T cell receptors and methods of use thereof

Abstract

The present disclosure relates to recombinant T cell receptors capable of binding to NY-ESO-1 epitopes and nucleic acid molecules encoding the same. In some embodiments, the nucleic acid molecules further comprise 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. Other aspects of the present disclosure relate to vectors comprising the nucleic acid molecules and cells comprising the recombinant TCRs, the nucleic acid molecules, or the vectors. Other aspects of the present disclosure relate to methods of using the recombinant TCRs, the nucleic acid molecules, the vectors, and the cells. In some embodiments, the methods comprise treating a cancer in a subject in need thereof.

Description

[0001] Cross-references to related applications

[0002] This PCT application claims priority to U.S. Provisional Application No. 62 / 813,639, filed March 4, 2019, which is incorporated herein by reference in its entirety.

[0003] References to sequence lists submitted electronically via EFS-WEB

[0004] The contents of the sequence list submitted electronically (name: 4285_001PC01_Seqlisting_ST25.txt, size: 23,578 bytes; and creation date: March 3, 2020) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure provides a recombinant T-cell receptor (“TCR”) that specifically binds to human NY-ESO-1 and its uses. Background of the Invention

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

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

[0009] This disclosure provides novel epitopes of the nonmutated antigen NY-ESO-1 and TCRs capable of specifically binding to said epitopes. These novel epitopes are associated with specific HLA alleles. The use of these tumor-responsive HLA-restricted NY-ESO-1 TCRs supports the applicability of broadened anti-NY-ESO-1 TCR gene therapy, particularly in immuno-oncology. Summary of the Invention

[0010] Certain aspects of this disclosure relate to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T-cell receptor (TCR) specifically binding to human NY-ESO-1 or its antigen-binding portion (“anti-NY-ESO-1 TCR”); 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, wherein the anti-NY-ESO-1 TCR cross-competitively binds to human NY-ESO-1 with a reference TCR comprising 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.

[0011] Certain aspects of this disclosure relate to a nucleic acid molecule comprising (i) a first nucleotide sequence encoding a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-NY-ESO-1 TCR”) that specifically binds to human NY-ESO-1; 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, wherein the anti-NY-ESO-1 TCR binds to the same or overlapping epitopes of human NY-ESO-1 as a reference TCR, the reference TCR comprising an α chain and a β chain, 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.

[0012] In some embodiments, the anti-NY-ESO-1 TCR binds to an epitope of NY-ESO-1 consisting of an amino acid sequence as listed in SEQ ID NO:13. In some embodiments, the HLA class I molecule is the HLA-C*03 allele. In some embodiments, the HLA class I molecule is selected from the HLA-C*03:02 allele, HLA-C*03:03 allele, HLA-C*03:04 allele, HLA-C*03:05 allele, and HLA-C*03:06 allele. In some embodiments, the HLA class I molecule is the HLA-C*03:03 allele.

[0013] In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:10.

[0014] In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:10. In some embodiments, the α chain CDR3 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:7.

[0015] In some embodiments, the α-chain CDR1 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:5. In some embodiments, the β-chain CDR1 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:8. In some embodiments, the α-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:6. In some embodiments, the β-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:9.

[0016] In some embodiments, the α-chain variable domain of the anti-NY-ESO-1 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-NY-ESO-1 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence listed in SEQ ID NO:2.

[0017] In some embodiments, the α-chain of the anti-NY-ESO-1 TCR further includes a constant region, wherein the constant region is different from the endogenous constant region of the α-chain. In some embodiments, the α-chain constant region of the anti-NY-ESO-1 TCR further includes a constant region, wherein the α-chain constant region comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity 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 that includes 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 β-chain of the anti-NY-ESO-1 TCR further includes a constant region, wherein the constant region is different from the endogenous constant region of the β-chain.

[0018] In some embodiments, the β-chain of the anti-NY-ESO-1 TCR 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 α-chain of the anti-NY-ESO-1 TCR comprises an amino acid sequence as listed in SEQ ID NO:1.

[0019] In some embodiments, the β-chain of the anti-NY-ESO-1 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.

[0020] 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.

[0021] In some implementations, the second nucleotide sequence encodes Cas9.

[0022] In some embodiments, the anti-NY-ESO-1 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.

[0023] 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.

[0024] 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-NY-ESO-1 TCR disclosed herein and the β-chain variable domain of the anti-NY-ESO-1 TCR disclosed herein. In some embodiments, the recombinant T-cell receptor (TCR) specifically binding to human NY-ESO-1 or its antigen-binding portion (“anti-NY-ESO-1 TCR”) cross-competitively binds to human NY-ESO-1 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-NY-ESO-1 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.

[0025] Certain aspects of this disclosure relate to a recombinant T-cell receptor (TCR) or its antigen-binding portion (“anti-NY-ESO-1 TCR”) that specifically binds to human NY-ESO-1, having the same or overlapping epitopes as a reference TCR binding to human NY-ESO-1; 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-NY-ESO-1 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-NY-ESO-1 TCR binds to an epitope of NY-ESO-1 consisting of an amino acid sequence as listed in SEQ ID NO:13.

[0026] In some embodiments, the epitope is complexed with an HLA class I molecule. In some embodiments, the HLA class I molecule is an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G allele. In some embodiments, the HLA class I molecule is the HLA-C*03 allele. In some embodiments, the HLA class I molecule is selected from the HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*03:05, and HLA-C*03:06 alleles. In some embodiments, the HLA class I molecule is the HLA-C*03:03 allele.

[0027] In some embodiments, the α-chain of the anti-NY-ESO-1 TCR includes variable domains containing α-chain CDR1, α-chain CDR2, and α-chain CDR3; and the β-chain of the anti-NY-ESO-1 TCR includes variable domains containing β-chain CDR1, β-chain CDR2, and β-chain CDR3; wherein the α-chain CDR3 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:7. In some embodiments, the β-chain CDR3 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:10.

[0028] In some embodiments, the α-chain of the anti-NY-ESO-1 TCR includes variable domains containing α-chain CDR1, α-chain CDR2, and α-chain CDR3; and the β-chain of the anti-NY-ESO-1 TCR includes variable domains containing β-chain CDR1, β-chain CDR2, and β-chain CDR3; wherein the β-chain CDR3 of the anti-NY-ESO-1 TCR contains the amino acid sequence as listed in SEQ ID NO:10. In some embodiments, the α-chain CDR3 of the anti-NY-ESO-1 TCR contains the amino acid sequence as listed in SEQ ID NO:7.

[0029] In some embodiments, the α-chain CDR1 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:5. In some embodiments, the β-chain CDR1 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:8. In some embodiments, the α-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:6. In some embodiments, the β-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:9.

[0030] In some embodiments, the α-chain variable domain of the anti-NY-ESO-1 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-NY-ESO-1 TCR comprises the amino acid sequence of the variable domain present in the amino acid sequence listed in SEQ ID NO:2.

[0031] 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.

[0032] 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.

[0033] In some embodiments, the α chain of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:1. In some embodiments, the β chain of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:2.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In some embodiments, the HLA class I molecule is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, the HLA class I molecule is HLA-C. In some embodiments, the HLA class I molecule is the HLA-C*03 allele. In some embodiments, the HLA class I molecule is selected from the HLA-C*03:02 allele, HLA-C*03:03 allele, HLA-C*03:04 allele, HLA-C*03:05 allele, and HLA-C*03:06 allele. In some embodiments, the HLA class I molecule is the HLA-C*03:03 allele. In some embodiments, the HLA class I molecule is the HLA-C*03:04 allele.

[0042] 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.

[0043] 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.

[0044] 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.

[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 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.

[0046] In some implementations, the T cells obtained from the human subject are tumor-infiltrating lymphocytes (TILs).

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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

[0051] Figure 1 This is a bar graph illustrating the number of C*03:04 / NY-ESO-1 T cells in melanoma TILs after stimulation with artificial APCs treated with overlapping peptide pulses. TILs were used as responding cells in the IFN-γELISPOT analysis. C*03:04-artificial APCs treated with overlapping peptide pulses to cover the full protein of NY-ESO-1 were used as stimulating cells. When stimulated with C*03:04-artificial APCs treated with overlapping peptide pulses derived from NY-ESO-1, TILs showed a higher proportion of cells sharing a common sequence. 91 YLAMPFATPM 100 Positive responses to two adjacent peptides (see also Table 5).

[0052] Figure 2A-2C C*03:04 / NY-ESO-1 for melanoma TIL 92-100 Graphical representation of multimer staining. Figure 2A The diagram shows the use of C*03:04 / NY-ESO-1 92-100 Multimers were used to stain TILs. C*03:04 / MAGE-A1 230-238 ( Figure 2B ) and C*03:04 / not swapped ( Figure 2C The polymer was used as a negative control. CD8 was displayed. + T cell multimer + Percentage of cells.

[0053] Figure 3 To show C*03:04 / NY-ESO-1 92-100 Bar graph for functional assessment of triglyceride endothelial cells (TILs) in multimer-positive melanoma. TILs are classified as C*03:04 / NY-ESO-1. 92-100 IFN-γ was generated in a specific manner. In the IFN-γ ELISPOT assay, TILs were used as responding cells. C*03:04-artificial APCs treated with the indicated peptide pulses were used as stimulating cells. MAGE-A1 was used. 230-238 Peptides were used as controls. Triples were performed, and the error bars show the standard deviation (SD). ***P < 0.001.

[0054] Figure 4A-4IFor use in the case of homopolymers, C*03:04 / NY-ESO-1 92-100 A graphical representation of positive staining in Jurkat76 / CD8 cells transduced with the TCR gene. This will be represented using C*03:04 / NY-ESO-1. 92-100 TCR( Figure 4B , 4E Jurkat 76 / CD8 cells transduced with 4H were treated with C*03:04 / NY-ESO-1 92-100 polymers ( Figure 4B Staining was performed using C*03:04 / HIVgag. 164-172 polymers ( Figure 4D , 4E (and 4F), C*07:02 / MAGE-A1 289-297 TCR (clones CL2); Figure 4C , 4F and 4I) and unexchanged polymers ( Figure 4G , 4H and 4I) and Jurkat 76 / CD8 cells not transduced with TCR ( Figure 4A , 4D (Compared to 4G) The presence of multimers was observed. + CD8 + Percentage of cells.

[0055] Figures 5A-5D For use in the case of homopolymers, C*03:04 / NY-ESO-1 92-100 TCR gene ( Figure 5B and 5D The graph represents the positive staining of transduced human primary T cells. It will be represented using C*03:04 / NY-ESO-1. 92-100 TCR-transduced primary T cells were treated with C*03:04 / NY-ESO-1 92-100 ( Figure 5B ) or C*03:04 / HIV gag 164-172 control polymer ( Figure 5D Staining. Untransduced primary T cells were used as a negative control. Figure 5A and 5C (Displays polymers) + CD8 + The percentage of T cells.

[0056] Figure 6 To illustrate, use C*03:04 / NY-ESO-1 60-72 A bar graph showing the strong reaction of TCR gene-transduced human primary T cells with homologous peptides presented by target class I molecules. In the IFN-γ ELISPOT analysis, C*03:04 / NY-ESO-1 will be used. 92-100Primary T cells transduced with the TCR gene or untransduced primary T cells (x-axis) were used as responding cells. HLA-C*03:04 transduced T2 cells (T2-C*03:04) were generated. NY-ESO-1 was used. 92-100 or HIV gag 164-172 T2 or T2-C*03:04 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<0.001.

[0057] Figure 7A and 7B To illustrate, use C*03:04 / NY-ESO-1 92-100 Graphical representation of TCR gene-transduced primary T cells recognizing tumor cells ( Figure 7A ) and its legend ( Figure 7B In IFN-γELISPOT analysis, C*03:04 / NY-ESO-1 was used. 92-100 Primary T cells transduced with the TCR gene or untransduced primary T cells were used as responding cells. After treatment with 100 ng / ml IFN-γ for 48 hours, if... Figure 7B ( Figure 7A The illustration (as indicated in the figure) shows that A375, SK-MEL-37, LM-MEL-53, and SK-MEL-21 cells, either untransduced or transduced with HLA-C*03:04 or NY-ESO-1, 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 8A-8E Graphical representation of the expression of NY-ESO-1, derived from the endogenous or transduced full-length gene. The expression of NY-ESO-1, derived from the endogenous or transduced full-length gene, in target cells was analyzed by intracellular flow cytometry after staining with anti-NY-ESO-1 mAb (hollow curve) and isotype control (solid curve).

[0059] Figures 9A-9D ΔNGFR in target cells transduced with the full-length HLA-C*03:04 gene labeled with ΔNGFR ( Figure 9B and 9D Graphical representation of expression in ). Surface expression of ΔNGFR in target cells transduced with the full-length HLA-C*03:04 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 9A and 9C ). Detailed Implementation

[0060] This disclosure relates to a TCR or its antigen-binding portion that specifically binds to an epitope on NY-ESO-1, 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 NY-ESO-1.

[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, “NY-ESO-1,” “New York esophageal squamous cell carcinoma 1,” “cancer-testis antigen 1B,” or “CTAG1B” refers to a tumor antigen expressed in multiple cancer types. NY-ESO-1 is a member of the cancer-testis antigen family, characterized by expression primarily limited to testicular germ cells and placental trophoblasts, with little or no expression in healthy adult somatic cells. NY-ESO-1 expression can be detected during embryonic development and is maintained in spermatogonia and primary spermatocytes. In females, NY-ESO-1 expression rapidly decreases in female oogonia. Low levels of NY-ESO-1 RNA have been detected in ovarian and endometrial tissues; however, NY-ESO-1 protein has not been found in these tissues. The NY-ESO-1 protein (SEQ ID NO: 52; Table 1) is an 18-kDa protein with 180 amino acids. See, for example, Thoma et al., Front. Immunol. 9:947 (2018).

[0077] Table 1. Amino acid sequence of NY-ESO-1

[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" can be abbreviated as PD, and as used herein, it 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 do not secrete 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 "a / 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 NY-ESO-1, 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 NY-ESO-1 epitope that binds to a TCR, and an HLA class I molecule complexed with a peptide containing the NY-ESO-1 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-NY-ESO-1TCR”) that specifically binds to human NY-ESO-1; 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-NY-ESO-1 TCR cross-competitively binds to human NY-ESO-1 with a reference TCR. In some embodiments, the anti-NY-ESO-1 TCR binds to the same or overlapping epitopes of human NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises an α chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7 (CAGMDSNYQLIW). In some embodiments, the anti-NY-ESO-1 TCR comprises a β chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10 (CASSLPLGYEQYF). 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-NY-ESO-1 TCR encoded by the first nucleotide sequence comprises an α-chain CDR1, wherein the α-chain CDR1 of the anti-NY-ESO-1 TCR comprises the amino acid sequence listed in SEQ ID NO:5 (YGATPY). In some embodiments, the anti-NY-ESO-1 TCR encoded by the first nucleotide sequence comprises a β-chain CDR1, wherein the β-chain CDR1 of the anti-NY-ESO-1 TCR comprises the amino acid sequence listed in SEQ ID NO:8 (MNHNS).

[0124] In some embodiments, the anti-NY-ESO-1 TCR encoded by the first nucleotide sequence comprises an α-chain CDR2, wherein the α-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence listed in SEQ ID NO:6 (YFSGDTLV). In some embodiments, the anti-NY-ESO-1 TCR encoded by the first nucleotide sequence comprises a β-chain CDR2, wherein the β-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence listed in SEQ ID NO:9 (SASEGT).

[0125] In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises a β-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:10. In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises an α-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:7. In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 TCR encoded by the first nucleotide also comprises a β-constant region that is an endogenous (e.g., naturally occurring) constant region different from 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-NY-ESO-1 TCR 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the anti-NY-ESO-1 TCR 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-NY-ESO-1 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-NY-ESO-1 TCR encoded by the first nucleotide sequence binds to the same epitope as the reference TCR. In some embodiments, the anti-NY-ESO-1 TCR binds to an epitope of NY-ESO-1 comprising the amino acid sequence listed in SEQ ID NO:13 (LAMPFATPM). In some embodiments, the anti-NY-ESO-1 TCR binds to an epitope of NY-ESO-1 consisting of an amino acid sequence as listed in SEQ ID NO:13. In some embodiments, the epitope consists of amino acid residues 92-100 (SEQ ID NO:52) of NY-ESO-1, for example, “NY-ESO-1”. 92-100 ".

[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 an HLA-C allele selected from the following: HLA-C*03:02 allele, HLA-C*03:03 allele, HLA-C*03:04 allele, HLA-C*03:05 allele, and HLA-C*03:06 allele. In some embodiments, the HLA-C allele is the HLA-C*03:02 allele. In some embodiments, the HLA-C allele is the HLA-C*03:03 allele. In some embodiments, the HLA-C allele is the HLA-C*03:04 allele. In some embodiments, the HLA-C allele is the HLA-C*03:05 allele. In some embodiments, the HLA-C allele is the HLA-C*03:06 allele.

[0138] In some implementations, HLA class 1 molecules are HLA-C alleles selected from the following groups: HLA-C*03:02:01; HLA-C*03:02:02:01; HLA-C*03:02:02:02; HLA-C*03:02:02:03; HLA-C*03:02:02:04; HLA-C*03:02:02:05; HLA-C*03:02:03; HLA-C*03:02:04; HLA-C*03:02:05; HLA-C*03:02:06; HLA-C*03:02:07; HLA-C*03:02:08; HLA-C*03:02:09; HLA-C*03:02:10; HLA-C*03:02:11; HLA-C*03:02:12; HLA-C*03:02:13; HLA-C*03:02:14; HLA-C*03:02:15; HLA-C*03:02:16; HLA-C*03:02:17; HLA-C*03:02:18; HLA-C*03:02:19; HLA-C*03:02:20; HLA-C*03:02:21; HLA-C*03:02:22; and any combination thereof. In some embodiments, HLA Class 1 molecules are HLA-C alleles selected from the following groups: HLA-C*03:03:01:01; HLA-C*03:03:01:02; HLA-C*03:03:01:03; HLA-C*03:03:01:04; HLA-C*03:03:01:05; HLA-C*03:03:01:06; HLA-C*03:03:01:07; HLA-C*03:03:01:08; HLA-C*03:03:01:09; HLA-C*03:03:01:10; HLA-C*03:03:01:11; HLA-C*03:03:01:12; HLA-C*03:03:01:13; HLA-C*0 3:03:01:14; HLA-C*03:03:02; HLA-C*03:03:03; HLA-C*03:03:04; HLA-C*03:0 3:05;HLA-C*03:03:06;HLA-C*03:03:07;HLA-C*03:03:08;HLA-C*03:03:09;HL A-C*03:03:10; HLA-C*03:03:11; HLA-C*03:03:12; HLA-C*03:03:13; HLA-C*03 :03:14; HLA-C*03:03:15; HLA-C*03:03:16; HLA-C*03:03:17; HLA-C*03:03:18;HLA-C*03:03:19;HLA-C*03:03:20;HLA-C*03:03:21;HLA-C*03:03:22;HLA-C*03 :03:23;HLA-C*03:03:24;HLA-C*03:03:25;HLA-C*03:03:26;HLA-C*03:03:27;H LA-C*03:03:28;HLA-C*03:03:29;HLA-C*03:03:30;HLA-C*03:03:31;HLA-C*03: 03:32;HLA-C*03:03:33;HLA-C*03:03:34;HLA-C*03:03:35;HLA-C*03:03:36;HLA -C*03:03:37;HLA-C*03:03:38;HLA-C*03:03:39;HLA-C*03:03:40;HLA-C*03:03 :41;HLA-C*03:03:42;HLA-C*03:03:43;HLA-C*03:03:44;HLA-C*03:03:45;HLA-C *03:03:46;HLA-C*03:03:47;HLA-C*03:03:48;HLA-C*03:03:49;HLA-C*03:03:5 0:HLA-C*03:03:51;HLA-C*03:03:52;HLA-C*03:03:53; 1. The main characteristics of HLA-C*03:04:01:01;HLA-C*03:0 :01:02;HLA-C*03:04:01:03;HLA-C*03:04:01:04;HLA-C*03:04: 01:05;HLA-C*03:04:01:06;HLA-C*03:04:01:07;HLA-C*03:04:0 1:08;HLA-C*03:04:01:09;HLA-C*03:04:01:10;HLA-C*03:04:01 :11;HLA-C*03:04:01:12;HLA-C*03:04:01:13;HLA-C*03:04:02; HLA-C*03:04:03;HLA-C*03:04:04;HLA-C*03:04:05;HLA-C*03:0 4:06;HLA-C*03:04:07;HLA-C*03:04:08;HLA-C*03:04:09;HLA-C *03:04:10;HLA-C*03:04:11;HLA-C*03:04:12;HLA-C*03:04:13;HLA-C*03:04:14;HLA-C*03:04:15;HLA-C*03:04:16;HLA-C*03:04: 17;HLA-C*03:04:18;HLA-C*03:04:19;HLA-C*03:04:20;HLA-C*03:0 4:21;HLA-C*03:04:22;HLA-C*03:04:23;HLA-C*03:04:24;HLA-C*0 3:04:25;HLA-C*03:04:26;HLA-C*03:04:27;HLA-C*03:04:28;HLA-C *03:04:29;HLA-C*03:04:30;HLA-C*03:04:31;HLA-C*03:04:32;HL A-C*03:04:33;ALL-C*03:04:34;A-A-C*03:04:35;A-C*03:04:36; HLA-C*03:04:37;HLA-C*03:04:38;HLA-C*03:04:39;HLA-C*03:04:4 0;HLA-C*03:04:41;HLA-C*03:04:42;HLA-C*03:04:43;HLA-C*03:04 :44;HLA-C*03:04:45;HLA-C*03:04:46;HLA-C*03:04:47;HLA-C*03 :04:48;HLA-C*03:04:49;HLA-C*03:04:50;HLA-C*03:04:51;HLA-C* 03:04:52;HLA-C*03:04:53;HLA-C*03:04:54;HLA-C*03:04:55;HLA -C*03:04:56;HLA-C*03:04:57;HLA-C*03:04:58;HLA-C*03:04:59;H LA-C*03:04:60;HLA-C*03:04:61;HLA-C*03:04:62;HLA-C*03:04:6 3;HLA-C*03:04:64;HLA-C*03:04:65;HLA-C*03:04:66;HLA-C*03:04 :67;HLA-C*03:04:68;HLA-C*03:04:69;HLA-C*03:04:70;HLA-C*03: 04:71;HLA-C*03:04:72;HLA-C*03:04:73;I don't want to lose my temper 1. The sequences of the HLA-C genes were as follows:HLA-C*03:05;HLA-C*03:06:01;C0-6:20HL

[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 / 0273213 A1, 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 the nucleic acid molecules disclosed herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a viral particle or virus. In some embodiments, the vector is a mammalian vector. In some embodiments, the vector is a bacterial vector.

[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 the recombinant T-cell receptor (TCR) of human NY-ESO-1 or its antigen-binding moiety (“anti-NY-ESO-1 TCR”). In some embodiments, the anti-NY-ESO-1 TCR is encoded by a nucleic acid molecule disclosed herein.

[0152] In some embodiments, the anti-NY-ESO-1 TCR cross-competitively binds to human NY-ESO-1 with a reference TCR. In some embodiments, the anti-NY-ESO-1 TCR binds to the same or overlapping epitopes of human NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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 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-NY-ESO-1 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-NY-ESO-1 TCR includes variable domains containing α-chain CDR1, α-chain CDR2, and α-chain CDR3; and the β-chain of the anti-NY-ESO-1 TCR includes variable domains containing β-chain CDR1, β-chain CDR2, and β-chain CDR3. In some embodiments, the anti-NY-ESO-1 TCR includes α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-NY-ESO-1 TCR includes 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-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:5. In some embodiments, the β-chain CDR1 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:8.

[0157] In some embodiments, the α-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:6. In some embodiments, the β-chain CDR2 of the anti-NY-ESO-1 TCR comprises the amino acid sequence as listed in SEQ ID NO:9.

[0158] In some embodiments, the anti-NY-ESO-1 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 α-chain amino acid sequence listed in SEQ ID NO:1. In some embodiments, the anti-NY-ESO-1 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 α-chain amino acid sequence listed in SEQ ID NO:1, wherein the anti-NY-ESO-1 TCR comprises an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-NY-ESO-1 TCR includes 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-NY-ESO-1 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 β-chain amino acid sequence listed in SEQ ID NO:2. In some embodiments, the anti-NY-ESO-1 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 β-chain amino acid sequence listed in SEQ ID NO:2, wherein the anti-NY-ESO-1 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the anti-NY-ESO-1 TCR includes 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises an α-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:7. In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 TCR encoded by the first nucleotide also 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 containing at least one, at least two, at least three, at least four, or at least five amino acid substitutions relative to the amino acid sequence of the constant region of the α-chain amino acid sequence listed in SEQ ID NO:1.

[0161] In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises a β-chain CDR3 containing the amino acid sequence as listed in SEQ ID NO:10. In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 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-NY-ESO-1 TCR comprises an α-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:7. In some embodiments, the anti-NY-ESO-1 TCR comprises an α-chain containing the amino acid sequence listed in SEQ ID NO:1.

[0163] In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 TCR comprises a β-chain CDR3 containing the amino acid sequence listed in SEQ ID NO:10. In some embodiments, the anti-NY-ESO-1 TCR comprises a β-chain containing the amino acid sequence listed in SEQ ID NO:2.

[0164] In some embodiments, the anti-NY-ESO-1 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-NY-ESO-1 TCR binds to the same epitope as the reference TCR. In some embodiments, the anti-NY-ESO-1 TCR binds to an epitope of NY-ESO-1 comprising the amino acid sequence listed in SEQ ID NO:13. In some embodiments, the anti-NY-ESO-1 TCR binds to an epitope of NY-ESO-1 consisting of an amino acid sequence as listed in SEQ ID NO:13 (LAMPFATPM). In some embodiments, the epitope consists of amino acid residues 92-100 (SEQ ID NO:52) of NY-ESO-1, for example, “NY-ESO-1”. 92-100 ".

[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 these 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-C alleles: HLA-C*03:02, HLA-C*03:03, HLA-C*03:04, HLA-C*03:05, and HLA-C*03:06. In some embodiments, the HLA-C allele is the HLA-C*03:02 allele. In some embodiments, the HLA-C allele is the HLA-C*03:03 allele. In some embodiments, the HLA-C allele is the HLA-C*03:04 allele. In some embodiments, the HLA-C allele is the HLA-C*03:05 allele. In some embodiments, the HLA-C allele is the HLA-C*03:06 allele.

[0169] In some implementations, HLA class 1 molecules are HLA-C alleles selected from the following groups: HLA-C*03:02:01; HLA-C*03:02:02:01; HLA-C*03:02:02:02; HLA-C*03:02:02:03; HLA-C*03:02:02:04; HLA-C*03:02:02:05; HLA-C*03:02:03; HLA-C*03:02:04; HLA-C*03:02:05; HLA-C*03:02:06; HLA-C*03:02:07; HLA-C*03:02:08; HLA-C*03:02:09; HLA-C*03:02:10; HLA-C*03:02:11; HLA-C*03:02:12; HLA-C*03:02:13; HLA-C*03:02:14; HLA-C*03:02:15; HLA-C*03:02:16; HLA-C*03:02:17; HLA-C*03:02:18; HLA-C*03:02:19; HLA-C*03:02:20; HLA-C*03:02:21; HLA-C*03:02:22; and any combination thereof. In some embodiments, HLA Class 1 molecules are HLA-C alleles selected from the following groups: HLA-C*03:03:01:01; HLA-C*03:03:01:02; HLA-C*03:03:01:03; HLA-C*03:03:01:04; HLA-C*03:03:01:05; HLA-C*03:03:01:06; HLA-C*03:03:01:07; HLA-C*03:03:01:08; HLA-C*03:03:01:09; HLA-C*03:03:01:10; HLA-C*03:03:01:11; HLA-C*03:03:01:12; HLA-C*03:03:01:13; HLA-C*0 3:03:01:14; HLA-C*03:03:02; HLA-C*03:03:03; HLA-C*03:03:04; HLA-C*03:0 3:05;HLA-C*03:03:06;HLA-C*03:03:07;HLA-C*03:03:08;HLA-C*03:03:09;HL A-C*03:03:10; HLA-C*03:03:11; HLA-C*03:03:12; HLA-C*03:03:13; HLA-C*03 :03:14; HLA-C*03:03:15; HLA-C*03:03:16; HLA-C*03:03:17; HLA-C*03:03:18;HLA-C*03:03:19;HLA-C*03:03:20;HLA-C*03:03:21;HLA-C*03:03:22;HLA-C*03 :03:23;HLA-C*03:03:24;HLA-C*03:03:25;HLA-C*03:03:26;HLA-C*03:03:27;H LA-C*03:03:28;HLA-C*03:03:29;HLA-C*03:03:30;HLA-C*03:03:31;HLA-C*03: 03:32;HLA-C*03:03:33;HLA-C*03:03:34;HLA-C*03:03:35;HLA-C*03:03:36;HLA -C*03:03:37;HLA-C*03:03:38;HLA-C*03:03:39;HLA-C*03:03:40;HLA-C*03:03 :41;HLA-C*03:03:42;HLA-C*03:03:43;HLA-C*03:03:44;HLA-C*03:03:45;HLA-C *03:03:46;HLA-C*03:03:47;HLA-C*03:03:48;HLA-C*03:03:49;HLA-C*03:03:5 0:HLA-C*03:03:51;HLA-C*03:03:52;HLA-C*03:03:53; 1. The main characteristics of HLA-C*03:04:01:01;HLA-C*03:0 :01:02;HLA-C*03:04:01:03;HLA-C*03:04:01:04;HLA-C*03:04: 01:05;HLA-C*03:04:01:06;HLA-C*03:04:01:07;HLA-C*03:04:0 1:08;HLA-C*03:04:01:09;HLA-C*03:04:01:10;HLA-C*03:04:01 :11;HLA-C*03:04:01:12;HLA-C*03:04:01:13;HLA-C*03:04:02; HLA-C*03:04:03;HLA-C*03:04:04;HLA-C*03:04:05;HLA-C*03:0 4:06;HLA-C*03:04:07;HLA-C*03:04:08;HLA-C*03:04:09;HLA-C *03:04:10;HLA-C*03:04:11;HLA-C*03:04:12;HLA-C*03:04:13;HLA-C*03:04:14;HLA-C*03:04:15;HLA-C*03:04:16;HLA-C*03:04: 17;HLA-C*03:04:18;HLA-C*03:04:19;HLA-C*03:04:20;HLA-C*03:0 4:21;HLA-C*03:04:22;HLA-C*03:04:23;HLA-C*03:04:24;HLA-C*0 3:04:25;HLA-C*03:04:26;HLA-C*03:04:27;HLA-C*03:04:28;HLA-C *03:04:29;HLA-C*03:04:30;HLA-C*03:04:31;HLA-C*03:04:32;HL A-C*03:04:33;ALL-C*03:04:34;A-A-C*03:04:35;A-C*03:04:36; HLA-C*03:04:37;HLA-C*03:04:38;HLA-C*03:04:39;HLA-C*03:04:4 0;HLA-C*03:04:41;HLA-C*03:04:42;HLA-C*03:04:43;HLA-C*03:04 :44;HLA-C*03:04:45;HLA-C*03:04:46;HLA-C*03:04:47;HLA-C*03 :04:48;HLA-C*03:04:49;HLA-C*03:04:50;HLA-C*03:04:51;HLA-C* 03:04:52;HLA-C*03:04:53;HLA-C*03:04:54;HLA-C*03:04:55;HLA -C*03:04:56;HLA-C*03:04:57;HLA-C*03:04:58;HLA-C*03:04:59;H LA-C*03:04:60;HLA-C*03:04:61;HLA-C*03:04:62;HLA-C*03:04:6 3;HLA-C*03:04:64;HLA-C*03:04:65;HLA-C*03:04:66;HLA-C*03:04 :67;HLA-C*03:04:68;HLA-C*03:04:69;HLA-C*03:04:70;HLA-C*03: 04:71;HLA-C*03:04:72;HLA-C*03:04:73;I don't want to lose my temper 1. The sequences of the HLA-C genes were as follows:HLA-C*03:05;HLA-C*03:06:01;C0-6:20HL

[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 HLA-C alleles selected from the following: HLA-C*03:02 allele, HLA-C*03:03 allele, HLA-C*03:04 allele, HLA-C*03:05 allele, and HLA-C*03:06 allele. In some embodiments, the HLA-C allele is the HLA-C*03:02 allele. In some embodiments, the HLA-C allele is the HLA-C*03:03 allele. In some embodiments, the HLA-C allele is the HLA-C*03:04 allele. In some embodiments, the HLA-C allele is the HLA-C*03:05 allele. In some embodiments, the HLA-C allele is the HLA-C*03:06 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] Some 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 NY-ESO-1 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 NY-ESO-1 antigen specificity against the HLA-C*03:04 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.

[0212] Since pHLA multimer production requires peptides with known and precise sequences, high-throughput screening for novel epitope peptides using pHLA multimer-based strategies is neither direct nor practical. In addition to structure-based analyses using pHLA multimers, functional analyses can be applied to determine T-cell antigen specificity. Functional analyses were performed using artificial antigen-presenting cells (APCs) that can process and handle longer peptides and act as stimulators to present epitope peptides via class I molecules. HLA-C*03:04-artificial APCs were pulsed with overlapping peptides (Table 5) covering the full protein of NY-ESO-1 and used as stimulators in the cytokine ELISPOT assay. When stimulated with C*03:04-artificial APCs pulsed with overlapping peptides derived from NY-ESO-1, C*03:04 showed significant differences in IFN-γ ELISPOT assays. + Melanoma TILs show pairs with shared sequences 91 YLAMPFATPM 100 Positive responses of three adjacent peptides ( Figure 1 Using a series of mutant deletion peptides, the minimum required peptide epitopes presented by the C*03:04 molecule were identified. 92 LAMPFATPM 100 Importantly, the C*03:04 / NY-ESO-192-100 multimer successfully stained up to 18.2% of polyclonal amplified TILs, thus demonstrating that C*03:04 / NY-ESO-1 92- 100 T cells are the dominant TIL population. Figure 2A-2C According to ELISPOT analysis, multimer-positive T cells secrete detectable IFN-γ in an HLA-restricted peptide-specific manner. Figure 3 ).

[0213] Table 5. NY-ESO-1 overlapping peptides.

[0214]

[0215]

[0216] Collect multimer-positive anti-tumor T cells and molecularly clone their TCR genes ( Figure 4A-4I (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 reconstructing on primary T cells, C*03:04 / NY-ESO-1 92-100 TCR-transduced T cells were successfully stained with homologous multimers. Figures 5A-5D ) and NY-ESO-1 presented by surface C*03:04 molecules 92-100 Peptide strong reaction ( Figure 6 Importantly, these cells were able to recognize tumor cells that naturally express the NY-ESO-1 gene and whose peptides were untreated with C*03:04 matching. Although both the A375 and SK-MEL-37 melanoma cell lines were negative for C*03:04, they endogenously expressed the NY-ESO-1 gene. When the C*03:04 molecule is ectopically expressed, the C*03:04 / NY-ESO-1 ratio is significantly increased. 92-100 TCR-transduced T cells successfully recognized both melanoma cell lines. Furthermore, SK-MEL-21 melanoma cells lacking endogenous NY-ESO-1 expression became resistant to C*03:04 / NY-ESO-1 upon transduction of the full-length NY-ESO-1 gene. 92-100 TCR-transduced T cells are reactive ( Figures 7A-7B (8A-8E and 9A-9D). These results clearly demonstrate that C*03:04 / NY-ESO-1 92-100 TCR-transduced T cells showed a strong affinity for recognizing tumor cells, and the cloned C*03:04 / NY-ESO-1 92-100 TCRs are tumor-responsive.

[0217] The use of a newly cloned tumor-reactive C*03:04-restricted NY-ESO-1 TCR gene can broaden the applicability of anti-NY-ESO-1TCR gene therapy to patients with HLA-A*02:01-positive cancers.

[0218] method

[0219] Cell samples

[0220] Peripheral blood samples were obtained from healthy donors after approval by the institutional review board. 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. A375, SK-MEL-21, SK-MEL-37, and LM-MEL-53 are melanoma cell lines. The aforementioned melanoma cell lines (except LM-MEL-53) were grown in DMEM supplemented with 10% FBS and 50 μg / ml gentamicin (Invitrogen). K562, T2, Jurkat 76, and LM-MEL-53 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.

[0221] peptides

[0222] The synthetic peptides were dissolved in DMSO to a concentration of 50 μg / ml. The peptides used were 20-meric overlapping peptides covering the full protein of NY-ESO-1 and C*03:04-restricted NY-ESO-1. 92-100 (LAMPFATPM), MAGE-A1 230-238 (SAYGEPRKL) and HIV gag 164-172 (YVDRFFKTL) peptide. Using MAGE-A1. 230-238 and HIV gag 164-172 Peptides were used as negative controls.

[0223] Gene

[0224] The HLA-C*03:04 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 an anti-NGFR monoclonal antibody (mAb). The full-length NY-ESO-1 gene was cloned from Me275 cells via RT-PCR according to the published sequence (SEQ ID NO:52). The TCR gene was cloned using the SMARTer RACE cDNA amplification kit (Takara Bio) via 5' rapid amplification of the cDNA ends (RACE) PCR. For cloning the TCRα gene, cDNA was amplified for the first round of PCR using supplied 5'-RACE primers and 3'-TCRα untranslated region primers (5'-GGAGAGTTCCCTCTGTTTGGAGAG-3'; SEQ ID NO:57). A second round of PCR was performed using modified 5'-RACE primers (5'-GTGTGGTGGTACGGGAATTCAAGCAGTGGTATCAACGCAGAGT-3'; SEQ ID NO: 58) and 3'-TCRα primers (5'-ACCACTGTGCTGGCGGCCGCTCAGCTGGACCACAGCCGCAGCG-3'; SEQ ID NO: 59). For cloning the TCRβ gene, the first round of PCR was performed using the supplied 5'-RACE primers and βC region-specific reverse primers 3'-Cβ-1 (5'-ATCGTCGACCACTGTGCTGGCGGCCGCTCGAGTTCCAGGGCTGCCTTCAGAAATCC-3'; SEQ ID NO: 60) and 3'-Cβ-2 (5'-GACCACTGTGCTGGCGGCCGCTCGAGCTAGCCTCTGGAATCCTTTCTCTTGACCATTGC-3'; SEQ ID NO: 61) to amplify the cDNA. A second round of PCR was performed using modified 5'-RACE primers and βC region-specific reverse primers. The allele names for the TCRα and β genes are based on the unique gene terminology of the international ImMunoGeneTics information system. http: / / www.imgt.org All genes were cloned into the pMX retroviral vector and transduced using a retroviral system based on 293GPG cells.

[0225] transfectants

[0226] 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)). The TCR gene was transduced into human primary T cells using retroviral supernatant derived from PG13. The TCR gene was transfected into the 293GPG cell line using TransIT293 (Mirus Bio). NY-ESO-1 was transduced using a full-length NY-ESO-1 gene retrovirus. - SK-MEL-21 cells were used to generate SK-MEL-21 / NY-ESO-1 cells. The expression of transduced NY-ESO-1 was assessed by flow cytometry after staining with anti-NY-ESO-1 mAb (clone D1Q2U; Cell Signaling Technology).

[0227] HLA-C*03:04 retrovirus transduction was used to transduce HLA-C*03:04. - A375 and SK-MEL-37 cells were used to generate A375 / C*03:04 and SK-MEL-37 / C*03:04 cells. The HLA-C*03:04 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.

[0228] Flow cytometry and cell sorting

[0229] Cell surface molecules were stained with PC5-conjugated anti-CD8 mAb (clone B9.11; Beckman Coulter), FITC-conjugated anti-NGFR (clone ME20.4; Biolegend), and APC / Cy7-conjugated anti-CD3 (clone UCHT1; Biolegend). Dead cells were identified using the LIVE / DEAD Fixable Aqua Dead Cell Stainkit (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 FACS Aria II (BD Biosciences).

[0230] Cytokine ELISPOT Analysis

[0231] IFN-γ ELISPOT assays were performed as previously described (see, for example, Kagoya et al., Nat. Commun. 9:1915 (2018); Anczurowski et al., Sci. Rep. 8:4804 (2018); and Yamashita et al., Nat Commun. 8:15244 (2017)). PVDF plates (Millipore, Bedford, MA) were coated with a capture mAb (1-D1K; MABTECH, Mariemont, OH), and T cells were added to each well with 2 x 10 T 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).

[0232] Primary CD8 transduced using cloned TCR + T cell expansion

[0233] 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.

[0234] Production of pHLA polymers in mammalian cells

[0235] 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 Genes and β2m genes were individually transduced into HEK293T cells. 43 Category I, which enables ectopic expression to mature soluble affinity. Q115E -K b Stable 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 incubated overnight at 37°C with 100-1000 μg / ml of the target class I restriction peptide 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 -Kb Molecular concentration.

[0236] pHLA polymer staining

[0237] 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 inoculation with R-phycoerythrin-conjugated AffiniPure Fab fragment goat anti-mouse IgG1 (Jackson Immuno Research Laboratories) at 4°C for 15 minutes. Next, wash the cells three times and co-stain with anti-CD8 mAb at 4°C for 15 minutes. Finally, use a live / dead fixable dead cell staining kit to identify dead cells.

[0238] Statistical analysis

[0239] 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> 4285.001PC01 / C-K / BMD <150> US 62 / 813,639 <151> 2019-03-04 <160> 61 <170> PatentIn version 3.5 <210> 1 <211> 273 <212> PRT <213> Artificial sequence <220> <223> α-chain amino acid sequence <400> 1 Met Leu Leu Glu Leu Ile Pro Leu Leu Gly Ile His Phe Val Leu Arg 1 5 10 15 Thr Ala Arg Ala Gln Ser Val Thr Gln Pro Asp Ile His Ile Thr Val 20 25 30 Ser Glu Gly Ala Ser Leu Glu Leu Arg Cys Asn Tyr Ser Tyr Gly Ala 35 40 45 Thr Pro Tyr Leu Phe Trp Tyr Val Gln Ser Pro Gly Gln Gly Leu Gln 50 55 60 Leu Leu Leu Lys Tyr Phe Ser Gly Asp Thr Leu Val Gln Gly Ile Lys 65 70 75 80 Gly Phe Glu Ala Glu Phe Lys Arg Ser Gln Ser Ser Phe Asn Leu Arg 85 90 95 Lys Pro Ser Val His Trp Ser Asp Ala Ala Glu Tyr Phe Cys Ala Gly 100 105 110 Met Asp Ser Asn Tyr Gln Leu Ile Trp Gly Ala Gly Thr Lys Leu Ile 115 120 125 Ile Lys 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> 311 <212> PRT <213> Artificial sequence <220> <223> β-chain amino acid sequence <400> 2 Met Ser Ile Gly Leu Leu Cys Cys Val Ala Phe Ser Leu Leu Trp Ala 1 5 10 15 Ser Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Asn Ser Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Ala Ser Glu Gly Thr Thr Asp Lys Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Leu Asn Lys Arg Glu Phe Ser Leu Arg 85 90 95 Leu Glu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Leu Pro Leu Gly Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175 Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300 Arg Lys Asp Ser Arg Gly Glx 305 310 <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 Tyr Gly Ala Thr Pro Tyr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> α CDR2 <400> 6 Tyr Phe Ser Gly Asp Thr Leu Val 1 5 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> α CDR3 <400> 7 Cys Ala Gly Met Asp Ser Asn Tyr Gln Leu Ile Trp 1 5 10 <210> 8 <211> 5 <212> PRT <213> Artificial sequence <220> <223> β CDR1 <400> 8 Met Asn His Asn Ser 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial sequence <220> <223> β CDR2 <400> 9 Ser Ala Ser Glu Gly Thr 1 5 <210> 10 <211> 13 <212> PRT <213> Artificial sequence <220> <223> β CDR3 <400> 10 Cys Ala Ser Ser Leu Pro Leu Gly Tyr Glu Gln Tyr Phe 1 5 10 <210> 11 <400> 11 000 <210> 12 <400> 12 000 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Epitope <400> 13 Leu Ala Met Pro Phe Ala Thr Pro Met 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> Nucleotide sequence of the α chain <400> 17 atgctcctgg agcttatccc actgctgggg atacattttg tcctgagaac tgccagagcc 60 cagtcagtga cccagcctga catccacatc actgtctctg aaggagcctc actggagttg 120 (注:原文中<{ 和<{220>疑似格式错误,已按原样保留并翻译)agatgtaact attcctatgg ggcaacacct tatctcttct ggtatgtcca gtcccccggc 180 caaggcctcc agctgctcct gaagtacttt tcaggagaca ctctggttca aggcattaaa 240 ggctttgagg ctgaatttaa gaggagtcaa tcttcttca atctgaggaa accctctgtg 300 cattggagtg atgctgctga gtacttctgt gccggcatgg atagcaacta tcagttaatc 360 tggggcgctg ggaccaagct aattataaag ccagatatcc agaaccctga ccctgccgtg 420 taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480 tctcaacaa 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> 933 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence of β chain <400> 18 atgagcatcg ggctcctgtg ctgtgtggcc ttttctctcc tgtgggcaag tccagtgaat 60 gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 120 cagtgtgccc aggatatgaa ccataactcc atgtactggt atcgacaaga cccaggcatg 180 ggactgaggc tgatttatta ctcagcttct gagggtacca ctgacaaagg agaagtcccc 240 aatggctaca atgtctccag attaaacaaa cgggagttct cgctcaggct ggagtcggct 300 gctccctccc agacatctgt gtacttctgt gccagcagtc tccctctagg ctacgagcag 360 tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 420 gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480 gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540 aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600 aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660 ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720 acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780 gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840 gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900 gccatggtca agagaaagga ttccagaggc tag 933 <210> 19 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 19 Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 1 5 I0 15 Gly Pro Gly Gly 20 <210> 20 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 20 Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro 1 5 10 15 Gly Ile Pro Asp 20 <210> twenty one <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty one Ser Thr Gly Asp Ala Asp Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly 1 5 10 15 Pro Gly Gly Asn 20 <210> twenty two <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty two Asp Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala 1 5 10 15 Gly Gly Pro Gly 20 <210> twenty three <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty three Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu 1 5 10 15 Ala Gly Ala Thr 20 <210> twenty four <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty four Gly Pro Gly Gly Asn Ala Gly Gly Pro Gly Glu Ala Gly Ala Thr Gly 1 5 10 15 Gly Arg Gly Pro 20 <210> 25 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 Ala Gly Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg 1 5 10 15 Gly Ala Gly Ala 20 <210> 26 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala 1 5 10 15 Arg Ala Ser Gly 20 <210> 27 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Gly Gly Arg Gly Pro Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly Pro 1 5 10 15 Gly Gly Gly Ala 20 <210> 28 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 28 Arg Gly Ala Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro 1 5 10 15 Arg Gly Pro His 20 <210> 29 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 29 Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro His Gly 1 5 10 15 Gly Ala Ala Ser 20 <210> 30 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 30 Pro Gly Gly Gly Ala Pro Arg Gly Pro His Gly Gly Ala Ala Ser Gly 1 5 10 15 Leu Asn Gly Cys 20 <210> 31 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 31 Pro Arg Gly Pro His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys 1 5 10 15 Arg Cys Gly Ala 20 <210> 32 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 32 Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg 1 5 10 15 Gly Pro Glu Ser 20 <210> 33 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 33 Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala Arg Gly Pro Glu Ser Arg 1 5 10 15 Leu Leu Glu Phe 20 <210> 34 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 34 Cys Arg Cys Gly Ala Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr 1 5 10 15 Leu Ala Met Pro 20 <210> 35 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 35 Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 1 5 10 15 Ala Thr Pro Met 20 <210> 36 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 36 Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu 1 5 10 15 Ala Glu Leu Ala 20 <210> 37 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 37 Tyr Leu Ala Met Pro Phe Ala Thr Pro Met Glu Ala Glu Leu Ala Arg 1 5 10 15 Arg Ser Leu Ala 20 <210> 38 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 38 Phe Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln 1 5 10 15 Asp Ala Pro Pro 20 <210> 39 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 39 Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp Ala Pro Pro Leu 1 5 10 15 Pro Val Pro Gly 20 <210> 40 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 40 Arg Arg Ser Leu Ala Gln Asp Ala Pro Pro Leu Pro Val Pro Gly Val 1 5 10 15 Leu Leu Lys Glu 20 <210> 41 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 41 Gln Asp Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe 1 5 10 15 Thr Val Ser Gly 20 <210> 42 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 42 Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn 1 5 10 15 Ile Leu Thr Ile 20 <210> 43 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 43 Val Leu Leu Lys Glu Phe Thr Val Ser Gly Asn Ile Leu Thr Ile Arg 1 5 10 15 Leu Thr Ala Ala 20 <210> 44 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 44 Phe Thr Val Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp 1 5 10 15 His Arg Gln Leu 20 <210> 45 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 45 Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln Leu Gln 1 5 10 15 Leu Ser Ile Ser 20 <210> 46 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 46 Arg Leu Thr Ala Ala Asp His Arg Gln Leu Gln Leu Ser Ile Ser Ser 1 5 10 15 Cys Leu Gln Gln 20 <210> 47 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 47 Asp His Arg Gln Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu 1 5 10 15 Ser Leu Leu Met 20 <210> 48 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 48 Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met Trp 1 5 10 15 Ile Thr Gln Cys 20 <210> 49 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 49 Ser Cys Leu Gln Gln Leu Ser Leu Leu Met Trp Ile Thr Gln Cys Phe 1 5 10 15 Leu Pro Val Phe 20 <210> 50 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 50 Leu Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu 1 5 10 15 Ala Gln Pro Pro 20 <210> 51 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 51 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser 1 5 10 15 Gly Gln Arg Arg 20 <210> 52 <211> 180 <212> PRT <213> Artificial sequence <220> <223> NY-ESO-1 amino acid sequence <400> 52 Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp 1 5 10 15 Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly 20 25 30 Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala 35 40 45 Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro 50 55 60 His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala 65 70 75 80 Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe 85 90 95 Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp 100 105 110 Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val 115 120 125 Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln 130 135 140 Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met 145 150 155 160 Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser 165 170 175 Gly Gln Arg Arg 180 <210> 53 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> siRNA-TCRa-1 <400> 53 guaaggauuc ugauguguat t 21 <210> 54 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> siRNA-TCRa-2 <400> 54 uacacaucag aauccuuact t 21 <210> 55 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> siRNA-TCRb-1 <400> 55 ccaccauccu cuaugagaut t 21 <210> 56 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> siRNA-TCRb-2 <400> 56 aucucauaga ggaugguggt t 21 <210> 57 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> 3-TCR α-untranslated region primers <400> 57 ggagagttcc ctctgtttgg agag 24 <210> 58 <211> 43 <212> DNA <213> Artificial sequence <220> <223> Modified 5'-RACE primers <400> 58 gtgtggtggt acgggaattc aagcagtggt atcaacgcag agt 43 <210> 59 <211> 43 <212> DNA <213> Artificial sequence <220> <223> 3'-TCR-a primer <400> 59 accactgtgc tggcggccgc tcagctggac cacagccgca gcg 43 <210> 60 <211> 56 <212> DNA <213> Artificial sequence <220> <223> β-C region specific reverse primer 3-C β-1 <400> 60 atcgtcgacc actgtgctgg cggccgctcg agttccaggg ctgccttcag aaatcc 56 <210> 61 <211> 59 <212> DNA <213> Artificial sequence <220> <223> β-C region specific reverse primer 3-C β-2 <400> 61 gaccactgtg ctggcggccg ctcgagctag cctctggaat cctttctctt gaccattgc 59

Claims

1. A nucleic acid molecule, said nucleic acid molecule comprising (i) A first nucleotide sequence encoding an anti-NY-ESO-1 TCR that specifically binds to a human NY-ESO-1 epitope consisting of the amino acid sequence shown in SEQ ID NO: 13, wherein the TCR is a recombinant T cell receptor or its antigen-binding moiety, and wherein the epitope is complexed with the HLA class I molecule HLA-C*03 allele; wherein the anti-NY-ESO-1 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; and wherein: (a) The amino acid sequence of the β chain CDR3 against NY-ESO-1TCR is shown in SEQ ID NO:10; (b) The amino acid sequence of the β-chain CDR2 against NY-ESO-1TCR is shown in SEQ ID NO:9; (c) The amino acid sequence of the β chain CDR1 against NY-ESO-1TCR is shown in SEQ ID NO:8; (d) The amino acid sequence of the α chain CDR3 against NY-ESO-1TCR is shown in SEQ ID NO:7; (e) The amino acid sequence of the α-chain CDR2 against NY-ESO-1TCR is shown in SEQ ID NO:6; (f) The amino acid sequence of the α-chain CDR1 against NY-ESO-1TCR 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-C*03 allele is selected from the HLA-C*03:02 allele, HLA-C*03:03 allele, HLA-C*03:04 allele, HLA-C*03:05 allele and HLA-C*03:06 allele.

3. The nucleic acid molecule as described in claim 1, wherein... (i) The α-chain variable domain of the anti-NY-ESO-1TCR contains the amino acid sequence of the variable domain present in the amino acid sequence listed in SEQ ID NO:1; (ii) The β-chain variable domain of the anti-NY-ESO-1TCR contains the amino acid sequence of the variable domain present in the amino acid sequence 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 amino acid sequence of the α chain of the anti-NY-ESO-1TCR is shown in SEQ ID NO:1; The amino acid sequence of the β chain of the anti-NY-ESO-1TCR is shown in SEQ ID NO:2; or (iii)(i) and (ii) both.

5. The nucleic acid molecule according to any one of claims 1 to 4, wherein the second nucleotide sequence (i) One or more siRNAs for reducing the expression of endogenous TCR, wherein the one or more siRNAs are complementary to a target sequence within a nucleotide sequence encoding a constant region of the endogenous TCR; (ii) Encode Cas9; or (iii)(i) and (ii) both.

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

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

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

9. The vector as described in claim 7, wherein the vector is a retroviral vector.

10. The vector of claim 7, wherein the vector is selected from the group consisting of: adenovirus vectors, lentivirus vectors, Sendai virus vectors, baculovirus vectors, Epstein-Barr virus vectors, multivaccinia virus vectors, vaccinia virus vectors, herpes simplex virus vectors, hybrid vectors, and adeno-associated virus (AAV) vectors.

11. The vector as described in claim 7, wherein the vector is a lentiviral vector.

12. A cell comprising a nucleic acid molecule according to any one of claims 1 to 6 or a vector according to any one of claims 7 to 11.

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

14. The cell of claim 12, wherein the cell is a T cell.

15. The cell of claim 12, wherein the cell is a natural killer cell.

16. The cell of claim 12, wherein the cell is a natural killer T cell or an ILC cell.

17. Use of the cells of any one of claims 12 to 16 in the preparation of a medicament for treating melanoma in a subject of 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 with a nucleic acid molecule of any one of claims 1 to 6 or a vector of any one of claims 7 to 11.

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 (NK) cell.

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