodiments, provided herein are T lymphocytes engineered to express immunocompetent CARs. Engineered cells can be produced by any suitable method in the art. In some embodiments, T lymphocytes are engineered to express / display immunocompetent TCRs obtained by the methods described herein (e.g., stimulation, capture, sequencing). In some embodiments, T lymphocytes are engineered to express / display immunocompetent CARs obtained by the methods described herein (e.g., stimulation, capture, sequencing).

[0095] In some embodiments, provided herein are nucleic acids and nucleic acid sequences encoding immunoactive TCR (or immunoactive CAR) as described above and cells having such nucleic acids. In some embodiments, the nucleic acid molecule is a recombinant nucleic acid molecule. In some embodiments, the nucleic acid molecule is synthetic. Nucleic acids encoding immunoactive TCR and portions thereof may include DNA, RNA, PNA (peptide nucleic acid) and their hybrids.

[0096] In some embodiments, the nucleic acid encoding the immunocompetent TCR and portions thereof comprises one or more regulatory sequences. For example, a promoter, a transcription enhancer and / or a sequence that allows induction of expression of the polynucleotides of the present disclosure may be employed. In some embodiments, the nucleic acid molecule is transcribed by an appropriate vector comprising a chimeric gene that allows transcription of the nucleic acid molecule in a cell.

[0097] In some embodiments, the nucleic acid molecule is a recombinant chimeric nucleic acid molecule comprising any of the above nucleic acid molecules, alone or in combination. In some embodiments, the nucleic acid molecule is part of a vector.

[0098] In some embodiments, provided herein are vectors comprising nucleic acid molecules described herein (e.g., encoding immunologically active TCRs and portions thereof). Many suitable vectors are known to those skilled in the art of molecular biology, and the selection of vectors will depend on the desired function and include plasmids, cosmids, viruses, phages, and other vectors commonly used in genetic engineering. Various plasmids and vectors can be constructed using methods well known to those skilled in the art; see, for example, the techniques described in: Sambrook et al. (1989) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989), (1994); incorporated by reference as a whole. Alternatively, the polynucleotides and vectors disclosed herein are reconstituted in liposomes for delivery to target cells. Cloning vectors can be used to isolate individual sequences of DNA. Related sequences can be transferred to expression vectors that need to express specific polypeptides. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, and pOP13CAT.

[0099] In some embodiments, the vector comprises a nucleic acid sequence that is operably linked to a regulatory sequence of a nucleic acid sequence encoding an immunologically active TCR and portions thereof. Such regulatory sequences (control elements) are known to the skilled person and may include a promoter, a splice cassette, a translation initiation codon, and an insertion site for introducing an insert into the vector. In a specific embodiment, the nucleic acid molecule is operably linked to the expression control sequence that allows expression in a eukaryotic or prokaryotic cell.

[0100] In some embodiments, the vector is a viral vector, such as a lentiviral vector or an adenovirus-associated vector.

[0101] In some embodiments, nucleic acids and / or vectors are used in cells to express encoded polypeptides (e.g., immunologically active TCRs and portions thereof, etc.) in the cells. Nucleic acid molecules or vectors containing DNA sequences encoding any of the immunologically active TCRs described herein are introduced into cells, which in turn produce the polypeptides. The described nucleic acid molecules and vectors can be designed to be introduced directly into cells or via liposomes or viral vectors (e.g., adenovirus, retrovirus).

[0102] According to the above, the present invention provides a method for obtaining a vector comprising a nucleic acid molecule encoding a polypeptide sequence described herein (e.g., an immunologically active TCR and portions thereof), particularly a plasmid, cosmid, virus, and phage commonly used in genetic engineering. In some embodiments, the vector is an expression vector and / or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses can be used to deliver polynucleotides and / or vectors to a target cell population. Recombinant vectors can be constructed using methods well known to those skilled in the art. Well-known methods transfer vectors into host cells, and the methods vary depending on the cell host type.

[0103] In some embodiments, cells are provided herein, comprising host cells transformed or transfected with a vector as defined above (e.g., encoding an immunologically active TCR as described herein). The host cell can be produced by introducing at least one of the above-mentioned vectors or at least one of the above-mentioned nucleic acid molecules into a host cell. The presence of at least one vector or at least one nucleic acid molecule in the host can mediate the expression of genes encoding the above-mentioned immunologically active TCR and parts thereof. The nucleic acid molecules or vectors introduced into the host cell can be incorporated into the host genome or they can be maintained outside the chromosome.

[0104] In some embodiments, methods are provided herein, the methods comprising culturing the host cell defined above under conditions that allow the introduction of nucleic acids and / or vectors. In some embodiments, methods are provided herein, the methods comprising culturing the host cell defined above under conditions that allow the expression of constructs (e.g., comprising immunocompetent TCRs or portions thereof). In specific embodiments, cultured cells are provided to a subject (e.g., a subject from whom the original cell is obtained, a second subject, etc.). Conditions for culturing cells with expression constructs are known in the art.

[0105] In some embodiments, the lymphocytes for engineering according to the embodiments herein come from any suitable source. For example, the source of lymphocytes is a subject (e.g., a subject to be treated, a healthy subject, etc.). Lymphocytes can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. In some embodiments, lymphocytes (e.g., cytotoxic T cells) of a specific type desired by the embodiments described herein are obtained by appropriate methods. In some embodiments, lymphocytes expressing specific markers are obtained by known methods (e.g., cell sorting). In some embodiments, cells are cultured after separation. In some embodiments, cells are engineered using the methods described herein.

[0106] In some embodiments, the compositions herein (e.g., engineered lymphocytes, antibodies, vaccines, nucleic acid molecules, vectors, etc.), alone or in any combination, are administered using standard delivery systems and methods and, in at least some aspects, together with a pharmaceutically acceptable carrier or excipient. In the case of a nucleic acid molecule or vector, it can be stably incorporated into the genome of the subject.

[0107] In some embodiments, methods and compositions for preventing, treating or ameliorating cancer are provided, the methods comprising administering to a subject in need thereof an effective amount of a composition as contemplated herein and / or produced by a method as contemplated herein (e.g., engineered lymphocytes, antibodies, vaccines, nucleic acid molecules, vectors, etc.). When administering cells, the engineered cells are administered to the treatment site or can be concentrated at the treatment site (e.g., cell type, tissue type, etc.).

[0108] Non-limiting examples of cancers that can be treated with the compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testicles, tongue, or uterus. Furthermore, cancer may specifically be of the following histological types, although they are not limited thereto: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocellular carcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocellular carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine carcinoma; sebaceous carcinoma; cerumenic gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; theca cell tumor, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus tumor; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; Wilms' tumor; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner's tumor tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian thyroid tumor, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant;Lymphangiosarcoma; Osteosarcoma; Juxtacortical osteosarcoma; Chondrosarcoma; Chondroblastoma, malignant; Mesodermal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Neurothecoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease disease); Hodgkin's lymphoma; granulomatous tumor; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative enteropathy; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid leukemia; and hairy cell leukemia. In some embodiments, the cancer is melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate cancer), pancreatic cancer (e.g., adenocarcinoma), breast cancer, colon cancer, gallbladder cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other malignancies. In some embodiments, the cancer is a solid tumor cancer. ;

[0109] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are used together with one or more combination therapies for treating cancer. In some embodiments, one or more chemotherapies and / or immunotherapies are co-administered with the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein. In some embodiments, one or more chemotherapeutic agents and / or immunotherapies are provided as combination therapies, with or without (known) synergistic effects.

[0110] The disclosure further encompasses co-administration schemes with other compounds that act via immune cells, such as targeted toxins or other blocking or functional antibodies or compounds. The co-administration clinical scheme may encompass co-administration with the administration of another component simultaneously, before or after the administration of another component. Specific combination therapies include chemotherapy, radiation, surgery, hormone therapy or other immunotherapy types. Many chemotherapeutic agents are currently known in the art and can be used in combination with the compounds of the present invention. In some embodiments, the chemotherapeutic agent is selected from the group consisting of: mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response regulators, anti-hormones, angiogenesis inhibitors and anti-androgens.

[0111] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with one or more chemotherapeutic agents. Chemotherapy for co-administration herein includes all classes of chemotherapeutic agents, such as alkylating agents, antimetabolites, plant alkaloids, antibiotics, hormone agents, and other anticancer drugs. Specific agents include, for example, albumin-bound paclitaxel (abraxane), altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, and the like. il), busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, fuldarabine, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin and vinblastin, or any analog or derivative variant of any of the foregoing agents, and combinations thereof. In some embodiments, chemotherapy is employed before, during, and / or after administration of the therapeutic compositions (eg, engineered lymphocytes, antibodies, vaccines, etc.) and methods herein.

[0112] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with radiation therapy, and methods of radiation therapy are known in the art. In some embodiments, radiation therapy is used before, during, and / or after the administration of the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein.

[0113] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with non-immune targeted therapies, such as agents that inhibit WNT, p53, and / or signaling pathways. Other examples include agents that inhibit tyrosine kinases, BRAF, STAT3, c-met, regulate gene expression, induce cell death, or block angiogenesis. Examples of specific agents include imatinib mesylate, imatinib succinate, succinimidyl transmembrane ... mesylate, dasatinib, nilotinib, bosutinib, lapatinib, gefinitib, erlotinib, tensirolimus, everolimus, vemurafenib, crizotinib, vorinostat, romidepsin, bexarotene, alitrionin, tretionin, bortezomib, carfilzomib, pralatrexate, sorafenib, sunitinib, pazopanib, regorafenib, or cabozantinib. In some embodiments, non-immune targeted therapies are employed before, during, and / or after administration of the engineered lymphocytes.

[0114] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with immunotherapy. Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be, for example, antibodies that are specific to some markers on the surface of tumor cells. An antibody alone can be used as a therapy effector, or it can supplement other cells to actually achieve cell killing. Antibodies can also prevent cancer immune escape or immunosuppression. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.), and are only used as targeting agents. Alternatively, effectors can be lymphocytes that carry surface molecules that interact directly or indirectly with tumor cell targets. In some embodiments, immunotherapy is used before, during, and / or after the administration of engineered lymphocytes.

[0115] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with gene therapy, wherein the therapeutic polynucleotides are administered before, after, or simultaneously with the engineered lymphocytes described herein. A variety of expression products are contemplated, including cell proliferation inducers, cell proliferation inhibitors, or programmed cell death regulators.

[0116] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are administered before, during, and / or after surgery. Surgery includes resections that physically remove, excise, and / or destroy all or a portion of cancerous tissue. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electroresection, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the embodiments herein may be used in conjunction with the removal of surface cancer, precancerous or accompanying amounts of normal tissue.

[0117] In some embodiments, the therapeutic compositions (eg, engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with other agents that enhance the therapeutic efficacy of the treatment.

[0118] In some embodiments, the co-administered agent is formulated into a single dose and / or composition. In some embodiments, the co-administered agent is in a single dose and / or composition. In some embodiments of applying a single dose and / or composition, dosage and / or composition are applied at intervals simultaneously, continuously or within a period of time (e.g., <30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more time or any suitable range between).

[0119] In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are provided as part of a kit or system, along with one or more additional components such as instructions, an administration device, additional therapeutic agents, diagnostic agents, research agents, etc.

[0120] experiment

[0121] Materials and Methods

[0122] Peptides

[0123] 9-mer and 10-mer peptides were synthesized by using standard solid phase synthesis methods and purified by reverse phase high performance liquid chromatography (HPLC) (see Tables 1a-e). The purity (>90%) and identity of the peptides were determined by analytical HPLC and mass spectrometry, respectively. The peptides were dissolved in dimethyl sulfoxide to 20 mg / ml and stored at -80°C.

[0124] Table 1. Amino acid sequences of peptides used to establish peptide-specific CTL

[0125] Table 1a. List of HLA-A*24:02 restricted peptides used to establish peptide-specific CTL

[0126] Peptide name Amino acid sequence SEQ ID NO CDCA5-A24-10-232 EWAAAMNAEF 1 CDH3-A24-10-807 DYLNEWGSRF 2 FOXM1-A24-9-262 IYTWIEDHF 3 HJURP-A24-9-408 KWLISPVKI 4 INHBB-A24-9-180 LYLKLLPYV 5 KIF20A-A24-10-66 KVYLRVRPLL 6 MELK-A24-9-87_7N EYCPGGNLF 7 NEIL3-A24-9-545 EWADLSFPF 8 RNF43-A24-9-721 NSQPVWLCL 9 SEMA5B-A24-10-290 AYDIGLFAYF 10 SMYD3-A24-9-197 QYCFECDCF 11 TOPK-A24-10-289 SYQKVIELFS 12 UBE2T-A24-9-60 RYPFEPPQI 13 VANGL1-A24-9-443 RYLSAGPTL 14 VEGFR1-A24-9-1084 SYGVLLWEI 15 VEGFR2-A24-9-169 RFVPDGNRI 16 WDHD1-A24-9-844 GYSNTATEW 17 WDRPUH-A24-9-314 IYRVSFTDF 18

[0127] Table 1b. List of HLA-A*02:01 restricted peptides used to establish peptide-specific CTL

[0128]

[0129]

[0130] Table 1c. List of HLA-A*11:01 restricted peptides used to establish peptide-specific CTL

[0131] Peptide name Amino acid sequence SEQ ID NO CDCA1-A11-9-219 KTKRLNELK 36 DEPDC1v1-A11-9-627 MSQNVDMPK 37 KIF20A-A11-9-45 VVSTSLEDK 38 MPHOSPH1-A11-10-1546 STSFEISRNK 39

[0132] Table 1d. List of HLA-A*33:03 restricted peptides used to establish peptide-specific CTL

[0133] Peptide name Amino acid sequence SEQ ID NO CDCA1-A33-9-43 EVLHMIYMR 40 FOXM1-A33-9-308 WTIHPSANR 41 MPHOSPH1-A33-9-608 EFTQYWAQR 42 VEGFR2-A33-9-114 YVYVQDY 43

[0134] Table 1e. List of HLA-A*03:01 restricted peptides used to establish peptide-specific CTL

[0135] Peptide name Amino acid sequence SEQ ID NO KOC1-A03-10-120 AVVNVTYSSK 44

[0136] Cell lines

[0137] TISI (HLA-A*24:02, B-lymphoblastoid cell line) was purchased from the International Histocompatibility Working Group. T2 (HLA-A*02:01, B-lymphoblastoid cell line), EB-3 (HLA-A3 / Aw32, B-lymphoblastoid cell line), Jiyoye (HLA-A32, B-lymphoblastoid cell line), SW480 (HLA-A*24:02, colorectal adenocarcinoma), HCC1143 (HLA-A*31:01, breast cancer), BT549 (HLA-A*02:01, breast cancer) and C1R (lacking HLA-A and HLA-B, B-lymphoblastoid cells) were purchased from the American Type Culture Collection (Rockville, MD). All cells were cultured according to the recommendations of their respective depositors.

[0138] Generation of C1R cells stably expressing HLA class I

[0139] In addition to TISI and T2, C1R cells transfected with human leukocyte antigen (HLA) were used as stimulator cells. The cDNA encoding the open reading frame of HLA class I (A*24:02, A*02:01, A*11:01, A*33:03 or A*03:01) was amplified by PCR and inserted into the expression vector. C1R cells were transfected with HLA class I expression vectors and cultured for 14 days in the presence of G418 (Invitrogen, Carlsbad, CA). G418-resistant monocytes and feeder cells were plated into 96-well cell culture plates (Corning, NY) containing culture medium supplemented with G418 and further cultured for 30 days. The expression of transfected HLA class I on C1R cells was confirmed by flow cytometry analysis.

[0140] In vitro CTL induction

[0141] Monocyte-derived dendritic cells (DC) are used as antigen presenting cells to induce cytotoxic T lymphocytes (CTLs) that respond to peptides presented on HLA class I. DCs were generated in vitro (reference 37; incorporated by reference in its entirety). Peripheral blood mononuclear cells (PBMCs) were separated from the blood of healthy volunteers by Ficoll-Paque PLUS (GE Healthcare). Monocytes (adherent cells in PBMCs) were cultured in the presence of 1000 IU / ml of granulocyte-macrophage colony stimulating factor (R&D Systems, Minneapolis, MN) and 1000 IU / ml of interleukin (IL)-4 (R&D System) in AIM-V medium (Invitrogen) containing 2% heat-inactivated human serum (AIM-V / 2% HS medium) to induce DCs. After seven days of culture, monocyte-derived DCs were pulsed with 20 μg / ml of synthetic peptides in AIM-V medium for 3 hours at 37°C in the presence of 3 μg / ml of β-2-microglobulin. These peptide-pulsed DCs were inactivated by X-ray irradiation (20 Gy) and mixed at a 1:20 ratio with autologous CD8+ T cells obtained from PBMCs using a CD8 positive isolation kit (Thermo Fisher Scientific, Carlsbad, CA). These cultures were set up in 48-well cell culture plates (Corning). Each well contained 1.5×10 4 Peptide-pulsed DCs, 3×10 5 CD8+T cells and 10ng / ml IL-7 (R&D System). The day after tomorrow (day 2), IL-2 (Novartis) was added to the culture at a final concentration of 20IU / ml. On days 7 and 14, CD8+T cells were further stimulated with autologous peptide-pulsed DCs. DCs were prepared in the same manner as described above each time. Peptide-specific IFN-γ production of CD8+T cells was tested by ELISPOT assay on day 21 (references 38-39; incorporated by reference in their entirety).

[0142] Expansion culture

[0143] After limiting dilution, CD8+ T cells were expanded using the rapid expansion method (reference 40; incorporated by reference in its entirety). EB-3 and Jiyoye were treated with mitomycin C and used as feeder cells. CD8+ T cells were incubated with feeder cells (5×10 6Cells) and 40ng / ml of anti-CD3 antibody were cultured in 25ml AIM-V / 5% HS medium. The next day (day 1), 3000IU of IL-2 was added to the culture. On days 5, 8 and 11, half the volume of the medium was replaced with fresh AIM-V / 5% HS medium containing 60IU / ml IL-2. Between days 14 and 16, peptide-specific IFN-γ production of CD8+T cells was tested by ELISA (references 38-39; incorporated by reference in their entirety).

[0144] Detection of peptide-specific IFN-γ

[0145] To examine peptide-specific IFN-γ production by CD8+ T cells, ELISPOT assay or ELISA was performed. Peptide-pulsed T2, TIS1, or C1R cells expressing HLA class I were prepared (1 × 10 4 Cells) were used as stimulator cells. CD8+ T cells were used as responder cells. IFN-γ ELISPOT assay and IFN-γ ELISA were performed according to the manufacturer's procedures (BD Biosciences, San Jose, CA).

[0146] Evaluation of the cytotoxic activity of CTLs against cancer cells by time-lapse recording

[0147] CTL and TCR engineered T cells were pretreated with IL-2 (100 U / mL) for 16 h. Target cells were pretreated with IFN-γ (100 U / mL) for 48 h before the experiment. Cells were incubated with 1 ug / mL Calcein AM (Dojindo, Kumamoto, Japan) for 30 min. After washing with PBS three times, 2 × 10 4 target cells and 2×10 5 FOXM1 / UBE2T-specific CTL or 4×10 5 TCR engineered T cells were mixed into sterile 16-well Lab-Tek Chamber Slide coverslips (Thermo Scientific). Time-lapse recording was performed by an inverted microscope Axio Vert.A1TL (Zeiss, Oberkochen, Germany). Live and dead cells were quantified using the ImageJ program (National Institutes of Health, Bethesda, MD).

[0148] T cell receptor sequencing

[0149] Determine TCR sequence (reference 41; incorporated as a whole by reference). Total RNA is extracted from amplified or right-handed multimer-positive T cells. Use SMART library construction kit (Clontech, Mountain View, CA) to synthesize cDNA with common 5'-RACE adapters. Fusion PCR is performed using a forward primer corresponding to the SMART adapter sequence and a reverse primer corresponding to the constant region of each of TCRA or TCRB to amplify TCRA or TCRB cDNA. After adding the Illumina index sequence with a barcode using Nextera index kit (Illumina, San Diego, CA), the prepared library is sequenced by 300bp double-end reads on MiSeq (Illumina). The resulting sequence reads (reference 41; incorporated as a whole by reference) are analyzed using Tcrip software. The sequence is also confirmed by Sanger sequencing using fusion PCR products as templates (Thermo Scientific).

[0150] TCR engineered T cells

[0151] TCRA and TCRB sequences were codon optimized and cloned into pMP71-PRE (references 18, 42; incorporated by reference in their entirety). To maximize TCR expression, modified murine TCRA and TCRB constant domains were used. Transient retroviral supernatants were produced and PBMCs from donors were transduced (reference 18; incorporated by reference in their entirety). Anti-human TCRβV antibodies were used to assess the expression of TCRs. Under conditions suitable for TCR engineered T cells for FOXM1 and UBE2T, staining was performed with APC-conjugated anti-mouse TCRβ monoclonal antibodies (H57-597, eBioscience, San Diego, CA), followed by incubation with anti-APC microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions, and only transduction of transduced TCR engineered T cells was performed. In order to increase the number of T cells transduced with the desired TCR that is not occupied by the antibody, the condition is based on the number of cells sorted under five different conditions of peak fluorescence intensity and by comparing antibody dilution. Antibody staining at ratios of 1:2000 (0.1 ug / mL) and 1:4000 (0.05 ug / mL) were determined to be suitable for sorting TCR-engineered T cells against FOXM1 and UBE2T, respectively.

[0152] result

[0153] Peptide-specific CTL

[0154] CTL clones specific for HLA-A*24:02, HLA-A*02:01, HLA-A*11:01, HLA-A*33:03 or HLA-A*03:01 restricted peptides from Tables 1a-e were induced. CTLs were captured with HLA dextro-multimers of each peptide, and the TCR sequences of these cells were determined (see Tables 2a-e for the CDR3 amino acid sequences of the TCRs in peptide-specific CTLs). All 44 CTL clones were evaluated for peptide-specific IFN-γ production by ELISA using HLA-expressing cells with or without the peptides.

[0155] Table 2. Amino acid sequences of CDR3 of TCR in peptide-specific CTL

[0156] Table 2a. List of major CDR3 sequences of CTL clones specific for HLA-A*24:02 restricted peptides

[0157]

[0158]

[0159] Table 2b. List of major CDR3 sequences of CTL clones specific for HLA-A*02:01 restricted peptides

[0160]

[0161]

[0162] Table 2c. List of major CDR3 sequences of CTL clones specific for HLA-A*11:01 restricted peptides

[0163]

[0164]

[0165] Table 2d. List of major CDR3 sequences of CTL clones specific for HLA-A*33:03 restricted peptides

[0166]

[0167] Table 2e. List of major CDR3 sequences of CTL clones specific for HLA-A*03:01 restricted peptides

[0168]

[0169] Induction of FOXM1- and UBE2T-derived peptide-specific CTLs with cytotoxic activity against cancer cells

[0170] Induction of CTL clones specific for peptides derived from FOXM1 and UBE2T (references 19-20; incorporated by reference in their entirety). Highly immunogenic short peptides derived from FOXM1 and UBE2T (e.g., IYTWIEDHF (SEQ ID NO: 3) and RYPFEPPQI (SEQ ID NO: 13), respectively) were identified by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay, which can induce HLA-A*24:02 restricted CTLs from PBMCs of healthy donors. After obtaining CTL clones by limiting dilution, it was confirmed that these FOXM1 and UBE2T specific CTLs produced IFN-γ when exposed to antigen-presenting C1R cells expressing HLA-A*24:02 (C1R-A24 cells) pulsed with specific peptides, while no or low IFN-γ production was detected in the absence of peptide stimulation of C1R-A24 cells ( Figure 1A ), indicating that the established FOXM1- and UBE2T-specific CTLs specifically recognized the HLA-A*24:02-restricted peptide.

[0171] When FOXM1 and UBE2T protein levels were examined in cancer cell lines by western blot analysis ( Figure 4 ), the cytotoxic activity of FOXM1 and UBE2T-specific CTLs against several cancer cell lines was examined by a time-lapse recording system. FOXM1 and UBE2T-specific CTLs showed extremely strong cytotoxic activity against SW480 cells expressing high levels of HLA-A24 and FOXM1 and UBE2T proteins. Almost no cytotoxicity was observed against HLA-A24-negative cancer cell lines HCC1143 and BT549 cells ( Figure 1B and 1C ). The results clearly demonstrated the HLA-restricted cytotoxic activity of antigen-specific T cells against cancer cells.

[0172] Generation of FOXM1 and UBE2 T-specific TCR-engineered T cells

[0173] Subsequently, the TCRA and TCRB chains of these FOXM1 and UBE2T-specific CTLs were sequenced by TCR repertoire analysis using next-generation sequencing ( Figure 2A Both CTL clones showed monoclonal TCR repertoires ( Figure 2A). By DNA sequencing, dominant TCRA and TCRB CDR3 clonotypes were identified for FOXM1-CTL (CACPIMWGSNYKLTF (SEQ ID NO: 49) and CASSLRVHEQYF (SEQ ID NO: 50)) and UBE2T-CTL (CAMREGRNFNKFYF (SEQ ID NO: 69) and CASSLSGGPNEQFF (SEQ ID NO: 70)). The cDNA information was used to construct a vector expressing the TCR, cloned into a lentiviral vector, and TCR-engineered T cells that recognize FOXM1 and UBE2T were generated. Transduction efficiency was measured by TCRvβ-specific antibodies (representative staining data are shown in Figure 2B ). For the assay, only TCR-transduced cells were transduced.

[0174] Cytotoxic activity of FOXM1- and UBE2-specific TCR-engineered T cells

[0175] It is then assessed whether the TCR engineered T cells kill cancer cells as the original CTL clones, e.g. Figure 1B and 1C T cells engineered with TCRs targeting FOXM1 and UBE2T exerted a significant killing effect on HLA-A24-positive SW480 cells and reduced cell viability by 47.5% and 39.3% during the initial five hours, but had no effect on HLA-A24-negative HCC1143 cells ( Figures 3A-3D When co-cultured with C1R-A24 cells pulsed with the corresponding peptides, TCR-engineered T cells showed peptide-specific IFN-γ production in an ELISPOT assay ( Figure 3E and 3F ).

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Sequence Listing <110> University of Chicago <120> Screening of T lymphocytes for cancer-specific antigens <130> UCHI-35360 / WO-1 / ORD <150> US 62 / 569,215 <151> 2017-10-06 <160> 132 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Glu Trp Ala Ala Ala Met Asn Ala Glu Phe 1 5 10 <210> 2 <211> 10 <212> PRT <213> Homo sapiens <400> 2 Asp Tyr Leu Asn Glu Trp Gly Ser Arg Phe 1 5 10 <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 Ile Tyr Thr Trp Ile Glu Asp His Phe 1 5 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Lys Trp Leu Ile Ser Pro Val Lys Ile 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Leu Tyr Leu Lys Leu Leu Pro Tyr Val 1 5 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <400> 6 Lys Val Tyr Leu Arg Val Arg Pro Leu Leu 1 5 10 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <400> 7 Glu Tyr Cys Pro Gly Gly Asn Leu Phe 1 5 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8 Glu Trp Ala Asp Leu Ser Phe Pro Phe 1 5 <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400> 9 Asn Ser Gln Pro Val Trp Leu Cys Leu 1 5 <210> 10 <211> 10 <212> PRT <213> Homo sapiens <400> 10 Ala Tyr Asp Ile Gly Leu Phe Ala Tyr Phe 1 5 10 <210> 11 <211> 9 <212> PRT <213> Homo sapiens <400> 11 Gln Tyr Cys Phe Glu Cys Asp Cys Phe 1 5 <210> 12 <211> 10 <212> PRT <213> Homo sapiens <400> 12 Ser Tyr Gln Lys Val Ile Glu Leu Phe Ser 1 5 10 <210> 13 <211> 9 <212> PRT <213> Homo sapiens <400> 13 Arg Tyr Pro Phe Glu Pro Pro Gln Ile 1 5 <210> 14 <211> 9 <212> PRT <213> Homo sapiens <400> 14 Arg Tyr Leu Ser Ala Gly Pro Thr Leu 1 5 <210> 15 <211> 9 <212> PRT <213> Homo sapiens <400> 15 Ser Tyr Gly Val Leu Leu Trp Glu Ile 1 5 <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Arg Phe Val Pro Asp Gly Asn Arg Ile 1 5 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Gly Tyr Ser Asn Thr Ala Thr Glu Trp 1 5 <210> 18 <211> 9 <212> PRT <213> Homo sapiens <400> 18 Ile Tyr Arg Val Ser Phe Thr Asp Phe 1 5 <210> 19 <211> 10 <212> PRT <213> Homo sapiens <400> 19 Ser Ile Ala Gly Gly Gln Ile Leu Ser Val 1 5 10 <210> 20 <211> 9 <212> PRT <213> Homo sapiens <400> 20 Ser Leu Gln Lys Ala Leu His His Leu 1 5 <210> 21 <211> 10 <212> PRT <213> Homo sapiens <400> 21 Thr Leu Leu Ser Ile Tyr Ile Asp Gly Val 1 5 10 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> twenty two Val Val Cys Ser Lys Leu Thr Glu Val 1 5 <210> twenty three <211> 10 <212> PRT <213> Homo sapiens <400> twenty three Ile Leu Val Val Cys Gly Tyr Ile Thr Val 1 5 10 <210> twenty four <211> 9 <212> PRT <213> Homo sapiens <400> twenty four Leu Leu Ile Gly Ser Thr Ser Tyr Val 1 5 <210> 25 <211> 9 <212> PRT <213> Homo sapiens <400> 25 Ala Leu Val Trp Leu Ile Asp Cys Ile 1 5 <210> 26 <211> 9 <212> PRT <213> Homo sapiens <400> 26 Leu Leu Phe Asp Glu Tyr His Lys Leu 1 5 <210> 27 <211> 10 <212> PRT <213> Homo sapiens <400> 27 Tyr Ile Tyr Asp Leu Phe Val Pro Val Ser 1 5 10 <210> 28 <211> 9 <212> PRT <213> Homo sapiens <400> 28 Arg Ile Ile Cys Glu Ala His Lys Val 1 5 <210> 29 <211> 9 <212> PRT <213> Homo sapiens <400> 29 Phe Gln Asn Ser Pro Pro Ala Ser Val 1 5 <210> 30 <211> 9 <212> PRT <213> Homo sapiens <400> 30 Arg Leu Ala Phe Asp Ile Met Arg Val 1 5 <210> 31 <211> 10 <212> PRT <213> Homo sapiens <400> 31 Thr Thr Met Trp Arg Ala Thr Thr Thr Val 1 5 10 <210> 32 <211> 9 <212> PRT <213> Homo sapiens <400> 32 Ala Leu Tyr Gly Arg Ala Leu Arg Val 1 5 <210> 33 <211> 9 <212> PRT <213> Homo sapiens <400> 33 Ile Thr Asp Gln Tyr Ile Tyr Met Val 1 5 <210> 34 <211> 9 <212> PRT <213> Homo sapiens <400> 34 Gly Leu Gly Pro Gly Leu Leu Gly Val 1 5 <210> 35 <211> 9 <212> PRT <213> Homo sapiens <400> 35 Lys Cys Leu Asp Phe Ser Leu Val Val 1 5 <210> 36 <211> 9 <212> PRT <213> Homo sapiens <400> 36 Lys Thr Lys Arg Leu Asn Glu Leu Lys 1 5 <210> 37 <211> 9 <212> PRT <213> Homo sapiens <400> 37 Met Ser Gln Asn Val Asp Met Pro Lys 1 5 <210> 38 <211> 9 <212> PRT <213> Homo sapiens <400> 38 Val Val Ser Thr Ser Leu Glu Asp Lys 1 5 <210> 39 <211> 10 <212> PRT <213> Homo sapiens <400> 39 Ser Thr Ser Phe Glu Ile Ser Arg Asn Lys 1 5 10 <210> 40 <211> 9 <212> PRT <213> Homo sapiens <400> 40 Glu Val Leu His Met Ile Tyr Met Arg 1 5 <210> 41 <211> 9 <212> PRT <213> Homo sapiens <400> 41 Trp Thr Ile His Pro Ser Ala Asn Arg 1 5 <210> 42 <211> 9 <212> PRT <213> Homo sapiens <400> 42 Glu Phe Thr Gln Tyr Trp Ala Gln Arg 1 5 <210> 43 <211> 9 <212> PRT <213> Homo sapiens <400> 43 Ile Tyr Val Tyr Val Gln Asp Tyr Arg 1 5 <210> 44 <211> 10 <212> PRT <213> Homo sapiens <400> 44 Ala Val Val Asn Val Thr Tyr Ser Ser Lys 1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 45 Cys Ala Ala Leu Asp Ser Asn Tyr Gln Leu Ile Trp 1 5 10 <210> 46 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 46 Cys Ala Ser Ser Lys Asn Gly Gly Ser Tyr Lys Asn Glu Gln Phe Phe 1 5 10 15 <210> 47 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 47 Cys Ala Met Arg Glu Val Leu Ser Gly Gly Gly Ala Asp Gly Leu Thr 1 5 10 15 Phe <210> 48 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 48 Cys Ala Ser Ser Pro Leu Ile Asp Thr Asn Gln Pro Gln His Phe 1 5 10 15 <210> 49 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 49 Cys Ala Cys Pro Ile Met Trp Gly Ser Asn Tyr Lys Leu Thr Phe 1 5 10 15 <210> 50 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 50 Cys Ala Ser Ser Leu Arg Val His Glu Gln Tyr Phe 1 5 10 <210> 51 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 51 Cys Ala Met Arg Glu Ala Leu Ser Tyr Asn Thr Asp Lys Leu Ile Phe 1 5 10 15 <210> 52 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 52 Cys Ala Ser Arg Glu Tyr Lys Asn Glu Gln Phe Phe 1 5 10 <210> 53 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 53 Cys Ala Pro Ser Gly Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe 1 5 10 15 <210> 54 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 54 Cys Ala Ser Ser Phe Ser Ile Asp Thr Gln Tyr Phe 1 5 10 <210> 55 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 55 Cys Ala Val Ile Gly Gly Gly Ser Asn Tyr Gln Leu Ile Trp 1 5 10 <210> 56 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 56 Cys Ala Ser Ser Pro Ser Pro Leu Asp Trp Glu Thr Gln Tyr Phe 1 5 10 15 <210> 57 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 57 Cys Ala Gly Arg Asn Ser Gly Thr Tyr Lys Tyr Ile Phe 1 5 10 <210> 58 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 58 Cys Ala Ser Ser Leu Gly Thr Pro Lys Glu Thr Gln Tyr Phe 1 5 10 <210> 59 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 59 Cys Ala Ala Arg Gly Tyr Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe 1 5 10 15 <210> 60 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 60 Cys Ala Ser Arg Gln Gly Gly Thr Pro Leu His Phe 1 5 10 <210> 61 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 61 Cys Ala Val Arg Arg Gly Asn Gln Phe Tyr Phe 1 5 10 <210> 62 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 62 Cys Ala Ser Ser Leu Ala Leu Gln Gly Met Val Ser Thr Glu Ala Phe 1 5 10 15 Phe <210> 63 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 63 Cys Ala Val Asp Met Trp Ser Gln Gly Asn Leu Ile Phe 1 5 10 <210> 64 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 64 Cys Ala Ser Ser Leu Gly Thr Gly Asp Tyr Glu Gln Tyr Phe 1 5 10 <210> 65 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 65 Cys Ala Val Arg Asp Ile Glu Ala Gly Gly Ser Tyr Ile Pro Thr Phe 1 5 10 15 <210> 66 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 66 Cys Ala Ser Ser Val Gly Trp Thr Ser Ser Tyr Glu Gln Tyr Phe 1 5 10 15 <210> 67 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 67 Cys Ala Val Glu Ala Gly Tyr Ser Thr Leu Thr Phe 1 5 10 <210> 68 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 68 Cys Ala Ser Gly Ala Phe Phe 1 5 <210> 69 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 69 Cys Ala Met Arg Glu Gly Arg Asn Phe Asn Lys Phe Tyr Phe 1 5 10 <210> 70 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 70 Cys Ala Ser Ser Leu Ser Gly Gly Pro Asn Glu Gln Phe Phe 1 5 10 <210> 71 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 71 Cys Ala Met Arg Glu Val Thr Gly Asn Gln Phe Tyr Phe 1 5 10 <210> 72 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 72 Cys Ala Ser Ser Gln Lys Ser Gly Pro Leu Lys Arg Gln Pro Gln His 1 5 10 15 Phe <210> 73 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 73 Cys Ala Val Arg Ala Gly Ala Gly Asn Met Leu Thr Phe 1 5 10 <210> 74 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 74 Cys Ala Ser Ser Ile Asp Gly Leu Ala Gly Glu Gln Tyr Phe 1 5 10 <210> 75 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 75 Cys Ala Met Ser Gln Tyr Gly Asn Lys Leu Val Phe 1 5 10 <210> 76 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 76 Cys Ala Ser Ser Glu Ile Arg Asn Ala Tyr Glu Gln Tyr Phe 1 5 10 <210> 77 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 77 Cys Ala Val Arg Gly Gly Ser Asn Tyr Gln Leu Ile Trp 1 5 10 <210> 78 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 78 Cys Ala Ser Ser Ser Ser Ser Ser Gly Thr Pro Trp Asn Glu Gln Phe Phe 1 5 10 15 <210> 79 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 79 Cys Ala Thr Val Asn Asp Tyr Lys Leu Ser Phe 1 5 10 <210> 80 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 80 Cys Ala Ser Ser Leu Val Leu Gly Arg Asn Thr Glu Ala Phe Phe 1 5 10 15 <210> 81 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 81 Cys Leu Val Gly Asp Arg Gln Ala Gly Thr Ala Leu Ile Phe 1 5 10 <210> 82 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 82 Cys Ser Val Glu Gly Ser Leu Gly Gly Arg Asp Glu Gln Phe Phe 1 5 10 15 <210> 83 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 83 Cys Ala Met Arg Glu Arg Ser Gly Gly Ser Tyr Ile Pro Thr Phe 1 5 10 15 <210> 84 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 84 Cys Ala Ser Lys Gly Thr Gly Gln Lys Glu Thr Gln Tyr Phe 1 5 10 <210> 85 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 85 Cys Ala Glu Thr Asp Thr Thr Ser Gly Thr Tyr Lys Tyr Ile Phe 1 5 10 15 <210> 86 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 86 Cys Ala Ser Ser Leu Phe Ala Gln Ser Ser Tyr Lys Asn Glu Gln Phe 1 5 10 15 Phe <210> 87 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 87 Cys Ala Ala Ser Ala Glu Gly Ala Gly Gly Thr Ser Tyr Gly Lys Leu 1 5 10 15 Thr Phe <210> 88 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 88 Cys Ala Ser Ser Leu Leu Lys Asn Thr Glu Ala Phe Phe 1 5 10 <210> 89 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 89 Cys Ala Val His Asp Asn Tyr Gly Gln Asn Phe Val Phe 1 5 10 <210> 90 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 90 Cys Ala Ser Ser Leu Gly Thr Gly Asn Glu Gln Tyr Phe 1 5 10 <210> 91 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 91 Cys Ala Thr Ile Arg Lys Leu Thr Gly Asn Gln Phe Tyr Phe 1 5 10 <210> 92 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 92 Cys Ala Ser Ser Arg Trp Lys Gly Gln Gly Leu His Thr Gly Glu Leu 1 5 10 15 Phe Phe <210> 93 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 93 Cys Ala Met Arg Glu Gly Gln Ala Gly Thr Ala Leu Ile Phe 1 5 10 <210> 94 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 94 Cys Ala Ser Ser Leu Arg Gln Gly Arg Asp Thr Gln Tyr Phe 1 5 10 <210> 95 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 95 Cys Ala Ala Ser Ala Gly Asn Tyr Gly Gln Asn Phe Val Phe 1 5 10 <210> 96 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 96 Cys Ala Ser Ser Ser Asp Arg Thr Ala Phe Phe 1 5 10 <210> 97 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 97 Cys Ala Val Asn Glu Pro Tyr Lys Leu Ser Phe 1 5 10 <210> 98 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 98 Cys Ala Ser Ser Phe Thr Lys Asn Glu Gln Tyr Phe 1 5 10 <210> 99 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 99 Cys Ala Met Arg Thr Gly Gly Lys Leu Ile Phe 1 5 10 <210> 100 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 100 Cys Ala Trp Ser Val Gly Gln Gly Val Arg Glu Thr Gln Tyr Phe 1 5 10 15 <210> 101 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 101 Cys Ala Glu Asn Leu Ala Arg Gly Gly Asn Lys Leu Thr Phe 1 5 10 <210> 102 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 102 Cys Ala Thr Ser Arg Asp Leu Phe Gly Asp Glu Gln Phe Phe 1 5 10 <210> 103 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 103 Cys Ala Gly Cys Pro Phe Arg Asp Asp Lys Ile Ile Phe 1 5 10 <210> 104 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 104 Cys Ala Ser Ser Leu Ala Gly Glu Glu Thr Gln Tyr Phe 1 5 10 <210> 105 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 105 Cys Ala Leu Asn Asn Ala Gly Asn Met Leu Thr Phe 1 5 10 <210> 106 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 106 Cys Ala Ser Thr Leu Arg Gly Trp Ser Thr Gly Glu Leu Phe Phe 1 5 10 15 <210> 107 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 107 Cys Ile Val Arg Ala Tyr Tyr Gly Gly Ala Thr Asn Lys Leu Ile Phe 1 5 10 15 <210> 108 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 108 Cys Ala Ser Ser Gln Ala Arg Met Gly Asn Gly Glu Leu Phe Phe 1 5 10 15 <210> 109 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 109 Cys Ala Glu Ser Gly Tyr Thr Gly Ala Asn Asn Leu Phe Phe 1 5 10 <210> 110 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 110 Cys Ala Ser Ser Ser Ala Arg Gln Gly Thr Asp Thr Gln Tyr Phe 1 5 10 15 <210> 111 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 111 Cys Ala Thr Asp Phe Asn Ala Gly Asn Met Leu Thr Phe 1 5 10 <210> 112 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 112 Cys Ala Ser Ser Pro Asp Arg Glu Ile Thr Asp Thr Gln Tyr Phe 1 5 10 15 <210> 113 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 113 Cys Ala Ala Ser Gly Arg Ala Gly Ala Asn Asn Leu Phe Phe 1 5 10 <210> 114 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 114 Cys Ser Ala Gly Val Ala Gly Gly Arg Pro Asp Thr Gln Tyr Phe 1 5 10 15 <210> 115 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 115 Cys Ala Val Ser Glu Ser Asp Ser Gly Tyr Ala Leu Asn Phe 1 5 10 <210> 116 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 116 Cys Ala Ser Ser Leu Gly Ile Asp Ser Gly Tyr Gly Tyr Thr Phe 1 5 10 15 <210> 117 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 117 Cys Ala Asp Val Ser Arg Asp Asp Lys Ile Ile Phe 1 5 10 <210> 118 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 118 Cys Ser Ala Leu Ala Gly Gly Asp Pro Tyr Glu Gln Tyr Phe 1 5 10 <210> 119 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 119 Cys Ala Met Arg Glu Gly Arg Ser Glu Val Ile Phe 1 5 10 <210> 120 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 120 Cys Ala Ser Ser Ser Tyr Asn Glu Gln Phe Phe 1 5 10 <210> 121 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 121 Cys Ala Glu Asn Gln Lys Gly Gly Lys Leu Ile Phe 1 5 10 <210> 122 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 122 Cys Ala Ser Ser Tyr Ser Arg Gly Thr Asn Thr Gly Glu Leu Phe Phe 1 5 10 15 <210> 123 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 123 Cys Ala Gly Gln Asp Asn Asn Asp Met Arg Phe 1 5 10 <210> 124 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 124 Cys Ala Ser Thr Ala Trp Gly Ala Asn Thr Glu Ala Phe Phe 1 5 10 <210> 125 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 125 Cys Ala Val Asn Ala Asn Thr Asp Lys Leu Ile Phe 1 5 10 <210> 126 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 126 Cys Ser Ala Trp Glu Arg Thr Ser Leu Phe Glu Gln Tyr Phe 1 5 10 <210> 127 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 127 Cys Leu Val Gly Arg Asp Asn Ala Gly Asn Met Leu Thr Phe 1 5 10 <210> 128 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 128 Cys Ala Ser Gly Thr Asp Thr Asp Thr Gln Tyr Phe 1 5 10 <210> 129 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 129 Cys Ala Gly Asp Pro Asp Ser Gly Asn Thr Pro Leu Val Phe 1 5 10 <210> 130 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 130 Cys Ala Ser Ser Val Gly Leu Thr Val Thr Asn Thr Glu Ala Phe Phe 1 5 10 15 <210> 131 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 131 Cys Ala Met Ser Ala Thr Glu Gly Arg Asp Asn Tyr Gly Gln Asn Phe 1 5 10 15 Val Phe <210> 132 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 132 Cys Ala Ser Gly Phe Tyr Thr Gly Val Ser Thr Glu Ala Phe Phe 1 5 10 15

Claims

1. A method for generating cytotoxic engineered lymphocytes, the method comprising: introducing a vector into a host lymphocyte, the vector comprising a nucleic acid sequence encoding a T cell receptor (TCR) alpha and beta chain comprising an amino acid sequence pair selected from the group consisting of SEQ ID NOs: 49 and 50 and 69 and 70; as well as Cultivating the host lymphocytes under conditions such that the nucleic acid sequences encoding T cell receptor (TCR) α and β chains are expressed in the vector and a TCR comprising α and β chains comprising an amino acid sequence pair selected from the group consisting of SEQ ID NOs: 49 and 50 and 69 and 70 is displayed on the engineered lymphocytes; wherein the engineered lymphocyte recognizes an antigen presenting cell displaying MHC that is bound to a peptide comprising an antigen sequence comprising a sequence selected from the group consisting of: SEQ ID NO: 3 and SEQ ID NO:

13.

2. Engineered lymphocytes produced by the method of claim 1.

3. The engineered lymphocyte of claim 2, wherein the engineered lymphocyte is a CD8+ cytotoxic lymphocyte.

4. Use of the engineered lymphocytes according to claim 2 or 3 in the preparation of a composition for treating HLA-A24 positive cancer.

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