T cell receptors and methods of use thereof

Abstract

To provide T cell receptors and methods of use thereof.SOLUTION: The present disclosure is directed to recombinant T cell receptors capable of binding a gp100 epitope and nucleic acid molecules encoding the same. In some embodiments, the nucleic acid molecules further comprise a second nucleotide sequence, where the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of an endogenous TCR. Other aspects of the disclosure are directed to vectors comprising the nucleic acid molecules and to cells comprising the recombinant TCRs, the nucleic acid molecules or the vectors. Still other aspects of the disclosure are directed to methods of using the same. In some embodiments, the methods comprise treating a cancer in a subject in need thereof.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 813,647, filed March 4, 2019, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (Name: 4285_005PC01_Seqlisting_ST25.txt, Size: 56,525 bytes; and Creation Date: March 3, 2020) are incorporated herein by reference in their entirety.

[0003] Field of the Disclosure The present disclosure provides recombinant T cell receptors ("TCRs") that specifically bind human gp100 and uses thereof. [Background technology]

[0004] Immunotherapy has emerged as an important tool in the fight against various diseases, including cancer. T cell therapy is at the forefront of immunotherapy development, and adoptive transfer of antitumor T cells has been shown to induce clinical responses in cancer patients. Many T cell therapies target mutated tumor antigens, but most neoantigens are not shared and are unique to each patient.

[0005] The number of potential non-mutated antigens outnumbers the number of mutated antigens by several orders of magnitude. Elucidation of T cell epitopes derived from shared antigens could facilitate the development of effective and safe adoptive T cell therapies that are readily accessible to larger cohorts of cancer patients. However, the sheer number of non-mutated antigens and the high polymorphism of HLA genes may hinder a comprehensive analysis of the specificity of anti-tumor T cell responses to non-mutated antigens.

[0006] The present disclosure provides novel epitopes for the non-mutated antigen gp100 and TCRs that can specifically bind to the epitopes. These novel epitopes are associated with specific HLA alleles. The use of these tumor-reactive HLA-restricted gp100 TCRs will broaden the scope of application of anti-gp100 TCR gene therapy, particularly in immuno-oncology. Summary of the Invention [Means for solving the problem]

[0007] Certain aspects of the present disclosure relate to nucleic acid molecules comprising: (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits expression of an endogenous TCR, and the anti-gp100 TCR cross-competes with a reference TCR comprising an alpha chain and a beta chain for binding to human gp100, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0008] Certain aspects of the present disclosure relate to nucleic acid molecules comprising: (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence, or the polypeptide encoded by the second nucleotide sequence, inhibits expression of an endogenous TCR, and the anti-gp100 TCR binds to the same epitope or an overlapping epitope of human gp100 as a reference TCR comprising an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:2.

[0009] In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, this epitope is complexed with an HLA class I molecule.

[0010] In some embodiments, the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. In some embodiments, the HLA class I molecule is an HLA-C*06 allele. In some embodiments, the HLA class I molecule is selected from an HLA-C*06:015 allele, an HLA-C*06:02 allele, an HLA-C*06:03 allele, an HLA-C*06:04 allele, an HLA-C*06:05 allele, an HLA-C*06:06 allele, an HLA-C*06:07 allele, and an HLA-C*06:086 allele. In some embodiments, the HLA class I molecule is an HLA-C*06:02 allele.

[0011] In some embodiments, the anti-gp100 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising alpha chain CDR1, alpha chain CDR2, and alpha chain CDR3; and wherein the beta chain comprises a variable domain comprising beta chain CDR1, beta chain CDR2, and beta chain CDR3; and wherein the alpha chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0012] In some embodiments, the anti-gp100 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising alpha chain CDR1, alpha chain CDR2, and alpha chain CDR3; and wherein the beta chain comprises a variable domain comprising beta chain CDR1, beta chain CDR2, and beta chain CDR3; and wherein the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the alpha chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0013] In some embodiments, the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0014] In some embodiments, the alpha chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the beta chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO: 2.

[0015] In some embodiments, the alpha chain of the anti-gp100 TCR further comprises a constant region, which is different from the endogenous constant region of the alpha chain. In some embodiments, the alpha chain of the anti-gp100 TCR further comprises a constant region, which comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region present in the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the alpha chain constant region comprises an amino acid sequence that comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the beta chain of the anti-gp100 TCR further comprises a constant region, which is different from the endogenous constant region of the beta chain.

[0016] In some embodiments, the beta chain of the anti-gp100 TCR further comprises a constant region, wherein the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region present in the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the beta chain constant region comprises an amino acid sequence that comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the alpha chain of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 1.

[0017] In some embodiments, the beta chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the second nucleotide sequence is one or more siRNAs that reduce expression of endogenous TCRs.

[0018] In some embodiments, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding the constant region of an endogenous TCR. In some embodiments, the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 53-56.

[0019] In some embodiments, the second nucleotide sequence encodes Cas9.

[0020] In some embodiments, the anti-gp100 TCR comprises an alpha chain constant region, a beta chain constant region, or both; wherein the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions in the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0021] Certain aspects of the present disclosure relate to vectors comprising the nucleic acid molecules disclosed herein. In some embodiments, the vector is a viral vector, a mammalian vector, or a bacterial vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, a hybrid vector, and an adeno-associated virus (AAV) vector. In some embodiments, the vector is a lentivirus.

[0022] Certain aspects of the present disclosure relate to a T cell receptor (TCR) or antigen-binding portion thereof comprising the alpha chain variable domain of an anti-gp100 TCR disclosed herein and the beta chain variable domain of an anti-gp100 TCR disclosed herein. In some embodiments, a recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR") cross-competes with a reference TCR for binding to human gp100; wherein the reference TCR comprises an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1, and the beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the anti-gp100 TCR comprises an alpha chain and a beta chain, the alpha chain comprising a constant region, and the beta chain comprising a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the set of amino acid sequences set forth in SEQ ID NO: 1, or (ii) the beta chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence of SEQ ID NO: 2.

[0023] Certain aspects of the present disclosure relate to a recombinant T cell receptor (TCR) or antigen-binding portion thereof (an "anti-gp100 TCR") that specifically binds to human gp100 and binds to the same epitope of human gp100 as a reference TCR or an overlapping epitope; wherein the reference TCR comprises an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1, and the beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the anti-gp100 TCR comprises an alpha chain and a beta chain, the alpha chain comprising a constant region, and the beta chain comprising a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 1, or (ii) the beta chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 consisting of the amino acid sequence set forth in SEQ ID NO:13.

[0024] In some embodiments, the epitope is complexed with an HLA class I molecule. In some embodiments, the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. In some embodiments, the HLA class I molecule is an HLA-C*06 allele. In some embodiments, the HLA class I molecule is selected from an HLA-C*06:01 allele, an HLA-C*06:02 allele, an HLA-C*06:03 allele, an HLA-C*06:04 allele, an HLA-C*06:05 allele, an HLA-C*06:06 allele, an HLA-C*06:07 allele, and an HLA-C*06:08 allele. In some embodiments, the HLA class I molecule is an HLA-C*06:02 allele.

[0025] In some embodiments, the alpha chain of an anti-gp100 TCR comprises a variable domain comprising alpha chain CDR1, alpha chain CDR2, and alpha chain CDR3; wherein the beta chain of the anti-gp100 TCR comprises a variable domain comprising beta chain CDR1, beta chain CDR2, and beta chain CDR3; and wherein the alpha chain CDR3 of the anti-gp100 comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0026] In some embodiments, the alpha chain of the anti-gp100 TCR comprises a variable domain comprising alpha chain CDR1, alpha chain CDR2, and alpha chain CDR3; wherein the beta chain of the anti-gp100 TCR comprises a variable domain comprising beta chain CDR1, beta chain CDR2, and beta chain CDR3; and wherein the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the alpha chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0027] In some embodiments, the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0028] In some embodiments, the alpha chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the beta chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO: 2.

[0029] In some embodiments, the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of the constant region present in the amino acid sequence set forth in SEQ ID NO:1.

[0030] In some embodiments, the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of the constant region present in the amino acid sequence set forth in SEQ ID NO:2.

[0031] In some embodiments, the alpha chain of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the beta chain of the anti-gp100 TCR comprises the amino acid sequence as set forth in SEQ ID NO: 2.

[0032] Certain aspects of the present disclosure relate to bispecific TCRs comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a TCR or antigen-binding portion thereof disclosed herein, or a TCR or antigen-binding portion thereof disclosed herein. In some embodiments, the first antigen-binding domain comprises a single-chain variable fragment ("scFv"). In some embodiments, the second antigen-binding domain specifically binds to a protein expressed on the surface of a T cell. In some embodiments, the second antigen-binding domain specifically binds to CD3. In some embodiments, the second antigen-binding domain comprises an scFv. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are covalently linked or joined. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are linked by a peptide bond.

[0033] Certain aspects of the present disclosure relate to cells comprising a nucleic acid molecule disclosed herein, a vector disclosed herein, a TCR disclosed herein, a recombinant TCR disclosed herein, or a bispecific TCR disclosed herein. In some embodiments, the cell further expresses CD3. In some embodiments, the cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or an ILC cell.

[0034] Certain aspects of the present disclosure relate to methods of treating cancer in a subject in need thereof, comprising administering to the subject a cell disclosed herein. In some embodiments, the cancer is selected from the group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, cervical cancer, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, and cancer of the penis. cancer of the lung, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of the foregoing cancers.

[0035] In some embodiments, the cancer is recurrent or refractory. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is advanced cancer. In some embodiments, the cancer is metastatic.

[0036] In some embodiments, the cells are obtained from a subject. In some embodiments, the cells are obtained from a donor other than the subject. In some embodiments, the subject is preconditioned (pretreated) prior to administration of the cells. In some embodiments, the preconditioning comprises administering to the subject a chemotherapy, a cytokine, a protein, a small molecule, or any combination thereof. In some embodiments, the preconditioning comprises administering an interleukin. In some embodiments, the preconditioning comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, the preconditioning comprises administering a preconditioning agent selected from the group consisting of cyclophosphamide, fludarabine, vitamin C, an AKT inhibitor, ATRA, rapamycin, or any combination thereof. In some embodiments, the preconditioning comprises administering cyclophosphamide, fludarabine, or both.

[0037] Certain aspects of the present disclosure relate to methods of engineering antigen-targeted cells, comprising transducing cells collected from a subject in need of T cell therapy with a nucleic acid disclosed herein or a vector disclosed herein. In some embodiments, the antigen-targeted cells further express CD3. In some embodiments, the cells are T cells or natural killer (NK) cells.

[0038] A particular aspect of the present disclosure relates to an HLA class I molecule complexed with a peptide, the HLA class I molecule comprising an α1 domain, an α2 domain, an α3 domain, and a β2m, the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 14.

[0039] In some embodiments, the HLA class I molecule is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, the HLA class I molecule is HLA-C. In some embodiments, the HLA class I molecule is the HLA-C*06 allele. In some embodiments, the HLA class I molecule is selected from the HLA-C*06:01 allele, the HLA-C*06:02 allele, the HLA-C*06:03 allele, the HLA-C*06:04 allele, the HLA-C*06:05 allele, and the HLA-C*06:06 allele. In some embodiments, the HLA class I molecule is the HLA-C*06:02 allele. In some embodiments, the HLA class I molecule is the HLA-C*03:03 allele.

[0040] In some embodiments, the HLA class I molecule is a monomer. In some embodiments, the HLA class I molecule is a dimer. In some embodiments, the HLA class I molecule is a trimer. In some embodiments, the HLA class I molecule is a tetramer. In some embodiments, the HLA class I molecule is a pentamer.

[0041] Certain aspects of the present disclosure relate to antigen-presenting cells (APCs) comprising the HLA class I molecules disclosed herein. In some embodiments, the HLA class I molecules are expressed on the surface of the APCs.

[0042] Certain aspects of the present disclosure relate to methods of enriching a target population of T cells obtained from a human subject, the method comprising contacting the T cells with an HLA class I molecule disclosed herein or an APC disclosed herein, wherein after the contacting, the enriched T cell population comprises a greater number of T cells capable of binding to HLA class I molecules relative to the number of T cells capable of binding to HLA class I molecules prior to the contacting.

[0043] Certain aspects of the present disclosure relate to a method of enriching a target population of T cells obtained from a human subject, the method comprising contacting T cells in vitro with a peptide, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 13, and wherein after said contacting, the enriched T cell population comprises a greater number of T cells capable of targeting tumor cells relative to the number of T cells capable of targeting tumor cells prior to said contacting.

[0044] In some embodiments, the T cells obtained from the human subject are tumor infiltrating lymphocytes (TILs).

[0045] Certain aspects of the present disclosure relate to methods of treating a tumor in a subject in need thereof, comprising administering to the subject an enriched population of T cells disclosed herein.

[0046] Certain aspects of the present disclosure relate to a method of enhancing cytotoxic T cell-mediated targeting of cancer cells in a subject suffering from cancer, comprising administering to the subject a peptide having the amino acid sequence set forth in SEQ ID NO: 13.

[0047] Certain aspects of the present disclosure relate to a cancer vaccine comprising a peptide having the amino acid sequence set forth in SEQ ID NO:13.

[0048] Certain aspects of the present disclosure relate to methods for selecting T cells capable of targeting tumor cells, comprising contacting a population of in vitro isolated T cells with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the T cells are tumor-infiltrating lymphocytes (TILs). [Brief explanation of the drawings]

[0049] [Figure 1]This is a bar graph showing the number of C*06:02 / gp100 T cells in melanoma TILs after stimulation with artificial APCs pulsed with overlapping peptides. TILs stimulated once with C*06:02-artificial APCs pulsed with overlapping peptides to cover the entire gp100 protein were used as responder cells for IFN-γ ELISPOT assays. C*06:02-artificial APCs pulsed with overlapping peptides derived from gp100 were used as stimulator cells. Following a single controlled peptide-specific stimulation, TILs reacted positively to two adjacent peptides with the shared sequence 186VTVYHRRGSRSYVPL200. (See also Table 5.) [Figure 2] A–D are graphical representations of C*06:02 / gp100190-198 multimer staining of melanoma TILs. TILs were stimulated once with C*06:02-artificial APCs pulsed with gp100190HRRGSRSY198 peptide. Data are shown for C*06:02 / gp100190-198 (A–B) or control C*06:02 / HIV nef120-128 (C–D) multimer staining before stimulation (day 0; A and C) and 14 days after stimulation (day 14; B and D). The percentage of multimer+ cells among CD8+ T cells is shown. [Figure 3] This is a bar graph showing functional evaluation of C*06:02 / gp100190-198 multimer-positive melanoma TILs. IFN-γ production by TILs in a C*06:02 / gp100190-198-specific manner after one peptide-specific stimulation. TILs stimulated once with C*06:02-artificial APCs pulsed with the gp100190-198 peptide were used as responder cells for IFN-γ ELISPOT analysis. C*06:02-artificial APCs pulsed with the indicated peptides were used as stimulator cells. HIV nef120-128 and gp100190-197 peptides were used as controls. Experiments were performed in triplicate, and error bars indicate SD. **P<0.01, ***P<0.001. [Figure 4]A-I are graphic representations of positive staining of Jurkat 76 / CD8 cells transduced with the C*06:02 / gp100190-198 TCR gene with the cognate multimer. Jurkat 76 / CD8 cells transduced with the C*06:02 / gp100190-198 TCR (B, E, and H) were stained with the C*06:02 / gp100190-198 multimer (B). Jurkat 76 / CD8 cells transduced with C*06:02 / HIV nef120-128 multimers (D, E, and F), C*06:02 / non-exchange multimers (G, H, and I), and C*07:02 / MAGE-A1289-297 TCR (clone CL2; C, F, and I) and untransduced (A, D, and G) TCRs were used as controls. The percentages of multimer+CD8+ cells are shown. [Figure 5] A-D are graphical representations of positive staining of human primary T cells transduced with the C*06:02 / gp100190-198 TCR gene (B and D) with the cognate multimer. Primary T cells transduced with the C*06:02 / gp100190-198 TCR were stained with C*06:02 / gp100190-198 (B) or the C*06:02 / HIVnef120-128 control multimer (D). Non-transduced primary T cells were used as negative controls (A and C). The percentage of multimer+ CD8+ T cells is shown. [Figure 6] This is a bar graph showing that human primary T cells transduced with the C*06:02 / gp100190-198 TCR gene strongly react with the cognate peptide presented by the target class I molecule. Primary T cells transduced with the C*06:02 / gp100190-198 TCR gene or untransduced primary T cells were used as responder cells in IFN-γ ELISPOT assays. HLA-null artificial APCs or C*06:02 artificial APCs pulsed with gp100190-198 or HIV nef120-128 peptide (control) were used as stimulator cells. Experiments were performed in triplicate, and error bars indicate SD. **P<0.01, ***P<0.001. [Figure 7](A) Graphical representation showing that primary T cells transduced with the C*06:02 / gp100190-198 TCR gene recognize tumor cells. Primary T cells transduced with the C*06:02 / gp100190-198 TCR gene or untransduced primary T cells were employed as responder cells in IFN-γ ELISPOT assays. As indicated in (B) (legend to A), untransduced or HLA-C*06:02- or gp100-transduced Malme-3M, SK-MEL-28, and A375 cells were employed as stimulator cells. Experiments were performed in triplicate, and error bars indicate SD. **P<0.01, ***P<0.001. [Figure 8] A to D are graphical representations of the expression of gp100 derived from endogenous or transduced full-length genes. Expression of gp100 derived from endogenous or transduced full-length genes in target cells was analyzed via intracellular flow cytometry after staining with anti-gp100 mAb (open curve) and isotype control (filled curve). [Figure 9] (A-D) Graphical representation of ΔNGFR expression in Malme-3M (A and B) and SK-MEL-28 (C and D) target cells transduced with the full-length HLA-C*06:02 gene tagged with ΔNGFR. Surface expression of ΔNGFR in target cells transduced with the full-length HLA-C*06:02 gene tagged with ΔNGFR was analyzed by flow cytometry after staining with anti-NGFR mAb (open curve) and isotype control (closed curve). ΔNGFR alone was used as a control (A and C). DETAILED DESCRIPTION OF THE INVENTION

[0050] The present disclosure relates to a TCR or antigen-binding portion thereof that specifically binds to an epitope on gp100, a nucleic acid molecule encoding the same, and a cell comprising the TCR or nucleic acid molecule. Some embodiments of the present disclosure relate to a method of treating cancer in a subject in need thereof, comprising administering the cells to the subject. Another embodiment of the present disclosure relates to an HLA class I molecule complexed with a peptide comprising an epitope of gp100.

[0051] I. Terminology In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise stated herein, each of the following terms shall have the meaning indicated below. Additional definitions are set forth throughout this application.

[0052] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0053] Furthermore, when used herein, "and / or" shall be construed as a specific disclosure of each of two particular features or components, with or without the other. Thus, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0054] The term "about" is used herein to mean approximately, roughly, roughly, or roughly. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value, by a variance of 10%, above or below (greater than or less than).

[0055] Whenever an embodiment is described herein with the word "comprising," it is to be understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0057] Units, prefixes, and symbols are shown in their International System of Units (SI) accepted format. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which can be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0058] "Administering" refers to the physical introduction of a factor into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration," as used herein, refers to a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epithelial, or mucosal routes of administration, such as intranasally, intravaginally, rectally, sublingually, or topically, and administration can be, for example, single, multiple, and / or over one or more extended periods of time.

[0059] As used herein, the term "T cell receptor" (TCR) refers to a heteromeric cell surface receptor that can specifically interact with a target antigen. As used herein, "TCR" includes, but is not limited to, naturally occurring and non-naturally occurring TCRs; full-length TCRs and their antigen-binding portions; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells and are responsible for T cell recognition and targeting of antigen-presenting cells (APCs). Antigen-presenting cells (APCs) display fragments of foreign proteins (antigens) complexed with major histocompatibility complexes (MHCs; herein also referred to as complexed with HLA molecules, e.g., HLA class 1 molecules). The TCR recognizes and binds the antigen:HLA complex, recruiting CD3 (expressed by T cells) and activating the TCR. The activated TCR initiates downstream signaling and immune responses, including the destruction of EPCs.

[0060] Generally, a TCR may comprise two chains, an alpha chain and a beta chain (or less commonly, a gamma chain and a delta chain), interconnected by disulfide bonds. Each chain comprises a variable domain (an alpha chain variable domain and a beta chain variable domain) and a constant region (an alpha chain constant region and a beta chain constant region). The variable domains are located distal to the cell membrane, and the variable domains interact with antigen. The constant regions are located proximal to the cell membrane. A TCR may further comprise a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses the transmembrane region and cytoplasmic tail, if present, as well as the conventional "constant region."

[0061] Variable domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are flanked by more conserved regions called framework regions (FRs). Each alpha and beta chain variable domain contains three CDRs and four FRs (FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4). Each variable domain contains a binding domain that interacts with antigen. Although all three CDRs of each chain are involved in antigen binding, CDR3 is considered to be the primary antigen-binding region. CDR1 also interacts with antigen, while CD2 is thought to primarily recognize the HLA complex.

[0062] Unless explicitly stated, and unless the context dictates otherwise, the term "TCR" also includes antigen-binding fragments or portions of any TCR disclosed herein, including monovalent and bivalent fragments or portions, and single-chain TCRs. The term "TCR" is not limited to naturally occurring TCRs bound to the surface of T cells. As used herein, the term "TCR" further refers to a TCR described herein expressed on the surface of a cell other than a T cell (e.g., a cell that naturally expresses or is modified to express CD3 as described herein), or a TCR described herein that does not include a cell membrane (e.g., an isolated TCR or a soluble TCR).

[0063] An "antigen-binding molecule," "portion of a TCR," or "TCR fragment" refers to any portion of a TCR that is less than the entire TCR. An antigen-binding molecule may include the complementarity-determining regions (CDRs) of an antigen.

[0064] "Antigen" refers to any molecule, e.g., a peptide, that can elicit an immune response or be bound by a TCR. As used herein, "epitope" refers to a portion of a polypeptide that can elicit an immune response or be bound by a TCR. The immune response may include antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will readily understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Antigens and / or epitopes can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens and / or epitopes can be specific to a particular tissue, such as cancer cells, or they can be broadly expressed. Furthermore, fragments of larger molecules can function as antigens. In one embodiment, the antigen is a tumor antigen. An epitope can be present on a longer polypeptide (e.g., a protein), or it can exist as a fragment of a longer polypeptide. In some embodiments, the epitope is complexed with a major histocompatibility complex (MHC; also referred to herein as complexed with an HLA molecule, e.g., an HLA class 1 molecule).

[0065] As used herein, "gp100," "glycoprotein 100," "melanocyte protein PMEL," or "ME20M" refers to a tumor antigen expressed, for example, in melanoma. gp100 is a 661-amino acid hydrophobic glycoprotein with a molecular weight of 70 kD (GenBank accession number NM_006928). See, for example, Eisenberg et al., Cell Imunol. 266(1):98-103 (2010). In vivo, gp100 is involved in melanosome maturation from stage I to stage II. As used herein, gp100 refers not only to the full-length standard sequence but also to variants and fragments thereof. Known variants of gp100 are provided at www.uniprot.org (UniProtKB-P40967, last accessed March 1, 2019). [Table 1]

[0066] As used herein, the term "HLA" refers to human leukocyte antigens. HLA genes encode human major histocompatibility complex (MHC) proteins. MHC proteins are expressed on the surface of cells and are involved in activating immune responses. HLA class I genes encode MHC class I molecules that are expressed on the surface of cells in complexes with peptide fragments of self or non-self proteins (antigens). T cells expressing TCR and CD3 recognize the antigen:MHC class I complex and initiate an immune response to target and destroy antigen-presenting cells that present non-self proteins.

[0067] As used herein, "HLA class I molecule" or "HLA class I molecule" refers to the protein product of a wild-type or variant HLA class I gene that encodes an MHC class I molecule. Thus, "HLA class I molecule" and "MHC class I molecule" are used interchangeably herein.

[0068] MHC class I molecules contain two protein chains: an alpha chain and a β2-microglobulin (β2m) chain. Human β2m is encoded by the B2M gene. The amino acid sequence of β2m is shown in SEQ ID NO: 16 (Table 2). The alpha chain of MHC class I molecules is encoded by the HLA gene complex. The HLA complex is located within the 6p21.3 region of the short arm of human chromosome 6 and contains more than 220 genes with diverse functions. HLA genes are highly diverse, with more than 20,000 HLA alleles and associated alleles known in the art, including more than 15,000 HLA class I alleles, encoding thousands of HLA proteins (e.g., hla.alleles.org, last accessed February 27, 2019). The HLA complex contains at least three genes encoding MHC class I alpha chain proteins: HLA-A, HLA-B, and HLA-C. Additionally, HLA-E, HLA-F, and HLA-G encode proteins that associate with MHC class I molecules. [Table 2]

[0069] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual. For example, autologous T cell therapy involves administering T cells isolated from the same subject to a subject. The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species. For example, allogeneic T cell transplantation involves administering T cells obtained from a donor other than the subject to a subject.

[0070] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, and other white blood cell malignancies. In some embodiments, the methods of the present invention are used to treat cancers of the human body, including, for example, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular melanoma, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue The present invention may be used to reduce tumor size in tumors resulting from sarcoma, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, other B-cell malignancies, and combinations of the above cancers. Certain cancers may respond to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancer refers to cancer that cannot be cured by surgical intervention, the cancer does not respond initially to chemotherapy or radiation therapy, or the cancer becomes less responsive over time.

[0071] As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall survival or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with tumors.Anti-tumor effect can also refer to the prevention of tumor development, for example, vaccination.

[0072] The term "progression-free survival," sometimes abbreviated as PFS, as used herein, refers to the time from the date of treatment to the date of disease progression according to the revised IWG Response Criteria for Malignant Lymphoma or death from any cause.

[0073] As used herein, "disease progression" or "progressive disease," which may be abbreviated as PD, refers to the worsening of one or more symptoms associated with a particular disease. For example, disease progression in a subject with cancer may include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[0074] "Duration of response," which may be abbreviated as DOR, as used herein, refers to the time from a subject's first objective response to the date of confirmed disease progression according to the revised IWG Response Criteria for Malignant Lymphoma, or death.

[0075] The term "overall survival," which may be abbreviated as OS, is defined as the time from the date of treatment to the date of death.

[0076] As used herein, a "cytokine" is a non-antibody protein released by one cell in response to contact with a specific antigen, which interacts with a second cell and mediates a response in the second cell. Cytokines may be endogenously expressed by a cell or administered to a subject. Cytokines may be released from immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines may induce various responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, proinflammatory cytokines, effector, and acute phase proteins. For example, homeostatic cytokines, such as interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and proinflammatory cytokines may promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placenta growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0077] A "chemokine" is a type of cytokine that mediates chemotaxis, or directional movement, of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0078] Other examples of analytes and cytokines of the present invention include, but are not limited to, chemokine (C-C motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9, IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, granulocyte colony-stimulating factor ( G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), CD154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

[0079] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject against disease manifestations or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of the disease, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0080] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocyte and a major component of the innate immune system. NK cells reject tumor- and virus-infected cells. They function through the process of apoptosis, or programmed cell death. They were called "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without the involvement of antibodies). T cell receptors (TCRs) distinguish T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytotoxic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem memory T cells such as naive cells), and (ii) naive T cells. SCM The cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting many functional attributes characteristic of memory cells; (ii) central memory T CMThe cells express L-selectin and CCR7 and secrete IL-2 but not IFNγ or IL-4, and (iii) effector memory T EM These include T cells (which do not express L-selectin or CCR7 but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence their name.

[0081] The terms "genetically engineered" or "engineered" refer to methods of modifying the genome of a cell, including, but not limited to, deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some embodiments, the modified cells are lymphocytes, e.g., T cells or modified cells that express CD3, which can be obtained from either a patient or a donor. The cells can be modified to express an exogenous construct, such as a T cell receptor (TCR) disclosed herein, that is integrated into the genome of the cell. In some embodiments, the cells are modified to express CD3.

[0082] "Immune response" refers to the actions of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies (Abs), cytokines, and complement) produced by any of these cells or the liver that result in the elimination of invading pathogens from the body of a vertebrate and / or the selective targeting, binding, damage, and destruction of pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0083] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or recurring with a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy includes adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.

[0084] Cells used in the immunotherapies described herein can be derived from sources known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using numerous techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety. Immunotherapy can also include administering modified cells to a subject, wherein the modified cells express CD3 and TCR as disclosed herein. In some embodiments, the modified cells are not T cells.

[0085] As used herein, a "patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0086] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, as well as longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. The polypeptide may be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0087] As used herein, "stimulation" refers to a primary response induced by the binding of a stimulatory molecule to its cognate ligand, which mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, such as a T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, MHC class I molecules loaded with peptides, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0088] The terms "conditioning" and "preconditioning" are used interchangeably herein to refer to preparing a patient in need of T cell therapy for the appropriate condition. Conditioning, as used herein, includes, but is not limited to, reducing the number of endogenous lymphocytes, removing cytokine sinks, increasing serum levels of one or more homeostatic cytokines or proinflammatory factors, enhancing effector function of T cells administered after conditioning, enhancing the activation and / or availability of antigen-presenting cells, or any combination thereof prior to T cell therapy. In one embodiment, "conditioning" comprises increasing the serum levels of one or more cytokines, such as interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), gamma-inducible protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), placenta growth factor (PLGF), C-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), or any combination thereof. In another embodiment, "conditioning" comprises increasing the serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0089] "Treatment" or "treating" a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, symptom severity, or recurrence of a symptom, complication, or condition, or biochemical manifestations associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0090] The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of the list or any of the listed members.

[0091] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within one standard deviation or more than one standard deviation, per practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any number between 2.7 mg and 3.3 mg (for 10%). Furthermore, particularly with respect to biological systems or processes, the term can mean a value up to an order of magnitude or up to five times greater. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of that particular value or composition.

[0092] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0093] Various aspects of the invention are described in further detail in the following subsections.

[0094] II. Disclosed Compositions The present disclosure relates to T cell receptors (TCRs) or antigen-binding portions thereof that specifically bind to epitopes on gp100, nucleic acid molecules encoding the same, and cells comprising the TCRs or nucleic acid molecules. Some aspects of the present disclosure relate to methods of treating cancer in a subject in need thereof, comprising administering to the subject cells comprising a TCR described herein. Other aspects of the present disclosure relate to epitopes of gp100 bound by the TCRs, and HLA class I molecules complexed with peptides comprising epitopes of gp100.

[0095] T cell receptors, or TCRs, are molecules found on the surface of T cells, or T lymphocytes, that are responsible for recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules. The binding between TCRs and antigenic peptides is relatively low affinity and degenerate: many TCRs recognize the same antigenic peptide, and many antigenic peptides are recognized by the same TCR.

[0096] The TCR is composed of two different protein chains (i.e., it is a heterodimer). In humans, in 95% of T cells, the TCR is composed of alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), whereas in 5% of T cells, the TCR is composed of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively). This ratio changes during ontogeny and in pathological conditions (e.g., leukemia). It also varies between species. Orthologs of four loci have been mapped in various species. Each locus can generate a variety of polypeptides with constant and variable regions.

[0097] When the TCR engages an antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0098] II.A. Nucleic acid molecules Certain aspects of the present disclosure relate to nucleic acid molecules comprising: (i) a first nucleotide sequence encoding a recombinant TCR or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence, or the polypeptide encoded by the second nucleotide sequence, inhibits expression of an endogenous TCR. In some embodiments, the second nucleotide sequence is a non-naturally occurring sequence. In other embodiments, the second nucleotide sequence is synthetic. In still other embodiments, the second nucleotide sequence comprises a sequence that targets a nucleotide sequence encoding an endogenous TCR. In some embodiments, the anti-gp100 TCR cross-competes with a reference TCR for binding to human gp100. In some embodiments, the anti-gp100 TCR binds to the same epitope on human gp100 as the reference TCR or an overlapping epitope.

[0099] In some embodiments, the reference TCR comprises an alpha chain and a beta chain; wherein the alpha chain comprises complementarity determining region 1 (CDR1), CDR2, and CDR3; and wherein the beta chain comprises CDR1, CDR2, and CDR3; and wherein the reference TCR comprises the alpha chain CDR3 set forth in SEQ ID NO:7 and the beta chain CDR3 set forth in SEQ ID NO:10. In some embodiments, the alpha chain CDR1, CDR2, and CDR3 sequences are present in the amino acid sequence set forth in SEQ ID NO:1, and the reference TCR comprises the beta chain CDR1, CDR2, and CDR3 sequences present in the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO:1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO:2. [Table 3-1] [Table 3-2]

[0100] II.A.1. TCR Encoded by the First Nucleotide Sequence The present disclosure relates to a TCR encoded by a first nucleotide sequence described herein. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain and a beta chain, wherein the alpha chain comprises a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; and wherein the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3. In some embodiments, the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence (CVVRGMDSSYKLIF) set forth in SEQ ID NO:7. In some embodiments, the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence (CATSSEDSSNQPQHF) set forth in SEQ ID NO:10. In some embodiments, the non-CDR regions of the alpha and / or beta chains are further modified, e.g., by substitution or mutation of one, two, three, four, five, or six amino acids, such that the alpha and / or beta chain is not naturally occurring. In some embodiments, the substitutions or mutations may improve the TCRs described herein in various ways, e.g., binding affinity, binding specificity, stability, viscosity, or any combination thereof.

[0101] In some embodiments, the anti-gp100 encoded by the first nucleotide sequence The TCR comprises an alpha chain CDR1, wherein the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence (VSPFSN) set forth in SEQ ID NO: 5. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain CDR1, wherein the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence (MTFSENT) set forth in SEQ ID NO: 8.

[0102] In some embodiments, the anti-gp100 encoded by the first nucleotide sequence The TCR comprises an alpha chain CDR2, wherein the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence (LNHNV) set forth in SEQ ID NO: 6. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain CDR2, wherein the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence (YYDKDF) set forth in SEQ ID NO: 9.

[0103] In some embodiments, the anti-gp100 encoded by the first nucleotide sequence The TCR comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, and the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.

[0104] In some embodiments, the anti-gp100 encoded by the first nucleotide sequence The TCR comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2, and the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain variable domain present in the amino acid sequence set forth in SEQ ID NO: 2.

[0105] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant domain of the alpha chain amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain constant domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, and the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence further comprises an alpha constant region that differs from the endogenous (e.g., naturally occurring) constant region of the alpha chain. In some embodiments, the alpha chain constant region comprises an amino acid sequence that comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1.

[0106] In some embodiments, the anti-gp100 encoded by the first nucleotide sequence The TCR comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2, and the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain constant region present in the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence further comprises a beta constant region that differs from the endogenous, e.g., naturally occurring, constant region of the beta chain. In some embodiments, the beta chain constant region comprises an amino acid sequence that comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:2.

[0107] In certain embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 1, and the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1.

[0108] In certain embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain comprising the amino acid sequence shown in SEQ ID NO:2.

[0109] In some embodiments, the anti-gp100 encoded by the first nucleotide sequence The TCR comprises an alpha chain constant region, a beta chain constant region, or both; wherein the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions in the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0110] II.A.2. Epitopes In some embodiments, the anti-gp100 encoded by the first nucleotide sequence The TCR binds to the same epitope as the reference TCR. In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 comprising the amino acid sequence set forth in SEQ ID NO: 13 (HRRGSRSYV). In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the epitope is amino acid residues 190-198 of gp100 (SEQ ID NO: 52), e.g., "gp100 190-198 "

[0111] In certain embodiments, the epitope is complexed with an HLA class I molecule. The human leukocyte antigen (HLA) system (human major histocompatibility complex [MHC]) is an important part of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules specialized for presenting antigenic peptides to the T cell receptor (TCR) on T cells. (See also Overview of the Immune System.) MHC molecules that present antigens (Ags) are divided into two major classes: class I MHC molecules and class II MHC molecules.

[0112] Class I MHC molecules are present as transmembrane glycoproteins on the surface of all nucleated cells. Intact class I molecules consist of an alpha heavy chain bound to a beta2-microglobulin molecule. This heavy chain comprises a transmembrane region with two peptide-binding domains, an Ig-like domain, and a cytoplasmic tail. The heavy chain of class I molecules is encoded by genes in the HLA-A, HLA-B, and HLA-C loci. T cells expressing CD8 molecules react with class I MHC molecules. These lymphocytes often have cytotoxic functions and must be able to recognize infected cells. Because all nucleated cells express class I MHC molecules, all infected cells can function as antigen-presenting cells for CD8 T cells (CD8 binds to the non-polymorphic portion of the class I heavy chain). Some class I MHC genes encode non-classical MHC molecules, such as HLA-G (which may play a role in protecting the fetus from the maternal immune response) and HLA-E (which presents peptides to specific receptors on natural killer [NK] cells).

[0113] In some embodiments, the HLA class I molecule is selected from HLA-A, HLA-B, and HLA-C alleles. In some embodiments, the HLA class I molecule is selected from HLA-E, HLA-F, and HLA-G alleles. In certain embodiments, the HLA class I molecule is an HLA-A allele. In certain embodiments, the HLA class I molecule is an HLA-B allele. In certain embodiments, the HLA class I molecule is an HLA-C allele.

[0114] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any known allele may be used in the present disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last visited February 27, 2019). In some embodiments, the HLA class 1 molecule is an HLA-C allele selected from HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*07, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18. In certain embodiments, the HLA-C allele is an HLA-C*06:02 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:03 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:04 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:05 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:06 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:07 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:08 allele.

[0115] The new version of HLA 1 also appears on the screen The name HLA-C is referred to as HLA-C*06:02:0 1:01、HLA-C*06:02:01:02、HLA-C*06:02:01:03、HLA-C*06:02:01:04、HLA- C*06:02:01:05、HLA-C*06:02:01:06、HLA-C*06:02:01:07、HLA-C*06:02: 01:08、HLA-C*06:02:01:09、HLA-C*06:02:01:10、HLA-C*06:02:01:11、HLA -C*06:02:01:12、HLA-C*06:02:01:13、HLA-C*06:02:01:14、HLA-C*06:02 :01:15、HLA-C*06:02:01:16、HLA-C*06:02:01:17、HLA-C*06:02:03、HLA-C *06:02:04、HLA-C*06:02:05、HLA-C*06:02:06、HLA-C*06:02:07、HLA-C*0 6:02:08、HLA-C*06:02:09、HLA-C*06:02:10、HLA-C*06:02:11、HLA-C*06:0 2:12、HLA-C*06:02:13、HLA-C*06:02:14、HLA-C*06:02:15、HLA-C*06:02: 16、HLA-C*06:02:17、HLA-C*06:02:18、HLA-C*06:02:19、HLA-C*06:02:20、 HLA-C*06:02:21、HLA-C*06:02:22、HLA-C*06:02:23、HLA-C*06:02:24、HL A-C*06:02:25、HLA-C*06:02:26、HLA-C*06:02:27、HLA-C*06:02:28、HLA-C *06:02:29、HLA-C*06:02:30、HLA-C*06:02:31、HLA-C*06:02:32、HLA-C*0 6:02:33、HLA-C*06:02:34、HLA-C*06:02:35、HLA-C*06:02:36、HLA-C*06:0 2:37、HLA-C*06:02:38、HLA-C*06:02:39、HLA-C*06:02:40、HLA-C*06:02: 41、HLA-C*06:02:42、HLA-C*06:02:43、HLA-C*06:02:44、HLA-C*06:02:45、HLA-C*06:02:46、HLA-C*06:02:47、HLA-C*06:02:48、HLA-C*06:02:49、HLA-C*06:02:50:01、HLA-C*06:02:50:02、HLA-C*06:02:51、HLA-C*06:02:52、HLA-C*06:02:53、HLA-C*06:02:54、HLA-C*06:02:55、HLA-C*06:02:56、HLA-C*06:02:57、HLA-C* 06:02:58, HLA-C*06:02:59, HLA-C*06:02:60, HLA-C*06:02:61, HLA-C*06:02:62, HLA-C*06:02:63, HLA-C*06:02:64, HLA-C*06:02:65, HLA-C*06:02:66, HLA-C*06:02:67, HLA-C*06:02:68, HLA-C*06:02:69, HLA-C*06:02:70, and HLA-C*06:02:71. The new HLA 1 has also been activated HLA-C is on Facebook:HLA-C*06:03 :01、HLA-C*06:03:02、HLA-C*06:04:01、HLA-C*06:04:02、HLA-C*06:05、H LA-C*06:06、HLA-C*06:07、HLA-C*06:08、HLA-C*06:09:01、HLA-C*06:09: 02、HLA-C*06:10、HLA-C*06:100、HLA-C*06:101、HLA-C*06:102:01、HLA-C* 06:102:02、HLA-C*06:103、HLA-C*06:104、HLA-C*06:105、HLA-C*06:106: 01、HLA-C*06:106:02、HLA-C*06:107、HLA-C*06:108、HLA-C*06:109、HLA- C*06:11、HLA-C*06:110、HLA-C*06:111、HLA-C*06:112、HLA-C*06:113、HL A-C*06:114、HLA-C*06:115、HLA-C*06:116、HLA-C*06:117、HLA-C*06:118、 HLA-C*06:119、HLA-C*06:12、HLA-C*06:120、HLA-C*06:121、HLA-C*06:12 2、HLA-C*06:123、HLA-C*06:124、HLA-C*06:125、HLA-C*06:126、HLA-C*06 :127:01:01、HLA-C*06:127:01:02、HLA-C*06:127:02、HLA-C*06:128、HLA -C*06:129、HLA-C*06:13、HLA-C*06:130、HLA-C*06:131、HLA-C*06:132:01 、HLA-C*06:132:02、HLA-C*06:133、HLA-C*06:134、HLA-C*06:135、HLA-C* 06:136、HLA-C*06:137、HLA-C*06:138、HLA-C*06:139、HLA-C*06:14、HLA-C *06:140、HLA-C*06:141、HLA-C*06:142、HLA-C*06:143、HLA-C*06:144、HL A-C*06:145、HLA-C*06:146、HLA-C*06:147、HLA-C*06:148、HLA-C*06:149、HLA-C*06:15, HLA-C*06:150, HLA-C*06:151, HLA-C*06:152, HLA-C*06:153, HLA-C*06:154, HLA-C*06:155:01:01, HLA-C*06:155:01:02, HLA-C*06:156, HLA-C*06:157, HLA-C*06:158, HLA-C*06:159, HLA-C*06:160, HLA-C*06:161, HLA-C*06:162, HLA-C*06:163, HLA-C*06:164, HLA-C*06:165, HLA -C*06:166, HLA-C*06:167, HLA-C*06:168, HLA-C*06:169, HLA-C*06:16, HLA-C*06:17, HLA-C*06:170, HLA-C*06:171:01:01, HLA-C*06:171:01:02, HLA-C*06:172, HLA-C*06:173, HLA-C*06:174, HLA-C*06:175, HLA-C*06:176, HLA-C*06:177, HLA-C*06:178, HLA-C*06:179, HLA-C*06:18, HLA-C*06 :180, HLA-C*06:181, HLA-C*06:182, HLA-C*06:183, HLA-C*06:184, HLA-C*06:185, HLA-C*06:186, HLA-C*06:187, HLA-C*06:188, HLA-C*06:189, HLA-C*06:19, HLA-C*06:190, HLA-C*06:191, HLA-C*06:192, HLA-C*06:193, HLA-C*06:194, HLA-C*06:195, HLA-C*06:196, HLA-C*06:197, HLA-C*06:1 98、HLA-C*06:199、HLA-C*06:20、HLA-C*06:200、HLA-C*06:201、HLA-C*06:202、HLA-C*06:203、HLA-C*06:204、HLA-C*06:205、HLA-C*06:206、HLA-C*06:207、HLA-C*06:208、HLA-C*06:209、HLA-C*06:21、HLA-C*06:210、HLA-C*06:211、HLA-C*06:212、HLA-C*06:213、HLA-C*06:214、HLA-C*06:215、HLA-C*06:216, HLA-C*06:217, HLA-C*06:218, HLA-C*06:219, HLA-C*06:22, HLA-C*06:220, HLA-C*06:221, HLA-C*06:222, HLA-C*06:223, HLA-C*06:224, HLA-C*06:225, HLA-C*06:226, HLA-C*06:227, HLA-C*06:228, HLA-C*06:229, HLA-C*06:23, HLA-C*06:230, HLA-C*06:231, HLA-C*06:232, HLA- C*06:233、HLA-C*06:234、HLA-C*06:235、HLA-C*06:236、HLA-C*06:237、HLA-C*06:238、HLA-C*06:239、HLA-C*06:24、HLA-C*06:240、HLA-C*06:241、HLA-C*06:242、HLA-C*06:243、HLA-C*06:244、HLA-C*06:245、HLA-C*06:246、HLA-C*06:247、HLA-C*06:248、HLA-C*06:249、HLA-C*06:25、HLA-C*06 :250、HLA-C*06:251、HLA-C*06:26、HLA-C*06:27、HLA-C*06:28、HLA-C*06:29、HLA-C*06:30、HLA-C*06:31、HLA-C*06:32、HLA-C*06:33、HLA-C*06:34:01、HLA-C*06:34:02、HLA-C*06:35、HLA-C*06:36、HLA-C*06:37、HLA-C*06:38、HLA-C*06:39、HLA-C*06:40、HLA-C*06:41、HLA-C*06:42:01、HLA-C* 06:42:02, HLA-C*06:43:01, HLA-C*06:43:02, HLA-C*06:44, HLA-C*06:45, HLA-C*06:46, HLA-C*06:47, HLA-C*06:48, HLA-C*06:49, HLA-C*06:50, HLA-C*06:51, HLA-C*06:52, HLA-C*06:53:01, HLA-C*06:53:02, HLA-C*06:54, HLA-C*06:55, HLA-C*06:56, HLA-C*06:57, HLA-C*06:58, HLA-C*06:59,HLA-C*06:60, HLA-C*06:61, HLA-C*06:62, HLA-C*06:63, HLA-C*06:64, HLA-C*06:65, HLA-C*06:66, HLA-C*06:67, HLA-C*06:68, HLA-C*06:69, HLA-C*06:70:01, HLA-C*06:70:02, HLA-C*06:71, HLA-C*06:72, HLA-C*06:73, HLA-C*06:74, HLA-C*06:75, HLA-C*06:76:01, HLA-C*06:76:02, HLA-C*06:77, HLA-C*06:78, HLA-C*06:79, HLA-C*06:80, HLA-C*06:81, HLA-C*06:82, HLA-C*06:83, HLA-C*06:84, HLA-C*06:85, HLA-C*06:86, HLA-C*06:87, HLA-C*06:88, HLA-C*06:89, HLA-C*06:90, HLA-C*06:91, HLA-C*06:92, HLA-C*06:93, HLA-C*06:94, HLA-C*06:95, HLA-C*06:96, HLA-C*06:97, HLA-C*06:98, HLA-C*06:99.,

[0116] II.A.3 Second nucleotide sequence The second nucleotide sequence of the nucleic acid molecule disclosed herein may be any sequence or may encode any polypeptide capable of inhibiting expression of an endogenous TCR. In some embodiments, the second nucleotide sequence is one or more siRNAs. In some embodiments, the one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding the constant region of an endogenous TCR. In certain embodiments, the one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding the constant region of a wild-type human TCR. In some embodiments, the one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding the constant region of the alpha chain of a wild-type TCR. In some embodiments, the one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding the constant region of the beta chain of a wild-type TCR. In some embodiments, the one or more siRNAs comprise (i) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding the constant region of the alpha chain of a wild-type TCR, and (ii) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding the constant region of the beta chain of a wild-type TCR.

[0117] In some embodiments, the one or more siRNAs comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 53-56 (Table 4). In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs are complementary to target sequences within the nucleotide sequence encoding the constant region of the alpha chain of a wild-type TCR, and wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 53 and 54. [Table 4]

[0118] In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs are complementary to target sequences within the nucleotide sequence encoding the constant region of the beta chain of the wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 55 and 56. In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes one or more siRNAs, wherein the one or more siRNAs comprise: (i) one or more siRNAs that are complementary to target sequences within the nucleotide sequence encoding the constant region of the alpha chain of the wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 53 and 54; and (ii) one or more siRNAs that are complementary to target sequences within the nucleotide sequence encoding the constant region of the beta chain of the wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 55 and 56.

[0119] In some embodiments, the second nucleotide sequence of the nucleic acid molecule comprises SEQ ID NOs: 53-56. In some embodiments, the second nucleotide sequence comprises SEQ ID NOs: 53-56, wherein one or more of SEQ ID NOs: 53-56 are separated by one or more nucleic acids that do not encode an siRNA. In certain embodiments, the one or more siRNAs are selected from the siRNAs disclosed in U.S. Publication No. 2010 / 0273213 A1, which is incorporated by reference in its entirety.

[0120] In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes a protein, where the protein is capable of inhibiting expression of an endogenous, e.g., wild-type, TCR. In some embodiments, the second nucleotide sequence encodes Cas9.

[0121] II.A.3 Vectors Certain aspects of the present disclosure relate to vectors comprising the nucleic acid molecules disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral particle or virus. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a bacterial vector.

[0122] In certain embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, and an adeno-associated virus (AAV) vector. In certain embodiments, the vector is an AAV vector. In some embodiments, the vector is a lentivirus. In certain embodiments, the vector is an AAV vector. In some embodiments, the vector is a Sendai virus. In some embodiments, the vector is a hybrid vector. Examples of hybrid vectors that may be used in the present disclosure are described in Huang and Kamihira, Biotechnol. Adv. 31(2):208-23(2103).

[0123] II.B. Recombinant T Cell Receptors (TCRs) Certain aspects of the present disclosure relate to recombinant T cell receptors (TCRs) or antigen-binding portions thereof that specifically bind to human gp100 ("anti-gp100 TCRs"). In some embodiments, the anti-gp100 TCRs are encoded by nucleic acid molecules disclosed herein.

[0124] In some embodiments, the anti-gp100 TCR cross-competes with the reference TCR for binding to human gp100. In some embodiments, the anti-gp100 TCR binds to the same epitope of human gp100 as the reference TCR or an overlapping epitope. In some embodiments, the reference TCR comprises an alpha chain and a beta chain, and the alpha chain is composed of the reference TCR and comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the beta chain of the reference TCR comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0125] In some embodiments, the anti-gp100 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region and the beta chain comprises a constant region; wherein the alpha chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR comprises an alpha chain and a beta chain, wherein the alpha chain constant region comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the constant region of the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1. The TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region and the beta chain comprises a constant region; wherein the beta chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the beta chain comprising the amino acid sequence set forth in SEQ ID NO:2.

[0126] In some embodiments, the anti-gp100 TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises a constant region and the beta chain comprises a constant region; wherein (i) the alpha chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the alpha chain comprising the amino acid sequence set forth in SEQ ID NO:1; and (ii) the beta chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the beta chain comprising the amino acid sequence set forth in SEQ ID NO:2.

[0127] In some embodiments, the alpha chain of the anti-gp100 TCR comprises a variable domain comprising alpha chain CDR1, alpha chain CDR2, and alpha chain CDR3; and the beta chain of the anti-gp100 TCR comprises a variable domain comprising beta chain CDR1, beta chain CDR2, and beta chain CDR3. In some embodiments, the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0128] In some embodiments, the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0129] In some embodiments, the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0130] In some embodiments, an anti-gp100 TCR comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, an anti-gp100 TCR comprises an alpha chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, and the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, an anti-gp100 TCR comprises an alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.

[0131] In some embodiments, an anti-gp100 TCR comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, an anti-gp100 TCR comprises a beta chain variable domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the variable domain of the beta chain amino acid sequence set forth in SEQ ID NO: 2, and the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, an anti-gp100 TCR comprises a beta chain variable domain present in the beta chain amino acid sequence set forth in SEQ ID NO: 2.

[0132] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence further comprises an alpha chain constant region, a beta chain constant region, or both an alpha chain constant region and a beta chain constant region. In some embodiments, the anti-gp100 TCR comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR comprises an alpha chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO: 1, and the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR comprises an alpha chain constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide The TCR further comprises an alpha chain constant region that differs from the endogenous, e.g., naturally occurring, constant region of the alpha chain. In some embodiments, the alpha chain constant region comprises an amino acid sequence that contains at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the alpha chain amino acid sequence set forth in SEQ ID NO:1.

[0133] In some embodiments, the anti-gp100 TCR comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR comprises a beta chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the constant region of the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-gp100 TCR comprises a beta chain constant region present in the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence further comprises a beta constant region that differs from the endogenous, e.g., naturally occurring, constant region of the beta chain. In some embodiments, the beta chain constant region comprises an amino acid sequence that includes at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the beta chain amino acid sequence set forth in SEQ ID NO:2.

[0134] In certain embodiments, the anti-gp100 TCR comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR comprises an alpha chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the alpha chain amino acid sequence set forth in SEQ ID NO: 1, and the anti-gp100 TCR comprises an alpha chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1.

[0135] In certain embodiments, the anti-gp100 TCR comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the anti-gp100 TCR comprises a beta chain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the beta chain amino acid sequence set forth in SEQ ID NO: 2, wherein the anti-gp100 TCR comprises a beta chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-gp100 TCR comprises a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0136] In some embodiments, the anti-gp100 TCR comprises an alpha chain constant region, a beta chain constant region, or both; wherein the alpha chain constant region, the beta chain constant region, or both, comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions in the target sequence relative to the corresponding amino acid sequence of the endogenous TCR.

[0137] II.B.2. Epitopes In some embodiments, the anti-gp100 TCR binds to the same epitope as the reference TCR. In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the epitope is amino acid residues 190-198 of gp100 (SEQ ID NO: 52), e.g., "gp100 190-198 "

[0138] In certain embodiments, the epitope is complexed with an HLA class I molecule. In some embodiments, the HLA class I molecule is selected from HLA-A, HLA-B, and HLA-C alleles. In some embodiments, the HLA class I molecule is selected from HLA-E, HLA-F, and HLA-G alleles. In certain embodiments, the HLA class I molecule is an HLA-A allele. In certain embodiments, the HLA class I molecule is an HLA-B allele. In certain embodiments, the HLA class I molecule is an HLA-C allele.

[0139] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any known allele may be used in the present disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last visited February 27, 2019). In some embodiments, the HLA class 1 molecule is an HLA-C allele selected from HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*07, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18. In certain embodiments, the HLA-C allele is an HLA-C*06:01 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:02 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:03 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:04 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:05 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:06 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:07 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:08 allele.

[0140] The new version of HLA 1 also appears on the screen The name HLA-C is referred to as HLA-C*06:02:0 1:01、HLA-C*06:02:01:02、HLA-C*06:02:01:03、HLA-C*06:02:01:04、HLA- C*06:02:01:05、HLA-C*06:02:01:06、HLA-C*06:02:01:07、HLA-C*06:02: 01:08、HLA-C*06:02:01:09、HLA-C*06:02:01:10、HLA-C*06:02:01:11、HLA -C*06:02:01:12、HLA-C*06:02:01:13、HLA-C*06:02:01:14、HLA-C*06:02 :01:15、HLA-C*06:02:01:16、HLA-C*06:02:01:17、HLA-C*06:02:03、HLA-C *06:02:04、HLA-C*06:02:05、HLA-C*06:02:06、HLA-C*06:02:07、HLA-C*0 6:02:08、HLA-C*06:02:09、HLA-C*06:02:10、HLA-C*06:02:11、HLA-C*06:0 2:12、HLA-C*06:02:13、HLA-C*06:02:14、HLA-C*06:02:15、HLA-C*06:02: 16、HLA-C*06:02:17、HLA-C*06:02:18、HLA-C*06:02:19、HLA-C*06:02:20、 HLA-C*06:02:21、HLA-C*06:02:22、HLA-C*06:02:23、HLA-C*06:02:24、HL A-C*06:02:25、HLA-C*06:02:26、HLA-C*06:02:27、HLA-C*06:02:28、HLA-C *06:02:29、HLA-C*06:02:30、HLA-C*06:02:31、HLA-C*06:02:32、HLA-C*0 6:02:33、HLA-C*06:02:34、HLA-C*06:02:35、HLA-C*06:02:36、HLA-C*06:0 2:37、HLA-C*06:02:38、HLA-C*06:02:39、HLA-C*06:02:40、HLA-C*06:02: 41、HLA-C*06:02:42、HLA-C*06:02:43、HLA-C*06:02:44、HLA-C*06:02:45、HLA-C*06:02:46、HLA-C*06:02:47、HLA-C*06:02:48、HLA-C*06:02:49、HLA-C*06:02:50:01、HLA-C*06:02:50:02、HLA-C*06:02:51、HLA-C*06:02:52、HLA-C*06:02:53、HLA-C*06:02:54、HLA-C*06:02:55、HLA-C*06:02:56、HLA-C*06:02:57、HLA-C* 06:02:58, HLA-C*06:02:59, HLA-C*06:02:60, HLA-C*06:02:61, HLA-C*06:02:62, HLA-C*06:02:63, HLA-C*06:02:64, HLA-C*06:02:65, HLA-C*06:02:66, HLA-C*06:02:67, HLA-C*06:02:68, HLA-C*06:02:69, HLA-C*06:02:70, and HLA-C*06:02:71. The new HLA 1 has also been activated HLA-C is on Facebook:HLA-C*06:03 :01、HLA-C*06:03:02、HLA-C*06:04:01、HLA-C*06:04:02、HLA-C*06:05、H LA-C*06:06、HLA-C*06:07、HLA-C*06:08、HLA-C*06:09:01、HLA-C*06:09: 02、HLA-C*06:10、HLA-C*06:100、HLA-C*06:101、HLA-C*06:102:01、HLA-C* 06:102:02、HLA-C*06:103、HLA-C*06:104、HLA-C*06:105、HLA-C*06:106: 01、HLA-C*06:106:02、HLA-C*06:107、HLA-C*06:108、HLA-C*06:109、HLA- C*06:11、HLA-C*06:110、HLA-C*06:111、HLA-C*06:112、HLA-C*06:113、HL A-C*06:114、HLA-C*06:115、HLA-C*06:116、HLA-C*06:117、HLA-C*06:118、 HLA-C*06:119、HLA-C*06:12、HLA-C*06:120、HLA-C*06:121、HLA-C*06:12 2、HLA-C*06:123、HLA-C*06:124、HLA-C*06:125、HLA-C*06:126、HLA-C*06 :127:01:01、HLA-C*06:127:01:02、HLA-C*06:127:02、HLA-C*06:128、HLA -C*06:129、HLA-C*06:13、HLA-C*06:130、HLA-C*06:131、HLA-C*06:132:01 、HLA-C*06:132:02、HLA-C*06:133、HLA-C*06:134、HLA-C*06:135、HLA-C* 06:136、HLA-C*06:137、HLA-C*06:138、HLA-C*06:139、HLA-C*06:14、HLA-C *06:140、HLA-C*06:141、HLA-C*06:142、HLA-C*06:143、HLA-C*06:144、HL A-C*06:145、HLA-C*06:146、HLA-C*06:147、HLA-C*06:148、HLA-C*06:149、HLA-C*06:15, HLA-C*06:150, HLA-C*06:151, HLA-C*06:152, HLA-C*06:153, HLA-C*06:154, HLA-C*06:155:01:01, HLA-C*06:155:01:02, HLA-C*06:156, HLA-C*06:157, HLA-C*06:158, HLA-C*06:159, HLA-C*06:160, HLA-C*06:161, HLA-C*06:162, HLA-C*06:163, HLA-C*06:164, HLA-C*06:165, HLA -C*06:166, HLA-C*06:167, HLA-C*06:168, HLA-C*06:169, HLA-C*06:16, HLA-C*06:17, HLA-C*06:170, HLA-C*06:171:01:01, HLA-C*06:171:01:02, HLA-C*06:172, HLA-C*06:173, HLA-C*06:174, HLA-C*06:175, HLA-C*06:176, HLA-C*06:177, HLA-C*06:178, HLA-C*06:179, HLA-C*06:18, HLA-C*06 :180, HLA-C*06:181, HLA-C*06:182, HLA-C*06:183, HLA-C*06:184, HLA-C*06:185, HLA-C*06:186, HLA-C*06:187, HLA-C*06:188, HLA-C*06:189, HLA-C*06:19, HLA-C*06:190, HLA-C*06:191, HLA-C*06:192, HLA-C*06:193, HLA-C*06:194, HLA-C*06:195, HLA-C*06:196, HLA-C*06:197, HLA-C*06:1 98、HLA-C*06:199、HLA-C*06:20、HLA-C*06:200、HLA-C*06:201、HLA-C*06:202、HLA-C*06:203、HLA-C*06:204、HLA-C*06:205、HLA-C*06:206、HLA-C*06:207、HLA-C*06:208、HLA-C*06:209、HLA-C*06:21、HLA-C*06:210、HLA-C*06:211、HLA-C*06:212、HLA-C*06:213、HLA-C*06:214、HLA-C*06:215、HLA-C*06:216, HLA-C*06:217, HLA-C*06:218, HLA-C*06:219, HLA-C*06:22, HLA-C*06:220, HLA-C*06:221, HLA-C*06:222, HLA-C*06:223, HLA-C*06:224, HLA-C*06:225, HLA-C*06:226, HLA-C*06:227, HLA-C*06:228, HLA-C*06:229, HLA-C*06:23, HLA-C*06:230, HLA-C*06:231, HLA-C*06:232, HLA- C*06:233、HLA-C*06:234、HLA-C*06:235、HLA-C*06:236、HLA-C*06:237、HLA-C*06:238、HLA-C*06:239、HLA-C*06:24、HLA-C*06:240、HLA-C*06:241、HLA-C*06:242、HLA-C*06:243、HLA-C*06:244、HLA-C*06:245、HLA-C*06:246、HLA-C*06:247、HLA-C*06:248、HLA-C*06:249、HLA-C*06:25、HLA-C*06 :250、HLA-C*06:251、HLA-C*06:26、HLA-C*06:27、HLA-C*06:28、HLA-C*06:29、HLA-C*06:30、HLA-C*06:31、HLA-C*06:32、HLA-C*06:33、HLA-C*06:34:01、HLA-C*06:34:02、HLA-C*06:35、HLA-C*06:36、HLA-C*06:37、HLA-C*06:38、HLA-C*06:39、HLA-C*06:40、HLA-C*06:41、HLA-C*06:42:01、HLA-C* 06:42:02, HLA-C*06:43:01, HLA-C*06:43:02, HLA-C*06:44, HLA-C*06:45, HLA-C*06:46, HLA-C*06:47, HLA-C*06:48, HLA-C*06:49, HLA-C*06:50, HLA-C*06:51, HLA-C*06:52, HLA-C*06:53:01, HLA-C*06:53:02, HLA-C*06:54, HLA-C*06:55, HLA-C*06:56, HLA-C*06:57, HLA-C*06:58, HLA-C*06:59,HLA-C*06:60, HLA-C*06:61, HLA-C*06:62, HLA-C*06:63, HLA-C*06:64, HLA- C*06:65, HLA-C*06:66, HLA-C*06:67, HLA-C*06:68, HLA-C*06:69, HLA-C*06: 70:01, HLA-C*06:70:02, HLA-C*06:71, HLA-C*06:72, HLA-C*06:73, HLA-C*0 6:74, HLA-C*06:75, HLA-C*06:76:01, HLA-C*06:76:02, HLA-C*06:77, HLA-C* 06:78, HLA-C*06:79, HLA-C*06:80, HLA-C*06:81, HLA-C*06:82, HLA-C*06:8 3, HLA-C*06:84, HLA-C*06:85, HLA-C*06:86, HLA-C*06:87, HLA-C*06:88, HLA -C*06:89, HLA-C*06:90, HLA-C*06:91, HLA-C*06:92, HLA-C*06:93, HLA-C*0 6:94, HLA-C*06:95, HLA-C*06:96, HLA-C*06:97, HLA-C*06:98, HLA-C*06:99. ,

[0141] II.B.3. Bispecific T cell receptor (TCR) Certain aspects of the present disclosure relate to bispecific TCRs comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a TCR or antigen-binding portion thereof disclosed herein. In some embodiments, the first antigen-binding domain comprises a single-chain variable fragment ("scFv").

[0142] In some embodiments, the second antigen-binding domain specifically binds to a protein expressed on the surface of a T cell. Any protein expressed on the surface of a T cell can be targeted by the bispecific antibodies disclosed herein. In certain embodiments, the protein expressed on the surface of a T cell is not expressed by other cells. In some embodiments, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells. In some embodiments, the protein expressed on the surface of a T cell is expressed on the surface of one or more other human immune cells, but not on the surface of a human non-immune cell. In some embodiments, the second antigen-binding domain specifically binds to a protein expressed on the surface of a T cell selected from CD3, CD2, CD5, CD6, CD8, CD11a (LFA-1α), CD43, CD45, and CD53. In certain embodiments, the second antigen-binding domain specifically binds to CD3. In some embodiments, the second antigen-binding domain comprises an scFv.

[0143] In some embodiments, the first and second antigen-binding domains are linked or attached by a covalent bond, hi some embodiments, the first and second antigen-binding domains are linked by a peptide bond.

[0144] II.C. TCR-expressing cells Certain aspects of the present disclosure relate to cells comprising a nucleic acid molecule disclosed herein, a vector disclosed herein, a recombinant TCR disclosed herein, a bispecific TCR disclosed herein, or any combination thereof. Any cell may be used in the present disclosure.

[0145] In certain embodiments, the cell expresses CD3. Expression of CD3 can be naturally occurring, e.g., CD3 is expressed from a nucleic acid sequence endogenously expressed by the cell. For example, T cells and natural killer (NK) cells naturally express CD3. Thus, in some embodiments, the cell is a T cell or a natural killer cell. In certain embodiments, the cell is a T cell selected from a natural killer T (NKT) cell and an innate lymphoid cell (ILC).

[0146] In some embodiments, the T cells are isolated from a human subject. In some embodiments, the human subject is the same subject who will ultimately receive the T cell therapy. In other embodiments, the subject is a donor subject, and the donor subject is not the same subject who will receive the T cell therapy.

[0147] In some embodiments, the cell is a cell that does not naturally express CD3, wherein the cell has been modified to express CD3. In some embodiments, the cell comprises a transgene encoding CD3, wherein the transgene is expressed by the cell. In some embodiments, the cell comprises a transgene encoding a protein that activates expression of endogenous CD3 by the cell. In some embodiments, the cell comprises a transgene encoding a protein or siRNA that inhibits an inhibitor of CD3 expression in the cell. In some embodiments, the transgene is integrated into the genome of the cell. In some embodiments, the transgene is not integrated into the genome of the cell.

[0148] In some embodiments, the cells modified to express CD3 are isolated from a human subject. In some embodiments, the human subject is the same subject who will ultimately receive the cell therapy. In other embodiments, the subject is a donor subject, and the donor subject is not the same subject who will receive the cell therapy.

[0149] II.D. HLA class I molecules Certain aspects of the present disclosure relate to an HLA class I molecule complexed with a peptide, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the peptide consists of the amino acid sequence set forth in SEQ ID NO: 13.

[0150] In some embodiments, the HLA class I molecule is HLA-A, HLA-B, or HLA-C. In some embodiments, the HLA class I molecule is HLA-E, HLA-F, or HLA-G. In some embodiments, the HLA class I molecule is an HLA-C allele selected from HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*07, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18. In certain embodiments, the HLA-C allele is an HLA-C*06:01 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:02 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:03 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:04 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:05 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:06 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:07 allele. In certain embodiments, the HLA-C allele is an HLA-C*06:08 allele. In some embodiments, the HLA allele is any HLA allele disclosed herein, e.g., above.

[0151] In some embodiments, the HLA class I molecule comprises an alpha chain and a β2m. In some embodiments, the alpha chain comprises an α1 domain, an α2 domain, and an α3 domain. In some embodiments, the β2m comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the sequence of the alpha chain is selected from any HLA protein sequence available at hla.alleles.org (last accessed February 27, 2019).

[0152] In some embodiments, the HLA class I molecule is a monomer. In some embodiments, the HLA class I molecule is a dimer. In some embodiments, the HLA class I molecule is a multimer. In some embodiments, the HLA class I molecule is a trimer. In some embodiments, the HLA class I molecule is a tetramer. In some embodiments, the HLA class I molecule is a pentamer.

[0153] Certain aspects of the present disclosure relate to antigen-presenting cells (APCs) comprising any of the HLA class I molecules disclosed herein. In certain embodiments, the APCs express HLA class I molecules on their surface. In certain embodiments, the APCs comprise two or more HLA class I molecules disclosed herein.

[0154] II.D. Vaccines Certain aspects of the present disclosure disclose a cancer vaccine comprising a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the cancer vaccine comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the vaccine further comprises one or more excipients. In some embodiments, the vaccine further comprises one or more additional peptides. In some embodiments, the one or more additional peptides comprise one or more additional epitopes.

[0155] III. Methods of the Disclosure Certain aspects of the present disclosure relate to methods of treating cancer in a subject in need thereof. Other aspects of the present disclosure relate to methods of engineering antigen-targeted cells. Other aspects of the present disclosure relate to methods of enriching a target population of T cells obtained from a human subject.

[0156] III.A. Cancer Treatment Methods Certain aspects of the present disclosure relate to methods of treating cancer in a subject in need thereof, comprising administering to the subject a nucleic acid molecule disclosed herein, a recombinant TCR disclosed herein, a bispecific TCR disclosed herein, an epitope disclosed herein, or an HLA class I molecule disclosed herein, or a vector or cell comprising any of the above.

[0157] In some embodiments, the cancer is melanoma, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or intraocular melanoma, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, cancer of the fallopian tubes, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, thyroid cancer ... The cancer is selected from sarcoma of tissues, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of these cancers. In some embodiments, the cancer is melanoma.

[0158] In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory. In some embodiments, the cancer is progressive. In some embodiments, the cancer is metastatic.

[0159] In some embodiments, the methods disclosed herein treat cancer in a subject. In some embodiments, the methods disclosed herein reduce the severity of one or more symptoms of cancer. In some embodiments, the methods disclosed herein reduce the size or number of tumors resulting from cancer. In some embodiments, the methods disclosed herein extend the overall survival of a subject relative to a subject not provided with a method disclosed herein. In some embodiments, the methods disclosed herein increase the progression-free survival of a subject relative to a subject not provided with a method disclosed herein. In some embodiments, the methods disclosed herein result in a partial response in a subject. In some embodiments, the methods disclosed herein result in a complete remission in a subject.

[0160] In some embodiments, the methods disclosed herein include treating cancer in a subject in need thereof, comprising administering to the subject a cell described herein, wherein the cell comprises a nucleic acid molecule disclosed herein, a vector disclosed herein, a recombinant TCR disclosed herein, and / or a bispecific antibody disclosed herein. In some embodiments, the cell is a T cell. In some embodiments, the cell is a cell that has been modified to express CD3.

[0161] In some embodiments, the cells, e.g., T cells, are obtained from the subject. In some embodiments, the cells, e.g., T cells, are obtained from a donor other than the subject.

[0162] In some embodiments, the subject is pretreated (preconditioned) prior to administering the cells. Preconditioning can include any substance that promotes T cell function and / or survival. In some embodiments, preconditioning includes administering to the subject chemotherapy, cytokines, proteins, small molecules, or any combination thereof. In some embodiments, preconditioning includes administering an interleukin. In some embodiments, preconditioning includes administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, preconditioning includes administering cyclophosphamide, fludarabine, or both. In some embodiments, preconditioning includes administering vitamin C, an AKT inhibitor, ATRA (vesanoid, tretinoin), rapamycin, or any combination thereof.

[0163] III.B. Methods for Engineering Antigen-Targeted Cells Certain aspects of the present disclosure relate to methods for engineering antigen-targeted cells. In some embodiments, the antigen is gp100 antigen. In some embodiments, the method includes transducing a cell with a nucleic acid molecule disclosed herein or a vector disclosed herein. The cell can be any cell described herein. In some embodiments, the cell is a T cell described herein. In some embodiments, the cell is a cell that has been modified to express CD3 as described herein. In some embodiments, the cell, e.g., a T cell, is obtained from a subject in need of T cell therapy. In some embodiments, the cell is obtained from a donor other than the subject in need of T cell therapy. In some embodiments, the cell is a T cell or a natural killer cell.

[0164] III. Methods for Enriching Target Populations of CT Cells Certain aspects of the present disclosure relate to methods for enriching a target population of T cells obtained from a human subject. In some embodiments, the method comprises contacting the T cells with an HLA class I molecule as disclosed herein. In some embodiments, the method comprises contacting the T cells with an APC as disclosed herein. In some embodiments, after contacting, the enriched population of T cells comprises a greater number of T cells capable of binding to the HLA class I molecule relative to the number of T cells capable of binding to the HLA class I molecule before contacting.

[0165] In some embodiments, the method comprises contacting T cells in vitro with a peptide comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the method comprises contacting T cells in vitro with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, after contacting, the enriched population of T cells comprises a greater number of T cells capable of binding to HLA class I molecules relative to the number of T cells capable of binding to HLA class I molecules before contacting.

[0166] Some aspects of the present disclosure relate to methods for selecting T cells capable of targeting tumor cells. In some embodiments, the methods comprise contacting a population of in vitro isolated T cells with a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the T cells are obtained from a human subject.

[0167] The T cells obtained from the human subject can be any T cells disclosed herein. In some embodiments, the T cells obtained from the human subject are tumor-infiltrating lymphocytes (TILs).

[0168] In some embodiments, the method further comprises administering the enriched T cells to a human subject, in some embodiments, the subject is preconditioned as described herein prior to receiving the T cells.

[0169] All of the various aspects, embodiments, and options described herein may be combined in any and all variations.

[0170] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0171] Having generally described the present disclosure, a further understanding can be obtained by reference to the examples provided herein, which are for illustrative purposes only and are not intended to be limiting. [Example]

[0172] Example 1 TILs were isolated from patients with metastatic melanoma and then polyclonally expanded in vitro to examine their gp100 antigen specificity for the HLA-C*06:02 allele. A combination of structure-based and functional assays using peptide / HLA (pHLA) multimers was used to measure Ag-specific T cell responses.

[0173] Because pHLA multimer production requires the use of peptides with known, precise sequences, it is not straightforward or practical to perform high-throughput screening of new epitope peptides using a pHLA multimer-based strategy. In addition to structural analyses using pHLA multimers, functional analyses can be applied to determine the antigen specificity of T cells. We performed functional assays using artificial antigen-presenting cells (APCs), which can incorporate and process longer peptides and present epitope peptides via class I molecules as stimulatory cells. C*06:02-artificial APCs were pulsed with overlapping peptides to cover the entire gp100 protein (Table 5) and used as stimulators in cytokine ELISPOT assays. Following one controlled stimulation with C*06:02-artificial APCs pulsed with overlapping peptides derived from gp100, C*06:02 +Melanoma TILs expressed a shared sequence in IFN-γ ELISPOT assays. 186 VTVYHRRGSRSYVPL 200 A positive reaction was observed for two adjacent peptides containing the C*06:02 molecule (Figure 1). Using a series of mutant deletion peptides, we identified the minimal required peptide epitope presented by the C*06:02 molecule. 190 HRRGSRSYV 198 We determined the CD8 + HLA-C*06:02 / gp100 accounts for 1.2% of T cells 190-198 T cells were identified (Fig. 2). Multimer-positive T cells secreted detectable IFN-γ in an HLA-restricted peptide-specific manner by ELISPOT analysis (Fig. 3). [Table 5-1] [Table 5-2] [Table 5-3]

[0174] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 4, SEQ ID NOs: 1 and 2). The antigen specificity and functional reactivity of the cloned TCR were verified by multimer staining and ELISPOT assay of TCR-reconstituted T cells. When reconstituted with primary T cells, C*06:02 / gp100 190-198 TCR-transduced T cells were successfully stained with cognate multimers of gp100 (Fig. 5), indicated by surface C*06:02 molecules. 190-198 The cells reacted strongly with the peptide (Figure 6). Importantly, these cells were able to recognize C*06:02-matched and peptide-unpulsed tumor cells that naturally express the gp100 gene (Figure 7). Both Malme-3M and SK-MEL-28 melanoma cell lines are negative for C*06:02 but endogenously express the gp100 gene. When the C*06:02 molecule was ectopically expressed, both melanoma cell lines showed a strong C*06:02 / gp100 response.190-198 Furthermore, A375 melanoma cells, which lack endogenous expression of gp100, were successfully recognized by TCR-transduced T cells only when transduced with the precursor gp100 gene. 190-198 These results suggest that C*06:02 / gp100 may be a potential therapeutic option for TCR-transduced T cells. 190-198 TCR-transduced T cells have sufficient affinity to recognize tumor cells, and the cloned C*06:02 / gp100 190-198 This clearly demonstrates that TCRs are tumor reactive.

[0175] gp100 is one of the most promising and widely studied shared antigens for bispecific T cell engager (BiTE) therapy. Clinical trials targeting gp100 are underway in patients with metastatic uveal melanoma, using IMCgp100, a bispecific biologic consisting of a soluble TCR recognizing the gp100 antigen fused to an scFV anti-CD3, which redirects T cell lysis of melanoma cells expressing gp100 in the context of HLA-A*02:01 molecules. The use of the newly cloned tumor-reactive C*06:02-restricted gp100 TCR gene may broaden the applicability of gp100-targeted BiTE therapy beyond HLA-A*02:01-positive cancer patients.

[0176] method

[0177] Cell samples

[0178] Peripheral blood samples were obtained from healthy donors after institutional review board approval. Mononuclear cells were obtained by density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). K562 is an erythroleukemia cell line deficient in HLA expression. Jurkat 76 is a T-cell leukemia cell line lacking TCR and CD8 expression. Malme-3M cell line was grown in IMDM supplemented with 20% FBS and 50 μg / ml gentamicin. SK-MEL-28 and A375 cell lines were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen). K562, T2, and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg / ml gentamicin. TILs isolated from metastatic melanoma patients were expanded in vitro.

[0179] peptide

[0180] The synthetic peptides were dissolved in DMSO at 50 μg / ml. The peptides used were the whole gp100 protein (Table 1) and the C*06:02 restricted gp100. 190-198 (HRRGSRSYV; SEQ ID NO: 13), gp100 190-197 (HRRGSRSY; SEQ ID NO: 194), and HIV nef 120-128 (YFPDWQNYT; SEQ ID NO: 63) peptide. 190-197 and HIV nef 120-128 The peptide was used as a negative control.

[0181] gene

[0182] The HLA-C*06:02 gene was fused to a truncated form of the human nerve growth factor receptor (ΔNGFR) via an internal ribosome entry site. ΔNGFR-transduced cells were isolated using an anti-NGFR monoclonal antibody (mAb). The full-length gp100 gene was purchased from Dharmacon (Lafayette, CO). The TCR gene was cloned by 5'-rapid amplification of cDNA ends (RACE) PCR using the SMARTer RACE cDNA Amplification Kit (Takara Bio). The 5'-RACE PCR product was cloned into a retroviral vector and sequenced. All genes were cloned into the pMX retroviral vector and transduced using the 293GPG cell-based retroviral system.

[0183] Transformants (transfectants)

[0184] Jurkat 76 / CD8 cells were transduced with individual TCRα and TCRβ genes. TCR transfectants derived from Jurkat 76 / CD8 were purified (purity >95%) using CD3 Microbeads (Miltenyi Biotec). K562-based artificial APCs expressing various HLA class I genes individually as single HLA alleles in combination with CD80 and CD83 have been previously reported (Butler and Hirano, Immunol. Rev. 257:191-209 (2014); Hirano et al., Clin. Cancer Res. 12:2967-75 (2006)). Retroviral supernatant from PG13 was used to transduce TCR genes into human primary T cells. TransIT293 (Mirus Bio) was used to transfect TCR genes into the 293GPG cell line. Gp100 - A375 cells were retrovirally transduced with the full-length gp100 gene to generate A375 / gp100. Expression of the transduced gp100 was assessed by flow cytometry after staining with anti-gp100 mAb (clone 7E3; LifeSpanBiosciences). HLA-C*06:02 -Malme-3M and SK-MEL-28 cells were retrovirally transduced with HLA-C*06:02 to generate Malme-3M / C*06:02 and SK-MEL-28 / C*06:02 cells. The HLA-C*06:02 gene was tagged with the ΔNGFR gene as described above, resulting in the ΔNGFR + The cells were purified (>95% pure) and used in subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0185] Flow cytometry and cell sorting

[0186] Cell surface molecules were visualized using a PC5-conjugated anti-CD8 mAb (clone B9.11; Beckman Cells were stained with FITC-conjugated anti-NGFR (clone ME20.4; Biolegend), and APC / Cy7-conjugated anti-CD3 (clone UCHT1; Biolegend). Dead cells were identified with the LIVE / DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies). For intracellular staining, cells were fixed and permeabilized using the Cytofix / Cytoperm kit (BD Biosciences). Stained cells were analyzed by flow cytometry (BD Biosciences), and data analysis was performed using FlowJo (Tree Star). Cell sorting was performed using a FACS Aria II (BD Biosciences).

[0187] Cytokine ELISPOT analysis

[0188] IFN-γ ELISPOT assays were performed. PVDF plates (Millipore, Bedford, MA) were coated with a capture mAb (1-D1K; MABTECH, Mariemont, OH), and T cells were cultured at 2 × 10 per well in the presence or absence of peptide. 4The plates were incubated with target cells at 37°C for 20–24 hours. Plates were then washed and incubated with biotin-conjugated detection mAb (7-B6-1; MABTECH). IFN-γ spots were then developed by adding HRP-conjugated SA (Jackson ImmunoResearch). The reaction was stopped by thorough rinsing with cold tap water. ELISPOT plates were scanned and counted using an ImmunoSpot plate reader and ImmunoSpot version 5.0 software (Cellular Technology Limited, Shaker Heights, OH).

[0189] CD8 in an HLA-restricted peptide-specific manner + Expansion of TILs

[0190] CD8 + TILs are CD8 + T cells were purified by negative magnetic selection using a T Cell Isolation Kit (Miltenyi Biotec). C*06:02-artificial APCs were pulsed with 10 μg / mL of gp100 peptide for 6 hours. Artificial APCs were then irradiated at 200 Gy, washed, and added to TILs at an effector-to-target (E:T) ratio of 20:1. Starting the following day, 10 IU / ml IL-2 (Novartis), 10 ng / ml IL-15 (Peprotech), and 30 ng / ml IL-21 (Peprotech) were added to the cultures every 3 days.

[0191] Primary CD8 transduced with cloned TCR + T cell proliferation

[0192] CD8 +T cells were purified by negative magnetic selection using a Pan T Cell Isolation Kit (Miltenyi Biotec). Purified T cells were stimulated with 200 Gy-irradiated artificial APC / mOKT3 at an E:T ratio of 20:1. Starting the following day, activated T cells were retrovirally transduced with the cloned TCR gene by centrifugation at 1,000 g for 1 hour at 32°C for three consecutive days. The following day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR-transduced T cells. The culture medium was replenished every 2–3 days.

[0193] Human cell-based production of pHLA multimers

[0194] The affinity-matured HLA class I gene contains a Glu(E) residue instead of a Gln(Q) residue at position 115 in the α2 domain and a mouse K(III) residue instead of the α3 domain of HLA class I. b By fusing the extracellular domain of the affinity-matured HLA class I gene with a Gly-SER(GS) flexible linker followed by a 6xHis tag, we were able to generate soluble HLA class I antibodies. Q115E -K b HEK293T cells were transfected with various soluble HLA class I antigens along with the β2m gene using a 293GPG cell-based retroviral system. Q115E -K b The genes were individually transduced with soluble affinity maturation class I Q115E -K b Stable HEK293T cells ectopically expressing HLA class I (HLA class II) were grown to confluence and then their medium was replaced. After 48 hours, the conditioned medium was harvested and either used immediately or frozen until further use. Soluble HLA class II (HLA class I) proteins produced by HEK293T transfectants were identified. IQ115E -K b The supernatant containing peptide was mixed with 100-1000 μg / ml of the desired class I restricted peptide overnight at 37°C for in vitro peptide exchange. Q115E -K bdimerization using anti-His mAb (clone AD1.1.10; Abcam) conjugated to a fluorescent dye such as phycoerythrin (PE) at a molar ratio of 2:1 for 2 h at room temperature or overnight at 4 °C. IQ115E -K b The concentration of the molecules was measured by specific ELISA using an anti-pan class I mAb (clone W6 / 32, in-house) and an anti-His tag biotinylated mAb (clone AD1.1.10, R&D systems) as capture and detection Abs, respectively.

[0195] pHLA multimer staining

[0196] T cells (1 x 10 5 ) were incubated in the presence of 50 nM dasatinib (LC Laboratories) for 30 min at 37°C. Next, cells were washed and incubated with 5-10 μg / ml of multimer for 30 min at room temperature. AffiniPure Fab fragment goat anti-mouse IgG1 conjugated to R-phycoerythrin (Jackson ImmunoResearch Laboratories) was added for 15 min at 4°C. Cells were then washed three times and co-stained with anti-CD8 mAb for 15 min at 4°C. Dead cells were finally identified using LIVE / DEAD Fixable Aqua Dead Cell Immunoprecipitation (DEAD). Identification was performed using a Stain kit.

[0197] statistical analysis

[0198] Statistical analysis was performed using GraphPad Prism 5.0e. To determine whether two groups were significantly different for a particular variable, analysis was performed using Welch's t-test (two-tailed). A P value of less than 0.05 was considered significant. In an embodiment of the present invention, for example, the following items are provided: (Item 1) (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits expression of an endogenous TCR; A nucleic acid molecule wherein the anti-gp100 TCR cross-competes with a reference TCR comprising an alpha chain and a beta chain for binding to human gp100, wherein the alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and the beta chain comprises the amino acid sequence set forth in SEQ ID NO: 2. (Item 2) (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR"); and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits expression of an endogenous TCR. and the second nucleotide sequence A nucleic acid molecule wherein the anti-gp100 TCR binds to the same epitope of human gp100 as or an overlapping epitope with a reference TCR comprising an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2. (Item 3) 3. The nucleic acid molecule of item 1 or 2, wherein the anti-gp100 TCR binds to an epitope of gp100 consisting of the amino acid sequence set forth in SEQ ID NO: 13. (Item 4) 4. The nucleic acid molecule of item 2 or 3, wherein the epitope is complexed with an HLA class I molecule. (Item 5) 5. The nucleic acid molecule of item 4, wherein the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. (Item 6) 5. The nucleic acid molecule of item 4, wherein the HLA class I molecule is an HLA-C*06 allele. (Item 7) 7. The nucleic acid molecule of any one of items 4 to 6, wherein the HLA class I molecule is selected from the HLA-C*06:015 allele, the HLA-C*06:02 allele, the HLA-C*06:03 allele, the HLA-C*06:04 allele, the HLA-C*06:05 allele, the HLA-C*06:06 allele, the HLA-C*06:07 allele, and the HLA-C*06:086 allele. (Item 8) 8. The nucleic acid molecule of any one of items 4 to 7, wherein the HLA class I molecule is an HLA-C*06:02 allele. (Item 9) the anti-gp100 TCR comprises an alpha chain and a beta chain; the alpha chain comprises a variable region comprising alpha chain CDR1, alpha chain CDR2, and alpha chain CDR3; the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; 9. The nucleic acid molecule according to any one of items 1 to 8, wherein the alpha chain CDR3 comprises the amino acid sequence shown in SEQ ID NO:7. (Item 10) 10. The nucleic acid molecule of item 9, wherein the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10. (Item 11) the anti-gp100 TCR comprises an alpha chain and a beta chain, the alpha chain comprising a variable region comprising alpha chain CDR1, alpha chain CDR2, and alpha chain CDR3; the beta chain comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; 9. The nucleic acid molecule of any one of items 1 to 8, wherein the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO: 10. (Item 12) 12. The nucleic acid molecule of item 11, wherein the alpha chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:7. (Item 13) 13. The nucleic acid molecule of any one of items 9 to 12, wherein the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:5. (Item 14) 14. The nucleic acid molecule of any one of items 9 to 13, wherein the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:8. (Item 15) 15. The nucleic acid molecule of any one of items 9 to 14, wherein the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:6. (Item 16) 16. The nucleic acid molecule of any one of items 9 to 15, wherein the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:9. (Item 17) 17. The nucleic acid molecule of any one of items 9 to 16, wherein the alpha chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence shown in SEQ ID NO: 1. (Item 18) 18. The nucleic acid molecule of any one of items 9 to 17, wherein the beta chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of a variable domain present in the amino acid sequence shown in SEQ ID NO:2. (Item 19) 19. The nucleic acid molecule of any one of items 9 to 18, wherein the alpha chain of the anti-gp100 TCR further comprises a constant region, wherein the constant region is different from the endogenous constant region of the alpha chain. (Item 20) 20. The nucleic acid molecule of any one of items 9 to 19, wherein the alpha chain of the anti-gp100 TCR comprises a constant region, and the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO: 1. (Item 21) 21. The nucleic acid molecule of any one of items 19 or 20, wherein the alpha chain constant region comprises an amino acid sequence that contains at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the alpha chain amino acid sequence set forth in SEQ ID NO:1. (Item 22) 22. The nucleic acid molecule of any one of items 9 to 21, wherein the beta chain of the anti-gp100 TCR further comprises a constant region, wherein the constant region is different from the endogenous constant region of the beta chain. (Item 23) 23. The nucleic acid molecule of any one of Items 9 to 22, wherein the beta chain of the anti-gp100 TCR further comprises a constant region, and the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a constant region present in the amino acid sequence set forth in SEQ ID NO:2. (Item 24) 24. The nucleic acid molecule of any one of items 22 or 23, wherein the beta chain constant region comprises an amino acid sequence containing at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid chain sequence set forth in SEQ ID NO:2. (Item 25) 25. The nucleic acid molecule of any one of items 9 to 24, wherein the alpha chain of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO: 1. (Item 26) 26. The nucleic acid molecule of any one of items 9 to 25, wherein the beta chain of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:2. (Item 27) 27. The nucleic acid molecule of any one of items 1 to 26, wherein the second nucleotide sequence is one or more siRNAs that reduce expression of an endogenous TCR. (Item 28) 28. The nucleic acid molecule of item 27, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding the constant region of the endogenous TCR. (Item 29) 29. The nucleic acid molecule of any one of items 27 or 28, wherein the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 53 to 56. (Item 30) 30. The nucleic acid molecule of any one of items 1 to 29, wherein the second nucleotide sequence encodes Cas9. (Item 31) 31. The nucleic acid molecule of any one of items 1 to 30, wherein the anti-gp100 TCR comprises an alpha chain constant region, a beta chain constant region, or both; and wherein the alpha chain constant region, the beta chain constant region, or both comprise an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 substitutions within the target sequence relative to the corresponding amino acid sequence of an endogenous TCR. (Item 32) 32. A vector comprising the nucleic acid molecule according to any one of items 1 to 31. (Item 33) 33. The vector of item 32, which is a viral vector, a mammalian vector, or a bacterial vector. (Item 34) 34. The vector according to item 32 or 33, which is a retroviral vector. (Item 35) 35. The vector according to any one of Items 32 to 34, wherein the vector is selected from the group consisting of an adenovirus vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, a hybrid vector, and an adeno-associated virus (AAV) vector. (Item 36) 36. The vector according to any one of items 32 to 35, which is a lentivirus. (Item 37) A T cell receptor (TCR) or an antigen-binding portion thereof, comprising the alpha chain variable domain of the anti-gp100 TCR according to any one of items 9 to 31 and the beta chain variable domain of the anti-gp100 TCR according to any one of items 9 to 31. (Item 38) a recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR") and cross-competes with a reference TCR for binding to human gp100; said reference TCR comprising an alpha chain and a beta chain, said alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and said beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2; said anti-gp100 TCR comprising an alpha chain and a beta chain, said alpha chain comprising a constant region and said beta chain comprising a constant region; (i) the alpha chain constant region is present in the amino acid sequence shown in SEQ ID NO: 1; At least one, at least two, at least three, at least four, or comprises an amino acid sequence with at least five amino acid substitutions, or (ii) the beta chain constant region corresponds to the constant region present in the amino acid sequence of SEQ ID NO: 2; and a recombinant T cell receptor (TCR) or antigen-binding portion thereof, comprising an amino acid sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions. (Item 39) a recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds to human gp100 (an "anti-gp100 TCR"), which binds to the same epitope on human gp100 as the reference TCR or an overlapping epitope; the reference TCR comprises an alpha chain and a beta chain, the alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and the beta chain comprising the amino acid sequence set forth in SEQ ID NO: 2; said anti-gp100 TCR comprising an alpha chain and a beta chain, said alpha chain comprising a constant region and said beta chain comprising a constant region; (i) the alpha chain constant region is a constant region present in the amino acid sequence set forth in SEQ ID NO: 1; comprises an amino acid sequence that has at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to a common region; or (ii) the recombinant T cell receptor (TCR) or an antigen-binding portion thereof, wherein the beta chain constant region comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence set forth in SEQ ID NO:2. (Item 40) 40. The anti-gp100 TCR of any one of items 38 or 39, which binds to an epitope of gp100 consisting of the amino acid sequence shown in SEQ ID NO: 13. (Item 41) 41. The anti-gp100 TCR of any one of items 39 or 40, wherein the epitope is complexed with an HLA class I molecule. (Item 42) 42. The anti-gp100 TCR of item 41, wherein the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. (Item 43) 43. The anti-gp100 TCR of any one of items 41 or 42, wherein the HLA class I molecule is the HLA-C*06 allele. (Item 44) 44. The anti-gp100 TCR according to any one of items 41 to 43, wherein the HLA class I molecule is selected from the HLA-C*06:01 allele, the HLA-C*06:02 allele, the HLA-C*06:03 allele, the HLA-C*06:04 allele, the HLA-C*06:05 allele, the HLA-C*06:06 allele, the HLA-C*06:07 allele, and the HLA-C*06:08 allele. (Item 45) 45. The anti-gp100 TCR of any one of items 41 or 44, wherein the HLA class I molecule is the HLA-C*06:02 allele. (Item 46) the alpha chain of the anti-gp100 TCR comprises a variable domain comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; the beta chain of the anti-gp100 TCR comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; 46. ​​The anti-gp100 TCR according to any one of items 38 to 45, wherein the alpha chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:7. (Item 47) The beta chain CDR3 of the anti-gp100 TCR is 47. The anti-gp100 TCR according to item 46, comprising the amino acid sequence (Item 48) the alpha chain of the anti-gp100 TCR comprises a variable domain comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3; the beta chain of the anti-gp100 TCR comprises a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; 46. ​​The anti-gp100 TCR according to any one of items 38 to 45, wherein the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:10. (Item 49) 49. The anti-gp100 TCR of item 48, wherein the alpha chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:7. (Item 50) 50. The anti-gp100 TCR of item 49, wherein the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:5. (Item 51) 51. The anti-gp100 TCR according to any one of items 46 to 50, wherein the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:8. (Item 52) 52. The anti-gp100 TCR according to any one of items 46 to 51, wherein the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:6. (Item 53) 53. The anti-gp100 TCR according to any one of items 46 to 52, wherein the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:9. (Item 54) 54. The anti-gp100 TCR according to any one of items 46 to 53, wherein the alpha chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence shown in SEQ ID NO: 1. (Item 55) 55. The anti-gp100 TCR according to any one of items 46 to 54, wherein the beta chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of a variable domain present in the amino acid sequence shown in SEQ ID NO:2. (Item 56) 56. The anti-gp100 TCR of any one of Items 38 to 55, wherein the alpha chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of the constant region present in the amino acid sequence shown in SEQ ID NO: 1. (Item 57) 57. The anti-gp100 TCR according to any one of Items 38 to 56, wherein the beta chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of the constant region present in the amino acid sequence set forth in SEQ ID NO:2. (Item 58) 58. The anti-gp100 TCR according to any one of items 38 to 57, wherein the alpha chain of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:1. (Item 59) 59. The anti-gp100 TCR according to any one of items 38 to 58, wherein the beta chain of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:2. (Item 60) 59. A bispecific TCR comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises the TCR or an antigen-binding portion thereof according to Aspect 37, or the TCR or an antigen-binding portion thereof according to any one of Aspects 38 to 59. (Item 61) 61. The bispecific TCR of item 60, wherein the first antigen-binding domain comprises a single-chain variable fragment ("scFv"). (Item 62) 62. The bispecific TCR of any one of items 60 or 61, wherein the second antigen-binding domain specifically binds to a protein expressed on the surface of a T cell. (Item 63) 63. The bispecific TCR according to any one of items 60 to 62, wherein the second antigen-binding domain specifically binds to CD3. (Item 64) 64. The bispecific TCR according to any one of items 60 to 63, wherein the second antigen-binding domain comprises an scFv. (Item 65) 65. The bispecific TCR according to any one of items 60 to 64, wherein the first antigen-binding domain and the second antigen-binding domain are linked or bound by a covalent bond. (Item 66) 66. The bispecific TCR according to any one of items 60 to 65, wherein the first antigen-binding domain and the second antigen-binding domain are linked by a peptide bond. (Item 67) A cell comprising the nucleic acid molecule of any one of Items 1 to 31, the vector of any one of Items 32 to 36, the TCR of Item 37, the recombinant TCR of any one of Items 38 to 59, or the bispecific TCR of any one of Items 60 to 66. (Item 68) The cell of paragraph 67, further expressing CD3. (Item 69) 69. The cell of item 67 or 68, selected from the group consisting of a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or an ILC cell. (Item 70) 70. A method of treating cancer in a subject in need thereof, comprising administering to the subject the cells according to any one of items 67 to 69. (Item 71) Item 70. The method according to item 70, wherein the cancer is selected from the group consisting of melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, cervical cancer, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, and cancer of the penis. the cancer is selected from the group consisting of cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of the foregoing cancers. (Item 72) Item 70 or 71, wherein the cancer is refractory or recurrent. The method described in paragraph . (Item 73) 73. The method of any one of items 70 to 72, wherein the cancer is locally advanced. (Item 74) 74. The method according to any one of items 70 to 73, wherein the cancer is advanced. (Item 75) 75. The method of any one of items 70 to 74, wherein the cancer is metastatic. (Item 76) 76. The method of any one of items 70 to 75, wherein the cells are obtained from the subject. (Item 77) 77. The method of any one of items 70 to 76, wherein the cells are obtained from a donor other than the subject. (Item 78) 78. The method of any one of items 70 to 77, wherein the subject is preconditioned prior to administration of the cells. (Item 79) 79. The method of any one of items 68-78, wherein the preconditioning comprises administering to the subject chemotherapy, cytokines, proteins, small molecules, or any combination thereof. (Item 80) 80. The method of item 78 or 79, wherein the preconditioning comprises administering an interleukin. (Item 81) 81. The method of any one of items 78 to 80, wherein the preconditioning comprises administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. (Item 82) 82. The method of any one of items 78-81, wherein the preconditioning comprises administering a preconditioning agent selected from the group consisting of cyclophosphamide, fludarabine, vitamin C, an AKT inhibitor, ATRA, rapamycin, or any combination thereof. (Item 83) 83. The method of any one of items 78 to 82, wherein the preconditioning comprises administering cyclophosphamide, fludarabine, or both. (Item 84) 37. A method of engineering antigen-targeted cells, comprising transducing cells collected from a subject in need of T cell therapy with the nucleic acid molecule of any one of items 1 to 31 or the vector of any one of items 32 to 36. (Item 85) 85. The method of claim 84, wherein the antigen-targeted cells further express CD3. (Item 86) 86. The method of any one of paragraphs 84 or 85, wherein the cell is a T cell or a natural killer (NK) cell. (Item 87) An HLA class I molecule complexed with a peptide, wherein the HLA class I molecule comprises an α1 domain, an α2 domain, an α3 domain, and a β2m, and the peptide consists of the amino acid sequence shown in SEQ ID NO: 14. (Item 88) 88. The HLA class I molecule of item 87, which is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. (Item 89) 89. The HLA class I molecule of any one of items 87 or 88, which is HLA-C. (Item 90) 90. The HLA class I molecule of any one of items 87 to 89, which is an HLA-C*06 allele. (Item 91) 91. The HLA class I molecule of any one of items 87 to 90, wherein the HLA class I molecule is selected from the HLA-C*06:01 allele, the HLA-C*06:02 allele, the HLA-C*06:03 allele, the HLA-C*06:04 allele, the HLA-C*06:05 allele, and the HLA-C*06:06 allele. (Item 92) 92. The HLA class I molecule according to any one of items 87 to 91, wherein the HLA class I molecule is an HLA-C*06:02 allele. (Item 93) 93. The HLA class I molecule according to any one of items 87 to 92, wherein the HLA class I molecule is an HLA-C*03:03 allele. (Item 94) 94. The HLA class I molecule of any one of items 87 to 93, which is a monomer. (Item 95) 94. The HLA class I molecule of any one of items 87 to 93, which is a dimer. (Item 96) 94. The HLA class I molecule of any one of items 87 to 93, which is a trimer. (Item 97) 94. The HLA class I molecule of any one of items 87 to 93, which is a tetramer. (Item 98) 94. The HLA class I molecule of any one of items 87 to 93, which is a pentamer. (Item 99) An antigen-presenting cell (APC) comprising the HLA class I molecule according to any one of items 87 to 98. (Item 100) Item 99. The APC of item 99, wherein the HLA class I molecule is expressed on the surface of the APC. (Item 101) 10. A method for enriching a target population of T cells obtained from a human subject, the method comprising contacting the T cells with an HLA class I molecule according to any one of items 87 to 98 or an APC according to item 99 or 100, wherein after said contacting, the enriched T cell population comprises a greater number of T cells capable of binding to HLA class I molecules relative to the number of T cells capable of binding to HLA class I molecules before said contacting. (Item 102) 1. A method for enriching a target population of T cells obtained from a human subject, comprising contacting the T cells in vitro with a peptide, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 13, and wherein following said contacting, the enriched T cell population comprises a greater number of T cells capable of targeting tumor cells relative to the number of T cells capable of targeting tumor cells prior to said contacting. (Item 103) 103. The method of any one of paragraphs 101 or 102, wherein the T cells obtained from the human subject are tumor-infiltrating lymphocytes (TILs). (Item 104) 103. A method of treating a tumor in a subject in need thereof, comprising administering to said subject the enriched T cells according to any one of items 101-102. (Item 105) A method for enhancing cytotoxic T cell-mediated targeting of cancer cells in a subject suffering from cancer, comprising administering to the subject a peptide having the amino acid sequence set forth in SEQ ID NO: 13. (Item 106) A cancer vaccine comprising a peptide having the amino acid sequence shown in SEQ ID NO:13. (Item 107) A method for selecting T cells capable of targeting tumor cells, comprising contacting a population of isolated T cells in vitro with a peptide consisting of the amino acid sequence set forth in SEQ ID NO:11. (Item 108) 108. The method of claim 107, wherein the T cells are tumor-infiltrating lymphocytes (TILs).

Claims

1. (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or antigen-binding portion thereof ("anti-gp100 TCR") that specifically binds to an HLA class I molecule complexed with a peptide portion of human gp100, wherein the anti-gp100 TCR comprises an alpha chain and a beta chain, the alpha chain comprising a variable region comprising an alpha chain CDR1, an alpha chain CDR2, and an alpha chain CDR3, and the beta chain comprising a variable domain comprising a beta chain CDR1, a beta chain CDR2, and a beta chain CDR3; (a) the beta chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10; (b) the beta chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:9; (c) the beta chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:6; (d) the alpha chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:7; (e) the alpha chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:8; and (f) the alpha chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:5; the first nucleotide sequence; (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits expression of an endogenous TCR; and A nucleic acid molecule comprising:

2. The nucleic acid molecule of claim 1, wherein the peptide portion of human gp100 consists of the amino acid sequence shown in SEQ ID NO:

13.

3. 3. The nucleic acid molecule of claim 2, wherein the HLA class I molecule is selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G alleles.

4. 4. The nucleic acid molecule of claim 3, wherein the HLA class I molecule is an HLA-C*06 allele optionally selected from an HLA-C*06:01 allele, an HLA-C*06:02 allele, an HLA-C*06:03 allele, an HLA-C*06:04 allele, an HLA-C*06:05 allele, an HLA-C*06:06 allele, an HLA-C*06:07 allele, and an HLA-C*06:08 allele.

5. (i) the alpha chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO: 1; (ii) the beta chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence set forth in SEQ ID NO:2; or (iii) both (i) and (ii); A nucleic acid molecule according to any one of claims 1 to 4.

6. The nucleic acid molecule of any one of claims 1 to 5, wherein the alpha chain of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:

1.

7. The nucleic acid molecule of any one of claims 1 to 6, wherein the beta chain of the anti-gp100 TCR comprises the amino acid sequence shown in SEQ ID NO:

2.

8. 8. The nucleic acid molecule of any one of claims 1 to 7, wherein the alpha chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 1 and the beta chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:

2.

9. the second nucleotide sequence is (i) one or more siRNAs that reduce expression of an endogenous TCR, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding the constant region of the endogenous TCR; (ii) encodes Cas9; or (iii) both (i) and (ii); A nucleic acid molecule according to any one of claims 1 to 8.

10. The nucleic acid molecule of claim 9, wherein the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 53-56.

11. A vector comprising the nucleic acid molecule of any one of claims 1 to 10.

12. A cell comprising the nucleic acid molecule of any one of claims 1 to 10 or the vector of claim 11, wherein the cell is optionally selected from a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or an ILC cell.

13. A composition for treating cancer in a subject in need thereof, comprising the cells of claim 12.

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