A2 / NY-ESO-1 specific T cell receptor and uses thereof

Abstract

This application provides genetically modified T cell receptors (TCRs) specific for epitopes of the cancer antigen NY-ESO-1. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells including, but not limited to, genetically engineered cells, and pharmaceutical compositions, are also provided. This application further provides the use of such modified T cell receptors (TCRs) and related compositions for cancer immunotherapy (e.g., adoptive cell therapy).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62 / 819,988, filed March 18, 2019, the disclosure of which is incorporated herein by reference in its entirety. [Technical field]

[0002] The present invention relates to genetically modified T cell receptors (TCRs) specific for epitopes derived from the cancer antigen NY-ESO-1. The present invention relates to related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and populations of cells, including but not limited to genetically engineered cells, and pharmaceutical compositions. The present invention further relates to the use of such modified TCRs and related compositions for cancer immunotherapy. [Background technology]

[0003] Cancer immunotherapy has established itself as a viable alternative and powerful complement to standard treatment approaches such as cytotoxic chemotherapy, radiation therapy, and surgery by harnessing and / or enhancing the patient's own immune response against cancer cells (Non-Patent Document 1). Among its many aspects, adoptive T cell therapy has previously proven its efficacy in the clinic (Non-Patent Document 2). In this type of cancer treatment, patients are transferred with ex vivo expanded autologous tumor-infiltrating lymphocytes that can naturally recognize and kill tumor targets (Non-Patent Document 3). Alternatively, the patient's peripheral blood T cells can be genetically modified to recognize tumor cells via ectopic expression of tumor-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) prior to expansion and adoptive transfer. NY-ESO-1 TCR-based (Non-Patent Document 4) and CD19 CAR-based (Non-Patent Document 5) T cell therapies are extraordinary examples of cancer immunotherapy that have shown great promise in the clinic in solid and hematological malignancies, respectively.

[0004] NY-ESO-1 or New York esophageal squamous cell carcinoma 1 is a tumor antigen expressed by various tumors (Non-Patent Document 6). Class I human leukocyte antigen (HLA) molecules of these cancerous cells present a peptide from this antigen, including a nine amino acid fragment from positions 157 to 165 (SLLMWITQC (SEQ ID NO: 8)). Thus, NY-ESO-1 157-165 An example of an epitope-HLA-A2 complex is NY-ESO-1. 157-165 The present invention provides a cancer marker that can be targeted by a T cell receptor (TCR) in adoptive cell transfer therapy, in which autologous cells transduced with a nucleic acid encoding a TCR specific for the epitope (SLLMWITQC (SEQ ID NO: 8)) are transferred to a patient. However, for that purpose, it would be desirable for the TCR to have a higher affinity for the peptide-HLA complex than the native TCR specific for that complex.

[0005] Because TCRs against “self” tumor antigens may have lower affinity than viral epitope-specific TCRs, e.g., due to thymic negative selection, there is a need to develop higher affinity TCRs that maintain specificity (i.e., do not cross-react with healthy tissue) while allowing for higher activity.

[0006] Despite being a potentially very powerful immunotherapy strategy, many patients do not show clinical benefit from adoptive T cell therapy or are unable to maintain long-term responses, and as a result, its application remains limited. Although treatment-related toxicity remains a major concern (Non-Patent Document 7), the main reason why adoptively transferred T cells are unable to achieve effective tumor control is the immunosuppressive microenvironment of the tumor itself (Non-Patent Document 8). Tumors deploy many strategies to evade detection by the immune system. Among them are altered chemokine expression profiles that prevent T cell chemoattraction; strengthened physical barriers by collagen fortification of surrounding tissues and promotion of abnormal intratumoral vasculature that prevents proper T cell extravasation; establishment of a metabolic microenvironment unfavorable to T cell function, characterized by low glucose levels; and suppression of T cell function by regulatory T cells (T reg) and myeloid-derived suppressor cells (MDSCs) to the tumor microenvironment, or polarization of others to acquire an immunosuppressive phenotype, such as M2 macrophages; and finally, direct inhibition of T cell function via upregulation of immune checkpoint receptors and other membrane-bound or soluble factors with similar properties (Non-Patent Document 10).

[0007] These obstacles can be overcome by combinatorial therapeutic strategies that enhance the activity of adoptively transferred T cells by addressing various aspects of the immunosuppressive tumor microenvironment. Currently, a myriad of approaches are being tested at preclinical and clinical levels, such as immune checkpoint blocking antibodies, cytokine therapy, metabolic enzyme inhibitors, agents that normalize tumor vasculature, and inhibitory immune cell depleting antibodies, in combination with chemotherapy and radiotherapy (Non-Patent Document 10). Alternative, more precise and targeted approaches include fine-tuning of transferred T cells via genetic modification with molecules that directly affect T cell activity or target the tumor microenvironment. Engineering T cells with chemokine receptors has been shown to promote T cell trafficking to tumor sites, CARs targeting VEGF receptors result in normalization of the vasculature, and overexpression or integration of costimulatory ligands into CARs gives transferred T cells an added edge. Furthermore, targeting of T cell-associated checkpoint ligands can alleviate T cell suppression via tumor-derived checkpoints (Non-Patent Document 11).

[0008] It is not surprising that most engineering-based strategies aim to directly improve T cell activity (Non-Patent Document 12), but no efforts have been described to date to modify adoptively transferred T cells with agents that could exploit or enhance the potentially beneficial activities of other tumor-resident immune cells. Tumor-associated macrophages and granulocytes have emerged as key players in establishing or hindering successful anti-tumor responses, and stimulation of their anti-tumor versus tumor-promoting properties may be of great importance (Non-Patent Documents 13 and 14). Towards the same direction, immune attraction and stimulation of dendritic cells (DCs) with ectopically expressed tumor-derived soluble factors has been shown to elicit potent anti-tumor responses mediated by activated endogenous T cells (Non-Patent Document 15). These observations highlight the need for suitable T cell engineering candidate agents that can effectively harness the anti-tumor potential of the aforementioned tumor-resident immune cell lineages.

[0009] The invention disclosed herein addresses this and other related needs. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Mellman, I., G. Coukos, and G. Dranoff, Nature, 2011. 480: p. 480 [Non-Patent Document 2] Restifo, NP, ME Dudley, and SA Rosenberg, Nature Reviews Immunology, 2012. 12: p. 269 [Non-Patent Document 3] Rosenberg, SA, et al., Clin Cancer Res, 2011. 17(13): p. 4550-7 [Non-Patent Document 4] Robbins, PF, et al., J Clin Oncol, 2011. 29(7): p. 917-24

Non-licensed Document 5

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0011] There is a great need in the art to develop TCRs with higher affinity for cancer antigens. 157-165 This and other needs are addressed by providing epitope-specific engineered TCRs, as well as related compositions and methods for using such TCRs for cancer immunotherapy (e.g., adoptive cell therapy). [Means for solving the problem]

[0012] In one aspect, provided herein is a polynucleotide encoding a modified T cell receptor (TCR) or a functional fragment thereof, wherein the modified TCR comprises a single amino acid substitution within complementarity determining region (CDR) 2 of the β chain of the modified TCR compared to CDR2 of the β chain of the unsubstituted wild type (WT) TCR.

[0013] In some embodiments, the modified TCR β chain sequence comprises an amino acid sequence that is at least 80% identical to the unsubstituted WT TCR β chain, or a functional fragment thereof, outside the CDR2 region of the modified TCR β chain, or a functional fragment thereof.

[0014] In some embodiments, the modified TCR β chain or functional fragment thereof comprises the amino acid sequence of an unsubstituted WT TCR β chain or functional fragment thereof having a single amino acid substitution in the CDR2 region (i.e., comprises an amino acid sequence of an unsubstituted WT TCR β chain or functional fragment thereof having a single amino acid substitution in the CDR2 region).

[0015] In some embodiments, the beta chain of the unsubstituted WT TCR comprises the amino acid sequence of SEQ ID NO:1.

[0016] In some embodiments, the single amino acid substitution is at residue 50, 51, 53, or 55 relative to the WT TCR. In some embodiments, the single amino acid substitution is at residue 53 or 55 relative to the WT TCR. In some embodiments, the single amino acid substitution is I53E, I53F, I53W, or D55E.

[0017] In some embodiments, the modified TCR binds to a cancer antigen with higher binding affinity than the WT TCR. In some embodiments, the cancer antigen is NY-ESO-1. 157-165 epitope (SEQ ID NO:8).

[0018] In some embodiments, the binding affinity of the modified TCR to the cancer antigen is about 5 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. In some embodiments, the binding affinity of the modified TCR to the cancer antigen is about 10 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. In some embodiments, the binding affinity of the modified TCR to the cancer antigen is about 25 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. In some embodiments, the binding affinity of the modified TCR to the cancer antigen is about 40 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. In some embodiments, the binding affinity of the modified TCR to the cancer antigen is about 40 to about 60 times higher than the binding affinity of the WT TCR to the cancer antigen. In some embodiments, the binding affinity of the modified TCR to the cancer antigen is about 40 to about 50 times higher than the binding affinity of the WT TCR to the cancer antigen. In some embodiments, the binding affinity of the modified TCR to the cancer antigen is about 50 times higher than the binding affinity of the WT TCR to the cancer antigen.

[0019] In some embodiments, the dissociation constant (K D) is between about 0.30 and about 4.5 μM. In some embodiments, the dissociation constant (K D ) is between about 0.30 and about 2 μM. In some embodiments, the dissociation constant (K D ) is between about 2 μM and about 3 μM. In some embodiments, the dissociation constant (K D In some embodiments, the dissociation constant (K D ) is about 0.41 μM. In some embodiments, the dissociation constant (K D ) is approximately 3.89 μM.

[0020] In some embodiments, the modified TCR comprises the amino acid sequence of any one of SEQ ID NOs: 2 to 5, or a functional fragment thereof, or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 5.

[0021] In some embodiments, the modified TCR is encoded by the nucleotide sequence of any one of SEQ ID NOs: 11-14, or a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 11-14.

[0022] In some embodiments, the polynucleotide is operably linked to at least one regulatory element for expression of the modified TCR. In some embodiments, the at least one regulatory element is a promoter.

[0023] In various embodiments, the polynucleotide is a DNA molecule.

[0024] In various embodiments, the polynucleotide is an RNA molecule or a derivative thereof.

[0025] In another aspect, provided herein is a recombinant vector comprising a polynucleotide described herein, wherein the polynucleotide is operably linked to at least one regulatory element for expression of a modified T cell receptor (TCR).

[0026] In some embodiments, the vector is a viral vector, hi some embodiments, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphavirus vector, a herpes viral vector, or a vaccinia viral vector.

[0027] In some embodiments, the vector is a non-viral vector.

[0028] In another aspect, provided herein is a modified T cell receptor (TCR) comprising a modified TCR beta chain or a functional fragment thereof encoded by a polynucleotide described herein.

[0029] In another aspect, provided herein is a modified T cell receptor (TCR) comprising: a) a modified TCR beta chain, or a functional fragment thereof, encoded by a polynucleotide described herein, and b) an alpha chain, or a functional fragment thereof.

[0030] In another aspect, provided herein is a modified T cell receptor (TCR) comprising the CDRs of the β chain of a modified TCR encoded by a polynucleotide described herein.

[0031] In another aspect, provided herein is a modified T cell receptor (TCR) comprising: a) a functional fragment of a beta chain of the modified TCR, the functional fragment comprising the CDRs of the beta chain encoded by a polynucleotide described herein; and b) a functional fragment of an alpha chain, the functional fragment comprising the CDRs of the alpha chain.

[0032] In some embodiments, the functional fragment of a) further comprises a constant region of a TCR beta chain, and / or the functional fragment of b) further comprises a constant region of a TCR alpha chain. In one embodiment, any of the constant regions are of human origin. In one embodiment, any of the constant regions are of murine origin.

[0033] In some embodiments the alpha chain comprises the alpha chain of a WT TCR or a functional fragment thereof. In one embodiment the alpha chain of a WT TCR comprises the amino acid sequence of SEQ ID NO:7.

[0034] In some embodiments, the modified TCR comprises the amino acid sequence of any one of SEQ ID NOs: 2-5, or a fragment thereof, or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2-5.

[0035] In another aspect, provided herein is a modified T cell receptor (TCR) comprising an alpha chain comprising the amino acid sequence of SEQ ID NO:7 and a beta chain comprising the amino acid sequence of any one of SEQ ID NOs:2-5.

[0036] In another aspect, provided herein is an isolated host cell (isolated host cell) comprising a modified T cell receptor (TCR) described herein.

[0037] In another aspect, provided herein is an isolated host cell comprising a polynucleotide described herein. In some embodiments, the polynucleotide is operably linked to at least one regulatory element capable of mediating expression of a modified T cell receptor (TCR) in the host cell.

[0038] In another aspect, provided herein is an isolated host cell comprising a vector described herein.

[0039] In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a lymphoid cell. In some embodiments, the lymphoid cell is a T cell. In some embodiments, the lymphoid cell is a natural killer (NK) cell.

[0040] In various embodiments, the host cells are obtained from peripheral blood mononuclear cells (PBMCs), tumor draining lymph nodes, or tumor infiltrates.

[0041] In various embodiments, the host cells are activated and / or expanded ex vivo.

[0042] In various embodiments, the host cell is an allogeneic cell.

[0043] In various embodiments, the host cells are autologous cells.

[0044] In various embodiments, the host cell is isolated from a subject having a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer expresses the cancer antigen NY-ESO-1 on the surface of the cell. 157-165 The epitope (SEQ ID NO: 8) is presented. In some embodiments, the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.

[0045] In various embodiments, the host cells are further engineered to express one or more exogenous molecules, hi some embodiments, the one or more exogenous molecules are immune signaling molecules.

[0046] In some embodiments, the immune signaling molecule is a cytokine.

[0047] In some embodiments, the immune signaling molecule is a chemokine.

[0048] In some embodiments, the immune signaling molecule is a growth factor, hi one embodiment, the growth factor is granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0049] Also provided herein is an isolated host cell comprising a T cell receptor (TCR) or a functional fragment thereof that binds to a cancer antigen, the host cell being further engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the cancer antigen is NY-ESO-1. 157-165 epitope (SEQ ID NO: 8). In some embodiments, the T cell receptor (TCR) is a WT TCR. In one embodiment, the β chain of the WT TCR comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In one embodiment, the α chain of the WT TCR comprises the amino acid sequence of SEQ ID NO: 7.

[0050] In some embodiments, the amino acid sequence of GM-CSF comprises SEQ ID NO: 21, 34 or 15, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 21, 34 or 15. In some embodiments, the nucleotide sequence encoding GM-CSF comprises SEQ ID NO: 22, 35 or 16, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 22, 35 or 16.

[0051] In some embodiments, the one or more exogenous molecules is a soluble receptor.

[0052] In some embodiments, the one or more exogenous molecules is a ligand.

[0053] In some embodiments, the one or more exogenous molecules are antigen binding proteins, hi some embodiments, the antigen binding protein is an antibody or an antibody fragment.

[0054] In some embodiments, the one or more exogenous molecules is a phosphodiesterase. In some embodiments, the phosphodiesterase is PDE4B2.

[0055] Also provided herein is an isolated host cell comprising a T cell receptor (TCR) or a functional fragment thereof that binds to a cancer antigen, the host cell being further engineered to express PDE4B2. In some embodiments, the cancer antigen is NY-ESO-1. 157-165 epitope (SEQ ID NO: 8). In some embodiments, the T cell receptor (TCR) is a WT TCR. In one embodiment, the β chain of the WT TCR comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In one embodiment, the α chain of the WT TCR comprises the amino acid sequence of SEQ ID NO: 7.

[0056] In some embodiments, the amino acid sequence of PDE4B2 comprises SEQ ID NO: 27 or 29, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 27 or 29. In some embodiments, the nucleotide sequence encoding PDE4B2 comprises SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 28 or 30.

[0057] In some embodiments, the one or more exogenous molecules are cell surface receptors. In some embodiments, the cell surface receptor is a chimeric antigen receptor. In some embodiments, the cell surface receptor is a cancer antigen NY-ESO-1 157-165 This is a T cell receptor that does not bind to the epitope (SEQ ID NO:8).

[0058] In another aspect, provided herein is a bifunctional molecule comprising a modified T cell receptor (TCR) or functional fragment thereof described herein and an immune effector polypeptide that specifically binds to a cell surface protein on a T cell. In some embodiments, the immune effector polypeptide comprises an antibody or an antibody fragment. In some embodiments, the immune effector polypeptide comprises a single chain variable region fragment (scFv).

[0059] In some embodiments, the immune effector polypeptide specifically binds to CD3. In some embodiments, the immune effector polypeptide comprises an antibody or antibody fragment derived from OKT3, UCHT-1, BMA031, or 12F6. In some embodiments, the immune effector polypeptide comprises an antibody or antibody fragment (e.g., scFV) derived from OKT3. In some embodiments, the immune effector polypeptide comprises an antibody or antibody fragment (e.g., scFV) derived from UCHT-1. In some embodiments, the immune effector polypeptide comprises an antibody or antibody fragment (e.g., scFV) derived from BMA031. In some embodiments, the immune effector polypeptide comprises an antibody or antibody fragment (e.g., scFV) derived from 12F6.

[0060] In another aspect, provided herein is a pharmaceutical composition comprising a host cell as described herein, or a bifunctional molecule as described herein, and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the composition is used in adoptive cell transfer therapy.

[0061] In another aspect, provided herein is a method of making a host cell described herein, comprising genetically engineering a host cell with a polynucleotide described herein or a vector described herein.

[0062] In another aspect, provided herein is a method of genetically engineering a host cell to express a modified T cell receptor (TCR) or functional fragment thereof described herein. In some embodiments, the method comprises genetically engineering the host cell with a polynucleotide described herein or a vector described herein. In some embodiments, the genetic engineering step is performed via viral gene delivery. In some embodiments, the genetic engineering step is performed via non-viral gene delivery. In some embodiments, the method is performed ex vivo. In some embodiments, the method further comprises ex vivo activation and / or expansion of the host cell.

[0063] In some embodiments of the cell engineering method, the modified TCR comprises the amino acid sequence of any one of SEQ ID NOs: 2-5, or a fragment thereof, or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2-5.

[0064] In some embodiments of the cell engineering method, the method further comprises genetically engineering the host cell to further express one or more exogenous molecules. In some embodiments, the one or more exogenous molecules are immune signaling molecules. In some embodiments, the exogenous molecule is a cytokine, a chemokine, a growth factor, a soluble receptor, a ligand, a phosphodiesterase, an antigen binding protein, or a cell surface receptor.

[0065] In some embodiments of the cell engineering method, the growth factor is granulocyte macrophage colony stimulating factor (GM-CSF). In one embodiment, the amino acid sequence of GM-CSF comprises SEQ ID NO: 21, 34 or 15, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 21, 34 or 15. In one embodiment, the nucleotide sequence encoding GM-CSF comprises SEQ ID NO: 22, 35 or 16, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 22, 35 or 16.

[0066] In some embodiments of the cell engineering method, the phosphodiesterase is PDE4B2. In one embodiment, the amino acid sequence of PDE4B2 comprises SEQ ID NO: 27 or 29, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 27 or 29. In one embodiment, the nucleotide sequence encoding PDE4B2 comprises SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 28 or 30.

[0067] In some embodiments of the cell engineering methods, the antigen binding protein is an antibody or an antibody fragment.

[0068] In some embodiments of the cell engineering methods, the cell surface receptor is a chimeric antigen receptor, or a cancer antigen NY-ESO-1. 157-165 This is a T cell receptor that does not bind to the epitope (SEQ ID NO:8).

[0069] In various embodiments of the cell engineering method, the host cell is a mammalian cell. In some embodiments, the host cell is a lymphoid cell. In some embodiments, the lymphoid cell is a T cell. In some embodiments, the lymphoid cell is a natural killer (NK) cell.

[0070] In various embodiments of the cell engineering methods, host cells are obtained from peripheral blood mononuclear cells (PBMCs), tumor-draining lymph nodes or tumor infiltrates.

[0071] In various embodiments, the host cells are activated and / or expanded ex vivo.

[0072] In various embodiments, the host cell is an allogeneic cell.

[0073] In various embodiments, the host cells are autologous cells.

[0074] In various embodiments, the host cell is isolated from a subject having a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer expresses the cancer antigen NY-ESO-1 on the surface of the cell. 157-165 The epitope (SEQ ID NO: 8) is presented. In some embodiments, the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.

[0075] In another aspect, provided herein is a method of stimulating or enhancing an immune response in a mammal in need thereof, comprising administering to the mammal an effective amount of a lymphoid cell comprising a modified T cell receptor (TCR) described herein, a host cell described herein, a bifunctional molecule described herein, a composition described herein, or a host cell produced by the methods described herein.

[0076] In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a lymphoid cell comprising an engineered T cell receptor (TCR) described herein, a host cell described herein, a bifunctional molecule described herein, a composition described herein, or a host cell produced by a method described herein. In some embodiments, the cells of the cancer express the cancer antigen NY-ESO-1 on their surface. 157-165 The epitope (SEQ ID NO: 8) is presented.

[0077] In some embodiments of the methods of treatment, the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.

[0078] In some embodiments, the method of treatment comprises: a) isolating T cells from a subject or mammal; b) genetically modifying said T cells ex vivo with a polynucleotide described herein or a vector described herein; c) optionally expanding and / or activating said T cells before, after, or during step b); and d) introducing the genetically modified T cells into a subject or mammal; Includes.

[0079] In various embodiments, the subject or mammal is a human.

[0080] These and other aspects of the invention will become apparent to those skilled in the art in the following description, claims, and drawings. [Brief description of the drawings]

[0081] [Figure 1A] Figures 1A-1E show the amino acid sequences of the α and β chains of the NY-ESO TCR mutant: Figure 1A shows the amino acid sequences of the TCR BC1 α and β chains. [Figure 1B] FIG. 1B shows the amino acid sequences of the α and β chains of TCR I53E. [Figure 1C] FIG. 1C shows the amino acid sequences of the α and β chains of TCR I53F. [Figure 1D] FIG. 1D shows the amino acid sequences of the α and β chains of TCR I53W. [Figure 1E] FIG. 1E shows the amino acid sequences of the α and β chains of TCR D55E. [Figure 2A] Figures 2A and 2B show flow cytometry analysis of CD4+ T cells transduced with various NY-ESO TCR mutants. Figure 2A shows flow cytometry TCRVb13.1 analysis of CD4+ T cells transduced with various NY-ESO TCR mutants. [Figure 2B]FIG. 2B shows flow cytometry HLA-A2.1 NY-ESO tetramer analysis of CD4+ T cells transduced with various NY-ESO TCR variants. [Figure 3A] Figures 3A and 3B show flow cytometry analysis of CD8+ T cells transduced with various NY-ESO TCR mutants. Figure 3A shows flow cytometry TCRVb13.1 analysis of CD8+ T cells transduced with various NY-ESO TCR mutants. [Figure 3B] FIG. 3B shows flow cytometry HLA-A2.1 NY-ESO tetramer analysis of CD8+ T cells transduced with various NY-ESO TCR mutants. [Figure 4A] Figures 4A and 4B show functional avidity analysis of NY-ESO-specific TCR transgenic CD4+ and CD8+ T cells. Figure 4A shows the production of interleukin-2 by CD4+ T cells transduced with various NY-ESO TCR variants. The target cells used were HLA-A2.1 positive cell line T2 loaded with serial 10-fold dilutions of NY-ESO157-165 peptide. "NT" refers to non-transduced cells. [Figure 4B] Figure 4B shows the production of interferon gamma (IFN-γ) by CD8+ T cells transduced with various NY-ESO TCR mutants. The target cells used were the HLA-A2.1 positive cell line T2 loaded with serial 10-fold dilutions of the NY-ESO157-165 peptide. "NT" refers to non-transduced cells. [Diagram 5] Figure 5 shows IL-2 production of NY-ESO TCR transgenic CD4+ cells. The target cells used were Me275 (a tumor cell line that naturally expresses HLA / A2-NY-ESO-1), A2008-A2 NLM and OVCAR5 NLM (cells engineered to express HLA-A2-NY-ESO-1), and NA8, A2008-A2 and OVCAR5 (tumor cell lines that do not express HLA / A2-NY-ESO-1). [Figure 6] Figure 6 shows IL-2 production of NY-ESO TCR transgenic CD4+ cells. The target cells used were the HLA.A2.1 positive, NY-ESO negative cell line NA8, and the HLA-A2.1 positive, NY-ESO positive cell lines Me275, A375, Saos-2 and U266. [Figure 7] Figure 7 shows IFN-γ production of NY-ESO TCR transgenic CD8+ cells. The target cells used were Me275 (a tumor cell line that naturally expresses HLA / A2-NY-ESO-1), A2008-A2 NLM and OVCAR5 NLM (cells engineered to express HLA-A2-NY-ESO-1), and NA8, A2008-A2 and OVCAR5 (tumor cell lines that do not express HLA / A2-NY-ESO-1). [Figure 8] Figure 8 shows IFN-γ production of NY-ESO TCR transgenic CD8+ cells. The target cells used were the HLA.A2.1 positive, NY-ESO negative cell line NA8, and the HLA-A2.1 positive, NY-ESO positive cell lines Me275, A375, Saos-2 and U266. [Figure 9] Figure 9 shows IL-2 production of NY-ESO TCR transgenic CD4+ cells (CD4+ cells expressing wild-type BC1 TCR, I53E, and I53F) in the context of peptide-pulsed target cells. Co-culture of CD4+ T cells with peptide-loaded T2 target cells for 24 hours. [Figure 10] Figure 10 shows IFN-γ production of NY-ESO TCR transgenic CD8+ cells (CD8+ cells expressing wild-type BC1 TCR, I53E, and I53F) in the context of peptide-pulsed target cells. 24 hour co-culture of CD4+ T cells with peptide-loaded target cells. [Figure 11]Figure 11 shows IFN-γ production of CD8+ T cells transduced with I53F, 153W, and 1G4LY TCR against various tumor cell lines. The target cells used were NY-ESO negative cell lines U87MG, OVCAR3, A431, A673, SKOV3, RD-ES, SK-N-AS, and HT-29, and NY-ESO positive cell line A375. [Figure 12] Figure 12 shows cell killing activity (measured by IncuCyte) by CD8+ cells expressing wild-type BC1 TCR, I53E TCR, and I53F TCR at various time points. "NT" refers to non-transduced cells. [Figure 13A] Figures 13A and 13B show in vivo evaluation of NY-ESO TCR transgenic cells using the WINN assay. Figure 13A shows a schematic diagram of the assay. HLA-A2+ / NY-ESO+ tumor cell line Me275 was mixed with NY-ESO transduced T cells and injected subcutaneously (sc) into the right flank of NSG mice. CD4+ and CD8+ percentages were 30% and 70%, respectively. Tumor size was measured twice a week. [Figure 13B] FIG. 13B shows the results of the assay. [Figure 14A] Figures 14A and 14B show the in vivo evaluation of NY-ESO TCR transgenic cells using NSG mice. Figure 14A shows a schematic diagram of the assay. On day 0 (d0), 0.5x106 Me275 cells were injected subcutaneously (sc) into the flank of NSG mice. When the tumor size was approximately 50-100 mm3, 10x106 NY-ESO-specific T cells were injected peritumorally on days 10 (d10) and 13 (d13). The percentages of CD4+ and CD8+ T cells were 30% and 70%, respectively. [Figure 14B] FIG. 14B shows the results of the assay. [Figure 15A]Figures 15A and 15B show the in vivo evaluation of NY-ESO TCR transgenic cells using NSG mice. Figure 15A shows a schematic of the assay. On day 0 (d0), 5x106 Me275 cells were injected subcutaneously (sc) into the flank of NSG mice. When tumors reached approximately 50-100 mm3 in size, 10x106 NY-ESO-specific T cells were injected intravenously (iv) on days 10 (d10) and 13 (d13). The percentages of CD4+ and CD8+ T cells were 30% and 70%, respectively. [Figure 15B] FIG. 15B shows the results of the assay. [Figure 16A] Figures 16A and 16B show the in vivo evaluation of NY-ESO TCR transgenic cells using NSG mice. Figure 16A shows a schematic of the assay. On day 0 (d0), 5x106 Me275 cells were injected subcutaneously (sc) into the flank of NSG mice. When tumors reached approximately 50-100 mm3 in size, various numbers of NY-ESO-specific T cells were injected intravenously (iv) on days 10 (d10) and 13 (d13). The percentages of CD4+ and CD8+ T cells were 30% and 70%, respectively. [Figure 16B] FIG. 16B shows the results of the assay. [Figure 17A] Figures 17A-17C show that human T cells can be efficiently co-engineered to stably express NY-ESO-1 TCR and secrete mouse GM-CSF. Figure 17A shows a schematic of the retroviral mouse GM-CSF construct and the lentiviral NY-ESO-1 TCR construct. [Figure 17B] Figure 17B shows representative contour plots of human CD8+ and CD4+ T cells 7 days after viral transduction. Expression of NY-ESO-1 TCR was confirmed using HLA-A2 restricted NY-ESO-1157-165 tetramer, while expression of mouse GM-CSF was tracked by detection of surface Thy1.1 reporter protein. "NT" refers to non-transduced T cells. [Figure 17C]Figure 17C shows that secreted mouse GM-CSF can be detected by ELISA in the supernatants of transduced CD8+ T cell cultures. "NT" refers to non-transduced T cells. [Figure 18A] Figures 18A and 18B show that secreted mouse GM-CSF does not affect the activity of human T cells. Detection of IFNγ secreted by NY-ESO-1 TCR engineered T cells is based on the recognition of HLA-A2+NY-ESO-1+ tumor cells. There is no effect of mouse GM-CSF on IFNγ levels. Figures 18A and 18B show the same experiment. "NT" stands for non-transformed. Statistical significance was determined by one-way ANOVA. ****p<0.0001. [Figure 18B] Figure 18B shows that NY-ESO-1 TCR engineered T cells can readily kill HLA-A2+NY-ESO-1+ tumor cells, and their cytotoxic activity is not affected by mouse GM-CSF. "NT" stands for non-transformed. Statistical significance was determined by one-way ANOVA. ****p<0.0001. [Figure 19A] Figures 19A-19C demonstrate that T cell-derived murine GM-CSF secreted in the tumor microenvironment can significantly enhance control of tumor growth by NY-ESO-1-specific T cells in the NSG human melanoma xenograft mouse model. Figure 19A shows tumor growth in mice by Winn assay. Human NY-ESO-1 TCR-expressing T cells co-engineered to secrete murine GM-CSF can effectively delay engraftment and establishment of Me275 melanoma cells compared to control T cells expressing only NY-ESO-1 TCR. "NT" refers to non-transduced T cells. Statistical significance of in vivo responses was determined by one-way ANOVA or unpaired t-tests for individual time points. ***p<0.001. [Figure 19B]Figure 19B shows the survival rate of mice by Winn assay. 40% of mice treated with NY-ESO-1 TCR-expressing mouse GM-CSF-secreting T cells were tumor-free. Statistical significance of survival curves was determined by Mantel-Cox log-rank test. **p<0.01. [Figure 19C] FIG. 19C shows that adoptive transfer of 2×107 NY-ESO-1 TCR-expressing mouse GM-CSF-secreting T cells showed better tumor control of established Me275 tumors compared to NY-ESO-1 TCR alone. "NT" refers to non-transduced T cells. Statistical significance of in vivo responses was determined by one-way ANOVA or unpaired t-test for individual time points. ***p<0.001. [Figure 20A] FIG. 20A provides an exemplary protein and nucleotide sequence for mouse GM-CSF. [Figure 20B] FIG. 20B provides an exemplary protein and nucleotide sequence for human GM-CSF. [Figure 21] Figure 21 shows that primary human T cells can be effectively transduced to express exogenous phosphodiesterase 4B2 (PDE4B2) and co-transduced to express exogenous PDE4B2 and NY-ESO-1 TCR variant β-I53F. Flow cytometry results are shown for expression of exogenous PDE4B2 or NY-ESO-1 TCR variant β-I53F on day 7 in untransduced, PDE4B2-transduced, and PDE4B2&NY-ESO-1 TCR-transduced CD4+ and CD8+ T cells. [Figure 22]Figure 22 shows that overexpression of PDE4B2 prevents the accumulation of intracellular cAMP. Shown are intracellular cAMP levels in resting CD4 T cells transduced with eGFP (gray line) or PDE4B2 (black line) upon exposure to forskolin (Fsk) or PGE2 for 1 h. After treatment, T cells were lysed and cAMP levels were quantified by ELISA. Results shown are from a single experiment in which all cAMP measurements were performed in triplicate. Error bars represent standard deviation (SD). [Figure 23A] Figures 23A and 23B show Th-1 cytokine production by T cells transduced with PDE4B2. Figure 23A shows the production of IFNγ or TNF-α by resting CD4 T cells transduced with eGFP or PDE4B2, as determined by intracellular cytokine staining (ICS) 7 hours after stimulation with (solid line: stimulated) or without (dotted line: unstimulated) plate-bound αCD3 and soluble αCD28 in the presence of forskolin (Fsk) or PGE2. The percentage of IFNγ+ or TNF-α+ cells among live CD45+eGFP+ lymphocytes (gray line) or live CD45+exogenous PDE4B2+ lymphocytes (black line) is shown, as well as representative plots. Results shown are from a single experiment performed in duplicate for all conditions. Error bars represent SD. [Figure 23B] Figure 23B shows the secretion of IFNγ by resting CD8 T cells transduced with eGFP, PDE4B2, eGFP&NY-ESO-1 TCR, or PDE4B2-&NY-ESO-1 TCR in response to 48 hours of co-culture with NY-ESO-1-presenting melanoma cells A375 in the presence of PGE2 or forskolin, as measured by ELISA. IFNγ concentrations in the co-culture supernatants are shown. Error bars represent SD. [Figure 24]Figure 24 shows that overexpression of PDE4B2 promotes proliferation under conditions inducing intracellular cAMP accumulation. The proliferative capacity of CD4 T cells transduced with eGFP (grey) or PDE4B2 (black) was determined by BrDU incorporation assay. T cells were restimulated for 48 h in the presence of PGE2 or forskolin (Fsk) with (solid line: stimulated) or without (dotted line: unstimulated) plate-bound αCD3. The percentage of BrDU+ cells among live CD45+ lymphocytes (left graph), the BrDU MFI of these populations (middle graph) as well as representative plots are shown. Results shown are from a single experiment performed in duplicate for all conditions. Error bars represent SD. [Diagram 25] Figure 25 shows that overexpression of PDE4B2 promotes cytotoxicity in the presence of PGE2. The ability of resting CD4 (left graph) or CD8 (left graph) T cells transduced with eGFP (open triangles), PDE4B2 (open circles), eGFP&NY-ESO-1 TCR (closed triangles), or PDE4B2&NY-ESO-1 TCR (closed circles) to suppress the spread of NY-ESO-1-presenting melanoma cells A375 in the presence (black line) or absence (gray line) of PGE2 was assessed by IncuCyte. The values ​​of red object area corresponding to mm2 occupied by the nuclei of A375 cells per well are represented. The results shown are from a single experiment in which all conditions were performed in triplicate. Four different planes per well were incorporated in the analysis. Error bars represent SD. [Figure 26] FIG. 26 provides exemplary protein sequences for human and mouse PDE4B2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] The present invention generally relates to the cancer antigen NY-ESO-1 157-165 Provided is a modified T cell receptor (TCR) with improved binding affinity to an epitope.

[0083] The present invention also provides related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and cell populations, including but not limited to engineered cells. Additionally, the present invention provides engineered cells comprising the modified TCR. Further provided by the present invention are pharmaceutical compositions related to the modified TCRs and cells of the present invention.

[0084] Additionally, the present invention provides methods for genetic engineering of lymphoid cells that express modified TCRs. Further provided by the present invention are methods for genetic engineering of lymphoid cells that express modified TCRs, as well as additional receptors (chimeric antigen receptors, or cancer antigen NY-ESO-1 157-165 The present invention relates to a method for the production of a cellular endothelial cell lineage comprising administering to a subject a therapeutic agent that is capable of cellular function by ...

[0085] Also provided by the invention is a method of treating cancer in a subject, comprising administering to a subject suffering from such cancer an effective amount of lymphoid cells presenting a modified TCR of the invention.Also provided by the invention is a method of stimulating or enhancing an immune response in a mammal, comprising administering to the mammal an effective amount of the genetically engineered lymphoid cells of the invention.

[0086] In one aspect, provided herein are engineered lymphoid lineage cells expressing modified TCR and cytokines and / or chemokines from the colony-stimulating factor cytokine / chemokine family. Members of the colony-stimulating factor cytokine / chemokine family are known to act on most of these myeloid cells and regulate their activity (Ushach, I. and A. Zlotnik, Journal of Leukocyte Biology, 2016. 100: p. 481-489). Specifically, granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to play an immunostimulatory role in the biology of monocytes, macrophages, granulocytes, and dendritic cells (DCs). GM-CSF was initially described as a hematopoietic growth factor, but has emerged as a key immune regulator in many pathological conditions, including autoimmune diseases and cancer. GM-CSF is secreted by cells of both hematopoietic (i.e., macrophages, natural killer (NK) cells, activated T cells) and non-hematopoietic (e.g., endothelial and fibroblast) origin. Although undetectable in the serum of healthy individuals, GM-CSF levels rapidly increase during inflammation (Becher, B., S. Tugues, and M. Greter, Immunity, 2016. 45: p. 963-973). GM-CSF exerts its pleiotropic effects through its cognate receptor, the GM-CSF receptor (GM-CSF-R), which is found primarily in cells of the monocyte / macrophage and granulocytic lineages, as well as DCs. Upon ligand binding, the GM-CSF receptor transmits signals related to cell survival, proliferation, differentiation, and activation (Hercus, TR, et al., Blood. 2009. 114: p. 1289-1298). By controlling the fate of such professional antigen-presenting cells (APCs), GM-CSF can indirectly regulate the activity of T cells, thus acting as a bridge between adaptive and innate immunity (Shi, Y., et al., Cell Res. 2006. 16(2):126-33).

[0087] In cancer treatment, GM-CSF has played a central role in supportive care of cancer patients as well as accelerating and enhancing recovery of the bone marrow compartment of the immune system after chemotherapy and / or stem cell transplantation regimens (Arellano, M. and S. Lonial, Biologics. 2008. 2(1):13-27). Moreover, due to its pivotal role in DC development and differentiation, GM-CSF is at the core of DC-based immunotherapy, either in the form of GM-CSF-secreting cancer vaccines that activate DCs (Gupta, R. and LA Emens. Discovery medicine, 2010. 10(50): p. 52-60) or adoptive transfer of GM-CSF-activated / skewed DCs as primary immunotherapy (Mookerjee, A., M. Graciotti, and L. Kandalaft, BioImpacts: BI, 2018. 8(3): p. 211-221). DCs have also emerged as key mediators of robust antitumor responses in preclinical studies using tumors forcibly overexpressing GM-CSF (Shi, FS, et al., Cancer Gene Ther, 1999. 6(1): p. 81-8) or after administration of soluble GM-CSF in cancer patients (Nasi, ML, et al., Cytokines Cell Mol Ther, 1999. 5(3): p. 139-44). Despite promising results, the use of GM-CSF as a single adjuvant therapy in the clinic has proven to be rather insufficient (Lawson, DH, et al., J Clin Oncol, 2015. 33(34): p. 4066-76) and has been limited by the appearance of dose-related toxicity (Antman, KS, et al., N Engl J Med, 1988. 319(10): p. 593-8). Interestingly, another series of studies showed that tumor-derived GM-CSF upregulates CD11b in the tumor microenvironment. + Gr-1 +We reveal a GM-CSF-driven immunosuppressive mechanism of antitumor response that is responsible for immune attraction of bone marrow-derived immunosuppressive cells (MDSCs), which in turn promotes tumor evasion (Pylayeva-Gupta, Y., et al., Cancer Cell, 2012. 21: p. 836-847).

[0088] To maximize the benefits of adoptive T cell transfer strategies and exploit the immunomodulatory anti-cancer properties of GM-CSF while avoiding unwanted side effects and off-target toxicity, a combinatorial approach was developed in which T cells were genetically co-engineered to ectopically express a high affinity NY-ESO-1 specific TCR and GM-CSF. One embodiment of T cells expressing NY-ESO-1 specific TCR and GM-CSF is illustrated in Example 6 below. Human T cells efficiently secrete fully functional soluble GM-CSF without affecting proliferative capacity or anti-tumor activity, and can upregulate human NY-ESO-1 in vivo. + It has been shown to induce a potent antitumor response against melanoma tumors.

[0089] In another aspect, provided herein is a genetically engineered lymphoid cell expressing a modified TCR and a phosphodiesterase. In some embodiments, the phosphodiesterase is phosphodiesterase 4B2 (PDE4B2). Cyclic AMP (cAMP), an intracellular second messenger, functions as a potent immunosuppressive signaling molecule in T cells and is involved in the upregulation of prostaglandin E2 (PGE 2 ), adenosine, and regulatory T cell function. Overexpression of phosphodiesterase reduces cAMP signaling and upregulates PGE 2It is possible to enhance the resistance of anti-tumor T cells to the inhibition of suppressive factors such as. See U.S. Patent No. 9,976,121 and Schmetterer, KG., Front. Immunol., 30 July 2019, which are incorporated by reference in their entirety for all purposes.

[0090] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0091] As used herein, the term "antigen" is a molecule and / or substance capable of specifically binding to an antibody or generating peptide fragments recognized by a T-cell receptor and / or capable of inducing an immune response. An antigen may contain one or more "epitopes". In certain embodiments, an antigen has several epitopes. An epitope is recognized by an antibody or lymphocyte in the context of an MHC molecule. In various embodiments, the antigen is NY-ESO-1. In various embodiments, the epitope is NY-ESO-1. 157-165 It is.

[0092] As used herein, the term "tumor antigen" or "cancer antigen", used interchangeably, is broadly defined as an antigen that is specifically expressed by or associated with a tumor or cancer cell, such as overexpressed or aberrantly expressed antigens, antigens produced by oncogenic viruses, carcinoembryonic antigens, modified cell surface glycolipid and glycoprotein antigens, cell type-specific differentiation antigens, etc. A tumor antigen present on the surface of a cancer cell is an antigen that is not present on the surface of an individual's normal somatic cells, i.e., the antigen is exposed to the immune system in the cancer cell but not in the normal somatic cells. An antigen may be expressed on the cell surface of a tumor cell, where it is recognized by components of the humoral immune system, such as B lymphocytes (B cells). Intracellular tumor antigens are processed into short peptide fragments that form complexes with major histocompatibility complex (MHC) molecules and are presented on the cell surface of cancer cells, where they are recognized by T lymphocytes (T cells) or T cell receptors (TCR) of natural killer cells. Preferably, a tumor antigen is one that is not expressed by normal cells, or at least not to the same level as tumor cells.

[0093] The term "functional fragment" as used herein refers to a fragment of a polypeptide or protein, or a polynucleotide encoding the polypeptide or protein, that retains at least one function of the full-length polypeptide or protein. A functional fragment may comprise an amino acid sequence of at least 5 contiguous amino acid residues, at least 6 contiguous amino acid residues, at least 7 contiguous amino acid residues, at least 8 contiguous amino acid residues, at least 9 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 11 contiguous amino acid residues, at least 12 contiguous amino acid residues, at least 13 contiguous amino acid residues, at least 14 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of the full-length polypeptide or protein. A functional fragment of a polypeptide or protein may retain one, two, three, four, five, or more functions of a full-length protein or polypeptide. For example, a functional fragment of a TCR that immunospecifically binds to a particular antigen (or epitope) may retain the ability to immunospecifically bind to that antigen (or epitope). In some embodiments, a functional fragment of a TCR comprises one or more complementarity determining regions (CDRs) of the α and / or β chain of the TCR. In some embodiments, a functional fragment of a TCR comprises the α and / or β chain of the TCR.

[0094] The term "variant" as used herein refers to a modified polypeptide, protein, or polynucleotide that has substantial or significant sequence identity or similarity with a wild-type polypeptide, protein, or polynucleotide. A variant may retain the same biological activity or may have an altered (e.g., improved, reduced, or eliminated) biological activity compared to the wild-type polypeptide, protein, or polynucleotide of the variant. A variant may include an insertion, deletion, or substitution of at least one amino acid residue or nucleotide.

[0095] The term "antigen binding protein" refers to any protein that binds to an antigen or polypeptide of interest or a fragment thereof. The protein can be either naturally derived or synthetic. Examples of antigen binding proteins include antibodies; polypeptides or fragments derived from antibodies, such as single chain variable fragments (scFv), Fab, Fab', F(ab'). 2 , and Fv fragments; polypeptides derived from T cell receptors, such as, for example, TCR variable domains; secreted factors (e.g., cytokines, growth factors) that can be artificially fused to signaling domains (e.g., "zytokines"); and any ligand or receptor fragment that binds to an antigen of interest (e.g., CD27, NKG2D). Combinatorial libraries can also be used to identify peptides that bind with high affinity to therapeutic targets.

[0096] As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, and antibody fragments (e.g., single chain antibodies, Fab fragments, Fv fragments, single chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2' fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies, triabodies, decabodies, and other domain antibodies (e.g., Holt, LJ, et al., Trends Biotechnol. (2003), 21, 11, 484-490)). The term "antibody" also refers to covalent diabodies, such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910. Antibodies useful in the methods described herein include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0097] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell so as to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthetic RNA and DNA molecules, transposons, phages, viruses, and the like. In certain embodiments, the vector is a viral vector, such as, but not limited to, an adenovirus, an adeno-associated virus, an alphavirus, a herpes virus, a lentivirus, a retrovirus, or a vaccinia vector.

[0098] The term "regulatory element" refers to any cis-acting genetic element that controls some aspect of the expression of a nucleic acid sequence. In some embodiments, the term "promoter" essentially includes the minimal sequence required to initiate transcription. In some embodiments, the term "promoter" includes sequences that initiate transcription, and also includes sequences that can upregulate or downregulate transcription, commonly referred to as "enhancer elements" and "repressor elements," respectively. In some embodiments, the promoter is a lymphocyte-specific promoter.

[0099] The term "operably linked" as used herein means that a nucleotide sequence is placed in a functional relationship with another nucleotide sequence. For example, when a coding sequence is operably linked to a promoter sequence, this generally means that the promoter can promote the transcription of the coding sequence. Operable linked means that the linked DNA sequences are usually adjacent, and when necessary to link two protein coding regions, adjacent and in reading frame. However, because enhancers can function when several kilobases away from the promoter, and intron sequences can be of variable length, some nucleotide sequences may be operably linked but not adjacent.

[0100] The terms "T cell" and "T lymphocyte" are interchangeable and are used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, e.g., T helper 1 (Thl) or T helper 2 (Th2) cells. T cells can be T helper cells (HTl; CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4 + CD8 + The population of T cells may be T cells, or other subsets of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells. Also included is "NKT cells," which refers to a specialized population of T cells that express the semi-invariant αβ T cell receptor, but also express various molecular markers typically associated with NK cells, such as NK1.1. NKT cells include NK1.1 + and NK1.1 - , and CD4 + , CD4 - , CD8 + , and CD8 - These include NKT cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CDId. NKT cells can have either protective or harmful effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. They also include "gamma-delta T cells (γδ T cells)", which refers to a special population that is a small subset of T cells that have a separate TCR on their surface; unlike the majority of T cells whose TCR is composed of two glycoprotein chains called the α-TCR chain and the β-TCR chain, the TCR on γδ T cells is composed of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immune regulation, are an important source of IL-17, and have robust CD8 + They are known to induce cytotoxic T cell responses. They also include "regulatory T cells" or "Tregs," which refer to T cells that suppress aberrant or excessive immune responses and play a role in immune tolerance. Treg cells are typically CD4+ cells that express the transcription factor Foxp3. + T cells, IL-10-producing CD4 + The T cells may also include transcription factor Foxp3 negative regulatory T cells.

[0101] The terms "natural killer cells" and "NK cells" are used interchangeably and are used synonymously herein. As used herein, NK cells are cells that express the CD16 + CD56 + and / or CD57 + TCR - NK refers to a differentiated lymphocyte with a phenotype characterized by the ability to bind to and kill cells that do not express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, to bind to and kill tumor cells or other diseased cells that express ligands for NK activating receptors, and to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0102] As used herein, "exogenous" refers to any molecule that is not derived from a particular cell as found in nature. An exogenous molecule can be expressed from a nucleic acid molecule that has been introduced into a host cell by artificial or natural means.

[0103] The terms "treat" or "treatment" of a condition, disorder or state include: (1) preventing, delaying or reducing the occurrence and / or likelihood of the appearance of at least one clinical or asymptomatic symptom of the condition, disorder or state in a subject who may be suffering from or predisposed to the condition, disorder or state, but who has not yet experienced or displayed a clinical or asymptomatic symptom of the condition, disorder or state; or (2) inhibiting the condition, disorder or state, i.e., arresting, reducing or delaying the occurrence of the disease or its recurrence, or at least one clinical or asymptomatic symptom thereof; or (3) relieving the disease, i.e., causing regression of the condition, disorder or state, or at least one clinical or asymptomatic symptom thereof. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0104] The term "effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would have been effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug used, the method of administration, etc.

[0105] The phrase "pharmacologically acceptable" as used in connection with the compositions described herein refers to molecular species and other components of such compositions that are physiologically tolerable and do not normally produce undesirable reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmacologically acceptable" means approved by a regulatory agency of the U.S. Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more specifically, humans.

[0106] The terms "patient," "individual," "subject," and "animal" are used interchangeably herein and refer to mammals, including but not limited to humans and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models. In a preferred embodiment, the subject is a human.

[0107] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solutions, saline solutions, and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of a binder (for compressed tablets), a glidant, an encapsulating agent, a flavoring agent, and a coloring agent. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0108] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0109] The term "about" or "approximately" includes being within a statistically meaningful range of values. Such ranges may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the term "about" or "approximately" depends on the particular system being tested and can be readily understood by one of ordinary skill in the art.

[0110] The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each separate value and each endpoint within that range, unless otherwise stated herein, and each separate value and endpoint is incorporated into the specification as if each separate value and endpoint were individually set forth herein.

[0111] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are available, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ.

[0112] Engineered T-cell receptors In one aspect, the present invention provides a modified T cell receptor (TCR) or a functional fragment thereof, wherein the modified TCR comprises a single amino acid substitution within complementarity determining region (CDR) 2 of the β chain of the modified TCR compared to CDR2 of the β chain of the unsubstituted wild type (WT) TCR.

[0113] In one embodiment, the invention provides a modified T cell receptor (TCR) comprising an amino acid sequence having only a single amino acid substitution located in the complementarity determining region (CDR) 2 of the beta chain of the TCR in the amino acid sequence of a wild type (WT) TCR, wherein the modified TCR retains the antigen specificity of the WT TCR and has a higher binding affinity for a cancer antigen compared to the WT TCR, and comprises the amino acid sequence of SEQ ID NO: 1 except for the single amino acid substitution located in CDR2 of SEQ ID NO: 1.

[0114] In certain embodiments, the binding affinity of the modified TCR to a cancer antigen is about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or more than 100-fold higher than the binding affinity of the WT TCR to a cancer antigen.

[0115] Dissociation constant (K D ) can be used to evaluate the binding affinity of the modified TCR to the cancer antigen. In certain embodiments, the dissociation constant (K D ) is between about 0.1 and about 10 μM. As a non-limiting example, the dissociation constant (K D ) can be about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.7 μM, about 2 μM, about 2.2 μM, about 2.5 μM, about 2.7 μM, about 3 μM, about 3.2 μM, about 3.5 μM, about 3.7 μM, about 3.9 μM, about 4 μM, about 4.2 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM.

[0116] In one embodiment, the invention provides a modified T cell receptor (TCR) that comprises the amino acid sequence of a wild type (WT) TCR except for a single amino acid substitution located in the complementarity determining region (CDR) 2 of the β chain of the TCR, wherein the modified TCR retains the antigen specificity of the WT TCR as compared to the WT TCR and is capable of targeting the cancer antigen NY-ESO-1. 157-165 It has a higher binding affinity to the epitope (SEQ ID NO:8) and comprises the amino acid sequence of SEQ ID NO:1 but with a single amino acid substitution located in CDR2 of SEQ ID NO:1.

[0117] In certain embodiments, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 2- to about 100-fold higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. As non-limiting examples, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, or more than 100-fold higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In various embodiments, the epitope is NY-ESO-1. 157-165 It is.

[0118] In one embodiment, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 5 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In one embodiment, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 10 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In one embodiment, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 25 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In one embodiment, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 40 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In one embodiment, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 40 to about 60 times higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In one embodiment, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 40 to about 50 times higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In one embodiment, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 50 times higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. In various embodiments, the epitope is NY-ESO-1. 157-165 It is.

[0119] Dissociation constant (K D ) can be used to evaluate the binding affinity of the modified TCR to the cancer antigen NY-ESO-1. In certain embodiments, the dissociation constant (K D ) is between about 0.1 and about 10 μM. As a non-limiting example, the dissociation constant (K D) can be about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.7 μM, about 2 μM, about 2.2 μM, about 2.5 μM, about 2.7 μM, about 3 μM, about 3.2 μM, about 3.5 μM, about 3.7 μM, about 3.9 μM, about 4 μM, about 4.2 μM, about 4.5 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM, or about 10 μM. In various embodiments, the epitope is NY-ESO-1 157-165 It is.

[0120] In one embodiment, the dissociation constant (K D In one embodiment, the dissociation constant (K D ) is between about 0.3 and about 2 μM. In one embodiment, the dissociation constant (K D In one embodiment, the dissociation constant (K D In one embodiment, the dissociation constant (K D In one embodiment, the dissociation constant (K D In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is about 3.9 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is about 3.9 μM. D ) is about 3.89 μM. In various embodiments, the epitope is NY-ESO-1 157-165 It is.

[0121] In certain embodiments, the modified TCR comprises a single amino acid substitution located at residue 53 of CDR2 of the β chain of the TCR. The single amino acid substitution may include, but is not limited to, I53E, I53F and I53W.

[0122] In certain embodiments, the modified TCR comprises a single amino acid substitution located at residue 55 of CDR2 of the β chain of the TCR. The single amino acid substitution may include, but is not limited to, D55E.

[0123] In certain embodiments, the modified TCR comprises a single amino acid substitution located at residue 50, 51, or 52 of CDR2 of the β chain of the TCR. The single amino acid substitution may include, but is not limited to, G50V, G50A, A51D, A51E, or G52Q.

[0124] In certain embodiments, the modified TCR beta chain sequence comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an unsubstituted WT TCR beta chain or functional fragment thereof outside the CDR2 region of the modified TCR beta chain or functional fragment thereof. In one embodiment, the modified TCR beta chain or functional fragment thereof comprises an amino acid sequence with a single amino acid substitution in the CDR2 region in the amino acid sequence of the unsubstituted WT TCR beta chain or functional fragment thereof. In one embodiment, the unsubstituted WT TCR beta chain comprises the amino acid sequence of SEQ ID NO:1.

[0125] In certain embodiments, the modified TCR comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 2-5. In one embodiment, the modified TCR comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the modified TCR comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified TCR comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In one embodiment, the modified TCR comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In any of the above embodiments, the modified TCR comprises a single amino acid substitution within CDR2 of the β chain compared to the β chain of the unsubstituted WT TCR (e.g. as set forth in SEQ ID NO: 1).

[0126] In certain embodiments, the modified TCR comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 5. In one embodiment, the modified TCR comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, the modified TCR comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified TCR comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the modified TCR comprises the amino acid sequence of SEQ ID NO: 5.

[0127] In certain embodiments, the modified TCR comprises an amino acid sequence encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs: 11-14. In one embodiment, the modified TCR comprises an amino acid sequence encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the modified TCR comprises an amino acid sequence encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 12. In one embodiment, the modified TCR comprises an amino acid sequence encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 13.In one embodiment, the modified TCR comprises an amino acid sequence encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 14. In any of the above embodiments, the modified TCR comprises a single amino acid substitution within CDR2 of the β chain compared to the β chain of the unsubstituted WT TCR (e.g. as set forth in SEQ ID NO: 1).

[0128] In certain embodiments, the modified TCR comprises an amino acid sequence encoded by a nucleotide sequence selected from any one of SEQ ID NOs: 11-14. In one embodiment, the modified TCR comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the modified TCR comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12. In one embodiment, the modified TCR comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 13. In one embodiment, the modified TCR comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 14.

[0129] In one aspect, the invention provides an engineered T cell receptor (TCR) comprising an α chain comprising the amino acid sequence of SEQ ID NO:7, and a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs:2-5. In one embodiment, the engineered T cell receptor (TCR) comprises an α chain comprising the amino acid sequence of SEQ ID NO:7, and a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the modified T cell receptor (TCR) comprises an α chain comprising the amino acid sequence of SEQ ID NO: 7, and a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified T cell receptor (TCR) comprises an α chain comprising the amino acid sequence of SEQ ID NO: 7, and a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 7, and a beta chain comprising an amino acid sequence having 75% sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of SEQ ID NO: 5. In any of the above embodiments, the modified TCR comprises a single amino acid substitution within CDR2 of the beta chain compared to the beta chain of an unsubstituted WT TCR (e.g., as set forth in SEQ ID NO: 1).

[0130] In one aspect, the present invention provides a modified T cell receptor (TCR) comprising an α chain comprising the amino acid sequence of SEQ ID NO: 7, and a β chain comprising the amino acid sequence of any one of SEQ ID NOs: 2-5. In one embodiment, the modified T cell receptor (TCR) comprises an α chain comprising the amino acid sequence of SEQ ID NO: 7, and a β chain comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the modified T cell receptor (TCR) comprises an α chain comprising the amino acid sequence of SEQ ID NO: 7, and a β chain comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified T cell receptor (TCR) comprises an α chain comprising the amino acid sequence of SEQ ID NO: 7, and a β chain comprising the amino acid sequence of SEQ ID NO: 4. In one embodiment, the modified T cell receptor (TCR) comprises an α chain comprising the amino acid sequence of SEQ ID NO: 7, and a β chain comprising the amino acid sequence of SEQ ID NO: 5.

[0131] In one aspect, the invention provides a modified T cell receptor (TCR) comprising an α chain encoded by the nucleotide sequence of SEQ ID NO:9 and a β chain encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleotide sequence selected from any one of SEQ ID NOs:11-14. In one embodiment, the modified T cell receptor (TCR) comprises an α chain encoded by the nucleotide sequence of SEQ ID NO:9 and a β chain encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleotide sequence of sequence SEQ ID NO:11. In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain encoded by the nucleotide sequence of SEQ ID NO:9 and a beta chain encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:12. In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain encoded by the nucleotide sequence of SEQ ID NO:9 and a beta chain encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:13.In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain encoded by the nucleotide sequence of SEQ ID NO:9, and a beta chain encoded by a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:14. In any of the above embodiments, the modified TCR comprises a single amino acid substitution within CDR2 of the beta chain compared to the beta chain of an unsubstituted WT TCR (e.g., as set forth in SEQ ID NO:1).

[0132] In one aspect, the present invention provides a modified T cell receptor (TCR) comprising an α chain encoded by the nucleotide sequence of SEQ ID NO:9, and a β chain encoded by the nucleotide sequence of any of SEQ ID NOs:11-14. In one embodiment, the modified T cell receptor (TCR) comprises an α chain encoded by the nucleotide sequence of SEQ ID NO:9, and a β chain encoded by the nucleotide sequence of SEQ ID NO:11. In one embodiment, the modified T cell receptor (TCR) comprises an α chain encoded by the nucleotide sequence of SEQ ID NO:9, and a β chain encoded by the nucleotide sequence of SEQ ID NO:12. In one embodiment, the modified T cell receptor (TCR) comprises an α chain encoded by the nucleotide sequence of SEQ ID NO:9, and a β chain encoded by the nucleotide sequence of SEQ ID NO:13. In one embodiment, the modified T cell receptor (TCR) comprises an α chain encoded by the nucleotide sequence of SEQ ID NO:9, and a β chain encoded by the nucleotide sequence of SEQ ID NO:14.

[0133] The invention also provides related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and populations of cells, including, but not limited to, genetically engineered cells.

[0134] In one aspect, the invention provides an isolated polypeptide comprising a functional portion of a modified TCR as described herein, the functional portion comprising the variable regions of the α and β chains of the TCR, the functional portion comprising an amino acid substitution. In a particular embodiment, the isolated polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2-5.

[0135] In one aspect, the invention provides an isolated polypeptide comprising a functional portion of a modified TCR as described herein, the functional portion further comprising the constant regions of the α and β chains of the TCR. In certain embodiments, the constant regions are of human origin. In other embodiments, the constant regions are of murine origin.

[0136] In one aspect, the invention provides a nucleic acid sequence encoding any of the amino acid sequences of the modified TCRs described herein.

[0137] It will be understood that conservative amino acid substitutions can be introduced into any of the polypeptides described herein to achieve a polypeptide having, for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a reference sequence, and preferably retaining the activity of that sequence. Conservative amino acid substitutions, as known in the art and referred to herein, include replacing an amino acid in a protein with an amino acid having a similar side chain, for example, in terms of structure, size, and / or chemical properties. For example, amino acids within each of the following groups may be interchanged with other amino acids within the same group: amino acids having aliphatic side chains, including glycine, alanine, valine, leucine, and isoleucine; amino acids having non-aromatic hydroxyl-containing side chains, such as serine and threonine; amino acids having acidic side chains, such as aspartic acid and glutamic acid; amino acids having amide side chains, including glutamine and asparagine; basic amino acids, including lysine, arginine, and histidine; amino acids having aromatic ring side chains, including phenylalanine, tyrosine, and tryptophan; and amino acids having sulfur-containing side chains, including cysteine ​​and methionine. Additionally, amino acids having acidic side chains, such as aspartic acid and glutamic acid, are contemplated herein as interchangeable with amino acids having amide side chains, such as asparagine and glutamine.

[0138] Examples of sequences useful in various embodiments of the present invention are provided below.

[0139] BC1 WT β chain MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG * (SEQ ID NO:1)

[0140] I53E β chain MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGETDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG * (SEQ ID NO:2)

[0141] I53F β chain MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGFTDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG * (SEQ ID NO:3)

[0142] I53W β chain MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGWTDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG * (SEQ ID NO:4)

[0143] D55E β chain MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITEQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG * (SEQ ID NO:5)

[0144] A97L β chain MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGLAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISH TQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG * (SEQ ID NO:6)

[0145] BC1 WT α chain METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQTGGSYIPTFGRGTSLIVHPYIQN PDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS * (SEQ ID NO:7)

[0146] NY-ESO-1 157-165 peptide SLLMWITQC (SEQ ID NO: 8)

[0147] BC1 WT α chain ATGGAAACCCTGCTGGGCCTGCTGATCCTGTGGCTGCAGCTGCAGTGGGTGTCCAGCAAGCAGGAAGTGACCCAGATCCCTGCCGCCCTGAGCGTGCCCGAGGGCGAGAACCTGGTGCTGAACTGCAGCTTCACCGACAGCGCCATCTACAACCTGCAGTGGTTCCGGCAGGACCCCGGCAAGGGCCTGACCAGCCTGCTGCTGATCCAGAGCAGCCAGCGGGAGCAGACCAGCGGCAGGCTGAACGCCAGCCTGGACAAGAGCAGCGGCAGAAGCACCCTGTACATCGCCGCCAGCCAGCCCGGCGACTCCGCCACCTACCTGTGCGCCGTGCGGCCTCAGACCGGCGGCAGCTACATCCCCACCTTCGGCAGGGGCACCAGCCTGATCGTGCACCCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGTCCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTTTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATTCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGT(SEQ ID NO:9)

[0148] BC1 WT β chain ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCATCACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO: 10)

[0149] I53E β chain ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCGAGACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO: 11)

[0150] I53F β chain ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCTTCACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO: 12)

[0151] I53W β chain ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCTGGACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO: 13)

[0152] D55E β chain ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCATCACCGAGCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO: 14)

[0153] Genetic manipulation of cells In one aspect, the invention provides a method of genetic engineering of lymphoid cells to express a modified TCR as described herein. In one embodiment, lymphoid cells are genetically engineered to express a modified TCR comprising an amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2. In one embodiment, lymphoid cells are genetically engineered to express a modified TCR comprising an amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3. In one embodiment, the lymphoid cells are engineered to express a modified TCR comprising an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In one embodiment, the lymphoid cells are engineered to express a modified TCR comprising an amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. The lymphoid cells are obtained from PBMCs, tumor-draining lymph nodes, or tumor infiltrates. In certain embodiments, the genetically engineered lymphoid cells are further engineered to express one or more exogenous molecules.In certain embodiments, the genetically engineered lymphoid cells are further engineered to express one or more cell surface receptors, examples of which include chimeric antigen receptors and the cancer antigen NY-ESO-1. 157-165 In certain embodiments, the engineered lymphoid cells are further engineered to express one or more soluble proteins. Examples of soluble proteins include, but are not limited to, cytokines, chemokines, growth factors, soluble receptors, ligands, antibodies, antibody fragments, and antigen-binding domains. In certain embodiments, the engineered lymphoid cells are further engineered to express one or more additional receptors and soluble proteins.

[0154] In one aspect, the invention provides an engineered lymphoid cell expressing a modified TCR as described herein. In one embodiment, the engineered lymphoid cell expresses a modified TCR comprising an amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2. In one embodiment, the engineered lymphoid cell expresses a modified TCR comprising an amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3. In one embodiment, the engineered lymphoid cell expresses a modified TCR comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In one embodiment, the engineered lymphoid cell expresses a modified TCR comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In any of the above embodiments, the modified TCR comprises a single amino acid substitution within CDR2 of the β chain compared to the β chain of the unsubstituted WT TCR (eg as set forth in SEQ ID NO:1).

[0155] The lymphoid cells are obtained from PBMCs, tumor-draining lymph nodes, or tumor infiltrates. In certain embodiments, the genetically engineered lymphoid cells are further engineered to express additional receptors, including chimeric antigen receptors and the cancer antigen NY-ESO-1. 157-165 In certain embodiments, the engineered lymphoid cells are further engineered to express soluble proteins, including, but not limited to, T cell receptors that are not directed against the epitope (SEQ ID NO: 8). In certain embodiments, the engineered lymphoid cells are further engineered to express soluble proteins, including, but not limited to, cytokines, chemokines, growth factors, soluble receptors, ligands, antibodies, antibody fragments, and antigen-binding domains. In certain embodiments, the engineered lymphoid cells are further engineered to express additional receptors and soluble proteins.

[0156] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2.

[0157] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:3.

[0158] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising the amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4.

[0159] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5.

[0160] In various embodiments, the genetically engineered lymphoid cells express a modified TCR that includes a single amino acid substitution within CDR2 of the β chain compared to the β chain of the unsubstituted WT TCR (e.g., as set forth in SEQ ID NO:1).

[0161] In some embodiments, the engineered lymphoid cells provided herein can be engineered to further express one or more exogenous molecules.

[0162] Lymphoid cells are cells of the immune system that react specifically with antigens to produce specific cell products. Samples containing lymphoid cells can be obtained from a number of sources of subjects, including, but not limited to, tissues (including tumor tissues, virus-infected tissues, tissues at sites of inflammation, sites of lymphocytic infiltration and sites of leukocyte infiltration), thymus, tumor tissues (e.g., samples, fragments), or enzymatically digested tissues, dissociated / suspended cells, lymph node samples, or body fluid samples (e.g., blood, ascites, lymph). Exemplary tissues include skin, adipose tissue, cardiovascular tissues (veins, arteries, capillaries, valves, etc.), nervous tissue, bone marrow, breast, gastrointestinal, lung tissue, eye tissues (such as cornea and lens), cartilage, bone, and mucosal tissue.

[0163] The sample may be untreated, enzymatically treated, and / or dissociated / suspended to form a cell suspension. If the sample is enzymatically treated, non-limiting examples of enzymes that may be used include collagenase, dispase, hyaluronidase, liberase, and deoxyribonuclease (DNase).

[0164] In certain embodiments, lymphoid cells for use in the present invention include tumor-infiltrating immune cells. Tumor-infiltrating immune cells are composed of both mononuclear and polymorphonuclear immune cells (i.e., T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, etc.) in variable proportions. In certain embodiments, lymphocytes for use in the present invention include tumor-infiltrating lymphocytes (TILs). TILs are white blood cells that have left the bloodstream and migrated toward the tumor. TILs are often found in the tumor stroma and within the tumor itself.

[0165] In certain embodiments, lymphoid cells for use in the present invention comprise peripheral blood lymphocytes (PBLs). In certain embodiments, lymphoid cells for use in the present invention comprise T lymphocytes (T cells) and / or natural killer cells (NK cells).

[0166] In a particular embodiment, the lymphoid cells for use in the present invention are T cells. In a particular embodiment, the T cells are CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cell is a regulatory T cell.

[0167] In a particular embodiment, the lymphocytes for use in the present invention are NK cells. In a particular embodiment, the NK cells are CD16 + CD56 + and / or CD57 + NK cells are characterized by their ability to bind to and kill cells that do not express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0168] Suitable conditions for lymphocyte culture include an appropriate medium (e.g., Minimum Essential Medium (MEM), RPMI Medium 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM-V, DMEM, a-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, and either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in sufficient amounts for growth and expansion).

[0169] Examples of other additives for lymphocyte expansion include, but are not limited to, detergents, piasmanate, pH buffers such as HEPES, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental cultures and not in the culture of cells that will be infused into the subject. The target cells are kept under the necessary conditions to support growth, e.g., appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2). 2 ) will be maintained under

[0170] The expansion of lymphoid cells can be carried out using methods and conditions known in the art. In a particular embodiment, the expansion of lymphoid cells is carried out according to the method described in PCT / EP2018 / 080343.

[0171] Genetic engineering of lymphoid cells is accomplished by at least one of transfection, transduction, or transient cell membrane disruption (i.e., cell squeeze) to introduce at least one polynucleotide encoding a modified TCR into lymphoid cells. In certain embodiments, the polynucleotide is introduced into lymphoid cells by transducing a substantially homogenous cell population with a recombinant expression vector. Such vectors can be viral or non-viral vectors. Exemplary viral vectors for use in the present invention include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, or baculoviral vectors. In one embodiment, the viral vector for use in the present invention is a lentiviral vector.

[0172] Additional receptors may be introduced into the engineered lymphoid cells expressing the modified TCR, which may enhance the function of the lymphoid cells (e.g., anti-tumor function).

[0173] In certain embodiments, the additional receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs) typically have an antigen binding domain fused to an intracellular signaling domain capable of activating or stimulating immune cells. The extracellular binding domain of the CAR may be composed of a single chain variable region fragment (scFv) resulting from fusing the heavy and light chains of the variable regions of a mouse or humanized monoclonal antibody. Alternatively, scFvs derived from Fab's (e.g., not from antibodies obtained from a Fab library) may be used. The scFvs may be fused to a transmembrane domain and then fused to an intracellular signaling domain. The CAR may be a first, second, or third generation CAR. "First generation" CARs include those that provide only a CD3ζ (zeta) signal upon antigen binding. "Second generation" CARs include those that provide costimulation (e.g., CD28 or CD137) and activation (CD3ζ). "Third generation" CARs include those that provide multiple costimulation (e.g., CD28 and CD137) and activation (CD3ζ). CARs can specifically recognize cancer antigens.

[0174] In certain embodiments, the additional receptor is the cancer antigen NY-ESO-1 157-165 These TCRs are not specific for any particular epitope. 157-165 It can specifically recognize cancer antigens other than epitopes.

[0175] In certain embodiments, the cancer antigen may be selected from CD7, CD74, CDS, CEA, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor alpha, GD2, GD3, HER2, hTERT, IL-13R-a2, KDR, K-light chain, LeY, Ll cells, MAGE-Al, mesothelin, MUC1, MUC16, NKG2D ligand, NY-ESO-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, and WT-1.

[0176] The engineered lymphoid cells expressing the modified TCR can be engineered to express and secrete a soluble protein or proteins. Soluble proteins for use in the present invention include, but are not limited to, cytokines, chemokines, growth factors, soluble receptors, ligands, antibodies, antibody fragments, and antigen-binding domains and functional variants thereof.

[0177] Cytokines that may be expressed and / or secreted by the genetically engineered lymphoid cells described herein include interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-35 (IL-35), interleukin-36 (IL-36), interleukin-37 (IL-37), interleukin-38 (IL-38), interleukin-39 (IL-39), interleukin-39 (IL-39), interleukin-39 (IL-39), interleukin-39 (IL-39), interleukin-30 (IL-39), interleukin-31 (IL-39), interleukin-31 (IL-39), interleukin-32 (IL-39), interle These include, but are not limited to, interferon alpha (IFN-alpha or IFN-α), interferon beta (IFN-beta or IFN-β), interferon gamma (IFN-gamma or IFN-γ), transforming growth factor-beta (TGF-β), CCL19, and erythropoietin. In some embodiments, the cytokine expressed by the genetically engineered lymphoid cells is IL-2. In some embodiments, the cytokine expressed by the genetically engineered lymphoid cells is IFN-γ. In some embodiments, the cytokine expressed by the genetically engineered lymphoid cells is GM-CSF.

[0178] Chemokines that may be expressed and / or secreted by the genetically engineered lymphoid cells described herein include, but are not limited to, CXC-chemokines such as interleukin-8 (IL-8), neutrophil-activating protein-1 (NAP-1), neutrophil-activating protein-2 (NAP-2), GRO, GROβ, GROγ, ENA-78, GCP-2, IP-10, MIG, CXCL1, CXCL12, CXCL16, CXCL19, and PF4; and CC-chemokines such as RANTES, MIP-1α, MIP-2β, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, CCL5, and eotaxin. Suitable chemokines described in International Publication No. WO2000078334 (e.g., Table 1), the entire contents of which are incorporated herein by reference, are also contemplated by the present invention.

[0179] Growth factors that may be expressed and / or secreted by the genetically engineered lymphoid cells described herein include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, tumor necrosis factor, transforming growth factor, epidermal growth factor, stem cell factor, platelet-derived growth factor, nerve growth factor, fibroblast growth factor, insulin-like growth factor, growth hormone, interleukin-1 (IL-1), interleukin-2 (IL-2), keratinocyte growth factor, ciliary neurotrophic factor, Schwann cell-derived growth factor, vaccinia virus growth factor, bombyxin, neu differentiation factor, v-Sis, and glial growth factor.

[0180] Soluble receptors that may be expressed and / or secreted by the engineered lymphoid cells described herein include, but are not limited to, soluble cytokine receptors such as IL-1RI, IL-1RII, TNFRI, TNFRII, IFN-α / βR, IL-4 receptor, IL-6 receptor, IL-10 receptor, IL-11 receptor, IL-13 receptor, IL-18 binding protein, and TGF-β receptor; and soluble growth factor receptors such as soluble epidermal growth factor receptor (sEGFR), soluble vascular endothelial growth factor receptor and PD-1 ectodomain, soluble VEGFR-1 and SIRP-α molecules. Soluble receptors that may be expressed and / or secreted by the engineered lymphoid cells described herein may be further fused to CD28 endodomain or 41BB endodomain, or any other co-stimulatory endodomain known in the art.

[0181] Ligands that may be expressed and / or secreted by the genetically engineered lymphoid cells described herein include nerve growth factor (NGF), CD40L (CD154), CD137L / 4-1BBL, tumor necrosis factor alpha (TNFα), CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), Fas ligand agonist (FasL agonist), LAG3 ligand, VEGFR1 ligand, TIM3 ligand, TIGIT ligand, SIRP-alpha ligand, CD30L / CD153, tumor necrosis factor beta (TNFβ) / lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257 / B cell activating factor (BAFF) / Blys / THANK / Ta11-1, glucocorticoids (GLUCOC), and / or IL-11. These include, but are not limited to, tumor necrosis factor (TNF) ligands such as luteinoid-induced TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14); immunoglobulin superfamily ligands such as CD80 and CD86; ligands for Toll-like receptors (TLRs), 4-1BB ligands, agonists, OX40 ligand agonists, ICOS ligand agonists, Flt3 ligands, phosphodiesterase 4B2 (PDE4B2), phosphodiesterase 4A (PDE4A), phosphodiesterase 7A (PDE7A), phosphodiesterase 4C (PDE4C), and programmed cell death protein-1 (PD-1) ligands.

[0182] The antibodies that can be expressed and / or secreted by the genetically engineered lymphoid cells described herein include those that specifically bind to cancer antigens. Such cancer antigens can be selected from CD7, CD19, CD74, CDS, CEA, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor alpha, GD2, GD3, HER2, hTERT, IL-13R-a2, KDR, K-light chain, LeY, Ll cells, MAGE-Al, mesothelin, MUC1, MUC16, NKG2D ligand, NY-ESO-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, and WT-1. The amino acid sequences that specifically bind to the antigens are known in the art or can be prepared using methods known in the art.

[0183] In certain embodiments, the antibody is a bispecific antibody (e.g., a bispecific T cell antibody (BiTE)). A bispecific antibody is a recombinant synthetic antibody that contains two different antigen-binding domains. For example, one of the antigen-binding domains can target a cancer antigen and the other can bind to a lymphocyte activation molecule.

[0184] Antibody fragments or antigen-binding domains having affinity for cancer antigens can be expressed by the genetically engineered lymphoid cells described herein. Antibody fragments include, but are not limited to, single chain antibodies, Fab fragments, Fv fragments, single chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2' fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intrabodies, minibodies, diabodies, triabodies, and decabodies.

[0185] The engineered lymphoid cells expressing modified TCRs may be further engineered to express gene knockdowns of inhibitory / checkpoint molecules, including but not limited to, PD-1, CTLA-4, LAG-3, TIGIT, VISTA, TIM-3, and Cbl-b. In some embodiments, the engineered lymphocytes expressing modified TCRs that may be engineered to express gene knockdowns may be further engineered to express and secrete additional soluble proteins, such as, but not limited to, IL-2, IL-33, GM-CSF, CD40 agonists.

[0186] Methods of using such genetically engineered lymphoid cells in adoptive cell transfer therapy are also provided. In certain embodiments, the lymphoid cells can be autologous (or autologous), allogeneic, syngeneic, or xenogeneic to the subject. In certain embodiments, the lymphoid cells are autologous to reduce immune reactive responses against the lymphocytes when reintroduced into the subject for immunotherapy treatment.

[0187] In one embodiment, provided herein is a genetically engineered lymphoid cell that expresses a TCR and further expresses one or more proteins. In certain embodiments, at least one of the proteins is a soluble protein. The soluble protein can be, for example, a growth factor (e.g., GM-CSF). In certain embodiments, at least one of the proteins is an intracellular protein. The intracellular protein can be, for example, a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphoid cell can further express one or more cell surface receptors. In certain embodiments, the TCR is an exogenous wild-type TCR. In certain embodiments, the TCR is a modified TCR.

[0188] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a TCR comprising an amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1, and further expressing one or more soluble proteins. The soluble protein can be a growth factor. The growth factor can be GM-CSF. The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0189] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising an amino acid sequence of SEQ ID NO:2 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2, and further expressing one or more soluble proteins. The soluble protein can be a growth factor. The growth factor can be GM-CSF. The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0190] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising an amino acid sequence of SEQ ID NO: 3 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, and further expressing one or more soluble proteins. The soluble protein can be a growth factor. The growth factor can be GM-CSF. The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0191] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising an amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4, and further expressing one or more soluble proteins. The soluble protein can be a growth factor. The growth factor can be GM-CSF. The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0192] In one embodiment, provided herein is a genetically engineered lymphoid cell expressing a modified TCR comprising an amino acid sequence of SEQ ID NO:5 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5, and further expressing one or more soluble proteins. The soluble protein can be a growth factor. The growth factor can be GM-CSF. The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0193] In some embodiments, the GM-CSF is mouse GM-CSF or a functional fragment thereof. The UniProt identifier for mouse GM-CSF is UniProtKB-P01587.

[0194] In some embodiments, GM-CSF comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15. In one embodiment, GM-CSF comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the nucleotide sequence encoding GM-CSF comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16. In one embodiment, the nucleotide sequence encoding GM-CSF comprises the nucleotide sequence of SEQ ID NO: 16.

[0195] In some embodiments, a functional fragment of GM-CSF comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19. In one embodiment, a functional fragment of GM-CSF comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, a nucleotide sequence encoding a functional fragment of GM-CSF comprises the nucleotide sequence of SEQ ID NO:20, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:20. In one embodiment, the nucleotide sequence encoding a functional fragment of GM-CSF comprises the nucleotide sequence of SEQ ID NO:20.

[0196] In some embodiments, the GM-CSF is human GM-CSF or a functional fragment thereof. The UniProt identifier for human GM-CSF is UniProtKB-P04141.

[0197] In some embodiments, GM-CSF comprises the amino acid sequence of SEQ ID NO:21 or 34, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:21 or 34. In one embodiment, GM-CSF comprises the amino acid sequence of SEQ ID NO:21 or 34. In some embodiments, the nucleotide sequence encoding GM-CSF comprises the nucleotide sequence of SEQ ID NO:22 or 35, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:22 or 35. In one embodiment, the nucleotide sequence encoding GM-CSF comprises the nucleotide sequence of SEQ ID NO:22 or SEQ ID NO:35.

[0198] In some embodiments, a functional fragment of GM-CSF comprises the amino acid sequence of SEQ ID NO:25 or 36, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:25 or 36. In one embodiment, a functional fragment of GM-CSF comprises the amino acid sequence of SEQ ID NO:25 or 36. In some embodiments, a nucleotide sequence encoding a functional fragment of GM-CSF comprises the nucleotide sequence of SEQ ID NO:26 or 37, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:26 or 37. In one embodiment, the nucleotide sequence encoding a functional fragment of GM-CSF comprises the nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:37.

[0199] Examples of sequences useful for expressing GM-CSF or a functional fragment thereof are provided below.

[0200] Mouse GM-CSF (full protein sequence) MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK (SEQ ID NO: 15)

[0201] Mouse GM-CSF (full DNA sequence) ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCAGCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACATTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAAAAACCAGGCCAAAAA(SEQ ID NO: 16)

[0202] Mouse GM-CSF (protein sequence of signal peptide) MWLQNLLFLGIVVYSLS(SEQ ID NO: 17)

[0203] Mouse GM-CSF (DNA sequence of signal peptide) ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCA(SEQ ID NO: 18)

[0204] Mouse GM-CSF (protein sequence of active soluble GM-CSF) APTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK(SEQ ID NO: 19)

[0205] Mouse GM-CSF (DNA sequence of active soluble GM-CSF) GCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACATTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAAAAACCAGGCCAAAAA(SEQ ID NO: 20)

[0206] Human GM-CSF (full length protein 1) MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO: 21)

[0207] Human GM-CSF (full length DNA1) ATGTGGCTGCAATCTCTGCTGCTGCTGGGCACAGTGGCCTGTTCTATTAGCGCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCCAAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGAGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCAGACCAGACTGGAACTGTACAAGCAGGGCCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCTGAGACAAGCTGTGCCACACAGACCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA(SEQ ID NO:22)

[0208] Human GM-CSF (protein sequence of signal peptide) MWLQSLLLLGTVACSIS(SEQ ID NO:23)

[0209] Human GM-CSF (DNA sequence of signal peptide) ATGTGGCTGCAATCTCTGCTGCTGCTGGGCACAGTGGCCTGTTCTATTAGC(SEQ ID NO:24)

[0210] Human GM-CSF (protein sequence of active soluble GM-CSF1) APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO:25) (SEQ ID NO:25)

[0211] Human GM-CSF (DNA sequence of active soluble GM-CSF1) GCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCCAAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGAGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCAGACCAGACTGGAACTGTACAAGCAGGGCCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCTGAGACAAGCTGTGCCACACAGACCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA(SEQ ID NO: 26)

[0212] Human GM-CSF (full length protein 2) MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO: 34)

[0213] Human GM-CSF (full length DNA2) ATGTGGCTGCAATCTCTGCTGCTGCTGGGCACAGTGGCCTGTTCTATTAGCGCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCCAAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGAGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCAGACCAGACTGGAACTGTACAAGCAGGGCCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCTGAGACAAGCTGTGCCACACAGATCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA(SEQ ID NO: 35)

[0214] Human GM-CSF (protein sequence of active soluble GM-CSF2) APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO: 36)

[0215] Human GM-CSF (DNA sequence of active soluble GM-CSF2) GCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCCAAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGAGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCAGACCAGACTGGAACTGTACAAGCAGGG CCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCTGAGACAAGCTGTGCCACACAGATCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA (SEQ ID NO: 37)

[0216] In one embodiment, provided herein is a genetically engineered lymphoid cell that expresses a TCR comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1, and further expresses one or more intracellular proteins. The intracellular protein can be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0217] In one embodiment, provided herein is a genetically engineered lymphoid cell that expresses a modified TCR comprising the amino acid sequence of SEQ ID NO:2 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2, and further expresses one or more intracellular proteins. The intracellular protein can be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0218] In one embodiment, provided herein is a genetically engineered lymphoid cell that expresses a modified TCR comprising the amino acid sequence of SEQ ID NO: 3 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, and further expresses one or more intracellular proteins. The intracellular protein can be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0219] In one embodiment, provided herein is a genetically engineered lymphoid cell that expresses a modified TCR comprising the amino acid sequence of SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4, and further expresses one or more intracellular proteins. The intracellular protein can be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0220] In one embodiment, provided herein is a genetically engineered lymphoid cell that expresses a modified TCR comprising the amino acid sequence of SEQ ID NO:5 or a functional fragment thereof, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:5, and further expresses one or more intracellular proteins. The intracellular protein can be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphoid cell can further express one or more cell surface receptors.

[0221] In some embodiments, the genetically engineered lymphocyte disclosed herein expresses phosphodiesterase in addition to the modified TCR of the present invention.In some embodiments, the phosphodiesterase is phosphodiesterase 4B2 (PDE4B2), phosphodiesterase 4A (PDE4A), phosphodiesterase 7A (PDE7A) or phosphodiesterase 4C (PDE4C).In one embodiment, the phosphodiesterase is phosphodiesterase 4B2 (PDE4B2).

[0222] In one embodiment, PDE4B2 is human PDE4B2 or a functional fragment thereof.The GenInfo identifier of human PDE4B2 is 82799482, and the NCBI accession number is NP_001032416.

[0223] In some embodiments, PDE4B2 comprises the amino acid sequence of SEQ ID NO:27, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:27. In one embodiment, PDE4B2 comprises the amino acid sequence of SEQ ID NO:27. In some embodiments, the nucleotide sequence encoding PDE4B2 comprises the nucleotide sequence of SEQ ID NO:28, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:28. In one embodiment, the nucleotide sequence encoding PDE4B2 comprises the nucleotide sequence of SEQ ID NO:28.

[0224] In one embodiment, PDE4B2 is mouse PDE4B2 or a functional fragment thereof. The GenInfo identifier of mouse PDE4B2 is 295789129, and the NCBI accession number is NP_001171451.

[0225] In some embodiments, PDE4B2 comprises the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:29. In one embodiment, PDE4B2 comprises the amino acid sequence of SEQ ID NO:29. In some embodiments, the nucleotide sequence encoding PDE4B2 comprises the nucleotide sequence of SEQ ID NO:30, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:30. In one embodiment, the nucleotide sequence encoding PDE4B2 comprises the nucleotide sequence of SEQ ID NO:30.

[0226] Examples of sequences useful for expressing PDE4B2 or a functional fragment thereof are provided below.

[0227] Human PDE4B2 amino acid sequence (SEQ ID NO:27)

[0228] Nucleotide sequence of human PDE4B2

[0229] Amino acid sequence of mouse PDE4B2 (SEQ ID NO:29)

[0230] Nucleotide sequence of mouse PDE4B2

[0231] Bifunctional molecules One approach to overcome the lack of strong anti-tumor T cell immunity is the ex vivo genetic modification of tumor-targeting T cells by using affinity-enhancing receptors made from either the above T cell receptors or antibody-derived receptors.A complementary approach that does not require ex vivo manipulation of T cells includes the use of fusion proteins that combine tumor recognition and T cell engaging domains to redirect T cells to target tumors.The specificity and anti-tumor activity of such fusion proteins are described, for example, in Cancer Immunol Immunother (2013) 62:773-785, Nat Med. 2012 Jun;18(6):980-7, and U.S. Patent No. 7,763,718, each of which is incorporated herein by reference in its entirety for all purposes.

[0232] In one aspect, provided herein is a bifunctional molecule comprising a modified TCR or functional fragment thereof disclosed herein and a polypeptide that specifically binds to a cell surface protein on a T cell. Examples of cell surface proteins on T cells include, but are not limited to, CD2, CD3, CD4, CD8, CD44, CD45RA, CD45RB, CD45RO, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD16, CD28, and IL-2R.

[0233] In certain embodiments, the polypeptide is an immune effector polypeptide.

[0234] As used herein, the term "immune effector polypeptide" generally refers to any molecule that induces or stimulates an immune response through direct or indirect activation of the humoral or cellular arms of the immune system, such as by activating T cells. Examples of immune effector polypeptides include, but are not limited to, IL-1, IL-1α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-β, IFN-γ, TNFα, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD44 antibody, anti-CD45RA antibody, anti-CD45RB antibody, anti-CD45RO antibody, anti-CD49a antibody, anti-CD49b antibody, anti-CD49c antibody, anti-CD49d antibody, anti-CD49e antibody, anti-CD49f antibody, anti-CD16 antibody, anti-CD28 antibody, anti-IL-2R antibody, viral proteins and peptides, and bacterial proteins or peptides.

[0235] In some embodiments, the polypeptide comprises an antibody or an antibody fragment. Antibody fragments include, but are not limited to, single chain antibodies, Fab fragments, Fv fragments, single chain Fv fragments (scFv), bivalent antibody fragments such as (Fab)2' fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies, triabodies, and decabodies. In certain embodiments, the polypeptide comprises an scFv. In certain embodiments, the polypeptide is an immune effector polypeptide.

[0236] In some embodiments, the polypeptide specifically binds to CD3. In some embodiments, the polypeptide comprises an anti-CD3 antibody. Examples of anti-CD3 antibodies include, but are not limited to, OKT3, UCHT-1, BMA031, and 12F6. In some embodiments, the polypeptide comprises an scFv derived from an anti-CD3 antibody. In some embodiments, the polypeptide comprises an scFv derived from OKT3, UCHT-1, BMA031, or 12F6. In certain embodiments, the polypeptide specifically binds to CD3 is an immune effector polypeptide.

[0237] In certain embodiments, the N-terminus of the TCR is linked to the C-terminus of a polypeptide that specifically binds to a cell surface protein on a T cell. In certain embodiments, the C-terminus of the TCR is linked to the N-terminus of a polypeptide that specifically binds to a cell surface protein on a T cell. In certain embodiments, the TCR is a heterodimeric αβTCR polypeptide pair or a single chain αβTCR (scTCR) polypeptide, and the N-terminus of the α or β chain of the heterodimeric TCR polypeptide pair or the N-terminus of the scTCR polypeptide is linked to the C-terminal amino acid of a polypeptide that specifically binds to a cell surface protein on a T cell. In certain embodiments, the TCR is a heterodimeric αβTCR polypeptide pair or a single chain αβTCR polypeptide, and the C-terminus of the α or β chain of the heterodimeric TCR polypeptide pair or the C-terminus of the scTCR polypeptide is linked to the N-terminal amino acid of a polypeptide that specifically binds to a cell surface protein on a T cell.

[0238] The link between the TCR and the polypeptide that specifically binds to a cell surface protein of a T cell may be direct or indirect through a linker sequence. Linker sequences are usually flexible and are composed of amino acids such as glycine, alanine, serine, etc. that do not have bulky side chains that may limit their flexibility. The usable or optimal length of the linker sequence is easily determined for any given TCR bifunctional molecule. In some examples, the linker is less than about 12, e.g., less than about 10, or 5-10 amino acids in length.

[0239] In some embodiments of the bifunctional molecules described herein, the modified TCR or functional fragment thereof comprises a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 2-5. In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acids of SEQ ID NO: 5. In any of the above embodiments, the modified TCR or functional fragment thereof comprises a single amino acid substitution within CDR2 of the β chain compared to the β chain of an unsubstituted WT TCR (e.g. as set forth in SEQ ID NO: 1).

[0240] In certain embodiments of the bifunctional molecules described herein, the modified TCR or functional fragment thereof comprises a β chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 2-5. In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising the amino acid sequence of SEQ ID NO: 4. In one embodiment, the modified TCR or functional fragment thereof comprises a β chain comprising the amino acid sequence of SEQ ID NO: 5.

[0241] In certain embodiments of the bifunctional molecules described herein, the modified TCR or functional fragment thereof further comprises an alpha chain comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the modified TCR or functional fragment thereof further comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 7.

[0242] In various embodiments, the bifunctional molecules described herein may be used in combination with the host cells or pharmaceutical compositions described herein.

[0243] Treatment method In one aspect, provided herein is a method for treating cancer in a subject.The method comprises administering to a subject suffering from such cancer an effective amount of lymphoid cell presenting modified TCR of the present invention, or a pharmaceutical composition comprising such cell.Non-limiting examples of cancer treatable by the method described herein include, for example, 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.

[0244] In one aspect, provided herein is a method for stimulating or enhancing an immune response in a mammal, comprising administering to the mammal an effective amount of a genetically engineered lymphoid cell of the invention, or a pharmaceutical composition comprising such a cell.

[0245] Additional examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (e.g., osteosarcoma or rhabdomyosarcoma), and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), adenosquamous carcinoma, lung cancer (including, e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including, e.g., digestive tract cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, primary or metastatic melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, brain (including, e.g., high-grade glioma, diffuse pontine ... These include cancers of the head and neck, including glioma, ependymoma, neuroblastoma, or glioblastoma, and head and neck cancer, and associated metastases.Additional examples of tumors are described in The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition at internet website of Merck Manuals); and the SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.

[0246] In various embodiments, the cancer treatable by the methods described herein has cancer antigen NY-ESO-1 on the surface of its cells. 157-165 The epitope (SEQ ID NO: 8) is presented.

[0247] In some embodiments of any of the above methods of treatment, the composition is administered in a therapeutically effective amount. The dosage of the composition administered in the methods of the present invention will vary widely depending on the subject's physical parameters, frequency of administration, method of administration, clearance rate, etc. The initial dose may be higher, followed by a lower maintenance dose. To maintain an effective dosage level, the dose may be administered less frequently, such as weekly or biweekly, or may be divided into smaller doses and administered daily, semiweekly, etc. It is contemplated that a variety of doses may be effective to achieve in vivo persistence of modified host cells. It is also contemplated that a variety of doses may be effective to improve in vivo effector functions of modified host cells.

[0248] In some embodiments, a composition comprising an engineered host cell produced by the methods described herein comprises at least 10 2 ~10 10 cells / kg body weight, 10 5 ~10 9 cells / kg body weight, 10 5 ~10 8 cells / kg body weight, 10 5 ~10 7 cells / kg body weight, 10 7 ~10 9 cells / kg body weight, or 10 7 ~10 8 The host cells may be administered at a dosage of cells / kg body weight, including all integer values ​​within these ranges. The number of modified host cells will depend on the therapeutic use for which the composition is intended.

[0249] The modified host cells may be administered multiple times at the dosages listed above. The modified host cells may be allogeneic, syngeneic, xenogeneic, or autologous (autologous) to the patient undergoing treatment.

[0250] The compositions and methods described in this disclosure can be utilized in combination with other types of tumor treatments, such as chemotherapy, surgery, radiation therapy, gene therapy, and the like.

[0251] It is also contemplated that when used to treat various diseases / disorders, the compositions and methods of the present disclosure may be utilized with other therapeutic methods / medicines suitable for the same or similar disease / disorder. Such other therapeutic methods / medicines may be co-administered (simultaneously or sequentially) to produce additive or synergistic effects. The appropriate therapeutically effective dosage of each drug may be reduced due to additive or synergistic effects.

[0252] In some embodiments of any of the above therapeutic methods, the method further comprises administering to the subject one or more additional compounds selected from the group consisting of immunosuppressants, biologics, probiotics, prebiotics, and cytokines (e.g., GM-CSF, IFN, or IL-2).

[0253] In some embodiments, the methods described herein further include providing exogenous GM-CSF in addition to the GM-CSF produced by the immune cells to enhance the function of the immune cells expressing the chimeric cytokine receptor of the present disclosure. The exogenous GM-CSF can be provided, for example, by i) injection of the FDA-approved GM-CSF drug sargramostim (Leukine™), or ii) use of a non-viral or viral vector expressing GM-CSF (e.g., the FDA-approved oncolytic virus talimogene laherparepvec [TVEC, Imlygic™] expressing GM-CSF). These drugs can be given before, together with, or after administration (e.g., infusion) of the immune cells expressing the chimeric cytokine receptor of the present disclosure to the patient.

[0254] By way of non-limiting example, the present invention may be combined with other therapies that block inflammation (eg, via blockade of IL1, INFα / β, IL6, TNF, IL23, etc.).

[0255] The methods and compositions of the present invention can be combined with other immunomodulatory therapies, such as, for example, therapeutic vaccines (including but not limited to, GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to, agents that block CTLA4, PD1, LAG3, TIM3, etc.), or activators (including but not limited to, agents that enhance 4-1BB, OX40, etc.). The methods of the present invention can also be combined with other therapies that have the ability to modulate NKT function or stability, including, but not limited to, CD1d, CD1d fusion proteins, CD1d dimers or larger polymers of CD1d, either unloaded or loaded with antigen; CD1d-chimeric antigen receptor (CD1d-CAR); or any of the five known CD1 isomers present in humans (CD1a, CD1b, CD1c, CD1e). The methods of the present invention can also be combined with other therapies, such as midostaurin, enasidenib, or combinations thereof.

[0256] The therapeutic method of the present invention can be combined with additional immunotherapy and therapy. For example, when used to treat tumors, the composition of the present invention can be used in combination with conventional therapies, such as surgery, radiation therapy, chemotherapy, or a combination thereof, depending on the type of tumor, the patient's condition, other health problems, and various factors. In certain embodiments, other therapeutic agents useful for combination tumor treatment with the inhibitor of the present invention include antiangiogenic agents. Many antiangiogenic agents have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitor of metalloproteinases (TEMP1 and TEMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, and those listed in Carmeliet and Jain (2000). In one embodiment, the modified host cells of the invention can be used in combination with a VEGF antagonist or a VEGF receptor antagonist, such as an anti-VEGF antibody, a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, an anti-VEGFR neutralizing antibody, an inhibitor of VEGFR tyrosine kinase, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0257] Non-limiting examples of chemotherapeutic compounds that can be used in the combination treatments of the present disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacytidine, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclospor ... Phosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin , hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide These include rituximab, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0258] These chemotherapeutic compounds can be classified according to their mechanism of action, for example, into the following groups: antimetabolites / anticancer agents, e.g., pyrimidine analogues (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogues, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, e.g., natural products such as the vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents such as the taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine. disruptor), epidipodophyllotoxin (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, protease inhibitors, ricamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase, which systematically metabolizes L-asparagine and removes cells that do not have the ability to synthesize asparagine themselves); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, thrombolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents (anti-migratory agents, such as cyclosporine, ... agent); antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin ); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disrupting agents. ;

[0259] In various embodiments of the methods described herein, the subject is a human. The subject may be of any age or sex, and may be a juvenile or adult.

[0260] Pharmaceutical Compositions, Dosage Forms and Administration In one aspect, provided herein is a pharmaceutical composition comprising a lymphoid cell having a modified TCR as described herein and a pharma- ceutical acceptable carrier.

[0261] The carrier may be, for example, a solvent or dispersion medium, including water, saline, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride.

[0262] Pharmaceutical compositions based on populations of engineered lymphoid cells with modified TCRs as described herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The lymphoid cells can be formulated, for example, for administration by injection, parenteral, vaginal, rectal administration, or by direct administration to a tumor.

[0263] The pharmaceutical composition can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). The preparation for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers, optionally containing preservatives. The pharmaceutical composition can further be formulated as a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain other agents such as suspending agents, stabilizers and / or dispersing agents.

[0264] The dosage forms suitable for injection use may include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulations must be sterile and fluid. They must be stable under the conditions of manufacture and under specified storage parameters (e.g., refrigeration and freezing, etc.) and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0265] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. Dose ranges and frequency of administration may vary depending on the nature of the population of engineered lymphoid cells having modified TCRs as described herein, and the disease state, as well as the particular patient parameters and the route of administration used.

[0266] In some embodiments, the population of engineered lymphoid cells having a modified TCR described herein comprises at least about 10 7 ~about 10 12 The subject may be administered a dose in the range of 0.1 to 1.5 mg / kg. The more precise dose may depend on the subject to which it is administered. For example, if the subject is a young person, a lower dose may be required, and if the subject is an adult human subject, a higher dose may be required. In certain embodiments, the more precise dose may depend on the subject's body weight. EXAMPLES

[0267] The present invention is further described and demonstrated by the following examples. However, the use of these and any other examples in the specification is merely illustrative and in no way limits the scope and meaning of the present invention or any exemplified term. Similarly, the present invention is not limited to the specific preferred embodiments described herein. Indeed, upon reading this specification, many modifications and variations of the present invention will be apparent to those skilled in the art, and such variations can be made without departing from the spirit or scope of the present invention. Therefore, the present invention should be limited only by the appended claims and the full scope of equivalents to which those claims are entitled.

[0268] Example 1. Binding Affinity of Soluble Rationally Engineered BC1 TCR Variants material and method Amino acid substitutions in the TCR were designed by structure-based computer-aided protein engineering. Starting from the experimental structure of the wild-type TCR-p-MHC complex (PDB ID 2bnr), the contribution of each TCR and pMHC residue to the binding free energy was estimated using Molecular Mechanics-Generalized Born Surface Area (MM-GBSA). These results were used to determine sequence modifications that could increase the affinity of the TCR for pMHC. The latter were computationally introduced into the TCR structure and the resulting changes in binding free energy were estimated again using MM-GBSA. Sequence modifications predicted in silico to favorably increase the binding free energy were retained for experimental validation. Soluble TCRs were generated both in mammalian cell culture systems and from bacterial inclusion bodies. A direct titration ELISA on soluble pMHC was used to compare binding strength compared to the wild-type TCR. The TCRs of interest were generated from refolding of bacterial inclusion bodies of the α and β chains and characterized by surface plasmon resonance to measure affinity and kinetics.

[0269] The wild-type TCR, designated BC1, differs from the well-characterized TCR 1G4 by only four amino acids (two in the α chain and two in the β chain) (Chen, JL, et al., J Exp Med 2005. 201, 1243-1255, which is incorporated by reference in its entirety for all purposes). The BC1 TCR is of clinical interest because it was derived from an immunodominant clone from a long-term surviving cancer patient.

[0270] Production and purification of soluble TCR in HEK-293 cells The α- and β-chains of TCR BC1 were cloned separately into the expression vector pHYK8 under the control of the CMV promoter. Heterodimeric chain pairing was facilitated by an acidic-basic zipper following the strategy of Chang et al. (Chang, HC, et al., Proc Natl Acad Sci USA, 1994. 91, 11408-11412). The TCR β-chain was truncated after Cys242 and replaced with a flexible linker region, a thrombin site, an acidic zipper, and a HIS tag, and the α-chain was truncated after Cys209 and replaced with a linker region, a thrombin site, and a basic zipper. Soluble TCR was produced by cotransfection of the plasmids into HEK-293 cells with linear 25 kDa polyethylenimine. Transfected cells were cultured in suspension in Pro293 CDM medium (Lonza) supplemented with 4 mM valproic acid (to minimize acidification) for up to 7 days. Culture supernatants were collected by centrifugation and TCRs were purified using Ni-NTA agarose (Qiagen) according to the manufacturer's suggestions.

[0271] Production of soluble TCR and pMHC from bacterial inclusion bodies The TCR α and β chains (cloned into pGMT7) were produced as inclusion bodies using BL21(DE3)pLys bacterial cells, solubilized by dialysis and refolded as previously described (Boulter, JM, et al., Protein Eng 2003. 16, 707-711). The TCR was then concentrated and filtered, followed by centrifugation with Ni 2+Fast protein liquid chromatography HIS-tag purification was performed using immobilized metal chelating Sepharose (GE Healthcare) and imidazole elution. Prior to SPR analysis, samples were concentrated with 10 kDa MWCO spin filters (Millipore) and gel filtered using an S200 column into HBS-EP buffer (10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% (v / v) P20 surfactant) to remove aggregates. Biotinylated A2 / NY-ESO157-165 was prepared as previously described (Altman, JD, et al., Science, 1996. 274, 94-96).

[0272] Rationally developed TCR titration ELISA Biotinylated pMHC (A2 / NY-ESO157-165 complex) was captured on SA-coated plates (96-well, high-binding plates, Corning Life Sciences) blocked with 2% BSA in Tris-buffered saline (TBS, pH 7.4). Plates were thoroughly washed with TBS (0.1% Tween) between each step. After incubation with soluble TCR in TBS (1% BSA, 0.1% Tween) for 1.5 h at room temperature, free sites on SA were blocked with biotin. Bound TCR was immunoblotted with anti-β-chain TCR mAb (TCR 1151, Thermo Scientific, Rockford, IL, USA) diluted 1 / 1500 in TBS (1% BSA, 0.1% Tween), followed by HRP-conjugated goat anti-anti-mouse IgG-Ab (Thermo Scientific) diluted 1 / 1500 in TBS (0.1% Tween), and HRP-conjugated goat anti-anti-mouse IgG-Ab (Thermo Scientific) diluted 1 / 1500 in TBS (0.1% Tween). 2 O 2 Detection was performed using HRP detection with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in citrate-phosphate buffer containing 0.1% EDTA. Plates were read at OD405-490 after 30 minutes. ELISA was repeated at least three times for each TCR.

[0273] In Table 1, binding of WT BC1 TCR is defined as "+++", binding greater than WT BC1 TCR is indicated as "++++", binding less than WT is indicated as "++", very weak binding is indicated as "+" and no binding is indicated as "-". [Table 1]

[0274] Surface plasmon resonance (SPR) of rationally developed TCRs SPR was performed at 25°C on a BIAcore 3000 and SA-coated CM5 sensor chips (BIACore, GE Healthcare). The flow cell was loaded with 200 response units of biotinylated pMHC at a rate of 10 μl / min to ensure uniform distribution. Streptavidin (SA) free sites were blocked with biotin and a reference cell was used as a control for bulk refractive index changes upon injection of sample solutions. For kinetic analysis, 6–8 serial dilutions of TCR were injected over the loaded chip at 50–100 μl / min. Data are representative from one of at least two independent experiments performed in duplicate or triplicate and give the best Chi2 value. k for each TCR was calculated using the 100 μl / min quantification. on value and k Off Values ​​were calculated assuming 1:1 Langmuir binding and data were analyzed using BIAevaluation 4.1 and a global fit algorithm. D k Off / k 0n It was calculated by:

[0275] In Table 2, " * " indicates that A97L and DMb are TCRs that confer optimal functional activity to T cells previously published (Irving, M., et al., JBC 2012. 287, 23068-23078, which is incorporated herein by reference in its entirety for all purposes). ** " is 1G4, NY-ESO-1 157-165(Dunn, SM, et al., Protein Sci 2006. 15, 710-721; Robbins, PF, et al., J Clin Oncol 2011. 29, 917-924; Rapoport, AP, et al., Nat Med 2015. 21, 914-921; Chen, JL, J Exp Med 2005. 201, 1243-1255, each of which is incorporated herein by reference in its entirety for all purposes). *** " indicates that 1G4LY is a high affinity variant of 1G4 that has been used in clinical trials (Dunn, SM, et al., Protein Sci 2006. 15, 710-721; Robbins, PF, et al., J Clin Oncol 2011. 29, 917-924; Rapoport, AP, et al., Nat Med 2015. 21, 914-921; Chen, JL, J Exp Med 2005. 201, 1243-1255, each of which is incorporated herein by reference in its entirety for all purposes). Mutations in the BC1 TCR variant are highlighted in bold. [Table 2]

[0276] Results and Discussion As described above, by rational design (MM-GBSA calculations) (Zoete, V. et al., JMR 2010. 23, 142-152; Zoete, V., and Michielin, O. Proteins 2007. 67, 1026-1047; Zoete, V., et al., Proteins 2005. 61, 79-93), we have developed HLA-A2-NY-ESO T cells with increased affinity and enhanced function compared to primary human T cells. 157-165A panel of TCRs was developed (Zoete, V. et al., JMR 2010. 23, 142-152; Schmid, DA, et al., Journal of immunology 2010. 184, 4936-4946; Irving, M., et al., JBC 2012. 287, 23068-23078).

[0277] As shown in Tables 1 and 2, the TCR panel consisted of HLA-A2-NY-ESO 157-165 Table 1 shows the direct binding of TCRs to pMHC by titration ELISA, and Table 2 shows the affinities and kinetics determined for a subset of these TCRs. As highlighted in bold in Table 1, there are several single amino acid substituted TCRs that bind pMHC with higher affinity than the WT TCR, indicated by "++++" for binding. As shown in Table 2, at least two different single amino acid substitutions at residue 53 of the WT BC1 TCR (I53E, and I53F) show higher binding affinity (K) when compared to the WT BC1 TCR. D WT>I53E>I53F), and I53F results in HLA-A2-NY-ESO-1 157-165 It has the highest affinity for

[0278] CD4 engineered with several different BC1 TCR variants stained with anti-TCR BV13.1 Ab and fluorescently labeled tetramers + and CD8 + Flow cytometry analysis of the cells (Figures 2A-2B and 3A-3B) shows that all variants tested (I53E, I53F, I53W, D55E, and DMb) show expression comparable to wild type (BC1 TCR-transduced cells) and the positive controls A97L and 1G4LY.

[0279] Example 2. Cytokine production by T cells expressing NY-ESO-1 TCR variants material and method Cell line culture 293T and Jurkat cell lines were purchased from ATCC. All cell lines were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mmol / l L-glutamine, 100 μg / ml penicillin, and 100 U / ml streptomycin. The 293T cell line was used for packaging and preparation of lentivirus.

[0280] Cloning of TCR α and β chains and lentivirus generation Both TCRa23 and TCRb13.1 ORFs were engineered into the lentiviral vector pRRL, in which most of the U3 region of the 3′ long terminal repeat was deleted, resulting in a self-inactivating 3′ long terminal repeat (SIN). The TCRa and TCRb chains were separated by a picornavirus-derived 2A sequence.

[0281] Lentivirus was produced by transient transfection of 293T cells using TurboFect. Briefly, 293T cells were co-transfected with the lentiviral vector pRRL encoding the NY-ESO TCRa and TCRb chains and lentiviral helper plasmids (R8.74 and pMD2G). 48 hours after transfection, lentiviral supernatants were harvested, filtered, and concentrated by ultracentrifugation. The pellets were resuspended in an appropriate volume of RPMI 10% FBS and stored at -80°C or used directly.

[0282] A total of 0.5x10 6 CD4 + or CD8 + T cells were cultured at 1 × 10 6The cells were stimulated with αCD3 / αCD28 beads for 18–20 h. On day 5, the beads were removed and T cells were cultured in the presence of 10 ng / ml IL-7 and 10 ng / ml IL-15. TCR expression was determined on day 6 and later time points post-transduction by flow cytometry analysis. Antibodies used were PE-labeled human TCRVb13.1, and APC-labeled anti-human CD4 and anti-human CD8 antibodies. NY-ESO 157-165 Correct folding of the introduced TCR was detected using a PE-labeled HLA-A2 tetramer presenting . Stained cells were analyzed using FlowJo software.

[0283] cell line HLA-A2.1 + / NY-ESO + Melanoma cell lines Mw275 and A375, and the NY-ESO negative cell line NA8 were cultured in IMDM supplemented with 10% FBS and antibiotics (100 IE / ml penicillin and 100 μg / ml streptomycin). Saos-2 (osteosarcoma) and U266 (B cells), both HLA-A2.1 / NY-ESO + , as well as OVCAr3 (ovary) and SKO# (ovary), both NY-ESO negative, were cultured in RPMI supplemented with 10% FBS and antibiotics.

[0284] The NY-ESO-negative cell lines, U87MG (brain), A431 (skin), A673 (sarcoma), RD-ES (sarcoma), HT-29 (lung), and SK-N-AS (neuroblastoma), were cultured in DMEM supplemented with 10% FBS and antibiotics.

[0285] Purification and transduction of primary T cells Primary human T cells were isolated from peripheral blood mononuclear cells (PBMCs) of buffy coats derived from healthy donors. All blood samples were collected with the donors' informed consent and genetically manipulated following ethical approval from the canton of Vaud (Switzerland). Total PBMCs were obtained via Lymphoprep (Axonlab) separation solution using a standard protocol of centrifugation and CD4 T cells were isolated using a negative selection kit combined with magnetic bead separation (easySEP, Stem Cell Technology). + and CD8 + T cells were isolated. T cells were cultured in complete medium (RPMI1640 with Glutamax supplemented with 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate (Invitrogen, Lifetechnologies)) and stimulated with anti-CD3 and anti-CD28 monoclonal antibody (mAB)-coated beads (Lifetechnologies) at a T cell:bead ratio of 1:2. 12–24 h after activation, T cells were transduced with lentiviral particles at a multiplicity of infection (MOI) of approximately 5–10. Human recombinant interleukin-2 (h-IL2; Glaxo) was added every other day to obtain a final concentration of 50 IU / ml by day 5 (+5) after stimulation. At this point, magnetic beads were removed and h-IL15 was added at 10 ng / mL (Miltenyi Biotec GmbH) without further addition of IL2. 0.5~1x10 for magnification 6 A cell density of 1000 ng / ml was maintained. Transduced TCR was measured by fluorescent multimer staining 5 days after transduction. Prior to all functional assays, resting engineered T cells were adjusted to identical transgene expression.

[0286] Cytokine production Cytokine release assays were performed in duplicate in 96-well round-bottom plates in a final volume of 200 μl of RPMI medium, with 5x10 cells per well. 4 of target cells and 5x10 4This was performed by co-culturing T cells from 1000- and 1000-mL of IFN-γ and IL-2 from 1000- and 1000-mL of ...

[0287] Results and Discussion To assess the influence of TCR binding parameters on cytokine production, we used CD4 + and CD8 + Cells were collected from NY-ESO 157-165 Serial 10-fold dilutions of peptides were incubated with loaded T2 cells. For IL-2 produced in response to activation via the TCR, secretion was observed in engineered CD4+ cells expressing TCRs I53F, I53W, D55E, DMb, and 1G4LY. + The maximum was reached in engineered CD4 cells expressing TCR I53F, I53W, D55E, and DMb. + The cells appear to perform better in terms of IL2 production when compared to the WT TCR, and their function is similar to the 1G4LY TCR (Figure 4A). The engineered TCR variant, I53E, showed similar function to the WT TCR in this assay. Under the same conditions, non-transduced CD4 + No IL-2 was produced by the cells (Fig. 4A).

[0288] IL-2 is most efficiently produced by healthy donor (HD) T cells engineered to express TCR I53F in tumor T cell lines that naturally express HLA / A2-NY-ESO-1 (Me275) or engineered to express HLA / A2-NY-Eso-1 (OVCAR5 NLM and A2008-A2-NLM), compared to non-transduced (NT) cells (negative control), wild-type BC1 TCR-transduced cells, and cells transduced to express TCR I53E and A97L, with no cross-reactivity with tumor cell lines that do not express HLA / A2-NY-ESO-1 (OVCAR5 and A2008-A2 and NA8) (Figure 5).

[0289] IL-2 inhibited the expression of CD4 T cells engineered to express TCR I53F and I53W in Me275, Saos-2, and U266 cells compared to non-transduced (NT) cells (negative control). + It is most efficiently produced by T cells. CD4 engineered to express TCR DMb and 1G4LY + Compared to other NY-ESO-positive cell lines in which IL-2 production by T cells is not significant, CD4 + IL-2 production by T cells. IL-2 was secreted by CD4 T cells engineered to express TCR I53E and D55E in both NY-ESO positive cell lines. + It was not produced by CD4 TCR-transduced cells, wild-type BC1 TCR-transduced cells, or cells transduced to express TCR A97L (Figure 6). + In T cells, A2 / NY-ESO-NY-1 157-165 Recognition of CD4 T cells is dependent on the CD8 receptor. In this case, CD4 T cells transduced with the WT BC1 TCR + T cells do not appear to respond to NY-ESO epitopes bound to MHC class I. However, CD4 +When T cells were transduced with TCR variants (i.e., I53F and 153W TCRs) that have higher binding affinity to the NY-ESO epitope bound to MHC class I (compared to the WT BC1 TCR), higher IL-2 production by these engineered T cells was observed, indicating less CD8 co-receptor dependency of I53F TCR-transduced cells.

[0290] In Figure 9, IL-2 inhibited the expression of CD4 T cells engineered to express TCR I53F compared to cells engineered to express wild type BC1 TCR and I53E TCR. + It is most efficiently produced by CD4 T cells engineered with TCR I53F compared to wild-type BC1 TCR engineered cells and cells engineered to express I53E. + T cells tend to be less CD8 coreceptor dependent (Figure 9), and the same trend is seen in Figure 5 when comparing IL-2 production by I53F TCR expressing cells with wild-type BC1 TCR engineered cells, cells engineered to express I53E, and cells expressing the A97LT CR.

[0291] For IFN-γ produced in response to TCR-mediated activation, engineered CD8 expressing TCRs A97L, I53E, I53F, I53W, D55E, DMb, and 1G4LY were used. + The cells performed similarly to WT BC1 TCR in this assay. Under the same conditions, non-transduced CD8 + No IFN-γ was produced by the cells (Fig. 4B ).

[0292] IFN-γ is efficiently produced by healthy donor (HD) T cells engineered to express TCRs A97L, I53E, and I53F in tumor T cell lines that naturally express HLA / A2-NY-ESO-1 (Me275) or that were engineered to express HLA / A2-NY-ESO-1 (OVCAR5 NLM and A2008-A2-NLM) compared to non-transduced (NT) cells (negative control). There is no cross-reactivity with tumor cell lines that do not express HLA / A2-NY-ESO-1 (OVCAR5 and A2008-A2 and NA8). IFN-γ production by a panel of affinity-enhanced BC1 TCR variants was similar to T cells engineered to express wild-type BC1 TCR (Figure 7). In FIG. 10, IFN-γ inhibits CD8 T cells engineered to express the I53F TCR compared to cells expressing wild-type BC1 and I53E TCRs. + It is most efficiently produced by cells.

[0293] IFN-γ was expressed by CD8 cells engineered to express the TCR I53F, D55E, and DMb in Me275 cells; I53F and DMb in A375 cells; I53F, I53W, D55E, and DMb in Saos-2 cells; and I53F, I53W, D55E, and DMb in U266 cells. + IFN-γ is most efficiently produced by CD8 T cells engineered to express A97L (a positive control mutant of the wild-type BC1 TCR) and 1G4LY (a positive control mutant of the wild-type 1G4 TCR) in all NY-ESO positive cell lines. + IFN-γ is efficiently produced by CD8 T cells engineered to express wild-type BC1 TCR in all NY-ESO positive cell lines except A375. + It was produced at modest levels by T cells (Fig. 8 ).

[0294] CD8 engineered to express I53F TCR when using the NY-ESO positive cell line A375 +T cells showed similar levels of IFN-γ production as 1G4LY, but 153W produced less IFN-γ compared to both I53F and 1G4(LY). There was no production of IFN-γ when NY-ESO negative cell lines were used (Figure 11).

[0295] Example 3. Cytotoxic activity of T cells expressing NY-ESO-1 TCR variants NY-ESO TCR-specific CD8 + The cytotoxic activity of T cells was measured using IncuCyte. 4 T2 target cells were plated at 2.5 / 10 per well in the presence of cytotoxic red reagent (Essen Biosciences, Ann Arbor, Michigan, USA). 4 NY-ESO tetramer-positive CD8 + T cells were co-cultured and images were taken every 2 hours using IncuCyte zoom software.

[0296] CD8 transduced with I53F and I53E + The cytotoxic activity of T cells was determined by CD8 + This was similar to the cytotoxic activity of T cells (Figure 12).

[0297] Example 4. Antitumor activity of T cells expressing NY-ESO-1 TCR variants Immediately before subcutaneous injection into NOD SCID γKO mice (NSG), 3x10 6 Me275 cells 6 / 10 6 A Winn assay was performed in which 100% of the NY-ESO-specific T cells were mixed with 100% of the CD4 + T cells and 70% CD8 + The tumors were composed of T cells. The tumor size was measured twice a week. The tumor volume was 1000 mm 3 When the sigma-induced hypercalcaemia was reached, the mice were sacrificed.

[0298] As seen in FIG. 13B, both A97L and I53F transduced T cells reduced tumor size to approximately 0 mm for 43 days. 3I53E-transduced T cells performed similarly to wild-type BC1 TCR-transduced T cells in this assay. With non-transduced T cells, tumor size was approximately 700 mm at day 43. 3 reached a size of

[0299] Example 5. Antitumor activity of T cells expressing NY-ESO-1 TCR variants 5x10 in NSG mice 6 Me275 cells were injected into the tumors with a tumor size of 50–100 mm. 3 When it reaches 10x10 6 NY-ESO-specific T cells were injected twice peritumor or intravenously (days 10 and 13). T cells were 30% CD4 + T cells and 70% CD8 + The tumor size was measured twice a week with a caliper. 3 When the sigma-induced hypercalcaemia was reached, the mice were sacrificed.

[0300] A97L and I53F transduced T cells, when administered peritumorally, reduced tumor size by 100 mm for 30 days. 3 I53E-transduced T cells performed similarly to wild-type BC1 TCR-transduced T cells in this assay. With non-transduced T cells, tumors grew to approximately 275 mm at day 30. 3 (Figure 14B).

[0301] When administered intravenously, T cells transduced with the I53F TCR (obtained from healthy donor 1) were effective in keeping tumors at a negligible size over a period of 50 days (Figure 15B, left). When T cells from another healthy donor (healthy donor 2) were used to transduce with the I53F TCR, such transduced cells were able to keep tumors at approximately 350 mm over a period of 65 days. 3 was efficient in keeping the concentration below 100 mM (Figure 15B, right).

[0302] When administered intravenously, T cells transduced separately with I53F and 1G4LY (obtained from healthy donor 3) were both effective and showed a similar profile in terms of maintaining smaller tumor volume size with increasing titration compared to non-transduced T cells (Figure 16B).

[0303] Example 6. Modified T cells expressing NY-ESO-1 TCR and GM-CSF material and method mouse NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were housed in-house under specific pathogen-free (SOPF) conditions and all animal experiments were performed under specific pathogen-free (SPF) conditions in the animal facility of the University of Lausanne. All experiments were approved by the veterinary authorities of the canton of Vaud and were performed in accordance with Swiss federal law.

[0304] cell line Wild type parent A375(HLA-A2 + , NY-ESO-1 + ), MelAvl3 (HLA-A2 + , NY-ESO-1 - ) and NA8 (HLA-A2 + , NY-ESO-1 - ) melanoma, H1650 (HLA-A2 + , NY-ESO-1 - ) non-small cell lung cancer, and LN-18 (HLA-A2 + , NY-ESO-1 + ) Glioblastoma cell line was purchased from ATCC. Me275 (HLA-A2 + , NY-ESO-1 + The OVCAR5 (HLA-A2) melanoma cell line was kindly provided by Prof. D. Speiser (University of Lausanne, Lausanne branch of the Ludwig Institute for Cancer Research) and was engineered to stably express luciferase in order to follow its activity in vivo. + , NY-ESO-1 -) and A2008-A2 (HLA-A2 + , NY-ESO-1 - ) Ovarian cancer cell lines were obtained from the University of Pennsylvania and engineered to stably express the full-length NY-ESO-1 cancer / testis family tumor antigen sequence, as well as mCherry and luciferase to track their activity in vitro and in vivo, respectively. All melanoma cell lines were maintained in IMDM+Glutamax (Thermo) supplemented with 10% FCS and 1% penicillin / streptomycin, while the remaining cell lines were maintained in RPMI (Thermo) supplemented with 10% FCS and 1% penicillin / streptomycin.

[0305] Molecular cloning The entire sequence of mouse CSF2 (GM-CSF) cDNA (Uniprot:P01587) was synthesized by Invitrogen. The sequence was then cloned into the pMSGV retroviral vector (Hughes, Human Gene Therapy, 2005;16:457-472) followed by a Thy1.1-T2A (CD90.1) reporter gene cassette using PCR and standard molecular cloning techniques to generate the pMSGV-Thy1.1-T2A-mGM-CSF retroviral plasmid. pMSGV-Thy1.1-T2A was used as a control vector.

[0306] Flow cytometry APC anti-human CD8, APC anti-human CD4, and APC anti-mouse Thy1.1 (CD90.1) were purchased from BioLegend. NY-ESO-1 TCR tetramers were produced in-house by the Peptide and Tetramer facility at the University of Lausanne. FITC anti-human MCSP was purchased from Miltenyi Biotec. APC anti-mouse CD45, APCCy7 anti-mouse F4 / 80, and PECy7 anti-mouse CD11b were provided and / or produced in-house by the Flow Cytometry Facility, FBM, LICR / UNIL. Data acquisition was performed on an LSRII flow cytometer (BD Biosciences) and data were analyzed with FlowJo (TreeStar).

[0307] Retrovirus / lentivirus production To generate retroviral particles, HEK293T cells were cotransfected with the pMSGV transfer plasmid and the retroviral packaging plasmids pMD-Gag / Pol and pMD RD114 (Milone et al., 2018 Leukemia 32, 1529-1541) feline endogenous viral envelope glycoprotein. Culture supernatants were collected 24, 48, and 72 hours after transfection and concentrated by ultracentrifugation at 24,000xg for 2 hours. The concentrated virus was stored at -80°C until use. Viral titers and MOIs were determined by expression of the Thy1.1 reporter gene in HEK293T cells.

[0308] Human T cell stimulation and lentiviral / retroviral transduction Healthy donor apheresis products were purchased from Transfusion Interregionale CRS SA, Epalinges, Switzerland, with written consent under an approved university ethics committee protocol. PBMCs were prepared using Lymphoprep (StemCell Technologies) density gradient centrifugation and CD8 or CD4 T cells were negatively isolated using CD8 or CD4 magnetic microbeads (Miltenyi) according to the manufacturer's protocol. Isolated CD8 and CD4 T cells were stimulated with anti-CD3 / CD28 beads (Invitrogen) at a bead:T cell ratio of 2:1 in the presence of human IL-2 (GlaxoSmithKline).

[0309] Lentiviral / retroviral transduction of human T cells Lentiviral transduction of T cells was performed 24 h after activation by adding viral particles directly to the medium (MOI 20) and was enhanced by the simultaneous addition of Lentiboost (SirionBiotech). Retroviral transduction of T cells was performed 48 h after activation. Briefly, T cells were transferred onto Retronectin-coated plates that had previously been spun with retroviral particles at 2000xg for 1.5 h. The next day, T cells were removed from the Retronectin-coated plates. CD3 / CD28 beads were removed 5 days after activation and T cells were subsequently cultured at 0.5–1x10 until downstream use. 6 T cells / ml were maintained in RPMI 1640-Glutamax (ThermoFisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisher Scientific), 1% penicillin / streptomycin, 10 ng / ml human IL-7 (Miltenyi), and 10 ng / ml IL-15 (Miltenyi).

[0310] IFNγ production / T cell cytotoxicity assay 10 5 of resting T cells (4:1 ratio of CD8 + :CD4 +) in complete medium for 10 5 The cells were co-cultured with 100% T cell lineages for 48 h. T cell numbers were normalized based on transduction efficiency, and non-transduced T cells were added when necessary to achieve similar T cell frequencies between conditions. After 48 h, IFNγ levels in collected cell-free culture supernatants were measured by ELISA (ThermoFisher Scientific) according to the manufacturer's protocol. T cell cytotoxicity was determined by flow cytometric analysis of cells and annexin V in the cultures. + / DAPI + The results were compared between the percentage of annexin V in cultures containing only tumor cells and the percentage of annexin V in cultures containing only tumor cells. + / DAPE + Normalized to the percentage of

[0311] Transgenic mouse GM-CSF production by human T cells T cells (CD8 or CD4) were cultured at 10 6 T cells / ml were cultured for 24 hours in serum-free medium supplemented with 10 ng / ml IL-7 / IL-15. Viability and cell number were previously determined using a hemocytometer. T cell numbers were normalized based on transduction efficiency, and non-transduced T cells were added as needed to achieve similar T cell frequencies between conditions. After 24 hours, mouse GM-CSF levels in collected cell-free supernatants were measured by ELISA (ThermoFisher Scientific) according to the manufacturer's protocol.

[0312] Generation of bone marrow-derived macrophages (BMDMs) Total bone marrow cells were isolated by flushing the femurs and tibias of NSG mice. Macrophages were generated by incubating total bone marrow cells for 7 days in DMEM+Glutamax (Thermo) medium supplemented with 10% FCS, 1% penicillin / streptomycin, 50 μM β-mercaptoethanol, and 50 ng / mL mouse M-CSF (Peprotech) and harvesting the adherent fraction. Medium was refreshed on days 3 and 6.

[0313] Winn assay 5x10 immunizations were administered to the flanks of 6-12 week old male NSG mice. 6 (or the number indicated) of NY-ESO-1 TCR-expressing murine GM-CSF-secreting human T cells (4:1 CD8 + :CD4 + ) or 5x10 premixed with an equivalent number of mock-transduced mouse GM-CSF-secreting human T cells. 6 Me275 melanoma cells were inoculated subcutaneously at 100 x 100 cm. Tumor growth was monitored by caliper measurements twice a week and calculated according to the formula V = 1 / 2(L x W 2 Tumor volume was calculated using the mean diameter (L) of the tumor (W) (where L is the largest longitudinal diameter and W is the largest transverse diameter). 3 Mice were sacrificed when they reached 100 mg / kg, lost >20% of their original body weight, or became weakened and moribund. Each group consisted of ≥5 mice.

[0314] Xenograft model 5x10 immunization was administered to the flank of 6-12 week old male NSG mice. 6 Me275 melanoma cells were inoculated subcutaneously. Concurrently, human T cells were activated, transduced, and expanded as described above. Approximately 2 weeks after inoculation, tumors grew to 50–100 mm 3 T cells were adoptively transferred into mice when the total number of T cells reached 1x10 per injection. 7 NY-ESO-1 TCR-expressing mouse GM-CSF-secreting human T cells (4:1 CD8 + :CD4 + ) or an equivalent number of mock-transduced mice with GM-CSF-secreting human T cells were administered twice on days 13 and 15. Tumor growth was monitored by caliper measurements as above.

[0315] Results and Discussion A schematic of the retroviral murine GM-CSF and lentiviral NY-ESO-1 TCR constructs used in this study is shown in Figure 17A. Seven days after viral transduction, human CD8 + and CD4 +Expression of NY-ESO-1 TCR and mouse GM-CSF by T cells was confirmed by flow cytometry (Figure 17B). Transduced CD8 + Secreted mouse GM-CSF can be detected by ELISA in the supernatants of T cell cultures (FIG. 17C), demonstrating that human T cells can be efficiently co-engineered to stably express the NY-ESO-1 TCR and secrete mouse GM-CSF.

[0316] IFNγ secretion by NY-ESO-1 TCR-engineered T cells was significantly associated with HLA-A2 + NY-ESO-1 + Tumor cells can be detected based on their recognition (Figure 18A). There is no effect of mouse GM-CSF on IFNγ levels. T cells engineered with NY-ESO-1 TCR express HLA-A2 + NY-ESO-1 + Tumor cells can be easily killed, and their cytotoxic activity is not affected by mouse GM-CSF (FIG. 18B), indicating that secreted mouse GM-CSF does not affect the activity of human T cells.

[0317] Human NY-ESO-1 TCR-expressing T cells co-engineered to secrete mouse GM-CSF can effectively delay the engraftment and establishment of Me275 melanoma cells compared to control T cells expressing only NY-ESO-1 TCR (Figure 19A). This is largely reflected in mouse survival, with 40% of mice treated with NY-ESO-1 TCR-expressing mouse GM-CSF-secreting T cells being tumor-free (Figure 19B). 7 Adoptive transfer of NY-ESO-1 TCR-expressing murine GM-CSF-secreting T cells from NSG mice demonstrated superior tumor control of established Me275 tumors compared to NY-ESO-1 TCR alone (Figure 19C), indicating that T cell-derived murine GM-CSF secreted in the tumor microenvironment can significantly enhance the control of tumor growth by NY-ESO-1-specific T cells in the NSG human melanoma xenograft mouse model.

[0318] Example 7. Engineered T cells expressing NY-ESO-1 TCR "I53F" variant and PDE4B2 material and method Molecular cloning To overexpress the human PDE4B2 homologue in human T cells, we used the lentiviral vector pRRL used in Bobisse, S. et al. Cancer Res. 69, 9385-9394 (2009). In particular, the sequence cloned downstream of the human PGK promoter and Kozak element (GCCACC (SEQ ID NO: 31)) encodes human WT PDE4B2 (NCBI Reference Sequence: NP_001032416.1; SEQ ID NO: 27 in FIG. 26) and is fused via a DGGG (SEQ ID NO: 32) linker to the following amino acid sequence: GKPIPNPLLGLDSTGGSGGGKPIPNPLLGLDSTGSGSGSGKPIPNPLLGLDST (SEQ ID NO: 33). This sequence contains three repeats of the V5 tag epitope and two linkers used in Reddy Chichili et al., Protein Sci. 22, 153-167 (2013). For engineering control T cells, the same pRRL vector backbone was used and an eGFP coding sequence was introduced downstream of the human PGK promoter and Kozak element. To confer NY-ESO-1 antigen specificity to T cells, Lamers, CHJ et al. Cancer Gene Ther. 15, 268-274 (2008) was used, which encodes the α and β chains of the NY-ESO-1 TCR variant β-I53F. All introduced DNA sequences were codon-optimized for protein expression in human cells.

[0319] 2. Preparation of Concentrated Lentivirus or Retrovirus For the purpose of generating viral particles, HEK-293T cells were transfected. Briefly, for the generation of lentiviral particles, 18 μg of gag / pol-encoding R8.74 plasmid, 7 μg of envelope protein-encoding VSVG plasmid, and 15 μg of transgene-encoding pRRL vector were diluted in 120 μl of TurboFect (R0532, Thermo Fisher Scientific) premixed with 3 mL of Opti-MEM (11058-021, Thermo Fisher Scientific). Similarly, for the generation of retroviral particles, 18 μg of gag / pol-encoding PegPam, 7 μg of envelope protein-encoding RD114 plasmid, and 22 μg of SFG retroviral vector were diluted in 120 μl of TurboFect (R0532, Thermo Fisher Scientific) premixed with 3 mL of Opti-MEM. Of note, the TurboFect / Opti-MEM solution was incubated for 5 min at room temperature before being used for dilution of packaging / transgene plasmids. Subsequently, the transfection mix was added on top of fresh R10 medium consisting of RPMI 1640 (11875085, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and penicillin / streptomycin (4-01F00-H, Bioconcept) in a T150 tissue culture flask containing 90–95% confluent HEK-293T cells after 30 min of incubation at room temperature. After 24 h, the culture supernatant was replaced with fresh R10, which was harvested after another 24 h and subjected to filtration (45 μm) and centrifugation at 24000 g for 2 h. Finally, the viral particles were resuspended in 400 μl of R10 before being flash frozen on dry ice and placed at -80 °C.

[0320] Isolation, transduction and culture of T cells PBMCs from healthy donors were isolated on day 0 by standard Ficoll-Paque centrifugation and CD4+ / -100% IgG antibodies were isolated using the respective negative isolation kits (130-096-533, 130-096-495 Miltenyi) according to the manufacturer's instructions. + or CD8 + T cells were purified. Immediately after isolation, T cells were stimulated with anti-CD3 / CD28 coated microbeads (11132D, Thermo Fisher Scientific) in R10 at a ratio of 2 beads per T cell in the presence of 50 U / mL recombinant human IL2 (gift from Glaxo SmithKline). 0.5x10 6 T cells / well, 1 / 10 6 T cells were activated at a concentration of 1:100 T cells / mL. For lentiviral transduction, 18-22 h after stimulation in the presence of LentiBOOST™ (Sirion Biotech GmbH), which was used at a dilution of 1:500. 6100 μl of concentrated lentivirus was added per well containing 100 T cells. Retroviral transduction occurred in untreated 48-well cell culture plates. Specifically, such wells were coated with 20 ng / mL RetroNectin (T100B, Takara) solution in PBS overnight at 4 °C, then washed with PBS and subsequently blocked with R10 for 30 min at 37 °C. After washing the wells with PBS, 50 μl of concentrated retrovirus was added together with 50 μl of R10 and the plates were centrifuged at 2000 g for 90 min at 25 °C. To perform retroviral co-transduction, T cells previously exposed to lentivirus were transferred to RetroNectin / retrovirus-coated wells within 40–44 h after stimulation and spun down at 260 g for 10 min. Such T cells were transferred to tissue culture-treated wells 24 h after retroviral transduction. R10 supplemented with IL2 (50 U / mL) to support T cell expansion was added to the cultures until day 5, when T cell activation microbeads were removed. From day 5 onwards, R10 supplemented with 10 ng / mL recombinant human IL7 and IL15 (130-095-764, 130-095-362, Miltenyi) was added to the T cell cultures. Transduction efficiency was assessed by FACS on day 7, and T cells were used for intracellular cAMP quantification or functional assays between days 8–21.

[0321] Measurement of cAMP Resting T cells were washed and incubated with various concentrations of forskolin or PGE 2 T cells were incubated at 1x10 6 T cells / mL, but 2.5x10 per condition 6 T cells were used. Subsequently, T cells were lysed with 250 μl of 0.1 M HCl, and their intracellular cAMP content was assessed with a direct cAMP ELISA kit (ADI-900-066, Enzo Life Sciences) according to the manufacturer's instructions.

[0322] Flow cytometry All FACS data were acquired on an LCRII flow cytometer (BD) and analyzed using FlowJo software. For dead cell exclusion, fixable aqua dead dye L34965 or L34975 (Invitrogen) were used according to the manufacturer's instructions, while the following antibodies were used for T cell staining: CD45: Pac Blue (304029, BioLegend), anti-V5 tag: FITC (R96325, Thermo Fisher Scientific), anti-V5 tag: Dy650 (NBP2-52653C, Novus biologicals), Vb13.1: PE (IM2292, BD), IFNγ: PeCy7 (502527, BioLegend), TNF-α: PE (502909, BioLegend), anti-BrDU: APC (12-5071-42, Thermo Fisher Scientific). 50x10 cells per well to assess intracellular cytokine production by FACS 3 live T cells were stimulated with a combination of plate-coated aCD3 (5 μg / mL) and soluble aCD28 (2 μg / mL) for 7 h in round-bottom 96-well plates. Golgi stop (554724, BD) was added to the wells at a dilution of 1:400 1.5 h after the start of the assay to prevent cytokine secretion. T cells were fixed and permeabilized using a standard fixation / permeabilization kit (554714, BD) according to the manufacturer's instructions before assessing transduction efficiency or the ability to produce cytokines. T cell proliferation was quantified using a BrdU staining kit for flow cytometry (8817-6600-42, Thermo Fisher Scientific). Briefly, 1x10 per well were incubated with 1x10 PBS. 5 of live T cells were activated for 48 h in flat-bottom 96-well plates with plate-coated aCD3 (5 μg / mL) and BrdU was added to wells at a working concentration of 10 μM 7 h prior to the end of the assay.

[0323] Quantification of IFNγ secretion Resting CD8 T cells transduced with GFP, PDE4B2, eGFP&NY-ESO-1 TCR, or PDE4B2&NY-ESO-1 TCR were treated with various concentrations of PGE. 2 The presence of IFNγ in the coculture supernatant was evaluated using an ELISA kit (88-7316-88, Thermo Fisher Scientific) according to the manufacturer's instructions. 5 A375 cells, 1x10 5 TCR + of live T cells or the corresponding number of eGFP- / PDE4B2-transduced T cells were added per well.

[0324] Cytotoxicity assay Resting CD4 or CD8 T cells transduced with eGFP, PDE4B2, eGFP&NY-ESO-1 TCR, or PDE4B2&NY-ESO-1 TCR were incubated with PGE 2 The cells were co-cultured with A375 cells for 47 or 44 hours in the presence or absence of 1x10 4 A375 tumor cells, as well as 25x10 3 TCR + The experiments were performed in flat-bottom 96-well plates with either live T cells or a corresponding number of eGFP- / PDE4B2-transduced T cells added per well. The A375 tumor cells used were engineered to express the nuclear fluorescent protein mCherry to allow for direct detection within the well.

[0325] Results and Discussion Expression of exogenous PDE4B2 or co-expression of exogenous PDE4B2 and NY-ESO-1 TCR variant β-I53F in primary human T cells was confirmed by flow cytometry (Figure 21). Because the NY-ESO-1 TCR contains the Vβ13.1 variant of the TCR β chain, the presence of the former can be inferred, although not entirely accurately, by detection of Vβ13.1.

[0326] Intracellular cAMP levels in resting CD4+ T cells transduced with eGFP or PDE4B2 were measured using Fsk or PGE 2 The levels of cAMP in T cells were measured after 1 hour of exposure to PDE4B2. After treatment, T cells were lysed and cAMP levels were quantified by ELISA. The results show that overexpression of PDE4B2 prevents the accumulation of intracellular cAMP (Figure 22).

[0327] The ability of resting CD4+ T cells transduced with eGFP or PDE4B2 to produce IFNγ or TNF-α was assessed by PGE 2 or forskolin, and stimulated with or without plate-bound aCD3 and soluble aCD28 for 7 h, as determined by intracellular cytokine staining (ICS). 2 IFNγ secretion by resting CD8 T cells transduced with eGFP, PDE4B2, eGFP&NY-ESO-1 TCR, or PDE4B2&NY-ESO-1 TCR in response to 48 h of coculture with NY-ESO-1-presenting melanoma cells A375 in the presence of IFNγ or forskolin was assessed by ELISA. Both results show that overexpression of PDE4B2 promotes Th-1 cytokine production under conditions that induce intracellular cAMP accumulation (Figures 23A, 23B).

[0328] The ability of CD4+ T cells transduced with eGFP or PDE4B2 to proliferate was determined by BrDU incorporation assay. T cells were transduced with PGE 2 or forskolin, and restimulated with or without plate-bound aCD3 for 48 h. The results show that overexpression of PDE4B2 promotes proliferation under conditions that induce intracellular cAMP accumulation (FIG. 24).

[0329] Resting CD4 or CD8 T cells transduced with eGFP, PDE4B2, eGFP&NY-ESO-1 TCR, or PDE4B2&NY-ESO-1 TCR expressed PGE 2The ability of PDE4B2 to suppress the growth of NY-ESO-1-presenting melanoma cells A375 in the presence or absence of PGE was evaluated by IncuCyte. As shown in FIG. 25, overexpression of PDE4B2 suppresses the growth of NY-ESO-1-presenting melanoma cells A375 in the presence or absence of PGE. 2 Promotes T cell cytotoxicity in the presence of

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[0331] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such variations are intended to be included within the scope of the appended claims.

[0332] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein. Finally, preferred embodiments of the present invention are described in sections. [Embodiment 1] A polynucleotide encoding a modified T cell receptor (TCR) or a functional fragment thereof, wherein the modified TCR comprises a single amino acid substitution within the complementarity determining region (CDR)2 of the β chain of the modified TCR compared to CDR2 of the β chain of an unsubstituted wild type (WT) TCR. [Embodiment 2] 2. The polynucleotide of embodiment 1, wherein the modified TCR beta chain sequence comprises an amino acid sequence that is at least 80% identical to the unsubstituted WT TCR beta chain, or a functional fragment thereof, outside the CDR2 region of the modified TCR beta chain, or a functional fragment thereof. [Embodiment 3] 3. The polynucleotide of embodiment 1 or 2, wherein the modified TCR beta chain or functional fragment thereof comprises an amino acid sequence having a single amino acid substitution in the CDR2 region in the amino acid sequence of a non-substituted WT TCR beta chain or functional fragment thereof. [Embodiment 4] 4. The polynucleotide according to any one of embodiments 1 to 3, wherein the β chain of the unsubstituted WT TCR comprises the amino acid sequence of SEQ ID NO:1. [Embodiment 5] 5. The polynucleotide of any of embodiments 1 to 4, wherein the single amino acid substitution occurs at residue 50, 51, 53, or 55 relative to the WT TCR. [Embodiment 6] 6. The polynucleotide according to any of embodiments 1 to 5, wherein said single amino acid substitution occurs at residue 53 or 55 relative to the WT TCR. [Embodiment 7] 7. The polynucleotide according to any one of embodiments 1 to 6, wherein the single amino acid substitution is I53E, I53F, I53W, or D55E. [Embodiment 8] 8. The polynucleotide of any one of embodiments 1 to 7, wherein the modified TCR binds to a cancer antigen with a higher binding affinity than a WT TCR. [Embodiment 9] The cancer antigen is NY-ESO-1 157-165 The polynucleotide of embodiment 8, which is an epitope (SEQ ID NO: 8). [Embodiment 10] 10. The polynucleotide according to embodiment 8 or 9, wherein the binding affinity of the modified TCR to the cancer antigen is about 5 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. [Embodiment 11] 11. The polynucleotide of embodiment 10, wherein the binding affinity of the modified TCR to the cancer antigen is about 10 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. [Embodiment 12] 12. The polynucleotide of embodiment 11, wherein the binding affinity of the modified TCR to the cancer antigen is about 25 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. [Embodiment 13] 13. The polynucleotide of embodiment 12, wherein the binding affinity of the modified TCR to the cancer antigen is about 40 to about 75 times higher than the binding affinity of the WT TCR to the cancer antigen. [Embodiment 14] 14. The polynucleotide of embodiment 13, wherein the binding affinity of the modified TCR to the cancer antigen is about 40 to about 60 times higher than the binding affinity of the WT TCR to the cancer antigen. [Embodiment 15] 15. The polynucleotide of embodiment 14, wherein the binding affinity of the modified TCR to the cancer antigen is about 40 to about 50 times higher than the binding affinity of the WT TCR to the cancer antigen. [Embodiment 16] 10. The polynucleotide of embodiment 8 or 9, wherein the binding affinity of the modified TCR to the cancer antigen is about 50-fold higher compared to the binding affinity of the WT TCR to the cancer antigen. [Embodiment 17] The dissociation constant (K D 10. The polynucleotide of embodiment 8 or 9, wherein the mAb is between about 0.30 μM and about 4.5 μM. [Embodiment 18] The dissociation constant (K D 18. The polynucleotide of embodiment 17, wherein the CpG concentration is between about 0.30 μM and about 2 μM. [Embodiment 19] The dissociation constant (K D 18. The polynucleotide of embodiment 17, wherein the CpG concentration is between about 2 μM and about 3 μM. [Embodiment 20] The dissociation constant (K D 18. The polynucleotide of embodiment 17, wherein the CpG concentration is between about 3 μM and about 4 μM. [Embodiment 21] The dissociation constant (K D 20. The polynucleotide of embodiment 17, wherein the CpG2+ signal is about 0.41 μM. [Embodiment 22] The dissociation constant (K D 18. The polynucleotide of embodiment 17, wherein the CpG2+ signal is about 3.89 μM. [Embodiment 23] A polynucleotide according to any one of embodiments 1 to 22, wherein the modified TCR comprises an amino acid sequence of any one of SEQ ID NOs: 2 to 5, or a functional fragment thereof, or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 5. [Embodiment 24] A polynucleotide according to any one of embodiments 1 to 23, wherein the modified TCR is encoded by a nucleotide sequence of any one of SEQ ID NOs: 11 to 14, or a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 11 to 14. [Embodiment 25] 25. A polynucleotide according to any one of embodiments 1 to 24, operably linked to at least one regulatory element for expression of the modified TCR. [Embodiment 26] 26. The polynucleotide of embodiment 25, wherein the at least one regulatory element is a promoter. [Embodiment 27] 27. A polynucleotide according to any one of embodiments 1 to 26, which is a DNA molecule. [Embodiment 28] 27. The polynucleotide according to any one of embodiments 1 to 26, which is an RNA molecule or a derivative thereof. [Embodiment 29] A recombinant vector comprising a polynucleotide according to any one of embodiments 1 to 28, wherein the polynucleotide is operably linked to at least one regulatory element for expression of a modified T cell receptor (TCR). [Embodiment 30] 30. The vector of embodiment 29, which is a viral vector. [Embodiment 31] The vector of embodiment 30, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphavirus vector, a herpes viral vector, or a vaccinia viral vector. [Embodiment 32] 30. The vector of embodiment 29, which is a non-viral vector. [Embodiment 33] A modified T cell receptor (TCR) comprising a modified TCR β chain or a functional fragment thereof encoded by a polynucleotide according to any one of embodiments 1 to 28. [Embodiment 34] a) a β chain of a modified TCR encoded by a polynucleotide according to any one of embodiments 1 to 29, or a functional fragment thereof; and b) α chain or a functional fragment thereof A modified T cell receptor (TCR). [Embodiment 35] A modified T cell receptor (TCR) comprising the CDRs of the β chain of a modified TCR encoded by a polynucleotide according to any one of embodiments 1 to 28. [Embodiment 36] a) a functional fragment of a β chain of a modified TCR, the functional fragment comprising the CDRs of the β chain encoded by a polynucleotide according to any one of embodiments 1 to 28; and b) a functional fragment of the α chain, the functional fragment comprising the CDRs of the α chain A modified T cell receptor (TCR). [Embodiment 37] A modified TCR as described in embodiment 36, wherein the functional fragment of a) further comprises a constant region of a TCR beta chain and / or the functional fragment of b) further comprises a constant region of a TCR alpha chain. [Embodiment 38] 38. A modified TCR as described in embodiment 37, wherein any of the constant regions is of human origin. [Embodiment 39] A modified TCR as described in embodiment 37, wherein any of the constant regions is of mouse origin. [Embodiment 40] A modified TCR according to any one of embodiments 34 to 39, wherein the alpha chain comprises the alpha chain of a WT TCR or a functional fragment thereof. [Embodiment 41] A modified TCR as described in embodiment 40, wherein the alpha chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:7. [Embodiment 42] A modified TCR described in any one of embodiments 33 to 41, comprising an amino acid sequence of any one of SEQ ID NOs: 2 to 5 or a fragment thereof, or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 5. [Embodiment 43] A modified T cell receptor (TCR), comprising an α chain comprising the amino acid sequence of SEQ ID NO:7, and a β chain comprising any one of the amino acid sequences of SEQ ID NOs:2 to 5. [Embodiment 44] An isolated host cell comprising a modified T cell receptor (TCR) according to any one of embodiments 33 to 43. [Embodiment 45] An isolated host cell comprising a polynucleotide according to any one of embodiments 1 to 28. [Embodiment 46] 46. ​​The isolated host cell of embodiment 45, wherein the polynucleotide is operably linked to at least one regulatory element capable of mediating expression of the modified T cell receptor (TCR) in the host cell. [Embodiment 47] An isolated host cell comprising a vector according to any one of embodiments 29 to 32. [Embodiment 48] 48. An isolated host cell according to any one of embodiments 44 to 47, which is a mammalian cell. [Embodiment 49] An isolated host cell according to any one of embodiments 44 to 48, which is a lymphocytic cell. [Embodiment 50] 50. The isolated host cell of embodiment 49, wherein the lymphoid cell is a T cell. [Embodiment 51] 50. The isolated host cell of embodiment 49, wherein the lymphoid cell is a natural killer (NK) cell. [Embodiment 52] 52. The isolated host cell according to any of embodiments 48 to 51, which is obtained from a peripheral blood mononuclear cell (PBMC), a tumor-draining lymph node, or a tumor infiltrate. [Embodiment 53] 53. An isolated host cell according to any of embodiments 44 to 52, which has been activated and / or expanded ex vivo. [Embodiment 54] 54. The isolated host cell according to any one of embodiments 44 to 53, which is an allogeneic cell. [Embodiment 55] 54. The isolated host cell according to any one of embodiments 44 to 53, which is an autologous cell. [Embodiment 56] 56. The isolated autologous host cell of embodiment 55, which is isolated from a subject having a disease. [Embodiment 57] 57. The isolated autologous host cell of embodiment 56, wherein the disease is cancer. [Embodiment 58] The cancer expresses the cancer antigen NY-ESO-1 on the surface of its cells. 157-165 58. An isolated autologous host cell as described in embodiment 57, which presents the epitope (SEQ ID NO: 8). [Embodiment 59] The isolated autologous host cell of embodiment 57 or 58, wherein the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer. [Embodiment 60] 60. An isolated host cell according to any of embodiments 44 to 59, further engineered to express one or more exogenous molecules. [Embodiment 61] 61. The isolated host cell of embodiment 60, wherein the one or more exogenous molecules are immune signaling molecules. [Embodiment 62] 62. The isolated host cell of embodiment 61, wherein the immune signaling molecule is a cytokine. [Embodiment 63] 62. The isolated host cell of embodiment 61, wherein the immune signaling molecule is a chemokine. [Embodiment 64] 62. The isolated host cell of embodiment 61, wherein the immune signaling molecule is a growth factor. [Embodiment 65] 65. The isolated host cell of embodiment 64, wherein the growth factor is granulocyte-macrophage colony-stimulating factor (GM-CSF). [Embodiment 66] An isolated host cell comprising a T cell receptor (TCR) or a functional fragment thereof that binds to a cancer antigen, the isolated host cell being further engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF). [Embodiment 67] The cancer antigen is NY-ESO-1 157-165 67. The isolated host cell of embodiment 66, wherein the epitope is (SEQ ID NO: 8). [Embodiment 68] 68. The isolated host cell of embodiment 66 or 67, wherein the T cell receptor (TCR) is a WT TCR. [Embodiment 69] 69. An isolated host cell according to any of embodiments 66 to 68, wherein the β chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1. [Embodiment 70] 70. An isolated host cell according to any of embodiments 66 to 69, wherein the alpha chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:7. [Embodiment 71] 71. The isolated host cell according to any one of embodiments 65 to 70, wherein the amino acid sequence of GM-CSF comprises SEQ ID NO: 21, 34 or 15, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 21, 34 or 15. [Embodiment 72] 72. The isolated host cell according to any one of embodiments 65 to 71, wherein the nucleotide sequence encoding GM-CSF comprises SEQ ID NO: 22, 35 or 16, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 22, 35 or 16. [Embodiment 73] 62. The isolated host cell of embodiment 60 or 61, wherein the one or more exogenous molecules are soluble receptors. [Embodiment 74] 62. The isolated host cell of embodiment 60 or 61, wherein the one or more exogenous molecules are ligands. [Embodiment 75] 62. The isolated host cell of embodiment 60 or 61, wherein the one or more exogenous molecules are antigen binding proteins. [Embodiment 76] 76. The isolated host cell of embodiment 75, wherein the antigen binding protein is an antibody or an antibody fragment. [Embodiment 77] 61. The isolated host cell of embodiment 60, wherein the one or more exogenous molecules is a phosphodiesterase. [Embodiment 78] 78. The isolated host cell of embodiment 77, wherein the phosphodiesterase is PDE4B2. [Embodiment 79] An isolated host cell comprising a T cell receptor (TCR) or a functional fragment thereof that binds to a cancer antigen, the isolated host cell being further engineered to express PDE4B2. [Embodiment 80] The cancer antigen is NY-ESO-1 157-165 80. The isolated host cell of embodiment 79, wherein the epitope is (SEQ ID NO:8). [Embodiment 81] 81. The isolated host cell of embodiment 79 or 80, wherein the T cell receptor (TCR) is a WT TCR. [Embodiment 82] 82. An isolated host cell according to any of embodiments 79 to 81, wherein the β chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1. [Embodiment 83] 83. An isolated host cell according to any of embodiments 79 to 82, wherein the alpha chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:7. [Embodiment 84] 84. An isolated host cell according to any of embodiments 78 to 83, wherein the amino acid sequence of PDE4B2 comprises SEQ ID NO: 27 or 29, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 27 or 29. [Embodiment 85] An isolated host cell according to any of embodiments 78 to 84, wherein the nucleotide sequence encoding PDE4B2 comprises SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 28 or 30. [Embodiment 86] 61. The isolated host cell of embodiment 60, wherein the one or more exogenous molecules are cell surface receptors. [Embodiment 87] 87. The isolated host cell of embodiment 86, wherein the cell surface receptor is a chimeric antigen receptor. [Embodiment 88] The cell surface receptor is a cancer antigen NY-ESO-1 157-165 87. The isolated host cell of embodiment 86, wherein the T cell receptor does not bind to the epitope (SEQ ID NO: 8). [Embodiment 89] A bifunctional molecule comprising a modified T cell receptor (TCR) or a functional fragment thereof according to embodiments 33 to 43, and an immune effector polypeptide that specifically binds to a cell surface protein on a T cell. [Embodiment 90] 90. The bifunctional molecule of embodiment 89, wherein said immune effector polypeptide comprises an antibody or an antibody fragment. [Embodiment 91] 91. The bifunctional molecule of embodiment 89 or 90, wherein said immune effector polypeptide comprises a single chain variable region fragment (scFv). [Embodiment 92] 92. The bifunctional molecule of any of embodiments 89 to 91, wherein the immune effector polypeptide specifically binds to CD3. [Embodiment 93] 93. The bifunctional molecule of embodiment 92, wherein the immune effector polypeptide comprises an antibody or antibody fragment derived from OKT3, UCHT-1, BMA031, or 12F6. [Embodiment 94] A pharmaceutical composition comprising a host cell according to any of embodiments 44 to 88, or a bifunctional molecule according to any of embodiments 89 to 93, and a pharma- ceutically acceptable carrier and / or excipient. [Embodiment 95] A pharmaceutical composition according to embodiment 94, for use in adoptive cell transfer therapy. [Embodiment 96] A method for producing a host cell according to any of embodiments 44 to 88, comprising genetically engineering a host cell with a polynucleotide according to any of embodiments 1 to 28 or a vector according to any of embodiments 29 to 32. [Embodiment 97] A method for genetically engineering a host cell to express a modified T cell receptor (TCR) or a functional fragment thereof according to any one of embodiments 33 to 43. [Embodiment 98] The method according to embodiment 97, comprising genetically engineering a host cell with a polynucleotide according to any of embodiments 1 to 28 or a vector according to any of embodiments 29 to 32. [Embodiment 99] The method of any one of embodiments 96 to 98, wherein the genetic manipulation step is carried out via viral gene delivery. [Embodiment 100] The method of embodiment 99, wherein the genetic manipulation step is carried out via non-viral gene delivery. [Embodiment 101] The method according to any one of embodiments 96 to 100, which is carried out ex vivo. [Embodiment 102] 102. The method of any of embodiments 96-101, further comprising activating and / or expanding the host cells ex vivo. [Embodiment 103] A method according to any one of embodiments 96 to 102, wherein the modified TCR comprises an amino acid sequence of any one of SEQ ID NOs: 2 to 5 or a fragment thereof, or an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2 to 5. [Embodiment 104] The method of any of embodiments 96 to 103, further comprising genetically engineering the host cell to further express one or more exogenous molecules. [Embodiment 105] 105. The method of embodiment 104, wherein the one or more exogenous molecules are immune signaling molecules. [Embodiment 106] The method of embodiment 104 or 105, wherein the exogenous molecule is a cytokine, chemokine, growth factor, soluble receptor, ligand, phosphodiesterase, antigen-binding protein, or cell surface receptor. [Embodiment 107] The method of embodiment 106, wherein the growth factor is granulocyte-macrophage colony-stimulating factor (GM-CSF). [Embodiment 108] 108. The method of embodiment 107, wherein the amino acid sequence of GM-CSF comprises SEQ ID NO: 21, 34 or 15, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 21, 34 or 15. [Embodiment 109] 109. The method of embodiment 107 or 108, wherein the nucleotide sequence encoding GM-CSF comprises SEQ ID NO: 22, 35 or 16, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 22, 35 or 16. [Embodiment 110] The method of embodiment 106, wherein the phosphodiesterase is PDE4B2. [Embodiment 111] The method of embodiment 110, wherein the amino acid sequence of PDE4B2 comprises SEQ ID NO: 27 or 29, or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 27 or 29. [Embodiment 112] 112. The method of embodiment 110 or 111, wherein the nucleotide sequence encoding PDE4B2 comprises SEQ ID NO: 28 or 30, or a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 28 or 30. [Embodiment 113] 107. The method of embodiment 106, wherein the antigen binding protein is an antibody or an antibody fragment. [Embodiment 114] The cell surface receptor is a chimeric antigen receptor or a cancer antigen NY-ESO-1 157-165 The method described in embodiment 106, wherein the T cell receptor does not bind to the epitope (sequence number 8). [Embodiment 115] The method according to any one of embodiments 96 to 114, wherein the host cell is a mammalian cell. [Embodiment 116] A method according to any one of embodiments 96 to 115, wherein the host cell is a lymphoid cell. [Embodiment 117] The method of embodiment 116, wherein the lymphoid cell is a T cell. [Embodiment 118] The method of embodiment 116, wherein the lymphoid cells are natural killer (NK) cells. [Embodiment 119] The method according to any of embodiments 115 to 118, wherein the host cells are obtained from peripheral blood mononuclear cells (PBMCs), tumor-draining lymph nodes, or tumor infiltrates. [Embodiment 120] 120. The method of any of embodiments 96-119, wherein the host cells are activated and / or expanded ex vivo. [Embodiment 121] The method according to any one of embodiments 96 to 120, wherein the host cell is an allogeneic cell. [Embodiment 122] The method according to any one of embodiments 96 to 120, wherein the host cell is an autologous cell. [Embodiment 123] 123. The method according to any one of embodiments 96 to 122, wherein the host cell is isolated from a subject having a disease. [Embodiment 124] The method of embodiment 123, wherein the disease is cancer. [Embodiment 125] The cancer cells express the cancer antigen NY-ESO-1 on their surface. 157-165 The method described in embodiment 124, which presents the epitope (sequence number 8). [Embodiment 126] The method of embodiment 124 or 125, wherein the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer. [Embodiment 127] A method for stimulating or enhancing an immune response in a mammal in need thereof, comprising administering to the mammal an effective amount of a lymphoid cell comprising a modified T cell receptor (TCR) according to any of embodiments 33 to 43, a host cell according to any of embodiments 44 to 88, a bifunctional molecule according to any of embodiments 89 to 93, a composition according to embodiment 94 or 95, or a host cell produced by the method according to any of embodiments 96 to 126. [Embodiment 128] A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a lymphoid cell comprising a modified T cell receptor (TCR) according to any of embodiments 33 to 43, a host cell according to any of embodiments 44 to 88, a bifunctional molecule according to any of embodiments 89 to 93, a composition according to embodiment 94 or 95, or a host cell produced by the method according to any of embodiments 96 to 126. [Embodiment 129] The cancer cells express the cancer antigen NY-ESO-1 on their surface. 157-165 The method described in embodiment 128, which presents the epitope (sequence number 8). [Embodiment 130] The method of embodiment 128 or 129, wherein the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, urinary bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer. [Embodiment 131] a) isolating T cells from a subject or mammal; b) genetically modifying said T cells ex vivo using a polynucleotide according to any of embodiments 1 to 28 or a vector according to any of embodiments 29 to 32; c) optionally expanding and / or activating said T cells before, after or during step b); d) introducing the genetically modified T cells into the subject or mammal; The method according to any one of embodiments 128 to 130, comprising: [Embodiment 132] The method according to any one of embodiments 128 to 131, wherein the subject or mammal is a human.

Claims

1. A polynucleotide encoding a modified T cell receptor (TCR) or a functional fragment thereof, the modified TCR comprising an α chain comprising the amino acid sequence of SEQ ID NO:7 and a β chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3, the β chain comprising a complementarity determining region (CDR) 1 having the amino acid sequence NHE, a CDR2 having the amino acid sequence of SEQ ID NO:56, and a CDR3 having the amino acid sequence of SEQ ID NO:62, the modified TCR encoding a cancer antigen NY-ESO-1 １５７－１６５ A polynucleotide that binds to the epitope (SEQ ID NO:8).

2. 2. The polynucleotide of claim 1, wherein the binding affinity of the modified TCR to the cancer antigen is 5-75 times higher than the binding affinity of the WT TCR to the cancer antigen.

3. The dissociation constant (K Ｄ 2. The polynucleotide of claim 1, wherein the .alpha.-amino acid salt is between 0.30 μM and 4.5 μM.

4. The polynucleotide of any one of claims 1 to 3, wherein the beta chain of the modified TCR is encoded by a nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

12.

5. A polynucleotide according to any one of claims 1 to 4, operably linked to at least one regulatory element for expression of the modified TCR.

6. The polynucleotide of claim 5 , wherein the at least one regulatory element is a promoter.

7. 7. The polynucleotide of any one of claims 1 to 6, which is (i) a DNA molecule, or (ii) an RNA molecule or a derivative thereof.

8. 8. A recombinant vector comprising the polynucleotide of any one of claims 1 to 7, wherein the polynucleotide is operably linked to at least one regulatory element for expression of a modified T cell receptor (TCR).

9. A modified T cell receptor (TCR) encoded by the polynucleotide of any one of claims 1 to 7.

10. 10. The modified T cell receptor (TCR) of claim 9, comprising an alpha chain comprising the amino acid sequence of SEQ ID NO:7, and a beta chain comprising the amino acid sequence of SEQ ID NO:

3.

11. (i) a modified T cell receptor (TCR) according to claim 9 or 10; (ii) a polynucleotide according to any one of claims 1 to 7; or (iii) the vector according to claim 8; 2. An isolated host cell comprising:

12. 12. The isolated host cell of claim 11, further engineered to express one or more exogenous molecules, wherein the one or more exogenous molecules are an immune signaling molecule, a soluble receptor, an antigen binding protein, or a phosphodiesterase.

13. 13. The isolated host cell of claim 12, wherein the one or more exogenous molecules is granulocyte-macrophage colony-stimulating factor (GM-CSF) or PDE4B2.

14. 11. A bifunctional molecule comprising a modified T cell receptor (TCR) of claim 9 or 10 and an immune effector polypeptide that specifically binds to a cell surface protein on a T cell.

15. A pharmaceutical composition comprising a host cell according to any one of claims 11 to 13, or a bifunctional molecule according to claim 14, and a pharma- ceutically acceptable carrier and / or excipient.

16. A method for producing ex vivo a host cell according to any one of claims 11 to 13, comprising genetically engineering a host cell with a polynucleotide according to any one of claims 1 to 7 or a vector according to claim 8.

17. 16. A composition for stimulating or enhancing an immune response in a mammal in need thereof, comprising a host cell according to any one of claims 11 to 13, a bifunctional molecule according to claim 14, or a pharmaceutical composition according to claim 15.

18. 16. A composition for treating cancer in a subject in need thereof, comprising a host cell according to any one of claims 11 to 13, a bifunctional molecule according to claim 14, or a pharmaceutical composition according to claim 15.

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