Composition of NY-ESO-1-specific T cell receptors restricted to multiple major histocompatibility complex molecules

Abstract

To provide a composition of NY-ESO-1-specific T cell receptors restricted on multiple major histocompatibility complex molecules.SOLUTION: Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. The classical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across diverse cancer types. It has also been targeted with an A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in diverse HLA haplotypes, we isolated TCRs specific for NY-ESO-1 presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we have developed an approach to extend applicability of TCR gene therapies targeting NY-ESO-1 to application to patient populations beyond those expressing HLA-A2.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of pending and commonly assigned U.S. Provisional Patent Application No. 62 / 727,485, filed September 5, 2018, and entitled "Composition of NY-ESO-1-Specific T Cell Receptors Restricted to Multiple Major Histocompatibility Complex Molecules," the contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with United States government support under grant numbers CA132681 and CA197633 from the National Institutes of Health. The United States government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on August 28, 2019, is titled 30435_364-WO-U1_SL.txt and is 101,375 bytes in size.

[0004] Technical Field The present invention relates to methods and materials useful in αβ T cell receptor gene therapy. [Background technology]

[0005] The αβ T cell receptor (TCR) determines the unique specificity of each naive T cell. Following recruitment of CD3 signaling proteins to the T cell surface, the TCR monitors peptide ligands presented by major histocompatibility complex (MHC) molecules on the surface of nucleated cells. The specificity of the TCR for a peptide-MHC complex is determined by both the presented MHC molecule and the presented peptide. The MHC locus (also known in humans as the human leukocyte antigen (HLA) locus) is the largest multiallelic locus in the human genome, containing over 18,000 MHC class I and II alleles whose frequencies vary widely among ethnic subgroups (1, 2). Ligands presented by MHC class I molecules are primarily derived from proteasomal cleavage of endogenously expressed antigens. Infected and cancerous cells present peptides that CD8+ T cells recognize as foreign or abnormal, resulting in T cell-mediated killing of the presenting cells.

[0006] T cells can be engineered to kill tumor cells through the introduction of tumor-reactive αβ TCR genes (3). The key to this approach is that the patient expresses MHC alleles that are restricted by the therapeutic TCR, and the target peptide is derived from a tumor-associated or tumor-specific antigen. Private (patient-specific) neoantigens resulting from tumor-specific mutations are a potential source of such targets (4). However, achieving personalized TCR gene therapy is complex because it requires identifying mutations through sequencing, isolating patient-specific TCRs that are reactive to the mutations, and then genetically engineering the patient's T cells as desired. This is even more challenging for tumors with low mutation burden, where inaccessible tumors cannot even be sequenced and where neoantigens are few or absent (5). For these last tumor types in particular, targeting public (non-patient-specific) tumor-restricted antigens using commercially available TCRs remains a viable option.

[0007] The first public antigen targeted by TCR gene therapy in the clinic was the melanocyte antigen MART1 / Melan-A, which induced objective responses in two of 15 metastatic melanoma patients (6). Using a higher-affinity MART1-reactive TCR (F5), the response rate increased to 30%, but it also resulted in various side effects, including vitiligo, uveitis, and transient hearing loss, due to MART1 expression on healthy melanocytes in the skin, eyes, and middle ear (7). T cell therapies targeting other public antigens have also resulted in similar morbidity or other serious adverse events due to on-target / off-tumor reactivity. For example, targeting carcinoembryonic antigen (CEA) can cause severe colitis in patients with metastatic colorectal cancer due to reactivity with normal colon tissue (8). More seriously, T cell therapies targeting ERBB2 or MAGE-A3, respectively, have resulted in death due to the lack of expression of the target antigen (or similar variants) in vital organs (9, 10). Thus, these studies highlight the importance of identifying public antigens that are strictly tumor-specific (11), especially when using well-expressed, high-affinity target receptors (7, 12), which are necessary for successful therapy. NY-ESO-1, the product of the CTAG1B gene, is a potential target for existing TCR gene therapy. As a common cancer-testis antigen, NY-ESO-1 is not expressed in normal non-germ cell tissues, but is aberrantly expressed in numerous tumors (13). The frequency of aberrant expression ranges from 10% to 50% in solid tumors, 25% to 50% in melanoma, and up to 80% in synovial sarcoma (13-18). High expression is observed in aggressive metastatic tumor tissues (14, 15, 19). Furthermore, NY-ESO-1 is highly immunogenic and promotes spontaneous and vaccine-induced T cell immune responses against multiple epitopes presented by various MHC alleles (20-23). ​​Consequently, HLA-A *The epitope NY-ESO-1157-165 (SLLMWITQC, SEQ ID NO: 36) presented by 02:01 targeted the allogeneic 1G4 TCR in a gene therapy trial, demonstrating objective response rates of 55% and 61% in patients with metastatic melanoma and synovial sarcoma, respectively, with no targeting-related adverse events (24, 25). Targeting this same A2-restricted epitope in lentiviral-mediated TCR gene therapy in patients with multiple myeloma also resulted in a 70% complete or near-complete response without significant safety concerns (26). Unfortunately, however, the majority of patients who responded to treatment relapsed within a few months, and loss of heterozygosity at the MHCI locus indicates that tumors are HLA-A-restricted. * It has been reported as a mechanism of escape from adoptive T cell therapy targeting 02:01 / NY-ESO-1157-165 (27). Thus, NY-ESO-1 is a tumor-specific, immunogenic public antigen that is expressed in a variety of tumor types and can be safely targeted in the clinic, but is easily lost when targeted via a single HLA subtype. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Robinson J, et al. (2015) The IPD and IMGT / HLA database: allele variant databases. Nucleic Acids Res 43(Database issue):D423-431. [Non-patent document 2] Gonzalez-Galarza FF, et al. (2015) Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations. Nucleic Acids Res 43(Database issue):D784-788. [Non-patent document 3] Johnson LA, et al. (2006) Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J Immunol 177(9):6548-6559. [Non-patent document 4] Schumacher TN & Schreiber RD (2015) Neoantigens in cancer immunotherapy. Science 348(6230):69-74. [Non-Patent Document 5] Bethune MT & Joglekar AV (2017) Personalized T cell-mediated cancer immunotherapy: progress and challenges. Current opinion in biotechnology 48:142-152. [Non-patent document 6] Morgan RA, et al. (2006) Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314(5796):126-129. [Non-Patent Document 7] Johnson LA, et al. (2009) Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 114(3):535-546.

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[0009] For the reasons stated above, there is a need in the art for additional methods and materials useful for NY-ESO-1 TCR gene therapy. [Means for solving the problem]

[0010] As mentioned above, T lymphocytes can be engineered to express tumor-specific T cell receptor (TCR) genes and thereby kill cancer cells. This approach—called TCR gene therapy—is effective, but can cause serious adverse events if the target is also expressed in healthy, non-cancerous tissues. NY-ESO-1 is a tumor-specific antigen that has been successfully and safely targeted via TCR gene therapy in melanoma, synovial sarcoma, and myeloma. However, previous studies have shown that HLA-A * Because previous studies focused on a single NY-ESO-1-derived epitope presented by 02:01, their application was limited to patients expressing that allele. As described below, we developed a novel TCR that collectively recognizes multiple NY-ESO-1-derived epitopes presented by multiple MHC alleles. This provides a general approach to expand targeted immunotherapy to more diverse MHC haplotypes.

[0011] Embodiments of the present invention include modified CD8 T cells containing nucleic acids encoding specific αβ T cell receptor polypeptides. + The present invention includes methods and materials for generating and using T cells. Embodiments of the invention include, for example, polynucleotides placed in vectors, where the polynucleotides encode Vα T cell receptor polypeptides and / or Vβ T cell receptor polypeptides. In a typical embodiment, Vα / Vβ T cell receptors, including Vα T cell receptor polypeptides and / or Vβ T cell receptor polypeptides, are expressed in a manner similar to that of CD8 T cells. + When expressed on T cells, CD8 + The heterologous Vα / Vβ T cell receptor expressed on the surface of T cells recognizes the NY-ESO-1 peptide associated with human leukocyte antigen A2, human leukocyte antigen B07, human leukocyte antigen B18, or human leukocyte antigen C03. In exemplary embodiments of the invention disclosed herein, the heterologous T cell receptor comprises a Vα / Vβ T cell receptor designated "3A1," "4A2," "5G6," "9D2," "1E4," "2B8," or "3C7."

[0012] Additionally, embodiments of the invention include numerous different TCR nucleic acids and polypeptides disclosed herein (e.g., αβ TCR nucleic acids and polypeptides encoding TCRs designated "3A1," "4A2," "5G6," "9D2," "1E4," "2B8," and "3C7"). For example, embodiments of the present invention include TCR Va and / or TCR Vβ polynucleotides: a polynucleotide encoding at least a 3A1 TCR Va polypeptide (SEQ ID NO:3); a polynucleotide encoding at least a 3A1 TCR Vβ polypeptide (SEQ ID NO:4); a polynucleotide encoding at least a 4A2 TCR Va polypeptide (SEQ ID NO:7); a polynucleotide encoding at least a 4A2 TCR Vβ polypeptide (SEQ ID NO:37); a polynucleotide encoding at least a 5G6 TCR Va polypeptide (SEQ ID NO:10); a polynucleotide encoding at least a 5G6 TCR Vβ polypeptide (SEQ ID NO:11); a polynucleotide encoding at least a 9D2 TCR Va polypeptide (SEQ ID NO:14); a polynucleotide encoding at least a 9D2 TCR Vβ polypeptide (SEQ ID NO:15); a polynucleotide encoding at least a 1E4 TCR Va polypeptide (SEQ ID NO:18); a polynucleotide encoding at least a 1E4 TCR Vβ polypeptide (SEQ ID NO:19); a polynucleotide encoding at least a 2B8 TCR Va polypeptide (SEQ ID NO:22); a polynucleotide encoding at least a 2B8 TCR Vβ polypeptide (SEQ ID NO:23); a polynucleotide encoding at least a 3C7 TCR Va polypeptide (SEQ ID NO:26); or a polynucleotide encoding at least a 3C7 TCR Va polypeptide (SEQ ID NO:27). The compositions of matter include one or more polynucleotides (typically located in one or more vectors) that encode a polynucleotide encoding a TCR Vβ polypeptide (SEQ ID NO:27). In typical embodiments of the invention, these polynucleotides further encode additional amino acids, such as constant regions of the alpha and / or beta polypeptides, a TM domain, a short cytoplasmic tail, etc.In an exemplary embodiment of the invention, a composition comprises polynucleotides encoding TCR Vα polypeptides together with polynucleotides encoding TCR Vβ polypeptides, and such polynucleotides are placed into one or more vectors such that the Vα / Vβ TCRs are expressed in mammalian cells (e.g., CD8. + T cells), and this expressed heterologous Vα / Vβ TCR recognizes the NY-ESO-1 peptide associated with human leukocyte antigens.

[0013] In another aspect, the present invention provides a method for administering nucleic acids encoding the TCR polypeptides disclosed herein to T cells (e.g., CD8 T cells expressing the NY-ESO-1 antigen) obtained from an individual diagnosed with cancer. + T cells) + In another aspect, the present invention provides a modified CD8 T cell produced according to the methods described herein. + In another aspect, the present invention includes a method of treating a disease or condition characterized by expression of NY-ESO-1. This treatment methodology can be achieved by using the modified CD8 T cells described herein. + The method comprises administering an effective amount of T cells(s) to a subject in need thereof. In general embodiments of the invention, the subject has cancer. In specific embodiments of the invention, the cancer cells form a solid tumor. In some embodiments of the invention, the cancer is melanoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer.

[0014] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and specific examples, while merely illustrative of some embodiments of the present invention, are not intended to be limiting and are for illustrative purposes. Numerous changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the present invention includes all such modifications. In certain embodiments, for example, the following are provided: (Item 1) A polynucleotide for placement into a vector: the polynucleotide encodes a Vα T cell receptor polypeptide and / or a Vβ T cell receptor polypeptide; and The Vα T cell receptor polypeptide and / or the Vα / Vβ T cell receptor containing the Vβ T cell receptor polypeptide are administered to CD8 + When expressed on T cells, the Vα / Vβ T cell receptor: human leukocyte antigen A2; human leukocyte antigen B07; human leukocyte antigen B18; or Human leukocyte antigen C03, The polynucleotide recognizing the NY-ESO-1 peptide associated with (Item 2) the T cell receptor: 3A1 T cell receptor; 4A2 T cell receptor; 5G6 T cell receptor; 9D2 T cell receptor; 1E4 T cell receptor; 2B8 T-cell receptor; or 2. The polynucleotide of item 1, comprising the 3C7 T cell receptor. (Item 3) wherein the vector: (a) a polynucleotide encoding the 3A1 Vα polypeptide (SEQ ID NO:3); (b) a polynucleotide encoding a 3A1 Vβ polypeptide (SEQ ID NO:4); (c) a polynucleotide encoding the 4A2 Vα polypeptide (SEQ ID NO:7); (d) a polynucleotide encoding a 4A2 Vβ polypeptide (SEQ ID NO: 37); (e) a polynucleotide encoding a 5G6 Vα polypeptide (SEQ ID NO: 10); (f) a polynucleotide encoding a 5G6 Vβ polypeptide (SEQ ID NO: 11); (g) a polynucleotide encoding a 9D2 Vα polypeptide (SEQ ID NO: 14); (h) a polynucleotide encoding a 9D2 Vβ polypeptide (SEQ ID NO: 15); (i) a polynucleotide encoding the 1E4 Vα polypeptide (SEQ ID NO: 18); (j) a polynucleotide encoding the 1E4 Vβ polypeptide (SEQ ID NO: 19); (k) a polynucleotide encoding a 2B8 Vα polypeptide (SEQ ID NO: 22); (l) a polynucleotide encoding a 2B8 Vβ polypeptide (SEQ ID NO: 23); (m) a polynucleotide encoding a 3C7 Vα polypeptide (SEQ ID NO: 26); or (n) The polynucleotide according to item 2, comprising at least one of the following: a polynucleotide encoding a 3C7 Vβ polypeptide (SEQ ID NO: 27). (Item 4) The vector is a CD8 + 4. The polynucleotide of item 3, comprising a polynucleotide sequence that regulates expression of the polypeptide in T cells. (Item 5) 5. The polynucleotide according to item 4, wherein the vector is a Sendai virus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or a lentivirus vector. (Item 6) 2. The polynucleotide of item 1, wherein the vector comprises a polynucleotide encoding a Vα polypeptide or a polynucleotide encoding a Vβ polypeptide. (Item 7) The vector comprises a polynucleotide encoding a Vα polypeptide together with a polynucleotide encoding a Vβ polypeptide positioned on the vector, such that the Vα / Vβ T cell receptor (TCR) is transduced to CD8 + 2. The polynucleotide of item 1, which is expressed on the surface of T cells. (Item 8) 8. A composition of matter comprising a host cell transduced with a vector according to any one of items 1 to 7. (Item 9) The host cells are human CD8 + 9. The composition of item 8, wherein the composition is a T cell. (Item 10) 10. The composition according to item 9, wherein the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients selected from the group consisting of buffering agents, antibacterial agents, tonicity adjusting agents, wetting agents, detergents, and pH adjusting agents. (Item 11) CD8 + T cells are obtained from an individual diagnosed with cancer that expresses the NY-ESO-1 antigen; and CD8 + T cells are transduced with a vector comprising a polypeptide encoding a TCR Vα polypeptide together with a polynucleotide encoding a TCR Vβ polypeptide, thereby transducing the heterologous TCR into CD8 + The composition of item 10, wherein the heterologous TCR is expressed on the surface of a T cell, and the heterologous TCR recognizes the NY-ESO-1 peptide associated with a human leukocyte antigen expressed on the surface of a cell of the cancer. (Item 12) Item 12. The composition of item 11, wherein the vector is a retroviral vector. (Item 13) 1. A method for killing cancer cells expressing the NY-ESO-1 antigen, comprising: + 10. The method of claim 9, wherein the cancer cells are treated with the CD8 T cells under conditions that allow the NY-ESO-1 peptide to be expressed on the surface of T cells and recognize the NY-ESO-1 peptide associated with a human leukocyte antigen expressed on the surface of the cancer cells, thereby recognizing and killing the cancer cells. + The method further comprising combining the antibody with T cells. (Item 14) The method further comprises: + 14. The method of item 13, which is carried out in vivo in a patient infused with T cells. (Item 15) Item 14. The method of item 13, wherein the cancer cells form a solid tumor. (Item 16) Item 14. The method of item 13, wherein the cancer cells are neuroblastoma cells, myeloma cells, metastatic melanoma cells, synovial sarcoma cells, bladder cancer cells, esophageal cancer cells, hepatocellular carcinoma cells, head and neck cancer cells, non-small cell lung cancer cells, ovarian cancer cells, prostate cancer cells, or breast cancer cells. (Item 17) The method comprises: providing a first modified CD8 targeting NY-ESO-1 peptide associated with a first human leukocyte antigen; + T cells are targeted to a second CD8 T cell receptor NY-ESO-1 peptide associated with a second human leukocyte antigen. + 14. The method of item 13, comprising administering in combination with T cells. (Item 18) A polynucleotide according to item 1 or a CD8 according to item 7 for the manufacture of a medicament for the treatment of cancer. + Use of T cells. (Item 19) the polynucleotide comprising: (a) a polynucleotide encoding the 3A1 Vα polypeptide (SEQ ID NO:3); (b) a polynucleotide encoding a 3A1 Vβ polypeptide (SEQ ID NO:4); (c) a polynucleotide encoding the 4A2 Vα polypeptide (SEQ ID NO:7); (d) a polynucleotide encoding a 4A2 Vβ polypeptide (SEQ ID NO: 37); (e) a polynucleotide encoding a 5G6 Vα polypeptide (SEQ ID NO: 10); (f) a polynucleotide encoding a 5G6 Vβ polypeptide (SEQ ID NO: 11); (g) a polynucleotide encoding a 9D2 Vα polypeptide (SEQ ID NO: 14); (h) a polynucleotide encoding a 9D2 Vβ polypeptide (SEQ ID NO: 15); (i) a polynucleotide encoding the 1E4 Vα polypeptide (SEQ ID NO: 18); (j) a polynucleotide encoding the 1E4 Vβ polypeptide (SEQ ID NO: 19); (k) a polynucleotide encoding a 2B8 Vα polypeptide (SEQ ID NO: 22); (l) a polynucleotide encoding a 2B8 Vβ polypeptide (SEQ ID NO: 23); (m) a polynucleotide encoding a 3C7 Vα polypeptide (SEQ ID NO: 26); or (n) the use according to Item 18, comprising at least one of the following polynucleotides: (a) a polynucleotide encoding a 3C7 Vβ polypeptide (SEQ ID NO: 27); (Item 20) 20. The use of item 19, wherein the cancer is melanoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer. [Brief explanation of the drawings]

[0015] [Figure 1A] This paper describes the expansion and isolation of NY-ESO-1-specific T cell clones. PBMCs were obtained from a patient with metastatic melanoma. A representative HLA-A2+, HLA-Cw3+ donor T cell cloning strategy is shown. The expansion and testing methods for identifying NY-ESO-1-reactive T cell clones are outlined. PBMCs were incubated with 28 NY-ESO-1 18-mer peptides (overlapping by 12 amino acids), expanded for 10 days, and then restimulated with individual peptides in the presence of BFA. Epitopes presented by the patient's MHC alleles are colored red, blue, and green for peptides containing the complete epitope sequence. [Figure 1B] This paper describes the expansion and isolation of NY-ESO-1-specific T cell clones. PBMCs were obtained from a patient with metastatic melanoma. A representative HLA-A2+, HLA-Cw3+ donor T cell cloning method is shown. Representative flow cytometry measurements of intracellular IFN-γ staining in PBMCs expanded by restimulation with individual NY-ESO-1-derived 18-mer peptides are shown. [Figure 1C]This paper describes the expansion and isolation of NY-ESO-1-specific T cell clones. PBMCs were obtained from patients with metastatic melanoma. A representative HLA-A2+, HLA-Cw3+ donor T cell cloning method is shown. A repopulation method using individual 9-mer or 10-mer peptides tested to elicit T cell responses is outlined. [Figure 1D] This paper describes the expansion and isolation of NY-ESO-1-specific T cell clones. PBMCs were obtained from a patient with metastatic melanoma. A representative HLA-A2+, HLA-Cw3+ donor T cell cloning method is shown. Representative flow cytometry data showing the NY-ESO-1-reactive subpopulation of CD3+CD8+ T cells before sorting are shown. Sorted cells were expanded in the presence of IL-2 and irradiated autologous PBMCs. [Figure 1E] This paper describes the expansion and isolation of NY-ESO-1-specific T cell clones. PBMCs were obtained from a patient with metastatic melanoma. A representative HLA-A2+, HLA-Cw3+ donor T cell cloning method is shown. Representative flow cytometry data are provided showing the NY-ESO-1-reactive subpopulation of CD3+CD8+ T cells after sorting. [Figure 2A] This paper provides a disclosure regarding the cloning and functional screening of NY-ESO-1-specific T cell receptors. A schematic diagram of the functional TCR cloning method is shown. For each TCR, two constructs incorporating human or mouse TCR constant domains were prepared. [Figure 2B]

[0003] Disclosure is provided regarding the cloning and functional screening of NY-ESO-1-specific T cell receptors. The protein sequence of NY-ESO-1 and the epitopes relevant to this study are shown (SEQ ID NO: 28). [Figure 2C]This paper provides a disclosure regarding the cloning and functional screening of NY-ESO-1-specific T cell receptors. Flow cytometry histograms are shown comparing HLA-A2 / NY157-165 dextramers binding in HEK293T cells transfected with vector backbone alone, the previously reported 1G4 TCR, and a novel A2-restricted NY-ESO-1-specific TCR. [Figure 2D] We provide a disclosure regarding the cloning and functional screening of NY-ESO-1-specific T cell receptors. Flow cytometry histograms comparing peptide-MHC dextramers binding in HEK293T cells transfected with vector backbone alone or novel NY-ESO-1-specific TCRs restricted to MHC alleles other than HLA-A2 are shown. Transfection experiments were performed twice, each in duplicate. Representative histograms are shown. [Figure 3A]

[0023] Figure 1 provides a disclosure regarding the function of A2-restricted NY-ESO-1-specific TCRs. An overlay of representative flow cytometry plots comparing A2 / NY-ESO-1157-165 dextramer binding on Jurkat cells expressing A2-restricted TCRs with human or mouse constant domains and on CD8+ Jurkat cells is shown. [Figure 3B] A2 provides functional disclosure of the NY-ESO-1-specific TCR restricted to A. As in A, dextramer-binding mean fluorescence intensity measurements are shown from two independent experiments. [Figure 3C] A2 provides functional disclosure of the restricted NY-ESO-1-specific TCR. B shows the ratio of dextramer-bound mean fluorescence intensity measurements from two independent experiments. [Figure 3D]This figure provides a functional description of the A2-restricted NY-ESO-1-specific TCR. An ELISA measuring IL-2 secretion from TCR-transduced Jurkat cells after 48 hours of co-incubation with K562 target cells expressing A2 / MART26-35 or A2 / NY-ESO-1157-165 single-chain trimers is shown. Experiments were repeated three times, with two technical replicates each. Mean values ​​± SD of a representative experiment are shown. [Figure 3E] This figure provides a functional description of the A2-restricted NY-ESO-1-specific TCR. An ELISA measuring IFN-γ secretion from TCR-transduced PBMCs after 48 hours of co-incubation with the melanoma cell line M257 or an A2+ derivative is shown. Experiments were repeated at least three times, with two technical replicates each. Mean values ​​± SD of a representative experiment are shown. [Figure 3F] This provides a functional disclosure of NY-ESO-1-specific TCRs restricted to A2. IncuCyte measurements of total area of ​​green objects over time are shown as a measure of TCR-transduced T cell-mediated killing of GFP+ A2+ M257 cells. Mean ± SD for four technical replicates is shown. [Figure 4A] This paper provides a disclosure regarding the in vivo antitumor effects of NY-ESO-1 TCR-engineered human T cells. (A and B) show (A) a schematic diagram of the experimental design for generating NY-ESO-1 TCR-engineered human T cells and (B) a schematic diagram of the experimental design for studying the antitumor effects of these engineered T cells in an NSG mouse human prostate tumor xenograft model. PBMC: peripheral blood mononuclear cells; NSG: immunodeficient NOD / SCID / γc- / - mice. [Figure 4B]This paper provides a disclosure regarding the in vivo antitumor effects of NY-ESO-1 TCR-engineered human T cells. (A and B) show (A) a schematic diagram of the experimental design for generating NY-ESO-1 TCR-engineered human T cells and (B) a schematic diagram of the experimental design for studying the antitumor effects of these engineered T cells in an NSG mouse human prostate tumor xenograft model. PBMC: peripheral blood mononuclear cells; NSG: immunodeficient NOD / SCID / γc- / - mice. [Figure 4C] We provide a disclosure regarding the in vivo anti-tumor efficacy of NY-ESO-1 TCR-engineered human T cells. (C) shows representative flow cytometry plots to characterize engineered human T cells present in the peripheral blood of experimental mice 14 days after adoptive T cell transfer. [Figure 4D] (D) provides a time course showing the persistence of engineered human T cells (gated as LNGFR+ hCD45+) in the peripheral blood of experimental mice. [Figure 4E] (E) shows mean fluorescence intensity measurements of mouse TCRs 14 days after adoptive T cell transfer. [Figure 4F] This provides a disclosure regarding the in vivo antitumor efficacy of NY-ESO-1 TCR-engineered human T cells. (F) shows the mean fluorescence intensity measurements of HLA-A2 / NY-ESO-1 dextramers of engineered human T cells in the peripheral blood of experimental mice 14 days after adoptive T cell transfer. [Figure 4G] This paper provides a disclosure regarding the in vivo anti-tumor efficacy of NY-ESO-1 TCR-engineered human T cells. (G and H) show cross-sectional area measurements of (G) PC-3 / HLA-A2 tumors and (H) PC-3 / HLA-A2 / NY-ESO-1 tumors. [Figure 4H]This paper provides a disclosure regarding the in vivo anti-tumor efficacy of NY-ESO-1 TCR-engineered human T cells. (G and H) show cross-sectional area measurements of (G) PC-3 / HLA-A2 tumors and (H) PC-3 / HLA-A2 / NY-ESO-1 tumors. [Figure 4I] This provides a disclosure regarding the in vivo anti-tumor effects of NY-ESO-1 TCR-engineered human T cells. Immunohistology images showing representative tumor sections are shown. CD3+ cells are stained red. Scale bar in top panel: 500 μm; scale bar in bottom panel: 50 μm. [Figure 4J] This paper provides a disclosure of the in vivo antitumor efficacy of NY-ESO-1 TCR-engineered human T cells. The percentage of CD3+ cell area relative to total tumor cross-sectional area is shown. Representative values ​​from two experiments. Data are expressed as mean ± SEM (n=4-5). ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by one-way ANOVA. [Figure 5A] This provides a functional description of NY-ESO-1-specific TCRs restricted to MHC alleles other than HLA-A2. Representative flow cytometry plot overlays comparing specific dextramer binding by Jurkat and CD8+ Jurkat cells expressing novel TCRs with human or mouse constant domains are shown. [Figure 5B] (B) shows dextramer-bound mean fluorescence intensity measurements from two independent experiments as in (A), providing a disclosure regarding the function of NY-ESO-1-specific TCRs restricted to MHC alleles other than HLA-A2. [Figure 5C] This provides information on the function of NY-ESO-1-specific TCRs restricted to MHC alleles other than HLA-A2. (C) shows the ratio of the dextramer-bound mean fluorescence intensity measurements from two independent experiments in (B). [Figure 5D]Disclosures are provided regarding the function of NY-ESO-1-specific TCRs restricted to MHC alleles other than HLA-A2. (D and E) Provide ELISAs measuring (D) IL-2 secretion from TCR-transduced Jurkat cells or (E) IFN-γ secretion from TCR-transduced PBMCs after 48 hours of co-incubation with K562 target cells expressing the single-chain trimer observed therein. [Figure 5E] Disclosures are provided regarding the function of NY-ESO-1-specific TCRs restricted to MHC alleles other than HLA-A2. (D and E) Provide ELISAs measuring (D) IL-2 secretion from TCR-transduced Jurkat cells or (E) IFN-γ secretion from TCR-transduced PBMCs after 48 hours of co-incubation with K562 target cells expressing the single-chain trimer observed therein. [Figure 6A] We provide a disclosure regarding targeting of the NY-ESO-1 epitope in a manner that is restricted to multiple MHC alleles, expanding the application of TCR gene therapy and stabilizing against loss of heterozygosity at MHC loci. T cells transduced with LNGFR alone, the A2-restricted 3A1 TCR, or the B7-restricted 1E4 TCR—or a 1:1 mixture of 3A1-transduced and 1E4-transduced T cells—were co-incubated with HLA-A2+eGFP+ target cells, HLA-B7+eGFP+ target cells, or a 1:1 mixture of target cells for 48 hours. (A and B) ELISAs measuring IFN-γ secretion from TCR-transduced PBMCs after 48 hours of co-incubation with (A) the M257 tumor cell line or (B) the PC-3 tumor cell line engineered to express eGFP and HLA-A*02:01 or HLA-B*07:02. The M257 line expresses endogenous NY-ESO-1. Experiments were repeated three times, with two technical replicates each. Mean values ​​± SD of a representative experiment are shown. [Figure 6B]We provide a disclosure regarding targeting of the NY-ESO-1 epitope in a manner that is restricted to multiple MHC alleles, expanding the application of TCR gene therapy and stabilizing against loss of heterozygosity at MHC loci. T cells transduced with LNGFR alone, the A2-restricted 3A1 TCR, or the B7-restricted 1E4 TCR—or a 1:1 mixture of 3A1-transduced and 1E4-transduced T cells—were co-incubated with HLA-A2+eGFP+ target cells, HLA-B7+eGFP+ target cells, or a 1:1 mixture of target cells for 48 hours. (A and B) ELISAs measuring IFN-γ secretion from TCR-transduced PBMCs after 48 hours of co-incubation with (A) the M257 tumor cell line or (B) the PC-3 tumor cell line engineered to express eGFP and HLA-A*02:01 or HLA-B*07:02. The M257 line expresses endogenous NY-ESO-1. Experiments were repeated three times, with two technical replicates each. Mean values ​​± SD of a representative experiment are shown. [Figure 6C] We provide a disclosure regarding targeting the NY-ESO-1 epitope in a manner that restricts it to multiple MHC alleles, expanding the application of TCR gene therapy and stabilizing it against loss of heterozygosity at MHC loci. T cells transduced with LNGFR alone, the A2-restricted 3A1 TCR, or the B7-restricted 1E4 TCR—or a 1:1 mixture of 3A1- and 1E4-transduced T cells—were co-incubated with HLA-A2+eGFP+ target cells, HLA-B7+eGFP+ target cells, or a 1:1 mixture of target cells for 48 hours. (C and D) show T cell-mediated killing of (C) M257 tumor cell line derivatives and (D) PC-3 tumor cell line derivatives measured over time using IncuCyte live cell analysis. The total area of ​​green objects (indicating tumor cell density) measured at each time point over 48 hours was normalized for each treatment compared to treatment with LNGFR-transduced T cells. Experiments were repeated three times, with each replicate repeated four or eight times. Results from a representative experiment of eight replicates are shown. [Figure 6D] We provide a disclosure regarding targeting the NY-ESO-1 epitope in a manner that restricts it to multiple MHC alleles, expanding the application of TCR gene therapy and stabilizing it against loss of heterozygosity at MHC loci. T cells transduced with LNGFR alone, the A2-restricted 3A1 TCR, or the B7-restricted 1E4 TCR—or a 1:1 mixture of 3A1- and 1E4-transduced T cells—were co-incubated with HLA-A2+eGFP+ target cells, HLA-B7+eGFP+ target cells, or a 1:1 mixture of target cells for 48 hours. (C and D) show T cell-mediated killing of (C) M257 tumor cell line derivatives and (D) PC-3 tumor cell line derivatives measured over time using IncuCyte live cell analysis. The total area of ​​green objects (indicating tumor cell density) measured at each time point over 48 hours was normalized for each treatment compared to treatment with LNGFR-transduced T cells. Experiments were repeated three times, with each replicate repeated four or eight times. Results from a representative experiment of eight replicates are shown. [Figure 7A] Disclosure is provided regarding the determination of the EC50 of NY-ESO-1-specific TCRs. (A and B) Provide ELISAs measuring IFN-γ secretion from PBMCs transduced with TCRs pulsed with various concentrations of (A) MART126-35 or (B) NYESO1157-165 peptides after 48 hours of co-incubation with K562 cells engineered to express HLA-A*02:01. [Figure 7B] Disclosure is provided regarding the determination of the EC50 of NY-ESO-1-specific TCRs. (A and B) Provide ELISAs measuring IFN-γ secretion from PBMCs transduced with TCRs pulsed with various concentrations of (A) MART126-35 or (B) NYESO1157-165 peptides after 48 hours of co-incubation with K562 cells engineered to express HLA-A*02:01. [Figure 7C]Disclosure is provided for the determination of the EC50 of NY-ESO-1-specific TCRs. (C-E) ELISAs are provided measuring IFN-γ secretion from TCR-transduced PBMCs pulsed with various concentrations of the indicated peptides after 48 hours of co-incubation with K562 cells engineered to express (C) HLA-B*07:02, (D) HLA-B*18:01, or (E) HLA-C*03:04. Mean values ​​± SD of two technical replicates are shown. EC50 values ​​determined by nonlinear curve fitting and associated error are shown. [Figure 7D] Disclosure is provided for the determination of the EC50 of NY-ESO-1-specific TCRs. (C-E) ELISAs are provided measuring IFN-γ secretion from TCR-transduced PBMCs pulsed with various concentrations of the indicated peptides after 48 hours of co-incubation with K562 cells engineered to express (C) HLA-B*07:02, (D) HLA-B*18:01, or (E) HLA-C*03:04. Mean values ​​± SD of two technical replicates are shown. EC50 values ​​determined by nonlinear curve fitting and associated error are shown. [Figure 7E] Disclosure is provided for the determination of the EC50 of NY-ESO-1-specific TCRs. (C-E) ELISAs are provided measuring IFN-γ secretion from TCR-transduced PBMCs pulsed with various concentrations of the indicated peptides after 48 hours of co-incubation with K562 cells engineered to express (C) HLA-B*07:02, (D) HLA-B*18:01, or (E) HLA-C*03:04. Mean values ​​± SD of two technical replicates are shown. EC50 values ​​determined by nonlinear curve fitting and associated error are shown. [Figure 8A]Disclosures regarding the establishment of xenograft tumor lines and the function of input T cells for in vivo experiments are provided. (A) ELISA measuring IFN-γ secretion from TCR-transduced PBMCs after 48 hours of co-incubation with derivatives of the PC-3 prostate cancer cell line engineered to express (left) HLA-A*02:01 and the NY-ESO-1 full protein, (center) HLA-A*02:01 alone, or (right) the NY-ESO-1 full protein alone. Mean values ​​± SD of two technical replicates are shown. [Figure 8B] (B) provides disclosure regarding the establishment of xenograft tumor lines and the function of input T cells for in vivo experiments. (C) provides an ELISA comparing IFN-γ secretion from TCR-transduced PBMCs after 48 hours of co-incubation with the indicated M257 or PC-3 target cells. Four days after transduction, TCR-transduced PBMCs were sorted for CD3+ / LNGFR+ and then expanded for 13 days before co-incubation / ELISA assays and in vivo experiments. Shown is the mean ± SD of a representative experiment with two technical replicates. [Figure 8C] We provide disclosure regarding the establishment of xenograft tumor lines and the function of input T cells for in vivo experiments. (C) shows flow cytometry contour plots comparing transduction (LNGFR+) levels in PBMCs transduced with TCRs used in in vivo experiments. DETAILED DESCRIPTION OF THE INVENTION

[0016] In describing the embodiments, reference may be made to the accompanying drawings, which constitute a part of this specification and which show, by way of illustration, specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the invention. Many of the techniques and procedures described or referenced herein are well understood and commonly used by those skilled in the art. Unless otherwise specified, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those skilled in the art to which the invention pertains. In some instances, terms having commonly understood meanings are defined herein for the sake of clarity and / or ready reference, and the inclusion of such definitions herein should not be construed as necessarily substantially different from what is commonly understood in the art.

[0017] NY-ESO-1 is a typical example of a cancer-testis antigen whose expression is restricted to germ cells and placental cells and re-expressed in tumor cells. NY-ESO-1 expression has been reported in a wide range of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer. Their ability to elicit spontaneous humoral and cellular immune responses, combined with their restricted expression pattern, make them excellent candidate targets for cancer immunotherapy. See, e.g., Thomas et al., Front Immunol. 2018;9: 947. doi: 10.3389 / fimmu.2018.00947.

[0018] The disclosure herein demonstrates the achievement of two important goals related to methods and materials useful in NY-ESO-1 TCR gene therapy. First, because TCRs with high potency and affinity are effective, we have demonstrated that A2 / NY-ESO-1 TCRs can be expressed with sensitivity equal to or greater than that of the clinically employed 1G4 TCR. 157-165We sought to identify novel TCRs targeting HLA-A. Because high-affinity TCRs can be cross-reactive (28-30), we established a protocol to isolate antigen-reactive TCRs directly from patient blood. Two of these novel TCRs demonstrated comparable or superior sensitivity to 1G4 in tumor-killing assays, both in vitro and in vivo. Second, to broaden the clinical utility of NY-ESO-1 as a TCR gene therapy target, we used our isolation protocol to identify TCRs targeting HLA-A. * We identified TCRs that target NY-ESO-1 epitopes presented by common MHC alleles other than 02:01. Targeting multiple NY-ESO-1 epitopes will enable treatment of a wider patient population and potentially lead to more potent tumor-eliminating therapy.

[0019] As described herein, the present invention provides methods and materials for producing and using modified T cells containing nucleic acids encoding specific T cell receptor polypeptides. As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in T cell activation in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of an alpha (α) chain and a beta (β) chain, although in some cells, TCRs are composed of gamma and delta chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but differ in anatomical location and function. Each chain consists of two extracellular domains: a variable domain and a constant domain. Embodiments of the present invention include several different TCR alpha / beta nucleic acids and the polypeptides they encode (e.g., TCR nucleic acids and encoded polypeptides for TCRs designated "3A1," "4A2," "5G6," "9D2," "1E4," "2B8," and "3C7").

[0020] Embodiments of the present invention include compositions of matter comprising one or more vectors containing the TCR polynucleotides disclosed herein. A "vector" is a composition of matter containing an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors.

[0021] Generally, the vector is an expression vector. As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter. In this context, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate the recombinant polynucleotide.

[0022] Embodiments of the invention include, for example, polynucleotides placed in expression vectors, where the polynucleotides encode Vα T cell receptor polypeptides and / or Vβ T cell receptor polypeptides. In such embodiments, Vα / Vβ T cell receptors, including Vα T cell receptor polypeptides and / or Vβ T cell receptor polypeptides, are expressed by CD8 + When expressed on T cells, the Vα / Vβ T cell receptor recognizes the NY-ESO-1 peptide in the context of human leukocyte antigen A2, human leukocyte antigen B07, human leukocyte antigen B18, or human leukocyte antigen C03. In practical embodiments of the invention disclosed herein, modified CD8 + The T cell receptor includes the 3A1 T cell receptor, the 4A2 T cell receptor, the 5G6 T cell receptor, the 9D2 T cell receptor, the 1E4 T cell receptor, the 2B8 T cell receptor, or the 3C7 T cell receptor.

[0023] In a general embodiment of the invention, the vector comprises at least one of: a polynucleotide encoding a 3A1 TCR Vα polypeptide (SEQ ID NO:3); a polynucleotide encoding a 3A1 TCR Vβ polypeptide (SEQ ID NO:4); a polynucleotide encoding a 4A2 TCR Vα polypeptide (SEQ ID NO:7); a polynucleotide encoding a 4A2 TCR Vβ polypeptide (SEQ ID NO:37); a polynucleotide encoding a 5G6 TCR Vα polypeptide (SEQ ID NO:10); a polynucleotide encoding a 5G6 TCR Vβ polypeptide (SEQ ID NO:11); a polynucleotide encoding a 9D2 TCR Vα polypeptide (SEQ ID NO:14); a polynucleotide encoding a 9D2 TCR Vβ polypeptide (SEQ ID NO:15); a polynucleotide encoding a 1E4 TCR Vα polypeptide (SEQ ID NO:18); a polynucleotide encoding a 1E4 TCR Vβ polypeptide (SEQ ID NO:19); a polynucleotide encoding a 2B8 TCR Vα polypeptide (SEQ ID NO:22); a polynucleotide encoding a 2B8 TCR Vβ polypeptide (SEQ ID NO:23); a polynucleotide encoding a 3C7 TCR Vα polypeptide (SEQ ID NO:26); or a polynucleotide encoding a 3C7 TCR Vβ polypeptide (SEQ ID NO:27). Table 1 below discloses exemplary polynucleotide sequences encoding these TCR polypeptides.

[0024] Generally, the compositions of the invention comprise one or more Vα / Vβ polypeptides, e.g., TCR The polynucleotides encoding Vα polypeptides are included together with polynucleotides encoding TCR Vβ polypeptides, such that the Vα / Vβ TCRs are expressed in mammalian cells (e.g., CD8 +T cells), whose Vα / Vβ TCR recognizes the HLA-associated NY-ESO-1 peptide. As used herein, the terms "transduced" or "transfected" or "transformed" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transduced" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. A cell includes the primary subject cell and its progeny.

[0025] In another aspect, the present invention provides a method for administering one or more nucleic acids (e.g., nucleic acids placed in a lentiviral vector) encoding a TCR disclosed herein to T cells (e.g., CD8 T cells expressing the NY-ESO-1 antigen obtained from a person diagnosed with cancer). + The present invention also includes methods of generating modified T cells comprising introducing into a T cell an endogenous T cell receptor (e.g., a modified T cell having a knocked-out endogenous T cell receptor and an exogenous / introduced T cell receptor that recognizes the NY-ESO-1 peptide in the context of an HLA). As used herein, the term "knockdown" refers to suppressing gene expression of one or more genes. As used herein, the term "knockout" refers to eliminating gene expression of one or more genes.

[0026] The modified T cells described herein can be included in a composition for use in a therapeutic regimen. The composition can include a pharmaceutical composition and can further include a pharmaceutically acceptable carrier. A therapeutically effective amount of a pharmaceutical composition containing the modified T cells can be administered. The pharmaceutical compositions of the present invention can include the modified T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include a buffer, such as neutral buffered saline or phosphate buffered saline; a carbohydrate, such as glucose, mannose, sucrose, or dextran or mannitol; a protein; a polypeptide; or an amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

[0027] Adoptive immunotherapy using T cells bearing antigen-specific TCRs has therapeutic potential in the treatment of cancer. + Genetic engineering of T cells has the advantage of redirecting T cells to a selected antigen, such as the NY-ESO-1 antigen. In this regard, in one aspect, the invention includes a method of stimulating a T cell-mediated immune response against a target cell or tissue in a subject, the method comprising administering to the subject an effective amount of a modified CD8 + In this embodiment, the administration of CD8 T cells is + T cells are modified as described elsewhere herein. Also, embodiments of the present invention include multiple modified CD8 T cells targeting multiple NY-ESO-1 epitopes. + For example, embodiments of the present invention include administering at least two different modified CD8 T cells. + a first modified CD8 T cell targeting NY-ESO-1 peptide associated with a first human leukocyte antigen; + T cells and a second CD8 targeting the NY-ESO-1 peptide associated with a second human leukocyte antigen. + and administering a combination of T cells.

[0028] Embodiments of the present invention include methods of treating a disease or condition characterized by expression of the common cancer-testis antigen NY-ESO-1. The treatment methodology comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising the modified T cells described herein. The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human. In general embodiments of the present invention, the human has a cancer that expresses the NY-ESO-1 antigen. In some embodiments of the present invention, the cancer cells form a solid tumor. In exemplary embodiments of the invention, the cancer cells are neuroblastoma cells, myeloma cells, metastatic melanoma cells, synovial sarcoma cells, bladder cancer cells, esophageal cancer cells, hepatocellular carcinoma cells, head and neck cancer cells, non-small cell lung cancer cells, ovarian cancer cells, prostate cancer cells, or breast cancer cells.

[0029] Related embodiments of the invention include methods for preventing and / or treating an individual diagnosed with, suspected of having, or at risk for developing or recurring cancer, wherein the cancer comprises cancer cells expressing the NY-ESO-1 antigen. The method comprises administering to the individual engineered human T cells comprising a recombinant polynucleotide encoding a TCR, wherein the T cells are capable of directly recognizing cancer cells expressing the NY-ESO-1 antigen, and wherein the direct recognition of the cancer cells comprises HLA class II-restricted binding of the TCR to the NY-ESO-1 antigen expressed by the cancer cells.

[0030] The engineered CD8 + With regard to the use of T cells, this method generally involves the use of CD8 + This involves administering an effective amount of a composition comprising T cells to an individual in need thereof (e.g., by intravenous or intraperitoneal injection). Suitable pharmaceutical compositions may be adapted for any appropriate route of administration, such as parenteral (such as subcutaneous, intramuscular, or intravenous), enteral (such as oral or rectal), inhalation, or intranasal. Such compositions may be prepared by any method known in the pharmaceutical art, for example, by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0031] In another aspect, the invention provides a method for treating a disease or condition characterized by expression of NY-ESO-1 in a subject in need thereof, comprising administering to the subject a polynucleotide described herein or a modified CD8 + In an exemplary embodiment of the invention, the disease is a cancer that expresses the NY-ESO-1 antigen, such as melanoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer.

[0032] There has been considerable development in this field, and numerous methods and materials known in the art can be adapted for use with the invention disclosed herein, such as those disclosed in U.S. Patent Publication Nos. 20190247432, 20190119350, 20190002523, 20190002522, 20180371050, 20180057560, 20170029483, 20160024174, and 20150141347, the contents of which are incorporated by reference. [Example]

[0033] Further aspects and embodiments of the present invention are provided in the following examples.

[0034] Example 1: Expansion and isolation of NY-ESO-1-specific T cell clones. We previously reported the presence of T cells reactive to various NY-ESO-1-derived epitopes in the blood of patients with metastatic melanoma (22). To enrich for these reactive T cells, we stimulated the proliferation of patient peripheral blood mononuclear cells (PBMCs) with a panel of 28 overlapping 18-mer peptides that collectively comprise the complete NY-ESO-1 protein sequence (Figure 1A). We then restimulated the expanded cells with individual peptides and performed intracellular staining for IFN-γ to determine which peptides stimulated proliferation. We then analyzed the stimulatory peptides using a predictive algorithm to identify minimal epitopes associated with each patient's MHC haplotype (31) (Figure 1B). The reactive T cells were repopulated in the presence of individual 9- to 10-mer peptides corresponding to the immunostimulatory epitopes (Figure 1C) and then sorted via fluorescence-activated cell sorting (FACS) using cognate peptide-MHC tetramers (Figure 1D). Cell lines expanded from these single cell types were clonal and showed reactivity to their cognate epitopes (Fig. 1E). * 02:01 / NY-ESO-1 157-165 Four cell lines reacting with epitopes presented by HLA-B and HLA-C alleles were selected for further study.

[0035] Example 2: Cloning and screening of NY-ESO-1-specific TCRs Using a commercially available RT-PCR kit with predefined multiplex primers targeting all human TRAV and TRBV gene segments, we cloned paired TCRα and TCRβ genes from sorted single cells. The resulting Vα and Vβ cDNAs were subcloned into retroviral vector backbones carrying human or mouse TCR constant regions (Figure 2A). To verify the specificity of the cloned TCRs, we used CD3 + HEK293T cells were transfected with each fully human TCR and stained with peptide-MHC dextramer reagents targeting each NY-ESO-1 epitope (Figure 2B). All four HLA-A2-restricted TCRs showed the expected reactivity (Figure 2C). Although analyzed events were gated at similar transfection levels, the novel TCRs exhibited highly variable dextramer binding. While dextramer binding of the 9D2 TCR was barely discernible from background, the 3A1 TCR exhibited superior dextramer binding compared to the clinically used 1G4 TCR. Dextramer binding of the 4A2 and 5G6 TCRs was intermediate between that of 9D2 and 1G4.

[0036] In addition, we confirmed that three of the four TCRs restricted to MHC alleles other than HLA-A2 specifically bound to their targets (Figure 2D). 60-72 Specific 1E4 TCR, B18 / NY-ESO-1 88-96 specific 2B8 TCR or Cw3 / NY-ESO-1 96-104 Both transfected 293T cells expressing the specific 3C7 TCR bound to each dextramer but not to untransfected cells transfected with the 9G2 TCR - Cw3 / NY-ESO-1. 92-100 The cells were cloned from T cells reactive with HEK 111, and showed no detectable binding to the homologous dextramer compared to untransfected cells. A possible reason for this is that the HEK 111 cells were cloned from T cells reactive with HEK 111, and showed no detectable binding to the homologous dextramer compared to untransfected cells. 293T cells do not express the CD8 coreceptor. CD8 binds directly to MHCI, increasing the avidity of TCR-pMHC interactions and precluding engagement of low-affinity TCRs (32). Therefore, we utilized this TCR to further analyze CD8 dependency in Jurkat T cells.

[0037] Example 3: Functional characterization of A2-restricted NY-ESO-1-specific TCRs The sensitivity of TCR-transduced T cells is determined by the monomeric affinity (K) of the TCR for its cognate peptide-MHC. d The affinity of TCRs varies from approximately 0.1 to 400 μM (33) and depends on the density of TCRs on the cell surface (12). Transduced TCRs are expressed at a wide range of levels on the T cell surface, due to variations in the efficiency with which they fold, dimerize, and compete with endogenous TCRs for assembly with the restricted CD3 chain (a property termed TCR "strength") (34, 35). Thus, optimal cytotoxic function of TCR-transduced T cells correlates with TCR affinity and surface expression (3, 12), highlighting the importance of selecting high-affinity, efficiently derived TCRs for gene therapy (7).

[0038] Because high-affinity TCR-pMHC interactions are less dependent on CD8 engagement, we reasoned that high-affinity TCRs could be identified by comparing dextramer binding of TCR-transduced Jurkat T cells with and without CD8 coexpression. In addition, because the strength of surface expression of human TCRs can be enhanced through substitution with mouse constant domains (36), we evaluated the strength of each TCR expressed as a fully human or murine derivative. Cells transduced with vehicle alone or a mismatched TCR (MART1-specific F5 TCR) inhibited A2 / NY-ESO-1 TCRs. 157-165 In contrast, the well-established 1G4 TCR (K DCells transduced with 1G4 (p = 9.3 μM) bound the cognate dextramer, regardless of whether the 1G4 was fully human or murine, and regardless of the presence or absence of CD8. Mutation of 1G4 enhanced dextramer binding by the muTCR to 1.4-fold compared to the parental huTCR, representing a modest improvement in potency (Figures 3B and 3C). The presence of CD8 enhanced dextramer binding of the 1G4 muTCR by 3.8-fold. Dextramer binding by the novel TCRs 4A2 and 5G6 was similar to that of 1G4 in both magnitude and relative indices (Figures 3A–3C). The 3A1 TCR enhanced dextramer binding by 1.9-fold in the presence of CD8, indicating that this TCR binds A2 / NY-ESO-1 with greater affinity than 1G4. 157-165 This indicates that 1G4, 4A2, and 5G6 muTCR-transduced CD8 + Compared to cells (compare the gradient of the green population in Figure 3A), 3A1 muTCR-transduced CD8 + This is also supported by the reduced dependence of dextramer binding on intracellular CD8 levels. Finally, 9D2 showed no detectable binding to dextramer on Jurkat cells in the absence of CD8 and only weak binding upon coexpression of CD8. Murineization of 9D2 did not increase its binding to dextramer.

[0039] To compare the functional sensitivity of T cells expressing the novel A2 / NY-ESO-1-specific TCR, TCR-transduced Jurkat T cells were cultured in a 2000-well plate. * 02:01 / NY-ESO-1 157-165 , or A * 02:01 / MART1 27-35Secreted interleukin-2 (IL-2) was measured by co-incubation with K562 cells expressing either the single-chain trimer (38). All TCRs exhibited the expected peptide specificity: the control MART1-specific F5 TCR mediated IL-2 release in response to MART1 presentation alone, and all NY-ESO-1-specific TCRs mediated IL-2 release in response to NY-ESO-1 presentation alone (Fig. 3D). Mouserization improved the functional sensitivity of all TCRs except 1G4. Consistent with the dextramer staining results, 1G4 and 3A1 muTCRs performed better than 4A2 and 5G6 muTCRs. In contrast, despite weaker binding to dextramer, 9D2 exhibited high functional sensitivity to its cognate ligand, comparable to 3A1. To quantify this observation, we used various concentrations of NY-ESO-1. 157-165 , or MART1 27-35 The peptide was + We measured IFN-γ secreted from TCR-transduced primary T cells pulsed with K562 cells and then co-incubated with peptide-pulsed targets (Fig. 7A and 7B). As observed with the single-chain trimer target, 3A1, 9D2, and 1G4 inhibited IFN-γ secretion in a manner similar to that observed with NY-ESO-1. 157-165 The functional sensitivity of 9D2 was 10-fold greater than that of 4A2, whereas the MFI of 4A2-conjugated dextramer was 18-fold greater than that of 9D2 (Figures 3A and 3B). To assess responses to endogenously processed and presented antigens, TCR-transduced primary T cells were transfected with the human melanoma cell line A2. + M257 (Fig. 3E). In this case, T cells transduced with 3A1, 9D2, and 1G4 responded equally well to each other and with higher sensitivity than T cells transduced with 5G6 and 4A2. TCR-transduced T cells were also transduced with HLA-A. * Finally, in vitro cytotoxicity closely correlated with cytokine release: T cells expressing 9D2 or 3A1 responded to A2 +It most efficiently killed M257 tumor cells, followed by 1G4, 5G6, and, least efficiently, 4A2-transduced T cells ( Fig. 3F ).

[0040] To enable evaluation of TCR function in tumor xenograft models, we engineered the PC-3 human prostate cancer cell line to express NY-ESO-1 and HLA-A. * We then confirmed that this strain elicited functional responses from TCR-transduced T cells in an antigen-dependent and MHC-restricted manner (Figure 8A). + New York + The relative response to PC-3 was A2 + The TCRs were consistent with those induced by M257 (Figures 3E and 8B). Based on these results, we selected the 1G4, 3A1, and 9D2 muTCRs for further functional characterization in vivo. We transduced activated human PBMCs with vectors encoding the respective murine TCRs and a transduction marker (low-affinity nerve growth factor receptor (LNGFR)) (Figure 4A). We transduced CD3 + LNGFR + ) T cell sorting was performed (Figure 8C), and then irradiated NOD / SCID / γc mice previously inoculated with PC-3 / HLA-A2 (control) and PC-3 / HLA-A2 / NYESO (target) tumors in the contralateral flank were used. - / - These T cells were injected intravenously into the retroorbital plexus of (NSG) mice (Figure 4B). We then monitored T cell engraftment and tumor size 2 weeks after T cell injection until the end of the experiment.

[0041] T cells transduced with 1G4 or 9D2 TCRs proliferated persistently or minimally in peripheral blood, whereas T cells transduced with 3A1 proliferated significantly (Figures 4C and 4D). In contrast, T cells transduced with LNGFR alone contracted over the course of the experiment, suggesting that proliferation of TCR-transduced T cells was antigen-driven. Expression levels of murine TCRβ (mTCRβ) were stable over the time course of the experiment and comparable among T cells transduced with different murine TCRs (Figures 4C and 4E). * 02:01 / NY 157-165 Dextramer + The staining levels of each TCR-transduced T cell cohort were also stable over time, but as expected from the in vitro results, there were significant differences between TCRs. Approximately 90% of human T cells transduced with 1G4 or 3A1 showed dextramer staining with high MFI. + In contrast, only about 1% of 9D2-transduced T cells expressed dextramers. + The MFI of staining was not significantly different from that of LNGFR-transduced controls (Figures 4C and 4F). Nevertheless, T cells transduced with 1G4, 3A1, or 9D2 reduced tumor size comparably and antigen-specifically, whereas T cells transduced with LNGFR failed to control tumor growth (Figures 4G and 4H).

[0042] At the end of the experiment, we sacrificed the mice and analyzed tumors for T cell infiltration by immunohistochemistry. Immunohistochemical staining revealed that antigen-specific T cells infiltrated only target tumors in all cohorts treated with TCR-transduced T cells (Figures 4I and 4J). Infiltration was significantly more pronounced in mice treated with 3A1-transduced T cells than in mice treated with 1G4- or 9D2-transduced T cells.

[0043] Example 4: Functional characterization of NY-ESO-1-specific TCRs restricted to HLA-B and HLA-C alleles Most immunotherapies targeting NY-ESO-1 restrict NY-ESO-1 to A2. 157-165 To broaden the use of immunotherapy targeting NY-ESO-1, we cloned TCRs from four non-A2-restricted T cell clones and transfected CD3 + We verified NY-ESO-1 reactivity against three of these TCR-Cw3 / NY-ESO-1 in 293T (Figure 2D). 92-100 9G2-, cloned from reactive T cells, co-expressed CD8 but not Cw3 / NY-ESO-1, with respect to transduced Jurkat T cells. 92-100 The results did not confer specificity to Jurkat or CD8 (Fig. 5A, 5B), and the study was not pursued further. + Comparison of dextramer binding mediated by three TCRs expressed in Jurkat cells and validated as human or mouse TCRs clearly demonstrated differences in potency and affinity (Figures 5A, 5B, and 5C). 60-72 The -specific 1E4 TCR has high potency but low affinity and is equally expressed on the surface of Jurkat cells as huTCR or muTCR, but is not expressed on CD8 + In the presence of only these CD8 + Dextramer binding to 1E4-transduced cells was highly dependent on the level of CD8 expressed. In contrast, B18 / NY-ESO-1 88-96 The specific 2B8 TCR is CD8 + Although Cw3 / NY-ESO-1 bound to dextramer in the absence of TCR, binding was substantially stronger with the murine TCR. 96-104 The specific 3C7 TCR showed moderately strong surface expression and affinity index comparable to that of 2B8.

[0044] These differences in TCR potency and affinity were reflected in functional assays. For all three TCRs, murine TCR constant region murine expression increased IL-2 production from TCR-transduced Jurkat cells co-incubated with cognate target cells. However, this increase was only 1.6- and 3.0-fold greater than that of the fully human TCRs 1E4 and 3C7, respectively, and 18.6-fold greater for 2B8, consistent with the latter's lower potency (Figure 5D). In peptide titration assays, the 1E4 TCR conferred lower sensitivity to the cognate peptide on transduced CD8+ T cells than either 3C7 or 2B8 (Figures 7C, 7D, and 7E), consistent with the predicted lower affinity of 1E4 based on strictly CD8-dependent dextramer binding.

[0045] Primary PBMCs transduced with each TCR responded to presentation of NY-ESO-1-derived epitopes in a peptide-specific and MHC-restricted manner (Figure 5E). Therefore, we believe that TCR gene therapy using multiple MHC-restricted NY-ESO-1-specific TCRs may be broadly applicable across patient haplotypes and more robust for tumor escape via loss of heterozygosity at the MHC1 locus. To test this, we transduced NY-ESO-1-expressing human cancer cells with HLA-A2 or HLA-B7. We then co-incubated one or both of these tumor targets with human T cells transduced with the A2-restricted 3A1 TCR, T cells transduced with the B7-restricted 1E4 TCR, or a mixture of 3A1 and 1E4-transduced T cells (Figure 6). As expected, combined targeting using a mixture of 3A1- and 1E4-transduced T cells enabled recognition of tumor cell populations expressing both MHC alleles or only one MHC allele (Figures 6A and 6B). In contrast, T cells targeting a single NY-ESO-1 epitope did not respond to NY-ESO-1-expressing tumor cells lacking the cognate MHC allele. Furthermore, when tumor targets contained a mixture of cells expressing different MHC alleles (mimicking tumor heterogeneity due to haploinsufficiency), T cells targeting both NY-ESO-1 epitopes more completely killed tumor cells than T cells targeting either single epitope (Figures 6C and 6D).

[0046] Consideration T cell-mediated immunotherapy has made clinical inroads against previously intractable cancers. The two most successful immunotherapies are checkpoint blockade and adoptive transfer of cancer-specific T cells. Checkpoint blockade has elicited favorable clinical responses in response to the increasing incidence of tumor mutations (39-41), suggesting that the immune system does not detect nonsynonymous mutations unless they fortuitously generate neoepitopes presented by the patient's complement of MHC molecules. This interpretation is supported by the recent finding that checkpoint blockade improves overall survival in melanoma patients who are heterozygous for HLA-A, HLA-B, and HLA-C loci and therefore present a much more diverse array of epitopes than patients homozygous for one or more of these MHC I loci (42). The importance of a diversely targeted antitumor immune response is similarly supported by results from adoptive T cell therapy, which indicate that loss of heterozygosity is a mechanism by which tumors can evade monospecific immune recognition while continuing to express other immunogenic antigens (43). Therefore, these studies have revealed that diversified targeting of multiple epitopes presented by multiple MHC alleles is desirable for successful immunotherapy. Second, targeting multiple epitopes derived from tumor-specific public antigens may be a promising alternative, as it targets neoepitopes in cancers with low mutation incidence.

[0047] Identifying tumor-associated public antigens that mediate tumor regression without significant morbidity or mortality due to on-target, off-tumor T cell reactivity has proven challenging. NY-ESO-1 is: 1) expressed exclusively in cancer cells and immune-privileged germ cells; 2) expressed in numerous patients with a variety of tumor types; 3) possesses high-affinity ligands for multiple common MHC alleles; 4) has been well characterized and induces objective responses in patients with several tumor types without specificity-related adverse events; 5) the majority of studies have restricted NY-ESO-1 to A2. 157-165Since we focus on recruiting T cell responses only against epitopes, we chose to focus on public antigen targets based on the criteria that they are still underutilized.

[0048] We employed an antigen-specific expansion protocol to isolate NY-ESO-1-reactive T cells from the peripheral blood of patients with metastatic melanoma. Using this method, we cloned several HLA-A2-restricted TCRs and compared them with respect to surface expression strength, affinity (i.e., dependence of target binding on CD8), and function (antigen-induced cytokine release and tumor target killing). From four candidates, we identified two that recognized and killed NY-ESO-1-expressing cancer cells at levels comparable to or better than the clinically used 1G4 TCR. This expansion-based approach to identifying TCR candidates is ideal for targeting public epitopes because speed of isolation is not a critical parameter; once identified, these TCRs can be used as pre-existing target receptors in any patient expressing the required MHC alleles. We have also demonstrated antigen-specific expansion of neoantigen-reactive T cells from peripheral blood (44, 45). However, on-demand isolation of TCRs targeting private neoepitopes requires more rapid methods than those used here (e.g., direct capture of antigen-specific T cells from the blood or expansion protocols optimized for rapidity). Because IFN-γ release strongly correlates with cytotoxicity (46), its use as an alternative to more complex tumor xenograft assays allows for rapid evaluation of candidates.

[0049] One isolated HLA-A2 / NY-ESO-1-reactive TCR, 9D2, showed poor staining with cognate multimers but demonstrated significant functional avidity for cognate antigen-presenting target cells. This is consistent with the observation that multimer staining underestimates functional T cell subsets (47) and can be explained by a higher affinity threshold for multimer binding compared with the affinity threshold for T cell activation (48). However, another isolated A2-restricted TCR, 4A2, showed clear multimer staining but was poorly functional in cell-based assays, seemingly at odds with this affinity threshold explanation. We offer no explanation for this latter result, but both results caution against over-reliance on multimer staining to narrow down immunotherapeutic candidates.

[0050] HLA-A *The 02:01 allele is the most common MHC I allele in Caucasian (45%) and Hispanic (41%) populations in the United States, but is less common in Asian (15%) and African (16%) populations in the United States (2). These latter populations would be particularly well served by expanding the targeting of TCR gene therapy to a broader panel of targetable MHC alleles beyond HLA-A2. In addition to HLA-A2-restricted TCRs, we isolated and functionally characterized NY-ESO-1-specific TCRs restricted to various HLA-B and HLA-C alleles. In doing so, we demonstrated that TCR gene therapy can, in principle, be extended to a larger subset of patients / haplotypes, and that, when used in combination, TCRs recognizing multiple epitopes from the same antigen more potently kill tumors with heterogeneous MHC expression (e.g., due to somatic loss of heterozygosity). Over 80% of people across ethnic groups express at least one allele of three MHCI supertypes (A2, A3, and B7, two of which are described herein), and over 99% express at least one allele of nine MHCI supertypes (49). Thus, obtaining a panel of public antigen-specific TCR reagents that would enable comprehensive application of TCR gene therapy presents limited but surmountable challenges.

[0051] Materials and Methods material Peptides were obtained from Anaspec (Fremont, CA), Thermo Fisher Scientific, and Antibodies used for flow cytometry were purchased from BD Biosciences (San Jose, CA), BioLegend (San Diego, CA), or eBioscience (San Diego, CA). Fluorescent peptide-MHC multimers were purchased from TCMetrix (Epalinges, Switzerland) or prepared in-house from biotinylated monomers as described in (50) (NIH). (The primers were obtained from Tetramer Core, Atlanta, GA, or were heterologously expressed in E. coli, refolded, and biotinylated in-house as described in (51).) Primers were Integrated DNA Technologies (Coralville, IA). KOD polymerase master mix and polybrene were purchased from EMD Millipore (Darmstadt, Germany). Sequencing was performed at Retrogen Inc. (San Diego, CA). Anti-CD3 (OKT3) and anti-CD28 (CD28.2) activating antibodies were purchased from eBioscience. Cytokines were purchased from Peprotech, Inc. (Rocky Hill, NJ). BioT transfection reagent was purchased from Bioland Scientific (Paramount, CA). Cell culture medium, antibiotics, and fetal bovine serum were purchased from Corning (Corning, NY). Human AB serum was purchased from Omega Scientific (Tarzana, CA). Poly-L-lysine and PHA-L (phytohemagglutinin-L) were purchased from Sigma (St. Louis, MO).

[0052] cell Cell lines (293T / 17, Jurkat E6-1, and K562) were purchased from the American Type Culture Collection (Manassas, VA). 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with antibiotics (penicillin / streptomycin) and 10% (v / v) fetal bovine serum (FBS). Jurkat and K562 cells were cultured in RPMI 1640 medium supplemented with antibiotics, 10% (v / v) FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, 1× MEM NEAA, and 1 mM sodium pyruvate. Cells were split every 2–3 days to maintain adherent cells at subconfluence or nonadherent cells at <10 6 Cells were maintained at a density of 100 cells / mL. Jurkat and K562 cells were transduced with non-replicating viral vectors, analyzed by flow cytometry, and used directly in cell assays or sorted by FACS to establish derived cell lines as described therein. Primary human PBMCs used in functional assays were purchased from the CFAR Virology CoreLab at the UCLA AIDS Institute and stimulated, transduced, and cultured as previously described (52). T cells were expanded from PBMCs in freshly cytokine-supplemented T cell medium (AIM-V medium supplemented with 5% heat-inactivated human AB serum, 55 μM β-mercaptoethanol, and 4 mM L-glutamine). All cells were grown and assayed at 37°C in 5% atmospheric CO2.

[0053] NY-ESO-1-specific CD8 + Generation and culture of T lymphocyte clones CD8 specific for epitopes of NY-ESO-1 with various HLA restrictions + T lymphocyte clone (157-165 / HLA-A * 02:01(53), 60-72 / HLA-B * 07:02(21), 88-96 / HLA-B * 18:01(23), 92-100 / HLA-C *03:04(54), 96-104 / HLA-C * 03:04(22), 124-133 / HLA-C * NY-ESO-1 (NY-ESO-1) antibodies (03:04 (22)) were generated from HLA-typed melanoma patients. All selected patients had Grade III / IV metastatic melanoma and had previously documented ex vivo NY-ESO-1 responses to relevant T lymphocyte epitopes (55). Patient PBMCs were stimulated in the presence of 1 μM pooled peptides (Mimotopes), which contain a 28 × 18-mer with a 12-amino acid overlap that collectively spans the NY-ESO-1 protein sequence, and then cultured for 10 days in the presence of 25 IU / ml IL-2 (Peprotech).

[0054] On day 10, cells were restimulated with 1 μM of each peptide in the presence of brefeldin A, and the CD8 responses to each peptide were + T cell activation was determined by intracellular cytokine staining (ICS). Briefly, cells were labeled with fixable live / dead violet stain (Invitrogen) according to the manufacturer's instructions and then incubated with antibodies against CD3 and CD8 for 15 minutes at 4°C. Samples were washed and fixed with fixation / permeabilization reagent (BD biosciences) for 20 minutes at 4°C. Cells were stained with permeabilization / wash solution (BD biosciences) containing anti-IFNγ (eBiosciences) for 25 minutes at 4°C. The gating method was: SSC / LD - ;CD3 + / CD8 + ;CD8 + / IFNγ + Data from at least 100,000 stained cells were acquired using a FACSCanto and analyzed using FlowJo software. Data collection and analysis followed the MIATA guidelines (56).

[0055] NY-ESO-1-reactive T cells were expanded in the presence of the identified allogeneic 9-10 mer epitope, then labeled with fluorescent tetramers containing the relevant peptide and HLA molecules (TCMetrix, Epalinges, Switzerland), and single-cell sorted using a MoFlo cell sorter. Clones were re-expanded with pooled allogeneic healthy donor PBMCs, 1 μg / ml PHA-L, and 600 IU / ml IL-2 (Cetus) as feeder cells. Approximately 20 days later, 1-10 × 10 T cells were re-expanded in the presence of allogeneic PBMCs, PHA-L, and IL-2 as feeder cells, as described above. 3 Each clone was restimulated, and the specificity of the clones was confirmed by tetramer staining.

[0056] T lymphocyte clones / lines were cultured in RPMI 1640 medium supplemented with 2 mM Glutamax, 100 IU / ml penicillin, 100 μg / ml streptomycin, 20 mM HEPES, 1% non-essential amino acids, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, and 10% human serum (TCRPMI). IL-2 (100 IU / ml) was added and replaced every 3 days.

[0057] Cloning of TCR constructs Single NY-ESO-1-reactive T cells were sorted for antigen specificity using a FACS Aria II and then lysed by freeze-thawing in the presence of RNase inhibitors. Novel TCR variable genes were cloned from the sorted single T cells using a custom panel of human TCR variable region-specific primers with the Qiagen OneStep RT-PCR kit (Redwood City, CA), followed by a nested PCR amplification step. The amplified variable genes were incorporated into TCR expression cassettes containing either human or mouse TCR constant domains and a 2A ribosomal skip peptide linking the alpha and beta genes via assembly PCR and restriction enzyme-mediated cloning. A P2A-binding gene encoding a truncated form of the low-affinity nerve growth factor receptor (LNGFR) was also included in the cassette as an independent transfection / transduction marker. The antigen specificity and MHC restriction of the cloned TCRs were assessed in 293T cells cotransfected with TCR and CD3 genes as described (52).

[0058] Evaluation of TCR retrieval and dextramer binding in Jurkat T cells Jurkat T cells were transduced with MSGV-based retroviruses encoding each novel TCR in the format of LNGFRΔ-P2A-TCRα-F2A-TCRβ. Viruses were produced in 293T cells as described (52). For transduction, Jurkat T cells were centrifuged (1350 × g, 30°C, 90 min) with unconcentrated viral supernatant supplemented with 5 μg / mL polybrene. TCR-transduced Jurkat cells were stained with cognate pMHC dextramers for 15 min at room temperature, followed by co-staining with antibodies against LNGFR and CD8α for 15 min at 4°C. Stained cells were analyzed by flow cytometry using a FACSCanto analyzer. The data presented here are consistent with the results of the LNGFR analysis. + Gating is on transduced cells. Transduction efficiency was >95%.

[0059] PBMC activation and transduction Primary human PBMCs were purchased from the CFAR Virology Core Lab at the UCLA AIDS Institute. The same PBMC donor was used in all reported experiments. Primary human PBMCs were transduced with retroviruses encoding novel TCRs as described (52). Briefly, 2 days before viral transduction, a total of 1–2 × 10 PBMCs were transduced per well into 24-well plates coated with T cell medium containing anti-CD3 (clone OKT3), 1 μg / mL soluble anti-CD28 (clone CD28.2), and 300 U / mL IL-2. 6 Thawed PBMCs were activated. 48 hours after activation, the majority of the medium was replaced with unconcentrated retroviral supernatant supplemented with 10 μg / mL polybrene, and the cells were centrifuged at 1350 × g at 30°C for 90 minutes. After spinfection, the majority of the retroviral supernatant was replaced with fresh medium containing 300 U / mL IL-2 and 1 mg / mL anti-CD28. After repeated transduction for 24 hours, the cells were washed with 1× PBS and then returned to fresh medium containing a final 300 U / mL IL-2. After an additional 3–4 days of culture, the cells were used in antigen stimulation assays. On the day before or the day of co-incubation, PBMCs were analyzed by FACS to assess the expression levels of LNGFR, TCR, and / or pMHC multimer binding.

[0060] Functional co-culture assay - cytokine ELISA When Jurkat T cells were used as effectors, coculture was performed in RPMI supplemented with 10% FBS, 100 IU / ml penicillin, 100 μg / ml streptomycin, and 4 mM L-glutamine. Effector cells (50,000 TCR-transduced Jurkat T cells) were co-incubated with target cells (50,000 K562 cells transduced with the allogeneic or control single-chain trimer) in a 96-well flat-bottom plate. Supernatants from replicate wells were collected 44–48 h after coculture and analyzed by enzyme-linked immunosorbent assay (ELISA) as described below.

[0061] When primary PBMCs were used as effectors, they were co-cultured in T cell medium containing 300 U / mL IL-2. Effector cells (50,000 TCR-transduced PBMCs) were co-incubated with target cells (50,000 M257, PC-3, or K562 cells) in 96-well flat-bottom plates. In some experiments, target cells were pulsed with peptide. Supernatants from 2-8 replicate wells for each condition were collected 44-48 hours after co-culture and analyzed by enzyme-linked immunosorbent assay (ELISA) as described below.

[0062] For experiments in which target cells were titrated with pulsed peptide, lyophilized peptide was dissolved in DMSO to 10 mM and then further diluted with water to a working stock of 2 mM. At the time of use, the 2 mM stock solution was diluted to 250 μM with cell culture medium and then serially diluted 5-fold from 250 μM to 3.2 nM. Each serial dilution was added at 25 μL / well to a 96-well U-bottom plate, followed by the addition of 50,000 target cells in 100 μL of medium to obtain final peptide concentrations ranging from 50 μM to 0.64 nM. Cells were pulsed with peptide for 2 hours at 37°C. At the end of the incubation period, the cells were diluted with 100 μL of medium / well, centrifuged, and the supernatant removed. Cells were washed with 200 μL of medium and then resuspended in 100 μL of medium. Then, 50,000 PBMCs prepared in 100 μl of medium were added to each well and co-incubated.

[0063] Generally, ELISA results were converted to concentrations (ng / mL) by interpolation against a standard curve, and concentrations obtained from replicate ELISA assays were averaged. For ELISA analysis, supernatants were diluted 50–100 times. Occasionally, further dilutions were required to place the signal within the range of the standard curve. All reagents for ELISA analysis were from BD Biosciences: OptEIA Reagent Set B (550534) was used for dilutions and washes, and the OptEIA Human IFN-γ ELISA Kit (555142) and OptEIA Human IL-2 ELISA Kit (555190) were used to measure IFN-γ and IL-2 release, respectively.

[0064] Functional Co-culture Assay - IncuCyte Cell Killing Assay Prior to co-culture in the IncuCyte killing assay, 96-well flat-bottom plates were coated with 100 μl of PBS containing 0.001% poly-L-lysine for 1 hour at 37°C, washed twice with 200 μl PBS each, and briefly air-dried. Target cells were added and allowed to settle for 3 hours at room temperature before effector cell addition. For co-culture, typically 25,000 PBMCs and 25,000 target cells were used per well in a 96-well plate. For assays in which multiple effector populations (with different TCRs) or multiple targets (with different MHCs) were mixed, 25,000 of each cell type were used, yielding a total of 75,000 or 100,000 cells per well (for single / mixed or mixed / mixed, respectively). The total volume in all wells was adjusted to 200 μL. The total area of ​​green objects (μm 2 / well) was quantified, and the decrease was calculated as GFP + This was interpreted as target cell killing. Cells were imaged at two locations per well every 2 hours, and the two images were merged together into a single data point. For each effector / target combination, data points from 4–8 replicate cocultures were used to plot a graph curve and calculate the standard deviation.

[0065] animal NOD.Cg-PrkdcSCIDIL-2rgtm1Wjl / SzJ(NOD / SCID / IL-2Rg - / - NSG (non-steroidal anti-inflammatory) mice were purchased from the Jackson Laboratory and housed in the animal facility at the University of California, Los Angeles (UCLA). Adult (16-week-old) male mice were used for in vivo tumor inoculation experiments. All animal experiments were approved by the UCLA Institutional Animal Care and Use Committee.

[0066] Human prostate tumor xenograft mouse model For xenograft tumor transplants, 10 × 10 6 PC-3 / HLA-A2 cells (a PC-3 cell line overexpressing HLA-A2) were injected subcutaneously into one flank of each mouse, and 10 × 10 6 PC-3 / HLA-A2 / NY-ESO-1 cells (a PC-3 cell line overexpressing HLA-A2 and NY-ESO-1) were injected subcutaneously into the other flank. Mice were allowed to grow solid tumors over a one-week period. Eight days after tumor injection, mice were irradiated (100 rads) and subsequently injected with 8 x 10 NY-ESO-1 cells designed to express LNGFR alone or in combination with NY-ESO-1-specific TCRs (1G4, 3A1, or 9D2). 6 Purified T cells were injected intravenously into the retro-orbital plexus. Mice were bled on days 3, 7, 10, and 14 for flow cytometry analysis. On day 14, mice were euthanized and tumors were collected for immunohistological analysis.

[0067] immunohistology Solid tumors dissected from experimental mice were fixed in 10% neutral buffered formalin and embedded in paraffin for sectioning (4 mm thick), followed by hematoxylin and eosin (H / E) staining or antibody staining (for human CD3ε) using standard procedures (UCLA Translational Pathology Core Laboratory). These sections were imaged at 4x and 40x magnification using an Olympus BX51 upright microscope equipped with an Optronics Macrofire CCD camera (AU Optronics). These images were analyzed using Optronics Picture Frame software (AU Optronics) and Image J software (version 1.51J8). After color thresholding using the Image J human CD3 antibody, stained slides were analyzed for CD3. + The area was measured and quantified using the following parameters: thresholding method: default; threshold color: red; color space: HSB; brightness: 168-215.

[0068] statistical analysis Statistical analysis of tumor xenograft experiments was performed using one-way analysis of variance followed by Tukey's multiple comparison test. Data are presented as mean ± SEM. P < 0.05 was considered significant. ns, not significant. * , P < 0.05; ** , P < 0.01; *** , P < 0.001; **** P<0.0001. All statistical analyses were performed using GraphPad PRISM software (version 6.0).

[0069] Table 1: TCR α / β polynucleotide and polypeptide sequences The following disclosure provides polynucleotide sequences and the variable region TCR protein sequences they encode in various embodiments of the invention (e.g., the polynucleotide sequence of SEQ ID NO: 1 encodes the variable region TCR protein of SEQ ID NO: 3).

[0070] 3A1 TCR Vα DNA sequence GGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCTAGTGATGTAGGCATCTACTTCTGTGCTGGATTTCTGGATAGCAACTATCAGTTAATCTGGGGCGCTGGGACCAAGCTAATTATAAAGCCAGAT(SEQ ID NO: 1)

[0071] 3A1 TCR Vβ DNA sequence GAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCGCTAGCGGGTACCGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGAC(SEQ ID NO: 2)

[0072] 3A1 TCR Vα protein sequence GQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGFLDSNYQLIWGAGTKLIIKPD(SEQ ID NO: 3)

[0073] 3A1 TCR Vβ protein sequence EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASASGYRTDTQYFGPGTRLTVLED (SEQ ID NO: 4)

[0074] 4A2 TCR Vα DNA sequence GCTCAGTCAGTGGCTCAGCCGGAAGATCAGGTCAACGTTGCTGAAGGGAATCCTCTGACTGTGAAATGCACCTATTCAGTCTCTGGAAACCCTTATCTTTTTTGGTATGTTCAATACCCCAACCGAGGCCTCCAGTTCCTTCTGAAATACATCACAGGGGATAACCTGGTTAAAGGCAGCTATGGCTTTGAAGCTGAATTTAACAAGAGCCAAACCTCCTTCCACCTGAAGAAACCATCTGCCCTTGTGAGCGACTCCGCTTTGTACTTCTGTGCTGTGAGAGACAGTCGGTCTGGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCGGTCATACCAAAT (SEQ ID NO: 5)

[0075] 4A2 TCR Vβ DNA sequence GGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGG CGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTCCCCAAGGTTATGGGGGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGAC (SEQ ID NO: 6)

[0076] 4A2 TCR Vα protein sequence AQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDSRSGAGSYQLTFGKGTKLSVIPN (SEQ ID NO: 7)

[0077] 4A2 TCR Vβ protein sequence GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAPQGYGGTDTQYFGPGTRLTVLED (SEQ ID NO: 37)

[0078] 5G6 TCR Vα DNA sequence GATGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAAGAGCCTGTTCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATACATTGGTATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAGCAATGTGAACAACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTCCAGTACCTTGATCCTGCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACTGCATCCTGAGAACCTCTGGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCGGTCATACCAAAT(SEQ ID NO: 8)

[0079] 5G6 TCR Vβ DNA sequence AGTGCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAACCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGCATATATCTCTGCAGCGCGGGAGGAGCGGGAGCGTCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGAC(SEQ ID NO: 9)

[0080] 5G6 TCR Vα protein sequence DAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILRTSGAGSYQLTFGKGTKLSVIPN(SEQ ID NO: 10)

[0081] 5G6 TCR Vβ Protein Sequence SAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSAGGAGASDTQYFGPGTRLTVLED (SEQ ID NO: 11)

[0082] 9D2 TCR Vα DNA Sequence CAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTAGATGACAAGATCATCTTTGGAAAAGGGACACGACTTCATATTCTCCCCAAT (SEQ ID NO: 12)

[0083] 9D2 TCR Vβ DNA Sequence GATGCTGGAGTTATCCAGTCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATCCAGCCCTCAGAACCCAGGGACTCAGCTGTGTACTTCTGTGCCAGCAGTTTGGGACAGCCAAGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGAC (SEQ ID NO: 13)

[0084] 9D2 TCR Vα protein sequence QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVDDKIIFGKGTRLHILPN (SEQ ID NO: 14)

[0085] 9D2 TCR Vβ protein sequence DAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLGQPSTDTQYFGPGTRLTVLED (SEQ ID NO: 15)

[0086] 1E4 TCR Vα DNA sequence AAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGG AAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTCTGTGCTGTGAGTACTGCGTATTCAGGAGGAGGTGCTGACGGACTCACCTTTGGCAAAGGGACTCATCTAATCATCCAGCCCTAT (SEQ ID NO: 16)

[0087] 1E4 TCR Vβ DNA sequence GATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCT GCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCCCCCCGACTGTTCGGGTCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGAC (SEQ ID NO: 17)

[0088] 1E4 TCR Vα protein sequence KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVSTAYSGGGADGLTFGKGTHLIIQPY (SEQ ID NO: 18)

[0089] 1E4 TCR Vβ protein sequence DTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSPPTVRVYGYTFGSGTRLTVVED (SEQ ID NO: 19)

[0090] 2B8 TCR Vα DNA sequence GGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAAGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCGGACCCTAACTTTGGAAATGAGAAATTAACCTTTGGGACTGGAACAAGACTCACCATCATACCCAAT(SEQ ID NO: 20)

[0091] 2B8 TCR Vβ DNA sequence GAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTGAATCCCTTTGCAACTAATGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAGGAC(SEQ ID NO: 21)

[0092] 2B8 TCR Vα protein sequence GQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCADPNFGNEKLTFGTGTRLTIIPN(SEQ ID NO: 22)

[0093] 2B8 TCR Vβ Protein Sequence EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLNPFATNEKLFFGSGTQLSVLED (SEQ ID NO: 23)

[0094] 3C7 TCR Vα DNA Sequence GGACAAAACATTGACCAGCCCACTGAGATGACAGCTACGGAAGGTGCCATTGTCCAGATCAACTGCACGTACCAGACATCTGGGTTCAACGGGCTGTTCTGGTACCAGCAACATGCTGGCGAAGCACCTACATTTCTGTCTTACAATGTTCTGGATGGTTTGGAGGAGAAAGGTCGTTTTTCTTCATTCCTTAGTCGGTCTAAAGGGTACAGTTACCTCCTTTTGAAGGAGCTCCAGATGAAAGACTCTGCCTCTTACCTCTGTGCTGTGAGAGGCGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAACTGTAAGAGCAAAT (SEQ ID NO: 24)

[0095] 3C7 TCR Vβ DNA Sequence GATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCTCGATACACGGTGTCTCTGGGGCCAACGTCCTGACTTTCGGGGCCGGCAGCAGGCTGACCGTGCTGGAGGAC (SEQ ID NO: 25)

[0096] 3C7 TCR Vα protein sequence GQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVRGDYKLSFGAGTTVTVRAN (SEQ ID NO: 26)

[0097] 3C7 TCR Vβ protein sequence DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSSIHGVSGANVLTFGAGSRLTVLED (SEQ ID NO: 27)

[0098] NY-ESO-1 protein (Homo sapiens): GenBank: CAA05908.1 MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR (SEQ ID NO: 28)

[0099] The terms used in this disclosure, e.g., "A2 / NY-ESO-1 157-165 " refers to HLA A2 associated with the NY-ESO-1 peptide comprising amino acids 157-165 of the protein sequence set forth above (i.e., SLLMWITQC (SEQ ID NO: 36)).

[0100] The following sequences include polynucleotide embodiments of the invention that are placed in a vector.

[0101] aaaccctcttgcagttgcatccgacttgtggtctcgctgt

[0102] cgacttgtggtctcgctgttccttgggagggtctcctctg

[0103] PMTB1330 (MSGV-LNGR-P2A+HHD2 TCR mouse constant region

[0104] tgcagttgcatccgacttgtggtctcgctgttccttggga

[0105] cttgtggtctcgctgttccttgggagggtctcctctgagt

[0106] pMTB1333 (MSGV-LNGFR-P2A-C03NY96 TCR mouse constant region)

[0107] pMTB1289 (MSGV-LNGFR-P2A-GBA1 TCR mouse constant region)

[0108] Publications All publications mentioned herein (e.g., references cited herein by number) are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications cited herein are cited for their disclosure prior to the filing date of this application. No determination should be made that the inventors are not entitled to antedate a publication by virtue of an earlier priority date or dates. Further, actual publication dates may be different from those stated and require independent verification. The following references include descriptions of methods and materials in the art.

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[0110] Conclusion This concludes the description of exemplary embodiments of the invention. The foregoing description of one or more embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teachings.

Claims

1. A polynucleotide for placement into a vector: The polynucleotide encodes a 9D2 Vα T cell receptor polypeptide (SEQ ID NO: 14) and a 9D2 Vβ T cell receptor polypeptide (SEQ ID NO: 15); and The Vα T cell receptor polypeptide and the Vβ T cell receptor comprising the Vα / Vβ T cell receptor polypeptide are administered to CD8 ＋ When expressed on T cells, the Vα / Vβ T cell receptor: Human leukocyte antigen A2; human leukocyte antigen B07; Human leukocyte antigen B18; or human leukocyte antigen C03, The polynucleotide recognizing the NY-ESO-1 peptide associated with

2. The vector is a CD8 ＋ The polynucleotide of claim 1 , comprising a polynucleotide sequence that regulates expression of the polypeptide in T cells.

3. The polynucleotide of claim 2 , wherein the vector is a Sendai virus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or a lentivirus vector.

4. The Vα / Vβ T cell receptor (TCR) is a CD8 ＋ The polynucleotide of claim 1, which is expressed on the surface of a T cell.

5. A composition of matter comprising a host cell transfected with a vector comprising the polynucleotide of any one of claims 1 to 3.

6. The host cells are human CD8 ＋ The composition of claim 5 which is a T cell.

7. 7. The composition of claim 6, wherein the composition is a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients selected from the group consisting of buffering agents, antibacterial agents, tonicity adjusting agents, wetting agents, detergents, and pH adjusting agents.

8. The CD8 ＋ T cells are obtained from an individual diagnosed with cancer that expresses the NY-ESO-1 antigen; and The CD8 ＋ T cells are transduced with a vector comprising a polynucleotide encoding a TCR Vα polypeptide together with a polynucleotide encoding a TCR Vβ polypeptide, thereby transducing the heterologous TCR into CD8 ＋ The composition of claim 7, wherein the heterologous TCR is expressed on the surface of a T cell, and the heterologous TCR recognizes a NY-ESO-1 peptide associated with a human leukocyte antigen expressed on the surface of a cell of the cancer.

9. The composition of claim 8 , wherein the vector is a retroviral vector.

10. 10. The composition of claim 6 for use in a method of killing cancer cells expressing the NY-ESO-1 antigen, said method comprising: translating a heterologous TCR into said CD8 ＋ and (c) detecting the NY-ESO-1 peptide associated with a human leukocyte antigen (HLA) expressed on the surface of the cancer cells under conditions that allow the NY-ESO-1 peptide to be expressed on the surface of the cancer cells and thereby recognize and kill the cancer cells. ＋ and combining said composition with T cells.

11. The method comprises: ＋ The composition according to claim 10, characterized in that it is carried out in vivo in a patient infused with T cells.

12. The composition of claim 10 , wherein the cancer cells form a solid tumor.

13. 11. The composition of claim 10, wherein the cancer cells are neuroblastoma cells, myeloma cells, metastatic melanoma cells, synovial sarcoma cells, bladder cancer cells, esophageal cancer cells, hepatocellular carcinoma cells, head and neck cancer cells, non-small cell lung cancer cells, ovarian cancer cells, prostate cancer cells, or breast cancer cells.

14. The method comprises: providing a first modified CD8 antibody targeting a NY-ESO-1 peptide associated with a first human leukocyte antigen; ＋ T cells are targeted to a second CD8 T cell receptor mediated by the NY-ESO-1 peptide associated with a second human leukocyte antigen. ＋ The composition of claim 10, wherein the composition is administered in combination with T cells.

15. 10. Use of the polynucleotide of claim 1 or the composition of claim 6 for the manufacture of a medicament for the treatment of cancer.

16. 16. The use of claim 15, wherein the cancer is melanoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer.

17. A composition for the treatment of cancer, comprising the polynucleotide of claim 1 or the composition of claim 6.

18. 18. The composition of claim 17, wherein the cancer is melanoma, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, or breast cancer.

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