A2 / NY-ESO-1 specific T cell receptor and its use

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 819,988, filed on March 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] [0001.1] This application contains a sequence table submitted electronically in ASCII format, and its entire contents are incorporated herein by reference. The ASCII copy was created on July 16, 2020, named 252457_000009_SL.txt, and has a size of 65,448 bytes. Field of the Invention

[0005] The present invention relates to genetically modified T cell receptors specific for epitopes 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 cell populations, 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.

[0006] background

[0007] Cancer immunotherapy has become a viable alternative and powerful supplement to standard care methods such as cytotoxic chemotherapy, radiotherapy and surgery by utilizing and / or enhancing the patient's own immune response to cancer cells (Mellman, I., G. Coukos, and G. Dranoff, Nature, 2011. 480: p. 480). Among many aspects, adoptive T cell therapy has long proven its effectiveness in the clinic (Restifo, NP, MEDudley, and SA Rosenberg, Nature Reviews Immunology, 2012. 12: p. 269). In this type of cancer treatment, patients receive autologous ex vivo expanded tumor-infiltrating lymphocytes that can naturally recognize and kill their tumor targets (Rosenberg, SA et al., Clin Cancer Res, 2011. 17 (13): p. 4550-7). Alternatively, the patient's peripheral blood T cells can be genetically modified to recognize tumor cells by 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 (Robbins, PF et al., J Clin Oncol, 2011.29(7): p.917-24) and CD19 CAR-based (Kochenderfer, JN et al., Blood, 2012.119: p.2709-2720) T cell therapies are extraordinary examples of cancer immunotherapy, showing great promise in the clinic, for solid tumors and hematological malignancies, respectively.

[0008] NY-ESO-1 or New York esophageal squamous cell carcinoma 1 is a tumor antigen expressed by a range of tumors (Chen, YT et al., Proc Natl Acad Sci USA, 1997. 94(5): p. 1914-1918). The class I human leukocyte antigen (HLA) molecules of these cancer cells present peptides from this antigen, including a 9-amino acid fragment (SLLMWITQC (SEQ ID NO: 8)) from positions 157-165. Therefore, NY-ESO-1 157-165 The epitope-HLA-A2 complex provides a cancer marker that can be targeted by TCRs, for example in adoptive cell transfer therapy, in which patients receive cells engineered to encode for NY-ESO-1. 157-165 Autologous cells transduced with nucleic acid expressing a T cell receptor (TCR) specific for the epitope (SLLMWITQC (SEQ ID NO: 8)). However, for this purpose, it would be ideal if the TCR had a higher affinity for the peptide-HLA complex than the natural TCR specific for the complex.

[0009] Because TCRs directed against "self" tumor antigens may have lower affinity than TCRs specific for viral epitopes, for example due to thymic negative selection, there is a need in the art to develop TCRs that maintain specificity (i.e., do not cross-react with healthy tissue) and have higher affinity, which can have higher activity.

[0010] Although immunotherapy may be a very effective strategy, many patients still do not show clinical benefits of adoptive T cell therapy or cannot maintain a durable response, so its application remains limited. Although treatment-related toxicity remains a major concern (Gust, J. et al. Cancer Discovery, 2017.7: p.1404-1419), the main reason why adoptively transferred T cells cannot achieve effective tumor control is the immunosuppressive microenvironment of the tumor itself (Zou, W. Nature Reviews Cancer, 2005.5: p.263). Tumors use a variety of strategies to evade detection by the immune system. These include altering the chemokine expression profile to prevent T cell chemoattraction; enhancing the physical barrier by enhancing collagen in surrounding tissues and promoting aberrant intratumoral vasculature to prevent proper T cell extravasation; establishing a metabolic microenvironment that is unfavorable to T cell function, characterized by low glucose levels; chemoattraction of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment or polarization of other cells to acquire an immunosuppressive phenotype, such as M2 macrophages; and finally, direct inhibition of T cell function by upregulating immune checkpoint receptors and other membrane-bound or soluble factors with similar properties (Baruch, EN et al., Cancer, 2017. 123(S11): p. 2154-2162).

[0011] These obstacles can be overcome by combinatorial therapeutic strategies that enhance the activity of adoptively transferred T cells by addressing different aspects of the immunosuppressive tumor microenvironment. A myriad of approaches are currently being tested at the preclinical and clinical levels, including immune checkpoint blocking antibodies, cytokine therapy, inhibitors of metabolic enzymes, agents that normalize tumor vasculature, and antibodies that deplete suppressive immune cells in combination with chemotherapy and radiotherapy (Zhang, H. and J. Chen, Journal of Cancer, 2018.9(10): p.1773-1781). Another more sophisticated and targeted approach is to fine-tune the transferred T cells by genetically modifying them with molecules that directly affect T cell activity or target the tumor microenvironment. T cell processing with chemokine receptors has been shown to enhance T cell trafficking to tumor sites, CARs targeting VEGF receptors can lead to normalization of the vasculature, and overexpression or integration of co-stimulatory ligands in CARs provides additional advantages for transferred T cells. In addition, targeting T cell-associated checkpoint ligands may alleviate tumor-derived checkpoint-mediated T cell suppression (Kunert, A. and R. Debets, Current Opinion in Immunology, 2018. 51: p. 133-139).

[0012] Although it is not surprising that most engineering-based strategies aim to directly enhance T cell activity (Yoon, DH et al., International journal of molecular sciences, 2018. 19(2): p. 340), no efforts have been described to modify adoptively transferred T cells with agents that can harness 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 stimulating their anti-tumor versus pro-tumor properties may be very important (Noy, R. and JW Pollard, Immunity, 2014. 41(1): p. 49-61; Fridlender, ZG and SM Albelda, Carcinogenesis, 2012. 33(5): p. 949-55). In the same direction, immune attraction and stimulation of dendritic cells (DCs) by ectopically expressed tumor-derived soluble factors have been shown to elicit strong anti-tumor responses mediated by activated endogenous T cells (Mach, N. et al., Cancer Res, 2000. 60(12): p.3239-46). These observations emphasize the need for suitable T cell engineering candidate agents that can effectively utilize the anti-tumor potential of the above-mentioned tumor-resident immune cell lineages.

[0013] The invention disclosed herein addresses this need and other related needs. SUMMARY OF THE INVENTION

[0015] There is a great need in the art to develop TCRs with higher affinity for cancer antigens. The present invention provides a TCR for the cancer antigen NY-ESO-1. 157-165 Epitope-specific modified TCRs, and related compositions and methods for using such TCRs for cancer immunotherapy (e.g., adoptive cell therapy) address this need and other needs.

[0016] 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 the complementarity determining region (CDR) 2 of its β chain relative to the CDR2 of the β chain of an unsubstituted wild-type (WT) TCR.

[0017] In some embodiments, outside the CDR2 region of the β chain of the modified TCR or its functional fragment, the β chain sequence of the modified TCR comprises an amino acid sequence that is at least 80% identical to the β chain of the unsubstituted WT TCR or its functional fragment.

[0018] In some embodiments, the β chain of the modified TCR or a functional fragment thereof comprises the amino acid sequence of the β chain of an unsubstituted WT TCR or a functional fragment thereof, with a single amino acid substitution in the CDR2 region.

[0019] In some embodiments, the β chain of the unsubstituted WT TCR comprises the amino acid sequence of SEQ ID NO:1.

[0020] In some embodiments, the single amino acid substitution occurs at residue 50, 51, 53, or 55 relative to the WT TCR. In some embodiments, the single amino acid substitution occurs at residue 53 or 55 relative to the WT TCR. In some embodiments, the single amino acid substitution is I53E, I53F, I53W, or D55E.

[0021] In some embodiments, the modified TCR binds to a cancer antigen with a higher binding affinity than a WT TCR. In some embodiments, the cancer antigen is NY-ESO-1 157-165 epitope (SEQ ID NO: 8).

[0022] 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 modified TCR has a binding affinity for the cancer antigen that is about 40 to about 50 times higher than the binding affinity of the WT TCR for the cancer antigen. In some embodiments, the modified TCR has a binding affinity for the cancer antigen that is about 50 times higher than the binding affinity of the WT TCR for the cancer antigen.

[0023] In some embodiments, the dissociation constant (K) of the modified TCR for the cancer antigen is D ) is between about 0.30 and about 4.5 μM. In some embodiments, the dissociation constant (K) of the modified TCR for the cancer antigen is between about 0.30 and about 4.5 μM. D ) is between about 0.30 and about 2 μM. In some embodiments, the dissociation constant (K) of the modified TCR for the cancer antigen is between about 0.30 and about 2 μM. D ) is between about 2 μM and about 3 μM. In some embodiments, the dissociation constant (K) of the modified TCR for the cancer antigen is between about 2 μM and about 3 μM. D ) is between about 3 μM and about 4 μM. In some embodiments, the dissociation constant (K) of the modified TCR for the cancer antigen is between about 3 μM and about 4 μM. D ) is about 0.41 μM. In some embodiments, the dissociation constant (K) of the modified TCR for the cancer antigen is about 0.41 μM. D ) is about 3.89 μM.

[0024] In some embodiments, the modified TCR comprises an amino acid sequence shown in any one of SEQ ID NOs: 2-5, or a functional fragment thereof, or an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2-5.

[0025] In some embodiments, the modified TCR is encoded by a nucleotide sequence shown in any one of SEQ ID NOs: 11-14, or a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NOs: 11-14.

[0026] In some embodiments, the polynucleotide is operably linked to at least one regulatory element for expressing the modified TCR. In some embodiments, the at least one regulatory element is a promoter.

[0027] In various embodiments, the polynucleotide is a DNA molecule.

[0028] In various embodiments, the polynucleotide is an RNA molecule or a derivative thereof.

[0029] In another aspect, provided herein is a recombinant vector comprising a polynucleotide as described herein, wherein the polynucleotide is operably linked to at least one regulatory element for expressing the modified T cell receptor (TCR).

[0030] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphaviral vector, a herpes virus vector, or a vaccinia virus vector.

[0031] In some embodiments, the vector is a non-viral vector.

[0032] 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.

[0033] In another aspect, provided herein is a modified T cell receptor (TCR) comprising a) a β chain or a functional fragment thereof of the modified TCR encoded by a polynucleotide described herein, and b) an α chain or a functional fragment thereof.

[0034] In another aspect, provided herein is a modified T cell receptor (TCR) comprising the CDRs of the beta chain of the modified TCR encoded by the polynucleotides described herein.

[0035] In another aspect, provided herein is a modified T cell receptor (TCR) comprising a) a functional fragment of the β chain of the modified TCR, wherein the functional fragment comprises the CDR of the β chain encoded by the polynucleotide described herein, and b) a functional fragment of the α chain, wherein the functional fragment comprises the CDR of the α chain.

[0036] In some embodiments, the functional fragment of a) further comprises a constant region of a TCRβ chain, and / or the functional fragment of b) further comprises a constant region of a TCRα chain. In one embodiment, any constant region is human. In one embodiment, any constant region is mouse.

[0037] In some embodiments, the α chain comprises the α chain of a WT TCR or a functional fragment thereof. In one embodiment, the α chain of a WT TCR comprises the amino acid sequence of SEQ ID NO:7.

[0038] In some embodiments, the modified TCR comprises any one of the amino acid sequences in SEQ ID NOs: 2-5, or a fragment thereof, or an amino acid sequence having at least 80% sequence identity with any one of the sequences in SEQ ID NOs: 2-5.

[0039] In another aspect, provided herein is a modified T cell receptor (TCR) comprising an α chain comprising the amino acid sequence shown in SEQ ID NO: 7 and a β chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-5.

[0040] In another aspect, provided herein is an isolated host cell comprising a modified T cell receptor (TCR) described herein.

[0041] 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 that is capable of mediating the expression of the modified T cell receptor (TCR) in the host cell.

[0042] In another aspect, provided herein is an isolated host cell comprising a vector described herein.

[0043] 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.

[0044] In various embodiments, the host cells are obtained from peripheral blood mononuclear cells (PBMCs), tumor draining lymph nodes, or tumor infiltrates.

[0045] In various embodiments, the host cell has been activated and / or expanded ex vivo.

[0046] In various embodiments, the host cell is an allogeneic cell.

[0047] In various embodiments, the host cell is an autologous cell.

[0048] In various embodiments, the host cell is isolated from a subject suffering from a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer presents the cancer antigen NY-ESO-1 on its cell surface. 157-165 Epitope (SEQ ID NO: 8). In some embodiments, the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.

[0049] In various embodiments, the host cell is further engineered to express one or more exogenous molecules. In some embodiments, the one or more exogenous molecules are immune signaling molecules.

[0050] In some embodiments, the immune signaling molecule is a cytokine.

[0051] In some embodiments, the immune signaling molecule is a chemokine.

[0052] In some embodiments, the immune signaling molecule is a growth factor. In one embodiment, the growth factor is granulocyte-macrophage colony stimulating factor (GM-CSF).

[0053] Also provided herein is an isolated host cell comprising a T cell receptor (TCR) or a functional fragment thereof that binds a cancer antigen, wherein the host cell is 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 with SEQ ID NO:1. In one embodiment, the α chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:7.

[0054] 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.

[0055] In some embodiments, the one or more exogenous molecules are soluble receptors.

[0056] In some embodiments, the one or more exogenous molecules are ligands.

[0057] In some embodiments, the one or more exogenous molecules are antigen binding proteins. In some embodiments, the antigen binding protein is an antibody or antibody fragment.

[0058] In some embodiments, the one or more exogenous molecules are phosphodiesterases. In some embodiments, the phosphodiesterase is PDE4B2.

[0059] Also provided herein are isolated host cells comprising a T cell receptor (TCR) or a functional fragment thereof that binds a cancer antigen, wherein the host cell is 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 with SEQ ID NO:1. In one embodiment, the α chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:7.

[0060] 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.

[0061] 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 chimeric antigen receptor that does not bind to the cancer antigen NY-ESO-1. 157-165 T cell receptor epitope (SEQ ID NO: 8).

[0062] On the other hand, provided herein are bifunctional molecules comprising a modified T cell receptor or a functional fragment thereof as 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 fragment (scFv).

[0063] 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.

[0064] In another aspect, a pharmaceutical composition is provided herein, comprising a host cell as described herein or a bifunctional molecule as described herein, and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is used for adoptive cell transfer therapy.

[0065] In another aspect, the present invention provides a method for producing the host cell described herein, comprising genetically engineering the host cell using the polynucleotide described herein or the vector described herein.

[0066] On the other hand, provided herein is a method for genetically engineering host cells to express a modified T cell receptor (TCR) or its functional fragment as described herein. In some embodiments, the method includes genetically engineering the host cell with a polynucleotide described herein or a vector described herein. In some embodiments, the genetic engineering step is carried out by viral gene delivery. In some embodiments, the genetic engineering step is carried out by non-viral gene delivery. In some embodiments, the method is carried out in vitro. In some embodiments, the method further includes in vitro activation and / or amplification of the host cell.

[0067] In some embodiments of the cell engineering method, the modified TCR comprises any one of the amino acid sequences in SEQ ID NOs: 2-5, or a fragment thereof, or an amino acid sequence having at least 80% sequence identity with any one of the sequences in SEQ ID NOs: 2-5.

[0068] 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 molecules are cytokines, chemokines, growth factors, soluble receptors, ligands, phosphodiesterases, antigen binding proteins, or cell surface receptors.

[0069] 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 with 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 with SEQ ID NO: 22, 35 or 16.

[0070] 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.

[0071] In some embodiments of the cell engineering methods, the antigen binding protein is an antibody or an antibody fragment.

[0072] In some embodiments of the cell engineering method, the cell surface receptor does not bind to the cancer antigen NY-ESO-1 157-165 A chimeric antigen receptor or T cell receptor that is expressed on a substrate containing the nucleotide sequence of the present invention.

[0073] 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.

[0074] In various embodiments of the cell engineering methods, the host cells are obtained from peripheral blood mononuclear cells (PBMCs), tumor draining lymph nodes, or tumor infiltrates.

[0075] In various embodiments, the host cell has been activated and / or expanded ex vivo.

[0076] In various embodiments, the host cell is an allogeneic cell.

[0077] In various embodiments, the host cell is an autologous cell.

[0078] In various embodiments, the host cell is isolated from a subject suffering from a disease. In some embodiments, the disease is cancer. In some embodiments, the cancer presents the cancer antigen NY-ESO-1 on its cell surface. 157-165 Epitope (SEQ ID NO: 8). In some embodiments, the cancer is myeloma, melanoma, sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.

[0079] 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 lymphocytes 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 a method described herein.

[0080] In another aspect, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of lymphocytes, the lymphocytes 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 a method described herein. In some embodiments, the cancer cell presents the cancer antigen NY-ESO-1 on its surface. 157-165 epitope (SEQ ID NO: 8).

[0081] 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, bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.

[0082] In some embodiments, the method of treatment comprises:

[0083] a) isolating T cells from a subject or a mammal;

[0084] b) genetically modifying the T cell ex vivo using a polynucleotide described herein or a vector described herein;

[0085] c) optionally, expanding and / or activating said T cells before, after or during step b); and

[0086] d) introducing the genetically modified T cells into said subject or mammal.

[0087] In various embodiments, the subject or mammal is a human.

[0088] These and other aspects of the invention will be apparent to those of ordinary skill in the art from the following description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figures 1A-1E The amino acid sequences of the α and β chains of the NY-ESO TCR mutants are shown. Figure 1A The amino acid sequences of TCR BC1 and β chains are shown. Figure 1B The amino acid sequences of the TCR I53E α and β chains are shown. Figure 1C The amino acid sequences of the TCR I53 Fα and β chains are shown. Figure 1D The amino acid sequences of the TCR I53W α and β chains are shown. Figure 1E The amino acid sequences of the TCR D55E α and β chains are shown.

[0091] Figure 2A-2B Shown are flow cytometric analyses of CD4+ T cells transduced with different NY-ESO TCR mutants. Figure 2A Flow cytometry TCRVb13.1 analysis of CD4+ T cells transduced with different NY-ESO TCR mutants is shown. Figure 2B Shown is flow cytometry HLA-A2.1 NY-ESO tetramer analysis of CD4+ T cells transduced with different NY-ESO TCR mutants.

[0092] Figure 3A-3B Shown are flow cytometric analyses of CD8+ T cells transduced with different NY-ESO TCR mutants. Figure 3A Flow cytometry TCRVb13.1 analysis of CD8+ T cells transduced with different NY-ESO TCR mutants is shown. Figure 3B Flow cytometric HLA-A2.1 NY-ESO tetramer analysis of CD8+ T cells transduced with different NY-ESO TCR mutants is shown.

[0093] Figure 4A-4B Functional affinity analysis of NY-ESO-specific TCR transgenic CD4+ and CD8+ T cells is shown. Interleukin-2 (IL-2) produced by CD4+ T cells transduced with different NY-ESO TCR mutants Figure 4A ) and interferon-γ (IFN-g) produced by CD8+ T cells ( Figure 4B ). The target cells used were loaded with 10-fold serial dilutions of NY-ESO 157-165 Peptide-positive HLA-A2.1 cell line T2. "NT" refers to non-transduced cells.

[0094] Figure 5IL-2 production by NY-ESO TCR transgenic CD4+ cells is shown. The target cells used were Me275 (a tumor cell line naturally expressing HLA / A2-NY-ESO-1), A2008-A2 NLM and OVCAR5 NLM (genetically engineered cells expressing HLA-A2-NY-ESO-1), and NA8, A2008-A2 and OVCAR5 (tumor cell lines not expressing HLA / A2-NY-ESO-1).

[0095] Figure 6 IL-2 production by NY-ESO TCR transgenic CD4+ cells is shown. 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.

[0096] Figure 7 IFN-γ production by NY-ESO TCR transgenic CD8+ cells is shown. The target cells used were Me275 (a tumor cell line naturally expressing HLA / A2-NY-ESO-1), A2008-A2 NLM and OVCAR5 NLM (genetically engineered cells expressing HLA-A2-NY-ESO-1), and NA8, A2008-A2 and OVCAR5 (tumor cell lines not expressing HLA / A2-NY-ESO-1).

[0097] Figure 8 IFN-γ production by NY-ESO TCR transgenic CD8+ cells is shown. 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.

[0098] Fig. 9 Shown is IL-2 production by NY-ESO TCR transgenic CD4+ cells (CD4+ cells expressing wild-type BC1 TCR, I53E and I53F) in the presence of peptide-pulsed target cells.CD4+ T cells were co-cultured with peptide-loaded T2 target cells for 24 hours.

[0099] Fig.10 Shown is IFN-γ production by NY-ESO TCR transgenic CD8+ cells (CD8+ cells expressing wild-type BC1 TCR, I53E and I53F) in the presence of peptide-pulsed target cells.CD4+ T cells were co-cultured with peptide-loaded target cells for 24 hours.

[0100] Fig.11IFN-γ production by CD8+ T cells transduced with I53F, I53W and 1G4LY TCRs against different tumor cell lines is shown. The target cells used were the NY-ESO negative cell lines U87MG, OVCAR3, A431, A673, SKOV3, RD-ES, SK-N-AS and HT-29, and the NY-ESO positive cell line A375.

[0101] Fig.12 The cell killing activity (measured by IncuCyte) of CD8+ cells expressing wild-type BC1 TCR, I53E TCR and I53F TCR at different time points is shown. "NT" refers to non-transduced cells.

[0102] Figures 13A-13B In vivo evaluation of NY-ESO TCR transgenic cells using the WINN assay is shown. Fig.13A A schematic diagram of the assay is shown. Fig. 13B Results of the assay are shown. HLA-A2+ / NY-ESO+ tumor cell line Me275 was mixed with NY-ESO transduced T cells and injected subcutaneously into the right flank of NSG mice. The percentages of CD4+ and CD8+ were 30% and 70%, respectively. Tumor size was measured twice a week.

[0103] Figures 14A-14B In vivo evaluation of NY-ESO TCR transgenic cells using NSG mice is shown. Fig.14A A schematic diagram of the assay is shown. Fig. 14B The results of the assay are shown. On day 0, 5 × 10 6 Me275 cells were injected subcutaneously into the flank of NSG mice. When the tumor size was approximately 50-100 mm 3 At the same time, 10×10 6 The percentages of CD4+ and CD8+ T cells were 30% and 70%, respectively.

[0104] Figures 15A-15B In vivo evaluation of NY-ESO TCR transgenic cells using NSG mice is shown. Fig.15A A schematic diagram of the assay is shown. Fig. 15B The results of the assay are shown. On day 0, 5×10 6 Me275 cells were injected subcutaneously into the flank of NSG mice. When the tumor size was approximately 50-100 mm 3 On days 10 and 13, 10x10 6 The percentages of CD4+ and CD8+ T cells were 30% and 70%, respectively.

[0105] Figures 16A-16B In vivo evaluation of NY-ESO TCR transgenic cells using NSG mice is shown. Fig.16A A schematic diagram of the assay is shown. Fig. 16B The results of the assay are shown. On day 0, 5×10 6 Me275 cells were injected subcutaneously into the flank of NSG mice. When the tumor size was approximately 50-100 mm 3 Different numbers of NY-ESO-specific T cells were intravenously injected on days 10 and 13. The percentages of CD4+ and CD8+ T cells were 30% and 70%, respectively.

[0106] Figures 17A-17C We demonstrated that human T cells could be efficiently co-engineered to stably express the NY-ESO-1 TCR and secrete mouse GM-CSF. Fig.17A Schematic representation of the retroviral mouse GM-CSF and lentiviral NY-ESO-1 TCR constructs is depicted. Fig. 17B Representative contour plots of human CD8+ and CD4+ T cells at day 7 after viral transduction are shown. 157-165 The expression of NY-ESO-1 TCR was confirmed by tetramerization, whereas the expression of mouse GM-CSF was confirmed by detecting the surface Thy1.1 reporter protein. Fig. 17C It was shown that secreted mouse GM-CSF can be detected by ELISA in the supernatant of transduced CD8+ T cell cultures. "NT" refers to non-transduced T cells.

[0107] Figures 18A-18B It was demonstrated that secreted mouse GM-CSF had no effect on human T cell activity. Fig.18A The detection of IFNγ secreted by NY-ESO-1 TCR-engineered T cells was shown to be based on recognition of HLA-A2+NY-ESO-1+ tumor cells. Mouse granulocyte colony-stimulating factor had no effect on IFNγ levels. Fig.18B showed that NY-ESO-1 TCR-engineered T cells could readily kill HLA-A2+NY-ESO-1+ tumor cells and their cytotoxic activity was not affected by mouse GM-CSF. Fig.18A and 18B Refers to the same experiment. "NT" refers to non-transduced. Statistical significance was determined by one-way ANOVA. ***p<0.0001.

[0108] Figures 19A-19CWe demonstrated that T cell-derived mouse GM-CSF secreted in the tumor microenvironment could significantly enhance the control of tumor growth by NY-ESO-1-specific T cells in the NSG human melanoma xenograft mouse model. Fig.19A Tumor growth in mice from a Winn assay is shown. T cells expressing the human NY-ESO-1 TCR co-engineered to secrete mouse GM-CSF can effectively slow the engraftment and establishment of Me275 melanoma cells compared to control T cells expressing the NY-ESO-1 TCR alone. Fig.19B The survival percentages of mice from the Winn assay are shown. 40% of mice treated with T cells expressing the NY-ESO-1 TCR and secreting mouse GM-CSF were tumor-free. Fig.19C Compared with NY-ESO-1 TCR alone, adoptive transfer of 2×10 7 T cells expressing NY-ESO-1 TCR and secreting mouse GM-CSF showed better tumor control against established Me275 tumors. "NT" refers to non-transduced T cells. Statistical significance of in vivo responses was determined by one-way ANOVA or unpaired t-test. ***p<0.001. Statistical significance of survival curves was determined by Mantel-Cox log-rank test. ***p<0.01.

[0109] Figures 20A-20B Exemplary protein and nucleotide sequences are provided for mouse and human GM-CSF.

[0110] Fig.21 It was demonstrated that primary human T cells can be efficiently transduced to express exogenous phosphodiesterase 4B2 (PDE4B2), or co-transduced to express exogenous PDE4B2 and NY-ESO-1 TCR variant β-I53F. Uninduced, PDE4B2-transduced, and PDE4B2 & NY-ESO-1 TCR-transduced CD4+ and CD8+ T cells were described, and the expression of exogenous PDE4B2 or NY-ESO-1 TCR variant β-I53F was analyzed by flow cytometry at day 7.

[0111] Fig. 22 It was demonstrated that overexpression of PDE4B2 prevented intracellular cAMP accumulation. The figure shows that exposure to forskolin (Fsk) or PGE 2 Intracellular cAMP levels in resting eGFP (grey line) or PDE4B2 (black line) transduced CD4 T cells after 1 hour. After treatment, T cells were lysed and cAPM levels were quantified by ELISA. Results shown are from a single experiment where all cAMP measurements were performed in triplicate. Error bars represent standard deviation (SD).

[0112] Figures 23A-23B D Th-1 cytokine production by PDE4B2-transduced T cells is shown. Fig.23A showed that in the presence of forskolin or PGE 2 IFNγ or TNF-α production by resting eGFP- or PDE4B2-transduced CD4 T cells as determined by intracellular cytokine staining (ICS) after 7 h of stimulation (solid line) or without stimulation (dashed line) with plate-bound αCD3 and soluble αCD28 in the presence of αCD3+ or αCD28. The percentage of IFNγ+ or TNF-α+ cells in live CD45+eGFP+ (grey line) or live CD45+ exogenous PDE4B2+ (black line) lymphocytes and representative graphs are depicted. Results shown are from a single experiment in which all conditions were performed in duplicate. Error bars represent standard deviations. Fig. 23B Interferon gamma secretion by resting eGFP-, PDE4B2-, eGFP&NY-ESO-1 TCR-, or PDE4B2-&NY-ESO-1 TCR-transduced CD8 T cells co-cultured with NY-ESO-1 presenting melanoma cells A375 in the presence of PGE2 or forskolin for 48 hours is shown. Interferon gamma concentrations of co-culture supernatants are depicted. Error bars represent standard deviations.

[0113] Fig.24 Prove that PDE4B2 overexpression promotes proliferation under the condition of inducing intracellular cAMP accumulation. The proliferation capacity of CD4 T cells transduced by eGFP- (grey) or PDE4B2 (black) is determined by BrDU incorporation assay. In the presence of PGE2 or forskolin, T cells are re-stimulated for 48 hours with (solid line) or without (dashed line) plate-bound αCD3. The percentage of BrDU+ cells in live CD45+ lymphocytes (left figure), the BrDU MFI of these populations (middle figure) and representative figures are depicted. The results shown are from a single experiment, in which all conditions are performed in duplicate. Error bars represent standard deviations.

[0114] Fig.25 It was demonstrated that PDE4B2 overexpression in PGE 2 In the presence of PGE 2 (black line) or no PGE 2(grey line), the ability of resting eGFP (open triangles)-, PDE4B2 (open circles)-, eGFP & NY-ESO-1 TCR (closed triangles)-, or PDE4B2 & NY-ESO-1 TCR (closed circles)-transduced CD4 (left panel) or CD8 (right panel) T cells to inhibit the expansion of NY-ESO-1 presenting melanoma cells A375 was assessed by IncuCyte. Depicted are red object area values, which correspond to the mm2 occupied by the nuclei of A375 cells in each well. The results shown are from a single experiment, in which all conditions were performed in triplicate. Four different planes per well were included in the analysis. Error bars represent standard deviations.

[0115] Fig.26 Exemplary protein sequences of human and murine PDE4B2 are provided.

[0116] Detailed Description

[0117] The present invention generally provides a modified T cell receptor (TCR) that is responsive to the cancer antigen NY-ESO-1. 157-165 The epitope has improved binding affinity.

[0118] 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 genetically engineered cells. In addition, the present invention provides genetically engineered cells with modified TCRs. The present invention also provides pharmaceutical compositions related to the modified TCRs and cells of the present invention.

[0119] In addition, the present invention provides a method for genetically engineering lymphocytes that express modified TCRs. The present invention further provides genetically engineered lymphocytes that express modified TCRs and additional receptors, including but not limited to chimeric antigen receptors or receptors that do not target the cancer antigen NY-ESO-1. 157-165 Receptors for epitopes, or soluble proteins, include but are not limited to cytokines, chemokines, growth factors, soluble receptors, ligands, antibody fragments and antigen binding domains. Also provided herein are methods of using such genetically engineered lymphocytes in adoptive cell transfer therapy.

[0120] The present invention also provides a method for treating cancer in a subject. The method comprises administering to a subject suffering from such cancer an effective amount of lymphocytes presenting the modified TCR of the present invention. The present invention also provides a method for stimulating or enhancing an immune response in a mammal, the method comprising administering to the mammal an effective amount of genetically engineered lymphocytes of the present invention.

[0121] On the one hand, there is provided herein a genetically engineered lymphocyte expressing a modified TCR and cytokines and / or chemokines from the colony stimulating factor cytokine / chemokine family. It is well known that members of the colony stimulating factor cytokine / chemokine family act on most 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, originally described as a hematopoietic growth factor, has become a key immunomodulator for a variety of pathological conditions including autoimmune diseases and cancer. Cells of hematopoietic origin, i.e. macrophages, natural killer (NK) cells, activated T cells and cells of non-hematopoietic origin such as endothelial cells and fibroblasts, all secrete GM-CSF. GM-CSF is not detectable in the serum of healthy individuals, but GM-CSF levels rise rapidly 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) found primarily on monocytes / macrophages and granulocyte lineages as well as dendritic cells. Upon ligand binding, the GM-CSF receptor transduces signals associated with cell survival, proliferation, differentiation, and activation (Hercus, TR et al., Blood. 2009. 114: p. 1289-1298). By controlling the fate of this professional antigen presenting cell (APCs), GM-CSF can indirectly regulate the activity of T cells, thereby acting as a bridge between adaptive immunity and innate immunity (Shi, Y. et al., Cell Res. 2006. 16 (2): 126-33).

[0122] In cancer treatment, GM-CSF plays a central role in the supportive care of cancer patients and in accelerating and enhancing the recovery of the immune system myeloid compartment after chemotherapy and / or stem cell transplantation regimens (Arellano, M. and S. Lonial, Biologics. 2008. 2(1): 13-27). In addition, its key role in DC development and differentiation has placed GM-CSF at the core of DC-based immunotherapy, in the form of cancer vaccines that secrete GM-CSF that activates DCs (Gupta, R. and L. A. Emens. Discovery medicine, 2010. 10(50): p. 52-60) or adoptive transfer of GM-CSF-activated / biased dendritic cells as primary immunotherapy (Mookerjee, A., M. Graciotti, and L. Kandalaft, BioImpacts: BI, 2018. 8(3): p. 211-221). Dendritic cells have also been a key mediator of strong anti-tumor responses in preclinical studies using tumors that were forced to overexpress GM-CSF (Shi, FS et al., Cancer Gene Ther, 1999. 6 (1): p. 81-8) or after administration of soluble GM-CSF to cancer patients (Nasi, ML et al., Cytokines Cell Mol Ther, 1999. 5 (3): p. 139-44). Despite the encouraging results, the use of GM-CSF as a single adjuvant therapy in the clinic has proven to be quite unsatisfactory (Lawson, DH et al., J Clin Oncol, 2015. 33 (34): p. 4066-76) and is limited by the appearance of dose-related toxicity (Antman, KS et al., N Engl J Med, 1988. 319 (10): p. 593-8). Interestingly, another group of studies revealed an immunosuppressive mechanism of GM-CSF-driven anti-tumor response, in which tumor-derived GM-CSF is responsible for the immune attraction of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment, thereby promoting tumor escape (Pylayeva-Gupta, Y. et al., Cancer Cell, 2012. 21: p. 836-847).

[0123] In order to maximize the benefits of the adoptive T cell transfer strategy and take advantage of the immunomodulatory anticancer properties of GM-CSF while avoiding any adverse side effects and off-target toxicity, a combined 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 exemplified below in Example 6. The results showed that human T cells efficiently secreted fully functional soluble GM-CSF without affecting their proliferation capacity or anti-tumor activity, and induced a strong anti-tumor response against human NY-ESO-1+ melanoma in vivo.

[0124] On the other hand, genetically engineered lymphocytes expressing modified TCR and phosphodiesterase are provided herein. In some embodiments, the phosphodiesterase is phosphodiesterase 4B2 (PDE4B2). Intracellular second messenger cyclic AMP (cAMP) is an effective immunosuppressive signal molecule in T cells, which is upregulated by a variety of tumor-related inhibitors, including prostaglandin E2 (PGE2), adenosine and regulatory T cell functions. Overexpression of phosphodiesterase can reduce cAMP signal transduction and increase the resistance of anti-tumor T cells to inhibitory factors such as prostaglandin E2 inhibition. See U.S. Patent No. 9,976,121 and Schmetterer, KG., Front.Immunol., July 30, 2019, all of which are incorporated herein by reference.

[0125] definition

[0126] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0127] As used herein, the term "antigen" is a molecule and / or substance that is capable of specifically binding to an antibody or generating a peptide fragment recognized by a T cell receptor and / or inducing an immune response. An antigen may contain one or more "epitopes". In certain embodiments, an antigen has several epitopes. In the case of an MHC molecule, an epitope is recognized by an antibody or a lymphocyte. In various embodiments, the antigen is NY-ESO-1. In various embodiments, the epitope is NY-ESO-1. 157-165 .

[0128] As used herein, the interchangeable terms "tumor antigen" or "cancer antigen" are broadly defined as antigens specifically expressed by or associated with tumors or cancer cells, such as overexpressed or abnormally expressed antigens, antigens produced by oncogenic viruses, carcinoembryonic antigens, altered cell surface glycolipid and glycoprotein antigens, cell type specific differentiation antigens. Tumor antigens present on the surface of cancer cells are antigens that are not present on the surface of normal somatic cells of an individual, i.e., the antigen is exposed to the immune system in cancer cells but not to the immune system in normal somatic cells. The antigen can be expressed on the cell surface of tumor cells, where it is recognized by components of the humoral immune system such as B lymphocytes (B cells). Intracellular tumor antigens are processed into shorter 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, tumor antigens are antigens that are not expressed by normal cells, or at least not expressed at the same level as tumor cells.

[0129] As used herein, the term "functional fragment" refers to a fragment of a polypeptide or protein, or a polynucleotide encoding a polypeptide or protein, which retains at least one function of the full-length polypeptide or protein. A functional fragment may comprise at least 5 consecutive amino acid residues, at least 6 consecutive amino acid residues, at least 7 consecutive amino acid residues, at least 8 consecutive amino acid residues, at least 9 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 11 consecutive amino acid residues, at least 12 consecutive amino acid residues, at least 13 consecutive amino acid residues, at least 14 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues or at least 250 consecutive 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 the full-length protein or polypeptide. For example, a TCR functional fragment that immunospecifically binds to a specific antigen (or epitope) can retain the ability to immunospecifically bind to an antigen (or epitope). In some embodiments, the functional fragment of a TCR comprises one or more complementary determining regions of an α chain and / or a β chain of a TCR. In some embodiments, the functional fragment of a TCR comprises an α chain and / or a β chain of a TCR.

[0130] The term "variant" as used herein refers to a modified polypeptide, protein or polynucleotide that has substantial or significant sequence identity or similarity to a wild-type polypeptide, protein or polynucleotide. A variant may retain the same biological activity, or have an altered (e.g., improved, reduced or eliminated) biological activity relative to the wild-type polypeptide, protein or polynucleotide of which it is a variant. A variant may comprise an insertion, deletion, substitution of at least one amino acid residue or nucleotide.

[0131] The term "antigen binding protein" refers to any protein that binds to an antigen of interest or a polypeptide or fragment thereof. The protein may be of natural origin or may be synthetic. Examples of antigen binding proteins include, but are not limited to, 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 TCR variable domains; secreted factors (such as cytokines, growth factors), which may be artificially fused to signaling domains (such as "zytokines"); and any ligand or receptor fragment that binds to the antigen of interest (such as CD27, NKG2D). Combinatorial libraries can also be used to identify peptides that bind to therapeutic targets with high affinity.

[0132] 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 Fv (sdFv), intrabodies, minibodies, diabodies, triabodies, decabodies and other domain antibodies (e.g., Holt (2003), 21, 11, 484-490)). The term "antibody" also refers to covalent dyads, such as those disclosed in U.S. Patent Publication No. 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent 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.

[0133] The terms "vector", "cloning vector" and "expression vector" refer to vectors that can introduce DNA or RNA sequences (such as foreign genes) into host cells, thereby genetically modifying the host and promoting the expression (such as transcription and translation) of the introduced sequences. Vectors include plasmids, synthetic RNA and DNA molecules, transposons, phages, viruses, etc. In certain embodiments, the vector is a viral vector, such as, but not limited to, adenovirus, adeno-associated virus, alpha virus, herpes virus, lentivirus, retrovirus or vaccinia virus vectors.

[0134] 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" includes the substantially minimal sequence required to initiate transcription. In some embodiments, the term "promoter" includes sequences that initiate transcription, and in addition, also includes sequences that can upregulate or downregulate transcription, usually referred to as "enhancer elements" and "repressor elements", respectively. In some embodiments, the promoter is a lymphocyte-specific promoter.

[0135] As used herein, the term "operably linked" refers to a nucleotide sequence being placed in a functional relationship with another nucleotide sequence. For example, if a coding sequence is operably linked to a promoter sequence, this generally means that the promoter can promote transcription of the coding sequence. Operably linked means that the DNA sequences being linked are generally continuous and, when necessary to connect two protein coding regions, continuous and in reading frame. However, since enhancers can function when separated from promoters by thousands of bases, and intron sequences can be of variable length, some nucleotide sequences may be operably linked but not continuous.

[0136] The terms "T cell" and "T lymphocyte" are interchangeable and are used as synonyms 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 cells, such as T helper cells 1 (Th1) or T helper cells 2 (Th2). T cells can be helper T 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+T cells, or any other T cell subsets. Other illustrative T cell populations suitable for specific embodiments include primitive T cells and memory T cells. Also included are "NKT cells", which refer to specialized T cell populations expressing semi-invariant αβT cell receptors, but also express a variety of molecular markers commonly associated with natural killer cells, such as NK1.1. NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+ and CD8- cells. The TCR on NKT cells is unique because it recognizes glycolipid antigens presented by MHC I-like molecules CD Id. Since NKT cells can produce cytokines that promote inflammation or immune tolerance, they may have protective or harmful effects. Also included are "γ-δT cells" (γδT cells), which refer to a special small population of T cells with unique TCRs on their surface, and unlike most T cells in which the TCR consists of two glycoprotein chains (called α- and β-TCR chains), the TCR in γδT cells consists of γ chains and δ chains. γδT cells can play a role in immune surveillance and immune regulation, and have been found to be an important source of IL-17, inducing a powerful CD8+ cytotoxic T cell response. Also included are "regulatory T cells" or "Tregs", which refer to T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs cells are usually CD4+ T cells that are positive for the transcription factor Foxp3, and may also include regulatory T cells that are negative for the transcription factor Foxp3, which are CD4+ T cells that produce interleukin-10.

[0137] The terms "natural killer cells" and "NK cells" are used interchangeably and are used synonymously herein. As used herein, natural killer cells refer to differentiated lymphocytes having a CD 16+CD56+ and / or CD57+TCR- phenotype. NKs are characterized by the ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activating specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and the ability to release protein molecules called cytokines that can stimulate or inhibit immune responses.

[0138] As used herein, "exogenous" refers to any molecule that is not derived from a particular cell found in nature. Exogenous molecules can be expressed from nucleic acid molecules introduced into a host cell by artificial or natural means.

[0139] The term "treating" a condition, disorder or condition includes: (1) preventing, delaying or reducing the incidence and / or likelihood of the development of at least one clinical or subclinical symptom of the condition, disorder or condition in a subject who may have or is susceptible to the condition, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the condition, disorder or condition; or (2) inhibiting the condition, disorder or condition, i.e., preventing, reducing or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing regression of the condition, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0140] The term "effective" as used for dosage 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. Note 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 is effective when administered alone. The exact amount required varies from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease being treated, the specific drug or drugs used, the mode of administration, etc.

[0141] The phrase "pharmaceutically acceptable" used in conjunction with the compositions described herein refers to the molecular entities and other ingredients of such compositions that are physiologically tolerable and generally do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term "pharmaceutically acceptable" refers to drugs approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, especially humans.

[0142] The terms "patient", "individual", "subject" and "animal" are used interchangeably herein to 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.

[0143] The term "carrier" refers to a diluent, adjuvant, excipient or carrier that is administered with a compound. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including liquids of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or aqueous saline solutions and aqueous glucose 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 pills), 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.

[0144] 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 methods and / or steps that will become apparent to one of ordinary skill in the art upon reading this disclosure.

[0145] The term "about" or "approximately" includes being within a statistically significant range of a numerical value. Such a range can be within the order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The permissible variation encompassed by the term "about" or "approximately" depends on the specific system being studied and can be readily understood by those of ordinary skill in the art.

[0146] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value and endpoint falling within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0147] The practice of the present invention employs, 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 described in detail, 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.

[0148] Modified T cell receptor

[0149] In one aspect, the present invention provides a modified T cell receptor (TCR) or a functional fragment thereof, wherein the modified T cell receptor comprises a single amino acid substitution within the complementarity determining region (CDR) 2 of the modified T cell receptor β chain relative to the CDR2 of the unsubstituted wild-type (WT) T cell receptor β chain.

[0150] In one embodiment, the present invention provides a modified T cell receptor (TCR) comprising the amino acid sequence of a wild-type (WT) TCR and having no more than one amino acid substitution in the complementarity determining region (CDR) 2 of the TCR β chain, wherein the modified TCR retains the antigen specificity of the WT TCR and has a higher binding affinity for a cancer antigen than the WT TCR, and comprises the amino acid sequence of SEQ ID NO: 1 and having no more than one amino acid substitution in the CDR2 of SEQ ID NO: 1

[0151] In certain embodiments, the modified TCR has a binding affinity for the cancer antigen that 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 for the cancer antigen.

[0152] Dissociation constant (K D ) can be used to assess the binding affinity of the modified TCR to the cancer antigen. In certain embodiments, the dissociation constant (K) of the modified TCR for the cancer antigen D ) is between about 0.1 and about 10 μM. As a non-limiting example, the dissociation constant (K) of the modified TCR for the cancer antigen is 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.

[0153] In one embodiment, the invention provides a modified T cell receptor (TCR) comprising the amino acid sequence of a wild-type (WT) TCR having no more than one amino acid substitution located in the complementarity determining region (CDR) 2 of the TCR β chain, wherein the modified TCR retains the antigen specificity of the WT TCR and has a higher specificity for the cancer antigen NY-ESO-1 than the WT TCR. 157-165The binding affinity of the epitope (SEQ ID NO:8) is higher and comprises the amino acid sequence of SEQ ID NO:1, with no more than one amino acid substitution located in the CDR2 of SEQ ID NO:1.

[0154] In certain embodiments, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 is about 2-fold to about 100-fold higher than the binding affinity of the WT TCR to the cancer antigen NY-ESO-1. As a non-limiting example, the binding affinity of the modified TCR to the cancer antigen NY-ESO-1 can be 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 .

[0155] 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 .

[0156] Dissociation constant (K D) can be used to assess the binding affinity of the modified TCR to the cancer antigen NY-ESO-1. In certain embodiments, the dissociation constant (K) of the modified TCR to the cancer antigen NY-ESO-1 is D ) is between about 0.1 and about 10 μM. As a non-limiting example, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is 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 .

[0157] In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D ) is between about 0.3 and about 4.5 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D ) is between about 0.3 and about 2 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D ) is between about 2 and about 3 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D ) is between about 3 and about 4 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D ) is about 0.4 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D ) is about 0.4 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is about 0.4 μM. D ) is about 0.41 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D ) is about 3.9 μM. In one embodiment, the dissociation constant (K) of the modified TCR for the cancer antigen NY-ESO-1 is D) is about 3.89 μM. In various embodiments, the epitope is NY-ESO-1 157-165 .

[0158] In certain embodiments, the modified TCR comprises a single amino acid substitution located at residue 53 within CDR2 of the TCR β chain. The single amino acid substitution may include, but is not limited to, I53E, I53F, and I53W.

[0159] In certain embodiments, the modified TCR comprises a single amino acid substitution at residue 55 within CDR2 of the TCR beta chain. The single amino acid substitution may include, but is not limited to, D55E.

[0160] In certain embodiments, the modified TCR comprises a single amino acid substitution at residue 50, 51 or 52 within the TCR β chain CDR2. The single amino acid substitution may include, but is not limited to, G50V, G50A, A51D, A51E or G52Q.

[0161] In certain embodiments, the modified TCR β 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 the β chain of the unsubstituted WT TCR or its functional fragment outside the CDR2 region of the modified TCR β chain or its functional fragment. In one embodiment, the modified TCR β chain or its functional fragment comprises the amino acid sequence of the β chain of the unsubstituted WT TCR or its functional fragment, and the single amino acid substitution is located in the CDR2 region. In one embodiment, the unsubstituted WT TCR β chain comprises the amino acid sequence of SEQ ID NO: 1.

[0162] 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 with an amino acid sequence selected from any 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 with an 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 with 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 with 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, relative to an unsubstituted WT TCR β chain (e.g., as shown in SEQ ID NO: 1), the modified TCR comprises a single amino acid substitution located within CDR2 of the β chain.

[0163] In certain embodiments, the modified TCR comprises an amino acid sequence selected from any one of SEQ ID NOs: 2-5. 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.

[0164] 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 with a nucleotide sequence selected from any 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 with a 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 located within CDR2 of the β chain relative to an unsubstituted WT TCR β chain (e.g., as set forth in SEQ ID NO: 1).

[0165] 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.

[0166] In one aspect, the present invention provides a modified T cell receptor (TCR) comprising an alpha chain comprising an amino acid sequence as shown in SEQ ID NO: 7, and a beta chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 2-5 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. In one embodiment, the modified T cell receptor comprises an alpha chain comprising an amino acid sequence as shown in SEQ ID NO: 7, and a beta chain comprising an amino acid sequence as shown in SEQ ID NO: 2 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. In one embodiment, the modified T cell receptor comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 7, and a beta chain comprising 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 comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 7, and a beta chain comprising 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 comprises an alpha chain comprising an amino acid sequence as set forth in SEQ ID NO: 7, and a beta chain 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 beta chain relative to an unsubstituted WT TCR beta chain (e.g., as set forth in SEQ ID NO: 1).

[0167] On the one hand, the present invention provides a modified T cell receptor (TCR), comprising an alpha chain containing the amino acid sequence shown in SEQ ID NO:7 and a beta chain containing the amino acid sequence shown in any one of SEQ ID NO:2-5. In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain containing the amino acid sequence shown in SEQ ID NO:7 and a beta chain containing the amino acid sequence shown in SEQ ID NO:2. In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain containing the amino acid sequence shown in SEQ ID NO:7 and a beta chain containing the amino acid sequence shown in SEQ ID NO:3. In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain containing the amino acid sequence shown in SEQ ID NO:7 and a beta chain containing the amino acid sequence shown in SEQ ID NO:4. In one embodiment, the modified T cell receptor (TCR) comprises an alpha chain containing the amino acid sequence shown in SEQ ID NO:7 and a beta chain containing the amino acid sequence shown in SEQ ID NO:5.

[0168] In one aspect, the present invention provides a modified T cell receptor (TCR) comprising 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 with the nucleotide sequence shown in any one of SEQ ID NOs:11-14. 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 with the nucleotide sequence shown in 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 with the nucleotide sequence shown in 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 with the nucleotide sequence shown in 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 with the nucleotide sequence shown in 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 relative to an unsubstituted WT TCR beta chain (e.g., as set forth in SEQ ID NO: 1).

[0169] On the one hand, the present invention provides a modified T cell receptor (TCR), which comprises an alpha chain encoded by the nucleotide sequence of SEQ ID NO:9 and a beta chain encoded by the nucleotide sequence shown in any one of SEQ ID NO:11-14. 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 the nucleotide sequence of 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 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 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 the nucleotide sequence of SEQ ID NO:14.

[0170] 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 genetically engineered cells.

[0171] In one aspect, the invention provides an isolated polypeptide comprising a functional portion of a modified TCR as described herein, wherein the functional portion comprises the variable regions of the α and β chains of the TCR, and wherein the functional portion comprises the amino acid substitutions. In certain embodiments, the isolated polypeptide comprises an amino acid sequence selected from SEQ ID NO: 2-5.

[0172] In one aspect, the invention provides an isolated polypeptide comprising a functional portion of a modified TCR as described herein, wherein the functional portion further comprises the constant regions of the α and β chains of the TCR. In certain embodiments, the constant region is human. In other embodiments, the constant region is mouse.

[0173] In one aspect, the invention provides nucleic acid sequences encoding any of the amino acid sequences of the modified TCRs described herein.

[0174] It is understood that conservative amino acid substitutions can be introduced into any polypeptide described herein to obtain a polypeptide having, for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with a reference sequence, and preferably retaining the activity of the sequence. As known in the art and referred to herein, conservative amino acid substitutions include replacing amino acids in a protein with amino acids having similar side chains, for example, in terms of structure, size and / or chemical properties. For example, amino acids in the following groups can be interchanged with other amino acids in the same group: amino acids with aliphatic side chains, including glycine, alanine, valine, leucine and isoleucine; amino acids with non-aromatic hydroxyl-containing side chains, such as serine and threonine; amino acids with acidic side chains, such as aspartic acid and glutamic acid; amino acids with amide side chains, including glutamine and asparagine; basic amino acids, including lysine, arginine and histidine; amino acids with aromatic ring side chains, including phenylalanine, tyrosine and tryptophan; and amino acids with sulfur-containing side chains, including cysteine ​​and methionine. Furthermore, amino acids having acidic side chains, such as aspartic acid and glutamic acid, are considered herein to be interchangeable with amino acids having amide side chains, such as asparagine and glutamine.

[0175] Examples of sequences useful in various embodiments of the invention are provided below.

[0176] BC1 WT β chain

[0177] MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQK ATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*(SEQ ID NO:1)

[0178] I53E β-chain

[0179] MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGETDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*(SEQ ID NO:2)

[0180] I53F beta chain

[0181] MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGFTDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*(SEQ ID NO:3)

[0182] I53W beta chain

[0183] MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGWTDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*(SEQ ID NO:4)

[0184] D55E β-chain

[0185] MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITEQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGAAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*(SEQ ID NO:5)

[0186] A97L β-chain

[0187] MAPRLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGLAGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*(SEQ ID NO:6)

[0188] BC1 WT α-chain

[0189] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPQTGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*(SEQ ID NO:7)

[0190] NY-ESO-1 157-165 Peptide

[0191] SLLMWITQC(SEQ ID NO:8)

[0192] BC1 WT α-chain

[0193] ATGGAAACCCTGCTGGGCCTGCTGATCCTGTGGCTGCAGCTGCAGTGGGTGTCCAGCAAGCAGGAAGTGACCCAGATCCCTGCCGCCCTGAGCGTGCCCGAGGGCGAGAACCTGGTGCTGAACTGCAGCTTCACCGACAGCGCCATCTACAACCTGCAGTGGTTCCGGCAGGACCCCGGCAAGGGCCTGACCAGCCTGCTGCTGATCCAGAGCAGCCAGCGGGAGCAGACCAGCGGCAGGCTGAACGCCAGCCTGGACAAGAGCAGCGGCAGAAGCACCCTGTACATCGCCGCCAGCCAGCCCGGCGACTCCGCCACCTACCTGTGCGCCGTGCGGCCTCAGACCGGCGGCAGCTACATCCCCACCTTCGGCAGGGGCACCAGCCTGATCGTGCACCCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGTCCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTTTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATTCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGT(SEQ IDNO:9)

[0194] BC1 WT β-chain

[0195] ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCATCACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO:10)

[0196] I53E β-chain

[0197] ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCGAGACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO:11)

[0198] I53F β-chain

[0199] ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCTTCACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO:12)

[0200] I53W β-chain

[0201] ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCTGGACCGACCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO:13)

[0202] D55E β-chain

[0203] ATGGCCCCGCGGCTGCTGTGTTGTGCCGCCCTGAGCCTGCTGTGGGCCGGACCTGTGAACGCCGGCGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAAACCGGCCAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACAGGCAGGACCCCGGCATGGGCCTGCGGCTGATCCACTACAGCGTGGGAGCCGGCATCACCGAGCAGGGCGAGGTGCCCAACGGCTACAACGTGAGCAGAAGCACCACCGAGGACTTCCCCCTGAGGCTGCTGTCTGCCGCCCCTAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCTATGTGGGAGCCGCCGGAGAGCTGTTCTTCGGCGAGGGCAGCCGGCTGACCGTGCTGGAAGATCTGAACAAAGTGTTCCCCCCCGAAGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAAGTGCACAGCGGCGTGTCCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGATAGCAGATACTGCCTGAGCAGCCGGCTGAGAGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCTATGGTGAAGCGGAAGGACAGCAGGGGCTGA(SEQ ID NO:14).

[0204] Genetically engineered cells

[0205] In one aspect, the invention provides a method for genetically engineering lymphocytes expressing a modified TCR as described herein. In one embodiment, the lymphocytes 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, the lymphocytes are genetically engineered to express 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. In one embodiment, the lymphocytes are genetically engineered to express 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 lymphocytes are genetically 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 lymphocytes can be obtained from PBMCs, tumor draining lymph nodes, or tumor infiltrates. In certain embodiments, the genetically engineered lymphocytes are further engineered to express one or more exogenous molecules. In certain embodiments, the genetically engineered lymphocytes are further engineered to express one or more cell surface receptors. Examples of cell surface receptors include, but are not limited to, chimeric antigen receptors and non-cancer antigens NY-ESO-1. 157-165Epitope (SEQ ID NO:8) of T cell receptor. In certain embodiments, the genetically engineered lymphocytes 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 genetically engineered lymphocytes are further engineered to express one or more additional receptors and soluble proteins.

[0206] On the one hand, the invention provides genetically engineered lymphocytes expressing modified TCRs as described herein. In one embodiment, the genetically engineered lymphocytes express modified TCRs comprising SEQ ID NO: 2 amino acid sequences, or amino acid sequences with SEQ ID NO: 2 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. In one embodiment, the genetically engineered lymphocytes express 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 with SEQ ID NO: 3. In one embodiment, the genetically engineered lymphocytes express 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 with SEQ ID NO: 4. In one embodiment, the genetically engineered lymphocyte 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 located within CDR2 of the β chain relative to an unsubstituted WT TCR β chain (e.g., as shown in SEQ ID NO: 1).

[0207] The lymphoid cells can be obtained from PBMCs, tumor draining lymph nodes or tumor infiltrates. In certain embodiments, the genetically engineered lymphocytes are further engineered to express additional receptors. The additional receptors include but are not limited to chimeric antigen receptors and non-cancer antigens NY-ESO-1. 157-165 In some embodiments, the genetically engineered lymphocytes are further engineered to express soluble proteins. Soluble proteins include, but are not limited to, cytokines, chemokines, growth factors, soluble receptors, ligands, antibodies, antibody fragments, and antigen binding domains. In some embodiments, the genetically engineered lymphocytes are further engineered to express additional receptors and soluble proteins.

[0208] In one embodiment, provided herein is a genetically engineered lymphocyte 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.

[0209] In one embodiment, provided herein is a genetically engineered lymphocyte 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.

[0210] In one embodiment, provided herein is a genetically engineered lymphocyte 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.

[0211] In one embodiment, provided herein is a genetically engineered lymphocyte 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.

[0212] In various embodiments, the genetically engineered lymphocytes express a modified TCR comprising a single amino acid substitution within CDR2 of the beta chain relative to an unsubstituted WT TCR beta chain (e.g., as set forth in SEQ ID NO: 1).

[0213] In some embodiments, the genetically engineered lymphocytes provided herein can be genetically engineered to further express one or more exogenous molecules.

[0214] Lymphocytes are cells of the immune system that react specifically with antigens to produce specific cell products. Samples containing lymphocytes can be obtained from a variety of sources of the subject, including but not limited to, for example, tissues (including tumor tissues, virally infected tissues, tissues at sites of inflammation, sites of lymphocyte 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 such as veins, arteries, capillaries, valves; neural tissues, bone marrow, breast, gastrointestinal, lung tissues, eye tissues such as corneas and lenses, cartilage, bone, and mucosal tissues.

[0215] The sample can be untreated, enzymatically treated and / or dissociated / suspended to form a cell suspension. When the sample is enzymatically treated, non-limiting examples of enzymes that can be used include collagenase, dispase, hyaluronidase, liberase and deoxyribonuclease (DNase).

[0216] In certain embodiments, lymphocytes used in the present invention include tumor infiltrating immune cells. Tumor infiltrating immune cells are composed of mononuclear and polymorphonuclear immune cells (i.e., T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, etc.) in different proportions. In certain embodiments, lymphocytes used in the present invention include tumor infiltrating lymphocytes (TIL). TIL is a leukocyte that leaves the bloodstream and migrates to the tumor. TIL can usually be found in the tumor stroma and inside the tumor.

[0217] In certain embodiments, lymphocytes used in the present invention include peripheral blood lymphocytes (PBLs). In certain embodiments, lymphocytes used in the present invention include T lymphocytes (T cells) and / or natural killer cells (NK cells).

[0218] In certain embodiments, the lymphocytes used in the present invention are T cells. In certain embodiments, the T cells are CD8+ T cells. In certain embodiments, the T cells are CD4+ cells. In certain embodiments, the T cells are regulatory T cells.

[0219] In certain embodiments, the lymphocytes used in the present invention are natural killer (NK) cells. In certain embodiments, the natural killer cells are CD16+CD56+ and / or CD57+ natural killer cells. The characteristics of NK are that it can bind to and kill cells that cannot express "self" MHC / HLA antigens by activating specific cell lytic enzymes, can kill tumor cells or other diseased cells that express ligands of NK activation receptors, and can release protein molecules called cytokines that stimulate or inhibit immune responses.

[0220] Conditions suitable for culturing lymphocytes include an appropriate culture medium (e.g., Minimum Essential Medium (MEM), RPMI Media 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, which is either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or amounts of cytokines sufficient for growth and expansion).

[0221] Examples of other additives for lymphocyte expansion include, but are not limited to, surfactants, phosphatidates, pH buffers such as HEPES, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. Antibiotics (such as penicillin and streptomycin) are included only in experimental cultures and not in cell cultures to be infused into a subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO). 2 ).

[0222] The expansion of lymphocytes can be carried out using methods and conditions known in the art. In certain embodiments, the expansion of lymphocytes is carried out according to the method described in International Application No. PCT / EP2018 / 080343.

[0223] The genetic engineering of lymphocytes can be completed by at least one of transfection, transduction or temporary cell membrane rupture (i.e., cell squeezing) to introduce at least one polynucleotide encoding the modified TCR into lymphocytes. In certain embodiments, the above-mentioned polynucleotides are introduced into lymphocytes by transducing a substantially uniform cell population with a recombinant expression vector. This vector can be a viral vector or a non-viral vector. Exemplary viral vectors used in the present invention include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, or baculovirus vectors. In one embodiment, the viral vector used in the present invention is a lentiviral vector.

[0224] Additional receptors can be engineered into genetically engineered lymphocytes expressing modified TCRs. Having additional receptors on genetically engineered lymphocytes can enhance lymphocyte function (e.g., anti-tumor function).

[0225] In certain embodiments, the above-mentioned additional receptor is a chimeric antigen receptor. Chimeric antigen receptors (CARs) generally have an antigen binding domain that is fused to an intracellular signaling domain that can activate or stimulate immune cells. The extracellular binding domain of CAR can be composed of a single-chain variable fragment (scFv), which is derived from the variable heavy chain region and light chain region of a fused mouse or humanized monoclonal antibody. Alternatively, scFv from Fab (rather than from an antibody, such as an antibody obtained from a Fab library) can be used. ScFv can fuse a transmembrane region and then fuse an intracellular signaling region. CAR can be a first-generation, second-generation, or third-generation CAR. "First-generation" CARs include those that provide CD3ζ signals only when antigens bind. "Second-generation" CARs include CARs that provide co-stimulation (such as CD28 or CD137) and activation (CD3ζ) simultaneously. "Third-generation" CARs include CARs that provide multiple co-stimulation (such as CD28 and CD137) and activation (CD3ζ). CAR can specifically recognize cancer antigens.

[0226] In certain embodiments, the additional receptor is for the cancer antigen NY-ESO-1 157-165 Epitope-insensitive TCR. This TCR can specifically recognize epitopes other than NY-ESO-1. 157-165 Cancer antigens other than epitopes.

[0227] In certain embodiments, the cancer antigen can be selected from CD7, CD74, CDS, CEA, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER2, hTERT, IL-13R-a2, KDR, K-light chain, LeY, L1 cells, MAGE-Al, mesothelin, MUC1, MUC16, NKG2D ligand, NY-ESO-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2 and WT-1.

[0228] Genetically engineered lymphocytes expressing modified TCRs can be engineered to express and secrete one or more soluble 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.

[0229] Cytokines that can be expressed and / or secreted by the genetically engineered lymphocytes described herein include, but are not limited to, 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), ...2 17), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-33 (IL-33), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon alpha (IFN-alpha or IFN-α), interferon beta (IFN-beta or IFN-β), interferon gamma (IFN-gamma or IFN-γ), transfer growth factor beta (TGF-β), CCL19 and erythropoietin. In some embodiments, the cytokine expressed by the genetically engineered lymphocytes is interleukin-2. In some embodiments, the cytokine expressed by the genetically engineered lymphocytes is interferon-γ. In some embodiments, the cytokine expressed by the genetically engineered lymphocytes is GM-CSF.

[0230] Chemokines that can be expressed and / or secreted by the genetically engineered lymphocytes described herein include, but are not limited to, CXC chemokines, such as interleukin-8 (IL-8), neutrophil activation protein-1 (NAP-1), neutrophil activation protein-2 (NAP-2), GRO, GROβ, GROγ, ENA-78, GCP-2, IP-10, MIG, CXCL1, CXCL12, CXCL16, CXCL19, and PF4; and CC chemokines, RANTES, MIP-1α, MIP-2β, monocyte chemoattractant protein-1 (MCP-1), MCP-2, MCP-3, CCL5, and eosinophil chemotactic factor. Suitable chemokines described in International Publication No. WO2000078334A1 (e.g., Table 1) are also included within the scope of the present invention, and the entire text of International Publication No. WO2000078334 A1 is incorporated herein by reference.

[0231] Growth factors that may be expressed and / or secreted by the genetically engineered lymphocytes 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 growth factor, Schwann cell-derived growth factor, vaccinia virus growth factor, silkworm protein, neu differentiation factor, v-Sis, and ciliary growth factor.

[0232] Soluble receptors that can be expressed and / or secreted by genetically engineered lymphocytes 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 receptors (sEGFRs), soluble vascular endothelial growth factor receptors, and PD-1 ectodomains, as well as soluble VEGFR-1 and SIRP-α molecules. Soluble receptors that can be expressed and / or secreted by genetically engineered lymphocytes described herein can be further fused to CD28 endodomains or 41BB endodomains or any other costimulatory endodomains known in the art.

[0233] The ligands that can be expressed and / or secreted by the genetically engineered lymphocytes described herein include, but are not limited to, tumor necrosis factor (TNF) ligands, such as 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-α ligand, CD30L / CD153, tumor necrosis factor beta (TNFβ) / lymphotoxin-α (LTα), lymphotoxin-β (LTβ), CD2 57 / B cell activating factor (BAFF) / Blys / THANK / Ta11-1, glucocorticoid-induced TNF receptor ligand (GITRL) and TNF-related apoptosis-inducing ligand (TRAIL) and LIGHT (TNFSF14); immunoglobulin superfamily ligands such as CD80 and CD86; and ligands of 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.

[0234] Genetically engineered lymphocytes described herein may express and / or secrete antibodies including antibodies that specifically bind to cancer antigens. Such cancer antigens may be selected from CD7, CD19, CD74, CDS, CEA, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER2, hTERT, IL-13R-a2, KDR, K-light chain, LeY, L1 cells, MAGE-A1, mesothelin, MUC1, MUC16, NKG2D ligands, NY-ESO-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, and WT-1. The amino acid sequence that specifically binds to the antigen is known in the art, or can be prepared using methods known in the art.

[0235] In certain embodiments, the antibody is a bispecific antibody (e.g., a bispecific T cell antibody (BiT)). A bispecific antibody is a recombinant synthetic antibody containing two different antigen binding domains. For example, one of the antigen binding domains can target a cancer antigen, while the other binds to a lymphocyte activation molecule.

[0236] Antibody fragments or antigen binding domains with affinity for cancer antigens can be expressed by genetically engineered lymphocytes as 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.

[0237] Genetically engineered lymphocytes expressing modified TCRs can be engineered to further express knockouts 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, genetically engineered lymphocytes expressing modified TCRs that can be engineered to express knockouts can be further engineered to express and secrete additional soluble proteins, such as but not limited to IL-2, IL-33, GM-CSF, CD40 agonists, and the like.

[0238] Methods of using such genetically engineered lymphocytes in adoptive cell transfer therapy are also provided. In certain embodiments, the lymphocytes can be autologous, allogeneic, isogenic, or allogenic to the subject. In certain embodiments, the lymphocytes are autologous so as to reduce the immune response against the lymphocytes when reintroduced into the subject for immunotherapy.

[0239] In one embodiment, provided herein is a genetically engineered lymphocyte expressing TCR and further expressing 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 intercellular protein. The intercellular protein can be, for example, a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphocyte can further express one or more cell surface receptors. In certain embodiments, TCR is an exogenous wild-type TCR. In certain embodiments, TCR is a modified TCR.

[0240] In one embodiment, provided herein is a genetically engineered lymphocyte 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 may be a growth factor. The growth factor may be GM-CSF. The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0241] In one embodiment, provided herein is a genetically engineered lymphocyte expressing a modified TCR, the 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 with SEQ ID NO: 2, and further expressing one or more soluble proteins. The soluble protein may be a growth factor. The growth factor may be GM-CSF. The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0242] In one embodiment, provided herein is a genetically engineered lymphocyte expressing a modified TCR, the 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 with SEQ ID NO: 3, and further expressing one or more soluble proteins. The soluble protein may be a growth factor. The growth factor may be GM-CSF. The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0243] In one embodiment, provided herein is a genetically engineered lymphocyte expressing a modified TCR, the 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 with SEQ ID NO: 4, and further expressing one or more soluble proteins. The soluble protein may be a growth factor. The growth factor may be GM-CSF. The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0244] In one embodiment, provided herein is a genetically engineered lymphocyte expressing a modified TCR, the 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 with SEQ ID NO: 5, and further expressing one or more soluble proteins. The soluble protein may be a growth factor. The growth factor may be GM-CSF. The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0245] In some embodiments, the GM-CSF is mouse GM-CSF or a functional fragment thereof. The UniProt identifier of mouse GM-CSF is UniProtKB-P01587.

[0246] 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.

[0247] In some embodiments, the 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, the functional fragment of GM-CSF comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the nucleotide sequence encoding the 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 the functional fragment of GM-CSF comprises the nucleotide sequence of SEQ ID NO:20.

[0248] In some embodiments, the GM-CSF is human GM-CSF or a functional fragment thereof. The UniProt identifier of human GM-CSF is UniProtKB-P04141.

[0249] 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 35.

[0250] In some embodiments, the 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, the functional fragment of GM-CSF comprises the amino acid sequence of SEQ ID NO: 25 or 36. In some embodiments, the nucleotide sequence encoding the 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 the functional fragment of GM-CSF comprises the nucleotide sequence of SEQ ID NO: 26 or 37.

[0251] Examples of sequences that can be used to express GM-CSF or functional fragments thereof are provided below.

[0252] Mouse GM-CSF (full-length protein sequence)

[0253] MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK(SEQID NO:15)

[0254] Mouse GM-CSF (full length DNA sequence)

[0255] ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCAGCACCCACCCGCTCACCCATCACTGTCACCCGGCTTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACATTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACC CGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAAAAACCAGGCCAAAAA(SEQ ID NO:16)

[0256] Mouse GM-CSF (protein sequence of signal peptide)

[0257] MWLQNLLFLGIVVYSLS(SEQ ID NO:17)

[0258] Mouse GM-CSF (DNA sequence of signal peptide)

[0259] ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCA(SEQ ID NO:18)

[0260] Mouse GM-CSF (protein sequence of active soluble GM-CSF)

[0261] APTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK(SEQ ID NO:19)

[0262] Mouse GM-CSF (DNA sequence of active soluble GM-CSF)

[0263] GCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACATTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAAAAACCAGGCCAAAAA(SEQ ID NO:20)

[0264] Human GM-CSF (Full-length protein 1)

[0265] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO:21)

[0266] Human GM-CSF (Full-length DNA 1)

[0267] ATGTGGCTGCAATCTCTGCTGCTGCTGGGCACAGTGGCCTGTTCTATTAGCGCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCCAAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCA GACCAGACTGGAACTGTACAAGCAGGGCCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCTGAGACAAGCTGTGCCACACAGACCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA(SEQ ID NO:22)

[0268] Human GM-CSF (protein sequence of signal peptide)

[0269] MWLQSLLLLGTVACSIS(SEQ ID NO:23)

[0270] Human GM-CSF (DNA sequence of signal peptide)

[0271] ATGTGGCTGCAATCTCTGCTGCTGCTGGGCACAGTGGCCTGTTCTATTAGC(SEQ ID NO:24)

[0272] Human GM-CSF (protein sequence of active soluble GM-CSF 1)

[0273] APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQTITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO:25)

[0274] Human GM-CSF (DNA sequence of active soluble GM-CSF 1)

[0275] GCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCCAAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGAGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCAGACCAGACTGGAACTGTACAAGCAGGGCCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCTGAGACAAGCTGTGCCACACAGACCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA(SEQ ID NO:26)

[0276] Human GM-CSF (Full-length Protein 2)

[0277] MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO:34)

[0278] Human GM-CSF (Full-length Protein 2)

[0279] ATGTGGCTGCAATCTCTGCTGCTGCTGGGCACAGTGGCCTGTTCTATTAGCGCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCC AAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGAGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCA GACCAGACTGGAACTGTACAAGCAGGGCCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCT GAGACAAGCTGTGCCACACAGATCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA(SEQ ID NO:35)

[0280] GM-CSF (GM-CSF 2 injection)

[0281] APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE(SEQ ID NO:36)

[0282] GM-CSF (GM-CSF 2 in DNA)

[0283] GCCCCTGCCAGATCTCCATCTCCTAGCACACAGCCTTGGGAGCACGTGAACGCCATCCAAGAAGCCAGACGGCTGCTGAACCTGAGCAGAGATACAGCCGCCGAGATGAACGAGACAGTGGAAGTGATCAGCGAGATGTTCGACCTGCAAGAGCCTACCTGCCTGCAGACCAGACTGGAACTGTACAAGCAG GGCCTGAGAGGCAGCCTGACCAAGCTGAAAGGCCCTCTGACAATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACACCTGAGACAAGCTGTGCCACACAGATCATCACCTTCGAGAGCTTCAAAGAGAACCTGAAGGACTTCCTGCTGGTCATCCCCTTCGACTGCTGGGAGCCCGTGCAAGAA(SEQ ID NO:37)

[0284] In one embodiment, provided herein is a genetically engineered lymphocyte 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 intracellular proteins. The intracellular protein may be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0285] In one embodiment, provided herein is a genetically engineered lymphocyte expressing a modified TCR, the 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 with SEQ ID NO: 2, and further expressing one or more intracellular proteins. The intracellular protein may be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0286] In one embodiment, provided herein is a genetically engineered lymphocyte 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 with SEQ ID NO: 3, and further expressing one or more intracellular proteins. The intracellular protein may be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0287] In one embodiment, provided herein is a genetically engineered lymphocyte 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 with SEQ ID NO: 4, and further expressing one or more intracellular proteins. The intracellular protein may be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0288] In one embodiment, provided herein is a genetically engineered lymphocyte 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 with SEQ ID NO: 5, and further expressing one or more intracellular proteins. The intracellular protein may be a phosphodiesterase (e.g., PDE4B2). The genetically engineered lymphocyte may further express one or more cell surface receptors.

[0289] In some embodiments, the genetically engineered lymphocytes disclosed herein express, in addition to the modified TCRs of the present invention, a phosphodiesterase. 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).

[0290] In one embodiment, PDE4B2 is human PDE4B2 or a functional fragment thereof.Human PDE4B2 has a GenInfo identifier of 82799482 and a NCBI accession number of NP_001032416.

[0291] 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.

[0292] In one embodiment, the PDE4B2 is mouse PDE4B2 or a functional fragment thereof. Mouse PDE4B2 has a GenInfo identifier of 295789129 and a NCBI accession number of NP_001171451.

[0293] 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.

[0294] Examples of sequences that can be used to express PDE4B2 or functional fragments thereof are provided below.

[0295] Human PDE4B2 amino acid sequence

[0296] MKEHGGTFSSTGISGGSGDSAMDSLQPLQPNYMPVCLFAEESYQKLAMETLEELDWCLDQLETIQTYRSVSEMASNKFKRMLNRELTHLSEMSRSGNQVSEYISNTFLDKQNDVEIPSPTQKDREKKKKQQLMTQISGVKKLMHSSSLNNTSISRFGVNTENEDHLAKELEDLNKWGLNIFNVAGYSHNRPLTCIMYAIFQERDLLKTFRISSDTFITYMMTLEDHYHSDVAYHNSLHAADVAQSTHVLLSTPALDAVFTDLEILAAIFAAAIHDVDHPGVSNQFLINTNSELALMYNDESVLENHHLAVGFKLLQEEHCDIFMNLTKKQRQTLRKMVIDMVLATDMSKHMSLLADLKTMVETKKVTSSGVLLLDNYTDRIQVLRNMVHCADLSNPTKSLELYRQWTDRIMEEFFQQGDKERERGMEISPMCDKHTASVEKSQVGFIDYIVHPLWETWADLVQPDAQDILDTLEDNRNWYQSMIPQSPSPPLDEQNRDCQGLMEKFQFELTLDEEDSEGPEKEGEGHSYFSSTKTLCVIDPENRDSLGETDIDIATEDKSPVDT(SEQ ID NO:27)

[0297] Human PDE4B2 nucleotide sequence

[0298]

[0299] Mouse PDE4B2 amino acid sequence

[0300] MKEQGGTVSGAASSRGGGDSAMASLQPLQPNYLSVCLFPEESYQKLAMETLEELDWCLDQLETIQTYRSVSEMASNKFKRMLNRELTHLSEMSRSGNQVSEYISNTFLDKQNDVEIPSPTQKDREKKKKQQLMTQISGVKKLMHSSSLNNTSISRFGVNTENEDHLAKELEDLNKWGLNIFNVAGYSHNRPLTCIMYAIFQERDLLKTFKISSDTFVTYMMTLEDHYHSDVAYHNSLHAADVAQSTHVLLSTPALDAVFTDLEILAAIFAAAIHDVDHPGVSNQFLINTNSELALMYNDESVLENHHLAVGFKLLQEEHCDIFQNLTKKQRQTLRKMVIDMVLATDMSKHMSLLADLKTMVETKKVTSSGVLLLDNYTDRIQVLRNMVHCADLSNPTKSLELYRQWTDRIMEEFFQQGDKERERGMEISPMCDKHTASVEKSQVGFIDYIVHPLWETWADLVQPDAQDILDTLEDNRNWYQSMIPQSPSPPLDERSRDCQGLMEKFQFELTLEEEDSEGPEKEGEGHSYFSSTKTLCVIDPENRDSLEETDIDIATEDKSPIDT(SEQ ID NO:29)

[0301] Mouse PDE4B2 nucleotide sequence

[0302]

[0303] Bifunctional molecules

[0304] One method to overcome the lack of effective anti-tumor T cell immunity is to genetically modify T cells ex vivo to target tumors by using receptors with enhanced affinity generated from T cell receptors or antibody-derived receptors as described above. A complementary method that does not require in vitro manipulation of T cells includes the use of fusion proteins that combine tumor recognition and T cell engagement domains to redirect T cells to target tumors. The specificity and anti-tumor activity of this fusion protein are described in, for example, Cancer Immunol Immunother (2013) 62: 773-785, Nat Med. June 2012; 18 (6): 980-7), and U.S. Patents 7,763,718 and 10,130,721, which are incorporated herein by reference for all purposes.

[0305] In one aspect, bifunctional molecules are provided herein, comprising a modified TCR disclosed herein or a functional fragment thereof, 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.

[0306] In certain embodiments, the polypeptide is an immune effector polypeptide.

[0307] As used herein, the term "immune effector polypeptide" generally refers to any molecule that induces or stimulates an immune response by directly or indirectly activating the humoral or cellular branches 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 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD44 antibodies, anti-CD45RA antibodies, anti-CD45RB antibodies, anti-CD45RO antibodies, anti-CD49a antibodies, anti-CD49b antibodies, anti-CD49c antibodies, anti-CD49d antibodies, anti-CD49e antibodies, anti-CD49f antibodies, anti-CD16 antibodies, anti-CD28 antibodies, anti-IL-2R antibodies, viral proteins and peptides, and bacterial proteins or peptides.

[0308] In some embodiments, the polypeptide comprises an antibody or antibody fragment. Antibody fragments may 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. In a specific embodiment, the polypeptide comprises scFv. In certain embodiments, the polypeptide is an immune effector polypeptide.

[0309] 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 that specifically binds to CD3 is an immune effector polypeptide.

[0310] In certain embodiments, the N-terminus of the TCR is connected 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 connected 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, wherein the N-terminus of the α or β chain of the heterodimeric TCR polypeptide pair or the N-terminus of the scTCR polypeptide is connected 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, wherein the C-terminus of the α or β chain of the heterodimeric TCR polypeptide pair or the C-terminus of the scTCR polypeptide is connected to the N-terminal amino acid of a polypeptide that specifically binds to a cell surface protein on a T cell.

[0311] The connection of TCR and the polypeptide of cell surface protein on specific binding T cell can be direct, also can be indirectly through linker sequence.Linker sequence is usually flexible, because they are made up of amino acid such as glycine, alanine and serine that may not limit flexible large side chain.In the case of any given TCR bifunctional molecule, the available or optimal length of linker sequence is easy to determine.In some cases, linker length will be less than about 12, for example, less than about 10, or 5-10 amino acid.

[0312] In some embodiments of the bifunctional molecules described herein, the modified TCR or its functional fragment comprises a beta 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 with an amino acid sequence selected from any of SEQ ID NOs: 2-5. In one embodiment, the modified TCR or its functional fragment comprises a beta 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 with an amino acid sequence of SEQ ID NO: 2. In one embodiment, the modified TCR or its functional fragment comprises a beta 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 with the amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified TCR or its functional fragment comprises a beta 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 with the amino acid sequence of SEQ ID NO: 4. In one embodiment, the modified TCR or its functional fragment comprises a beta 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 with the amino acid sequence of SEQ ID NO: 5. In any of the above embodiments, relative to an unsubstituted WT TCR beta chain (e.g., as shown in SEQ ID NO: 1), the modified TCR or its functional fragment comprises a single amino acid substitution located within CDR2 of the beta chain.

[0313] In certain embodiments of the bifunctional molecules described herein, the modified TCR or its functional fragment 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 its functional fragment comprises a β chain comprising an amino acid sequence of SEQ ID NO: 2. In one embodiment, the modified TCR or its functional fragment comprises a β chain comprising an amino acid sequence of SEQ ID NO: 3. In one embodiment, the modified TCR or its functional fragment comprises a β chain comprising an amino acid sequence of SEQ ID NO: 4. In one embodiment, the modified TCR or its functional fragment comprises a β chain comprising an amino acid sequence of SEQ ID NO: 5.

[0314] In certain embodiments of the bifunctional molecules described herein, the modified TCR or its functional fragment 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 its functional fragment further comprises an alpha chain comprising the amino acid sequence of SEQ ID NO: 7.

[0315] In various embodiments, the bifunctional molecules described herein can be used in conjunction with the host cells or pharmaceutical compositions described herein.

[0316] Treatment

[0317] On the one hand, a method for treating cancer in a subject is provided herein. The method includes administering to a subject suffering from such cancer an effective amount of lymphocytes presenting the modified TCR of the present invention or a pharmaceutical composition comprising these cells. Non-limiting examples of cancers that can be treated by the methods 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.

[0318] In one aspect, the present invention provides a method for stimulating or enhancing an immune response in a mammal, the method comprising administering to the mammal an effective amount of the genetically engineered lymphocytes of the present invention or a pharmaceutical composition comprising the cells.

[0319] Other examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (eg, 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 cell carcinoma, lung cancer (e.g., including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach cancer or gastric cancer (e.g., including gastrointestinal cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal 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 (e.g., high-grade glioma, diffuse pontine glioma, ependymoma, neuroblastoma or glioblastoma) and head and neck cancer and related metastases. Other examples of tumors can be found 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 the Merck Manuals internet address); and SEER Program Coding and Staging Manual 2016, each of which is incorporated herein by reference in its entirety for all purposes.

[0320] In various embodiments, the cancers treatable by the methods described herein may present the cancer antigen NY-ESO-1 on their cell surface. 157-165 epitope (SEQ ID NO: 8).

[0321] In some embodiments of any of the above methods of treatment, the composition is administered in a therapeutically effective amount. Depending on the subject's physical parameters, dosing frequency, administration method, clearance rate, etc., the dosage of the composition administered in the method of the present invention will vary greatly. The initial dose may be large, followed by a smaller maintenance dose. The dose may be administered less frequently, such as once a week or every two weeks, or divided into smaller doses and administered daily, half a week, etc., to maintain an effective dose level. It is expected that multiple doses will effectively achieve the in vivo persistence of the modified host cell. It is also expected that multiple doses will effectively improve the in vivo effector function of the modified host cell.

[0322] In some embodiments, 10 2 Up to 10 10 cells / kg body weight, 10 5 Up to 10 9 cells / kg body weight, 10 5 Up to 10 8 cells / kg body weight, 10 5 Up to 10 7 cells / kg body weight, 10 7 Up to 10 9 cells / kg body weight or 10 7 Up to 10 8 The composition comprising the modified host cells prepared by the methods described herein is administered at a dosage of 1 cell / kg body weight, including all integer values ​​within these ranges. The number of modified host cells will depend on the intended therapeutic use of the composition.

[0323] The modified host cells may be administered multiple times at the doses listed above. The modified host cells may be allogeneic, syngeneic, xenogeneic or autologous to the patient being treated.

[0324] The compositions and methods described in the present disclosure can be used in conjunction with other types of tumor treatments, such as chemotherapy, surgery, radiation, gene therapy, and the like.

[0325] It is also contemplated that when used to treat various diseases / disorders, the compositions and methods of the present disclosure can be used together with other treatment methods / agents applicable to the same or similar diseases / disorders. Such other treatment methods / agents can be co-administered (simultaneously or sequentially) to produce an additive or synergistic effect. Due to such additive or synergistic effects, the appropriate therapeutically effective dose of each drug can be reduced.

[0326] In some embodiments of any of the above treatment methods, the method further comprises administering to the subject one or more additional compounds selected from immunosuppressants, biologics, probiotics, prebiotics, and cytokines (eg, GM-CSF, IFN, or IL-2).

[0327] In some embodiments, in addition to GM-CSF produced by immune cells, the methods described herein further include providing exogenous GM-CSF to enhance the function of immune cells expressing the chimeric cytokine receptor of the present disclosure. Exogenous GM-CSF can be provided by, for example, but not limited to, the following methods: i) injection of the FDA-approved GM-CSF drug Sargramostin (Leukine TM ), or ii) using non-viral or viral vectors to express GM-CSF (e.g., the FDA-approved oncolytic virus talimogene laherparepveec [TVEC, Imlygic TM ]). These agents can be administered before, during, or after the administration (eg, infusion) of immune cells expressing the chimeric cytokine receptor of the invention to the patient.

[0328] As a non-limiting example, the present invention can be combined with other therapies that block inflammation (e.g., by blocking IL1, INF alpha / beta, IL6, tumor necrosis factor, IL23, etc.).

[0329] The methods and compositions of the present invention can be combined with other immunomodulatory treatments, such as 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 treatment methods that have the ability to regulate NKT function or stability, including but not limited to CD1d, CD1d-fusion protein, CD1d dimer, or larger polymers of CD1d that are not loaded or loaded with antigen, CD1d-chimeric antigen receptor (CD1d-CAR) or any other isomer of the five known CD1 isomers present in humans (CD1a, CD1b, CD1c, CD1e). The methods of the present invention can also be used in combination with other treatment methods such as midostatin, etanercept, or a combination thereof.

[0330] The treatment methods of the present invention can be combined with additional immunotherapy and therapy. For example, when used to treat tumors, depending on the type of tumor, patient condition, other health problems and various factors, the composition of the present invention can be used in combination with conventional therapy, such as surgery, radiotherapy, chemotherapy or a combination thereof. In some aspects, other therapeutic agents that can be used to treat tumors in combination with the inhibitor of the present invention include anti-angiogenic agents. Many anti-angiogenic drugs have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor β, interferon α, soluble KDR and FLT-1 receptor, placental hyperplasia-related protein (placentalproliferin-related protein), and those listed by Carmeliet and Jain (2000). In one embodiment, the modified host cells of the present 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 VEGF receptor, a neutralizing anti-VEGFR antibody, an inhibitor of VEGFR tyrosine kinase, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranizumab).

[0331] Non-limiting examples of chemotherapeutic compounds useful in the combination therapy of the present disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, bcg, bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, actinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide , gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ferrinotecan, letrozole, leucovorin, leuprolide, levamisole, methoxyprogesterone, medroxyprogesterone, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole , octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, pofibrem, procarbazine, raltitrexed, rituximab, streptozotocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0332] These chemotherapeutic compounds can be classified into the following groups according to their mechanism of action: antimetabolites / antineoplastic agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimiotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine and vinorelbine), microtubule disrupting agents such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epipodophyllotoxins (etoposide, teniposide), DNA agents (actinomycin, amsacrine, anthracyclines, bleomycin); =Inhibitors such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and deprives the body of the ability to cells that synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (chloroethylamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamines (altretinoin and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptouracil) (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (letrozole, anastrozole); anticoagulants ( heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytics (e.g., tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; 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 (retinoic acid);mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, enoposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, hydrocortisone, prednisolone, hydrocortisone) and growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors. ;

[0333] In various embodiments of the methods described herein, the subject is a human. The subject can be a teenager or an adult of any age or gender.

[0334] Pharmaceutical compositions, dosage forms and administration

[0335] In one aspect, provided herein is a pharmaceutical composition comprising a lymphocyte having a modified TCR as described herein and a pharmaceutically acceptable carrier.

[0336] The carrier can be a solvent or dispersion medium containing, for example, water, saline, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and a vegetable oil. The action of microorganisms can be prevented by various antibacterial and antifungal agents known in the art. In many cases, it is preferred to include isotonic agents, such as sugars or sodium chloride.

[0337] Pharmaceutical compositions based on a population of genetically engineered lymphocytes with modified TCRs described herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. Lymphocytes can be formulated for administration by, for example, injection, parenteral, vaginal, rectal administration, or direct administration to a tumor.

[0338] The pharmaceutical composition can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Injectable formulations can be present in unit dosage form, for example in ampoules or multidose containers, optionally with the addition of preservatives. The pharmaceutical composition can be further formulated as a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain other agents, including suspending agents, stabilizers and / or dispersants.

[0339] Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions; formulations include 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 form must be sterile and must be fluid. It must remain stable under manufacturing conditions and certain storage parameters (such as refrigeration and freezing), and must be preserved to prevent the contaminating action of microorganisms such as bacteria and fungi.

[0340] After formulation, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The dosage range and frequency of administration can vary depending on the nature of the genetically engineered lymphocyte population with a modified TCR as described herein, the medical condition and parameters of the specific patient, and the route of administration used.

[0341] In some embodiments, a population of genetically engineered lymphocytes having a modified TCR described herein can be grown in an amount of about 10 7 to about 10 12 The dosage of the drug may be administered to the subject. A more accurate dosage also depends on the subject. For example, if the subject is a teenager, a lower dosage may be required, and if the subject is an adult subject, a higher dosage may be required. In certain embodiments, a more accurate dosage may depend on the subject's weight. Example

[0342] The present invention is also described and illustrated by the following examples. However, the use of these and other examples anywhere in the specification is merely illustrative and in no way limits the scope and meaning of the present invention or any exemplary term. Likewise, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention will be apparent to those skilled in the art who read this specification, and these changes may be made without departing from the spirit or scope of the present invention. Therefore, the present invention is limited only by the terms of the appended claims and the full scope of equivalents of these claims.

[0343] Example 1 Binding affinity of soluble rationally designed BC1 TCR variants

[0344] Materials and methods

[0345] TCR amino acid substitutions were designed by structure-based computer-assisted 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 identify sequence modifications that could increase the affinity of the TCR for pMHC. The latter were actually introduced in the TCR structure, and the resulting changes in binding free energy were again estimated using the MM-GBSA method. For experimental validation, the sequence modifications predicted by computer were retained to favorably increase the binding free energy. Soluble TCRs were produced both in mammalian cell culture systems and in bacterial inclusion bodies. A direct titration ELISA against soluble pMHC was used to compare the binding strength with that of the wild-type TCR. The TCRs of interest were produced by refolding of bacterial inclusion bodies of the α and β chains and characterized by surface plasmon resonance to measure affinity and kinetics.

[0346] The wild-type TCR was named BC1 and differs from the well-characterized TCR 1G4 by only 4 amino acids (2 in the α chain and 2 in the β chain) (Chen, JL et al., J Exp Med 2005.201, 1243-1255, the entire contents of which are incorporated herein by reference for all purposes). The BC1 TCR is clinically significant because it is derived from an immunodominant clone of a long-term surviving cancer patient. Production and purification of soluble TCR in HEK-293 cells

[0347] The α chain and β chain of TCR BC1 are cloned into expression vector pHYK8 respectively and are under the control of CMV promoter. According to the strategy of Chang et al. (Chang, HC et al., Proc Natl Acad Sci USA, 1994.91, 11408-11412), heterodimer chain pairing is promoted by acid-alkaline zipper. TCR β chain is truncated after Cys242, and replaces flexible joint area, thrombin site, acid zipper and HIS tag, and α chain is truncated after Cys209, and replaces joint area, thrombin site and alkaline zipper replacement. Soluble TCR is produced by cotransfection of plasmid and linear 25kDa polyethyleneimine into HEK-293 cells. Transfected cells are suspended and cultured for up to 7 days in Pro293 CDM culture medium (Lonza) with the addition of 4mM valproic acid (so that acidification is minimized). Culture supernatants were collected by centrifugation and TCRs were purified using Ni-NTA agarose (Qiagen) following the manufacturer's recommendations.

[0348] Production of soluble TCR and pMHC from bacterial inclusion bodies

[0349] BL21(DE3)pLys bacterial cells were used to produce TCR α and β chains (cloned into pGMT7) as inclusion bodies, which were solubilized and refolded by dialysis as previously described (Boulter, JM et al., Protein Eng 2003. 16, 707-711). The TCRs were then concentrated and filtered before being purified with Ni 2+ Fast protein liquid chromatography HIS-tag purification was performed by fixed metal chelate agarose gel (GE Healthcare) and imidazole elution. Before SPR analysis, the sample was concentrated on a 10 kDa MWCO spin filter (Millipore) and gel-filtered into HBS-EP buffer (10 mM HEPES, pH 7.4, 0.15 M sodium chloride, 3 mM ethylenediaminetetraacetic acid, 0.005% v / v P20 surfactant) using an S200 chromatographic column to remove aggregates. Biotinylated A2 / NY-ESO157-165 was prepared as previously described (Altman, JD et al., Science, 1996.274, 94-96).

[0350] Titration ELISA of Rationally Developed TCRs

[0351] Biotinylated pMHC (A2 / NY-ESO157-165 complex) was captured on SA-coated plates (96-well, high binding plates, Corning Life Sciences) and blocked with 2% bovine serum albumin in Tris-buffered saline (TBS, pH 7.4). The plates were washed thoroughly with 0.1% Tween in TBS between each step. After incubation for 1.5 h at room temperature with TBS, 1% bovine serum albumin, 0.1% Tween, free sites on SA were blocked with biotin and then incubated with soluble TCR in TBS, 1% BSA, 0.1% tween for 1.5 h at room temperature. Bound TCRs were detected 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-mouse IgG-Ab (Thermo Scientific) diluted 1 / 1500 in TBS, 0.1% tween, and HRP was detected with 2,2'-azido-bis(3-ethylbenzothiazolidine-6-sulfonic acid) containing H2OO2 in citrate-phosphate buffer. Plates were read at OD405-490 after 30 minutes. ELISA for each TCR was repeated at least three times.

[0352] In Table 1 , WT BC1 TCR binding is defined as “+++”, binding greater than WT BC1 TCR is shown as “++++”, binding less than WT is shown as “++”, very weak binding is shown as “+”, and no binding is shown as “-”.

[0353] Table 1 Direct ELISA binding of soluble rationally designed TCRs to plate-captured pMHC relative to binding of wild-type TCR BC1

[0354]

[0355]

[0356] Surface plasmon resonance (SPR) of rationally developed TCRs

[0357] SPR was performed on a BIAcore 3000 and SA-coated CM5 sensor chips (BIAcore, GE Healthcare) at 25 °C. The flow cell was loaded with 200 response units of biotinylated pMHC at a rate of 10 μl / min for uniform distribution. Free sites on streptavidin (SA) were blocked with biotin and reference cells were used as controls to prevent changes in the bulk refractive index after injection of the sample solution. For kinetic analysis, 6–8 serial dilutions of TCR were injected at 50–100 μl / min on the loaded chip. Data represent one experiment out of at least two independent experiments performed in duplicate or triplicate giving the best Chi2 value. Assuming 1:1 Langmuir binding, the k for each TCR was calculated. on and k off The data were analyzed using BIAevaluation4.1 and the global fitting algorithm. D By k off / k on calculate.

[0358] In Table 2, "*" indicates that A97L and DMb are previously disclosed TCRs that confer optimal functional activity to T cells (Irving, M. et al., JBC 2012.287, 23068-23078, the entire contents of which are incorporated herein by reference for all purposes). "**" indicates that 1G4 is derived from a TCR that binds to NY-ESO-1 157-165Wild-type TCRs isolated from patients with known characteristics (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 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 variants are highlighted in bold.

[0359] Table 2 Biocore analysis of selected rationally designed TCRs

[0360]

[0361]

[0362] Results and discussion

[0363] As described above, a panel of HLA-A2-NY-ESO was developed 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). 157-165 TCRs have been developed that have increased affinity and function compared to native human T cells (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).

[0364] As shown in Tables 1 and 2, the TCR panel showed that it was specific for HLA-A2-NY-ESO 157-165 Table 1 shows direct binding of TCRs to pMHC as determined 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, several TCRs with single amino acid substitutions bind to pMHC with higher affinity than the WT TCR described with "++++". As shown in Table 2, at least two different single amino acid substitutions at residue 53 of the WT BC1 TCR (I53E and I53F) result in higher binding affinity (K) than the WT BC1 TCR. D WT>I53E>I53F), among which I53F is positive for HLA-A2-NY-ESO-1 157-165 Has the highest affinity.

[0365] Flow cytometric analysis of engineered CD4+ and CD8+ cells with several different BC1 TCR variants stained with anti-TCR BV13.1 Ab and fluorescent tetramers ( Figure 2A-2B and Figure 3A-3B ) showed that all tested variants (I53E, I53F, I53W, D55E and DMb) expressed comparable to wild type (BC1 TCR-transduced cells) and positive controls A97L and 1G4LY.

[0366] Example 2 Cytokine production by T cells expressing NY-ESO-1 TCR variants

[0367] Materials and methods

[0368] Cell line culture

[0369] 293T and Jurkat cell lines were purchased from ATCC. All cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated bovine serum albumin, 2 mmol / l L-glutamine, 100 μg / ml penicillin, and 100 U / ml streptomycin. 293T cell line was used for lentiviral packaging and preparation.

[0370] Cloning of TCRα and TCRβ chains and lentiviral production

[0371] Both the TCRα23 and TCRβ13.1 open reading frames were integrated into the lentiviral vector pRRL, in which most of the U3 region of the 3' long terminal repeat was deleted, resulting in self-inactivation, or SIN, of the 3' long terminal repeat. The TCRα and TCRβ chains were separated by a picornavirus-derived 2A sequence.

[0372] Lentivirus was produced by transient transfection of 293T cells with TurboFect. Briefly, 293T cells were co-transfected with the lentiviral vector pRRL encoding NY-ESOTCRa and TCRb chains and lentiviral helper plasmids (R8.74 and pMD2G). The lentiviral supernatant was harvested 48 h after transfection, filtered, and concentrated by ultracentrifugation. The pellet was resuspended in an appropriate volume of RPMI10% PBS and stored at -80°C or used directly.

[0373] Before lentiviral transduction, 1×10 6 αCD3 / αCD28 beads stimulated a total of 0.5 × 10 6 CD4+ or CD8+ T cells were cultured for 18-20 hours. On day 5, 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 by flow cytometry on day 6 and later time points after transduction. Antibodies used were PE-labeled human TCRVb13.1, and APC-labeled anti-human CD4 and anti-human CD8. NY-ESO was presented 157-165 PE-labeled HLA-A2 tetramers were used to detect the correct folding of the introduced TCR. The stained cells were analyzed using FlowJo software.

[0374] Cell lines

[0375] HLA-A2.1 + / NY-ESO + Melanoma cell lines Mw275 and A375, and 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.

[0376] 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.

[0377] Purification and transduction of primary T cells

[0378] Primary human T cells were isolated from peripheral blood mononuclear cells derived from buffy coats of healthy donors. All blood samples were collected with informed consent from the donors and then genetically modified with the approval of the Ethics Committee of the Canton of Vaud. Total PBMCs were obtained by Lymphoprep (Axonlab) separation solution using a standard centrifugation protocol, and CD4+ and CD8+ T cells were isolated using a negative selection kit combined with magnetic bead separation (easySEP, Stem Celltechnology). T cells were cultured in complete medium (RPMI 1640 with Glutamax, supplemented with 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, 100 g / ml streptomycin sulfate) (Invitrogen, Lifetechnologies) and stimulated with beads coated with anti-CD3 and anti-CD28 monoclonal antibodies (Lifetechnologies) at a ratio of 1:2 T cells: beads. 12 to 24 hours 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; GlaxoSmithKline) was added every other day to obtain a final concentration of 50 IU / ml until 5 days after stimulation (day +5). At this time, the magnetic beads were removed and 10 mg / ml h-IL15 (Miltenyi Biotec GmbH) was added without adding IL2. The concentration was maintained at 0.5-1×10 6 The engineered T cells were expanded at a cell density of 10 cells / mL. The introduced TCR was measured by fluorescent multimer staining 5 days after transduction. Prior to all functional analyses, resting engineered T cells were conditioned for the same transgene expression.

[0379] Cytokine production

[0380] Cytokine release assays were performed by culturing 5 × 10 cells / well in a final volume of 200 μl of RPMI medium in 96-well round-bottom plates. 4 T cells and 5×10 4 The co-culture supernatants were collected 24 hours later and tested for the presence of interferon-γ and interleukin-2 using enzyme-linked immunosorbent assay kits according to the manufacturer's protocol (BioLegend). The reported values ​​represent the average of transduced T cells from four healthy donors (HD).

[0381] Results and discussion

[0382] To evaluate the effect of TCR binding parameters on cytokine production, CD4+ and CD8+ cells that were not transduced and transduced with different TCR variants and WTBC1 TCR were loaded with 10-fold serial dilutions of NY-ESO 157-165The engineered CD4+ cells expressing TCRs I53F, I53W, D55E, DMb, and 1G4LY were incubated with T2 cells containing peptides. The secretion of IL-2 produced in response to TCR-mediated activation was maximal with engineered CD4+ cells expressing TCRs I53F, I53W, D55E, and DMb. Compared with WT TCR, engineered CD4+ cells expressing TCRs I53F, I53W, D55E, and DMb appeared to function better in terms of IL2 production, and their function was similar to that of 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+ cells did not produce interleukin-2 ( Figure 4A ).

[0383] Among tumor T cell lines that naturally express HLA / A2-NY-ESO-1(Me275) or are genetically engineered to express HLA / A2-NY-Eso-1(OVCAR5 NLM and A2008-A2-NLM), healthy donor (HD) T cells engineered to express TCR I53E produced IL-2 most efficiently compared to non-transduced (NT) cells (negative control), wild-type BC1 TCR-transduced cells, and cells transduced to express TCR I53E, as well as cells transduced to express A97L. There was no cross-reactivity with tumor cell lines that do not express HLA / A2-NY-ESO-1(OVCAR5 and A2008-A2 and NA8) Figure 5 ).

[0384] CD4+ T cells expressing TCR I53F and I53W in Me275, Saos-2, and U266 cells produced interleukin-2 most efficiently compared to non-transduced (NT) cells (negative control). CD4+ T cells engineered to express TCR DMb and 1G4LY produced interleukin-2 in Saos-2 and U266 cells, compared to other NY-ESO-positive cell lines where CD4+ T cells engineered to express TCR DMb and 1G4LY did not produce IL-2 significantly. CD4+ cells engineered to express TCR I53E, D55E, cells transduced with wild-type BC1 TCR, and cells transduced to express TCR A97L did not produce IL-2 in any of the NY-ESO-positive cell lines (negative control). Figure 6 ). In CD4+ T cells transduced with WT BC1 TCR, A2 / NY-ESO-NY-1 157-165Recognition is CD8 receptor dependent. In this case, CD4+ T cells transduced with the WT BC1 TCR appeared to be unresponsive to the NY-ESO epitope bound to MHC class I. However, when CD4+ T cells were transduced with TCR variants (i.e., I53F and I53W TCRs) that had higher binding affinity (compared to the WT BC1 TCR) for the NY-ESO epitope bound to MHC class I, higher IL-2 production by these engineered T cells was observed, indicating that the I53F TCR-transduced cells were less dependent on the CD8 co-receptor.

[0385] exist Fig. 9 In the study, CD4+ T cells engineered to express TCR I53F produced IL-2 most efficiently compared to cells engineered to express wild-type BC1 TCR and I53E TCR. CD4+ T cells engineered with TCR I53F had a trend toward less CD8 co-receptor dependence when compared to cells engineered with wild-type BC1 TCR and cells engineered to express I53E ( Fig. 9 ), and when Figure 5 The same trend was observed when compared to wild-type BC1 TCR-engineered cells, cells engineered to express I53E, and cells expressing the A97L TCR.

[0386] Regarding IFNγ production in response to TCR-mediated activation, engineered CD8+ cells expressing TCRs A97L, I53E, I53F, I53W, D55E, DMb, and 1G4LY showed similar function to WT BC1 TCR in this assay. Under the same conditions, non-transduced CD8+ cells did not produce IFNγ ( Figure 4B ).

[0387] Healthy donor (HD) T cells engineered to express TCRs A97L, I53E, and I53F efficiently produced IFN-γ compared to non-transduced (NT) cells (negative controls) from tumor T cell lines naturally expressing HLA / A2-NY-ESO-1 (Me275) or genetically engineered to express HLA / A2-NY-ESO-1 (OVCAR5 NLM and A2008-A2-NLM). There was 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 the affinity-enhanced BC1 TCR variant group was similar to that of T cells engineered to express wild-type BC1 TCR ( Figure 7 ).exist Fig.10 In the assay, CD8+ cells engineered to express the I53F TCR produced IFN-γ most efficiently when compared to cells expressing the wild-type BC1 TCR and the I53E TCR.

[0388] CD8+ T cells engineered to express TCRs 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 produced IFN-γ most efficiently. 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) produced IFN-γ efficiently in all NY-ESO-positive cell lines. CD8+ T cells engineered to express the wild-type BC1 TCR produced IFN-γ at moderate levels in all NY-ESO-positive cell lines except A375. Figure 8 ).

[0389] When the NY-ESO-positive cell line A375 was used, CD8+ T cells engineered to express the I53F TCR produced similar levels of IFN-γ as 1G4Y, whereas I53W produced less IFN-γ compared to I53F and 1G4(LY). When the NY-ESO-negative cell line was used, there was no IFN-γ production ( Fig.11 ).

[0390] Example 3 Cytotoxic activity of T cells expressing NY-ESO-1 TCR variants

[0391] The cytotoxic activity of NY-ESO TCR-specific CD8+ T cells was measured using IncuCyte. 4 T2 target cells were collected with 2.5 × 10 4 NY-ESO tetramer-positive CD8+ T cells were co-cultured in the presence of cytotoxic red reagent (Essen Bioscience, Ann Arbor, Michigan, USA). Images were taken every 2 hours using IncuCyte zoom software.

[0392] The cytotoxic activity of CD8+ T cells transduced with I53F and I53E was similar to that of CD8+ cells expressing wild-type BC1 TCR ( Fig.12 ).

[0393] Example 4 Anti-tumor activity of T cells expressing NY-ESO-1 TCR variants

[0394] Shortly before subcutaneous injection into NOD SCID gamma KO mice (NSG), a Winn test was performed in which 3 × 106 Me275 cells and 6×10 6 NY-ESO-specific T cells were mixed. The T cells consisted of 30% CD4+ T cells and 70% CD8+ T cells. Tumor size was measured twice a week. Once the tumor volume reached 1000mm 3 , the mice were killed.

[0395] like Fig. 13B As shown, both A97L- and I53F-transduced T cells effectively maintained tumor size at approximately 0 mm 3 In this assay, I53E-transduced T cells performed similarly to wild-type BC1 TCR-transduced T cells. In non-transduced T cells, tumor size reached approximately 700 mm at day 43. 3 .

[0396] Example 5 Anti-tumor activity of T cells expressing NY-ESO-1 TCR variants

[0397] NSG mice were injected with 5 × 10 6 Me275 cells. Once the tumor reaches 50-100 mm 3 Size, will be 10×10 6 NY-ESO-specific T cells were injected peritumorally or intravenously twice (day 10 and day 13). The T cells consisted of 30% CD4+ T cells and 70% CD8+ T cells. Tumor size was measured twice a week with a caliper. Once the tumor reached 1000 mm 3 , the mice were killed.

[0398] When administered peritumorally, A97L- and I53F-transduced T cells effectively maintained tumor size to less than 100 mm 3 In this assay, I53E-transduced T cells performed similarly to T cells transduced with wild-type BC1 TCR. In non-transduced T cells, tumors reached approximately 275 mm at day 30. 3 Size( Fig. 14B ).

[0399] When administered intravenously, T cells transduced with the I53F TCR (obtained from a healthy donor 1) effectively maintained tumors at negligible size over 50 days ( Fig. 15B , left). When T cells from another healthy donor (Healthy Donor 2) were used for transduction with the I53F TCR, such transduced cells effectively maintained tumors less than approximately 350 mm for sixty-five days. 3 ( Fig. 15B ,right).

[0400] When administered intravenously, T cells transduced with I53F and 1G4LY, respectively (obtained from healthy donor 3), were both potent and, when increased in titer, had similar characteristics to untransduced T cells in terms of keeping tumor volume size low ( Fig. 16B ).

[0401] Example 6 Modified T cells expressing NY-ESO-1 TCR and GM-CSF

[0402] Materials and methods

[0403] Mouse

[0404] NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ(NSG) mice were bred in-house under Specific Opportunistic 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 performed in accordance with Swiss federal law.

[0405] Cell lines

[0406] 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 + ) melanoma cell line was kindly provided by Professor D. Speiser (Lausanne branch of Ludwig Institute for Cancer Research, University of Lausanne) and was engineered to stably express luciferase to track its in vivo activity. +,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 activities 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.

[0407] Molecular amplification

[0408] The mouse CSF2 (GM-CSF) cDNA full sequence (Uniprot: P01587) was synthesized by Invitrogen. The sequence was then cloned into the pMSGV retroviral vector (Hughes, Human Gene Therapy, 2005; 16: 457-472) using PCR and standard molecular cloning techniques, followed by the Thy1.1-T2A (CD90.1) reporter gene cassette, to generate the pMSGV-Thy1.1-T2A-mGM-CSF retroviral plasmid. pMSGV-Thy1.1-T2A was used as a control vector.

[0409] Flow cytometry

[0410] APC anti-human CD8, APC anti-human CD4, APC anti-mouse Thy1.1 (CD90.1) were purchased from BioLegend. NY-ESO-1 TCR tetramer was produced in-house at the University of Lausanne’s Peptide and Tetramer Factory. FITC anti-human MCSP was purchased from Miltenyi Biotec. APC anti-mouse CD45, APCCy7 anti-mouse F4 / 80, and PECy7 anti-mouse CD11b were kindly provided by the Flow Cytometry Facility, FBM, LICR / UNIL and / or produced in-house. Acquisition was performed on an LSRII flow cytometer (BD Biosciences) and data were analyzed on FlowJo (TreeStar).

[0411] Retroviral / lentiviral production

[0412] To produce retroviral particles, HEK293T cells were co-transfected with pMSGV transfer plasmid and retroviral packaging plasmids pMD-Gag / Pol and pMD RD114 (Milone et al, 2018 Leukemia 32, 1529-1541) feline endogenous viral envelope glycoproteins. Culture supernatants were collected 24 hours, 48 ​​hours, and 72 hours after transfection and concentrated by ultracentrifugation at 24,000 g × for 2 hours. The concentrated virus was stored at -80°C until use. Viral titers and MOIs were determined by Thy1.1 reporter gene expression in HEK293T cells.

[0413] Human T cell stimulation and lentiviral / retroviral transduction

[0414] Healthy donor apheresis products were purchased from Transfusion Interrégionale CRS SA, Epalinges, Switzerland, with written consent, according to an approved university institutional review board protocol. PBMCs were prepared using Lymphoprep (StemCell Technologies) density gradient centrifugation and negatively isolated CD8 or CD4 T cells using CD8 or CD4 magnetic microbeads (Miltenyi) following the manufacturer's protocol. Isolated CD8 or CD4 T cells were stimulated with anti-CD3 / CD28 beads (Invitrogen) at a 2:1 beads:T cell ratio in the presence of human interleukin-2 (GlaxoSmithKline).

[0415] Lentiviral / retroviral transduction of human T cells

[0416] Lentiviral transduction of T cells was performed 24 h after activation by adding viral particles directly to the culture medium (MOI 20) and was enhanced by the simultaneous addition of Lentiboost (Sirion Bitechnology). Retroviral transduction of T cells was performed 48 h after activation. Briefly, T cells were transferred to connexin-coated plates that had been previously spun with retroviral particles at 2000 × g for 1.5 h. The next day, T cells were removed from the connexin-coated plates. CD3 / CD28 beads were removed 5 days after activation and then resuspended in 0.5–1 × 10 6T cells were maintained at 100 T cells / ml in RPMI 1640-Glutamax (ThermoFisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (ThermoFisherScientific), 1% penicillin / streptomycin, 10 ng / ml human interleukin-7 (Miltenyi), and 10 ng / ml interleukin-15 (Miltenyi) until downstream use.

[0417] Interferon-γ production / T cell cytotoxicity assay

[0418] 10 5 resting T cells (4:1CD8+:CD4+) and 10 5 The tumor cells were co-cultured in complete medium for 48 h. The number of T cells was normalized according to the transduction efficiency, and untransduced T cells were added when necessary to obtain similar T cell frequencies under different conditions. After 48 h, interferon γ levels were determined in the collected cell-free culture supernatants by ELISA (ThermoFisherScientific) according to the manufacturer's protocol. T cell toxicity was determined by analyzing the cells by flow cytometry and was defined as the expression of annexin V in the culture medium. + / DAPI + Percentage of tumor cells. Results were normalized to Annexin V in cultures containing only tumor cells. + / DAPI + percentage.

[0419] Human T cells produce GM-CSF in transgenic mice

[0420] T cells (CD8 or CD4) were divided into 10 6 T cells / ml were cultured in serum-free medium supplemented with 10 ng / ml IL-7 / IL-15 for 24 h. Viability and cell number were previously determined using a hematocrit. T cell numbers were normalized according to transduction efficiency, and untransduced T cells were added when necessary to obtain similar T cell frequencies under different conditions. After 24 h, mouse GM-CSF levels in the collected cell-free supernatants were determined by ELISA (ThermoFisher Scientific) according to the manufacturer's protocol.

[0421] Generation of bone marrow-derived macrophages (BMDMs)

[0422] Whole bone marrow cells were isolated by flushing the femur and tibia of NSG mice. Macrophages were generated by incubating whole 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. The medium was changed on days 3 and 6.

[0423] Winn Test

[0424] Male NSG mice aged 6-12 weeks were subcutaneously inoculated in the flank with 5 × 10 6 5×10 (or other indicated amount) of human T cells expressing NY-ESO-1 TCR and secreting mouse GM-CSF (4:1 CD8+:CD4+) or an equal number of mock-transduced human T cells secreting mouse GM-CSF were mixed 6 Me275 melanoma cells. Tumor growth was monitored twice weekly by caliper measurement and the formula V = 1 / 2 (L x W 2 ) to calculate the tumor volume, where L is the maximum longitudinal diameter and width W is the maximum transverse diameter. 3 Mice were sacrificed when they lost more than 20% of their body weight or became weak and moribund. Each group consisted of ≥5 mice.

[0425] Xenograft Models

[0426] 5 × 10 6 Me275 melanoma cells. In parallel, human T cells were activated, transduced, and expanded as described above. When tumors reached 50–100 mm approximately 2 weeks after inoculation, 3 T cells were adoptively transferred into mice at 13 days and 15 days. T cells were injected twice with 1×10 7 NY-ESO-1 TCR expressing, mouse GM-CSF secreting human T cells (4:1 CD8+:CD4+) or an equal number of mock transduced mouse GM-CSF secreting human T cells were added. Tumor growth was monitored by caliper measurement as described above.

[0427] Results and discussion

[0428] Schematic diagram of the retroviral mouse GM-CSF and lentiviral NY-ESO-1 TCR constructs used in the study are shown in Fig.17A At day 7 after viral transduction, NY-ESO-1TCR and mouse GM-CSF expression in human CD8+ and CD4+ T cells was confirmed by flow cytometry ( Fig. 17B Secreted mouse GM-CSF can be detected in the supernatant of transduced CD8+ T cell cultures by ELISA ( Fig. 17C ). This demonstrates that human T cells can be efficiently co-engineered to stably express the NY-ESO-1 TCR and secrete mouse GM-CSF.

[0429] In recognition of HLA-A2 + NY-ESO-1 + After tumor cells, IFNγ secretion by NY-ESO-1TCR engineered T cells can be detected ( Fig.18A GM-CSF in mice had no effect on interferon-γ levels. NY-ESO-1 TCR-engineered T cells can readily kill HLA-A2 + NY-ESO-1 + tumor cells, and their cytotoxic activity was not affected by mouse GM-CSF ( Fig.18B ). This suggests that secreted mouse granulocyte colony-stimulating factor has no effect on human T cell activity.

[0430] Human NY-ESO-1 TCR-expressing T cells co-engineered to secrete mouse GM-CSF effectively slowed the engraftment and establishment of Me275 melanoma cells compared with control T cells expressing NY-ESO-1 TCR alone ( Fig.19A This was greatly reflected in the survival rate of the mice, with 40% of the mice treated with T cells expressing the NY-ESO-1 TCR and secreting mouse GM-CSF being tumor-free ( Fig.19B ). Compared with NY-ESO-1 TCR alone, adoptive transfer of 2×10 7 T cells expressing NY-ESO-1 TCR and secreting mouse GM-CSF showed better tumor control against established Me275 tumors ( Fig.19C ). This showed that in the NSG human melanoma xenograft mouse model, T cell-derived mouse GM-CSF secreted in the tumor microenvironment could significantly enhance the control of tumor growth by NY-ESO-1-specific T cells.

[0431] Example 7 Modified T cells expressing NY-ESO-1 TCR "I53F" variant and PDE4B2

[0432] Materials and methods

[0433] Molecular amplification

[0434] The lentiviral vector pRRL used in Bobisse, S. et al. Cancer Res. 69, 9385-9394 (2009) was used to overexpress human PDE4B2 homologues in human T cells. In particular, sequences cloned downstream of the human PGK promoter and the Kozak element (GCCACC (SEQ ID NO: 31)) encoding human WT PDE4B2 (NCBI reference sequence: NP_001032416.1; Fig.26 27) and connected to the following amino acid sequence via a DGGG (SEQ ID NO: 32) linker: GKPIPNPLLGLDSTGGSGGGKPIPNPLLGLDST GSGSGSGKPIPNPLLGLDST (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 the engineering of control T cells, the same pRRL vector backbone was used, in which the eGFP coding sequence was introduced downstream of the human PGK promoter and Kozak element. In order to confer NY-ESO-1 antigen specificity to T cells, the SFG retroviral vector used in Lamers, CHJ et al. Cancer Gene Ther. 15, 268-274 (2008) was used, which encodes the α chain and β chain of the NY-ESO-1 TCR variant β-I53F. All introduced DNA sequences were codon optimized for protein expression in human cells.

[0435] Concentrated lentiviral or retroviral production

[0436] HEK-293T cells were transfected for the purpose of producing viral particles. In short, for the production of lentiviral particles, 18 μg of the R8.74 plasmid encoding gag / pol, 7 μg of the VSVG plasmid encoding the envelope protein, and 15 μg of the pRRL vector encoding the transgene were diluted with 120 μl TurboFect (R0532, Thermo Fisher Scientific) premixed with 3 mL Opti-MEM (11058-021, Thermo Fisher Scientific). In a similar manner, in order to produce retroviral particles, 18 μg of the PegPam encoding gag / pol, 7 μg of the RD114 plasmid encoding the envelope protein, and 22 μg of the SFG retroviral vector were diluted with 120 μl TurboFect premixed with 3 mL Opti-MEM. It is worth noting that the TurboFect / Opti-MEM solution was incubated at room temperature for 5 minutes before being used to dilute the packaging / transgenic plasmid. Subsequently, the transfection mixture was incubated at room temperature for 30 minutes and then 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 24 hours, the culture supernatant was replaced with fresh R10 and harvested after another 24 hours, filtered (45 μm) and centrifuged at 24000 g for 2 hours. Finally, the viral particles were resuspended in 400 μl R10 and then quickly frozen with dry ice and placed at -80°C.

[0437] Isolation, transduction and culture of T cells

[0438] PBMCs from healthy donors were isolated on day 0 by standard Ficoll-Paque centrifugation according to the manufacturer's instructions and CD4 + or CD8 + T cells. After isolation, T cells in R10 were immediately stimulated with anti-CD3 / CD28-coated microbeads (11132D, Thermo Fisher Scientific) in the presence of 50 U / mL recombinant human IL2 (kindly provided by Glaxo SmithKline), 2 microbeads per T cell. T cells were activated in 48-well plates, 0.5 × 10 6 T cells at a concentration of 1 × 10 per ml 6For lentiviral transduction, after 18-22 hours of stimulation, add LentiBOOST TM (Sirion Biotech GmbH), 100 μl of concentrated lentivirus containing 0.5 × 10 6 T cells. Retroviral transduction occurred in the wells of an untreated 48-well cell culture plate. Specifically, the wells were coated with a 20 ng / ml solution of RetroNectin (T100B, Takara) at 4°C overnight before washing with PBS, then the wells were washed with PBS and blocked with R10 for 30 minutes at 37°C. After washing the wells with PBS, 50 μl of concentrated retrovirus and 50 μl of R10 were added, and the plates were centrifuged at 2000g for 90 minutes at 25 degrees. For retroviral co-transduction, T cells previously exposed to lentivirus were transferred to RetroNectin / retrovirus-coated wells within 40-44 hours after stimulation and spun at 260g for 10 minutes. 24 hours after retroviral transduction, such T cells were transferred to tissue culture treated wells. To support T cell expansion, R10 supplemented with IL2 (50 U / mL) was added to the cultures until day 5, the time point at which T cell activation beads were removed. R10 supplemented with 10 ng / mL recombinant human IL7 and IL15 (130-095-764, 130-095-362, Miltenyi) was added to T cell cultures starting on day 5. On day 7, transduction efficiency was assessed by flow cytometry, while T cells were used for intracellular cAMP quantification or functional assays at any time from day 8 to day 21.

[0439] Measurement of cAMP

[0440] Resting T cells were washed and exposed to different concentrations of forskolin or PGE 2 1 × 10 cells / mL in a 12-well plate. 6 T cells were treated at a concentration of 10 T cells, and 2.5 × 10 T cells were used per condition. 6 Subsequently, T cells were lysed with 250 μl of 0.1 M hydrochloric acid, and their intracellular cAMP content was assessed using a direct cAMP ELISA kit (ADI-900-066, Enzo Life Sciences) according to the manufacturer's instructions.

[0441] Flow cytometry

[0442] All flow cytometry data were acquired on an LCRII flow cytometer (BD) and analyzed using FlowJo software. Fixable aqueous dead dyes L34965 or L34975 (Invitrogen) were used for dead cell exclusion according to the manufacturer's instructions, and the following antibodies were used for T cell staining: CD45: Pac Blue (304029, BioLegend), anti-V5tag: FITC (R96325, Thermo Fisher Scientific), anti-V5tag: Dy650 (NBP2-52653C, Novusbiologicals), Vb13.1: PE (IM2292, BD), IFNγ: PeCy7 (502527, BioLegend), TNF-α: PE (502909, BioLegend), anti-BrDU: APC (12-5071-42, Thermo Fisher Scientific). To assess intracellular cytokine production by flow cytometry, 50 × 10 cells / well were activated with a combination of plate-coated aCD3 (5 μg / mL) and soluble αCD28 (2 μg / mL) in a round-bottom 96-well plate. 3 live T cells for 7 hours. To prevent cytokine secretion, Golgi stop (554724, BD) was added to the wells at a dilution of 1:400 1.5 hours after the start of the assay. Before evaluating the transduction efficiency of T cells or their ability to produce cytokines, T cells were fixed and permeabilized using a standard fixation / permeabilization kit (554714, BD) according to the manufacturer's instructions. BrdU staining kit for flow cytometry (8817-6600-42, Thermo Fisher Scientific) was used for T cell proliferation quantification. Briefly, 1×10 5 Live T cells were activated with plate-coated αCD3 (5 μg / mL) in flat-bottom 96-well plates for 48 h, and BrdU was added to the wells at a working concentration of 10 μM 7 h before termination of the assay.

[0443] Quantification of interferon-γ secretion

[0444] Resting eGFP-, PDE4B2-, eGFP&NY-ESO-1 TCR-, or PDE4B2&NY-ESO-1 TCR-transduced CD8 T cells and NY-ESO-1 presenting melanoma cells A375 were co-cultured with different concentrations of PGE2 or forskolin for 48 h, and the presence of IFNγ in the co-culture supernatants was assessed by ELISA kit (88-7316-88, Thermo Fisher Scientific) according to the manufacturer's instructions. Co-cultures were performed in flat-bottom 96-well plates, and 1 × 10 5 A375 cells and 1×10 5 TCR + Live T cells, or corresponding numbers of eGFP- / PDE4B2-transduced T cells.

[0445] Cytotoxicity assay

[0446] Resting eGFP-, PDE4B2-, eGFP&NY-ESO-1 TCR-, or PDE4B2&NY-ESO-1 TCR-transduced CD4 or CD8 T cells were expressed in the presence or absence of PGE 2 Co-culture with A375 cells for 47 or 44 hours. Co-culture was monitored by the IncuCyte instrument in a flat-bottom 96-well plate, and 1×10 4 A375 tumor cells and 25×10 3 TCR + Live T cells, or corresponding numbers of eGFP- / PDE4B2-transduced T cells. The A375 tumor cells used were engineered to express the nuclear fluorescent protein mCherry to enable direct detection in the wells.

[0447] Results and discussion

[0448] 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 ( Fig.21 ). Since NY-ESO-1 TCR contains the Vβ13.1 variant of the TCRβ chain, the presence of the former can be inferred by detecting Vβ13.1, although it is not completely accurate.

[0449] Exposure to Fsk or PGE 2 After 1 hour, the intracellular cAMP levels of resting eGFP- or PDE4B2-transduced CD4 T cells were measured. After treatment, T cells were lysed and cAMP levels were quantified by ELISA. The results showed that PDE4B2 overexpression prevented intracellular cAMP accumulation ( Fig. 22).

[0450] In PGE 2 The ability of resting eGFP- or PDE4B2-transduced CD4 T cells to produce IFNγ or TNF-α was determined by intracellular cytokine staining (ICS) after stimulation with or without plate-bound aCD3 and soluble aCD28 in the presence or absence of forskolin for 7 h. 2 IFN-γ secretion by resting eGFP-, PDE4B2-, eGFP&NY-ESO-1 TCR-, or PDE4B2&NY-ESO-1 TCR-transduced CD8 T cells in response to 48 h of co-culture with NY-ESO-1-presenting melanoma cells A375 in the presence or absence of forskolin. Both results suggest that PDE4B2 overexpression promotes Th-1 cytokine production under conditions that induce intracellular cAMP accumulation ( Fig.23A , 23B ).

[0451] The proliferation capacity of CD4 T cells transduced with eGFP or PDE4B2 was determined by BrDU incorporation assay. T cells were restimulated with or without plate-bound αCD3 in the presence of PGE2 or forskolin for 48 h. The results showed that PDE4B2 overexpression promoted proliferation under conditions that induced intracellular cAMP accumulation ( Fig.24 ).

[0452] The ability of resting eGFP-, PDE4B2-, eGFP & NY-ESO-1 TCR-, or PDE4B2 & NY-ESO-1 TCR-transduced CD4 or CD8 T cells to inhibit the expansion of NY-ESO-1 presenting melanoma cells A375 in the presence or absence of PGE2 was assessed by IncuCyte. Fig.25 As shown, PDE4B2 overexpression promoted T cell cytotoxicity in the presence of PGE2.

[0453] References

[0454] 1.Dunn,S.M.,Rizkallah,P.J.,Baston,E.,Mahon,T.,Cameron,B.,Moysey,R.,Gao,F.,Sami,M.,Boulter,J.,Li,Y.,and Jakobsen,B.K.(2006)Directed evolution ofhuman T cell receptor CDR2 residues by phage display dramatically enhancesaffinity for cognate peptide-MHC without increasing apparent cross-reactivity.Protein Sci 15,710-721.

[0455] 2.Robbins,P.F.,Morgan,R.A.,Feldman,S.A.,Yang,J.C.,Sherry,R.M.,Dudley,M.E.,Wunderlich,J.R.,Nahvi,A.V.,Helman,L.J.,Mackall,C.L.,Kammula,U.S.,Hughes,M.S.,Restifo,N.P.,Raffeld,M.,Lee,C.C.,Levy,C.L.,Li,Y.F.,El-Gamil,M.,Schwarz,S.L.,Laurencot,C.,and Rosenberg,S.A.(2011)Tumor regression in patients withmetastatic synovial cell sarcoma and melanoma using genetically engineeredlymphocytes reactive with NY-ESO-1.J Clin Oncol 29,917-924.

[0456] 3.Rapoport,A.P.,Stadtmauer,E.A.,Binder-Scholl,G.K.,Goloubeva,O.,Vogl,D.T.,Lacey,S.F.,Badros,A.Z.,Garfall,A.,Weiss,B.,Finklestein,J.,Kulikovskaya,I.,Sinha,S.K.,Kronsberg,S.,Gupta,M.,Bond,S.,Melchiori,L.,Brewer,J.E.,Bennett,A.D.,Gerry,A.B.,Pumphrey,N.J.,Williams,D.,Tayton-Martin,H.K.,Ribeiro,L.,Holdich,T.,Yanovich,S.,Hardy,N.,Yared,J.,Kerr,N.,Philip,S.,Westphal,S.,Siegel,D.L.,Levine,B.L.,Jakobsen,B.K.,Kalos,M.,and June,C.H.(2015)NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumoreffects in myeloma.Nat Med 21,914-921.

[0457] 4.Chen,J.L.,Stewart-Jones,G.,Bossi,G.,Lissin,N.M.,Wooldridge,L.,Choi,E.M.,Held,G.,Dunbar,P.R.,Esnouf,R.M.,Sami,M.,Boulter,J.M.,Rizkallah,P.,Renner,C.,Sewell,A.,van der Merwe,P.A.,Jakobsen,B.K.,Griffiths,G.,Jones,E.Y.,and Cerundolo,V.(2005)Structural and kinetic basis for heightenedimmunogenicity of T cell vaccines.J Exp Med 201,1243-1255.

[0458] 5.Zoete,V.,Irving,M.B.,and Michielin,O.(2010)MM-GBSA binding freeenergy decomposition and T cell receptor engineering.Journal of molecularrecognition.JMR 23,142-152.

[0459] 6.Zoete,V.,and Michielin,O.(2007)Comparison between computationalalanine scanning and per-residue binding free energy decomposition forprotein-protein association using MM-GBSA:application to the TCR-p-MHCcomplex.Proteins 67,1026-1047.

[0460] 7.Zoete,V.,Meuwly,M.,and Karplus,M.(2005)Study of the insulindimerization:binding free energy calculations and per-residue free energydecomposition.Proteins 61,79-93.

[0461] 8.Pettersen,E.F.,Goddard,T.D.,Huang,C.C.,Couch,G.S.,Greenblatt,D.M.,Meng,E.C.,and Ferrin,T.E.(2004)UCSF Chimera--a visualization system forexploratory research and analysis.J Comput Chem 25,1605-1612.

[0462] 9.Dull,T.,Zufferey,R.,Kelly,M.,Mandel,R.J.,Nguyen,M.,Trono,D.,andNaldini,L.(1998)A third-generation lentivirus vector with a conditionalpackaging system.J Virol 72,8463-8471.

[0463] 10.Schmid,D.A.,Irving,M.B.,Posevitz,V.,Hebeisen,M.,Posevitz-Fejfar,A.,Sarria,J.C.,Gomez-Eerland,R.,Thome,M.,Schumacher,T.N.,Romero,P.,Speiser,D.E.,Zoete,V.,Michielin,O.,and Rufer,N.(2010)Evidence for a TCR affinitythreshold delimiting maximal CD8 T cell function.Journal of immunology 184,4936-4946.

[0464] 11.Chang,H.C.,Bao,Z.,Yao,Y.,Tse,A.G.,Goyarts,E.C.,Madsen,M.,Kawasaki,E.,Brauer,P.P.,Sacchettini,J.C.,Nathenson,S.G.,and et al.(1994)A generalmethod for facilitating heterodimeric pairing between two proteins:application to expression of alpha and beta T-cell receptor extracellularsegments.Proc Natl Acad Sci U S A 91,11408-11412.

[0465] 12.Boulter,J.M.,Glick,M.,Todorov,P.T.,Baston,E.,Sami,M.,Rizkallah,P.,and Jakobsen,B.K.(2003)Stable,soluble T-cell receptor molecules forcrystallization and therapeutics.Protein Eng 16,707-711.

[0466] 13.Altman,J.D.,Moss,P.A.,Goulder,P.J.,Barouch,D.H.,McHeyzer-Williams,M.G.,Bell,J.I.,McMichael,A.J.,and Davis,M.M.(1996)Phenotypic analysis ofantigen-specific T lymphocytes.Science 274,94-96.

[0467] 14.Irving,M.,Zoete V.,Hebeisen M.,Schmid D.,Baumgartner P.,GuillaumeP.,Romero P.,Speiser D.,Luescher I.,Rufer N.,Michielin O.,(2012)Interplaybetween T Cell Receptor Binding Kinertics and the Level of Cognate PeptidePresented by Major Histocompatibility Complexes Governs CD8 + T CellResponsiveness.JBC 287,23068-23078.

[0468] 15.Chen,Y.T.,Scanlan,M.J.,Sahin,U.,Türeci,O.,Gure,A.O.,Tsang,S.,Williamson,B.,Stockert,E.,Pfreundschuh,M.,and Old L.J.(1997)A testicularantigen aberrantly expressed in human cancers detected by autologous antibodyscreening.Proc Natl Acad Sci U S A 94,1914-1918.

[0469] 16.Mellman,I.,G.Coukos,and G.Dranoff,Cancer immunotherapy comes ofage.Nature,2011.480:p.480.

[0470] 17.Restifo,N.P.,M.E.Dudley,and S.A.Rosenberg,Adoptive immunotherapyfor cancer:harnessing the T cell response.Nature Reviews Immunology,2012.12:p.269.

[0471] 18.Rosenberg,S.A.,et al.,Durable complete responses in heavilypretreated patients with metastatic melanoma using T-cell transferimmunotherapy.Clin Cancer Res,2011.17(13):p.4550-7.

[0472] 19.Robbins,P.F.,et al.,Tumor regression in patients with metastaticsynovial cell sarcoma and melanoma using genetically engineered lymphocytesreactive with NY-ESO-1.J Clin Oncol,2011.29(7):p.917-24.

[0473] 20.Kochenderfer,J.N.,et al.,B-cell depletion and remissions ofmalignancy along with cytokine-associated toxicity in a clinical trial ofanti-CD19 chimeric-antigen-receptor-transduced T cells.Blood,2012.119:p.2709-2720.

[0474] 21.Gust,J.,et al.,Endothelial Activation and Blood-Brain BarrierDisruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-TCells.Cancer Discovery,2017.7:p.1404-1419.

[0475] 22.Zou,W.,Immunosuppressive networks in the tumour environment andtheir therapeutic relevance.Nature Reviews Cancer,2005.5:p.263.

[0476] 23.Baruch,E.N.,et al.,Adoptive T cell therapy:An overview ofobstacles and opportunities.Cancer,2017.123(S11):p.2154-2162.

[0477] 24.Zhang,H.and J.Chen,Current status and future directions of cancerimmunotherapy.Journal of Cancer,2018.9(10):p.1773-1781.

[0478] 25.Kunert,A.and R.Debets,Engineering T cells for adoptive therapy:outsmarting the tumor.Current Opinion in Immunology,2018.51:p.133-139.

[0479] 26.Yoon,D.H.,et al.,Incorporation of Immune Checkpoint Blockade intoChimeric Antigen Receptor T Cells(CAR-Ts):Combination or Built-In CAR-T.International journal of molecular sciences,2018.19(2):p.340.

[0480] 27.Noy,R.and J.W.Pollard,Tumor-associated macrophages:from mechanismsto therapy.Immunity,2014.41(1):p.49-61.

[0481] 28.Fridlender,Z.G.and S.M.Albelda,Tumor-associated neutrophils:friendor foe?Carcinogenesis,2012.33(5):p.949-55.

[0482] 29.Mach,N.,et al.,Differences in dendritic cells stimulated in vivoby tumors engineered to secrete granulocyte-macrophage colony-stimulatingfactor or Flt3-ligand.Cancer Res,2000.60(12):p.3239-46.

[0483] 30.Ushach,I.and A.Zlotnik,Biological role of granulocyte macrophagecolony-stimulating factor(GM-CSF)and macrophage colony-stimulating factor(M-CSF)on cells of the myeloid lineage.Journal of Leukocyte Biology,2016.100:p.481-489.

[0484] 31.Becher,B.,S.Tugues,and M.Greter,GM-CSF:From Growth Factor toCentral Mediator of Tissue Inflammation.Immunity,2016.45:p.963-973.

[0485] 32.Hercus,T.R.,et al.,The granulocyte-macrophage colony-stimulatingfactor receptor:linking its structure to cell signaling and its role indisease.Blood.2009.114:p.1289-1298.

[0486] 33.Shi,Y.,et al.,Granulocyte-macrophage colony-stimulating factor(GM-CSF)and T-cell responses:what we do and don't know.Cell Res.2006.16(2):126-33.

[0487] 34.Arellano,M.and S.Lonial.Clinical uses of GM-CSF,a criticalappraisal and update.in Biologics:Targets&Therapy.Biologics.2008;2(1):13-27.

[0488] 35.Gupta,R.and L.A.Emens,GM-CSF-secreting vaccines for solid tumors:moving forward.Discovery medicine,2010.10(50):p.52-60.

[0489] 36.Mookerjee,A.,M.Graciotti,and L.Kandalaft,A cancer vaccine withdendritic cells differentiated with GM-CSF and IFNαand pulsed with a squaricacid treated cell lysate improves T cell priming and tumor growth control ina mouse model.BioImpacts:BI,2018.8(3):p.211-221.

[0490] 37.Shi,F.S.,et al.,Granulocyte-macrophage colony-stimulating factor(GM-CSF)secreted by cDNA-transfected tumor cells induces a more potentantitumor response than exogenous GM-CSF.Cancer Gene Ther,1999.6(1):p.81-8.

[0491] 38.Nasi,M.L.,et al.,Intradermal injection of granulocyte-macrophagecolony-stimulating factor(GM-CSF)in patients with metastatic melanomarecruits dendritic cells.Cytokines Cell Mol Ther,1999.5(3):p.139-44.

[0492] 39.Lawson,D.H.,et al.,Randomized,Placebo-Controlled,Phase III Trialof Yeast-Derived Granulocyte-Macrophage Colony-Stimulating Factor(GM-CSF)Versus Peptide Vaccination Versus GM-CSF Plus Peptide Vaccination VersusPlacebo in Patients With No Evidence of Disease After Complete SurgicalResection of Locally Advanced and / or Stage IV Melanoma:A Trial of the EasternCooperative Oncology Group-American College of Radiology Imaging NetworkCancer Research Group(E4697).J Clin Oncol,2015.33(34):p.4066-76.

[0493] 40.Antman,K.S.,et al.,Effect of recombinant human granulocyte-macrophage colony-stimulating factor on chemotherapy-inducedmyelosuppression.N Engl J Med,1988.319(10):p.593-8.

[0494] 41.Pylayeva-Gupta,Y.,et al.,Cancer Cell Oncogenic Kras-Induced GM-CSFProduction Promotes the Development of Pancreatic Neoplasia.Cancer Cell,2012.21:p.836-847.

[0495] 42. Bobisse, S. et al. Reprogramming T lymphocytes for melanoma adoptiveimmunotherapy by T-cell receptor gene transfer with lentiviral vectors. Cancer Res. 69, 9385-9394 (2009).

[0496] 43. Reddy Chichili, VP, Kumar, V. & Sivaraman, J. Linkers in the structuralbiology of protein-protein interactions. Protein Sci. 22, 153-167 (2013).

[0497] 44. Lamers, CH Jet al. Retroviral vectors for clinical immunogenetherapy are stable for up to 9 years. Cancer Gene Ther. 15, 268-274 (2008).

[0498] ***

[0499] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, other than those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0500] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification. Sequence Listing <110> Ludwig Institute for Cancer Research <120> A2 / NY-ESO-1 specific T cell receptor and its use <130> 252457.000009 <140> PCT / IB2020 / 000140 <141> 2020-03-17 <150> 62 / 819,988 <151> 2019-03-18 <160> 82 <170> PatentIn version 3.5 <210> 1 <211> 311 <212> PRT <213> Unknown <220> <221> source <223> / Remarks="Unknown description: BC1 WT β chain sequence" <400> 1 Met Ala Pro Arg Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Ala Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <210> 2 <211> 311 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 2 Met Ala Pro Arg Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Glu Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Ala Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <210> 3 <211> 311 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 3 Met Ala Pro Arg Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Phe Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Ala Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <210> 4 <211> 311 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 4 Met Ala Pro Arg Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Trp Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Ala Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <210> 5 <211> 311 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 5 Met Ala Pro Arg Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Glu Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Ala Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <210> 6 <211> 311 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 6 Met Ala Pro Arg Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Val Gly Leu Ala Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly 305 310 <210> 7 <211> 274 <212> PRT <213> Unknown <220> <221> Source <223> / Remark = "Unknown description: BC1 WT α-chain sequence" <400> 7 Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp 1 5 10 15 Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val 20 25 30 Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala 35 40 45 Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr 50 55 60 Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg 65 70 75 80 Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95 Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg 100 105 110 Pro Gln Thr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser 115 120 125 Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <210> 8 <211> 9 <212> PRT <213> Human <400> 8 Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5 <210> 9 <211> 822 <212> DNA <213> Unknown <220> <221> Source <223> / Remark = "Unknown description: BC1 WT α-chain sequence" <400> 9 atggaaaccc tgctgggcct gctgatcctg tggctgcagc tgcagtgggt gtccagcaag 60 caggaagtga cccagatccc tgccgccctg agcgtgcccg agggcgagaa cctggtgctg 120 aactgcagct tcaccgacag cgccatctac aacctgcagt ggttccggca ggaccccggc 180 aagggcctga ccagcctgct gctgatccag agcagccagc gggagcagac cagcggcagg 240 ctgaacgcca gcctggacaa gagcagcggc agaagcaccc tgtacatcgc cgccagccag 300 cccggcgact ccgccaccta cctgtgcgcc gtgcggcctc agaccggcgg cagctacatc 360 cccaccttcg gcaggggcac cagcctgatc gtgcacccct acatccagaa ccccgacccc 420 gccgtgtacc agctgcggga cagcaagtcc agcgacaaga gcgtgtgcct gttcaccgac 480 ttcgacagcc agaccaacgt gagccagagc aaggacagcg acgtgtacat caccgacaag 540 accgtgctgg acatgcggag catggacttc aagagcaaca gcgccgtggc ctggtccaac 600 aagagcgact tcgcctgcgc caacgccttc aacaacagca tcatccccga ggacaccttt 660 ttccccagcc ccgagagcag ctgcgacgtg aaactggtgg agaagagctt cgagaccgac 720 accaacctga acttccagaa cctgagcgtg atcggcttca gaattctgct gctgaaggtg 780 gccggcttca acctgctgat gaccctgcgg ctgtggagca gt 822 <210> 10 <211> 936 <212> DNA <213> Unknown <220> <221> Source <223> / Remark = "Unknown description: BC1 WT β-chain sequence" <400> 10 atggccccgc ggctgctgtg ttgtgccgcc ctgagcctgc tgtgggccgg acctgtgaac 60 gccggcgtga cccagacccc caagttccag gtgctgaaaa ccggccagag catgaccctg 120 cagtgcgccc aggacatgaa ccacgagtac atgagctggt acaggcagga ccccggcatg 180 ggcctgcggc tgatccacta cagcgtggga gccggcatca ccgaccaggg cgaggtgccc 240 aacggctaca acgtgagcag aagcaccacc gaggacttcc ccctgaggct gctgtctgcc 300 gcccctagcc agaccagcgt gtacttctgc gccagcagct atgtgggagc cgccggagag 360 ctgttcttcg gcgagggcag ccggctgacc gtgctggaag atctgaacaa agtgttcccc 420 cccgaagtgg ccgtgttcga gcccagcgag gccgagatca gccacaccca gaaggccacc 480 ctggtgtgtc tggccaccgg cttcttcccc gaccacgtgg agctgtcctg gtgggtgaac 540 ggcaaggaag tgcacagcgg cgtgtccacc gacccccagc ccctgaagga gcagcccgcc 600 ctgaacgata gcagatactg cctgagcagc cggctgagag tgagcgccac cttctggcag 660 aacccccgga accacttcag atgccaggtg cagttctacg gcctgagcga gaacgacgag 720 tggacccagg atagagccaa gcccgtgacc cagatcgtgt ccgccgaggc ctggggcaga 780 gccgactgcg gcttcaccag cgtgtcctac cagcagggcg tgctgagcgc cacaatcctg 840 tacgagatcc tgctgggcaa ggccacactg tacgccgtgc tggtgtccgc cctggtgctg 900 atggctatgg tgaagcggaa ggacagcagg ggctga 936 <210> 11 <211> 936 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Remark = "Description of artificial sequence: synthetic polynucleotide" <400> 11 atggccccgc ggctgctgtg ttgtgccgcc ctgagcctgc tgtgggccgg acctgtgaac 60 gccggcgtga cccagacccc caagttccag gtgctgaaaa ccggccagag catgaccctg 120 cagtgcgccc aggacatgaa ccacgagtac atgagctggt acaggcagga ccccggcatg 180 ggcctgcggc tgatccacta cagcgtggga gccggcgaga ccgaccaggg cgaggtgccc 240 aacggctaca acgtgagcag aagcaccacc gaggacttcc ccctgaggct gctgtctgcc 300 gcccctagcc agaccagcgt gtacttctgc gccagcagct atgtgggagc cgccggagag 360 ctgttcttcg gcgagggcag ccggctgacc gtgctggaag atctgaacaa agtgttcccc 420 cccgaagtgg ccgtgttcga gcccagcgag gccgagatca gccacaccca gaaggccacc 480 ctggtgtgtc tggccaccgg cttcttcccc gaccacgtgg agctgtcctg gtgggtgaac 540 ggcaaggaag tgcacagcgg cgtgtccacc gacccccagc ccctgaagga gcagcccgcc 600 ctgaacgata gcagatactg cctgagcagc cggctgagag tgagcgccac cttctggcag 660 aacccccgga accacttcag atgccaggtg cagttctacg gcctgagcga gaacgacgag 720 tggacccagg atagagccaa gcccgtgacc cagatcgtgt ccgccgaggc ctggggcaga 780 gccgactgcg gcttcaccag cgtgtcctac cagcagggcg tgctgagcgc cacaatcctg 840 tacgagatcc tgctgggcaa ggccacactg tacgccgtgc tggtgtccgc cctggtgctg 900 atggctatgg tgaagcggaa ggacagcagg ggctga 936 <210> 12 <211> 936 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Remark = "Description of artificial sequence: synthetic polynucleotide" <400> 12 atggccccgc ggctgctgtg ttgtgccgcc ctgagcctgc tgtgggccgg acctgtgaac 60 gccggcgtga cccagacccc caagttccag gtgctgaaaa ccggccagag catgaccctg 120 cagtgcgccc aggacatgaa ccacgagtac atgagctggt acaggcagga ccccggcatg 180 ggcctgcggc tgatccacta cagcgtggga gccggcttca ccgaccaggg cgaggtgccc 240 aacggctaca acgtgagcag aagcaccacc gaggacttcc ccctgaggct gctgtctgcc 300 gcccctagcc agaccagcgt gtacttctgc gccagcagct atgtgggagc cgccggagag 360 ctgttcttcg gcgagggcag ccggctgacc gtgctggaag atctgaacaa agtgttcccc 420 cccgaagtgg ccgtgttcga gcccagcgag gccgagatca gccacaccca gaaggccacc 480 ctggtgtgtc tggccaccgg cttcttcccc gaccacgtgg agctgtcctg gtgggtgaac 540 ggcaaggaag tgcacagcgg cgtgtccacc gacccccagc ccctgaagga gcagcccgcc 600 ctgaacgata gcagatactg cctgagcagc cggctgagag tgagcgccac cttctggcag 660 aacccccgga accacttcag atgccaggtg cagttctacg gcctgagcga gaacgacgag 720 tggacccagg atagagccaa gcccgtgacc cagatcgtgt ccgccgaggc ctggggcaga 780 gccgactgcg gcttcaccag cgtgtcctac cagcagggcg tgctgagcgc cacaatcctg 840 tacgagatcc tgctgggcaa ggccacactg tacgccgtgc tggtgtccgc cctggtgctg 900 atggctatgg tgaagcggaa ggacagcagg ggctga 936 <210> 13 <211> 936 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Remark = "Description of artificial sequence: synthetic polynucleotide" <400> 13 atggccccgc ggctgctgtg ttgtgccgcc ctgagcctgc tgtgggccgg acctgtgaac 60 gccggcgtga cccagacccc caagttccag gtgctgaaaa ccggccagag catgaccctg 120 cagtgcgccc aggacatgaa ccacgagtac atgagctggt acaggcagga ccccggcatg 180 ggcctgcggc tgatccacta cagcgtggga gccggctgga ccgaccaggg cgaggtgccc 240 aacggctaca acgtgagcag aagcaccacc gaggacttcc ccctgaggct gctgtctgcc 300 gcccctagcc agaccagcgt gtacttctgc gccagcagct atgtgggagc cgccggagag 360 ctgttcttcg gcgagggcag ccggctgacc gtgctggaag atctgaacaa agtgttcccc 420 cccgaagtgg ccgtgttcga gcccagcgag gccgagatca gccacaccca gaaggccacc 480 ctggtgtgtc tggccaccgg cttcttcccc gaccacgtgg agctgtcctg gtgggtgaac 540 ggcaaggaag tgcacagcgg cgtgtccacc gacccccagc ccctgaagga gcagcccgcc 600 ctgaacgata gcagatactg cctgagcagc cggctgagag tgagcgccac cttctggcag 660 aacccccgga accacttcag atgccaggtg cagttctacg gcctgagcga gaacgacgag 720 tggacccagg atagagccaa gcccgtgacc cagatcgtgt ccgccgaggc ctggggcaga 780 gccgactgcg gcttcaccag cgtgtcctac cagcagggcg tgctgagcgc cacaatcctg 840 tacgagatcc tgctgggcaa ggccacactg tacgccgtgc tggtgtccgc cctggtgctg 900 atggctatgg tgaagcggaa ggacagcagg ggctga 936 <210> 14 <211> 936 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Remark = "Description of artificial sequence: synthetic polynucleotide" <400> 14 atggccccgc ggctgctgtg ttgtgccgcc ctgagcctgc tgtgggccgg acctgtgaac 60 gccggcgtga cccagacccc caagttccag gtgctgaaaa ccggccagag catgaccctg 120 cagtgcgccc aggacatgaa ccacgagtac atgagctggt acaggcagga ccccggcatg 180 ggcctgcggc tgatccacta cagcgtggga gccggcatca ccgagcaggg cgaggtgccc 240 aacggctaca acgtgagcag aagcaccacc gaggacttcc ccctgaggct gctgtctgcc 300 gcccctagcc agaccagcgt gtacttctgc gccagcagct atgtgggagc cgccggagag 360 ctgttcttcg gcgagggcag ccggctgacc gtgctggaag atctgaacaa agtgttcccc 420 cccgaagtgg ccgtgttcga gcccagcgag gccgagatca gccacaccca gaaggccacc 480 ctggtgtgtc tggccaccgg cttcttcccc gaccacgtgg agctgtcctg gtgggtgaac 540 ggcaaggaag tgcacagcgg cgtgtccacc gacccccagc ccctgaagga gcagcccgcc 600 ctgaacgata gcagatactg cctgagcagc cggctgagag tgagcgccac cttctggcag 660 aacccccgga accacttcag atgccaggtg cagttctacg gcctgagcga gaacgacgag 720 tggacccagg atagagccaa gcccgtgacc cagatcgtgt ccgccgaggc ctggggcaga 780 gccgactgcg gcttcaccag cgtgtcctac cagcagggcg tgctgagcgc cacaatcctg 840 tacgagatcc tgctgggcaa ggccacactg tacgccgtgc tggtgtccgc cctggtgctg 900 atggctatgg tgaagcggaa ggacagcagg ggctga 936 <210> 15 <211> 141 <212> PRT <213> Mouse <400> 15 Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu 1 5 10 15 Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His 20 25 30 Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val 35 40 45 Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys 50 55 60 Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu 65 70 75 80 Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser 85 90 95 Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr 100 105 110 Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu 115 120 125 Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Gly Gln Lys 130 135 140 <210> 16 <211> 423 <212> DNA <213> Mouse <400> 16 atgtggctgc agaatttact tttcctgggc attgtggtct acagcctctc agcacccacc 60 cgctcaccca tcactgtcac ccggccttgg aagcatgtag aggccatcaa agaagccctg 120 aacctcctgg atgacatgcc tgtcacattg aatgaagagg tagaagtcgt ctctaacgag 180 ttctccttca agaagctaac atgtgtgcag acccgcctga agatattcga gcagggtcta 240 cggggcaatt tcaccaaact caagggcgcc ttgaacatga cagccagcta ctaccagaca 300 tactgccccc caactccgga aacggactgt gaaacacaag ttaccaccta tgcggatttc 360 atagacagcc ttaaaacctt tctgactgat atcccctttg aatgcaaaaa accaggccaa 420 aaa 423 <210> 17 <211> 17 <212> PRT <213> Mouse <400> 17 Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu 1 5 10 15 Ser <210> 18 <211> 51 <212> DNA <213> Mouse <400> 18 atgtggctgc agaatttact tttcctgggc attgtggtct acagcctctc a 51 <210> 19 <211> 124 <212> PRT <213> Mouse <400> 19 Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His Val 1 5 10 15 Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val Thr 20 25 30 Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys Lys 35 40 45 Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu Arg 50 55 60 Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser Tyr 65 70 75 80 Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr Gln 85 90 95 Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu Thr 100 105 110 Asp Ile Pro Phe Glu Cys Lys Lys Pro Gly Gln Lys 115 120 <210> 20 <211> 372 <212> DNA <213> Mouse <400> 20 gcacccaccc gctcacccat cactgtcacc cggccttgga agcatgtaga ggccatcaaa 60 gaagccctga acctcctgga tgacatgcct gtcacattga atgaagaggt agaagtcgtc 120 tctaacgagt tctccttcaa gaagctaaca tgtgtgcaga cccgcctgaa gatattcgag 180 cagggtctac ggggcaattt caccaaactc aagggcgcct tgaacatgac agccagctac 240 taccagacat actgcccccc aactccggaa acggactgtg aaacacaagt taccacctat 300 gcggatttca tagacagcct taaaaccttt ctgactgata tcccctttga atgcaaaaaa 360 ccaggccaaa aa 372 <210> 21 <211> 144 <212> PRT <213> Human <400> 21 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 <210> 22 <211> 432 <212> DNA <213> Human <400> 22 atgtggctgc aatctctgct gctgctgggc acagtggcct gttctattag cgcccctgcc 60 agatctccat ctcctagcac acagccttgg gagcacgtga acgccatcca agaagccaga 120 cggctgctga acctgagcag agatacagcc gccgagatga acgagacagt ggaagtgatc 180 agcgagatgt tcgacctgca agagcctacc tgcctgcaga ccagactgga actgtacaag 240 cagggcctga gaggcagcct gaccaagctg aaaggccctc tgacaatgat ggccagccac 300 tacaagcagc actgccctcc aacacctgag acaagctgtg ccacacagac catcaccttc 360 gagagcttca aagagaacct gaaggacttc ctgctggtca tccccttcga ctgctgggag 420 cccgtgcaag aa 432 <210> 23 <211> 17 <212> PRT <213> human <400> 23 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser <210> 24 <211> 51 <212> DNA <213> human <400> 24 atgtggctgc aatctctgct gctgctgggc acagtggcct gttctattag c 51 <210> 25 <211> 127 <212> PRT <213> human <400> 25 Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val 1 5 10 15 Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr 20 25 30 Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp 35 40 45 Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125 <210> 26 <211> 381 <212> DNA <213> Human <400> 26 gcccctgcca gatctccatc tcctagcaca cagccttggg agcacgtgaa cgccatccaa 60 gaagccagac ggctgctgaa cctgagcaga gatacagccg ccgagatgaa cgagacagtg 120 gaagccagac ggctgctgaa cctgagcaga gatacagccg ccgagatgaa cgagacagtg 120 gaagtgatca gcgagatgtt cgacctgcaa gagcctacct gcctgcagac cagactggaa 180 gaagtgatca gcgagatgtt cgacctgcaa gagcctacct gcctgcagac cagactggaa 180 ctgtacaagc agggcctgag aggcagcctg accaagctga aaggccctct gacaatgatg 240 ctgtacaagc agggcctgag aggcagcctg accaagctga aaggccctct gacaatgatg 240 gccagccact acaagcagca ctgccctcca acacctgaga caagctgtgc cacacagacc 300 gccagccact acaagcagca ctgccctcca acacctgaga caagctgtgc cacacagacc 300 atcaccttcg agagcttcaa agagaacctg aaggacttcc tgctggtcat ccccttcgac 360 atcaccttcg agagcttcaa agagaacctg aaggacttcc tgctggtcat ccccttcgac 360 tgctgggagc ccgtgcaaga a 381 tgctgggagc ccgtgcaaga a 381 <210> 27<210> 27 <211> 564<211> 564 <212> PRT<212> PRT <213> 人<213> Human <400> 27<400> 27 Met Lys Glu His Gly Gly Thr Phe Ser Ser Thr Gly Ile Ser Gly Gly Met Lys Glu His Gly Gly Thr Phe Ser Ser Thr Gly Ile Ser Gly Gly 1 5 10 15 1 5 10 15 Ser Gly Asp Ser Ala Met Asp Ser Leu Gln Pro Leu Gln Pro Asn Tyr Ser Gly Asp Ser Ala Met Asp Ser Leu Gln Pro Leu Gln Pro Asn Tyr 20 25 30 20 25 30 Met Pro Val Cys Leu Phe Ala Glu Glu Ser Tyr Gln Lys Leu Ala Met Met Pro Val Cys Leu Phe Ala Glu Glu Ser Tyr Gln Lys Leu Ala Met 35 40 45 35 40 45 Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile 50 55 60 50 55 60 Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg 65 70 75 80 Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly 85 90 95 Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn 100 105 110 Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys 115 120 125 Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His 130 135 140 Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn Thr 145 150 155 160 Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys Trp 165 170 175 Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro Leu 180 185 190 Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr 195 200 205 Phe Arg Ile Ser Ser Asp Thr Phe Ile Thr Tyr Met Met Thr Leu Glu 210 215 220 Asp His Tyr His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala Ala 225 230 235 240 Asp Val Ala Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Asp 245 250 255 Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala Ala 260 265 270 Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn 275 280 285 Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu 290 295 300 Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu His Cys 305 310 315 320 Asp Ile Phe Met Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys 325 330 335 Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser 340 345 350 Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser 355 360 365 Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu 370 375 380 Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser Leu 385 390 395 400 Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Gln 405 410 415 Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys 420 425 430 Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp 435 440 445 Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln Pro 450 455 460 Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp Tyr 465 470 475 480 Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Gln Asn 485 490 495 Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr Leu 500 505 510 Asp Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His Ser 515 520 525 Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg 530 535 540 Asp Ser Leu Gly Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser 545 550 555 560 Pro Val Asp Thr <210> 28 <211> 1692 <212> DNA <213> Human <400> 28 atgaaggaac acggcggcac ctttagcagc acaggcatct ctggtggcag cggcgatagc 60 gccatggatt ctctgcaacc cctgcagcct aactacatgc ccgtgtgcct gttcgccgag 120 gaaagctacc agaaactggc catggaaacc ctggaagaac tggactggtg cctggaccag 180 ctggaaacca tccagaccta cagatccgtg tccgagatgg ccagcaacaa gttcaagcgg 240 atgctgaaca gagagctgac ccacctgagc gagatgagcc gctctggaaa tcaggtgtcc 300 gagtatatca gcaacacctt cctggacaag cagaacgacg tggaaatccc atctcctaca 360 cagaaggacc gcgagaagaa gaaaaagcag cagctgatga cccagatcag cggcgtgaag 420 aaactgatgc acagcagcag cctgaacaac accagcatca gcagattcgg cgtgaacacc 480 gagaacgagg accacctggc caaagagctg gaagatctga acaaatgggg cctgaacatc 540 ttcaacgtgg ccggctacag ccaacaaga cccctgacct gcattatgta cgccatcttc 600 caagagcggg acctgctgaa aaccttccgg atcagcagcg acaccttcat cacctacatg 660 atgacccttg aggaccacta ccacagcgac gtggcctacc acaatagcct gcatgccgct 720 gatgtggccc agagcaca cgtgctgctg tctacaccag ctctggatgc cgtgttcacc 780 gacctggaaa ttctggccgc catctttgcc gccgctatcc acgatgttga tcaccccggc 840 gtgtccaacc agttcctgat caataccaac agcgagctgg ccctgatgta caacgacgag 900 agcgtgctgg aaaaccacca tctggccgtg ggcttcaagc tgctccaaga ggaacactgc 960 gacatcttca tgaacctgac caagaagcag cggcagaccc tgcggaagat ggtcatcgat 1020 atggtgctgg ccaccgacat gagcaagcac atgtctctgc tggccgacct gaaaaccatg 1080 gtcgagacaa agaaagtgac cagcagcgg gttctgctgc tggacaacta caccgacaga 1140 atccaggtgc tgagaaacat ggtgcactgc gccgatctga gcaaccccac caagagcctg 1200 gaactgtaca gacagtggac cgaccggatc atggagaagt tctttcagca aggcgacaaa 1260 gaacgcgagc gcggcatgga aatctcccca atgtgcgata agcacaccgc cagcgtggaa 1320 aagtcccaag tgggctttat cgactacatc gtgcaccctc tgtgggagac atgggccgat 1380 ctggttcagc ctgacgctca ggacatcctg gacacactgg aagataaccg gaactggtat 1440 cagagcatga tccctcagag cccctctcca cctctggatg agcagaacag agattgccag 1500 ggcctgatgg aaaagttcca gttcgagctg acactggacg aagaggactc tgagggcccc 1560 gagaaagaag gcgagggcca cagctacttc agcagcacaa agaccctgtg cgtgatcgac 1620 cccgagaaca gggatagcct gggcgagaca gacatcgaca ttgccaccga ggacaagagc 1680 cccgtggata ca 1692 <210> 29 <211> 564 <212> PRT <213> Mouse <400> 29 Met Lys Glu Gln Gly Gly Thr Val Ser Gly Ala Ala Ser Ser Arg Gly 1 5 10 15 Gly Gly Asp Ser Ala Met Ala Ser Leu Gln Pro Leu Gln Pro Asn Tyr 20 25 30 Leu Ser Val Cys Leu Phe Pro Glu Glu Ser Tyr Gln Lys Leu Ala Met 35 40 45 Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr Ile 50 55 60 Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys Arg 65 70 75 80 Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly 85 90 95 Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln Asn 100 105 110 Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys Lys 115 120 125 Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met His 130 135 140 Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn Thr 145 150 155 160 Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys Trp 165 170 175 Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro Leu 180 185 190 Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys Thr 195 200 205 Phe Lys Ile Ser Ser Asp Thr Phe Val Thr Tyr Met Met Thr Leu Glu 210 215 220 Asp His Tyr His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala Ala 225 230 235 240 Asp Val Ala Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu Asp 245 250 255 Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala Ala 260 265 270 Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn 275 280 285 Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu 290 295 300 Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu His Cys 305 310 315 320 Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg Lys 325 330 335 Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Ser 340 345 350 Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser 355 360 365 Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val Leu 370 375 380 Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser Leu 385 390 395 400 Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe Gln 405 410 415 Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys 420 425 430 Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp 435 440 445 Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln Pro 450 455 460 Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp Tyr 465 470 475 480 Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Arg Ser 485 490 495 Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr Leu 500 505 510 Glu Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly His Ser 515 520 525 Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn Arg 530 535 540 Asp Ser Leu Glu Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys Ser 545 550 555 560 Pro Ile Asp Thr <210> 30 <211> 1692 <212> DNA <213> Mouse <400> 30 atgaaggaac agggcggcac cgtgtctggc gccgcttcta gtagaggcgg aggcgatagc 60 gccatggcca gtctgcagcc actgcagccc aactacctga gcgtgtgcct gttccccgag 120 gaaagctacc agaaactggc tatggaaacc ctggaagaac tggactggtg cctggaccag 180 ctggaaacca tccagaccta cagatccgtg tccgagatgg ccagcaacaa gttcaagagg 240 atgctgaaca gagagctgac ccacctgagc gagatgagca gatccggcaa ccaggtgtcc 300 gagtatatca gcaacacctt cctggacaag cagaacgacg tggaaatccc cagccccacc 360 cagaaggacc gcgagaagaa gaaaaagcag cagctgatga cccagatcag cggcgtgaag 420 aaactgatgc acagcagcag cctgaacaac accagcatca gcagattcgg cgtgaacacc 480 gagaacgagg accacctggc caaagagctg gaagatctga acaaatgggg cctgaacatc 540 ttcaacgtgg ccggctacag ccacaacaga cccctgacct gcattatgta cgccatcttc 600 caggaacggg acctgctgaa aaccttcaag atcagcagcg acaccttcgt gacctacatg 660 atgacactgg aagatcacta ccacagcgac gtggcctacc acaactctct gcacgccgcc 720 gatgtggccc agagcactca tgtgctgctg agcacccctg ccctggacgc cgtgttcacc 780 gatctggaaa tcctggccgc tatcttcgcc gctgccatcc acgatgtgga ccaccctggc 840 gtgtccaacc agttcctgat caacacaaac agcgagctgg ccctgatgta caacgacgag 900 agcgtgctgg aaaaccacca tctggccgtg ggcttcaagc tgctgcagga agaacactgc 960 gacatctttc agaacctgac caagaagcag aggcagaccc tgagaaagat ggtcatcgac 1020 atggtgctgg ccaccgacat gagcaagcac atgtccctgc tggccgacct gaaaaccatg 1080 gtggaaacaa agaaagtgac cagctccggc gtgctgctgc tggacaacta caccgacaga 1140 atccaggtgc tgaggaacat ggtgcactgc gccgacctgt ccaaccccac caagagcctg 1200 gaactgtaca gacagtggac cgacaggatc atggaagagt tctttcagca aggcgacaaa 1260 gaacgcgaga ggggcatgga aatcagcccc atgtgcgaca agcacaccgc cagcgtggaa 1320 aagtcccaag tgggctttat cgactacatc gtgcaccccc tgtgggagac atgggccgat 1380 ctggtgcagc ctgacgccca ggacatcctg gacactctgg aagataaccg gaactggtat 1440 cagagcatga tcccccagag ccccagccct ccactggacg agagatccag agactgccag 1500 ggcctgatgg aaaagttcca gttcgagctg actctggaag aagaggacag cgagggcccc 1560 gagaaagaag gcgagggcca ctcttacttc agcagcacca agacactgtg cgtgatcgac 1620 cccgagaaca gggacagcct cgaagagact gacatcgaca ttgccaccga ggacaagagc 1680 cccatcgata ca 1692 <210> 31 <211> 6 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Remark = "Description of artificial sequence: synthetic oligonucleotide" <400> 31 gccacc 6 <210> 32 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 32 Asp Gly Gly Gly 1 <210> 33 <211> 53 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 33 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Gly Gly 1 5 10 15 Ser Gly Gly Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser 20 25 30 Thr Gly Ser Gly Ser Gly Ser Gly Lys Pro Ile Pro Asn Pro Leu Leu 35 40 45 Gly Leu Asp Ser Thr 50 <210> 34 <211> 144 <212> PRT <213> people <400> 34 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 <210> 35 <211> 432 <212> DNA <213> Human <400> 35 atgtggctgc aatctctgct gctgctgggc acagtggcct gttctattag cgcccctgcc 60 agatctccat ctcctagcac acagccttgg gagcacgtga acgccatcca agaagccaga 120 cggctgctga acctgagcag agatacagcc gccgagatga acgagacagt ggaagtgatc 180 agcgagatgt tcgacctgca agagcctacc tgcctgcaga ccagactgga actgtacaag 240 cagggcctga gaggcagcct gaccaagctg aaaggccctc tgacaatgat ggccagccac 300 tacaagcagc actgccctcc aacacctgag acaagctgtg ccacacagat catcaccttc 360 gagagcttca aagagaacct gaaggacttc ctgctggtca tccccttcga ctgctgggag 420 cccgtgcaag aa 432 <210> 36 <211> 127 <212> PRT <213> Human <400> 36 Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val 1 5 10 15 Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr 20 25 30 Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp 35 40 45 Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln 50 55 60 Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met 65 70 75 80 Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys 85 90 95 Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110 Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125 <210> 37 <211> 381 <212> DNA <213> Human <400> 37 gcccctgcca gatctccatc tcctagcaca cagccttggg agcacgtgaa cgccatccaa 60 gaagccagac ggctgctgaa cctgagcaga gatacagccg ccgagatgaa cgagacagtg 120 gaagtgatca gcgagatgtt cgacctgcaa gagcctacct gcctgcagac cagactggaa 180 ctgtacaagc agggcctgag aggcagcctg accaagctga aaggccctct gacaatgatg 240 gccagccact acaagcagca ctgccctcca acacctgaga caagctgtgc cacacagatc 300 atcaccttcg agagcttcaa agagaacctg aaggacttcc tgctggtcat ccccttcgac 360 tgctgggagc ccgtgcaaga a 381 <210> 38 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / Note="Unknown description: WT TCR (BC 1) sequence" <400> 38 Gln Ser Ser Gln 1 <210> 39 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 39 Gln Thr Ser Gln 1 <210> 40 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 40 Gln Ser Thr Gln 1 <210> 41 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 41 Gln Ser Asn Gln 1 <210> 42 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 42 Gln Ser Ser Trp 1 <210> 43 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 43 Gln Ser Ser Gln 1 <210> 44 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / Note="Unknown description: WT TCR (BC 1) sequence" <400> 44 Gln Thr Gly Gly 1 <210> 45 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 45 Gln Thr Gly Gly 1 <210> 46 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 46 Gln Glu Gly Gly 1 <210> 47 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 47 Gln Tyr Gly Gly 1 <210> 48 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 48 Gln Thr Gly Ala 1 <210> 49 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 49 Gln Thr Gly Ser 1 <210> 50 <211> 7 <212> PRT <213> Unknown <220> <221> source <223> / Note="Unknown description: WT TCR (BC 1) sequence" <400> 50 Val Gly Ala Gly Ile Thr Asp 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 51 Val Gly Ala Gly Ile Thr Asp 1 5 <210> 52 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 52 Val Val Ala Gly Ile Thr Asp 1 5 <210> 53 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 53 Val Gly Asp Gly Ile Thr Asp 1 5 <210> 54 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 54 Val Ala Asp Gly Ile Thr Asp 1 5 <210> 55 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 55 Val Gly Ala Gln Ile Thr Asp 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 56 Val Gly Ala Gly Phe Thr Asp 1 5 <210> 57 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 57 Val Gly Ala Gly Trp Thr Asp 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 58 Val Gly Ala Gly Glu Thr Asp 1 5 <210> 59 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 59 Val Gly Ala Gly Ile Thr Glu 1 5 <210> 60 <211> 7 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 60 Val Ala Glu Gly Ile Thr Asp 1 5 <210> 61 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / Note="Unknown description: WT TCR (BC 1) sequence" <400> 61 Tyr Val Gly Ala 1 <210> 62 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 62 Tyr Val Gly Ala 1 <210> 63 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 63 Asn Val Gly Ala 1 <210> 64 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 64 Tyr Leu Gly Ala 1 <210> 65 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 65 Tyr Ile Gly Ala 1 <210> 66 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 66 Tyr Val Ser Ala 1 <210> 67 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 67 Tyr Val Gly Val 1 <210> 68 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 68 Tyr Val Gly Ile 1 <210> 69 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 69 Tyr Val Gly Leu 1 <210> 70 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 70 Tyr Val Gly Asp 1 <210> 71 <211> 8 <212> PRT <213> Unknown <220> <221> source <223> / Note="Unknown description: WT (Lau155 BV13c1) 'BC1' sequence" <400> 71 Ser Val Gly Ala Gly Ile Thr Asp 1 5 <210> 72 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 72 Ser Val Gly Ala Gly Ile Thr Asp 1 5 <210> 73 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 73 Ser Val Gly Ala Gly Glu Thr Asp 1 5 <210> 74 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 74 Ser Val Gly Ala Gly Phe Thr Asp 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 75 Ser Val Gly Ala Gly Trp Thr Asp 1 5 <210> 76 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 76 Ser Val Gly Ala Gly Ile Thr Glu 1 5 <210> 77 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 77 Ser Val Ala Glu Gly Ile Thr Asp 1 5 <210> 78 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / Note="Unknown description: WT (Lau155 BV13c1) 'BC1' sequence" <400> 78 Gly Ala Ala Gly 1 <210> 79 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 79 Gly Leu Ala Gly 1 <210> 80 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 80 Gly Ala Ala Gly 1 <210> 81 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 81 Gly Asn Thr Gly 1 <210> 82 <211> 4 <212> PRT <213> Artificial sequence <220> <221> source <223> / Remarks="Description of artificial sequence: synthetic peptide" <400> 82 Gly Leu Tyr Gly 1

Claims

1. A modified T cell receptor (TCR) comprising a) a β chain of a T cell receptor, and b) an α chain of a TCR, wherein a) the β chain comprises a CDR1 represented by the amino acid sequence NHE, a CDR2 represented by any one of SEQ ID NOs: 56-58, and a CDR3 represented by the amino acid sequence YVGA (SEQ ID NO: 62); and b) the α chain comprises CDR1 of the α chain of TCR consisting of the amino acid sequence shown in SEQ ID NO:7, CDR2 of the amino acid sequence shown in QSSQ (SEQ ID NO:43), and CDR3 of the amino acid sequence shown in QTGG (SEQ ID NO:45).

2. The modified T cell receptor (TCR) of claim 1, wherein the modified T cell receptor comprises a single amino acid substitution within the complementarity determining region (CDR) 2 of the β chain of the modified T cell receptor relative to the CDR 2 of the β chain of the unsubstituted wild-type (WT) T cell receptor, wherein the single amino acid substitution is I53F, I53E or I53W, and the modified TCR binds to the cancer antigen NY-ESO-1 shown in the amino acid sequence SEQ ID NO: 8 157-165 Epitope.

3. The modified TCR according to claim 1, wherein the α chain of TCR and / or the β chain of TCR comprises a constant region of human origin or a constant region of mouse origin.

4. The modified TCR of any one of claims 1-3, wherein the α chain comprises the α chain of a WT TCR.

5. A modified TCR according to claim 4, wherein the α chain of the WT TCR comprises the amino acid sequence of SEQ ID NO:

7.

6. A modified TCR according to any one of claims 1-3, wherein the β chain of the modified TCR comprises an amino acid sequence shown in any one of SEQ ID NOs: 2-4, or a fragment thereof, or an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2-4.

7. The modified T cell receptor (TCR) according to claim 1, comprising an α chain comprising the amino acid sequence of SEQ ID NO: 7 and a β chain comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-4.

8. The modified T cell receptor (TCR) according to any one of claims 1 to 3, which binds to the cancer antigen NY-ESO-1 shown in the amino acid sequence SEQ ID NO: 8 157-165 Epitope.

9. The modified TCR of claim 2, wherein the β chain sequence of the modified TCR comprises an amino acid sequence that is at least 80% identical to the β chain of an unsubstituted WT TCR, outside of CDR2 of the β chain of the modified TCR.

10. The modified TCR of claim 2, wherein the β chain of the modified TCR comprises the amino acid sequence of the β chain of an unsubstituted WT TCR and has the single amino acid substitution I53F, I53E or I53W within the CDR2.

11. A modified TCR according to claim 2, wherein the β chain of the unsubstituted WT TCR comprises the amino acid sequence of SEQ ID NO:

1.

12. The modified TCR of claim 2, wherein the modified TCR binds to a cancer antigen with a higher binding affinity than the WT TCR.

13. The modified TCR of claim 12, wherein the binding affinity of the modified TCR to the cancer antigen is 5 to 75 times higher than the binding affinity of the WT TCR to the cancer antigen.

14. The modified TCR of claim 13, wherein the binding affinity of the modified TCR to the cancer antigen is 10 to 75 times higher than the binding affinity of the WT TCR to the cancer antigen.

15. The modified TCR of claim 14, wherein the binding affinity of the modified TCR to the cancer antigen is 25 to 75 times higher than the binding affinity of the WT TCR to the cancer antigen.

16. The modified TCR of claim 15, wherein the binding affinity of the modified TCR to the cancer antigen is 40 to 75 times higher than the binding affinity of the WT TCR to the cancer antigen.

17. The modified TCR of claim 16, wherein the binding affinity of the modified TCR to the cancer antigen is 40 to 60 times higher than the binding affinity of the WT TCR to the cancer antigen.

18. The modified TCR of claim 17, wherein the binding affinity of the modified TCR to the cancer antigen is 40 to 50 times higher than the binding affinity of the WT TCR to the cancer antigen.

19. The modified TCR of claim 12, wherein the modified TCR has a binding affinity for the cancer antigen that is 50-fold higher than the binding affinity of the WT TCR for the cancer antigen.

20. The modified TCR of claim 12, wherein the modified TCR has a dissociation constant (K) between 0.30 and 4.5 μM. D ) binds to the cancer antigen.

21. The modified TCR of claim 20, wherein the dissociation constant (K D ) between 0.30 and 2 μM.

22. The modified TCR of claim 20, wherein the dissociation constant (K D ) between 2 μM and 3 μM.

23. The modified TCR of claim 20, wherein the dissociation constant (K D ) between 3 μM and 4 μM.

24. The modified TCR of claim 20, wherein the dissociation constant (K D ) is 0.41 μM.

25. The modified TCR of claim 20, wherein the dissociation constant (K D ) is 3.89μM.

26. A modified TCR according to any one of claims 1-3, wherein the β chain of the modified TCR comprises an amino acid sequence shown in any one of SEQ ID NOs: 2-4, or an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2-4.

27. A polynucleotide encoding the modified T cell receptor (TCR) according to any one of claims 1-26.

28. The polynucleotide of claim 27, wherein the β chain of the modified TCR is encoded by a nucleotide sequence as shown in any one of SEQ ID NOs: 11-13 or a nucleotide sequence having at least 80% sequence identity with a sequence as shown in any one of SEQ ID NOs: 11-13.

29. The polynucleotide of claim 27, wherein the polynucleotide is operably linked to at least one regulatory element for expressing the modified TCR.

30. The polynucleotide of claim 29, wherein the at least one regulatory element is a promoter.

31. The polynucleotide of claim 27, which is a DNA molecule.

32. The polynucleotide of claim 27, which is an RNA molecule.

33. A recombinant vector comprising the polynucleotide of any one of claims 27-32, wherein the polynucleotide is operably linked to at least one regulatory element for expressing the modified T cell receptor (TCR).

34. The vector according to claim 33, which is a viral vector.

35. The vector of claim 34, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an alphaviral vector, a herpes virus vector, or a vaccinia virus vector.

36. The vector according to claim 33, wherein the vector is a non-viral vector.

37. The vector of claim 33, wherein the β chain of the modified TCR comprises the amino acid sequence of any one of SEQ ID NOs: 2-4, and the α chain of the modified TCR comprises the amino acid sequence of SEQ ID NO:

7.

38. An isolated host cell comprising the modified T cell receptor (TCR) of any one of claims 1-26 or the polynucleotide of any one of claims 27-32, or the vector of any one of claims 33-37.

39. The isolated host cell of claim 38, 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.

40. The isolated host cell of claim 38, wherein the host cell is a mammalian cell.

41. The isolated host cell of claim 38, wherein the host cell is a lymphoid cell.

42. The isolated host cell of claim 41, wherein the lymphoid cell is a T cell.

43. The isolated host cell of claim 41, wherein the lymphoid cell is a Natural Killer (NK) cell.

44. The isolated host cell of any one of claims 40-43, wherein the host cell is obtained from peripheral blood mononuclear cells (PBMC), tumor draining lymph nodes, or tumor infiltrates.

45. The isolated host cell of claim 38, wherein the host cell has been activated and / or expanded ex vivo.

46. ​​The isolated host cell of claim 38, wherein the host cell is an allogeneic cell.

47. The isolated host cell of claim 38, wherein the host cell is an autologous cell.

48. The isolated host cell of claim 47, wherein the host cell is isolated from a subject suffering from cancer.

49. The isolated host cell according to claim 48, wherein the cancer presents the cancer antigen NY-ESO-1 shown in the amino acid sequence SEQ ID NO: 8 on its cell surface. 157-165 Epitope.

50. The isolated host cell of claim 48 or claim 49, wherein the cancer is myeloma, melanoma, sarcoma, urinary bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, prostate cancer, bladder cancer, skin cancer, lung cancer, ovarian cancer, or brain cancer.

51. The isolated host cell of claim 38, further engineered to express one or more exogenous molecules.

52. The isolated host cell of claim 51, wherein the one or more exogenous molecules are immune signaling molecules.

53. The isolated host cell of claim 52, wherein the immune signaling molecule is a cytokine.

54. The isolated host cell of claim 52, wherein the immune signaling molecule is a chemokine.

55. The isolated host cell of claim 52, wherein the immune signaling molecule is a growth factor.

56. The isolated host cell of claim 55, wherein the growth factor is granulocyte-macrophage colony stimulating factor (GM-CSF).

57. The isolated host cell according to claim 56, wherein the amino acid sequence of GM-CSF is shown in SEQ ID NO: 21, 34 or 15.

58. The isolated host cell of claim 56, wherein the nucleotide sequence encoding GM-CSF is SEQ ID NO: 22, 35 or 16.

59. The isolated host cell of claim 51, wherein the one or more exogenous molecules are soluble receptors.

60. The isolated host cell of claim 51, wherein the one or more exogenous molecules is a ligand.

61. The isolated host cell of claim 51, wherein the one or more exogenous molecules are antigen binding proteins.

62. The isolated host cell of claim 61, wherein the antigen binding protein is an antibody or an antibody fragment.

63. The isolated host cell of claim 51, wherein the one or more exogenous molecules is a phosphodiesterase.

64. The isolated host cell of claim 63, wherein the phosphodiesterase is PDE4B2.

65. The isolated host cell of claim 38, wherein the host cell is further engineered to express PDE4B2.

66. The isolated host cell of claim 65, wherein the TCR comprises a beta chain comprising an amino acid sequence as shown in any one of SEQ ID NOs: 2-4 or an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2-4.

67. An isolated host cell according to claim 65, wherein the modified TCR comprises an alpha chain comprising the amino acid sequence of SEQ ID NO:

7.

68. The isolated host cell according to claim 64 or 65, wherein the amino acid sequence of PDE4B2 is shown in SEQ ID NO: 27 or 29.

69. The isolated host cell according to claim 64 or 65, wherein the nucleotide sequence encoding PDE4B2 is shown as SEQ ID NO: 28 or 30.

70. The isolated host cell of claim 51, wherein the one or more exogenous molecules are cell surface receptors.

71. The isolated host cell of claim 70, wherein the cell surface receptor is a chimeric antigen receptor.

72. The isolated host cell according to claim 70, wherein the cell surface receptor is a cancer antigen NY-ESO-1 that does not bind to the amino acid sequence shown in SEQ ID NO: 8 157-165 Epitope of the T cell receptor.

73. A pharmaceutical composition comprising the host cell of any one of claims 38-72 and a pharmaceutically acceptable carrier and / or excipient.

74. The pharmaceutical composition of claim 73, wherein the composition is used for adoptive cell transfer therapy.

75. A method for producing the host cell of any one of claims 38-72, comprising genetically engineering the host cell with the polynucleotide of any one of claims 27-32 or the vector of any one of claims 33-37.

76. A method of genetically engineering a host cell to express the modified T cell receptor (TCR) of any one of claims 1-26.

77. The method of claim 76, comprising genetically engineering a host cell with the polynucleotide of any one of claims 27-32 or the vector of any one of claims 33-37.

78. The method of any one of claims 75-77, wherein the genetic engineering step is performed by viral gene delivery.

79. The method of claim 78, wherein the genetic engineering step is performed by non-viral gene delivery.

80. The method of claim 76, wherein the method is performed ex vivo.

81. The method of claim 76, wherein the method further comprises activating and / or expanding the host cells ex vivo.

82. The method of claim 76, wherein the β chain of the modified TCR comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2-4, or a fragment thereof, or an amino acid sequence having at least 80% sequence identity with the sequence as shown in any one of SEQ ID NOs: 2-4.

83. The method of claim 76, further comprising genetically engineering the host cell to further express one or more exogenous molecules.

84. The method of claim 83, wherein the one or more exogenous molecules are immune signaling molecules.

85. The method of claim 83, wherein 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.

86. The method of claim 85, wherein the growth factor is granulocyte-macrophage colony stimulating factor (GM-CSF).

87. The method of claim 86, wherein the amino acid sequence of GM-CSF is shown in SEQ ID NO: 21, 34 or 15.

88. The method of claim 86, wherein the nucleotide sequence encoding GM-CSF is shown in SEQ ID NO: 22, 35 or 16.

89. The method of claim 85, wherein the phosphodiesterase is PDE4B2.

90. The method of claim 89, wherein the amino acid sequence of PDE4B2 is shown in SEQ ID NO: 27 or 29.

91. The method of claim 89, wherein the nucleotide sequence encoding PDE4B2 is shown as SEQ ID NO: 28 or 30.

92. The method of claim 85, wherein the antigen binding protein is an antibody or an antibody fragment.

93. The method of claim 85, wherein the cell surface receptor is a non-binding cancer antigen NY-ESO-1 157-165 A chimeric antigen receptor or T cell receptor that is expressed on the surface of the protein κB2 (SEQ ID NO: 8).

94. The method of claim 76, wherein the host cell is a mammalian cell.

95. The method of claim 76, wherein the host cell is a lymphoid cell.

96. The method of claim 95, wherein the lymphoid cells are T cells.

97. The method of claim 95, wherein the lymphoid cells are natural killer (NK) cells.

98. The method of any one of claims 94-97, wherein the host cells are obtained from peripheral blood mononuclear cells (PBMCs), tumor draining lymph nodes, or tumor infiltrates.

99. The method of claim 76, wherein the host cell has been activated and / or expanded ex vivo.

100. The method of claim 76, wherein the host cell is an allogeneic cell.

101. The method of claim 76, wherein the host cell is an autologous cell.

102. The method of claim 76, wherein the host cell is isolated from a subject suffering from cancer.

103. The method of claim 102, wherein the cancer comprises a cancer antigen NY-ESO-1 presenting an amino acid sequence of SEQ ID NO: 8 on its surface. 157-165 epitope of the cell.

104. The method of claim 102 or claim 103, 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.

105. The use of lymphoid cells in the preparation of a medicament for stimulating or enhancing an immune response in a mammal in need thereof, wherein the lymphoid cells comprise a modified T cell receptor (TCRs) as described in any one of claims 1-26, a host cell as described in any one of claims 38-72, a composition as described in claim 73 or claim 74, or a host cell produced by a method as described in any one of claims 76-104.

106. Use of lymphocytes in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the lymphocytes comprise a modified T cell receptor (TCR) as described in any one of claims 1-26, a host cell as described in any one of claims 38-72, a composition as described in claim 73 or claim 74, or a host cell produced by the method as described in any one of claims 76-104.

107. The use according to claim 106, wherein the cancer comprises a cancer antigen NY-ESO-1 presenting an amino acid sequence as shown in SEQ ID NO: 8 on its surface. 157-165 epitope of the cell.

108. The use according to claim 106, 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.

109. The use according to any one of claims 106-108, comprising: a) isolating T cells from the subject; b) genetically modifying the T cell ex vivo using the polynucleotide of any one of claims 27 to 32 or the vector of any one of claims 33 to 37; c) optionally, expanding and / or activating said T cells before, after or during step b); and d) introducing the genetically modified T cells into said subject.

110. The use according to any one of claims 106-108, wherein the subject is a mammal.

111. The use according to any one of claims 106-108, wherein the subject is a human.

Images