T cell receptors and methods of use thereof

Abstract

The present disclosure relates to recombinant T cell receptors capable of binding to the gp100 epitope and nucleic acid molecules encoding the recombinant T cell receptors. In some embodiments, the nucleic acid molecule further comprises a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCR. Other aspects of the present disclosure relate to vectors comprising the nucleic acid molecule and cells comprising the recombinant TCR, the nucleic acid molecule or the vector. Other aspects of the present disclosure relate to methods of using the recombinant TCR, the nucleic acid molecule, the vector and the cell. In some embodiments, the method comprises treating cancer in a subject in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 62 / 813,645, filed March 4, 2019, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically via EFS-WEB

[0004] The contents of the electronically submitted Sequence Listing (Name: 4285_004PC01_Seqlisting_ST25.txt, Size: 57,727 bytes; and Creation Date: March 3, 2020) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure provides recombinant T cell receptors ("TCRs") that specifically bind to human gplOO and uses thereof. Background of the Invention

[0007] Immunotherapy has emerged as a key tool in the fight against a variety of diseases, including cancer. T cell therapy is at the forefront of immunotherapeutic development, and adoptive transfer of anti-tumor T cells has been shown to induce clinical responses in cancer patients. While many T cell therapies target mutated tumor antigens, the vast majority of neoantigens are not shared but unique to each patient.

[0008] The number of potential non-mutated antigens exceeds the number of mutated antigens by several orders of magnitude. Elucidating T cell epitopes derived from commonly shared antigens could facilitate the robust development of effective and safe adoptive T cell therapies readily accessible to larger groups of cancer patients. However, the sheer number of non-mutated antigens and the high polymorphism of HLA genes may have hindered comprehensive analysis of the specificity of anti-tumor T cell responses to non-mutated antigens.

[0009] The present disclosure provides novel epitopes of the non-mutated antigen gp100 and TCRs that can specifically bind to these epitopes. These novel epitopes are associated with specific HLA alleles. The use of these tumor-reactive HLA-restricted gp100 TCRs supports the broadening applicability of anti-gp100 TCR gene therapy, particularly in immuno-oncology. Summary of the Invention

[0010] Certain aspects of the present disclosure relate to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or an antigen-binding portion thereof that specifically binds 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, wherein the anti-gp100 TCR cross-competes with a reference TCR for binding to human gp100, the reference TCR comprising an α chain and a β chain, and wherein the α chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and the β chain comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0011] Certain aspects of the present disclosure relate to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T cell receptor (TCR) or an antigen-binding portion thereof that specifically binds 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, wherein the anti-gp100 TCR binds to the same epitope or an overlapping epitope of human gp100 as a reference TCR, the reference TCR comprising an α chain and a β chain, wherein the α chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and the β chain comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0012] 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 complexed with an HLA class I molecule.

[0013] 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-B*40 allele. In some embodiments, the HLA class I molecule is selected from the group consisting of an HLA-B*40:01 allele, an HLA-B*40:02 allele, an HLA-B*40:03 allele, an HLA-B*40:04 allele, an HLA-B*40:05 allele, and an HLA-B*40:06 allele. In some embodiments, the HLA class I molecule is an HLA-B*40:01 allele.

[0014] In some embodiments, the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a variable region comprising an α chain CDR1, an α chain CDR2, and an α chain CDR3; and wherein the β chain comprises a variable domain comprising a β chain CDR1, a β chain CDR2, and a β chain CDR3; wherein the α chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the β chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0015] In some embodiments, the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a variable region comprising an α chain CDR1, an α chain CDR2, and an α chain CDR3; and wherein the β chain comprises a variable domain comprising a β chain CDR1, a β chain CDR2, and a β chain CDR3; wherein the β chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the α chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0016] In some embodiments, the α chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the β chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the α chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the β chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0017] In some embodiments, the α chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain set forth in SEQ ID NO: 1. In some embodiments, the β chain variable domain of the anti-gp100 TCR comprises the amino acid sequence of the variable domain set forth in SEQ ID NO: 2.

[0018] In some embodiments, the α chain of the anti-gp100 TCR further comprises a constant region, wherein the constant region is different from the endogenous constant region of the α chain. In some embodiments, the α chain of the anti-gp100 TCR further comprises a constant region, wherein the α 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: 1. In some embodiments, the α chain constant region comprises an amino acid sequence comprising 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 β chain of the anti-gp100 TCR further comprises a constant region, wherein the constant region is different from the endogenous constant region of the β chain.

[0019] 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 comprising 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 set forth in SEQ ID NO: 1.

[0020] 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 TCR.

[0021] In some embodiments, the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a 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.

[0022] In some embodiments, the second nucleotide sequence encodes Cas9.

[0023] In some embodiments, the anti-gp100 TCR comprises an α chain constant region, a β chain constant region, or both; and wherein the α chain constant region, the β 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 the endogenous TCR.

[0024] Certain aspects of the present disclosure relate to a vector comprising a nucleic acid molecule 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.

[0025] Certain aspects of the present disclosure relate to a T cell receptor (TCR), or an antigen binding portion thereof, comprising an alpha chain variable domain of an anti-gp100 TCR disclosed herein and a beta chain variable domain of an anti-gp100 TCR disclosed herein. In some embodiments, the recombinant T cell receptor (TCR) or antigen-binding portion thereof that specifically binds human gp100 ("anti-gp100 TCR") cross-competes for binding to human gp100 with a reference TCR; wherein the reference TCR comprises an α chain and a β chain, and wherein the α chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and the β chain comprises the amino acid sequence set forth in SEQ ID NO: 2; and wherein the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein (i) the α 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 present in the amino acid sequence set forth in SEQ ID NO: 1, or (ii) the β 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 present in the amino acid sequence set forth in SEQ ID NO: 2.

[0026] Certain aspects of the present disclosure relate to a recombinant T cell receptor (TCR), or antigen-binding portion thereof, that specifically binds human gp100 (an "anti-gp100 TCR") that binds to the same epitope of human gp100 as a reference TCR, or an overlapping epitope thereof; wherein the reference TCR comprises an α chain and a β chain, and wherein the α chain comprises the amino acid sequence set forth in SEQ ID NO: 1 and the β chain comprises the amino acid sequence set forth in SEQ ID NO: 2; and wherein the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein (i) the α 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 present in the amino acid sequence set forth in SEQ ID NO: 1, or (ii) the β 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 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.

[0027] 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-B*40 allele. In some embodiments, the HLA class I molecule is selected from the group consisting of an HLA-B*40:01 allele, an HLA-B*40:02 allele, an HLA-B*40:03 allele, an HLA-B*40:04 allele, an HLA-B*40:05 allele, and an HLA-B*40:06 allele. In some embodiments, the HLA class I molecule is an HLA-B*40:01 allele.

[0028] In some embodiments, the α chain of the anti-gp100 TCR comprises a variable domain comprising an α chain CDR1, an α chain CDR2, and an α chain CDR3; and wherein the β chain of the anti-gp100 TCR comprises a variable domain comprising a β chain CDR1, a β chain CDR2, and a β chain CDR3; wherein the anti-gp100 α chain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the β chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0029] In some embodiments, the α chain of the anti-gp100 TCR comprises a variable domain comprising an α chain CDR1, an α chain CDR2, and an α chain CDR3; and wherein the β chain of the anti-gp100 TCR comprises a variable domain comprising a β chain CDR1, a β chain CDR2, and a β chain CDR3; wherein the β chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the α chain CDR3 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0030] In some embodiments, the α chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the β chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the α chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the β chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0031] In some embodiments, the α 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 β 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.

[0032] In some embodiments, the α chain constant region comprises an amino acid sequence that has 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.

[0033] In some embodiments, the beta chain constant region comprises an amino acid sequence that has 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.

[0034] In some embodiments, the alpha chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the beta chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:2.

[0035] Certain aspects of the present disclosure relate to a bispecific TCR comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a TCR disclosed herein or its antigen-binding portion thereof or a TCR disclosed herein or its antigen-binding portion thereof. 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 connected or associated by a covalent bond. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are connected by a peptide bond.

[0036] Certain aspects of the present disclosure relate to a cell 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.

[0037] Certain aspects of the present disclosure relate to a method 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, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, 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, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile 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, kidney or ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma 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.

[0038] In some embodiments, the cancer is recurrent or refractory. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is advanced. In some embodiments, the cancer is metastatic.

[0039] 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 pretreated before administering the cells. In some embodiments, the pretreatment includes administering chemotherapy, cytokines, proteins, small molecules, or any combination thereof to the subject. In some embodiments, the pretreatment includes administering an interleukin. In some embodiments, the pretreatment includes administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, the pretreatment includes administering a pretreatment agent selected from the group consisting of: cyclophosphamide, fludarabine, vitamin C, AKT inhibitors, ATRA, rapamycin, or any combination thereof. In some embodiments, the pretreatment includes administering cyclophosphamide, fludarabine, or both.

[0040] Certain aspects of the present disclosure relate to a method for engineering a cell targeting an antigen, the method comprising transducing a cell 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 cell targeting the antigen further expresses CD3. In some embodiments, the cell is a T cell or a natural killer (NK) cell.

[0041] Certain aspects of the present disclosure relate to 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 β2m, and wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO:14.

[0042] 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-B. In some embodiments, the HLA class I molecule is the HLA-B*40 allele. In some embodiments, the HLA class I molecule is selected from the group consisting of the HLA-B*40:01 allele, the HLA-B*40:02 allele, the HLA-B*40:03 allele, the HLA-B*40:04 allele, the HLA-B*40:05 allele, and the HLA-B*40:06 allele. In some embodiments, the HLA class I molecule is the HLA-B*40:01 allele. In some embodiments, the HLA class I molecule is the HLA-B*40:02 allele.

[0043] 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.

[0044] Certain aspects of the present disclosure relate to an antigen presenting cell (APC) comprising an HLA class I molecule disclosed herein. In some embodiments, the HLA class I molecule is expressed on the surface of the APC.

[0045] Certain aspects of the present disclosure relate to a method of enriching a population of target 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 higher number of T cells capable of binding the HLA class I molecule relative to the number of T cells capable of binding the HLA class I molecule before the contacting.

[0046] Certain aspects of the present disclosure relate to a method of enriching a population of target T cells obtained from a human subject, the method comprising contacting the T cells in vitro with a peptide, wherein the peptide consists of the amino acid sequence as set forth in SEQ ID NO: 13, wherein after the contacting, the enriched T cell population comprises a higher number of T cells capable of targeting tumor cells relative to the number of T cells capable of targeting tumor cells before the contacting.

[0047] In some embodiments, the T cells obtained from the human subject are tumor infiltrating lymphocytes (TIL).

[0048] Certain aspects of the present disclosure relate to a method of treating a tumor in a subject in need thereof, the method comprising administering to the subject an enriched T cell population disclosed herein.

[0049] 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, the method comprising administering to the subject a peptide having the amino acid sequence as set forth in SEQ ID NO: 13.

[0050] 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.

[0051] Certain aspects of the present disclosure relate to a method of selecting T cells capable of targeting tumor cells, the method comprising contacting a population of isolated T cells with a peptide in vitro, wherein the peptide consists of the amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Bar graph illustrating the number of B*40:01 / gp100 T cells in melanoma TILs after stimulation with artificial APCs pulsed with overlapping peptides. In an IFN-γ ELISPOT assay, TILs stimulated once with B*40:01-artificial APCs pulsed with overlapping peptides encompassing the entire gp100 protein were used as responder cells. B*40:01-artificial APCs pulsed with overlapping peptides derived from gp100 were used as stimulator cells. Following a single controlled peptide-specific stimulation, TILs showed responses to genes with shared sequences. 446 STESITGSLGPLLDG 460 The positive responses of two adjacent peptides were detected. (See also Table 5).

[0053] Figures 2A-2D B*40:01 / gp100 of melanoma TIL 448-458 Graphical representation of multimeric staining. TILs were stained with gp100 448 ESITGSLGPLL 458 Peptide-pulsed B*40:01-aAPC were stimulated once. The data are shown for the period before stimulation (day 0; Figure 2A and 2C ) and 14 days after stimulation (day 14; Figure 2B and 2D )B*40:01 / gp100 448-458 ( Figures 2A-2B ) or control B*40:01 / HIV nef 92-100 ( Figures 2C-2D ) Data of multimer staining. Showing CD8 + Multimers in T cells + The percentage of cells.

[0054] Figure 3 To show B*40:01 / gp100 448-458 Bar graph of functional evaluation of multimer-positive melanoma TILs. After one peptide-specific stimulation, TILs expressed B*40:01 / gp100. 448-458 In the IFN-γ ELISPOT assay, gp100 was used to produce IFN-γ. 448-458TILs stimulated once with peptide-pulsed B*40:01-artificial APCs were used as responder cells. B*40:01-artificial APCs pulsed with the indicated peptides were used as stimulator cells. 92-100和 gp100 448-458 Peptide served as a control. Experiments were performed in triplicate, and error bars show SD. *P<0.05.

[0055] Figures 4A-4I For use in homomultimers B*40:01 / gp100 448-458 Graphical representation of positive staining of TCR gene transduced Jurkat76 / CD8 cells. 448-458 TCR( Figure 4B 、 4E and 4H) transduced Jurkat 76 / CD8 cells with B*40:01 / gp100 448-458 Polymers ( Figure 4B ) dyeing. Using B*40:01 / NY-ESO-1 125-133 Polymers ( Figure 4D 、 4E and 4F), B*40:01 / unexchanged multimers ( Figure 4G 、 4H and 4I) and B*40:01 / NY-ESO-1 125-133 TCR transduced and non-transduced ( Figure 4A 、 4D and 4G) Jurkat 76 / CD8 cells were used as controls. + CD8 + The percentage of cells.

[0056] Figures 5A-5D For use in homomultimers B*40:01 / gp100 448-458 TCR gene ( Figure 5B and 5D ) were transduced with B*40:01 / gp100. 448-458 TCR transduced primary T cells with B*40:01 / gp100 448-458 ( Figure 5B ) or B*40:01 / HIV nef 92-100 Control polymer ( Figure 5D ) staining. Untransduced primary T cells were used as negative control ( Figure 5A and 5C ). Show multimers + CD8 + The percentage of T cells.

[0057] Figure 6 For illustration, B*40:01 / gp100 448-458 Bar graph showing TCR gene-transduced primary human T cells that reacted strongly to the cognate peptide presented by the target class I molecule. In the IFN-γ ELISPOT assay, B*40:01 / gp100 448-458 TCR gene transduced primary T cells or non-transduced primary T cells were used as responder cells. HLA-B*40:01 transduced T2 cells (T2-B*40:01) were generated. 448-458 or HIV nef 92-100 Peptide (control)-pulsed T2-B*40:01 cells were used as stimulators. The experiment was performed in triplicate, and the error bars show SD. *P<0.05, **P<0.01.

[0058] Figure 7A For illustration, B*40:01 / gp100 448-458 Graphical representation of tumor cell recognition by primary T cells transduced with TCR genes. In IFN-γ ELISPOT assay, B*40:01 / gp100 was used. 448-458 TCR gene transduced primary T cells or non-transduced primary T cells were used as responder cells. Figure 7B ( Figure 7A As indicated in the figure legends, SK-MEL-28 and SK-MEL-37 cells, either untransduced or transduced with HLA-B*40:01 and / or gp100, were used as stimulators after 48 hours of treatment with 100 ng / ml IFNγ. Experiments were performed in triplicate, and error bars represent SD. *P < 0.05, **P < 0.01.

[0059] Figures 8A-8D Graphical representation of the expression of gp100 derived from endogenous or transduced full-length genes. The expression of gp100 derived from endogenous or transduced full-length genes in target cells was analyzed by intracellular flow cytometry after staining with anti-gp100 mAb (open curves) and isotype control (solid curves).

[0060] Figures 9A-9B ΔNGFR is expressed in target cells transduced with the full-length HLA-B*40:01 gene tagged with ΔNGFR ( Figure 9B ). Surface expression of ΔNGFR in target cells transduced with the full-length HLA-B*40:01 gene tagged with ΔNGFR was analyzed by flow cytometry after staining with anti-NGFR mAb (open curves) and isotype control (solid curves). ΔNGFR alone was used as a control ( Figure 9A ). DETAILED DESCRIPTION

[0061] The present disclosure relates to TCRs or antigen-binding portions thereof that specifically bind to an epitope on gp100, nucleic acid molecules encoding the TCRs, 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 the cells to the subject. Other aspects of the present disclosure relate to HLA class I molecules complexed with a peptide comprising an epitope of gp100.

[0062] I. Terminology

[0063] To make this disclosure more easily understood, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meanings set forth below. Additional definitions are set forth in this application.

[0064] It should be noted that the term "a / an" entity refers to one or more of said entity; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Thus, the terms "a / an," "one or more," and "at least one" are used interchangeably herein.

[0065] Furthermore, “and / or” when used herein should be considered to specifically disclose each of the two specified features or components with or without the other. Thus, the term “and / or” when used herein in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” when used in phrases such as “A, B, and / or C” is intended to cover each of the following: 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).

[0066] The term "about" is used herein to mean approximately, roughly, about, or in the vicinity of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" is used herein to modify a numerical value above or below the stated value by a deviation of 10% above or below (higher or lower).

[0067] It will be understood that wherever the word "comprising" is used herein to describe aspects, otherwise similar aspects described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press provide a comprehensive dictionary of many of the terms used in this disclosure for those of skill in the art.

[0069] Units, prefixes, and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Amino acid sequences are written from left to right in an amino to carboxyl orientation. The headings provided herein are not limitations of the various aspects of the present disclosure and may be given such headings by reference to the specification as a whole. Therefore, the terms defined immediately below are more fully defined by reference to this specification as a whole.

[0070] "Administer " refers to using any one of various methods and delivery systems known to those skilled in the art to physically introduce agent to the subject. Exemplary routes of administration for preparations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, through spinal column or other parenteral routes of administration, such as by injection or infusion. As used herein, phrase "parenteral administration" means the mode of administration except enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, through trachea, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. In some embodiments, preparations are administered via non-parenteral route (e.g., oral). Other non-parenteral routes include through local, epidermal or transmucosal routes of administration, such as intranasal, vaginal, rectal, sublingual or through topical administration. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0071] As used herein, the term "T cell receptor" (TCR) refers to a heterogeneous 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 antigen-binding portions thereof; 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. Antigen-presenting cells (APCs) display fragments of foreign proteins (antigens) compounded with major histocompatibility complexes (MHC; also referred to herein as HLA molecules, such as HLA class 1 molecules). TCR recognizes and binds to antigen:HLA complexes, and recruits CD3 (expressed by T cells), thereby activating TCRs. Activated TCRs initiate downstream signaling and immune responses, including the destruction of EPCs.

[0072] In general, TCRs may comprise two chains, an α chain and a β chain (or less commonly a γ chain and a δ chain), interconnected by a disulfide bond. Each chain comprises a variable domain (an α chain variable domain and a β chain variable domain) and a constant region (an α chain constant region and a β chain constant region). The variable domain is located at the distal end of the cell membrane, and the variable domain interacts with the antigen. The constant region is located at the proximal end of the cell membrane. TCRs may also comprise a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses the transmembrane region and the cytoplasmic tail (when present) as well as the traditional "constant region".

[0073] The variable domains can be further subdivided into regions with high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each α chain variable domain and β chain variable domain comprises three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Each variable domain contains a binding domain that interacts with an antigen. Although all three CDRs on each chain participate in antigen binding, CDR3 is believed to be the primary antigen binding region. CDR1 also interacts with antigens, while CD2 is believed to primarily recognize HLA complexes.

[0074] In the absence of explicit statements, and unless the context indicates otherwise, the term "TCR" also includes an antigen-binding fragment or antigen-binding portion of any TCR disclosed herein, and includes monovalent and bivalent fragments or portions, and single-chain TCRs. The term "TCR" is not limited to naturally occurring TCRs that bind to the surface of T cells. As used herein, the term "TCR" further refers to TCRs expressed on the surface of cells other than T cells (e.g., cells naturally expressing or modified to express CD3 as described herein), or TCRs described herein that do not contain a cell membrane (e.g., isolated TCRs or soluble TCRs).

[0075] "Antigen binding molecule," "portion of a TCR," or "TCR fragment" refers to any portion of a TCR that is smaller than the entire TCR. An antigen binding molecule may include an antigen complementarity determining region (CDR).

[0076] "Antigen" refers to any molecule, such as a peptide, that causes an immune response or can be bound by a TCR. As used herein, "epitope" refers to a portion of a polypeptide that causes an immune response or can be bound by a TCR. An immune response may involve the production of antibodies or the activation of specific immunocompetent cells or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually all proteins or peptides, can serve 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 certain tissue (such as a cancer cell), or they can be widely expressed. In addition, fragments of larger molecules can serve as antigens. In one embodiment, the antigen is a tumor antigen. The epitope can be present in a longer polypeptide (e.g., in a protein), or the epitope 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, such as an HLA class 1 molecule).

[0077] As used herein, "gp100", "glycoprotein 100", "melanocyte protein PMEL" or "ME20M" refers to a tumor antigen expressed in, for example, melanoma. gp100 is a hydrophobic glycoprotein of 661 amino acids 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 the maturation of melanosomes from phase I to phase II. As used herein, gp100 refers not only to the full-length canonical 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).

[0078] Table 1. gp100 amino acid sequence

[0079]

[0080] As used herein, the term "HLA" refers to human leukocyte antigens. HLA genes encode major histocompatibility complex (MHC) proteins in humans. MHC proteins are expressed on the surface of cells and are involved in the activation of immune responses. HLA class I genes encode MHC class I molecules, which are expressed on the surface of cells in the form of complexes with peptide fragments (antigens) of self or non-self proteins. T cells expressing TCR and CD3 recognize antigen:MHC class I complexes and initiate an immune response to target and destroy antigen-presenting cells displaying non-self proteins.

[0081] 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 encoding an MHC class I molecule. Thus, "HLA class I molecule" and "MHC class I molecule" are used interchangeably herein.

[0082] MHC class I molecules contain two protein chains: an α chain and a β2-microglobulin (β2m) chain. Human β2m is encoded by the B2M gene. The amino acid sequence of β2m is set forth in SEQ ID NO: 16 (Table 2). The α chain of the MHC class I molecule is encoded by the HLA gene complex. The HLA complex is located in the 6p21.3 region on the short arm of human chromosome 6 and contains more than 220 genes with various functions. The HLA gene is highly variable, with more than 20,000 HLA alleles and related alleles, including more than 15,000 HLA class I alleles known in the art, which encode thousands of HLA proteins, including more than 10,000 HLA class I proteins (see, for example, hla.alleles.org, last visited on February 27, 2019). There are at least three genes encoding MHC class I α chain proteins in the HLA complex: HLA-A, HLA-B, and HLA-C. Additionally, HLA-E, HLA-F, and HLA-G encode proteins that associate with MHC class I molecules.

[0083] Table 2. Amino acid sequence of human β2m

[0084]

[0085] The term "autologous" refers to any material derived from the same individual into which it is subsequently introduced. For example, autologous T-cell therapy involves administering to a subject T cells isolated from the same individual. The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species. For example, allogeneic T-cell transplantation involves administering to a subject T cells obtained from a donor other than the subject.

[0086] "Cancer" refers to a wide variety of diseases characterized by uncontrolled growth of abnormal cells in the body. Disordered cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" may include tumors. Examples of cancers treatable 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 can be used to reduce the size of a tumor originating from, for example, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, skin or intraocular melanoma, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, 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), esophageal cancer, small intestine cancer, cancer of the endocrine system , thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, 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, kidney or ureter cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, 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 such cancers. Specific cancers may respond to chemotherapy or radiation therapy or the cancer may be refractory. Refractory cancer refers to a cancer that is not amenable to surgical intervention and that does not initially respond to chemotherapy or radiation therapy or that becomes unresponsive over time.

[0087] As used herein, "anti-tumor effect" refers to a biological effect that can be in the form of a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease 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 a tumor. An anti-tumor effect can also refer to the prevention of tumorigenesis, such as a vaccine.

[0088] The term "progression-free survival," which may be abbreviated as PFS, as used herein refers to the time from the date of treatment to the date of disease progression according to the modified IWG Response Criteria for Malignant Lymphoma or death from any cause.

[0089] "Disease progression" or "progressive disease" may be abbreviated as PD, and as used herein, refers to the worsening of one or more symptoms associated with a particular disease. For example, disease progression in a subject suffering from cancer may include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[0090] "Duration of response," which may be abbreviated as DOR, as used herein refers to the period of time between a subject's first objective response and the date of confirmed disease progression according to the modified IWG response criteria for malignant lymphomas or death.

[0091] The term "overall survival" may be abbreviated as OS, which is defined as the time from the day of treatment to the day of death.

[0092] As used herein, "cytokine" refers to a non-antibody protein released by a cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. Cytokines can be expressed endogenously by cells or administered to a subject. Cytokines can be released by immune cells including macrophages, B cells, T cells and mast cells to propagate an immune response. Cytokines can induce various responses in receptor cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors and acute phase proteins. For example, homeostatic cytokines including interleukin (IL) 7 and IL-15 promote immune cell survival and proliferation, and proinflammatory cytokines can promote inflammatory responses. 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) γ. Examples of proinflammatory cytokines include, but are not limited to, IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-α, TNF-β, 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 placental 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).

[0093] "Chemokines" are a class of cytokines that mediate cellular chemotaxis or directional movement. 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 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-induced protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0094] Other examples of analytes and cytokines of the present invention include, but are not limited to, chemokine (CC 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) β, 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-related and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

[0095] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, prevents the onset of disease in a subject or promotes regression of disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the agent's activity in in vitro assays.

[0096] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells or B cells. NK cells are a class of cytotoxic / cell toxic lymphocytes representing the main components of the innate immune system. NK cells repel tumors and cells infected by viruses. They work through the process of apoptosis or programmed cell death. They are called "natural killers" because they do not need to be activated to kill cells. T cells play a major role in cell-mediated immunity (not involving 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, namely: helper T cells (such as CD4+ cells), cytotoxic T cells (also referred to as TC, cytotoxic T lymphocytes, CTLs, T-killer cells, cytolytic T cells, CD8+T cells or killer T cells), memory T cells ((i) stem cell-like memory T cells), and cytotoxic T cells. SCMCentral memory T cells (i.e., naive 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, and display many of the unique functional characteristics of memory cells); (ii) central memory T cells (i.e., naive cells) are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+; however, they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and display many of the unique functional characteristics of memory cells. CM cells express L-selectin and CCR7, secrete IL-2 but not IFNγ or IL-4, and (iii) however, effector memory T EM B cells do not express L-selectin or CCR7, but produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Treg, suppressor T cells or CD4+CD25+ regulatory T cells), natural killer T cells (NKT) and γδT cells. On the other hand, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens and play the role of antigen presenting cells (APCs) and become memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow from which the name of the B cell is derived.

[0097] The term "genetic engineering" or "engineering" refers to a method of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a portion thereof or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, such as a T cell or a modified cell expressing CD3, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct incorporated into the cell genome, such as a T cell receptor (TCR) disclosed herein. In some embodiments, the cell is modified to express CD3.

[0098] "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 produced by any of these cells or the liver (including Abs, cytokines, and complement) that result in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation in a vertebrate.

[0099] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of contracting a disease or suffering from recurrence of a disease by a method that includes inducing, enhancing, suppressing, or otherwise altering an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy may include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation.

[0100] The cells used in the immunotherapy described herein can be from any source known in the art. For example, T cells can be separated from a hematopoietic stem cell population in vitro, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained using methods known to those skilled in the art, such as FICOLL. TM A unit of blood collected from a subject by any number of techniques for separation and / or apheresis. Other 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 may also include administering modified cells to the subject, wherein the modified cells express CD3 and a TCR disclosed herein. In some embodiments, the modified cells are not T cells.

[0101] As used herein, "patient" includes any person suffering from cancer (eg, lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0102] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can comprise a sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the terms refer to short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include naturally occurring peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0103] As used herein, "stimulation" refers to the primary response induced by the binding of a stimulatory molecule to its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell, such as a T cell receptor (TCR) / CD3 complex. A "stimulatory ligand" is a molecule that specifically binds to a stimulatory molecule on a T cell when present on an antigen presenting cell (e.g., aAPC, dendritic cells, B cells, and similar cells), thereby mediating the primary response caused by the T cell, including but not limited to ligands for activation, initial immune response, proliferation, and similar reactions. 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.

[0104] The terms "treatment" and "pretreatment" are used interchangeably herein and indicate preparation of patients requiring T cell therapy for suitable situations. Treatment as used herein includes, but is not limited to, reducing the number of endogenous lymphocytes, removing the cytokine sink, increasing the serum content of one or more homeostatic cytokines or proinflammatory factors, enhancing the effector function of T cells administered after treatment, enhancing antigen presenting cell activation and / or availability, or any combination thereof. In one embodiment, "treatment" includes increasing the serum content of one or more cytokines, such as interleukin 7 (IL-7), interleukin 15 (IL-15), interleukin 10 (IL-10), interleukin 5 (IL-5), γ-induced protein 10 (IP-10), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), placental 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, "treating" comprises increasing serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0105] "Treatment" or "treating" of a subject refers to any type of intervention or treatment performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or disorders, or biochemical markers associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0106] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of the alternatives. As used herein, the indefinite article "a / a" should be understood to mean "one or more" of any recited or listed components.

[0107] The term "about" or "substantially comprising" refers to a value or composition as determined by a person of ordinary skill in the art within an acceptable error range of a particular value or composition, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "substantially comprising" can mean within 1 or more than 1 standard deviation according to the convention in the art. Alternatively, "about" or "substantially 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%). In addition, particularly with respect to biological systems or methods, the term can mean up to an order of magnitude or up to 5 times the value. When a specific value or composition is provided in the application and claims, unless otherwise stated, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range of the specific value or composition.

[0108] Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range described herein should be understood to include any integer value and, where appropriate, fractions thereof (such as tenths and hundredths of an integer) within the recited range.

[0109] Various aspects of the invention are described in more detail in the following subsections.

[0110] II. Compositions of the Disclosure

[0111] The present disclosure relates to T cell receptors (TCRs) or antigen-binding portions thereof that specifically bind to an epitope on gp100, nucleic acid molecules encoding the TCRs, 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 a cell comprising a TCR as described herein. Other aspects of the present disclosure relate to an epitope of gp100 that binds to a TCR, and an HLA class I molecule complexed with a peptide comprising the epitope of gp100.

[0112] The T cell receptor, or TCR, is a molecule present on the surface of T cells or T lymphocytes that is responsible for recognizing fragments of antigens that are peptides bound to major histocompatibility complex (MHC) molecules. The binding between the TCR and the antigenic peptide is relatively low affinity and degenerate: in other words, many TCRs recognize the same antigenic peptide and many antigenic peptides are recognized by the same TCR.

[0113] TCR is composed of two different protein chains (in other words, it is a heterodimer). In humans, in 95% of T cells, TCR is composed of α (α) chain and β (β) chain (encoded by TRA and TRB, respectively), but in 5% of T cells, TCR is composed of γ and δ (γ / δ) chain (encoded by TRG and TRD, respectively). This ratio changes during individual occurrence and in diseased states (such as leukemia). It is also different between species. Orthologs of 4 loci have been located in various species. Each locus can produce a variety of polypeptides with constant regions and variable regions.

[0114] When the TCR engages with an antigenic peptide and MHC (peptide / MHC), T lymphocytes are activated through signal transduction, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0115] II.A. Nucleic Acid Molecules

[0116] 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 a 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 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 or an overlapping epitope of human gp100 as the reference TCR.

[0117] In some embodiments, the reference TCR comprises an α chain and a β chain; wherein the α chain comprises a complementarity determining region 1 (CDR1), a CDR2, and a CDR3; wherein the β chain comprises CDR1, CDR2, and CDR3; and wherein the reference TCR comprises the α chain CDR3 listed in SEQ ID NO: 7 and the β chain CDR3 listed in SEQ ID NO: 10. In some embodiments, the α chain CDR1, CDR2, and CDR3 sequences are present in the amino acid sequence listed in SEQ ID NO: 1, and the reference TCR comprises the β chain CDR1, CDR2, and CDR3 sequences present in the amino acid sequence listed in SEQ ID NO: 2. In some embodiments, the reference TCR comprises an α chain and a β chain, wherein the α chain comprises the amino acid sequence as set forth in SEQ ID NO: 1 and the β chain comprises the amino acid sequence as set forth in SEQ ID NO: 2.

[0118] Table 3. α and β chain TCR sequences

[0119]

[0120]

[0121] II.A.1. TCR Encoded by the First Nucleotide Sequence

[0122] 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 α chain and a β chain, wherein the α chain comprises a variable domain comprising an α chain CDR1, an α chain CDR2, and an α chain CDR3; and wherein the β chain comprises a variable domain comprising a β chain CDR1, a β chain CDR2, and a β chain CDR3. In some embodiments, the anti-gp100 TCR comprises an α chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7 (CAEGDTGRRALTF). In some embodiments, the anti-gp100 TCR comprises a β chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (CASSPGASYEQYF). In some embodiments, the non-CDR regions in the α chain and / or β chain are further modified, e.g., by substitution or mutation of one, two, three, four, five, or six amino acids, such that the α chain and / or β chain is not naturally occurring. In some embodiments, substitutions or mutations can improve the TCRs described herein in various ways, such as binding affinity, binding specificity, stability, viscosity, or any combination thereof.

[0123] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α chain CDR1, wherein the α chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 5 (DSSSTY). In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β chain CDR1, wherein the β chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 8 (IFSNMDM).

[0124] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α chain CDR2, wherein the α chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 6 (MGHRA). In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β chain CDR2, wherein the β chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 9 (YSYEKL).

[0125] 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%, 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, wherein 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 the α chain variable domain present in the α chain amino acid sequence set forth in SEQ ID NO:1.

[0126] 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%, 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, 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 the beta chain variable domain present in the amino acid sequence set forth in SEQ ID NO:2.

[0127] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence further comprises an α chain constant region, a β chain constant region, or both an α chain constant region and a β chain constant region. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α chain constant region that has 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 α 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 α chain constant region that has 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 α chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-gp100 TCR comprises an α 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 the α chain constant region present in the α 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 α constant region that is different from an endogenous (e.g., naturally occurring) constant region of the α chain. In some embodiments, the α chain constant region comprises an amino acid sequence comprising at least 1, at least 2, at least 3, at least 4, or at least 5 amino acid substitutions relative to the amino acid sequence of the constant region of the α chain amino acid sequence set forth in SEQ ID NO: 1.

[0128] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a beta chain constant region that has 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 that has 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 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 is different from an 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.

[0129] 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, wherein 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.

[0130] 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 set forth in SEQ ID NO:2.

[0131] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α chain constant region, a β chain constant region, or both; and wherein the α chain constant region, the β 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 the endogenous TCR.

[0132] II.A.2. Epitopes

[0133] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence 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 (ESITGSLGPLL). 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 consists of amino acid residues 448-458 of gp100 (SEQ ID NO: 52), e.g., "gp100 448-458 ”.

[0134] In certain embodiments, the epitope is complexed with an HLA class I molecule. The human leukocyte antigen (HLA) system (major histocompatibility complex [MHC] in humans) is an important part of the immune system and is controlled by genes located on chromosome 6. It encodes cell surface molecules that are specialized to present antigenic peptides to the T cell receptor (TCR) on T cells. (See also Overview of the Immune System.) MHC molecules that present antigens (Ag) are divided into two major categories: class I MHC molecules and class II MHC molecules.

[0135] Class I MHC molecules are present on the surface of all nucleated cells in the form of transmembrane glycoproteins. Complete class I molecules are composed of an alpha heavy chain bound to a beta-2 microglobulin molecule. The heavy chain consists of two peptide-binding domains, an Ig-like domain, and a transmembrane region with a cytoplasmic tail. The heavy chains of class I molecules are encoded by genes at 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, which require them to be able to recognize any infected cell. Because every nucleated cell expresses class I MHC molecules, all infected cells can act 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 certain receptors on natural killer [NK] cells).

[0136] In some embodiments, the HLA class 1 molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C alleles. In some embodiments, the HLA class 1 molecule is selected from the group consisting of HLA-E, HLA-F, and HLA-G alleles. In certain embodiments, the HLA class 1 molecule is an HLA-A allele. In certain embodiments, the HLA class 1 molecule is an HLA-B allele. In certain embodiments, the HLA class 1 molecule is an HLA-C allele.

[0137] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any of the known alleles can be used in the present disclosure. An updated list of HLA alleles is available at hla.alleles.org / (last accessed February 27, 2019). In some embodiments, the HLA class 1 molecule is an HLA-B allele selected from the group consisting of: HLA-B*07, HLA-B*08, HLA-B*13, HLA-B*14, HLA-B*15, HLA-B*18, HLA-B*27, HLA-B*35, HLA-B*37, HLA-B*38, HLA-B*39, HLA-B*40, HLA-B*41, HLA-B*42, HLA-B*44, HLA-B*45, HLA-B*46, HLA-B*47, HLA-B*48, HLA-B*49, HLA-B*50, HLA-B*51, HLA-B*52, HLA-B*53, HLA-B*54, HLA-B*55, HLA-B*56, HLA-B*57, HLA-B*58, HLA-B*59, HLA-B*60, HLA-B*61, HLA-B*62, HLA-B*64, HLA-B*65, HLA-B*66, HLA-B*67, HLA-B*68, HLA-B*69, HLA-B*70, HLA-B*71, HLA-B*72, HLA-B*73, HLA-B*74, HLA-B*75, HLA-B*76, HLA-B*77, HLA-B*78, HLA-B*79, HLA-B*80, HLA-B*81 In certain embodiments, the HLA-B allele is an HLA-B*40:01 allele. In certain embodiments, the HLA-B allele is an HLA-B*40:02 allele. In certain embodiments, the HLA-B allele is an HLA-B*40:03 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:04 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:05 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:06 allele.

[0138] In certain embodiments, the HLA class I molecule is an HLA-B allele selected from the group consisting of: HLA-B*40:01:01, HLA-B*40:01:02:01, HLA-B*40:01:02:02, HLA-B*40:01:02:03, HLA-B*40:01:02:04, HLA-B*40:01:02:05, HLA-B*40:01:02:06, HLA-B*40:01:02:07, HLA-B*40:01:02:08, HLA-B*40:01:02:09, HLA-B*40:01:03, HLA-B*40:01:04, HLA-B*40:01:05, HLA-B*40:01:06, HLA-B*40:01:07, HLA-B*40:01:08, HLA-B*40:01:09, HLA-B*40:01:10, HLA-B*40:01:11, HLA-B*40:01:12, HLA-B*40:01:13, HLA-B*40:01:14, HLA-B*40:01:15, HLA-B*40:01:16, HLA-B*40:01:17, HLA-B*40:01:18, HLA-B*40:01:19, HLA-B*40:01:20, HLA-B*40:01:21, HLA-B*40:01:22, HLA-B*40:01:23, HLA-B*40:01:24, HLA-B*40:01:25, HLA-B*40:01:26, HLA-B*40:01:27, HLA-B*40:01:28, HLA-B*40:01:29, HLA-B*40:01:30, HLA-B*40:01:31, HLA-B*40:01:32, HLA-B*40:01:33, HLA-B*40:01:34, HLA-B*40:01:35, HLA-B*40:01:36, HLA-B*40:01:37, HLA-B*40:01:38, HLA-B*40:01:39, HLA-B*40:01:40, HLA-B*40:01:41, HLA-B*40:01:42, HLA-B*40:01:43, HLA-B*40:01:44, HLA-B*40:01:45, HLA-B*40:01:46, HLA-B*40:01:47, HLA-B*4�:01:48, HLA-B*40:01:49, HLA-B*40:01:50, HLA-B*40:01:51, HLA-B*40:01:52, HLA-B*40:01:53, HLA-B*40:01:54,HLA - B*40:01:55, HLA - B*40:01:56, HLA - B*40:01:57, HLA - B*40:01:58, HLA - B*40:01:59, and HLA - B*40:01:60. In some embodiments, the HLA class I molecule is an HLA - B allele selected from the group consisting of: HLA - B*40:02:01:01, HLA - B*40:02:01:02, HLA - B*40:02:01:03, HLA - B*40:02:01:04, HLA - B*40:02:01:05, HLA - B*40:02:01:06, HLA - B*40:02:01:07, HLA - B*40:02:01:08, HLA - B*40:02:02, HLA - B*40:02:03, HLA - B*40:02:04, HLA - B*40:02:05, HLA - B*40:02:06, HLA - B*40:02:07, HLA - B*40:02:08, HLA - B*40:02:09, HLA - B*40:02:10, HLA - B*40:02:11, HLA - B*40:02:12, HLA - B*40:0,2:13, HLA - B*40:02:14, HLA - B*40:02:15, HLA - B*40:02:16, HLA - B*40:02:17, HLA - B*40:02:18, HLA - B*40:02:19, HLA - B*40:02:20, HLA - B* / 40:02:21, HLA - B*40:02:22, HLA - B*40:02:23, HLA - B*40:02:24, HLA - B*40:02:25, HLA - B*40:02:26, HLA - B*40:03:01:01, HLA - B*40:03:01:02, HLA - B*40:04:01, HLA - B*40:04:02, HLA - B*40:05:01:01, HLA - B*40:05:01:02, HLA - B*40:06:01:01, HLA - B*40:06:01:02, HLA - B*40:06:02, HLA - B*40:06:03, HLA - B*40:06:04:01, HLA - B*40:06:04:02, HLA - B*40: / 06:05, HLA - B*40:06:06, HLA - B*40:06:07, HLA - B*40:06:08, HLA - B*40:06:09, HLA - B*40:06:10, HLA - B*40:06:11, HLA - B*40:06:12, HLA - B*40:06:13,SONG-B*40:06:14、SONG-B*40:06:15、SONG-B*40:06:16、SONG-B*40:06:17、SONG-B*40: 06:18、SONGS-B*40:07、SONGS-B*40:08、SONGS-B*40:09、SONGS-B*40:100、SONGS-B*40:101、SONGS-B*40:101、HL A-B*40:102、SONGS-B*40:103、SONGS-B*40:104、SONGS-B*40:105、SONGS-B*40:106、SONGS-B*4 0:107、SONGS-B*40:108、SONGS-B*40:109、SONGS-B*40:10:01:01、SONGS-B*40:10:01:02、SONGS-B*40:10:01:02、SONGS AB*40:10:02, SONG-B*40:110, SONG-B*40:111, SONG-B*40:112, SONG-B*40:113, SONG-B*40:114:01, SONG-B*40: 114:02, SONG-B*40:115, SONG-B*40:116, SONG-B*40:117, SONG-B*40:118, SONG-B*40:119, SONG-B*40:11:01, SONG -B*40:11:02、SONGS-B*40:12、SONGS-B*40:120、SONGS-B*40:121、SONGS-B*40:122、SONGS-B*40:123、SONGS-B*40:124:0 1, SONG-B*40:124:02, SONG-B*40:125:01, SONG-B*40:125:02, SONG-B*40:126, SONG-B*40:127, SONG-B*40:128, H LA-B*40:129、SONGS-B*40:13、SONGS-B*40:130:01、SONGS-B*40:130:02、SONGS-B*40:131、SONGS-B*40:132、SONGS-B*40 :133、SONGS-B*40:134、SONGS-B*40:135、SONGS-B*40:136、SONGS-B*40:137、SONGS-B*40:138、SONGS-B*40:139、SONGS-B*4 0:140, SONG-B*40:141, SONG-B*40:142, SONG-B*40:143, SONG-B*40:144, SONG-B*40:145, SONG-B*40:146, SONG-B* 40:147, SONG-B*40:148, SONG-B*40:149, SONG-B*40:14:01, SONG-B*40:14:02, SONG-B*40:14:03, SONG-B*40:15SONG-B*40:150, SONG-B*40:151, SONG-B*40:152, SONG-B*40:153, SONG-B*40:1 54、SONGS-B*40:155:01、SONGS-B*40:155:02、SONGS-B*40:156、SONGS-B*40:157、H LA-B*40:158、SONGS-B*40:159、SONGS-B*40:16、SONGS-B*40:160:01、SONGS-B*40: 160:02、SONGS-B*40:161、SONGS-B*40:162、SONGS-B*40:163、SONGS-B*40:164、SONGS- B*40:165, SONG-B*40:166, SONG-B*40:167, SONG-B*40:168, SONG-B*40:169, H LA-B*40:170、SONGS-B*40:171、SONGS-B*40:172、SONGS-B*40:173、SONGS-B*40:1 、SONGS-B*40:175、SONGS-B*40:176、SONGS-B*40:177、SONGS-B*40:178、SONGS-B*40: 179、SONGS-B*40:18、SONGS-B*40:180、SONGS-B*40:181、SONGS-B*40:182、SONGS-B*40 :183、SONGS-B*40:184、SONGS-B*40:185、SONGS-B*40:186:01、SONGS-B*40:186:0 、SONGS-B*40:187、SONGS-B*40:188、SONGS-B*40:189、SONGS-B*40:19、SONGS-B*40:1 0、SONGS-B*40:191、SONGS-B*40:192、SONGS-B*40:193、SONGS-B*40:194、SONGS-B*4 :195、SONGS-B*40:196、SONGS-B*40:197、SONGS-B*40:198、SONGS-B*40:199、SONGS-B* 40:200、SONGS-B*40:201、SONGS-B*40:202、SONGS-B*40:203、SONGS-B*40:204、SONGS -B*40:205、SONGS-B*40:206、SONGS-B*40:207、SONGS-B*40:208、SONGS-B*40:209、H LA-B*40:20:01:01, SONGS-B*40:20:01:02, SONGS-B*40:21, SONGS-B*40:210, HL A-B*40:211, SONG-B*40:212, SONG-B*40:213, SONG-B*40:214, SONG-B*40:2SONG-B*40:216, SONG-B*40:217, SONG-B*40:218, SONG-B*40:219, SONG-B*40:2 20、SONGS-B*40:221、SONGS-B*40:222、SONGS-B*40:223、SONGS-B*40:224、SONGS-B*4 :225、SONGS-B*40:226、SONGS-B*40:227、SONGS-B*40:228、SONGS-B*40:229、SONGS-B *40:2257、SONGS-B*40:23、SONGS-B*40:230、SONGS-B*40:231、SONGS-B*40:232、SONGS -B*40:233, SONG-B*40:234, SONG-B*40:235, SONG-B*40:236, SONG-B*40:237 SONG-B*40:238, SONG-B*40:239, SONG-B*40:24, SONG-B*40:240, SONG-B*40:2 、SONGS-B*40:242、SONGS-B*40:243、SONGS-B*40:244、SONGS-B*40:245、SONGS-B*40: 246、SONGS-B*40:247、SONGS-B*40:248、SONGS-B*40:249、SONGS-B*40:25、SONGS-B*40 :250、SONGS-B*40:251、SONGS-B*40:252、SONGS-B*40:253、SONGS-B*40:254、SONGS-B *40:255, SONG-B*40:256, SONG-B*40:257, SONG-B*40:258, SONG-B*40:259, SONG -B*40:26, SONG-B*40:260, SONG-B*40:261, SONG-B*40:262, SONG-B*40:263, H LA-B*40:264、SONGS-B*40:265、SONGS-B*40:266、SONGS-B*40:267、SONGS-B*40:2 、SONGS-B*40:269、SONGS-B*40:270、SONGS-B*40:271、SONGS-B*40:272、SONGS-B*40: 273、SONGS-B*40:274、SONGS-B*40:275、SONGS-B*40:276、SONGS-B*40:277、SONGS-B*4 0:278、SONGS-B*40:279、SONGS-B*40:27:01、SONGS-B*40:27:02、SONGS-B*40:28、SONGS-B*40:28、SONGS A-B*40:280, SONG-B*40:281, SONG-B*40:282, SONG-B*40:283, SONG-B*40:2SONG-B*40:285, SONG-B*40:286, SONG-B*40:287, SONG-B*40:288, SONG-B*40:289, SONG-B*40:29, SONG-B*40:290, SONG-B *40:291, SONG-B*40:292, SONG-B*40:293, SONG-B*40:294, SONG-B*40:295, SONG-B*40:296, SONG-B*40:297, SONG-B*40:2 98:01、SONGS-B*40:298:02、SONGS-B*40:299、SONGS-B*40:30、SONGS-B*40:300、SONGS-B*40:301、SONGS-B*40:302、SONGS-B*40:3 03、SONGS-B*40:304、SONGS-B*40:305、SONGS-B*40:306、SONGS-B*40:307、SONGS-B*40:308、SONGS-B*40:309、SONGS-B*40:31、SONGS -B*40:310, SONG-B*40:311, SONG-B*40:312, SONG-B*40:313, SONG-B*40:314, SONG-B*40:315, SONG-B*40:316, SONG-B*40 :317、SONGS-B*40:318、SONGS-B*40:319、SONGS-B*40:32、SONGS-B*40:320、SONGS-B*40:321、SONGS-B*40:322、SONGS-B*40:323、H LA-B*40:324、SONGS-B*40:325、SONGS-B*40:326、SONGS-B*40:327、SONGS-B*40:328、SONGS-B*40:329、SONGS-B*40:33、SONGS-B*4 0:330, SONG-B*40:331, SONG-B*40:332, SONG-B*40:333, SONG-B*40:334, SONG-B*40:335, SONG-B*40:336, SONG-B*40:337 98、SONGS-B*40:338、SONGS-B*40:339、SONGS-B*40:34、SONGS-B*40:340、H LA-B*40:341、SONGS-B*40:342、SONGS-B*40:343、SONGS-B*40:344、SONGS-B *40:345、SONGS-B*40:346、SONGS-B*40:347、SONGS-B*40:348、SONGS-B*40: 349. SONG-B*40:350, SONG-B*40:351, SONG-B*40:352, SONG-B*40:353.SONG-B*40:354, SONG-B*40:355, SONG-B*40:356, SONG-B*40:357, SONG-B*40:358, SONG-B*4 0:359、SONGS-B*40:35:01、SONGS-B*40:35:02、SONGS-B*40:36、SONGS-B*40:360、SONGS-B*40:361 86、SONGS-B*40:362、SONGS-B*40:363、SONGS-B*40:364、SONGS-B*40:365、SONGS-B*40:3 6. SONG-B*40:367, SONG-B*40:368, SONG-B*40:369, SONG-B*40:37, SONG-B*40:370, H LA-B*40:371, SONGS-B*40:372, SONGS-B*40:373, SONGS-B*40:374, SONGS-B*40:375, HL A-B*40:376, SONG-B*40:377, SONG-B*40:378, SONG-B*40:379, SONG-B*40:38, SONG-B *40:380、SONGS-B*40:381、SONGS-B*40:382、SONGS-B*40:383、SONGS-B*40:384、SONGS-B* 40:385、SONGS-B*40:386、SONGS-B*40:387、SONGS-B*40:388、SONGS-B*40:389、SONGS-B*40 :39、SONGS-B*40:390、SONGS-B*40:391、SONGS-B*40:392、SONGS-B*40:393、SONGS-B*40:3 94、SONGS-B*40:395、SONGS-B*40:396、SONGS-B*40:397、SONGS-B*40:398、SONGS-B*40:399 57、SONGS-B*40:40、SONGS-B*40:400、SONGS-B*40:401、SONGS-B*40:402、SONGS-B*40:403、SONGS-B*40:404 、SONGS-B*40:406、SONGS-B*40:407、SONGS-B*40:408、SONGS-B*40:409、SONGS-B*40:410、SONGS-B*40:411、 SONG-B*40:412, SONG-B*40:413, SONG-B*40:414, SONG-B*40:42, SONG-B*40:43, SONG-B*40:44, SONG- B*40:45, SONG-B*40:46, SONG-B*40:47, SONG-B*40:48, SONG-B*40:49, SONG-B*40:50, SONG-B*40:51.SONG-B*40:52, SONG-B*40:53, SONG-B*40:54, SONG-B*40:55, SONG-B*40:56, SONG-B*40:57, SONG-B*40: 58. SONG-B*40:59, SONG-B*40:60, SONG-B*40:61, SONG-B*40:62, SONG-B*40:63, SONG-B*40:64:01:01, H LA-B*40:64:01:02、SONGS-B*40:65、SONGS-B*40:66、SONGS-B*40:67、SONGS-B*40:68、SONGS-B*40:69、SONGS-B*40:69、SONGS-B *40:70:01、SONGS-B*40:70:02、SONGS-B*40:71、SONGS-B*40:72:01、SONGS-B*40:72:02、SONGS-B*40:73、SONGS -B*40:74、SONGS-B*40:75、SONGS-B*40:76、SONGS-B*40:77、SONGS-B*40:78、SONGS-B*40:79、SONGS-B*40:80、H LA-B*40:81, SONGS-B*40:82, SONGS-B*40:83, SONGS-B*40:84, SONGS-B*40:85, SONGS-B*40:86, SONGS-B*40:8 :01、SONGS-B*40:87:02、SONGS-B*40:88、SONGS-B*40:89、SONGS-B*40:90、SONGS-B*40:91、SONGS-B*40:92、SONGS -B*40:93, SONG-B*40:94, SONG-B*40:95, SONG-B*40:96, SONG-B*40:97, SONG-B*40:98, SONG-B*40:99.

[0139] II.A.3 Introductory snowflakes

[0140] The second nucleotide sequence of nucleic acid molecules disclosed herein can be any sequence capable of suppressing the expression of endogenous TCR or can encode any polypeptide capable of suppressing the expression of endogenous TCR. In some embodiments, the second nucleotide sequence is one or more siRNA. In some embodiments, the target sequence in the nucleotide sequence of the constant region of one or more siRNA and the constant region encoding endogenous TCR is complementary. In certain embodiments, the target sequence in the nucleotide sequence of the constant region of one or more siRNA and the constant region encoding wild-type human TCR is complementary. In some embodiments, the target sequence in the nucleotide sequence of the constant region of one or more siRNA and the α chain encoding wild-type TCR is complementary. In some embodiments, the target sequence in the nucleotide sequence of the constant region of one or more siRNA and the β chain encoding wild-type TCR is complementary. In some embodiments, one or more siRNAs include (i) one or more siRNAs complementary to the target sequence in the nucleotide sequence of the constant region encoding wild-type TCR and (ii) one or more siRNAs complementary to the target sequence in the nucleotide sequence of the constant region encoding wild-type TCR.

[0141] 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 a target sequence within a nucleotide sequence encoding a constant region of an α chain of a wild-type TCR, and wherein the one or more siRNAs comprise a nucleic acid sequence set forth in SEQ ID NOs: 53 and 54.

[0142] Table 4. siRNA sequences

[0143]

[0144]

[0145] 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 a target sequence within a nucleotide sequence encoding a constant region of a beta chain of a wild-type TCR, and 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 a target sequence within a nucleotide sequence encoding a constant region of an alpha chain of a 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 a target sequence within a nucleotide sequence encoding a constant region of a beta chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences set forth in SEQ ID NOs: 55 and 56.

[0146] 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 siRNA. In certain embodiments, the one or more siRNAs are selected from the siRNAs disclosed in U.S. Publication No. 2010 / 0273213A1, which is incorporated herein by reference in its entirety.

[0147] In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes a protein, wherein the protein is capable of inhibiting the expression of endogenous (e.g., wild-type) TCR. In some embodiments, the second nucleotide sequence encodes Cas9.

[0148] II.A.3 Vector

[0149] Certain aspects of the present invention 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 virion or virus. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a bacterial vector.

[0150] In certain embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of: adenoviral vector, lentivirus, Sendai virus, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector and adeno-associated virus (AAV) vector. In specific embodiments, the vector is an AAV vector. In some embodiments, the vector is a lentivirus. In specific embodiments, the vector is an AAV vector. In some embodiments, the vector is Sendai virus. In some embodiments, the vector is a hybrid vector. Examples of hybrid vectors that can be used in the present invention can be found in Huang and Kamihira, Biotechnol. Adv. 31 (2): 208-23 (2103), which is incorporated herein by reference in its entirety.

[0151] II.B. Recombinant T Cell Receptor (TCR)

[0152] Certain aspects of the invention relate to a recombinant T cell receptor (TCR) or antigen binding portion thereof that specifically binds human gp100 ("anti-gp100 TCR"). In some embodiments, the anti-gp100 TCR is encoded by a nucleic acid molecule disclosed herein.

[0153] 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 of human gp100 as the reference TCR, or to an overlapping epitope. In some embodiments, the reference TCR comprises an α chain and a β chain, and the α chain of the reference TCR comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the β chain of the reference TCR comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0154] In some embodiments, the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein the α 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 α chain comprising the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein the β 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 β chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0155] In some embodiments, the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a constant region, and wherein the β chain comprises a constant region; wherein (i) the α 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 α chain comprising the amino acid sequence set forth in SEQ ID NO: 1; and (ii) the β 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 β chain comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0156] In some embodiments, the α chain of the anti-gp100 TCR comprises a variable domain comprising an α chain CDR1, an α chain CDR2, and an α chain CDR3; and the β chain of the anti-gp100 TCR comprises a variable domain comprising a β chain CDR1, a β chain CDR2, and a β chain CDR3. In some embodiments, the anti-gp100 TCR comprises an α chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR comprises a β chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0157] In some embodiments, the α chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the β chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:8.

[0158] In some embodiments, the α chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the β chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO:9.

[0159] In some embodiments, the 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, the 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, wherein 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 the alpha chain variable domain present in the alpha chain amino acid sequence set forth in SEQ ID NO: 1.

[0160] In some embodiments, the anti-gp100 TCR comprises a beta chain variable domain that has 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 comprises a beta chain variable domain that has 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, 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 the beta chain variable domain present in the beta chain amino acid sequence set forth in SEQ ID NO: 2.

[0161] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide further comprises an α chain constant region, a β chain constant region, or both an α chain constant region and a β chain constant region. In some embodiments, the anti-gp100 TCR comprises an α chain constant region that has 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 α chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR comprises an α chain constant region that has 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 at least about 100% sequence identity to the constant region of the α chain amino acid sequence set forth in SEQ ID NO: 1, wherein the anti-gp100 TCR comprises an α chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the anti-gp100 TCR comprises an α chain constant region present in the α chain amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide further comprises an α constant region that is different from an endogenous (e.g., naturally occurring) constant region of the α chain. In some embodiments, the α 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 α chain amino acid sequence set forth in SEQ ID NO: 1.

[0162] In some embodiments, the anti-gp100 TCR comprises a beta chain constant region that has 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 that has 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 the beta chain constant region as 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 further comprises a beta constant region that is different from an 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 1, at least 2, at least 3, at least 4, or at least 5 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.

[0163] 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, wherein 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.

[0164] 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.

[0165] In some embodiments, the anti-gp100 TCR comprises an α chain constant region, a β chain constant region, or both; and wherein the α chain constant region, the β 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 the endogenous TCR.

[0166] II.B.2. Epitopes

[0167] In some embodiments, the anti-gp100 TCR binds to the same epitope as the reference TCR.

[0168] 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 consists of amino acid residues 448-458 of gp100 (SEQ ID NO: 52), e.g., "gp100 448-458 ”.

[0169] In certain embodiments, the epitope is complexed with an HLA class I molecule. In certain embodiments, the HLA class 1 molecule is selected from the group consisting of HLA-A, HLA-B, and HLA-C alleles. In certain embodiments, the HLA class 1 molecule is selected from the group consisting of HLA-E, HLA-F, and HLA-G alleles. In certain embodiments, the HLA class 1 molecule is an HLA-A allele. In certain embodiments, the HLA class 1 molecule is an HLA-B allele. In certain embodiments, the HLA class 1 molecule is an HLA-C allele.

[0170] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any of the known alleles can be used in the present invention. An updated list of HLA alleles is available at hla.alleles.org / (last accessed February 27, 2019). In some embodiments, the HLA class 1 molecule is an HLA-B allele selected from the group consisting of: HLA-B*07, HLA-B*08, HLA-B*13, HLA-B*14, HLA-B*15, HLA-B*18, HLA-B*27, HLA-B*35, HLA-B*37, HLA-B*38, HLA-B*39, HLA-B*40, HLA-B*41, HLA-B*42, HLA-B*44, HLA-B*45, HLA-B*46, HLA-B*47, HLA-B*48, HLA-B*49, HLA-B*50, HLA-B*51, HLA-B*52, HLA-B*53, HLA-B*54, HLA-B*55, HLA-B*56, HLA-B*57, HLA-B*58, HLA-B*59, HLA-B*60, HLA-B*61, HLA-B*62, HLA-B*64, HLA-B*65, HLA-B*66, HLA-B*67, HLA-B*68, HLA-B*69, HLA-B*70, HLA-B*71, HLA-B*72, HLA-B*73, HLA-B*74, HLA-B*75, HLA-B*76, HLA-B*77, HLA-B*78, HLA-B*79, HLA-B*80, HLA-B*81 In certain embodiments, the HLA-B allele is an HLA-B*40:01 allele. In certain embodiments, the HLA-B allele is an HLA-B*40:02 allele. In certain embodiments, the HLA-B allele is an HLA-B*40:03 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:04 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:05 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:06 allele.

[0171] In certain embodiments, the HLA class I molecule is an HLA-B allele selected from the group consisting of: HLA-B*40:01:01, HLA-B*40:01:02:01, HLA-B*40:01:02:02, HLA-B*40:01:02:03, HLA-B*40:01:02:04, HLA-B*40:01:02:05, HLA-B*40:01:02:06, HLA-B*40:01:02:07, HLA-B*40:01:02:08, HLA-B*40:01:02:09, HLA-B*40:01:03, HLA-B*40:01:04, HLA-B*40:01:05, HLA-B*40:01:06, HLA-B*40:01:07, HLA-B*40:01:08, HLA-B*40:01:09, HLA-B*40:01:10, HLA-B*40:01:11, HLA-B*40:01:12, HLA-B*40:01:13, HLA-B*40:01:14, HLA-B*40:01:15, HLA-B*40:01:16, HLA-B*40:01:17, HLA-B*40:01:18, HLA-B*40:01:19, HLA-B*40:01:20, HLA-B*40:01:21, HLA-B*40:01:22, HLA-B*40:01:23, HLA-B*40:01:24, HLA-B*40:01:25, HLA-B*40:01:26, HLA-B*40:01:27, HLA-B*40:01:28, HLA-B*40:0, HLA-B*40:01:30, HLA-B*40:01:31, HLA-B*40:01:32, HLA-B*40:01:33, HLA-B*40:01:34, HLA-B*40:01:35, HLA-B*40:01:36, HLA-B*40:01:37, HLA-B*40:01:38, HLA-B*40:01:39, HLA-B*40:01:40, HLA-B*40:01:41, HLA-B*40:01:42, HLA-B*40:01:43, HLA-B*40:01:44, HLA-B*40:01:45, HLA-B*40:01:46, HLA-B*40:01:47, HLA-B*40:01:48, HLA-B*40:01:49, HLA-B*40:01:50, HLA-B*40:01:51, HLA-B*40:01:52, HLA-B*40:01:53, HLA-B*40:01:54,HLA - B*40:01:55, HLA - B*40:01:56, HLA - B*40:01:57, HLA - B*40:01:58, HLA - B*40:01:59 and HLA - B*40:01:60. In some embodiments, the HLA class I molecule is an HLA - B allele selected from the group consisting of: HLA - B*40:02:01:01, HLA - B*40:02:01:02, HLA - B*40:02:01:03, HLA - B*40:02:01:04, HLA - B*40:02:01:05, HLA - B*40:02:01:06, HLA - B*40:02:01:07, HLA - B*40:02:01:08, HLA - B*40:02:02, HLA - B*40:02:03, HLA - B*40:02:04, HLA - B*40:02:05, HLA - B*40:02:06, HLA - B*40:02:07, HLA - B*40:02:08, HLA - B*40:02:09, HLA - B*40:02:10, HLA - B*40:02:11, HLA - B*40:02:12, HLA - B*40:02:13, HLA - B*40:02:14, HLA - B*40:02:15, HLA - B*40:02:16, HLA - B*40:02:17, HLA - B*40:02:18, HLA - B*40:02:19, HLA - B*40:02:20, HLA - B*40:02:21, HLA - B*40:02:22, HLA - B*40:02:23, HLA - B*40:02:24, HLA - B*40:02:25, HLA - B*40:02:26, HLA - B*40:03:01:01, HLA - B*40:03:01:02, HLA - B*40:04:01, HLA - B*40:04:02, HLA - B*40:05:01:01, HLA - B*40:05:01:02, HLA - B*40:06:01:01, HLA - B*40:06:01:02, HLA - B*40:06:02, HLA - B*40:06:03, HLA - B*40:06:04:01, HLA - B*40:06:04:02, HLA - B*40:06:05, HLA - B*40:06:06, HLA - B*40:06:07, HLA - B*40:06:08, HLA - B*40:06:09, HLA - B*40:06:10, HLA - B*40:06:11, HLA - B*40:06:12, HLA - B*40:06:13,SONG-B*40:06:14、SONG-B*40:06:15、SONG-B*40:06:16、SONG-B*40:06:17、SONG-B*40: 06:18、SONGS-B*40:07、SONGS-B*40:08、SONGS-B*40:09、SONGS-B*40:100、SONGS-B*40:101、SONGS-B*40:101、HL A-B*40:102、SONGS-B*40:103、SONGS-B*40:104、SONGS-B*40:105、SONGS-B*40:106、SONGS-B*4 0:107、SONGS-B*40:108、SONGS-B*40:109、SONGS-B*40:10:01:01、SONGS-B*40:10:01:02、SONGS-B*40:10:01:02、SONGS AB*40:10:02, SONG-B*40:110, SONG-B*40:111, SONG-B*40:112, SONG-B*40:113, SONG-B*40:114:01, SONG-B*40: 114:02, SONG-B*40:115, SONG-B*40:116, SONG-B*40:117, SONG-B*40:118, SONG-B*40:119, SONG-B*40:11:01, SONG -B*40:11:02、SONGS-B*40:12、SONGS-B*40:120、SONGS-B*40:121、SONGS-B*40:122、SONGS-B*40:123、SONGS-B*40:124:0 1, SONG-B*40:124:02, SONG-B*40:125:01, SONG-B*40:125:02, SONG-B*40:126, SONG-B*40:127, SONG-B*40:128, H LA-B*40:129、SONGS-B*40:13、SONGS-B*40:130:01、SONGS-B*40:130:02、SONGS-B*40:131、SONGS-B*40:132、SONGS-B*40 :133、SONGS-B*40:134、SONGS-B*40:135、SONGS-B*40:136、SONGS-B*40:137、SONGS-B*40:138、SONGS-B*40:139、SONGS-B*4 0:140, SONG-B*40:141, SONG-B*40:142, SONG-B*40:143, SONG-B*40:144, SONG-B*40:145, SONG-B*40:146, SONG-B* 40:147, SONG-B*40:148, SONG-B*40:149, SONG-B*40:14:01, SONG-B*40:14:02, SONG-B*40:14:03, SONG-B*40:15SONG-B*40:150, SONG-B*40:151, SONG-B*40:152, SONG-B*40:153, SONG-B*40:1 54、SONGS-B*40:155:01、SONGS-B*40:155:02、SONGS-B*40:156、SONGS-B*40:157、H LA-B*40:158、SONGS-B*40:159、SONGS-B*40:16、SONGS-B*40:160:01、SONGS-B*40: 160:02、SONGS-B*40:161、SONGS-B*40:162、SONGS-B*40:163、SONGS-B*40:164、SONGS- B*40:165, SONG-B*40:166, SONG-B*40:167, SONG-B*40:168, SONG-B*40:169, H LA-B*40:170、SONGS-B*40:171、SONGS-B*40:172、SONGS-B*40:173、SONGS-B*40:1 、SONGS-B*40:175、SONGS-B*40:176、SONGS-B*40:177、SONGS-B*40:178、SONGS-B*40: 179、SONGS-B*40:18、SONGS-B*40:180、SONGS-B*40:181、SONGS-B*40:182、SONGS-B*40 :183、SONGS-B*40:184、SONGS-B*40:185、SONGS-B*40:186:01、SONGS-B*40:186:0 、SONGS-B*40:187、SONGS-B*40:188、SONGS-B*40:189、SONGS-B*40:19、SONGS-B*40:1 0、SONGS-B*40:191、SONGS-B*40:192、SONGS-B*40:193、SONGS-B*40:194、SONGS-B*4 :195、SONGS-B*40:196、SONGS-B*40:197、SONGS-B*40:198、SONGS-B*40:199、SONGS-B* 40:200、SONGS-B*40:201、SONGS-B*40:202、SONGS-B*40:203、SONGS-B*40:204、SONGS -B*40:205、SONGS-B*40:206、SONGS-B*40:207、SONGS-B*40:208、SONGS-B*40:209、H LA-B*40:20:01:01, SONGS-B*40:20:01:02, SONGS-B*40:21, SONGS-B*40:210, HL A-B*40:211, SONG-B*40:212, SONG-B*40:213, SONG-B*40:214, SONG-B*40:2SONG-B*40:216, SONG-B*40:217, SONG-B*40:218, SONG-B*40:219, SONG-B*40:220, SONG-B*40:221, SONG-B*40:222, SONG-B *40:223, SONG-B*40:224, SONG-B*40:225, SONG-B*40:226, SONG-B*40:227, SONG-B*40:228, SONG-B*40:229, SONG-B*40:2 57 SONG-B*40:23, SONG-B*40:230, SONG-B*40:231, SONG-B*40:232, SONG-B*40:233, SONG-B*40:234, SONG-B*40:235, SONG-B* 40:236, SONG-B*40:237, SONG-B*40:238, SONG-B*40:239, SONG-B*40:24, SONG-B*40:240, SONG-B*40:241, SONG-B*40:242, HL A-B*40:243, SONG-B*40:244, SONG-B*40:245, SONG-B*40:246, SONG-B*40:247, SONG-B*40:248, SONG-B*40:249, SONG-B*40:2 5. SONG-B*40:250, SONG-B*40:251, SONG-B*40:252, SONG-B*40:253, SONG-B*40:254, SONG-B*40:255, SONG-B*40:256, SONG-B*4 0:257, SONG-B*40:258, SONG-B*40:259, SONG-B*40:26, SONG-B*40:260, SONG-B*40:261, SONG-B*40:262, SONG-B*40:263, SONG -B*40:264, SONG-B*40:265, SONG-B*40:266, SONG-B*40:267, SONG-B*40:268, SONG-B*40:269, SONG-B*40:270, SONG-B*40:271 、SONGS-B*40:272、SONGS-B*40:273、SONGS-B*40:274、SONGS-B*40:275、SONGS-B*40:276、SONGS-B*40:277、SONGS-B*40:278、SONGS-B*4 0:279, SONG-B*40:27:01, SONG-B*40:27:02, SONG-B*40:28, SONG-B*40:280, SONG-B*40:281, SONG-B*40:282, SONG-B*40:283SONG-B*40:284, SONG-B*40:285, SONG-B*40:286, SONG-B*40:287, SONG-B*40:288, SONG-B*40:289, SONG-B*40:29, SONG-B*4 0:290, SONG-B*40:291, SONG-B*40:292, SONG-B*40:293, SONG-B*40:294, SONG-B*40:295, SONG-B*40:296, SONG-B*40:297, H LA-B*40:298:01、SONGS-B*40:298:02、SONGS-B*40:299、SONGS-B*40:30、SONGS-B*40:300、SONGS-B*40:301、SONGS-B*40:302、HL A-B*40:303, SONG-B*40:304, SONG-B*40:305, SONG-B*40:306, SONG-B*40:307, SONG-B*40:308, SONG-B*40:309, SONG-B*40: 31. SONG-B*40:310, SONG-B*40:311, SONG-B*40:312, SONG-B*40:313, SONG-B*40:314, SONG-B*40:315, SONG-B*40:316, SONG -B*40:317, SONG-B*40:318, SONG-B*40:319, SONG-B*40:32, SONG-B*40:320, SONG-B*40:321, SONG-B*40:322, SONG-B*40:32 3, SONG-B*40:324, SONG-B*40:325, SONG-B*40:326, SONG-B*40:327, SONG-B*40:328, SONG-B*40:329, SONG-B*40:33, SONG-B *40:330, SONG-B*40:331, SONG-B*40:332, SONG-B*40:333, SONG-B*40:334, SONG-B*40:335, SONG-B*40:336, SONG-B*40:337 98、SONGS-B*40:338、SONGS-B*40:339、SONGS-B*40:34、SONGS-B*40:340、SONGS-B*40:341、SONGS-B*40:342、SONGS-B*40:343、SONGS-B*40:344 SONG-B*40:345, SONG-B*40:346, SONG-B*40:347, SONG-B*40:348, SONG-B*40:349, SONG-B*40:350, SONG-B*40:351, SONG-B*40:3SONG-B*40:353, SONG-B*40:354, SONG-B*40:355, SONG-B*40:356, SONG-B*40:357, SONG-B*40:358, H LA-B*40:359、SONGS-B*40:35:01、SONGS-B*40:35:02、SONGS-B*40:36、SONGS-B*40:360、SONGS-B*40:361 86、SONGS-B*40:362、SONGS-B*40:363、SONGS-B*40:364、SONGS-B*40:365、SONGS-B*40:3 6. SONG-B*40:367, SONG-B*40:368, SONG-B*40:369, SONG-B*40:37, SONG-B*40:370, H LA-B*40:371, SONGS-B*40:372, SONGS-B*40:373, SONGS-B*40:374, SONGS-B*40:375, HL A-B*40:376, SONG-B*40:377, SONG-B*40:378, SONG-B*40:379, SONG-B*40:38, SONG-B *40:380、SONGS-B*40:381、SONGS-B*40:382、SONGS-B*40:383、SONGS-B*40:384、SONGS-B* 40:385、SONGS-B*40:386、SONGS-B*40:387、SONGS-B*40:388、SONGS-B*40:389、SONGS-B*40 :39、SONGS-B*40:390、SONGS-B*40:391、SONGS-B*40:392、SONGS-B*40:393、SONGS-B*40:3 94、SONGS-B*40:395、SONGS-B*40:396、SONGS-B*40:397、SONGS-B*40:398、SONGS-B*40:399 57、SONGS-B*40:40、SONGS-B*40:400、SONGS-B*40:401、SONGS-B*40:402、SONGS-B*40:403、SONGS-B*40: 404、SON-B*40:406、SON-B*40:407、SON-B*40:408、SON-B*40:409、SON-B*40:410、SON-B*4 0:411、SONGS-B*40:412、SONGS-B*40:413、SONGS-B*40:414、SONGS-B*40:42、SONGS-B*40:43、SONGS-B*4 0:44, SONG-B*40:45, SONG-B*40:46, SONG-B*40:47, SONG-B*40:48, SONG-B*40:49, SONG-B*40:50HLA-B*40:51, HLA-B*40:52, HLA-B*40:53, HLA-B*40:54, HLA-B*40:55, HLA-B*40:56, HLA-B*40:57 , HLA-B*40:58, HLA-B*40:59, HLA-B*40:60, HLA-B*40:61, HLA-B*40:62, HLA-B*40:63, HLA-B*40:6 4:01:01, HLA-B*40:64:01:02, HLA-B*40:65, HLA-B*40:66, HLA-B*40:67, HLA-B*40:68, HLA-B*40: 69. HLA-B*40:70:01, HLA-B*40:70:02, HLA-B*40:71, HLA-B*40:72:01, HLA-B*40:72:02, HLA-B*40: 73. HLA-B*40:74, HLA-B*40:75, HLA-B*40:76, ​​HLA-B*40:77, HLA-B*40:78, HLA-B*40:79, HLA-B*40 :80, HLA-B*40:81, HLA-B*40:82, HLA-B*40:83, HLA-B*40:84, HLA-B*40:85, HLA-B*40:86, HLA-B*4 0:87:01, HLA-B*40:87:02, HLA-B*40:88, HLA-B*40:89, HLA-B*40:90, HLA-B*40:91, HLA-B*40:92, HLA-B*40:93, HLA-B*40:94, HLA-B*40:95, HLA-B*40:96, HLA-B*40:97, HLA-B*40:98 and HLA-B*40:99. ,

[0172] II.B.3. Bispecific T-cell receptors (TCRs)

[0173] Certain aspects of the present disclosure relate to a bispecific TCR comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a TCR disclosed herein, or an antigen binding portion thereof. In some embodiments, the first antigen binding domain comprises a single-chain variable fragment ("scFv").

[0174] 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 it is not expressed on the surface of human non-immune cells. In some embodiments, the second antigen-binding domain specifically binds to a protein expressed on the surface of a T cell selected from the group consisting of: 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.

[0175] In some embodiments, the first antigen binding domain and the second antigen binding domain are linked or associated by a covalent bond. In some embodiments, the first antigen binding domain and the second antigen binding domain are linked by a peptide bond.

[0176] II.C. TCR-Expressing Cells

[0177] 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 can be used in the present disclosure.

[0178] In certain embodiments, the cell expresses CD3. CD3 expression may be naturally occurring, for example, 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 natural killer T (NKT) cells and innate lymphoid cells (ILCs).

[0179] In some embodiments, the T cells are isolated from a human subject. In some embodiments, the human subject is the same subject that will ultimately receive the T cell therapy. In other embodiments, the subject is a donor subject, wherein the donor subject is not the same subject that will receive the T cell therapy.

[0180] 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 endogenous CD3 expression of the cell. In some embodiments, the cell comprises a transgene encoding a protein or siRNA that inhibits CD3 expression in the cell. In some embodiments, the transgene is incorporated into the genome of the cell. In some embodiments, the transgene is not incorporated into the genome of the cell.

[0181] 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 that will ultimately receive the cell therapy. In other embodiments, the subject is a donor subject, wherein the donor subject is not the same subject that will receive the cell therapy.

[0182] II.D. HLA class I molecules

[0183] 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.

[0184] 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 1 molecule is an HLA-B allele selected from the group consisting of: HLA-B*07, HLA-B*08, HLA-B*13, HLA-B*14, HLA-B*15, HLA-B*18, HLA-B*27, HLA-B*35, HLA-B*37, HLA-B*38, HLA-B*39, HLA-B*40, HLA-B*41, HLA-B*42, HLA-B*44, HLA-B*45, HLA-B*46, HLA-B*47, HLA-B*48, HLA-B*49, HLA-B*50, HLA-B*51, HLA-B*52, HLA-B*53, HLA-B*54, HLA-B*55, HLA-B*56, HLA-B*57, HLA-B*58, HLA-B*59, HLA-B*60, HLA-B*61, HLA-B*62, HLA-B*64, HLA-B*65, HLA-B*66, HLA-B*67, HLA-B*68, HLA-B*69, HLA-B*70, HLA-B*71, HLA-B*72, HLA-B*73, HLA-B*74, HLA-B*75, HLA-B*76, HLA-B*77, HLA-B*78, HLA-B*79, HLA-B*80, HLA-B*81, HLA-B*82, HLA-B*83, HLA-B In certain embodiments, the HLA-B allele is an HLA-B*40:01 allele. In certain embodiments, the HLA-B allele is an HLA-B*40:02 allele. In certain embodiments, the HLA-B allele is an HLA-B*40:03 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:04 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:05 allele. In certain embodiments, the HLA-B allele is the HLA-B*40:06 allele. In some embodiments, the HLA allele is any HLA allele disclosed herein, e.g., as above.

[0185] In some embodiments, an HLA class I molecule comprises an α chain and β2m. In some embodiments, the α chain comprises an α1 domain, an α2 domain, and an α3 domain. In some embodiments, 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 about 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 α chain is selected from any of the HLA protein sequences available at hla.alleles.org (last accessed February 27, 2019).

[0186] 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.

[0187] 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 an HLA class I molecule on the surface of the APCs. In certain embodiments, the APCs comprise more than one HLA class I molecule disclosed herein.

[0188] II.D. Vaccines

[0189] Certain aspects of the present disclosure relate to 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 other peptides. In some embodiments, the one or more other peptides comprise one or more other epitopes.

[0190] III. Methods of the Disclosure

[0191] 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 cells that target an antigen. Other aspects of the present disclosure relate to methods of enriching a target T cell population obtained from a human subject.

[0192] III.A. Methods of Treating Cancer

[0193] 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 foregoing.

[0194] In some embodiments, the cancer is selected from melanoma, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, skin or intraocular malignant melanoma, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, 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), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, In some embodiments, the cancer is melanoma.

[0195] In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory. In some embodiments, the cancer is advanced. In some embodiments, the cancer is metastatic.

[0196] 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 cancer symptoms. In some embodiments, the methods disclosed herein reduce the size or number of tumors derived from cancer. In some embodiments, relative to a subject for whom the methods disclosed herein are not provided, the methods disclosed herein increase the overall survival of the subject. In some embodiments, relative to a subject for whom the methods disclosed herein are not provided, the methods disclosed herein increase the progression-free survival of the subject. In some embodiments, the methods disclosed herein cause a partial response in a subject. In some embodiments, the methods disclosed herein cause a complete response in a subject.

[0197] 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 modified to express CD3.

[0198] In some embodiments, the cells (e.g., T cells) are obtained from a subject. In some embodiments, the cells (e.g., T cells) are obtained from a donor other than the subject.

[0199] In some embodiments, the subject is pretreated before administering the cells. Pretreatment may include any substance that contributes to T cell function and / or survival. In some embodiments, pretreatment includes administering chemotherapy, cytokines, proteins, small molecules, or any combination thereof to the subject. In some embodiments, pretreatment includes administering interleukins. In some embodiments, pretreatment includes administering IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or any combination thereof. In some embodiments, pretreatment includes administering cyclophosphamide, fludarabine, or both. In some embodiments, pretreatment includes administering vitamin C, AKT inhibitors, ATRA (vesanoid, retinoic acid), rapamycin, or any combination thereof.

[0200] III.B. Methods for Engineering Cells to Target Antigens

[0201] Certain aspects of the present disclosure relate to methods for engineering cells targeting antigens. In some embodiments, the antigen is a gp100 antigen. In some embodiments, the method comprises transducing cells with a nucleic acid molecule disclosed herein or a vector disclosed herein. The cell may be any cell described herein. In some embodiments, the cell is a T cell described herein. In some embodiments, the cell is a cell modified to express CD3 as described herein. In some embodiments, the cell (e.g., 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 a subject in need of T cell therapy. In some embodiments, the cell is a T cell or a natural killer cell.

[0202] III.C. Methods for Enriching Target T Cell Populations

[0203] Certain aspects of the present disclosure relate to methods for enriching a population of target T cells obtained from a human subject. In some embodiments, the methods comprise contacting the T cells with an HLA class I molecule disclosed herein. In some embodiments, the methods comprise contacting the T cells with an APC disclosed herein. In some embodiments, after contacting, the enriched T cell population comprises a higher 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.

[0204] In some embodiments, the method comprises contacting T cells in vitro with a peptide, wherein the peptide comprises 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, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, after contacting, the enriched T cell population comprises a higher 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.

[0205] Some aspects of the present disclosure relate to a method for selecting T cells capable of targeting tumor cells. In some embodiments, the method comprises contacting a population of isolated T cells with a peptide in vitro, wherein the peptide consists of an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the T cells are obtained from a human subject.

[0206] 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).

[0207] In some embodiments, the method further comprises administering the enriched T cells to the human subject.In some embodiments, the subject is pretreated as described herein prior to receiving the T cells.

[0208] The various aspects, embodiments, and options described herein may all be combined in any and all variations.

[0209] 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.

[0210] Having generally described the invention, a further understanding can be obtained by reference to the examples provided herein. These examples are for purposes of illustration only and are not intended to be limiting.

[0211] Example

[0212] Example 1

[0213] TILs were isolated from patients with metastatic melanoma, followed by polyclonal expansion in vitro, and their gp100 antigen specificity was examined for the HLA-B*40:01 allele. A combination of structure-based and functional assays using peptide / HLA (pHLA) multimers has been used to measure Ag-specific T cell responses.

[0214] Since pHLA multimer production requires the use of peptides with known exact sequences, it is not direct or practical to use a pHLA multimer-based strategy for high-throughput screening of new epitope peptides. In addition to structure-based analysis using pHLA multimers, functional analysis can also be applied to determine the antigenic specificity of T cells. Functional analysis was performed using artificial antigen-presenting cells (APCs), which can process and process longer peptides and present epitope peptides via class I molecules as stimulatory cells. B*40:01-artificial APCs were pulsed with overlapping peptides for covering the whole protein of gp100 (Table 5) and used as stimulators in cytokine ELISPOT assays. After a controlled stimulation with B*40:01-artificial APCs pulsed with overlapping peptides derived from gp100, B*40:01 + Melanoma TILs show pairs with shared sequences 446 STESITGSLGPLLDG 460 The positive response of two adjacent peptides ( Figure 1 Using a series of mutant deletion peptides, the minimum required peptide epitopes presented by the B*40:01 molecule were determined. 448 ESITGSLGPLL 458 . HLA-B*40:01 / gp100 was identified 448-458 T cells, which account for CD8 + 0.11% of T cells (Figure 2). After a controlled peptide-specific stimulation with B*40:01-artificial APC, B*40:01 / gp100 448-458 The frequency of T cells increased to 1.8%, excluding the possibility that the low staining percentage represented a false positive (Figure 2). According to ELISPOT analysis, multimer-positive T cells secreted detectable IFN-γ in an HLA-restricted peptide-specific manner ( Figure 3 ).

[0215] Table 5. gp100 overlapping peptides.

[0216]

[0217]

[0218] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Figure 4, SEQ ID NO: 1 and 2). The antigen specificity and functional reactivity of the cloned TCR were verified by multimer staining and ELISPOT assay of TCR reconstructed T cells. When reconstructed based on primary T cells, B*40:01gp100 448-458 TCR-transduced T cells successfully stained in the presence of homomultimers (Figure 5) and were associated with gp100 presented by surface B*40:01 molecules. 448-458 Strong peptide reaction ( Figure 6 Importantly, these cells were able to recognize B*40:01-matched tumor cells that naturally expressed the gp100 gene and were not pulsed with the peptide ( FIG7 ). SK-MEL-37 melanoma cells, which lack endogenous expression of both B*40:01 and gp100, became responsive to B*40:01 / gp100 only when both the B*40:01 and gp100 genes (but not either gene alone) were transduced. 448-458 TCR-transduced T cells were responsive (Figures 7-9). These results clearly demonstrate that B*40:01 / gp100 448-458 TCR-transduced T cells are fully enthusiastic in recognizing tumor cells and the cloned B*40:01 / gp100 448-458 TCRs are tumor-reactive.

[0219] Gp100 is one of the most promising and well-studied common antigens in bispecific T cell engager (BiTE) therapies, and a clinical trial targeting gp100 is underway in patients with metastatic uveal melanoma using IMCgp100, a bispecific biologic comprising a soluble TCR recognizing the gp100 antigen fused to an scFv anti-CD3, to redirect T cell lysis of gp100-expressing melanoma cells in the presence of HLA-A*02:01 molecules. The use of a newly cloned, tumor-reactive B*40:01-restricted gp100 TCR gene may broaden the applicability of gp100-targeted BiTE therapies beyond patients with HLA-A*02:01-positive cancers.

[0220] method

[0221] Cell samples

[0222] 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 erythroleukemic cell line with defective HLA expression. T2 is HLA-A*02:01 +T cell leukemia / B-LCL hybrid cell lines. Jurkat 76 is a T cell leukemia cell line lacking TCR and CD8 expression. 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 RPMI1640 supplemented with 10% FBS and 50 μg / ml gentamicin. TILs isolated from patients with metastatic melanoma were grown in vitro.

[0223] peptides

[0224] The synthetic peptides were dissolved in DMSO to 50 μg / ml. The peptides used were 20-mer overlapping peptides to cover the whole protein of gp100 (Table 1) and B*40:01 restricted gp100. 448-458 (ESITGSLGPLL; SEQ ID NO:13), gp100 449-458 (SITGSLGPLL; SEQ ID NO:194), NY-ESO-1 125-133 (EFTVSGNIL; SEQ ID NO: 195) and HIVnef 92-100 (KEKGGLEGL; SEQ ID NO: 63) peptide. Using gp100 449-458 NY-ESO-1 125-133 and HIV nef 92-100 Peptide served as a negative control.

[0225] Gene

[0226] The HLA-B*40:01 gene was fused to a truncated version 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). TCR genes were cloned by 5'-rapid amplification of cDNA ends (RACE) PCR using the SMARTer RACE cDNA amplification kit (Takara Bio). 5'-RACE PCR products were cloned into retroviral vectors and sequenced. All genes were cloned into pMX retroviral vectors and transduced using a 293GPG cell-based retroviral system.

[0227] transfectants

[0228] Jurkat 76 / CD8 cells were transduced with individual TCRα and TCRβ genes as previously reported. 42-44. Jurkat 76 / CD8-derived TCR transfectants were purified (>95% purity) using CD3 microbeads (Miltenyi Biotec). K562-based artificial APCs have previously been reported to express various HLA class I genes as single HLA alleles in combination with CD80 and CD83 (Butler and Hirano, Immunol. Rev. 257: 191-209 (2014); Hirano et al., Clin. Cancer Res. 12: 2967-75 (2006)). TCR genes were transduced into human primary T cells using PG13-derived retroviral supernatant. TCR genes were transfected into the 293GPG cell line using TransIT293 (Mirus Bio). Gp100 was retrovirally transduced with the full-length gp100 gene. - SK-MEL-37 cells were used to produce SK-MEL-37 / gp100. The expression of transduced gp100 was assessed by flow cytometry after staining with anti-gp100 mAb (clone 7E3; LifeSpan Biosciences). HLA-B*40:01 retrovirus was used to transduce HLA-B*40:01. - SK-MEL-37 cells were used to generate SK-MEL-37 / B*40:01 cells. The HLA-B*40:01 gene was tagged with the ΔNGFR gene as described above, and the ΔNGFR + The cells were purified (>95% purity) and used in subsequent experiments. The ΔNGFR gene alone was retrovirally transduced as a control.

[0229] Flow cytometry and cell sorting

[0230] Cell surface molecules were stained with PC5-conjugated anti-CD8 mAb (clone B9.11; Beckman Coulter), FITC-conjugated anti-NGFR (clone ME20.4; Biolegend), and APC / Cy7-conjugated anti-CD3 (clone UCHT1; Biolegend). Dead cells were identified using a LIVE / DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies). For intracellular staining, cells were fixed and permeabilized using a Cytofix / Cytoperm kit (BD Biosciences). Stained cells were analyzed by flow cytometry (BD Biosciences), and data were analyzed using FlowJo (Tree Star). Cell sorting was performed using a FACS Aria II (BD Bioscience).

[0231] Cytokine ELISPOT analysis

[0232] IFN-γ ELISPOT assays were performed. PVDF plates (Millipore, Bedford, MA) were coated with capture mAb (1-D1K; MABTECH, Mariemont, OH), and T cells were incubated at 2 x 10 cells per well in the presence or absence of peptide. 4 The target cells were incubated together at 37°C for 20-24 hours. The plates were then washed and incubated with biotin-conjugated detection mAb (7-B6-1; MABTECH). HRP-conjugated SA (Jackson ImmunoResearch) was then added and the IFN-γ spots were developed. The reaction was stopped by rinsing thoroughly with cold tap water. The ELISPOT plates were scanned and counted using an ImmunoSpot plate reader and ImmunoSpot version 5.0 software (Cellular Technology Limited, Shaker Heights, OH).

[0233] CD8 + TILs expand in an HLA-restricted peptide-specific manner

[0234] Using CD8 + CD8 T cell isolation kit (Miltenyi Biotec) was used to isolate CD8 T cells by negative magnetic selection. + TIL purification. B*40:01-artificial APCs were pulsed with 10 μg / mL gp100 peptide for 6 hours. The artificial APCs were then irradiated at 200 Gy, washed, and added to the TILs at an effector to target (E:T) ratio of 20:1. Starting from the second 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 three days.

[0235] Primary CD8 transduced with cloned TCR + T cell expansion

[0236] CD3 T cells were isolated by negative magnetic selection using a whole T cell isolation kit (Miltenyi Biotec). +T cell purification. Purified T cells were stimulated with 200 Gy irradiated aAPC / mOKT3 at an E:T ratio of 20:1. Starting on the second day, activated T cells were retrovirally transduced with the cloned TCR gene via centrifugation at 1,000 g for 1 hour at 32°C for 3 consecutive days. On the second day, 100 IU / ml IL-2 and 10 ng / ml IL-15 were added to the TCR-transduced T cells. Culture medium was replenished every 2-3 days.

[0237] Human cell-based production of pHLA multimers

[0238] The affinity-matured HLA class I gene was engineered to carry a Glu (E) residue instead of a Gln (Q) residue at position 115 of the α2 domain and a mouse Kp1 residue in place of the HLA class I α3 domain. b Soluble HLA class I was generated by sequentially fusing the extracellular domain of the affinity-matured HLA class I gene to a Gly-Ser (GS) flexible linker and a 6x His tag. Q115E -K b Gene. Using a 293GPG cell-based retroviral system with various soluble HLA class I Q115E -K b HEK293T cells were transduced with the β2m gene and the β2m gene. Q115E -K b Stable HEK293T cells were grown until confluent and the medium was then changed. Forty-eight hours later, the conditioned medium was harvested and used immediately or frozen until use. Q115E -K b The supernatant was mixed with 100-1000 μg / ml of the target class I restricted peptide at 37°C overnight for in vitro peptide exchange. The peptide-loaded soluble monomeric class I was stained with anti-His mAb (clone AD1.1.10; Abcam) conjugated to a fluorescent dye such as phycoerythrin (PE) at a 2:1 molar ratio. Q115E -K b Dimerization was performed at room temperature for 2 hours or at 4°C overnight. Functional soluble HLA class I was measured by specific ELISA using anti-whole class I mAb (clone W6 / 32, in-house) and anti-His tag biotinylated mAb (clone AD1.1.10, R&D systems) as capture and detection Abs, respectively. Q115E -K b The concentration of molecules.

[0239] pHLA multimer staining

[0240] T cells (1×10 5 ) and incubated at 37°C for 30 minutes. The cells were then washed and incubated with 5-10 μg / ml of the polymer at room temperature for 30 minutes, and R-phycoerythrin-conjugated AffiniPure Fab fragment goat anti-mouse IgG1 (Jackson ImmunoResearch Laboratories) was added for 15 minutes at 4°C. The cells were then washed three times and co-stained with anti-CD8 mAb for 15 minutes at 4°C. Dead cells were finally identified using a Live / Dead Fixable Dead Cell Stain Kit.

[0241] Statistical analysis

[0242] Statistical analysis was performed using GraphPad Prism 5.0e. To determine whether a given variable was significantly different between two groups, Welch's t test (two-sided) was used for analysis. P values ​​< 0.05 were considered significant. Sequence Listing <110> University Health Network <120> T cell receptors and methods of use thereof <130> 4285.004PC01 / CK / BMD <150> US 62 / 813,645 <151> 2019-03-04 <160> 195 <170> PatentIn version 3.5 <210> 1 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> α chain amino acid sequence <400> 1 Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu 1 5 10 15 Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser 20 25 30 Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser 35 40 45 Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu 50 55 60 Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln 65 70 75 80 Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg 85 90 95 Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu 100 105 110 Gly Asp Thr Gly Arg Arg Ala Leu Thr Phe Gly Ser Gly Thr Arg Leu 115 120 125 Gln Val Gln Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270 Serum Glx <210> 2 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> β-chain amino acid sequence <400> 2 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met 20 25 30 Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His 35 40 45 Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu 50 55 60 Met Phe Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro 65 70 75 80 Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His 85 90 95 Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Pro Gly Ala Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> αCDR2 <400> 6 Met Gly His Arg Ala 1 5 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> αCDR3 <400> 7 Cys Ala Glu Gly Asp Thr Gly Arg Arg Ala Leu Thr Phe 1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> βCDR1 <400> 8 Ile Phe Ser Asn Met Asp Met 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> βCDR2 <400> 9 Tyr Ser Tyr Glu Lys Leu 1 5 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> βCDR3 <400> 10 Cys Ala Ser Ser Pro Gly Ala Ser Tyr Glu Gln Tyr Phe 1 5 10 <210> 11 <400> 11 000 <210> 12 <400> 12 000 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Epitope <400> 13 Glu Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu 1 5 10 <210> 14 <400> 14 000 <210> 15 <400> 15 000 <210> 16 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> B2m amino acid sequence <400> 16 Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30 His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg Asp Met 115 <210> 17 <211> 822 <212> DNA / / 这里没有具体翻译内容,推测是原文中的一个占位符之类的,按照要求保留原样<213> Artificial Sequence <220> <223> Alpha chain nucleotide sequence <400> 17 atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60 agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120 ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180 ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240 agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300 cagactgggg actcagctat ctacttctgt gcagaggggg acacgggcag gagagcactt 360 acttttggga gtggaacaag actccaagtg caaccaaata tccagaaccc tgaccctgcc 420 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660 cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720 aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780 gggtttaatc tgctcatgac gctgcggctg tggtccagct ga 822 <210> 18 <211> 933 <212> DNA<​​​ <223> β-chain nucleotide sequence <400> 18 atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcagt tcccatagac 60 actgaagtta cccagacacc aaaacacctg gtcatgggaa tgacaaataa gaagtctttg 120 aaatgtgaac aacatatggg gcacagggct atgtattggt acaagcagaa agctaagaag 180 ccaccggagc tcatgtttgt ctacagctat gagaaactct ctataaatga aagtgtgcca 240 agtcgcttct cacctgaatg ccccaacagc tctctcttaa accttcacct acacgccctg 300 cagccagaag actcagccct gtatctctgc gccagcagcc caggggcatc ctacgagcag 360 tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 420 gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480 gtatgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540 aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600 aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660 ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720 acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780 gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catccttat 840 gagatcttgc tagggaagc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900 gccatggtca agaaagga ttccagaggc tag 933 <210> 19 <400> 19 000 <210> 20 <400> 20 000 <210> 21 <400> 21 000 <210> 22 <400> 22 000 <210> 23 <400> 23 000 <210> 24 <400> 24 000 <210> 25 <400> 25 000 <210> 26 <400> 26 000 <210> 27 <400> 27 000 <210> 28 <400> 28 000 <210> 29 <400> 29 000 <210> 30 <400> 30 000 <210> 31 <400> 31 000 <210> 32 <400> 32 000 <210> 33 <400> 33 000 <210> 34 <400> 34 000 <210> 35 <400> 35 000 <210> 36 <400> 36 000 <210> 37 <400> 37 000 <210> 38 <400> 38 000 <210> 39 <400> 39 000 <210> 40 <400> 40 000 <210> 41 <400> 41 000 <210> 42 <400> 42 000 <210> 43 <400> 43 000 <210> 44 <400> 44 000 <210> 45 <400> 45 000 <210> 46 <400> 46 000 <210> 47 <400> 47 000 <210> 48 <400> 48 000 <210> 49 <400> 49 000 <210> 50 <400> 50 000 <210> 51 <400> 51 000 <210> 52 <211> 661 <212> PRT <213> Artificial Sequence <220> <223> gp100 amino acid sequence <400> 52 Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly 1 5 10 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp Trp 20 25 30 Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu 35 40 45 Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly 50 55 60 Gln Val Ser Leu Lys Val Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala 65 70 75 80 Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly Ser Gln Lys Val 85 90 95 Leu Pro Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly 100 105 110 Ser Gln Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp 115 120 125 Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser 130 135 140 Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp 145 150 155 160 Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg 165 170 175 Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg 180 185 190 Gly Ser Arg Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala Phe Thr 195 200 205 Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln Leu Arg Ala 210 215 220 Leu Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe 225 230 235 240 Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu 245 250 255 Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg 260 265 270 Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala 275 280 285 Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser 290 295 300 Pro Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro 305 310 315 320 Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr 325 330 335 Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln 340 345 350 Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355 360 365 Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val 370 375 380 Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met 385 390 395 400 Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 405 410 415 Gln Val Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro 420 425 430 Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu 435 440 445 Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu 450 455 460 Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr 465 470 475 480 Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala 485 490 495 Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu 500 505 510 Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile 515 520 525 Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530 535 540 Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys Gly 545 550 555 560 Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser 565 570 575 Leu Ala Val Val Ser Thr Gln Leu Ile Met Pro Gly Gln Glu Ala Gly 580 585 590 Leu Gly Gln Val Pro Leu Ile Val Gly Ile Leu Leu Val Leu Met Ala 595 600 605 Val Val Leu Ala Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp 610 615 620 Phe Ser Val Pro Gln Leu Pro His Ser Ser Ser His Trp Leu Arg Leu 625 630 635 640 Pro Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu 645 650 655 Ser Gly Gln Gln Val 660 <210> 53 <211> 21 <212> Ms <213> Artificial Sequence <220> <223> siRNA-TCRa-1 <400> 53 guaaggauuc ugauguguat t 21 <210> 54 <211> 21 <212> Ms <213> Artificial Sequence <220> <223> siRNA-TCRa-2 <400> 54 uacacaucag aauccuuact t 21 <210> 55 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> siRNA TCRb 1 <400> 55 ccaccauccu cuaugagaut t 21 <210> 56 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> siRNA TCRb 2 <400> 56 aucucauaga ggaugguggt t 21 <210> 57 <400> 57 000 <210> 58 <400> 58 000 <210> 59 <400> 59 000 <210> 60 <400> 60 000 <210> 61 <400> 61 000 <210> 62 <400> 62 000 <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HIV nef92-100 <400> 63 Lys Glu Lys Gly Gly Leu Glu Gly Leu 1 5 <210> 64 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 64 Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly 1 5 10 15 Ala Leu Leu Ala 20 <210> 65 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 65 Lys Arg Cys Leu Leu His Leu Ala Val Ile Gly Ala Leu Leu Ala Val 1 5 10 15 Gly Ala Thr Lys 20 <210> 66 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 66 His Leu Ala Val Ile Gly Ala Leu Leu Ala Val Gly Ala Thr Lys Val 1 5 10 15 Pro Arg Asn Gln 20 <210> 67 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 67 Gly Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp 1 5 10 15 Trp Leu Gly Val 20 <210> 68 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 68 Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp Trp Leu Gly Val Ser 1 5 10 15 Arg Gln Leu Arg 20 <210> 69 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 69 Val Pro Arg Asn Gln Asp Trp Leu Gly Val Ser Arg Gln Leu Arg Thr 1 5 10 15 Lys Ala Trp Asn 20 <210> 70 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 70 Asp Trp Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg 1 5 10 15 Gln Leu Tyr Pro 20 <210> 71 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 71 Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln Leu Tyr Pro Glu 1 5 10 15 Trp Thr Glu Ala 20 <210> 72 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 72 Thr Lys Ala Trp Asn Arg Gln Leu Tyr Pro Glu Trp Thr Glu Ala Gln 1 5 10 15 Arg Leu Asp Cys 20 <210> 73 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 73 Arg Gln Leu Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp 1 5 10 15 Arg Gly Gly Gln 20 <210> 74 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 74 Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly Gln Val 1 5 10 15 Ser Leu Lys Val 20 <210> 75 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 75 Gln Arg Leu Asp Cys Trp Arg Gly Gly Gln Val Ser Leu Lys Val Ser 1 5 10 15 Asn Asp Gly Pro 20 <210> 76 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 76 Trp Arg Gly Gly Gln Val Ser Leu Lys Val Ser Asn Asp Gly Pro Thr 1 5 10 15 Leu Ile Gly Ala 20 <210> 77 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 77 Val Ser Leu Lys Val Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala Asn 1 5 10 15 Ala Ser Phe Ser 20 <210> 78 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 78 Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala Asn Ala Ser Phe Ser Ile 1 5 10 15 Ala Leu Asn Phe 20 <210> 79 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 79 Thr Leu Ile Gly Ala Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro 1 5 10 15 Gly Ser Gln Lys 20 <210> 80 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 80 Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly Ser Gln Lys Val 1 5 10 15 Leu Pro Asp Gly 20 <210> 81 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 81 Ile Ala Leu Asn Phe Pro Gly Ser Gln Lys Val Leu Pro Asp Gly Gln 1 5 10 15 Val Ile Trp Val 20 <210> 82 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 82 Pro Gly Ser Gln Lys Val Leu Pro Asp Gly Gln Val Ile Trp Val Asn 1 5 10 15 Asn Thr Ile Ile 20 <210> 83 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 83 Val Leu Pro Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn 1 5 10 15 Gly Ser Gln Val 20 <210> 84 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 84 Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly Ser Gln Val Trp 1 5 10 15 Gly Gly Gln Pro 20 <210> 85 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 85 Asn Asn Thr Ile Ile Asn Gly Ser Gln Val Trp Gly Gly Gln Pro Val 1 5 10 15 Tyr Pro Gln Glu 20 <210> 86 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 86 Asn Gly Ser Gln Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr 1 5 10 15 Asp Asp Ala Cys 20 <210> 87 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 87 Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp Ala Cys Ile 1 5 10 15 Phe Pro Asp Gly 20 <210> 88 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 88 Val Tyr Pro Gln Glu Thr Asp Asp Ala Cys Ile Phe Pro Asp Gly Gly 1 5 10 15 Pro Cys Pro Ser 20 <210> 89 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 89 Thr Asp Asp Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly 1 5 10 15 Ser Trp Ser Gln 20 <210> 90 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 90 Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys 1 5 10 15 Arg Ser Phe Val 20 <210> 91 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 91 Gly Pro Cys Pro Ser Gly Ser Trp Ser Gln Lys Arg Ser Phe Val Tyr 1 5 10 15 Val Trp Lys Thr 20 <210> 92 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 92 Gly Ser Trp Ser Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp 1 5 10 15 Gly Gln Tyr Trp 20 <210> 93 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 93 Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln 1 5 10 15 Val Leu Gly Gly 20 <210> 94 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 94 Tyr Val Trp Lys Thr Trp Gly Gln Tyr Trp Gln Val Leu Gly Gly Pro 1 5 10 15 Val Ser Gly Leu 20 <210> 95 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 95 Trp Gly Gln Tyr Trp Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser 1 5 10 15 Ile Gly Thr Gly 20 <210> 96 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 96 Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg 1 5 10 15 Ala Met Leu Gly 20 <210> 97 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 97 Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg Ala Met Leu Gly Thr 1 5 10 15 His Thr Met Glu 20 <210> 98 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 98 Ser Ile Gly Thr Gly Arg Ala Met Leu Gly Thr His Thr Met Glu Val 1 5 10 15 Thr Val Tyr His 20 <210> 99 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 99 Arg Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg 1 5 10 15 Arg Gly Ser Arg 20 <210> 100 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 100 Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg Gly Ser Arg Ser 1 5 10 15 Tyr Val Pro Leu 20 <210> 101 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 101 Val Thr Val Tyr His Arg Arg Gly Ser Arg Ser Tyr Val Pro Leu Ala 1 5 10 15 His Ser Ser Ser 20 <210> 102 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 102 Arg Arg Gly Ser Arg Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala 1 5 10 15 Phe Thr Ile Thr 20 <210> 103 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 103 Ser Tyr Val Pro Leu Ala His Ser Ser Ser Ala Phe Thr Ile Thr Asp 1 5 10 15 Gln Val Pro Phe 20 <210> 104 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 104 Ala His Ser Ser Ser Ala Phe Thr Ile Thr Asp Gln Val Pro Phe Ser 1 5 10 15 Val Ser Val Ser 20 <210> 105 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 105 Ala Phe Thr Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln 1 5 10 15 Leu Arg Ala Leu 20 <210> 106 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 106 Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln Leu Arg Ala Leu Asp 1 5 10 15 Gly Gly Asn Lys 20 <210> 107 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 107 Ser Val Ser Val Ser Gln Leu Arg Ala Leu Asp Gly Gly Asn Lys His 1 5 10 15 Phe Leu Arg Asn 20 <210> 108 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 108 Gln Leu Arg Ala Leu Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln 1 5 10 15 Pro Leu Thr Phe 20 <210> 109 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 109 Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe Ala 1 5 10 15 Leu Gln Leu His 20 <210> 110 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 110 His Phe Leu Arg Asn Gln Pro Leu Thr Phe Ala Leu Gln Leu His Asp 1 5 10 15 Pro Ser Gly Tyr 20 <210> 111 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 111 Gln Pro Leu Thr Phe Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Leu 1 5 10 15 Ala Glu Ala Asp 20 <210> 112 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 112 Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu 1 5 10 15 Ser Tyr Thr Trp 20 <210> 113 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 113 Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu Ser Tyr Thr Trp Asp 1 5 10 15 Phe Gly Asp Ser 20 <210> 114 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 114 Leu Ala Glu Ala Asp Leu Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser 1 5 10 15 Gly Thr Leu Ile 20 <210> 115 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 115 Leu Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser 1 5 10 15 Arg Ala Leu Val 20 <210> 116 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 116 Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg Ala Leu Val Val 1 5 10 15 Thr His Thr Tyr 20 <210> 117 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 117 Ser Gly Thr Leu Ile Ser Arg Ala Leu Val Val Thr His Thr Tyr Leu 1 5 10 15 Glu Pro Gly Pro 20 <210> 118 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 118 Ser Arg Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val 1 5 10 15 Thr Ala Gln Val 20 <210> 119 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 119 Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala Gln Val Val 1 5 10 15 Leu Gln Ala Ala 20 <210> 120 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 120 Leu Glu Pro Gly Pro Val Thr Ala Gln Val Val Leu Gln Ala Ala Ile 1 5 10 15 Pro Leu Thr Ser 20 <210> 121 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 121 Val Thr Ala Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys 1 5 10 15 Gly Ser Ser Pro 20 <210> 122 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 122 Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser Pro Val 1 5 10 15 Pro Gly Thr Thr 20 <210> 123 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 123 Ile Pro Leu Thr Ser Cys Gly Ser Ser Pro Val Pro Gly Thr Thr Asp 1 5 10 15 Gly His Arg Pro 20 <210> 124 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 124 Cys Gly Ser Ser Pro Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr 1 5 10 15 Ala Glu Ala Pro 20 <210> 125 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 125 Val Pro Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro Asn 1 5 10 15 Thr Thr Ala Gly 20 <210> 126 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 126 Asp Gly His Arg Pro Thr Ala Glu Ala Pro Asn Thr Thr Ala Gly Gln 1 5 10 15 Val Pro Thru Thr 20 <210> 127 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 127 Thr Ala Glu Ala Pro Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu 1 5 10 15 Val Val Gly Thr 20 <210> 128 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 128 Asn Thr Thr Ala Gly Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr 1 5 10 15 Pro Gly Gln Ala 20 <210> 129 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 129 Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr Pro Gly Gln Ala Pro 1 5 10 15 Thr Ala Glu Pro 20 <210> 130 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 130 Glu Val Val Gly Thr Thr Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser 1 5 10 15 Gly Thr Thr Ser 20 <210> 131 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 131 Thr Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val 1 5 10 15 Gln Val Pro Thr 20 <210> 132 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 132 Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln Val Pro Thr Thr 1 5 10 15 Glu Val Ile Ser 20 <210> 133 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 133 Ser Gly Thr Thr Ser Val Gln Val Pro Thr Thr Glu Val Ile Ser Thr 1 5 10 15 Ala Pro Val Gln 20 <210> 134 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 134 Val Gln Val Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met 1 5 10 15 Pro Thr Ala Glu 20 <210> 135 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 135 Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr Ala Glu Ser 1 5 10 15 Thr Gly Met Thr 20 <210> 136 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 136 Thr Ala Pro Val Gln Met Pro Thr Ala Glu Ser Thr Gly Met Thr Pro 1 5 10 15 Glu Lys Val Pro 20 <210> 137 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 137 Met Pro Thr Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val 1 5 10 15 Ser Glu Val Met 20 <210> 138 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 138 Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val Met Gly 1 5 10 15 Thr Thr Leu Ala 20 <210> 139 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 139 Pro Glu Lys Val Pro Val Ser Glu Val Met Gly Thr Thr Leu Ala Glu 1 5 10 15 Met Ser Thr Pro 20 <210> 140 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 140 Val Ser Glu Val Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu 1 5 10 15 Ala Thr Gly Met 20 <210> 141 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 141 Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met Thr 1 5 10 15 Pro Ala Glu Val 20 <210> 142 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 142 Glu Met Ser Thr Pro Glu Ala Thr Gly Met Thr Pro Ala Glu Val Ser 1 5 10 15 Ile Val Val Leu 20 <210> 143 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 143 Glu Ala Thr Gly Met Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser 1 5 10 15 Gly Thr Thr Ala 20 <210> 144 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 144 Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 1 5 10 15 Gln Val Thr Thr 20 <210> 145 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 145 Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala Gln Val Thr Thr Thr 1 5 10 15 Glu Trp Val Glu 20 <210> 146 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 146 Ser Gly Thr Thr Ala Ala Gln Val Thr Thr Thr Glu Trp Val Glu Thr 1 5 10 15 Thr Ala Arg Glu 20 <210> 147 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 147 Ala Gln Val Thr Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu 1 5 10 15 Pro Ile Pro Glu 20 <210> 148 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 148 Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro Ile Pro Glu Pro 1 5 10 15 Glu Gly Pro Asp 20 <210> 149 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 149 Thr Thr Ala Arg Glu Leu Pro Ile Pro Glu Pro Glu Gly Pro Asp Ala 1 5 10 15 Ser Ser Ile Met 20 <210> 150 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 150 Leu Pro Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser 1 5 10 15 Thr Glu Ser Ile 20 <210> 151 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 151 Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu Ser Ile Thr 1 5 10 15 Gly Ser Leu Gly 20 <210> 152 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 152 Ala Ser Ser Ile Met Ser Thr Glu Ser Ile Thr Gly Ser Leu Gly Pro 1 5 10 15 Leu Leu Asp Gly 20 <210> 153 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 153 Ser Thr Glu Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr 1 5 10 15 Ala Thr Leu Arg 20 <210> 154 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 154 Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu 1 5 10 15 Val Lys Arg Gln 20 <210> 155 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 155 Pro Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu Val Lys Arg Gln Val 1 5 10 15 Pro Leu Asp Cys 20 <210> 156 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 156 Thr Ala Thr Leu Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val 1 5 10 15 Leu Tyr Arg Tyr 20 <210> 157 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 157 Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr Gly 1 5 10 15 Ser Phe Ser Val 20 <210> 158 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 158 Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val Thr 1 5 10 15 Leu Asp Ile Val 20 <210> 159 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 159 Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln 1 5 10 15 Gly Ile Glu Ser 20 <210> 160 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 160 Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala 1 5 10 15 Glu Ile Leu Gln 20 <210> 161 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 161 Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala Glu Ile Leu Gln Ala 1 5 10 15 Val Pro Ser Gly 20 <210> 162 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 162 Gln Gly Ile Glu Ser Ala Glu Ile Leu Gln Ala Val Pro Ser Gly Glu 1 5 10 15 Gly Asp Ala Phe 20 <210> 163 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 163 Ala Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly Asp Ala Phe Glu 1 5 10 15 Leu Thr Val Ser 20 <210> 164 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 164 Ala Val Pro Ser Gly Glu Gly Asp Ala Phe Glu Leu Thr Val Ser Cys 1 5 10 15 Gln Gly Gly Leu 20 <210> 165 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 165 Glu Gly Asp Ala Phe Glu Leu Thr Val Ser Cys Gln Gly Gly Leu Pro 1 5 10 15 Lys Glu Ala Cys 20 <210> 166 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 166 Glu Leu Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met 1 5 10 15 Glu Ile Ser Ser 20 <210> 167 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 167 Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met Glu Ile Ser Ser Pro 1 5 10 15 Gly Cys Gln Pro 20 <210> 168 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 168 Pro Lys Glu Ala Cys Met Glu Ile Ser Ser Pro Gly Cys Gln Pro Pro 1 5 10 15 Ala Gln Arg Leu 20 <210> 169 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 169 Met Glu Ile Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys 1 5 10 15 Gln Pro Val Leu 20 <210> 170 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 170 Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val Leu Pro 1 5 10 15 Ser Pro Ala Cys 20 <210> 171 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 171 Pro Ala Gln Arg Leu Cys Gln Pro Val Leu Pro Ser Pro Ala Cys Gln 1 5 10 15 Leu Val Leu His 20 <210> 172 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 172 Cys Gln Pro Val Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln 1 5 10 15 Ile Leu Lys Gly 20 <210> 173 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 173 Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys Gly Gly 1 5 10 15 Ser Gly Thr Tyr 20 <210> 174 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 174 Gln Leu Val Leu His Gln Ile Leu Lys Gly Gly Ser Gly Thr Tyr Cys 1 5 10 15 Leu Asn Val Ser 20 <210> 175 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 175 Gln Ile Leu Lys Gly Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu 1 5 10 15 Ala Asp Thr Asn 20 <210> 176 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 176 Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser 1 5 10 15 Leu Ala Val Val 20 <210> 177 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 177 Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser Leu Ala Val Val Ser 1 5 10 15 Thr Gln Leu Ile 20 <210> 178 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 178 Leu Ala Asp Thr Asn Ser Leu Ala Val Val Ser Thr Gln Leu Ile Met 1 5 10 15 Pro Gly Gln Glu 20 <210> 179 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 179 Ser Leu Ala Val Val Ser Thr Gln Leu Ile Met Pro Gly Gln Glu Ala 1 5 10 15 Gly Leu Gly Gln 20 <210> 180 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 180 Ser Thr Gln Leu Ile Met Pro Gly Gln Glu Ala Gly Leu Gly Gln Val 1 5 10 15 Pro Leu Ile Val 20 <210> 181 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 181 Met Pro Gly Gln Glu Ala Gly Leu Gly Gln Val Pro Leu Ile Val Gly 1 5 10 15 Ile Leu Leu Val 20 <210> 182 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 182 Ala Gly Leu Gly Gln Val Pro Leu Ile Val Gly Ile Leu Leu Val Leu 1 5 10 15 Met Ala Val Val 20 <210> 183 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 183 Val Pro Leu Ile Val Gly Ile Leu Leu Val Leu Met Ala Val Val Leu 1 5 10 15 Ala Ser Leu Ile 20 <210> 184 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 184 Gly Ile Leu Leu Val Leu Met Ala Val Val Leu Ala Ser Leu Ile Tyr 1 5 10 15 Arg Arg Arg Leu 20 <210> 185 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 185 Leu Met Ala Val Val Leu Ala Ser Leu Ile Tyr Arg Arg Arg Leu Met 1 5 10 15 Lys Gln Asp Phe 20 <210> 186 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 186 Leu Ala Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp Phe Ser 1 5 10 15 Val Pro Gln Leu 20 <210> 187 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 187 Tyr Arg Arg Arg Leu Met Lys Gln Asp Phe Ser Val Pro Gln Leu Pro 1 5 10 15 His Ser Ser Ser 20 <210> 188 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 188 Met Lys Gln Asp Phe Ser Val Pro Gln Leu Pro His Ser Ser Ser His 1 5 10 15 Trp Leu Arg Leu 20 <210> 189 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 189 Ser Val Pro Gln Leu Pro His Ser Ser Ser His Trp Leu Arg Leu Pro 1 5 10 15 Arg Ile Phe Cys 20 <210> 190 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 190 Pro His Ser Ser Ser His Trp Leu Arg Leu Pro Arg Ile Phe Cys Ser 1 5 10 15 Cys Pro Ile Gly 20 <210> 191 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 191 His Trp Leu Arg Leu Pro Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu 1 5 10 15 Asn Ser Pro Leu 20 <210> 192 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 192 Pro Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu 1 5 10 15 Ser Gly Gln Gln 20 <210> 193 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide fragments <400> 193 Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu Ser 1 5 10 15 Gly Gln Gln Val 20 <210> 194 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> gp100449-458 <400> 194 Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu 1 5 10 <210> 195 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> NY-ESO-1125-133 <400> 195 Glu Phe Thr Val Ser Gly Asn Ile Leu 1 5

Claims

1. A nucleic acid molecule comprising (i) a first nucleotide sequence encoding an anti-gp100 TCR, the anti-gp100 TCR being a recombinant T cell receptor (TCR), or an antigen-binding portion thereof, that specifically binds an epitope of human gp100 consisting of the amino acid sequence set forth in SEQ ID NO: 13, wherein the epitope is complexed with an HLA class I molecule HLA-B*40 allele; wherein the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a variable domain comprising an α chain CDR1, an α chain CDR2, and an α chain CDR3; and wherein the β chain comprises a variable domain comprising a β chain CDR1, a β chain CDR2, and a β chain CDR3; and wherein: (a) The amino acid sequence of the β chain CDR3 of the anti-gp100 TCR is shown in SEQ ID NO: 10; (b) the amino acid sequence of the β chain CDR2 of the anti-gp100 TCR is shown in SEQ ID NO: 9; (c) the amino acid sequence of the β chain CDR1 of the anti-gp100 TCR is shown in SEQ ID NO: 6; (d) the amino acid sequence of the α chain CDR3 of the anti-gp100 TCR is shown in SEQ ID NO: 7; (e) the amino acid sequence of the α chain CDR2 of the anti-gp100 TCR is shown in SEQ ID NO: 8; and (f) the amino acid sequence of the α chain CDR1 of the anti-gp100 TCR is shown in SEQ ID NO: 5; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCR.

2. The nucleic acid molecule of claim 1 , wherein the HLA class I molecule HLA-B*40 allele is selected from the group consisting of an HLA-B*40:01 allele, an HLA-B*40:02 allele, an HLA-B*40:03 allele, an HLA-B*40:04 allele, an HLA-B*40:05 allele, and an HLA-B*40:06 allele.

3. The nucleic acid molecule of claim 1, wherein (i) the α 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 variable domain of the β chain 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).

4. The nucleic acid molecule of claim 3, wherein the α chain of the anti-gp100 TCR further comprises a constant region, wherein the constant region of the α chain comprises an amino acid sequence having at least 90% sequence identity to the constant region present in the amino acid sequence set forth in SEQ ID NO:

1.

5. The nucleic acid molecule of claim 3, wherein 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 90% sequence identity to the constant region present in the amino acid sequence set forth in SEQ ID NO:

2.

6. The nucleic acid molecule according to any one of claims 1 to 5, wherein (i) the α chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 1; (ii) the β chain of the anti-gp100 TCR comprises the amino acid sequence set forth in SEQ ID NO: 2; or (iii) Both (i) and (ii).

7. The nucleic acid molecule according to any one of claims 1 to 5, wherein the second nucleotide sequence (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 a constant region of the endogenous TCR; (ii) encoding Cas9; or (iii) Both (i) and (ii).

8. The nucleic acid molecule of claim 7, wherein the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 53-56.

9. A vector comprising the nucleic acid molecule according to any one of claims 1 to 8.

10. The vector according to claim 9, wherein the vector is a viral vector, a mammalian vector or a bacterial vector. The vector according to claim 9 , which is a retroviral vector.

12. The vector of claim 9, wherein the vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, 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. The vector according to claim 9 , which is a lentiviral vector.

14. A cell comprising the nucleic acid molecule of any one of claims 1 to 8 or the vector of any one of claims 9 to 13.

15. The cell of claim 14, further expressing CD3.

16. The cell of claim 14, which is a T cell.

17. The cell of claim 14, which is a natural killer T (NKT) cell or an ILC cell.

18. The cell of claim 14, which is a natural killer (NK) cell.

19. Use of the cell of any one of claims 14 to 16 in the preparation of a medicament for treating melanoma in a subject in need thereof.

20. The use according to claim 19, wherein the melanoma is relapsed or refractory.

21. The use of claim 19, wherein the melanoma is locally advanced.

22. The use of claim 19, wherein the melanoma is in an advanced stage.

23. The use of claim 19, wherein the melanoma is metastatic.

24. The use of claim 19, wherein the cell is obtained from the subject.

25. The use of claim 19, wherein the cells are obtained from a donor other than the subject.

26. A method of engineering cells that target an antigen, the method comprising transducing cells collected from a subject in need of T cell therapy with the nucleic acid molecule of any one of claims 1 to 8 or the vector of any one of claims 9 to 13.

27. The method of claim 26, wherein the antigen-targeted cells further express CD3.

28. The method of claim 26, wherein the cell is a T cell.

29. The method of claim 26, wherein the cells are natural killer (NK) cells.

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