T cell receptors and methods of use thereof

Abstract

This disclosure relates to a recombinant T-cell receptor capable of binding to the gp100 epitope and a nucleic acid molecule encoding said recombinant T-cell receptor. In some embodiments, the nucleic acid molecule further comprises a second nucleotide sequence, wherein said second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCRs. Other aspects of this disclosure relate to a vector comprising said nucleic acid molecule and a cell comprising said recombinant TCR, said nucleic acid molecule, or said vector. Other aspects of this disclosure relate to methods of using said recombinant TCR, said nucleic acid molecule, said vector, and said cell. In some embodiments, said methods include treating a subject with cancer in need.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims priority benefit of U.S. Provisional Application 62 / 813,651, filed March 4, 2019, which is incorporated by reference in its entirety.

[0003] REFERENCE TO SEQUENCE LISTING FILE SUBMITTED ELECTRONICALLY

[0004] The content of the electronically submitted sequence listing in ASCII text file (Name: 4285_007PC01_Seqlisting_ST25.txt, Size: 18,388 bytes; and Date of Creation: March 3, 2020) is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0005] The present disclosure provides recombinant T cell receptors (“TCRs”) that specifically bind human gp100 and uses thereof. BACKGROUND

[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 agent development, and the adoptive transfer of anti-tumor T cells has 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 orders of magnitude. Elucidating T cell epitopes derived from shared antigens can facilitate robust development of effective and safe adoptive T cell therapies readily accessible to a large cohort of cancer patients. However, the absolute number of non-mutated antigens and the high polymorphism of HLA genes can 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 capable of specifically binding the epitopes. These novel epitopes are associated with specific HLA alleles. The use of these tumor-reactive HLA-restricted gp100 TCRs supports broadening the applicability of anti-gp100 TCR gene therapy, particularly in immuno-oncology. SUMMARY

[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 to human gplOO ("anti-gplOO 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-gplOO TCR cross-competes for binding to human gplOO with a reference TCR, which reference TCR comprises an alpha chain and a beta chain, and wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as 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 to human gplOO ("anti-gplOO 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-gplOO TCR binds to the same epitope or an overlapping epitope of human gplOO as a reference TCR, which reference TCR comprises an alpha chain and a beta chain, wherein the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0012] In some embodiments, the anti-gplOO TCR binds to an epitope of gplOO consisting of an amino acid sequence as 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 nucleic acid molecule of claim 4, wherein 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 nucleic acid molecule of claim 4, wherein the HLA Class I molecule is an HLA-A*24 allele. In some embodiments, the nucleic acid molecule of any one of claims 4 to 6, wherein the HLA Class I molecule is selected from the group consisting of an HLA-A*24:01 allele, an HLA-A*24:02 allele, and an HLA-A*24:03 allele. In some embodiments, the nucleic acid molecule of any one of claims 4 to 7, wherein the HLA Class I molecule is an HLA-A*24:01 allele.

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

[0016] In some embodiments, the α-chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:5. In some embodiments, the β-chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:8. In some embodiments, the α-chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:6. In some embodiments, the β-chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as listed 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 present in the amino acid sequence listed 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 listed in SEQ ID NO:2.

[0018] In some embodiments, the anti-gp100 TCR α-chain further comprises a constant region, wherein said constant region is different from the endogenous constant region of said α-chain. In some embodiments, the anti-gp100 TCR α-chain further comprises a constant region, wherein said 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 with the constant region present in the amino acid sequence listed 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 listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR β-chain further comprises a constant region, wherein said constant region is different from the endogenous constant region of said β-chain.

[0019] In some embodiments, the anti-gp100 TCR β-chain further comprises a constant region, wherein the β-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 with the constant region present in the amino acid sequence listed in SEQ ID NO:2. In some embodiments, the β-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 listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR α-chain comprises an amino acid sequence as listed in SEQ ID NO:1.

[0020] In some embodiments, the β-chain of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:2. In some embodiments, the second nucleotide sequence is one or more siRNAs that reduce the expression of endogenous TCRs.

[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 NO:53-56.

[0022] In some implementations, 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, β-chain constant region, or both comprise an amino acid sequence having at least one, at least two, at least three, at least four, or at least five substituted amino acid sequences relative to the endogenous TCR within the target sequence.

[0024] Certain aspects of this disclosure relate to a vector comprising the nucleic acid molecules disclosed herein. In some embodiments, the vector is a viral vector, a mammalian vector, or a bacterial vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of: adenovirus vectors, lentiviruses, Sendai virus vectors, baculovirus vectors, Epstein-Barr viral vectors, multivaccinia virus vectors, vaccinia virus vectors, herpes simplex virus vectors, hybrid vectors, and adeno-associated virus (AAV) vectors. In some embodiments, the vector is a lentivirus.

[0025] Certain aspects of this disclosure relate to a T-cell receptor (TCR) or its antigen-binding portion comprising the α-chain variable domain of the anti-gp100 TCR disclosed herein and the β-chain variable domain of the anti-gp100 TCR disclosed herein. In some embodiments, the recombinant T-cell receptor (TCR) specifically binding to human gp100 or its antigen-binding portion (“anti-gp100 TCR”) cross-competitively binds 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 as listed in SEQ ID NO:1 and the β chain comprises the amino acid sequence as listed 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 constant region of the α chain 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 listed in SEQ ID NO:1, or (ii) the constant region of the β chain 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 of SEQ ID NO:2.

[0026] Certain aspects of this disclosure relate to a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-gp100 TCR”) that specifically binds to human gp100, having the same or overlapping epitopes as a reference TCR binding to human gp100; wherein the reference TCR comprises an α chain and a β chain, and wherein the α chain comprises an amino acid sequence as listed in SEQ ID NO:1 and the β chain comprises an amino acid sequence as listed 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 constant region of the α chain comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence listed in SEQ ID NO:1, or (ii) the constant region of the β chain comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region present in the amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 consisting of an amino acid sequence as listed 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 anti-gp100 TCR as described in claim 41 or 42, wherein the HLA class I molecule is the HLA-A*24 allele. In some embodiments, the anti-gp100 TCR as described in any one of claims 41 to 43, wherein the HLA class I molecule is selected from the HLA-A*24:01 allele, the HLA-A*24:02 allele, and the HLA-A*24:03 allele. In some embodiments, the anti-gp100 TCR as described in any one of claims 41 to 44, wherein the HLA class I molecule is the HLA-A*24:02 allele.

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

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

[0030] In some embodiments, the α-chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:5. In some embodiments, the β-chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:8. In some embodiments, the α-chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:6. In some embodiments, the β-chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as listed 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 listed 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 listed in SEQ ID NO:2.

[0032] In some embodiments, 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 with the amino acid sequence present in the constant region listed in SEQ ID NO:1.

[0033] In some embodiments, the β-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 with the amino acid sequence present in the constant region listed in SEQ ID NO:2.

[0034] In some embodiments, the α-chain of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:1. In some embodiments, the β-chain of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:2.

[0035] Certain aspects of this 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, or 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”). 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 scFv. In some embodiments, the first and second antigen-binding domains are covalently linked or associated. In some embodiments, the first and second antigen-binding domains are linked by a peptide bond.

[0036] Certain aspects of this disclosure relate to a cell comprising the nucleic acid molecules disclosed herein, the vectors disclosed herein, the TCRs disclosed herein, the recombinant TCRs disclosed herein, or the bispecific TCRs disclosed herein. In some embodiments, the cell further expresses CD3. In some embodiments, the cell is selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, or ILC cells.

[0037] Certain aspects of this disclosure relate to a method of treating a subject with cancer, the method comprising administering the cells disclosed herein to the subject. 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, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease. Diseases, 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 cancer, central nervous system (CNS) sarcoma, 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 thereof.

[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 prior to administration of 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 interleukins. 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 this disclosure relate to a method for engineering cells that target an antigen, the method comprising transducing cells collected from a subject requiring T-cell therapy using nucleic acids or vectors disclosed herein. In some embodiments, the antigen-targeting cells further express CD3. In some embodiments, the cells are T cells or natural killer (NK) cells.

[0041] Some aspects of this 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 a β2m domain, and wherein the peptide consists of an amino acid sequence as listed 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-A. In some embodiments, the HLA class I molecule is the HLA-A*24 allele. In some embodiments, the HLA class I molecule is selected from the HLA-A*24:01 allele, the HLA-A*24:02 allele, and the HLA-A*24:03 allele. In some embodiments, the HLA class I molecule is the HLA-A*24:01 allele. In some embodiments, the HLA class I molecule is the HLA-A*24:02 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 this disclosure relate to an antigen-presenting cell (APC) that contains HLA class I molecules disclosed herein. In some embodiments, the HLA class I molecules are expressed on the surface of the APC.

[0045] Some aspects of this disclosure relate to a method for 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 contact, the enriched population of T cells contains a higher number of T cells capable of binding the HLA class I molecule compared to the number of T cells capable of binding the HLA class I molecule before the contact.

[0046] Some aspects of this disclosure relate to a method for 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 an amino acid sequence as listed in SEQ ID NO:13, wherein after the contact, the enriched population of T cells contains a higher number of T cells capable of targeting tumor cells compared to the number of T cells capable of targeting tumor cells before the contact.

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

[0048] Some aspects of this disclosure relate to a method for treating a tumor in a subject in need, the method comprising administering to the subject an enriched population of T cells disclosed herein.

[0049] Some aspects of this disclosure relate to a method for enhancing cytotoxic T-cell-mediated cancer cell targeting in a subject with cancer, the method comprising administering to the subject a peptide having an amino acid sequence as listed in SEQ ID NO:13.

[0050] Some aspects of this disclosure relate to a cancer vaccine comprising a peptide having an amino acid sequence as listed in SEQ ID NO:13.

[0051] Some aspects of this disclosure relate to a method for selecting T cells capable of targeting tumor cells, the method comprising contacting a population of isolated T cells in vitro with a peptide, wherein the peptide comprises an amino acid sequence as listed in SEQ ID NO:11. In some embodiments, the T cells are tumor-infiltrating lymphocytes (TILs). Attached Figure Description

[0052] Figures 1A-1D This is a graphical representation of A*24:02 / gp100-intron 4 multimer staining in melanoma TILs. TILs were stimulated once with A*24:02-artificial APCs treated with a pulse of gp100-intron 4 peptide. The results are shown prior to stimulation (day 0). Figure 1A and 1C ) and 14 days after stimulation (day 14; Figure 1B and 1D A*24:02 / gp100-intron4( Figures 1A-1B Or refer to A*24:02 / HTLV-1 tax 301-309 polymers ( Figures 1C-1D The staining data shows CD8. + T cell multimer + Percentage of cells.

[0053] Figure 2 Bar graph illustrating the functional assessment of A*24:02 / gp100-intron 4 multimer-positive melanoma TILs. A*24:02-positive TILs produce IFN-γ in an HLA-A*24:02-restricted peptide-specific manner. TILs were used as responding cells in the IFN-γ ELISPOT assay. HLA-A*24:02-transduced T2 cells (T2-A*24:02) were generated. T2 or T2-A*24:02 cells treated with the indicated peptide pulse were used as stimulating cells. HTLV-1 titration was employed. 301-309 Peptides were used as controls. Triples were performed, and the error bars show the standard deviation (SD). **P < 0.01, ***P < 0.001.

[0054] Figures 3A-3I This image shows a graphical representation of positive staining in Jurkat 76 / CD8 cells transduced with the A*24:02 / gp100-intron 4 TCR gene under homopolymer conditions. The A*24:02 / gp100-intron 4 TCR gene will be used as the gene's signature. Figure 3B , 3E Jurkat 76 / CD8 cells transduced with A*24:02 / gp100-intron 4 multimer (and 3H) were used. Figure 3B Staining was performed using A*24:02 / HTLV-1 tax.301-309 polymers ( Figure 3D , 3E And 3F), A*24:02 / unexchangeable polymer ( Figure 3G , 3H and 3I) and use A*24:02 / MAGE-A4 143-151 TCR (cloning MA24); Figure 3C , 3F and 3I) transduction and non-transduction ( Figure 3A , 3D Jurkat76 / CD8 cells (and 3G) were used as controls. Multimers were observed. + CD8 + The percentage of T cells.

[0055] Figures 4A-4D For use in the case of homopolymers, the A*24:02 / gp100-intron 4 TCR gene ( Figure 4B and 4D The image shows a graph of positive staining in primary human T cells transduced with A*24:02 / gp100-intron 4. Primary T cells transduced with A*24:02 / gp100-intron 4 TCR are then labeled with A*24:02 / gp100-intron 4. Figure 4B ) or A*24:02 / HTLV-1 tax 301-309 control polymer ( Figure 4D Staining. Untransduced primary T cells were used as a negative control. Figure 4A and 4C (Displays polymers) + CD8 + The percentage of T cells.

[0056] Figure 5 To illustrate the strong response of human primary T cells transduced with the A*24:02 / gp100-intron 4 TCR gene to homologous peptides presented by target class I molecules, a bar graph is used. In the IFN-γ ELISPOT analysis, primary T cells transduced with or without the A*24:02 / gp100-intron 4 TCR gene were used as responding cells (x-axis). The gp100-intron 4-activated HTLV-1 tax... 301-309 T2 or T2-A*24:02 cells treated with peptide (control) pulses were used as stimulating cells. Triples were used in the experiment, and the error bar shows the SD. **P<0.01.

[0057] Figure 6AThis is a graphical representation illustrating the recognition of tumor cells by primary T cells transduced with the A*24:02 / gp100-intron 4 TCR gene. In the IFN-γELISPOT analysis, primary T cells transduced with or without the A*24:02 / gp100-intron 4 TCR gene were used as responding cells. Figure 6B ( Figure 6A The illustration (as indicated) shows that Malme-3M, SK-MEL-28, and A375 cells, either untransduced or transduced with HLA-A*24:02 and / or gp100-intron 4, were used as stimulating cells. Experiments were performed in triplicate, and error bars are shown for SD. *P<0.05, **P<0.01, ***P<0.001.

[0058] Figures 7A-7E Graphical representation of the expression of endogenous gp100 or transduced gp100-intron 4 gene. The expression of endogenous gp100 or transduced gp100-intron 4 gene in target cells was analyzed by intracellular flow cytometry after staining with anti-gp100 mAb (hollow curve) and isotype control (solid curve).

[0059] Figures 8A-8F Expression of ΔNGFR in target cells transduced with the full-length HLA-A*24:02 gene labeled with ΔNGFR ( Figure 8B , 8D The graphs of ΔNGFR and 8F are shown. Surface expression of ΔNGFR in target cells transduced with the full-length HLA-A*24:02 gene labeled with ΔNGFR was analyzed by flow cytometry after staining with anti-NGFR mAb (hollow curve) and isotype control (solid curve). ΔNGFR alone was used as a control ( Figure 8A , 8C and 8E). Detailed Implementation

[0060] This disclosure relates to a TCR or its antigen-binding portion that specifically binds to an epitope on gp100, a nucleic acid molecule encoding said TCR, and a cell containing said TCR or said nucleic acid molecule. Some aspects of this disclosure relate to methods of treating cancer in a subject of need, said methods including administering said cells to said subject. Other aspects of this disclosure relate to HLA class I molecules complexed with a peptide containing an epitope of gp100.

[0061] I. Terminology

[0062] To make this disclosure more readily understandable, certain terms are first defined. As used in this application, each of the following terms shall have the meaning set forth below unless expressly provided otherwise herein. Other definitions are set forth in this application.

[0063] It should be noted that the term "a / an" refers to one or more of the entities described; for example, "a nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a / an," "one or more / species," and "at least one / species" are used interchangeably herein.

[0064] Furthermore, when used herein, "and / or" should be considered as specifically disclosing each of the two specified features or components, in the presence or absence of the other. Therefore, 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 herein 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).

[0065] The term “about” is used herein to mean approximately, roughly, about, or near. When the term “about” is used in conjunction with a numerical range, it modifies the range by expanding the boundaries above and below the stated value. Generally, the term “about” is used herein to modify values ​​that are higher or lower than the stated value by a deviation of 10% upwards or downwards (higher or lower).

[0066] It should be understood that wherever the word “comprising” is used in this document to describe aspects, other similar aspects described in other ways by the terms “composed of” and / or “substantially composed of” are also provided.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, the 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 the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide those skilled in the art with comprehensive dictionaries of many terms used in this disclosure.

[0068] Units, prefixes, and symbols are represented in their form as accepted by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' direction. Amino acid sequences are written from left to right in an amino-to-carboxyl direction. The headings provided herein are not intended to limit the various aspects of this disclosure and may be taken as such by reference to the specification as a whole. Therefore, the terms defined below immediately are defined more fully by reference to this specification in its entirety.

[0069] "Administration" means the physical introduction of an agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracapsular, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route (e.g., orally). Other non-parenteral routes include local, transdermal, or transmucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. It can also be applied, for example, once, multiple times, and / or over one or more extended time periods.

[0070] As used herein, the term “T cell receptor” (TCR) refers to a heterogeneous cell surface receptor capable of specifically interacting with a target antigen. As used herein, “TCR” includes, but is not limited to, naturally occurring and non-naturally occurring TCRs; full-length TCRs and their antigen-binding portions; chimeric TCRs; TCR fusion constructs; and synthetic TCRs. In humans, TCRs are expressed on the surface of T cells and are responsible for T cell recognition and targeting by antigen-presenting cells. Antigen-presenting cells (APCs) display fragments of foreign proteins (antigens) that are complexed with the major histocompatibility complex (MHC; also referred to herein as complexed with HLA molecules, such as HLA class 1 molecules). TCRs recognize and bind to the antigen:HLA complex and recruit CD3 (expressed by T cells), thereby activating the TCR. Activated TCRs initiate downstream signaling and immune responses, including the disruption of EPCs.

[0071] Generally, a TCR may consist of two chains interconnected by disulfide bonds, an α-chain and a β-chain (or less commonly, a γ-chain and a δ-chain). Each chain contains variable domains (α-chain variable domains and β-chain variable domains) and constant regions (α-chain constant regions and β-chain constant regions). The variable domains are located at the distal end of the cell membrane and interact with the antigen. The constant regions are located at the proximal end of the cell membrane. A TCR may also contain a transmembrane region and a short cytoplasmic tail. As used herein, the term "constant region" encompasses both the transmembrane region and the cytoplasmic tail (when present) as well as the conventional "constant region".

[0072] The variable domains can be further subdivided into highly variable regions, called complementarity-determining regions (CDRs), which are scattered with more conserved regions called framework regions (FRs). Each α-chain and β-chain variable domain contains three CDRs and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Each variable domain contains a binding domain that interacts with the 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 the antigen, while CD2 is believed to primarily recognize the HLA complex.

[0073] Unless explicitly stated otherwise, and unless the context otherwise indicates, the term “TCR” also includes antigen-binding fragments or portions 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 bound 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 expressed or modified to express CD3 as described herein), or TCRs without a cell membrane as described herein (e.g., isolated TCRs or soluble TCRs).

[0074] "Antigen-binding molecule," "part of TCR," or "TCR fragment" refers to any portion of the TCR smaller than the whole. Antigen-binding molecules may include antigen complementarity-determining regions (CDRs).

[0075] "Antigen" refers to any molecule, such as a peptide, that elicits an immune response or is capable of binding to a TCR. As used herein, "epitope" refers to a portion of a polypeptide that elicits an immune response or is capable of binding to a TCR. An immune response may involve antibody production or activation of cells with specific immune activity, or both. Those skilled in the art will readily understand that any macromolecule, including virtually all proteins or peptides, can act as an antigen. Antigens and / or epitopes may be expressed endogenously, i.e., expressed from genomic DNA, or may be recombinantly expressed. Antigens and / or epitopes may be specific to a particular tissue (such as cancer cells), or they may be widely expressed. Furthermore, fragments of larger molecules may act as antigens. In one embodiment, the antigen is a tumor antigen. Epitopes may be present in longer polypeptides (e.g., proteins), or epitopes may be present as fragments of longer polypeptides. In some embodiments, the epitope is complexed with a major histocompatibility complex (MHC; also referred to herein as complexed with HLA molecules, such as HLA class 1 molecules).

[0076] 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 kDa (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 its variants and fragments. Known variants of gp100 are available at www.uniprot.org (UniProtKB-P40967; last accessed March 1, 2019).

[0077] Table 1. Amino acid sequence of gp100

[0078]

[0079] 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 cell surfaces and participate in the activation of immune responses. HLA class I genes encode MHC class I molecules, which are expressed on cell surfaces as 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 immune responses to target and destroy antigen-presenting cells displaying non-self proteins.

[0080] 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. Therefore, "HLA class I molecule" and "MHC class I molecule" are used interchangeably in this article.

[0081] MHC class I molecules consist of 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 described in SEQ ID NO:16 (Table 2). The α chain of MHC class I molecules 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 multiple functions. The HLA gene is highly variable, with more than 20,000 HLA alleles and associated 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 accessed February 27, 2019). At least three genes encoding MHC class I α chain proteins are present in the HLA complex: HLA-A, HLA-B, and HLA-C. In addition, HLA-E, HLA-F, and HLA-G encode proteins that associate with MHC class I molecules.

[0082] Table 2. Amino acid sequence of human β2m

[0083]

[0084] The term "autologous" means any material derived from the same individual as the individual into whom the material is subsequently introduced. For example, autologous T-cell therapy involves administering T cells isolated from the same individual to a subject. The term "allogeneic" means any material derived from an individual and subsequently introduced into another individual of the same species. For example, allogeneic T-cell transplantation involves administering T cells obtained from a donor other than the subject to a subject.

[0085] "Cancer" refers to a wide range of diseases characterized by the 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 can also metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors. Examples of cancers treatable by the methods of this invention include, but are not limited to, cancers of the immune system, including lymphoma, leukemia, and other white blood cell malignancies. In some embodiments, the method of the present invention can be used to reduce the size of tumors originating from, for example, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or ocular malignant melanoma, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or ocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric 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 bowel cancer, and endocrine system cancers. 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 tumor, central nervous system (CNS) spurs, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, 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 thereof. Certain cancers may be responsive to chemotherapy or radiation therapy, or the cancer may be refractory. Refractory cancers are those that are not suitable for surgical intervention and that initially do not respond to chemotherapy or radiation therapy or become unresponsive over time.

[0086] As used in this article, "antitumor effect" refers to biological effects that can exist in the following forms: reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, reduction in the number of metastases, increase in overall survival or progression-free survival, increase in life expectancy, or improvement in various physiological symptoms associated with tumors. Antitumor effect can also refer to the prevention of tumor development, such as through vaccines.

[0087] The term “progression-free survival” may be abbreviated as PFS, which, as used herein, refers to the time from the date of treatment to the date of disease progression or death from any cause according to the revised IWG Response Criteria for Malignant Lymphoma.

[0088] "Disease progression" or "progressive disease" may be abbreviated as PD, which, as used herein, refers to the worsening of one or more symptoms associated with a specific disease. For example, disease progression in a subject with cancer may include an increase in the number or size of one or more malignant lesions, tumor metastasis, and death.

[0089] "Duration of response" can be abbreviated as DOR, which, as used in this article, refers to the period between the date a subject first objectively responds and the date on which disease progression or death is confirmed according to the revised IWG response guidelines for malignant lymphoma.

[0090] The term "overall survival" can be abbreviated as OS, which is defined as the time from the date of treatment to the date of death.

[0091] 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 recipient cells. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL)7 and IL-15, promote immune cell survival and proliferation, and pro-inflammatory 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 pro-inflammatory 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).

[0092] Chemokines are a class of cytokines that mediate cellular chemotaxis or directed movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eosinophil chemokine-3, macrophage-derived chemokines (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), γ-inducible protein 10 (IP-10), and thymus and activation-regulating chemokines (TARC or CCL17).

[0093] Other examples of analytes and cytokines in this 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), and leukemia inhibitory factor (LIF). Oncogene M (OSM), CD154, lymphotoxin (LT)β, 4-1BB ligand (4-1BBL), proliferation-inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-associated ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF-associated and ApoL-associated leukocyte-expressed ligand 1 (TALL-1) or TNF-associated apoptosis-inducing ligand (TRAIL).

[0094] The “therapeutic effective amount,” “effective dose,” “effective amount,” or “therapeutic effective dose” of a drug or therapeutic agent is any amount by which a drug, when used alone or in combination with another therapeutic agent, prevents the onset of disease in a subject or promotes disease regression proven by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of injury or disability caused by the suffering caused by the disease. 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 that predict efficacy in humans, or by assaying activity in vitro using an assay agent.

[0095] 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 that represent a major component of the innate immune system. NK cells repel tumor cells 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 require activation to kill cells. T cells play a major role in cell-mediated immunity (not involving antibodies). The T cell receptor (TCR) distinguishes T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T-killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells ((i) stem cell-like memory T cells), and memory T cells ((i) stem cell-like memory T cells). SCMCells (such as naïve 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 exhibit many of the unique functional characteristics of memory cells; (ii) central memory T 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 (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKTs), and γδ T cells. On the other hand, B cells play a major role in humoral immunity (involving antibodies). B cells produce antibodies and antigens and act as antigen-presenting cells (APCs), and become memory B cells after activation through antigen-antigen interactions. In mammals, immature B cells form in the bone marrow, from which the name B cell originates.

[0096] The terms "genetic engineering" or "engineering" refer to methods of modifying the genome of a cell, including but not limited to deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof. In some embodiments, the modified cells are lymphocytes, such as T cells, or modified cells expressing CD3, which may be obtained from a patient or donor. Cells may be modified to express exogenous constructs incorporated into the cell's genome, such as the T cell receptor (TCR) disclosed herein. In some embodiments, cells are modified to express CD3.

[0097] "Immune response" refers to the action of cells of the immune system (such as T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which cause 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 cases of autoimmunity or pathological inflammation in vertebrates.

[0098] The term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting or relapsing from a disease by means of methods including inducing, enhancing, suppressing, or otherwise altering the 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.

[0099] Cells used in the immunotherapy described herein may be derived from any source known in the art. For example, T cells may be distinguishable from hematopoietic stem cell populations in vitro, or T cells may be obtained from the subject. T cells may be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from sources known to those skilled in the art, such as FICOLL. TM Any number of techniques for isolating and / or apheresis of blood components can be used to collect a unit of blood from a subject. 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 a subject, wherein the modified cells express CD3 and the TCR disclosed herein. In some embodiments, the modified cells are not T cells.

[0100] As used herein, “patient” includes anyone with cancer (such as lymphoma or leukemia). The terms “subject” and “patient” are used interchangeably in this document.

[0101] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a sequence that may contain a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to a short chain, which is also commonly referred to in the art, for example, as a peptide, oligopeptide, and oligomer; and to a longer chain, which is commonly referred to in the art, as a protein, 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, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0102] As used herein, “stimulus” refers to a major response induced by the binding of a stimulating molecule to its homologous ligand, wherein the binding mediates a signal transduction event. A “stimulating molecule” is a molecule on a T cell that specifically binds to a homologous stimulating ligand present on an antigen-presenting cell, such as the T cell receptor (TCR) / CD3 complex. A “stimulating ligand” is a ligand present on an antigen-presenting cell (e.g., aAPC, dendritic cells, B cells, and similar cells) that can specifically bind to a stimulating molecule on a T cell, thereby mediating a major response induced by the T cell, including but not limited to activation, initiation of an immune response, proliferation, and similar reactions. Stimulating ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, hyperagonist anti-CD28 antibodies, and hyperagonist anti-CD2 antibodies.

[0103] The terms “treatment” and “pretreatment” are used interchangeably herein and refer to preparation for a patient requiring T-cell therapy for an appropriate situation. Treatment as used herein includes, but is not limited to, reducing the number of endogenous lymphocytes prior to T-cell therapy, removing the cytokine sink, increasing serum levels of one or more homeostatic cytokines or pro-inflammatory 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 serum levels of one or more cytokines, such as interleukin-7 (IL-7), interleukin-15 (IL-15), interleukin-10 (IL-10), interleukin-5 (IL-5), γ-inducible 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, "treatment" includes increasing serum levels of IL-7, IL-15, IP-10, MCP-1, PLGF, CRP, or any combination thereof.

[0104] Treatment / treating of a subject refers to any type of intervention or treatment administered to a subject or to a subject with the aim of reversing, alleviating, improving, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or lesions or biochemical markers associated with the disease. In one implementation, treatment / treating includes partial remission. In another implementation, treatment / treating includes complete remission.

[0105] The use of substitution (e.g., "or") should be understood to mean one, both, or any combination of the substitutes. As used herein, the indefinite article "a / kind" should be understood to mean "one / kind or more / kinds" of any of the described or enumerated components.

[0106] The terms “about” or “substantially comprise” refer to a specific value or composition that, as determined by a person skilled in the art, is within an acceptable margin of error, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measuring system. For example, “about” or “substantially comprise” may mean within one or more standard deviations according to convention in the art. Alternatively, “about” or “substantially comprise” may mean a range of up to 10% (i.e., ±10%). For example, about 3 mg may include any number between 2.7 mg and 3.3 mg (for 10%). Furthermore, specifically in relation to biological systems or methods, the term may mean up to an order of magnitude or up to five times the value. When specific values ​​or compositions are provided in this application and claims, unless otherwise stated, the meaning of “about” or “substantially comprise” should be assumed to be within an acceptable margin of error for said specific value or composition.

[0107] Unless otherwise stated, as described herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range and (where appropriate) its fraction (such as tenths and percentes of an integer).

[0108] The various aspects of the invention are described in more detail in the following sections.

[0109] II. Compositions disclosed herein

[0110] This disclosure relates to a T-cell receptor (TCR) or its antigen-binding portion that specifically binds to an epitope on gp100, a nucleic acid molecule encoding said TCR, and a cell containing said TCR or said nucleic acid molecule. Some aspects of the invention relate to a method of treating cancer in a subject of need, the method comprising administering to the subject cells containing the TCR described herein. Other aspects of this disclosure relate to an epitope of gp100 that binds to a TCR, and an HLA class I molecule complexed with a peptide containing the gp100 epitope.

[0111] T-cell receptors, or TCRs, are molecules present on the surface of T cells or T lymphocytes that are responsible for recognizing fragments of antigens as peptides that bind to the major histocompatibility complex (MHC) molecule. The binding between TCRs and antigenic peptides has a relatively low affinity and is degenerate: in other words, many TCRs recognize the same antigenic peptides and many antigenic peptides are recognized by the same TCRs.

[0112] The TCR consists of two distinct protein chains (in other words, it is a heterodimer). In humans, in 95% of T cells, the TCR is composed of an α (α) chain and a β (β) chain (encoded by TRA and TRB, respectively), but in 5% of T cells, the TCR is composed of a γ and a δ (γ / δ) chain (encoded by TRG and TRD, respectively). This ratio varies during ontogeny and in disease states (such as leukemia). It also differs between species. Orthologs of four loci have been located in various species. Each locus can produce a variety of polypeptides with constant and variable regions.

[0113] When the TCR binds to the antigenic peptide and MHC (peptide / MHC), T lymphocytes are activated through signal transduction, which is a series of biochemical events mediated by related enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0114] II.A. Nucleic acid molecules

[0115] Certain aspects of this disclosure relate to nucleic acid molecules comprising (i) a first nucleotide sequence encoding a recombinant TCR or its antigen-binding portion (“anti-gp100 TCR”) that specifically binds to human gp100; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or a polypeptide encoded by the second nucleotide sequence inhibits the 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 of nucleotides targeting and encoding an endogenous TCR. In some embodiments, the anti-gp100 TCR cross-competitively binds to human gp100 with a reference TCR. In some embodiments, the anti-gp100 TCR binds to the same or overlapping epitopes of human gp100 as the reference TCR.

[0116] In some embodiments, the reference TCR comprises an α chain and a β chain; wherein the α chain comprises complementarity-determining regions 1 (CDR1), CDR2, and 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 listed in SEQ ID NO:1 and the β chain comprises the amino acid sequence listed in SEQ ID NO:2.

[0117] Table 3. TCR sequences of α-chain and β-chain

[0118]

[0119]

[0120]

[0121] II.A.1. TCR encoded by the first nucleotide sequence

[0122] This 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 variable domains containing α chain CDR1, α chain CDR2, and α chain CDR3; and wherein the β chain comprises variable domains containing β chain CDR1, β chain CDR2, and β chain CDR3. In some embodiments, the anti-gp100 TCR comprises an α chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7 (CAVATDSWGKLQF). In some embodiments, the anti-gp100 TCR comprises a β chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10 (CASSLLPEGTGRVSGYTF). In some embodiments, the non-CDR regions in the α chain and / or β chain are further modified, for example, by substitution or mutation of one, two, three, four, five, or six amino acids, such that the α chain and / or β chain are not naturally occurring. In some implementations, substitution or mutation can improve the TCR 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 listed in SEQ ID NO:5 (DSAIYN). 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 listed in SEQ ID NO:8 (IQSSQRE).

[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 listed in SEQ ID NO:6 (PRHDT). 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 listed in SEQ ID NO:9 (FYEKMQ).

[0125] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α-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 with the α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α-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 with the α-chain amino acid sequence listed in SEQ ID NO:1, wherein the anti-gp100 TCR comprises an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence includes the α-chain variable domain present in the α-chain amino acid sequence listed in SEQ ID NO:1.

[0126] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β-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 with the β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β-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 with the β-chain amino acid sequence listed in SEQ ID NO:2, wherein the anti-gp100 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence includes a β-chain variable domain present in the amino acid sequence listed in SEQ ID NO:2.

[0127] 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 encoded by the first nucleotide sequence comprises an α-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the α-chain amino acid sequence listed in SEQ ID NO:1, wherein the anti-gp100 TCR comprises an α-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:7. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α-chain constant region present in the α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence further comprises an α-chain constant region 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 one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the α-chain amino acid sequence listed in SEQ ID NO:1.

[0128] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the β-chain amino acid sequence listed in SEQ ID NO:2, wherein the anti-gp100 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β-chain constant region present in the amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide also comprises a β-constant region different from an endogenous (e.g., naturally occurring) constant region of the β-chain. In some embodiments, the β-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 of the β-chain amino acid sequence listed in SEQ ID NO:2.

[0129] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α-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 with the α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises an α-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 with the α-chain amino acid sequence listed in SEQ ID NO:1, wherein the anti-gp100 TCR comprises an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-gp100TCR encoded by the first nucleotide sequence comprises an α-chain containing the amino acid sequence listed in SEQ ID NO:1.

[0130] In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β-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 with the β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide sequence comprises a β-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 with the β-chain amino acid sequence listed in SEQ ID NO:2, wherein the anti-gp100 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the anti-gp100TCR encoded by the first nucleotide sequence comprises a β chain containing the amino acid sequence listed 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 one, at least two, at least three, at least four, or at least five substituted amino acid sequences relative to the endogenous TCR within the target sequence.

[0132] II.A.2. Epitope

[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 listed in SEQ ID NO:13 (VYFFLPDHL). In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 consisting of an amino acid sequence as listed in SEQ ID NO:13. In some embodiments, the epitope consists of intron 4 of gp100 (SEQ ID NO:52), for example, "gp100-intron 4".

[0134] In some implementations, the epitope is complexed with an HLA class I molecule. The human leukocyte antigen (HLA) system (the 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 T cell receptors (TCRs) on T cells. (See also Overview of the Immune System.) Antigen-presenting MHC molecules are divided into two main classes: class I MHC molecules and class II MHC molecules.

[0135] Class I MHC molecules exist as transmembrane glycoproteins on the surface of all nucleated cells. A complete class I molecule consists of an α-heavy chain bound to a β-2 microglobulin molecule. The heavy chain comprises two peptide-binding domains, an Ig-like domain, and a transmembrane region with a cytoplasmic tail. The heavy chain of class I molecules is encoded by genes at the HLA-A, HLA-B, and HLA-C loci. T cells expressing CD8 molecules respond to class I MHC molecules. These lymphocytes often possess cytotoxic functions, thus requiring the ability to recognize any infected cells. 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 plays a role in protecting the fetus from maternal immune responses) and HLA-E (which presents peptides to certain receptors on natural killer (NK) cells).

[0136] In some embodiments, HLA class 1 molecules are selected from HLA-A, HLA-B, and HLA-C alleles. In some embodiments, HLA class 1 molecules are selected from HLA-E, HLA-F, and HLA-G alleles. In some embodiments, HLA class 1 molecules are HLA-A alleles. In some embodiments, HLA class 1 molecules are HLA-B alleles. In some embodiments, HLA class 1 molecules are HLA-C alleles.

[0137] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any of the known alleles may be used in this 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 selected from the following HLA-A alleles: HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-A*25, HLA-A*26, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*32, HLA-A*33, HLA-A*34, HLA-A*36, HLA-A*43, HLA-A*66, HLA-A*68, HLA-A*69, HLA-A*74, and HLA-A*80. In some embodiments, the HLA-A allele is the HLA-A*24:01 allele. In some embodiments, the HLA-A allele is the HLA-A*24:02 allele. In some embodiments, the HLA-A allele is the HLA-A*24:03 allele. In some embodiments, the HLA-A allele is the HLA-A*24:11 allele. In some embodiments, the HLA-A allele is the HLA-A*24:23 allele. In some embodiments, the HLA-A allele is the HLA-A*24:24 allele. In some embodiments, the HLA-A allele is the HLA-A*24:25 allele. In some embodiments, the HLA-A allele is the HLA-A*24:26 allele.

[0138] In certain embodiments, the HLA class I molecule is an HLA-A allele selected from the group consisting of: HLA-A*24:02:01:01, HLA-A*24:02:01:02, HLA-A*24:02:01:03, HLA-A*24:02:01:04, HLA-A*24:02:01:05, HLA-A*24:02:01:06, HLA-A*24:02:01:07, HLA-A*24:02:01:08, HLA-A*24:02:01:09, HLA-A*24:02:01:10, HLA-A*24:02:01:11, HLA-A*24:02:01:12, HLA-A*24:02:01:13, HLA-A*24:02:01:14, HLA-A*24:02:01:15, HLA-A*24:02:01:16, HLA-A*24:02:01:17, HLA-A*24:02:01:18, HLA-A*24:02:01:19, HLA-A*24:02:01:20, HLA-A*24:02:02, HLA-A*24:02:03, HLA-A*24:02:04, HLA-A*24:02:05, HLA-A*24:02:06, HLA-A*24:02:07, HLA-A*24:02:08, HLA-A*24:02:09, HLA-A*24:02:10, HLA-A*24:02:100, HLA-A*24:02:101, HLA-A*24:02:102, HLA-A*24:02:103, HLA-A*24:02:104, HLA-A*24:02:105, HLA-A*24:02:106, HLA-A*24:02:107, HLA-A*24:02:108, HLA-A*24:02:109, HLA-A*24:02:11, HLA-A*24:02:110, HLA-A*24:02:111, HLA-A*24:02:112, HLA-A*24:02:113, HLA-A*24:02:114, HLA-A*24:02:115, HLA-A*24:02:12, HLA-A*24:02:13, HLA-A*24:02:14, HLA-A*24:02:15, HLA-A*24:02:16, HLA-A*24:02:17, HLA-A*24:02:18, HLA-A*24:02:19, HLA-A*24:02:20, HLA-A*24:02:21, HLA-A*24:02:22, HLA-A*24:02:23, HLA-A*24:02:24,HLA-A*24:02:25、HLA-A*24:02:26、HLA-A*24:02:27、HLA-A*24:02:28、HLA-A*24:02:29、HLA-A*24:02:30、HLA-A*24:02:31、HLA-A*24:02:32、HLA-A*24:02:33、HLA-A*24:02:34、HLA-A*24:02:35、HLA-A*24:02:36、HLA-A*24:02:37、HLA-A*24:02:38、HLA-A*24:02:39、HLA-A*24:02:40、HLA-A*2 4:02:41、HLA-A*24:02:42、HLA-A*24:02:43、HLA-A*24:02:44、HLA-A*24:02:45、HLA-A*24:02:46、HLA-A*24:02:47、HLA-A*24:02:48、HLA-A*24:02:49、HLA-A*24:02:50、HLA-A*24:02:51、HLA-A*24:02:52、HLA-A*24:02:53、HLA-A*24:02:54、HLA-A*24:02:55、HLA-A*24:02:56、HLA-A*24:02:57、 HLA-A*24:02:58、HLA-A*24:02:59、HLA-A*24:02:60、HLA-A*24:02:61、HLA-A*24:02:62、HLA-A*24:02:63、HLA-A*24:02:64、HLA-A*24:02:65、HLA-A*24:02:66、HLA-A*24:02:67、HLA-A*24:02:68、HLA-A*24:02:69、HLA-A*24:02:70、HLA-A*24:02:71、HLA-A*24:02:72、HLA-A*24:02:73、HLA-A*2 4:02:74、HLA-A*24:02:75、HLA-A*24:02:76、HLA-A*24:02:77、HLA-A*24:02:78、HLA-A*24:02:79、HLA-A*24:02:80、HLA-A*24:02:81、HLA-A*24:02:82、HLA-A*24:02:83、HLA-A*24:02:84、HLA-A*24:02:85、HLA-A*24:02:86、HLA-A*24:02:87、HLA-A*24:02:88、HLA-A*24:02:89、HLA-A*24:02:90、HLA-A*24:02:91, HLA-A*24:02:92, HLA-A*24:02:93, HLA-A*24:02:94, HLA-A*24:02:95, HLA-A*24:02:96, HLA-A*24:02:97, HLA-A*24:02:98 and HLA-A*24:02:99. In some embodiments, the class I HLA molecule is an HLA-A allele selected from the group consisting of: HLA-A*24:03:01:01, HLA-A*24:03:01:02, HLA-A*24:03:02, HLA-A*24:03:03, HLA-A*24:03:04, HLA-A*24:04, HLA-A*24:05:01, HLA-A*24:05:02, HLA-A*24:06, HLA-A*24:07:01, HLA-A*24:07:02, HLA-A*24:08, HLA-A*24:09, HLA-A*24:100, HLA-A*24:101, HLA-A*24:102, HLA-A*24:103, HLA-A*24:104, HLA-A*24:105, HLA-A*24:106, HLA-A*24:107, HLA-A*24:108, HLA-A*24:109, HLA-A*24:10:01, HLA-A*24:10:02, HLA-A*24:110, HLA-A*24:111, HLA-A*24:112, HLA-A*24:113, HLA-A*24:114, HLA-A*24:115, HLA-A*24:116, HLA-A*24:117, HLA-A*24:118, HLA-A*24:119, HLA-A*24:11, HLA-A*24:120, HLA-A*24:121, HLA-A*24:122, HLA-A*24:123, HLA-A*24:124, HLA-A*24:125, HLA-A*24:126, HLA-A*24:127, HLA-A*24:128, HLA-A*24:129, HLA-A*24:130, HLA-A*24:131, HLA-A*24:132, HLA-A*24:133, HLA-A*24:134, HLA-A*24:135:01, HLA-A*24:135:02, HLA-A*24:136, HLA-A*24:137, HLA-A*24:138, HLA-A*24:139, HLA-A*24:13:01, HLA-A*24:13:02, HLA-A*24:140, HLA-A*24:141HLA-A*24:142:01、HLA-A*24:142:02、HLA-A*24:143、HLA-A*24:144、HLA-A*24:145、HLA-A*24:146、HLA-A*24:147、HLA-A*24:148、HLA-A*24:149、HLA-A*24:14:01:01、HLA-A*24:14:01:02、HLA-A*24:14:01:03、HLA-A*24:15、HLA-A*24:150、HLA-A*24:151、HLA-A*24:152、HLA-A*24:153、HLA-A*24: 154、HLA-A*24:155、HLA-A*24:156、HLA-A*24:157、HLA-A*24:158、HLA-A*24:159、HLA-A*24:160、HLA-A*24:161、HLA-A*24:162、HLA-A*24:163、HLA-A*24:164、HLA-A*24:165、HLA-A*24:166、HLA-A*24:167、HLA-A*24:168、HLA-A*24:169、HLA-A*24:17、HLA-A*24:170、HLA-A*24:171、HLA-A*24:172: 01、HLA-A*24:172:02、HLA-A*24:173、HLA-A*24:174、HLA-A*24:175、HLA-A*24:176、HLA-A*24:177、HLA-A*24:178、HLA-A*24:179、HLA-A*24:18、HLA-A*24:180、HLA-A*24:181、HLA-A*24:182、HLA-A*24:183、HLA-A*24:184、HLA-A*24:185、HLA-A*24:186、HLA-A*24:187、HLA-A*24:188、HLA-A-24:18 9、HLA-A*24:19、HLA-A*24:190、HLA-A*24:191、HLA-A*24:192、HLA-A*24:193、HLA-A*24:194、HLA-A*24:195、HLA-A*24:196、HLA-A*24:197、HLA-A*24:198、HLA-A*24:199、HLA-A*24:200、HLA-A*24:201、HLA-A*24:202、HLA-A*24:203、HLA-A*24:204、HLA-A*24:205、HLA-A*24:206、HLA-A*24:207:01、HLA-A*24:207:02、HLA-A*24:208:01、HLA-A*24:208:02、HLA-A*24:209、HLA-A*24:20:01:01、HLA-A*24:20:01:02、HLA-A*24:210、HLA-A*24:212、HLA-A*24:213、HLA-A*24:214、HLA-A*24:215、HLA-A*24:216、HLA-A*24:217、HLA-A*24:218、HLA-A*24:219、HLA-A*24:21:01、HLA-A*24:21:02、HLA- A*24:21:03、HLA-A*24:22、HLA-A*24:220、HLA-A*24:221、HLA-A*24:222、HLA-A*24:223、HLA-A*24:224、HLA-A*24:225:01、HLA-A*24:225:02、HLA-A*24:226:01、HLA-A*24:226:02、HLA-A*24:227、HLA-A*24:228、HLA-A*24:229、HLA-A*24:23、HLA-A*24:230、HLA-A*24:231、HLA-A*24:232、HLA-A*2 4:233、HLA-A*24:234、HLA-A*24:235、HLA-A*24:236、HLA-A*24:237、HLA-A*24:238、HLA-A*24:239、HLA-A*24:24、HLA-A*24:240、HLA-A*24:241、HLA-A*24:242、HLA-A*24:243、HLA-A*24:244、HLA-A*24:245、HLA-A*24:246、HLA-A*24:247、HLA-A*24:248、HLA-A*24:249、HLA-A*24:25、HLA-A*24:2 50、HLA-A*24:251、HLA-A*24:252、HLA-A*24:253、HLA-A*24:254、HLA-A*24:255、HLA-A*24:256、HLA-A*24:257、HLA-A*24:258、HLA-A*24:259、HLA-A*24:26、HLA-A*24:260、HLA-A*24:261、HLA-A*24:262、HLA-A*24:263、HLA-A*24:264、HLA-A*24:265、HLA-A*24:266、HLA-A*24:267、HLA-A*24:268、HLA-A*24:269、HLA-A*24:27、HLA-A*24:270、HLA-A*24:271、HLA-A*24:272、HLA-A*24:273、HLA-A*24:274、HLA-A*24:275、HLA-A*24:276、HLA-A*24:277、HLA-A*24:278、HLA-A*24:279、HLA-A*24:28、HLA-A*24:280、HLA-A*24:281、HLA-A*24:282、HLA-A*24:283、HLA-A*24:284、HLA-A*24:285、HLA -A*24:286、HLA-A*24:287、HLA-A*24:288、HLA-A*24:289、HLA-A*24:29、HLA-A*24:29、HLA-A*24:290、HLA-A*24:291、HLA-A*24:292、HLA-A*24:293、HLA-A*24:294、HLA-A*24:295、HLA-A*24:296、HLA-A*24:297、HLA-A*24:298、HLA-A*24:299、HLA-A*24:30、HLA-A*24:300、HLA-A*24:301、HLA-A*24:302、HLA-A* 24:303、HLA-A*24:304、HLA-A*24:305、HLA-A*24:306、HLA-A*24:307、HLA-A*24:308、HLA-A*24:309、HLA-A*24:31、HLA-A*24:310:01、HLA-A*24:310:02、HLA-A*24:311、HLA-A*24:312、HLA-A*24:313:01、HLA-A*24:313:02、HLA-A*24:314、HLA-A*24:315、HLA-A*24:316、HLA-A*24:317、HLA-A*24: 318、HLA-A*24:319、HLA-A*24:32、HLA-A*24:320、HLA-A*24:321、HLA-A*24:322、HLA-A*24:323、HLA-A*24:324、HLA-A*24:325、HLA-A*24:326、HLA-A*24:327、HLA-A*24:328、HLA-A*24:329、HLA-A*24:33、HLA-A*24:330、HLA-A*24:331、HLA-A*24:332、HLA-A*24:333、HLA-A*24:334、HLA-A-24:335、HLA-A*24:336、HLA-A*24:337、HLA-A*24:338、HLA-A*24:339、HLA-A*24:34、HLA-A*24:340、HLA-A*24:341、HLA-A*24:342、HLA-A*24:343、HLA-A*24:344、HLA-A*24:345、HLA-A*24:346、HLA-A*24:347:01、HLA-A*24:347:02、HLA-A*24:348、HLA-A*24:349、HLA-A*24:35、HLA-A*24:350、HLA-A*24:351 ,HLA-A*24:352,HLA-A*24:353,HLA-A*24:354,HLA-A*24:355,HLA-A*24:356,HLA-A*24:357,HLA-A*24:358,HLA-A*24:359,HLA-A*24:360,HLA-A*24:361,HLA-A*24:362,HLA-A*24:363,HLA-A*24:364,HLA-A*24:365,HLA-A*24:366,HLA-A*24:367,HLA-A*24:368,HLA-A*24:369,HLA-A*24:36,HLA -A*24:37, HLA-A*24:370, HLA-A*24:371, HLA-A*24:372, HLA-A*24:373, HLA-A*24:374, HLA-A*24:375, HLA-A*24:376, HLA-A*24:377, HLA-A*24:378, HLA-A*24:379, HLA-A*24:38, HLA-A*24:380, HLA-A*24:381, HLA-A*24:382, HLA-A*24:383, HLA-A*24:384, HLA-A*24:385, HLA-A*24:386, HLA-A*24 :387、HLA-A*24:388、HLA-A*24:389、HLA-A*24:39、HLA-A*24:390、HLA-A*24:391、HLA-A*24:392、HLA-A*24:393、HLA-A*24:394、HLA-A*24:395、HLA-A*24:396、HLA-A*24:397、HLA-A*24:398、HLA-A*24:399、HLA-A*24:400、HLA-A*24:401、HLA-A*24:402、HLA-A*24:403、HLA-A*24:404、HLA-A*24:405,HLA-A*24:406、HLA-A*24:407、HLA-A*24:408、HLA-A*24:409、HLA-A*24:40、HLA-A*24:41、HLA-A*24:410、HLA-A*24:411、HLA-A*24:412、HLA-A*24:413、HLA-A*24:414、HLA-A*24:415、HLA-A*24:416、HLA-A*24:417、HLA-A*24:418、HLA-A*24:419、HLA-A*24:42、HLA-A*24:420、HLA-A*24:421、HLA-A* 24:422、HLA-A*24:423、HLA-A*24:424、HLA-A*24:425、HLA-A*24:426、HLA-A*24:427、HLA-A*24:428、HLA-A*24:429、HLA-A*24:43、HLA-A*24:430、HLA-A*24:431、HLA-A*24:432、HLA-A*24:433、HLA-A*24:44、HLA-A*24:45、HLA-A*24:46、HLA-A*24:47、HLA-A*24:48、HLA-A*24:49、HLA-A*24:50、HLA- A*24:51、HLA-A*24:52、HLA-A*24:53、HLA-A*24:54、HLA-A*24:55、HLA-A*24:56、HLA-A*24:57、HLA-A*24:58、HLA-A*24:59、HLA-A*24:60、HLA-A*24:61、HLA-A*24:62、HLA-A*24:63、HLA-A*24:64、HLA-A*24:66、HLA-A*24:67、HLA-A*24:68、HLA-A*24:69、HLA-A*24:70、HLA-A*24:71、HLA-A*24:72、HL A-A*24:73、HLA-A*24:74:01、HLA-A*24:74:02、HLA-A*24:75、HLA-A*24:76、HLA-A*24:77、HLA-A*24:78、HLA-A*24:79、HLA-A*24:80、HLA-A*24:81、HLA-A*24:82、HLA-A*24:83、HLA-A*24:84、HLA-A*24:85、HLA-A*24:86、HLA-A*24:87、HLA-A*24:88、HLA-A*24:89、HLA-A*24:90:01、HLA-A*24:90:02、HLA-A*24:91, HLA-A*24:92, HLA-A*24:93, HLA-A*24:94, HLA-A*24:95, HLA-A*24:96, HLA-A*24:97, HLA-A*24:98 and HLA-A*24:99. ,

[0139] II.A.3. Second nucleotide sequence

[0140] The second nucleotide sequence of the nucleic acid molecule disclosed herein may be any sequence capable of inhibiting the expression of endogenous TCR or any polypeptide capable of encoding the inhibition of endogenous TCR expression. In some embodiments, the second nucleotide sequence is one or more siRNAs. In some embodiments, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the endogenous TCR. In some embodiments, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the wild-type human TCR. In some embodiments, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the α chain of the wild-type TCR. In some embodiments, one or more siRNAs are complementary to a target sequence within the nucleotide sequence encoding a constant region of the β chain of the wild-type TCR. In some embodiments, one or more siRNAs comprise (i) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding a constant region of the α chain of the wild-type TCR and (ii) one or more siRNAs complementary to a target sequence within the nucleotide sequence encoding a constant region of the β chain of the wild-type TCR.

[0141] In some embodiments, one or more siRNAs comprise nucleotide sequences selected from the group consisting of SEQ ID NO:53-56 (Table 4). In some embodiments, a second nucleotide sequence of a nucleic acid molecule encodes one or more siRNAs, wherein said one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the α chain of a wild-type TCR, and wherein said one or more siRNAs comprise nucleic acid sequences listed in SEQ ID NO:53 and 54.

[0142] Table 4. siRNA sequences

[0143]

[0144] 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 the β chain of a wild-type TCR, and wherein the one or more siRNAs comprise the nucleic acid sequences listed in SEQ ID NO: 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 complementary to a target sequence within a nucleotide sequence encoding a constant region of the α chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences listed in SEQ ID NO: 53 and 54; and (ii) one or more siRNAs complementary to a target sequence within a nucleotide sequence encoding a constant region of the β chain of a wild-type TCR, wherein the one or more siRNAs comprise the nucleic acid sequences listed in SEQ ID NO: 55 and 56.

[0145] In some embodiments, the second nucleotide sequence of the nucleic acid molecule comprises SEQ ID NO:53-56. In some embodiments, the second nucleotide sequence comprises SEQ ID NO:53-56, wherein one or more of SEQ ID NO:53-56 are separated by one or more nucleic acids that do not encode siRNA. In some embodiments, the one or more siRNAs are selected from the siRNA disclosed in U.S. Publication No. 2010 / 0273213A1, which is incorporated herein by reference in its entirety.

[0146] In some embodiments, the second nucleotide sequence of the nucleic acid molecule encodes a protein capable of inhibiting the expression of an endogenous (e.g., wild-type) TCR. In some embodiments, the second nucleotide sequence encodes Cas9.

[0147] II.A.3. Carrier

[0148] Certain aspects of this invention relate to vectors comprising nucleic acid molecules disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral particle or virus. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a bacterial vector.

[0149] In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is selected from the group consisting of adenovirus vectors, lentiviruses, Sendai virus, baculovirus vectors, Epstein-Barr virus vectors, lactoviral vectors, vaccinia virus vectors, herpes simplex virus vectors, and adeno-associated virus (AAV) vectors. In a particular embodiment, the vector is an AAV vector. In some embodiments, the vector is a lentivirus. In a particular embodiment, 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 this invention can be found in Huang and Kamihira, Biotechnol. Adv. 31(2):208-23(2103), which is incorporated herein by reference in its entirety.

[0150] II.B. Recombinant T-cell receptor (TCR)

[0151] Certain aspects of this invention relate to the specific binding of a recombinant T-cell receptor (TCR) of human gp100 or its antigen-binding moiety (“anti-gp100 TCR”). In some embodiments, the anti-gp100 TCR is encoded by a nucleic acid molecule disclosed herein.

[0152] In some embodiments, the anti-gp100 TCR cross-competitively binds to human gp100 with a reference TCR. In some embodiments, the anti-gp100 TCR binds to the same or overlapping epitopes of human gp100 as the reference TCR. 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 as listed in SEQ ID NO:1. In some embodiments, the β chain of the reference TCR comprises the amino acid sequence as listed in SEQ ID NO:2.

[0153] 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 constant region of the α chain comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the α chain comprising the amino acid sequence listed 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 constant region of the β chain comprises an amino acid sequence having at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the β chain comprising the amino acid sequence listed 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 (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 listed 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 listed in SEQ ID NO:2.

[0155] In some embodiments, the α-chain of the anti-gp100 TCR includes variable domains containing α-chain CDR1, α-chain CDR2, and α-chain CDR3; and the β-chain of the anti-gp100 TCR includes variable domains containing β-chain CDR1, β-chain CDR2, and β-chain CDR3. In some embodiments, the anti-gp100 TCR includes an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-gp100 TCR includes a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10.

[0156] In some embodiments, the α-chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:5. In some embodiments, the β-chain CDR1 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:8.

[0157] In some embodiments, the α-chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:6. In some embodiments, the β-chain CDR2 of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO:9.

[0158] In some embodiments, the anti-gp100 TCR comprises an α-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 with the variable domain of the α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR comprises an α-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 with the variable domain of the α-chain amino acid sequence listed in SEQ ID NO:1, wherein the anti-gp100 TCR comprises an α-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:7. In some embodiments, the anti-gp100 TCR comprises an α-chain variable domain present in the α-chain amino acid sequence listed in SEQ ID NO:1.

[0159] In some embodiments, the anti-gp100 TCR comprises a β-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 with the variable domain of the β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR comprises a β-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 with the variable domain of the β-chain amino acid sequence listed in SEQ ID NO:2, wherein the anti-gp100 TCR comprises a β-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:10. In some embodiments, the anti-gp100 TCR comprises a β-chain variable domain present in the β-chain amino acid sequence listed in SEQ ID NO:2.

[0160] 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 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 with the constant region of the α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR comprises an α-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the α-chain amino acid sequence listed in SEQ ID NO:1, wherein the anti-gp100 TCR comprises an α-chain CDR3 containing the amino acid sequence as listed 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 listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide also comprises an α-constant region 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 containing at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the constant region of the α-chain amino acid sequence listed in SEQ ID NO:1.

[0161] In some embodiments, the anti-gp100 TCR comprises a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the constant region of the β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR comprises a β-chain constant region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the constant region of the β-chain amino acid sequence listed in SEQ ID NO:2, wherein the anti-gp100 TCR comprises a β-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:10. In some embodiments, the anti-gp100 TCR comprises the β-chain constant region present in the β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR encoded by the first nucleotide also includes a β constant region, which is an endogenous (e.g., naturally occurring) constant region different from the β chain. In some embodiments, the β chain constant region includes 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 β chain amino acid sequence listed in SEQ ID NO:2.

[0162] In some embodiments, the anti-gp100 TCR comprises an α-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 with the α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-gp100 TCR comprises an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-gp100 TCR comprises an α-chain containing the amino acid sequence listed in SEQ ID NO:1.

[0163] In some embodiments, the anti-gp100 TCR comprises a β-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 with the β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-gp100 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:2.

[0164] 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 one, at least two, at least three, at least four, or at least five substituted amino acid sequences relative to the endogenous TCR within the target sequence.

[0165] II.B.2. Epitope

[0166] In some embodiments, the anti-gp100 TCR binds to the same epitope as the reference TCR. In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 comprising the amino acid sequence listed in SEQ ID NO:13. In some embodiments, the anti-gp100 TCR binds to an epitope of gp100 consisting of an amino acid sequence as listed in SEQ ID NO:13. In some embodiments, the epitope consists of an amino acid residue intron 4 of gp100 (SEQ ID NO:52), for example, "gp100-intron 4".

[0167] In some embodiments, the epitope is complexed with an HLA class I molecule. In some embodiments, the HLA class I molecule is selected from HLA-A, HLA-B, and HLA-C alleles. In some embodiments, the HLA class I molecule is selected from HLA-E, HLA-F, and HLA-G alleles. In some embodiments, the HLA class I molecule is an HLA-A allele. In some embodiments, the HLA class I molecule is an HLA-B allele. In some embodiments, the HLA class I molecule is an HLA-C allele.

[0168] Many HLA-A, HLA-B, and HLA-C alleles are known in the art, and any of the known alleles may be used in this 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 selected from the following HLA-A alleles: HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-A*25, HLA-A*26, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*32, HLA-A*33, HLA-A*34, HLA-A*36, HLA-A*43, HLA-A*66, HLA-A*68, HLA-A*69, HLA-A*74, and HLA-A*80. In some embodiments, the HLA-A allele is the HLA-A*24:01 allele. In some embodiments, the HLA-A allele is the HLA-A*24:02 allele. In some embodiments, the HLA-A allele is the HLA-A*24:03 allele. In some embodiments, the HLA-A allele is the HLA-A*24:11 allele. In some embodiments, the HLA-A allele is the HLA-A*24:23 allele. In some embodiments, the HLA-A allele is the HLA-A*24:24 allele. In some embodiments, the HLA-A allele is the HLA-A*24:25 allele. In some embodiments, the HLA-A allele is the HLA-A*24:26 allele.

[0169] In certain embodiments, the HLA class I molecule is an HLA-A allele selected from the group consisting of: HLA-A*24:02:01:01, HLA-A*24:02:01:02, HLA-A*24:02:01:03, HLA-A*24:02:01:04, HLA-A*24:02:01:05, HLA-A*24:02:01:06, HLA-A*24:02:01:07, HLA-A*24:02:01:08, HLA-A*24:02:01:09, HLA-A*24:02:01:10, HLA-A*24:02:01:11, HLA-A*24:02:01:12, HLA-A*24:02:01:13, HLA-A*24:02:01:14, HLA-A*24:02:01:15, HLA-A*24:02:01:16, HLA-A*24:02:01:17, HLA-A*24:02:01:18, HLA-A*24:02:01:19, HLA-A*24:02:01:20, HLA-A*24:02:02, HLA-A*24:02:03, HLA-A*24:02:04, HLA-A*24:02:05, HLA-A*24:02:06, HLA-A*24:02:07, HLA-A*24:02:08, HLA-A*24:02:09, HLA-A*24:02:10, HLA-A*24:02:100, HLA-A*24:02:101, HLA-A*24:02:102, HLA-A*24:02:103, HLA-A*24:02:104, HLA-A*24:02:105, HLA-A*24:02:106, HLA-A*24:02:107, HLA-A*24:02:108, HLA-A*24:02:109, HLA-A*24:02:11, HLA-A*24:02:110, HLA-A*24:02:111, HLA-A*24:02:112, HLA-A*24:02:113, HLA-A*24:02:114, HLA-A*24:02:115, HLA-A*24:02:12, HLA-A*24:02:13, HLA-A*24:02:14, HLA-A*24:02:15, HLA-A*24:02:16, HLA-A*24:02:17, HLA-A*24:02:18, HLA-A*24:02:19, HLA-A*24:02:20, HLA-A*24:02:21, HLA-A*24:02:22, HLA-A*24:02:23, HLA-A*24:02:24,HLA-A*24:02:25、HLA-A*24:02:26、HLA-A*24:02:27、HLA-A*24:02:28、HLA-A*24:02:29、HLA-A*24:02:30、HLA-A*24:02:31、HLA-A*24:02:32、HLA-A*24:02:33、HLA-A*24:02:34、HLA-A*24:02:35、HLA-A*24:02:36、HLA-A*24:02:37、HLA-A*24:02:38、HLA-A*24:02:39、HLA-A*24:02:40、HLA-A*2 4:02:41、HLA-A*24:02:42、HLA-A*24:02:43、HLA-A*24:02:44、HLA-A*24:02:45、HLA-A*24:02:46、HLA-A*24:02:47、HLA-A*24:02:48、HLA-A*24:02:49、HLA-A*24:02:50、HLA-A*24:02:51、HLA-A*24:02:52、HLA-A*24:02:53、HLA-A*24:02:54、HLA-A*24:02:55、HLA-A*24:02:56、HLA-A*24:02:57、 HLA-A*24:02:58、HLA-A*24:02:59、HLA-A*24:02:60、HLA-A*24:02:61、HLA-A*24:02:62、HLA-A*24:02:63、HLA-A*24:02:64、HLA-A*24:02:65、HLA-A*24:02:66、HLA-A*24:02:67、HLA-A*24:02:68、HLA-A*24:02:69、HLA-A*24:02:70、HLA-A*24:02:71、HLA-A*24:02:72、HLA-A*24:02:73、HLA-A*2 4:02:74、HLA-A*24:02:75、HLA-A*24:02:76、HLA-A*24:02:77、HLA-A*24:02:78、HLA-A*24:02:79、HLA-A*24:02:80、HLA-A*24:02:81、HLA-A*24:02:82、HLA-A*24:02:83、HLA-A*24:02:84、HLA-A*24:02:85、HLA-A*24:02:86、HLA-A*24:02:87、HLA-A*24:02:88、HLA-A*24:02:89、HLA-A*24:02:90、HLA-A*24:02:91, HLA-A*24:02:92, HLA-A*24:02:93, HLA-A*24:02:94, HLA-A*24:02:95, HLA-A*24:02:96, HLA-A*24:02:97, HLA-A*24:02:98 and HLA-A*24:02:99. In some embodiments, the class I HLA molecule is an HLA-A allele selected from the group consisting of: HLA-A*24:03:01:01, HLA-A*24:03:01:02, HLA-A*24:03:02, HLA-A*24:03:03, HLA-A*24:03:04, HLA-A*24:04, HLA-A*24:05:01, HLA-A*24:05:02, HLA-A*24:06, HLA-A*24:07:01, HLA-A*24:07:02, HLA-A*24:08, HLA-A*24:09, HLA-A*24:100, HLA-A*24:101, HLA-A*24:102, HLA-A*24:103, HLA-A*24:104, HLA-A*24:105, HLA-A*24:106, HLA-A*24:107, HLA-A*24:108, HLA-A*24:109, HLA-A*24:10:01, HLA-A*24:10:02, HLA-A*24:110, HLA-A*24:111, HLA-A*24:112, HLA-A*24:113, HLA-A*24:114, HLA-A*24:115, HLA-A*24:116, HLA-A*24:117, HLA-A*24:118, HLA-A*24:119, HLA-A*24:11, HLA-A*24:120, HLA-A*24:121, HLA-A*24:122, HLA-A*24:123, HLA-A*24:124, HLA-A*24:125, HLA-A*24:126, HLA-A*24:127, HLA-A*24:128, HLA-A*24:129, HLA-A*24:130, HLA-A*24:131, HLA-A*24:132, HLA-A*24:133, HLA-A*24:134, HLA-A*24:135:01, HLA-A*24:135:02, HLA-A*24:136, HLA-A*24:137, HLA-A*24:138, HLA-A*24:139, HLA-A*24:13:01, HLA-A*24:13:02, HLA-A*24:140, HLA-A*24:141HLA-A*24:142:01、HLA-A*24:142:02、HLA-A*24:143、HLA-A*24:144、HLA-A*24:145、HLA-A*24:146、HLA-A*24:147、HLA-A*24:148、HLA-A*24:149、HLA-A*24:14:01:01、HLA-A*24:14:01:02、HLA-A*24:14:01:03、HLA-A*24:15、HLA-A*24:150、HLA-A*24:151、HLA-A*24:152、HLA-A*24:153、HLA-A*24: 154、HLA-A*24:155、HLA-A*24:156、HLA-A*24:157、HLA-A*24:158、HLA-A*24:159、HLA-A*24:160、HLA-A*24:161、HLA-A*24:162、HLA-A*24:163、HLA-A*24:164、HLA-A*24:165、HLA-A*24:166、HLA-A*24:167、HLA-A*24:168、HLA-A*24:169、HLA-A*24:17、HLA-A*24:170、HLA-A*24:171、HLA-A*24:172: 01、HLA-A*24:172:02、HLA-A*24:173、HLA-A*24:174、HLA-A*24:175、HLA-A*24:176、HLA-A*24:177、HLA-A*24:178、HLA-A*24:179、HLA-A*24:18、HLA-A*24:180、HLA-A*24:181、HLA-A*24:182、HLA-A*24:183、HLA-A*24:184、HLA-A*24:185、HLA-A*24:186、HLA-A*24:187、HLA-A*24:188、HLA-A-24:18 9、HLA-A*24:19、HLA-A*24:190、HLA-A*24:191、HLA-A*24:192、HLA-A*24:193、HLA-A*24:194、HLA-A*24:195、HLA-A*24:196、HLA-A*24:197、HLA-A*24:198、HLA-A*24:199、HLA-A*24:200、HLA-A*24:201、HLA-A*24:202、HLA-A*24:203、HLA-A*24:204、HLA-A*24:205、HLA-A*24:206、HLA-A*24:207:01、HLA-A*24:207:02、HLA-A*24:208:01、HLA-A*24:208:02、HLA-A*24:209、HLA-A*24:20:01:01、HLA-A*24:20:01:02、HLA-A*24:210、HLA-A*24:212、HLA-A*24:213、HLA-A*24:214、HLA-A*24:215、HLA-A*24:216、HLA-A*24:217、HLA-A*24:218、HLA-A*24:219、HLA-A*24:21:01、HLA-A*24:21:02、HLA- A*24:21:03、HLA-A*24:22、HLA-A*24:220、HLA-A*24:221、HLA-A*24:222、HLA-A*24:223、HLA-A*24:224、HLA-A*24:225:01、HLA-A*24:225:02、HLA-A*24:226:01、HLA-A*24:226:02、HLA-A*24:227、HLA-A*24:228、HLA-A*24:229、HLA-A*24:23、HLA-A*24:230、HLA-A*24:231、HLA-A*24:232、HLA-A*2 4:233、HLA-A*24:234、HLA-A*24:235、HLA-A*24:236、HLA-A*24:237、HLA-A*24:238、HLA-A*24:239、HLA-A*24:24、HLA-A*24:240、HLA-A*24:241、HLA-A*24:242、HLA-A*24:243、HLA-A*24:244、HLA-A*24:245、HLA-A*24:246、HLA-A*24:247、HLA-A*24:248、HLA-A*24:249、HLA-A*24:25、HLA-A*24:2 50、HLA-A*24:251、HLA-A*24:252、HLA-A*24:253、HLA-A*24:254、HLA-A*24:255、HLA-A*24:256、HLA-A*24:257、HLA-A*24:258、HLA-A*24:259、HLA-A*24:26、HLA-A*24:260、HLA-A*24:261、HLA-A*24:262、HLA-A*24:263、HLA-A*24:264、HLA-A*24:265、HLA-A*24:266、HLA-A*24:267、HLA-A*24:268、HLA-A*24:269、HLA-A*24:27、HLA-A*24:270、HLA-A*24:271、HLA-A*24:272、HLA-A*24:273、HLA-A*24:274、HLA-A*24:275、HLA-A*24:276、HLA-A*24:277、HLA-A*24:278、HLA-A*24:279、HLA-A*24:28、HLA-A*24:280、HLA-A*24:281、HLA-A*24:282、HLA-A*24:283、HLA-A*24:284、HLA-A*24:285、HLA -A*24:286、HLA-A*24:287、HLA-A*24:288、HLA-A*24:289、HLA-A*24:29、HLA-A*24:29、HLA-A*24:290、HLA-A*24:291、HLA-A*24:292、HLA-A*24:293、HLA-A*24:294、HLA-A*24:295、HLA-A*24:296、HLA-A*24:297、HLA-A*24:298、HLA-A*24:299、HLA-A*24:30、HLA-A*24:300、HLA-A*24:301、HLA-A*24:302、HLA-A* 24:303、HLA-A*24:304、HLA-A*24:305、HLA-A*24:306、HLA-A*24:307、HLA-A*24:308、HLA-A*24:309、HLA-A*24:31、HLA-A*24:310:01、HLA-A*24:310:02、HLA-A*24:311、HLA-A*24:312、HLA-A*24:313:01、HLA-A*24:313:02、HLA-A*24:314、HLA-A*24:315、HLA-A*24:316、HLA-A*24:317、HLA-A*24: 318、HLA-A*24:319、HLA-A*24:32、HLA-A*24:320、HLA-A*24:321、HLA-A*24:322、HLA-A*24:323、HLA-A*24:324、HLA-A*24:325、HLA-A*24:326、HLA-A*24:327、HLA-A*24:328、HLA-A*24:329、HLA-A*24:33、HLA-A*24:330、HLA-A*24:331、HLA-A*24:332、HLA-A*24:333、HLA-A*24:334、HLA-A-24:335、HLA-A*24:336、HLA-A*24:337、HLA-A*24:338、HLA-A*24:339、HLA-A*24:34、HLA-A*24:340、HLA-A*24:341、HLA-A*24:342、HLA-A*24:343、HLA-A*24:344、HLA-A*24:345、HLA-A*24:346、HLA-A*24:347:01、HLA-A*24:347:02、HLA-A*24:348、HLA-A*24:349、HLA-A*24:35、HLA-A*24:350、HLA-A*24:351 ,HLA-A*24:352,HLA-A*24:353,HLA-A*24:354,HLA-A*24:355,HLA-A*24:356,HLA-A*24:357,HLA-A*24:358,HLA-A*24:359,HLA-A*24:360,HLA-A*24:361,HLA-A*24:362,HLA-A*24:363,HLA-A*24:364,HLA-A*24:365,HLA-A*24:366,HLA-A*24:367,HLA-A*24:368,HLA-A*24:369,HLA-A*24:36,HLA -A*24:37, HLA-A*24:370, HLA-A*24:371, HLA-A*24:372, HLA-A*24:373, HLA-A*24:374, HLA-A*24:375, HLA-A*24:376, HLA-A*24:377, HLA-A*24:378, HLA-A*24:379, HLA-A*24:38, HLA-A*24:380, HLA-A*24:381, HLA-A*24:382, HLA-A*24:383, HLA-A*24:384, HLA-A*24:385, HLA-A*24:386, HLA-A*24 :387、HLA-A*24:388、HLA-A*24:389、HLA-A*24:39、HLA-A*24:390、HLA-A*24:391、HLA-A*24:392、HLA-A*24:393、HLA-A*24:394、HLA-A*24:395、HLA-A*24:396、HLA-A*24:397、HLA-A*24:398、HLA-A*24:399、HLA-A*24:400、HLA-A*24:401、HLA-A*24:402、HLA-A*24:403、HLA-A*24:404、HLA-A*24:405,HLA-A*24:406、HLA-A*24:407、HLA-A*24:408、HLA-A*24:409、HLA-A*24:40、HLA-A*24:41、HLA-A*24:410、HLA-A*24:411、HLA-A*24:412、HLA-A*24:413、HLA-A*24:414、HLA-A*24:415、HLA-A*24:416、HLA-A*24:417、HLA-A*24:418、HLA-A*24:419、HLA-A*24:42、HLA-A*24:420、HLA-A*24:421、HLA-A* 24:422、HLA-A*24:423、HLA-A*24:424、HLA-A*24:425、HLA-A*24:426、HLA-A*24:427、HLA-A*24:428、HLA-A*24:429、HLA-A*24:43、HLA-A*24:430、HLA-A*24:431、HLA-A*24:432、HLA-A*24:433、HLA-A*24:44、HLA-A*24:45、HLA-A*24:46、HLA-A*24:47、HLA-A*24:48、HLA-A*24:49、HLA-A*24:50、HLA- A*24:51、HLA-A*24:52、HLA-A*24:53、HLA-A*24:54、HLA-A*24:55、HLA-A*24:56、HLA-A*24:57、HLA-A*24:58、HLA-A*24:59、HLA-A*24:60、HLA-A*24:61、HLA-A*24:62、HLA-A*24:63、HLA-A*24:64、HLA-A*24:66、HLA-A*24:67、HLA-A*24:68、HLA-A*24:69、HLA-A*24:70、HLA-A*24:71、HLA-A*24:72、HL A-A*24:73、HLA-A*24:74:01、HLA-A*24:74:02、HLA-A*24:75、HLA-A*24:76、HLA-A*24:77、HLA-A*24:78、HLA-A*24:79、HLA-A*24:80、HLA-A*24:81、HLA-A*24:82、HLA-A*24:83、HLA-A*24:84、HLA-A*24:85、HLA-A*24:86、HLA-A*24:87、HLA-A*24:88、HLA-A*24:89、HLA-A*24:90:01、HLA-A*24:90:02、HLA-A*24:91, HLA-A*24:92, HLA-A*24:93, HLA-A*24:94, HLA-A*24:95, HLA-A*24:96, HLA-A*24:97, HLA-A*24:98 and HLA-A*24:99. ,

[0170] II.B.3. Bispecific T-cell receptor (TCR)

[0171] Certain aspects of this 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 the TCR disclosed herein or its antigen-binding portion. In some embodiments, the first antigen-binding domain comprises a single-stranded variable fragment (“scFv”).

[0172] In some embodiments, the second antigen-binding domain specifically binds to proteins expressed on the surface of T cells. Any protein expressed on the surface of T cells can be targeted by the bispecific antibodies disclosed herein. In some embodiments, the protein expressed on the surface of T cells is not expressed by other cells. In some embodiments, the protein expressed on the surface of T cells is expressed on the surface of one or more other human immune cells. In some embodiments, the protein expressed on the surface of T cells is expressed on the surface of one or more other human immune cells, but not on the surface of human non-immune cells. In some embodiments, the second antigen-binding domain specifically binds to proteins selected from the group consisting of CD3, CD2, CD5, CD6, CD8, CD11a (LFA-1α), CD43, CD45, and CD53 expressed on the surface of T cells. In some embodiments, the second antigen-binding domain specifically binds to CD3. In some embodiments, the second antigen-binding domain includes scFv.

[0173] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are linked or associated via covalent bonds. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are linked via peptide bonds.

[0174] II.C. Cells expressing TCR

[0175] Certain aspects of this disclosure relate to cells comprising the nucleic acid molecules disclosed herein, the vectors disclosed herein, the recombinant TCRs disclosed herein, the bispecific TCRs disclosed herein, or any combination thereof. Any cell may be used in this disclosure.

[0176] In some embodiments, the cells express CD3. CD3 expression can be naturally occurring, for example, CD3 is expressed from a nucleic acid sequence that is endogenously expressed by the cell. For example, T cells and natural killer (NK) cells naturally express CD3. Therefore, in some embodiments, the cells are T cells or natural killer cells. In some embodiments, the cells are T cells selected from natural killer T (NKT) cells and innate lymphoid cells (ILCs).

[0177] In some implementations, the T cells are isolated from a human subject. In some implementations, the human subject is the same subject who will ultimately receive the T-cell therapy. In other implementations, the subject is a donor subject, wherein the donor subject is not the same subject who will receive the T-cell therapy.

[0178] In some embodiments, the cells are cells that do not naturally express CD3, wherein the cells have been modified to express CD3. In some embodiments, the cells contain a transgene encoding CD3, wherein the transgene is expressed by the cells. In some embodiments, the cells contain a transgene encoding a protein encoding endogenous CD3 expression in activated cells. In some embodiments, the cells contain a transgene encoding a protein or siRNA encoding a CD3 expression inhibitor in the cells. In some embodiments, the transgene is incorporated into the cell's genome. In some embodiments, the transgene is not incorporated into the cell's genome.

[0179] In some embodiments, the cells modified to express CD3 are isolated from a human subject. In some embodiments, the human subject is the same subject who will ultimately receive the cell therapy. In other embodiments, the subject is a donor subject, wherein the donor subject is not the same subject who will receive the cell therapy.

[0180] II.D.HLA Class I molecules

[0181] Certain aspects of this disclosure relate to an HLA class I molecule complexed with a peptide, wherein the peptide comprises the amino acid sequence listed in SEQ ID NO:13. In some embodiments, the peptide consists of the amino acid sequence listed in SEQ ID NO:13.

[0182] In some embodiments, HLA class I molecules are HLA-A, HLA-B, or HLA-C. In some embodiments, HLA class I molecules are HLA-E, HLA-F, or HLA-G. In some embodiments, HLA class 1 molecules are selected from the following HLA-A alleles: HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*24, HLA-A*25, HLA-A*26, HLA-A*29, HLA-A*30, HLA-A*31, HLA-A*32, HLA-A*33, HLA-A*34, HLA-A*36, HLA-A*43, HLA-A*66, HLA-A*68, HLA-A*69, HLA-A*74, and HLA-A*80. In some embodiments, the HLA-A allele is the HLA-A*24:01 allele. In some embodiments, the HLA-A allele is the HLA-A*24:02 allele. In some embodiments, the HLA-A allele is the HLA-A*24:03 allele. In some embodiments, the HLA-A allele is the HLA-A*24:11 allele. In some embodiments, the HLA-A allele is the HLA-A*24:23 allele. In some embodiments, the HLA-A allele is the HLA-A*24:24 allele. In some embodiments, the HLA-A allele is the HLA-A*24:25 allele. In some embodiments, the HLA-A allele is the HLA-A*24:26 allele. In some embodiments, the HLA allele is any HLA allele disclosed herein, such as those described above.

[0183] In some embodiments, the HLA class I molecule comprises an α chain and a β2m. In some embodiments, the α chain comprises an α1 domain, an α2 domain, and an α3 domain. In some embodiments, the β2m comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the amino acid sequence listed 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).

[0184] In some embodiments, HLA class I molecules are monomers. In some embodiments, HLA class I molecules are dimers. In some embodiments, HLA class I molecules are polymers. In some embodiments, HLA class I molecules are trimers. In some embodiments, HLA class I molecules are tetramers. In some embodiments, HLA class I molecules are pentamers.

[0185] Certain aspects of this disclosure relate to antigen-presenting cells (APCs) that contain any of the HLA class I molecules disclosed herein. In some embodiments, the APC expresses an HLA class I molecule on its surface. In some embodiments, the APC contains more than one HLA class I molecule disclosed herein.

[0186] II.D. vaccine

[0187] Certain aspects of this disclosure relate to a cancer vaccine comprising a peptide having an amino acid sequence as listed in SEQ ID NO:13. In some embodiments, the cancer vaccine comprises a peptide consisting of an amino acid sequence listed 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.

[0188] III. The Method of This Disclosure

[0189] Some aspects of this disclosure relate to methods for treating cancer in subjects of need. Other aspects of this disclosure relate to methods for engineering cells that target antigens. Still other aspects of this disclosure relate to methods for enriching target T cell populations obtained from human subjects.

[0190] III.A. Methods of Treating Cancer

[0191] Certain aspects of this disclosure relate to methods for treating cancer in a subject in need, the methods comprising administering to the subject a nucleic acid molecule disclosed herein, a recombinant TCR disclosed herein, a bispecific TCR disclosed herein, an epitope disclosed herein, or an HLA class I molecule disclosed herein, or a vector or cell comprising any of the above.

[0192] In some implementation schemes, the cancer is selected from melanoma, bone cancer, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, cutaneous or ocular malignant melanoma, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric 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 bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, and adrenal cancer. 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 tumor, central nervous system (CNS) spurs, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), other B-cell malignancies, and combinations of the aforementioned cancers. In some embodiments, the cancer is melanoma.

[0193] In some implementations, the cancer is recurrent. In some implementations, the cancer is refractory. In some implementations, the cancer is advanced. In some implementations, the cancer is metastatic.

[0194] In some embodiments, the methods disclosed herein treat a subject's cancer. 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 originating from cancer. In some embodiments, the methods disclosed herein increase a subject's overall survival relative to a subject not provided with the methods disclosed herein. In some embodiments, the methods disclosed herein increase a subject's progression-free survival relative to a subject not provided with the methods disclosed herein. In some embodiments, the methods disclosed herein elicit a partial response in a subject. In some embodiments, the methods disclosed herein elicit a complete response in a subject.

[0195] In some embodiments, the methods disclosed herein include treating a subject with cancer, including administering the subject cells described herein, wherein the cells comprise the nucleic acid molecules disclosed herein, the vectors disclosed herein, the recombinant TCRs disclosed herein, and / or the bispecific antibodies disclosed herein. In some embodiments, the cells are T cells. In some embodiments, the cells are cells modified to express CD3.

[0196] In some embodiments, the cells (e.g., T cells) are obtained from the subject. In some embodiments, the cells (e.g., T cells) are obtained from a donor other than the subject.

[0197] In some embodiments, the subject is pretreated prior to cell administration. 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), rapamycin, or any combination thereof.

[0198] III.B. Methods for engineering cells targeting antigens

[0199] Certain aspects of this disclosure relate to methods for engineering cells targeting an antigen. In some embodiments, the antigen is the gp100 antigen. In some embodiments, the method includes transducing cells with a nucleic acid molecule or a vector disclosed herein. The cells may be any cells described herein. In some embodiments, the cells are T cells as described herein. In some embodiments, the cells are cells modified to express CD3 as described herein. In some embodiments, the cells (e.g., T cells) are obtained from a subject requiring T cell therapy. In some embodiments, the cells are obtained from a donor other than a subject requiring T cell therapy. In some embodiments, the cells are T cells or natural killer cells.

[0200] III.C. Methods for enriching target T cell populations

[0201] Certain aspects of this disclosure relate to methods for enriching a population of target T cells obtained from human subjects. In some embodiments, the method includes contacting T cells with the HLA class I molecules disclosed herein. In some embodiments, the method includes contacting T cells with the APCs disclosed herein. In some embodiments, after contact, the enriched population of T cells contains a higher number of T cells capable of binding HLA class I molecules compared to the number of T cells capable of binding HLA class I molecules before contact.

[0202] In some embodiments, the method includes contacting T cells in vitro with a peptide, wherein the peptide comprises the amino acid sequence listed in SEQ ID NO:13. In some embodiments, the method includes contacting T cells in vitro with a peptide, wherein the peptide consists of the amino acid sequence listed in SEQ ID NO:13. In some embodiments, after contact, the enriched T cell population comprises a higher number of T cells capable of binding HLA class I molecules compared to the number of T cells capable of binding HLA class I molecules before contact.

[0203] Some aspects of this disclosure relate to a method for selecting T cells capable of targeting tumor cells. In some embodiments, the method includes contacting a population of isolated T cells in vitro with a peptide, said peptide consisting of an amino acid sequence as listed in SEQ ID NO:13. In some embodiments, the T cells are obtained from a human subject.

[0204] The T cells obtained from human subjects can be any T cells disclosed herein. In some embodiments, the T cells obtained from human subjects are tumor-infiltrating lymphocytes (TILs).

[0205] In some embodiments, the method further includes administering enriched T cells to a human subject. In some embodiments, as described herein, the subject is pretreated prior to receiving the T cells.

[0206] All aspects, implementation schemes, and options described in this article can be combined in any and all variations.

[0207] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.

[0208] Having generally described the invention, further understanding can be obtained by referring to the embodiments provided herein. These embodiments are for illustrative purposes only and are not intended to be limiting.

[0209] Example

[0210] Example 1

[0211] TILs were isolated from patients with metastatic melanoma, followed by polyclonal expansion in vitro, and their gp100 antigen specificity against the HLA-A*24:02 allele was examined. A combination of structure-based and functional analyses using peptide / HLA (pHLA) multimers was used to measure antigen-specific T cell responses. T cells were stained using pHLA multimers and the previously known gp100-intron 4-peptide (Figure 1). TILs showed positivity for the A*24:02 / gp100-intron 4 multimer. According to ELISPOT analysis, multimer-positive T cells secreted detectable IFN-γ (IFN-γ) in an HLA-restricted peptide-specific manner. Figure 2 ).

[0212] Multimer-positive anti-tumor T cells were collected and their TCR genes were molecularly cloned (Fig. 3; SEQ ID NO: 1 and 2). The antigen specificity and functional reactivity of the cloned TCRs were verified by multimer staining and ELISPOT assay of TCR-reconstructed T cells. When reconstructed on primary T cells, T cells transduced with A*24:02 / gp100-intron 4 TCRs were successfully stained with homologous multimers (Fig. 4) and reacted strongly with the gp100-intron 4 peptide presented by the surface A*24:02 molecule. Figure 5 Importantly, these cells were able to recognize tumor cells that natively expressed the gp100 gene and were A*24:02-matched and whose peptides were not pulsed (Fig. 6). Although the Malme-3M and SK-MEL-28 melanoma cell lines were negative for A*24:02, they endogenously expressed the gp100 gene. When the A*24:02 molecule was ectopically expressed, T cells transduced with the A*24:02 / gp100-intron 4 TCR successfully recognized both melanoma cell lines. Furthermore, A375 melanoma cells lacking endogenous expression of both A*24:02 and gp100 became responsive to T cells transduced with the A*24:02 / gp100-intron 4 TCR when both A*24:02 and gp100-intron 4 genes (rather than either gene alone) were transduced (Figs. 6–8). These results clearly demonstrate that T cells transduced by A*24:02 / gp100-intron 4TCR are highly enthusiastic about recognizing tumor cells, and that the cloned A*24:02 / gp100-intron 4TCR is tumor-responsive.

[0213] Gp100 is one of the promising and well-studied common antigens in bispecific T-cell connective (BiTE) therapy, and clinical trials targeting gp100 have been conducted in patients with metastatic uveal melanoma using IMCgp100, a bispecific bispecific biopharmaceutical containing a soluble TCR that recognizes the gp100 antigen and is fused to scFv anti-CD3, thereby redirecting T-cell lysis of gp100-expressing melanoma cells in the presence of the HLA-A*02:01 molecule. The use of a newly cloned tumor-reactive A*24:02-restricted gp100-intron 4 TCR gene could broaden the applicability of gp100-targeting BiTE therapy beyond HLA-A*02:01-positive cancer patients.

[0214] method

[0215] Cell samples

[0216] Peripheral blood samples were obtained from healthy donors. Monocytes were obtained by density gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). K562 is an erythroleukemia cell line with defective HLA expression. T2 was HLA-A* 02:01. + T-cell leukemia / B-LCL heterozygous cell lines. Jurkat 76 is a T-cell leukemia cell line lacking TCR and CD8 expression. Melme-3M cell line was grown in IMDM supplemented with 20% FBS and 50 μg / ml gentamicin (Invitrogen). SK-MEL-28 and A375 cell lines were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen). K562, T2, and Jurkat 76 cell lines were cultured in RPMI 1640 supplemented with 10% FBS and 50 μg / ml gentamicin. TILs isolated from metastatic melanoma patients were grown in vitro.

[0217] peptides

[0218] The synthesized peptides were dissolved in DMSO to a concentration of 50 μg / ml. The peptides used were A*24:02-restricted gp100-intron 4 (VYFFLPDHL; SEQ ID NO:13) and HTLV-1 tax. 301-309 (SFHSLHLLF; SEQ ID NO: 194) peptide. Using HTLV-1tax 301-309 Peptides were used as negative controls.

[0219] Gene

[0220] The HLA-A*24:02 gene was fused to a truncated form of human nerve growth factor receptor (ΔNGFR) via an internal ribosome entry site. ΔNGFR-transduced cells were isolated using anti-NGFR (mAb). The full-length gp100 gene was purchased from Dharmacon (Lafayette, CO). The gp100 genomic DNA was isolated from SK-MEL-28 cells using the PureLink Genomic DNA Mini Kit (Invitrogen). The TCR gene was cloned using the SMARTer RACE cDNA amplification kit (Takara Bio) via 5'-rapid amplification (RACE) PCR. The 5'-RACE PCR product was cloned into a retroviral vector and sequenced. All genes were cloned into the pMX retroviral vector and transduced using a 293GPG cell-based retroviral system.

[0221] transfectants

[0222] Jurkat 76 / CD8 cells were transduced with individual TCRα and TCRβ genes. TCR transfectants derived from Jurkat 76 / CD8 were purified (>95% purity) using CD3 microbeads (Miltenyi Biotec). Previously, K562-based artificial APCs have been reported to express various HLA class I genes as single HLA alleles bound to 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 retroviral supernatant derived from PG13. TCR genes were transfected into the 293GPG cell line using TransIT293 (Mirus Bio). Gp100 was transduced using retroviruses from exons 1, 2, and 3 of the gp100 gene and intron 4. - A375 cells were used to generate A375 / gp100-intron 4 cells. The expression of transduced gp100-intron 4 cells was assessed by flow cytometry after staining with anti-gp100 mAb (clone 7E3; LifeSpan Biosciences). HLA-A*24:02 retrovirus was used for transduction. - Malme-3M, SK-MEL-28, and A375 cells were used to generate Malme-3M / A*24:02, SK-MEL-28 / A*24:02, and A375 / A*24:02 cells, respectively. The HLA-A*24:02 gene was labeled with the ΔNGFR gene as described above, and the ΔNGFR... +Cells were purified (>95% purity) and used in subsequent experiments. Retroviral transduction of the ΔNGFR gene alone served as a control.

[0223] Flow cytometry and cell sorting

[0224] Cell surface molecules were stained with PC5-conjugated anti-CD8 monoclonal antibody (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 the LIVE / DEAD Fixable AquaDead Cell Stain kit (Life Technologies). For intracellular staining, cells were fixed and permeabilized using the Cytofix / Cytoperm kit (BD Biosciences). Stained cells were analyzed by flow cytometry (BD Biosciences), and data were analyzed using FlowJo (Tree Star). Cell sorting was performed using FACSAria II (BD Biosciences).

[0225] Cytokine ELISPOT Analysis

[0226] IFN-γ ELISPOT assay was performed. PVDF plates (Millipore, Bedford, MA) were coated with capture mAbs (1-D1K; MABTECH, Mariemont, OH), and T cells were added to each well at 2 x 10⁻⁶ cells in the presence or absence of the peptide. 4 The target cells were incubated together at 37°C for 20–24 hours. The plate was then washed and incubated with a biotin-conjugated detection mAb (7-B6-1; MABTECH). HRP-conjugated SA (Jackson ImmunoResearch) was then added to visualize the IFN-γ spots. The reaction was stopped by thorough rinsing with cold tap water. The ELISPOT plate was scanned and counted using an ImmunoSpot reader and ImmunoSpot version 5.0 software (Cellular Technology Limited, Shaker Heights, OH).

[0227] CD8 + TIL is amplified in a HLA-restricted peptide-specific manner.

[0228] Use CD8 +T-cell separation kit (Miltenyi Biotec) uses negative magnetic selection to separate CD8 cells. + TIL purification. A*24:02-artificial APC was pulsed with 10 μg / mL gp100-intron tetrapeptide for 6 hours. The artificial APC was then irradiated at 200 Gy, washed, and added to the TIL at an effector to target (E:T) ratio of 20:1. Starting from day two, 10 IU / ml IL-2 (Novartis), 10 ng / ml IL-15 (Peprotech), and 30 ng / ml IL-21 (Peprotech) were added to the culture every three days.

[0229] Primary CD8 transduced using cloned TCR + T cell expansion

[0230] CD3 cells were isolated using a whole T-cell isolation kit (Miltenyi Biotec) via negative magnetic selection. + T cell purification. Purified T cells were stimulated with artificial APC / mOKT3 irradiated with 200 Gy at an E:T ratio of 20:1. Starting from the second day, activated T cells were transduced with a cloned TCR gene retrovirus after 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.

[0231] Based on the generation of pHLA polymers in human cells

[0232] HLA class I genetically engineered mice to carry a Glu(E) residue replacing the Gln(Q) residue at position 115 of the α2 domain and a mouse K gene replacing the HLA class I α3 domain. b The gene-derived α3 ​​domain. Soluble HLA class I genes were generated by sequentially fusing the extracellular domain of affinity-matured HLA class I genes with a Gly-Ser (GS) flexible linker and a 6x His tag. Q115E -K b Genes. Using a 293GPG cell-based retroviral system with various soluble HLA class I... Q115E -K b The gene and the β2m gene were individually transduced into HEK293T cells. This facilitated the ectopic expression of soluble affinity maturation in category I cells. Q115E -K bStable HEK293T cells were grown until confluence, and then the culture medium was changed. After 48 hours, the conditioned medium was harvested and used immediately or frozen until use. The HEK293T transfectants were used to produce cells containing soluble HLA class I... Q115E -K b The supernatant was mixed with 100-1000 μg / ml of the target class I restriction peptide overnight at 37°C for in vitro peptide exchange. Soluble class I peptide-loaded monomers were then exchanged using an 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 carried out for 2 hours at room temperature or overnight at 4°C. Functional soluble HLA class I mAbs (clone W6 / 32, internal) and anti-His-tagged biotinylated mAbs (clone AD1.1.10, R&Dsystems) were used as capture and detection abs, respectively, by specific ELISA to measure functional soluble HLA class I. Q115E -K b Molecular concentration.

[0233] pHLA polymer staining

[0234] In the presence of 50 nM dasatinib (LC laboratories), T cells (1 x 10) 5 Incubate at 37°C for 30 minutes. Then wash the cells and incubate with 5-10 μg / ml of polymer at room temperature for 30 minutes, followed by 15 minutes at 4°C with R-phycoerythrin-conjugated AffiniPure Fab fragment goat anti-mouse IgG1 (Jackson ImmunoResearch Laboratories). Next, wash the cells three times and co-stain with anti-CD8 mAb at 4°C for 15 minutes. Finally, identify dead cells using a live / dead fixable dead cell staining kit.

[0235] Statistical analysis

[0236] Statistical analysis was performed using GraphPad Prism 5.0e. To determine whether the given variables were significantly different between the two groups, Welch's t-test (two-tailed) was used. A p-value < 0.05 was considered significant. sequence list <110> University Health Network <120> T-cell receptors and their usage <130> 4285.007PC01 / C-K / BMD <150> US 62 / 813,651 <151> 2019-03-04 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of α chain <400> 1 Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp 1 5 10 15 Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val 20 25 30 Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala 35 40 45 Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr 50 55 60 Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg 65 70 75 80 Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95 Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Ala 100 105 110 Thr Asp Ser Trp Gly Lys Leu Gln Phe Gly Ala Gly Thr Gln Val Val 115 120 125 Val Thr Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg 130 135 140 Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser 180 185 190 Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe 195 200 205 Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser 210 215 220 Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn 225 230 235 240 Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu 245 250 255 Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 Glx <210> 2 <211> 324 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of β-chain <400> 2 Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Leu Leu Pro Glu Gly 115 120 125 Thr Gly Arg Val Ser Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr 130 135 140 Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe 145 150 155 160 Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val 165 170 175 Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp 180 185 190 Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro 195 200 205 Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser 210 215 220 Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe 225 230 235 240 Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr 245 250 255 Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp 260 265 270 Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val 275 280 285 Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu 290 295 300 Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg 305 310 315 320 Lys Asp Phe Glx <210> 3 <400> 3 000 <210> 4 <400> 4 000 <210> 5 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> α CDR1 <400> 5 Asp Ser Ala Ile Tyr Asn 1 5 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> α CDR2 <400> 6 Pro Arg His Asp Thr 1 5 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> α CDR3 <400> 7 Cys Ala Val Ala Thr Asp Ser Trp Gly Lys Leu Gln Phe 1 5 10 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> β CDR1 <400> 8 Ile Gln Ser Ser Gln Arg Glu 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> β CDR2 <400> 9 Phe Tyr Glu Lys Met Gln 1 5 <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <22223> β CDR3 <400> 10 Cys Ala Ser Ser Leu Leu Pro Glu Gly Thr Gly Arg Val Ser Gly Tyr 1 5 10 15 Thr Phe <210> 11 <400> 11 000 <210> 12 <400> 12 000 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <400> 13 Val Tyr Phe Phe Leu Pro Asp His Leu 1 5 <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> 819 <212> DNA <213> Artificial Sequence <220> <223> Alpha chain nucleotide sequence <400> 17 atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60 caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctc 120 aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180 aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240 cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300 cctggtgact cagccaccta cctctgtgct gtcgcaactg acagctgggg gaaattgcag 360 tttggagcag ggacccaggt tgtggtcacc ccagatatcc agaaccctga ccctgccgtg 420 taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480 tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 540 ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 600 gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 660 agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 720 ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780 tttaatctgc tcatgacgct gcggctgtgg tccagctga 819 <210> 18 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> β-chain nucleotide sequence <400> 18 atgcttagtc ctgacctgcc tgactctgcc tggaacacca ggctcctctg ccatgtcatg 60 ctttgtctcc tgggagcagt ttcagtggct gctggagtca tccagtcccc aagacatctg 120 atcaaagaaa agagggaaac agccactctg aaatgctatc ctatccctag acacgacact 180 gtctactggt accagcaggg tccaggtcag gacccccagt tcctcatttc gttttatgaa 240 aagatgcaga gcgataaagg aagcatccct gatcgattct cagctcaaca gttcagtgac 300 tatcattctg aactgaacat gagctccttg gagctggggg actcagccct gtacttctgt 360 gccagcagcc tcctaccgga agggacaggc cgtgtaagtg gctacacctt cggttcgggg 420 accaggttaa ccgttgtaga ggacctgaac aaggtgttcc cacccgaggt cgctgtgttt 480 gagccatcag aagcagagat ctcccacacc caaaaggcca cactggtgtg cctggccaca 540 ggcttcttcc ctgaccacgt ggagctgagc tggtgggtga atgggaagga ggtgcacagt 600 ggggtcagca cggacccgca gcccctcaag gagcagcccg ccctcaatga ctccagatac 660 tgcctgagca gccgcctgag ggtctcggcc accttctggc agaacccccg caaccacttc 720 cgctgtcaag tccagttcta cgggctctcg gagaatgacg agtggaccca ggatagggcc 780 aaacccgtca cccagatcgt cagcgccgag gcctggggta gagcagactg tggctttacc 840 tcggtgtcct accagcaagg ggtcctgtct gccaccatcc tctatgagat cctgctaggg 900 aaggccaccc tgtatgctgt gctggtcagc gcccttgtgt tgatggccat ggtcaagaga 960 aaggatttct ga 972 <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> DNA <213> Artificial Sequence <220> <223> siRNA‑TCRa‑1 <400> 53 guaaggauuc ugauguguat t 21 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> siRNA‑TCRa‑2 <400> 54 uacacaucag aauccuuact t 21 <210> 55 <211> 21 <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

Claims

1. A nucleic acid molecule, said nucleic acid molecule comprising (i) A first nucleotide sequence encoding a recombinant anti-gp100T cell receptor or its antigen-binding portion specifically binding to an epitope of human gp100 as shown in SEQ ID NO: 13, wherein the epitope is complexed with the HLA class I molecule HLA-A*24 allele, wherein the anti-gp100 TCR comprises an α chain and a β chain, wherein the α chain comprises a variable domain comprising α chain CDR1, α chain CDR2, and α chain CDR3; and wherein the β chain comprises a variable domain comprising β chain CDR1, β chain CDR2, and β chain CDR3; wherein: (a) The amino acid sequence of the β-chain CDR3 against gp100 TCR is shown in SEQ ID NO: 10; (b) The amino acid sequence of the β-chain CDR2 against gp100 TCR is shown in SEQ ID NO: 9; (c) The amino acid sequence of the β-chain CDR1 against gp100 TCR is shown in SEQ ID NO: 6; (d) The amino acid sequence of the α-chain CDR3 against gp100 TCR is shown in SEQ ID NO: 7; (e) The amino acid sequence of the α-chain CDR2 against gp100 TCR is shown in SEQ ID NO: 8; (f) The amino acid sequence of the α-chain CDR1 against gp100 TCR is shown in SEQ ID NO: 5; and (ii) a second nucleotide sequence, wherein the second nucleotide sequence or the polypeptide encoded by the second nucleotide sequence inhibits the expression of endogenous TCR.

2. The nucleic acid molecule as described in claim 1, wherein the HLA class I molecule HLA-A*24 allele is selected from the HLA-A*24:01 allele, the HLA-A*24:02 allele, and the HLA-A*24:03 allele.

3. The nucleic acid molecule as described in claim 1, wherein... (i) The α-chain of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO: 1; (ii) The β-chain of the anti-gp100 TCR comprises the amino acid sequence as listed in SEQ ID NO: 2; or (iii) (i) and (ii) both.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein the second nucleotide sequence (i) One or more siRNAs for reducing the expression of endogenous TCRs, 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) Encode Cas 9; or (iii) (i) and (ii) both.

5. The nucleic acid molecule of claim 4, wherein the one or more siRNAs comprise one or more nucleotide sequences selected from the group consisting of SEQ ID NO:53-56.

6. A vector comprising the nucleic acid molecule of any one of claims 1 to 5.

7. The vector as described in claim 6, wherein the vector is a viral vector, a mammalian vector, or a bacterial vector.

8. The vector as described in claim 6, wherein the vector is a retroviral vector.

9. The vector of claim 6, wherein the vector is selected from the group consisting of: adenovirus vector, lentivirus vector, Sendai virus vector, baculovirus vector, Epstein-Barr virus vector, lactovirus vector, vaccinia virus vector, herpes simplex virus vector, hybrid vector and adeno-associated virus vector.

10. The vector as described in claim 6, wherein the vector is a lentiviral vector.

11. A cell comprising a nucleic acid molecule according to any one of claims 1 to 5 or a vector according to any one of claims 6 to 10.

12. The cell of claim 11, wherein the cell further expresses CD3.

13. The cell of claim 11, wherein the cell is a T cell.

14. The cell of claim 11, wherein the cell is a natural killer cell.

15. The cell of claim 11, wherein the cell is a natural killer T cell.

16. The cell of claim 11, wherein the cell is an ILC cell.

17. Use of the cells of claim 11 in the preparation of a medicament for treating melanoma in a subject in need.

18. The use as described in claim 17, wherein the melanoma is recurrent or refractory.

19. The use as claimed in claim 17, wherein the melanoma is locally advanced.

20. The use as claimed in claim 17, wherein the melanoma is advanced.

21. The use as claimed in claim 17, wherein the melanoma is metastatic.

22. The use as claimed in claim 17, wherein the cells are obtained from the subject.

23. The use as claimed in claim 17, wherein the cells are obtained from a donor other than the subject.

24. A method for engineering cells targeting an antigen, the method comprising transducing cells collected from a subject requiring T-cell therapy using the nucleic acid molecule of claim 1.

25. The method of claim 24, wherein the cells targeting the antigen further express CD3.

26. The method of claim 24, wherein the cell is a T cell.

27. The method of claim 24, wherein the cell is a natural killer cell.

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