MAGEC2 immunogenic peptides, binding proteins that recognize MAGEC2 immunogenic peptides, and uses thereof

Abstract

Provided herein are MAGEC2 immunogenic peptides, binding proteins that recognize MAGEC2 immunogenic peptides, and uses thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 174,808, filed April 14, 2021, and U.S. Provisional Application No. 63 / 329,523, filed April 11, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Adoptive cell transfer (ACT) using engineered T cells has demonstrated great efficacy in treating certain types of liquid tumors and shows promise in treating solid tumors. T cell receptor engineered T cells (TCR-T) are T cells expressing a foreign TCR that recognizes antigens present on cancer cells. TCR-antigen interaction is a core component of the targeting mechanism that allows TCR-T cells to kill cancer cells. One of the challenges in the widespread testing and adoption of TCR-T therapy is the lack of TCR-antigen pairs that are applicable to a wide variety of patients and conditions. Although several TCRs and antigens have been explored in clinical trials, most antigens explored are specific to one HLA allele, namely HLA-A. * 02:01, limiting the number of patients eligible for therapy, and the cancer cells * 02:01 It also allows latent resistance to develop by mutating alleles.

[0003] In addition, the number of antigens explored is limited because the discovery of new TCR-antigen pairs is difficult, which usually requires the prediction of epitopes presented by MHC.On the other hand, such epitopes may not be immunogenic, making it difficult to identify reactive TCRs, or epitopes may not be processed and physiologically presented by cancer cells.Therefore, there is a great need in the art to identify TCR-antigen pairs in the context of various HLA alleles that are widely applicable in order to develop useful reagents for diagnosis, prognosis, treatment, and drug screening related to disorders characterized by antigen expression. Summary of the Invention

[0004] The present invention is based, at least in part, on the discovery of MAGEC2 immunogenic peptides and binding proteins that recognize such MAGEC2 immunogenic peptides based on an unbiased functional selection method used to discover antigens of TCR clonotypes identified from subjects with disorders associated with MAGEC2 expression (e.g., subjects suffering from melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer or bladder urothelial carcinoma). The identified TCRs are capable of binding to various HLA alleles (e.g., HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H ... * 07:02, and HLA-A * In the context of the MAGEC2 immunogenic peptides, such as those listed in Table 1, in the context of the MAGEC2 immunogenic peptides of the present invention, the present inventors have recognized MAGEC2 immunogenic peptides, such as those listed in Table 1, in the context of the MAGEC2 immunogenic peptides of the present invention ...

[0005] In one aspect, an immunogenic peptide is provided that comprises a peptide epitope selected from the peptide sequences listed in Table 1.

[0006] In another embodiment, an immunogenic peptide is provided that consists of a peptide epitope selected from the peptide sequences listed in Table 1.

[0007] Numerous embodiments are further provided which may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the immunogenic peptide is derived from MAGEC2 protein, and optionally the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length. In another embodiment, the immunogenic peptide is capable of eliciting an immune response against MAGEC2 and / or MAGEC2 expressing cells in a subject, and optionally the immune response is selected from the group consisting of: i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell proliferation, cytokine release and / or cytotoxic killing.

[0008] In yet another aspect, there is provided an immunogenic composition comprising at least one immunogenic peptide described herein.

[0009] Numerous embodiments are further provided which may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the immunogenic composition further comprises an adjuvant. In another embodiment, the immunogenic composition is capable of eliciting an immune response against MAGEC2 and / or MAGEC2 expressing cells in a subject, optionally the immune response being selected from the group consisting of: i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell proliferation, cytokine release and / or cytotoxic killing.

[0010] In yet another aspect, a composition is provided comprising a peptide epitope selected from the peptide sequences listed in Table 1 and an MHC molecule.

[0011] Numerous embodiments are further provided which may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule is an HLA-A * 02. HLA-A * 03. HLA-A * 01. HLA-A * 11. HLA-A * 24, and / or HLA-B * 07, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A * 0205, HLA-A * 0206, HLA-A * 0207, HLA-A * 0210, HLA-A * 0211, HLA-A * 0212, HLA-A * 0213, HLA-A * 0214, HLA-A * 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A * 0242, HLA-A * 0253, HLA-A * 0260, HLA-A * 0274 allele, HLA-A *0301, HLA-A * 0302, HLA-A * 0305, HLA-A * 0307, ​​HLA-A * 0101, HLA-A * 0102, HLA-A * 0103, HLA-A * 0116 allele, HLA-A * 1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104, HLA-A * 1105, HLA-A * 1119 allele, HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and HLA-B * 0721 allele.

[0012] In another aspect, there is provided a stable MHC-peptide complex comprising an immunogenic peptide as described herein in the context of an MHC molecule.

[0013] Numerous embodiments are further provided which may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule is an HLA-A * 02. HLA-A * 03. HLA-A * 01. HLA-A * 11. HLA-A * 24, and / or HLA-B * 07, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A * 0205, HLA-A * 0206, HLA-A * 0207, HLA-A * 0210, HLA-A * 0211, HLA-A * 0212, HLA-A * 0213, HLA-A * 0214, HLA-A * 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A * 0242, HLA-A * 0253, HLA-A * 0260, HLA-A * 0274 allele, HLA-A * 0301, HLA-A * 0302, HLA-A * 0305, HLA-A * 0307, ​​HLA-A * 0101, HLA-A* 0102, HLA-A * 0103, HLA-A * 0116 allele, HLA-A * 1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104, HLA-A * 1105, HLA-A * 1119 allele, HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and HLA-B * 0721 allele. In yet another embodiment, the peptide epitope and the MHC molecule are covalently linked and / or the alpha and beta chains of the MHC molecule are covalently linked. In another embodiment, the stable MHC-peptide complex comprises a detectable label, optionally, the detectable label is a fluorophore.

[0014] In yet another aspect, there is provided an immunogenic composition comprising a stable MHC-peptide complex as described herein and an adjuvant.

[0015] In yet another aspect, there is provided an isolated nucleic acid encoding an immunogenic peptide described herein, or a complement thereof.

[0016] In another aspect, a vector is provided that comprises the isolated nucleic acid described herein.

[0017] In yet another aspect, the cell line comprises a) an isolated nucleic acid as described herein; b) a vector as described herein; and / or c) produces one or more immunogenic peptides as described herein and / or displays one or more stable MHC-peptide complexes as described herein on its cell surface. A cell is provided, optionally wherein the cell is genetically engineered.

[0018] In yet another aspect, there is provided a device or kit comprising a) one or more immunogenic peptides as described herein, and / or b) one or more stable MHC-peptide complexes as described herein, optionally comprising a reagent for detecting binding of a) and / or b) to a binding protein, optionally wherein the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0019] In another aspect, there is provided a method for detecting T cells that bind to a stable MHC-peptide complex, comprising: a) contacting a sample comprising T cells with a stable MHC-peptide complex as described herein; and b) detecting binding of the T cells to the stable MHC-peptide complex, optionally further determining the percentage of stable MHC-peptide specific T cells that bind to the stable MHC-peptide complex, optionally wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

[0020] Numerous embodiments are further provided which may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the T cells are CD8+ T cells. In another embodiment, the detecting and / or determining is performed using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay. In yet another embodiment, the sample comprises T cells that have been in contact or are suspected of being in contact with one or more MAGEC2 proteins or fragments thereof.

[0021] In yet another aspect, there is provided a method for determining whether T cells have been exposed to MAGEC2, comprising: a) incubating a cell population comprising T cells with an immunogenic peptide described herein or a stable MHC-peptide complex described herein; and b) detecting a presence or level of reactivity, wherein the presence or higher level of reactivity compared to a control level indicates that the T cells have been exposed to MAGEC2, and optionally the cell population comprising T cells is obtained from a subject.

[0022] In yet another aspect, there is provided a method for predicting clinical outcome in a subject suffering from a disorder characterized by MAGEC2 expression, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein; and b) comparing the presence or level of reactivity with that from a control, wherein the control is obtained from a subject with a good clinical outcome, and wherein a presence or higher level of reactivity in the subject sample compared to the control indicates that the subject will have a good clinical outcome.

[0023] In another aspect, there is provided a method of assessing the effectiveness of a therapy for a disorder characterized by MAGEC2 expression, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more of the immunogenic peptides described herein or one or more of the stable MHC-peptide complexes described herein in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject; and b) determining the presence or level of reactivity between one or more of the immunogenic peptides described herein or one or more of the stable MHC-peptide complexes described herein and T cells obtained from the subject present in a second sample obtained from the subject after providing the therapy to the subject, wherein a presence or higher level of reactivity in the second sample compared to the first sample is an indication that the therapy is effective in treating a disorder characterized by MAGEC2 expression in the subject.

[0024] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the level of responsiveness is indicated by a) the presence of binding, and / or b) T cell activation and / or effector function, optionally T cell activation or effector function being T cell proliferation, killing or cytokine release. In another embodiment, the method further comprises repeating steps a) and b) at a subsequent time point, optionally, between the first time point and the subsequent time point, the subject has undergone a treatment to ameliorate a disorder characterized by MAGEC2 expression. In yet another embodiment, the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay. In yet another embodiment, the control level is a reference number. In another embodiment, the control level is the level of a subject without a disorder characterized by MAGEC2 expression.

[0025] In yet another aspect, a method for preventing and / or treating a disorder characterized by MAGEC2 expression in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0026] In yet another aspect, methods are provided for identifying a peptide binding molecule, or antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, the method comprising: a) providing a cell presenting a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule on its surface; b) determining binding of a plurality of candidate peptide binding molecules, or antigen-binding fragments thereof, to the peptide epitope in the context of the MHC molecule on the cell; and c) identifying one or more peptide binding molecules, or antigen-binding fragments thereof, that bind to the peptide epitope in the context of the MHC molecule.

[0027] Numerous embodiments are further provided which may be applied to any aspect encompassed by the invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, step a) comprises contacting an MHC molecule on the surface of a cell with a peptide epitope selected from the peptide sequences listed in Table 1. In another embodiment, step a) comprises expressing a peptide epitope selected from the peptide sequences listed in Table 1 in a cell using a vector comprising a heterologous sequence encoding the peptide epitope.

[0028] In another aspect, there is provided a method of identifying a peptide binding molecule, or antigen-binding fragment thereof, that binds to a peptide epitope selected from a peptide sequence listed in Table 1, comprising: a) providing a peptide epitope, alone or in the context of an MHC molecule, that comprises a peptide epitope selected from a peptide sequence listed in Table 1, alone or in a stable MHC-peptide complex; b) determining binding of a plurality of candidate peptide binding molecules, or antigen-binding fragments thereof, to the peptide or stable MHC-peptide complex; and c) identifying one or more peptide binding molecules, or antigen-binding fragments thereof, that bind to the peptide epitope or stable MHC-peptide complex, where optionally the MHC or MHC-peptide complex is as described herein.

[0029] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the plurality of candidate peptide-binding molecules comprises an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain. In another embodiment, the plurality of candidate peptide-binding molecules comprises at least 2, 5, 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9The plurality of candidate peptide-binding molecules comprises one or more different candidate peptide-binding molecules from a sample from a subject or a population of subjects; or the plurality of candidate peptide-binding molecules comprises one or more candidate peptide-binding molecules comprising a mutation in a parent scaffold peptide-binding molecule from a sample from a subject. In yet another embodiment, the subject or population of subjects a) is not afflicted with a disorder characterized by MAGEC2 expression and / or has recovered from a disorder characterized by MAGEC2 expression, or b) is afflicted with a disorder characterized by MAGEC2 expression. In another embodiment, the subject or population of subjects has been administered a composition described herein. In yet another embodiment, the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally the mammal is a human, a primate or a rodent. In yet another embodiment, the subject is an animal model of a disorder characterized by MAGEC2 expression, an HLA transgenic mouse, and / or a human TCR transgenic mouse. In another embodiment, the sample comprises peripheral blood mononuclear cells (PBMCs), T cells and / or CD8+ memory T cells.

[0030] In yet another aspect, there is provided a peptide-binding molecule or antigen-binding fragment thereof identified according to the methods described herein, which is optionally an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0031] In yet another aspect, there is provided a method of treating a disorder characterized by MAGEC2 expression in a subject, comprising administering to the subject a therapeutically effective amount of engineered T cells expressing a peptide binding molecule or antigen binding fragment thereof that i) binds to a peptide epitope selected from the sequences listed in Table 1, ii) is identified according to the methods described herein, and / or iii) binds to a stable MHC-peptide complex comprising a peptide epitope selected from the sequences listed in Table 1 in the context of an MHC molecule, optionally wherein the peptide binding molecule or antigen binding fragment thereof is an antibody, an antigen binding fragment of an antibody, a TCR, an antigen binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain, and optionally wherein the MHC or MHC-peptide complex is as described herein.

[0032] Numerous embodiments are further provided which may be applied to any aspect encompassed by the invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, T cells are isolated from a) a subject, b) a donor not afflicted with a disorder characterized by MAGEC2 expression, or c) a donor who has recovered from a disorder characterized by MAGEC2 expression.

[0033] In another aspect, there is provided a method of treating a disorder characterized by MAGEC2 expression in a subject, comprising transfusing antigen-specific T cells into the subject, wherein the antigen-specific T cells are generated by: a) stimulating immune cells from the subject with a composition described herein; and b) expanding the antigen-specific T cells in vitro or ex vivo, optionally wherein i) the immune cells are isolated from the subject prior to stimulating the immune cells, and / or ii) the immune cells comprise PBMCs, T cells, CD8+ T cells, naive T cells, central memory T cells and / or effector memory T cells.

[0034] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the agent is contacted under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, the immunogenic peptide, the stable MHC-peptide complex, the T cell receptor and / or the immune cell. In another embodiment, the peptide epitope, the immunogenic peptide, the stable MHC-peptide complex and / or the T cell receptor are expressed by a cell, and the cell is expanded and / or isolated during one or more steps. In yet another embodiment, the disorder characterized by MAGEC2 expression is a cancer or its recurrence, optionally the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma. In yet another embodiment, the subject is an animal model of a disorder characterized by MAGEC2 expression and / or a mammal, optionally the mammal is a human, a primate or a rodent.

[0035] In yet another aspect, a binding protein that binds to a polypeptide comprising an immunogenic peptide sequence described herein, an immunogenic peptide described herein, and / or a stable MHC-peptide complex described herein, and is optionally an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain. The binding protein is provided,

[0036] Numerous embodiments are further provided that may be applied to any aspect encompassed by the invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the binding protein comprises a) a TCR alpha chain CDR sequence having at least about 80% identity with a TCR alpha chain CDR sequence selected from the group consisting of TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence having at least about 80% identity with a TCR beta chain CDR sequence selected from the group consisting of TCR beta chain CDR sequences listed in Table 2, and the binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, and optionally, its binding affinity is about 5×10 -4 K below M d In another embodiment, the binding protein has a) a TCR alpha chain variable (V α ) TCR V domain sequences selected from the group consisting of α TCR V domain sequence with at least about 80% identity α and / or b) a TCR beta chain variable (V β ) TCR V domain sequences selected from the group consisting of β TCR V domain sequence with at least about 80% identity β The binding protein comprises a domain sequence and is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In yet another embodiment, the binding protein comprises a) a TCR alpha chain sequence having at least about 80% identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence having at least about 80% identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, and the binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4K below M d In yet another embodiment, the binding protein comprises a) a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, and the binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In yet another embodiment, the binding protein provided has a) a TCR alpha chain variable (V α ) TCR V domain sequences selected from the group consisting of α and / or b) a TCR beta chain variable (V β ) TCR V domain sequences selected from the group consisting of β The binding protein comprises a domain sequence and is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In another embodiment, the binding protein provided comprises a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In another embodiment, the TCR comprises 1) a TCR alpha chain CDR, a TCR V α 1) the TCR beta chain CDRs, TCR V domain, and / or the TCR alpha chain are encoded by a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2; and / or 2) the TCR beta chain CDRs, TCR V domain, and / or the TCR alpha chain are encoded by a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2; βThe domain, and / or the TCR beta chain are encoded by a TRBV, TRBJ and / or TRBC gene or fragment thereof selected from the group of TRBV, TRBJ and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has up to 5 amino acid substitutions, insertions, deletions or combinations thereof compared to a cognate reference CDR sequence listed in Table 2. In yet another embodiment, the binding protein is chimeric, humanized or human. In yet another embodiment, the binding protein comprises a binding domain having a transmembrane domain and an effector domain that is intracellular. In another embodiment, the TCR alpha chain and the TCR beta chain are covalently linked, optionally, the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide. In yet another embodiment, the TCR alpha chain and / or the TCR beta chain are covalently linked to a substructure, optionally, the covalently linked substructure comprises an affinity tag or label. In yet another embodiment, the affinity tag is selected from the group consisting of CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, Halo tag, HA tag, Flag tag, His tag, biotin tag and V5 tag, and / or the label is a fluorescent protein. In another embodiment, the covalently linked moiety is selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or an antigen-binding fragment thereof. In yet another embodiment, the binding protein binds to a pMHC complex on the cell surface. In yet another embodiment, the MHC or MHC-peptide complex is as described herein. In another embodiment, binding of the binding protein to the MAGEC2 peptide-MHC (pMHC) complex elicits an immune response, optionally the immune response is selected from the group consisting of i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell proliferation, cytokine release and / or cytotoxic killing. In yet another embodiment, the binding protein binds to a MAGEC2 immunogenic peptide-MHC (pMHC) complex at a concentration of about 1×10 -4M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, or about 1 x 10 -12 K below M d In yet another embodiment, the binding protein has a higher binding affinity to peptide-MHC (pMHC) compared to a known T cell receptor, and optionally the higher binding affinity is at least 1.05-fold higher. In another embodiment, the binding protein induces a higher T cell proliferation, cytokine release and / or cytotoxic killing when contacted with a target cell having heterozygous expression of MAGEC2 compared to a known T cell receptor, and optionally the induction is at least 1.05-fold higher. As used herein, reference to fold change can, in some embodiments, be in comparison to any reference mode of interest, for example, in comparison to a different binding protein; in comparison to the same binding protein in a different context, such as expression of the same binding protein in different immune cells, at different levels, in combination with other agents described herein; and similar comparisons. In yet another embodiment, the cytotoxic killing is the cytotoxic killing of a target cancer cell. In yet another embodiment, the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer, and bladder urothelial carcinoma. In another embodiment, the binding protein is ALKDVEERV / HLA-A * 02. LLFGLALIEV / HLA-A *02, SESIKKKVL / HLA-B * 44, and ASSTLYLVF / HLA-B * 57 does not bind to a peptide-MHC (pMHC) complex selected from the group consisting of:

[0037] In yet another aspect, there is provided a TCR alpha and / or beta chain selected from the group consisting of the TCR alpha and beta chain sequences listed in Table 2.

[0038] In another aspect, an isolated nucleic acid molecule is provided that i) hybridizes under stringent conditions to the complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, ii) has a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, and / or iii) has a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide listed in Table 2, optionally comprising 1) a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2, and / or 2) a TRBV, TRBJ and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ and TRBC genes listed in Table 2.

[0039] Numerous embodiments are further provided that may be applied to any aspect encompassed by the invention and / or may be combined with any other embodiment described herein For example, in one embodiment, the nucleic acid is codon optimized for expression in a host cell.

[0040] In yet another aspect, there is provided a vector comprising the isolated nucleic acid described herein, optionally wherein i) the vector is a cloning vector, an expression vector or a viral vector, and / or ii) the vector comprises a vector sequence listed in Table 3.

[0041] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the vector further comprises a nucleic acid sequence encoding CD8α and / or CD8β. In another embodiment, the nucleic acid sequence encoding CD8α or CD8β is operably linked to a nucleic acid encoding a tag. In yet another embodiment, the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding CD8α or CD8β such that the tag is fused to the N-terminus of CD8α or CD8β. In yet another embodiment, the tag is a CD34 enrichment tag. In another embodiment, the isolated nucleic acid described herein and the nucleic acid sequence encoding CD8α and / or CD8β are interconnected with an internal ribosome entry site or with a nucleic acid sequence encoding a self-cleaving peptide. In yet another embodiment, the self-cleaving peptide is P2A, E2A, F2A or T2A.

[0042] In yet another aspect, there is provided a host cell comprising an isolated nucleic acid described herein, the host cell comprising a vector described herein, and / or expressing a binding protein described herein, optionally wherein the cell is genetically engineered.

[0043] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the host cell comprises a chromosomal gene knockout of a TCR gene, an HLA gene, or both. In another embodiment, the host cell comprises a knockout of an HLA gene selected from alpha 1 macroglobulin gene, alpha 2 macroglobulin gene, alpha 3 macroglobulin gene, beta 1 microglobulin gene, beta 2 microglobulin gene, and combinations thereof. In yet another embodiment, the host cell comprises a knockout of a TCR gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and combinations thereof. In yet another embodiment, the host cell expresses CD8 alpha and / or CD8 beta, optionally fused to a CD34 enrichment tag. In another embodiment, the host cell is enriched using the CD34 enrichment tag. In yet another embodiment, the host cell is a hematopoietic progenitor cell, a peripheral blood mononuclear cell (PBMC), a cord blood cell, or an immune cell. In yet another embodiment, the immune cell is a T cell, a cytotoxic lymphocyte, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte progenitor cell, a cytotoxic lymphocyte stem cell, a CD4 + T cells, CD8 +The host cell is a T cell, a CD4 / CD8 double negative T cell, a gamma delta (γδ) T cell, a natural killer (NK) cell, a NK-T cell, a dendritic cell, or a combination thereof. In yet another embodiment, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof. In another embodiment, the T cell is a primary T cell, or a cell of a T cell line. In yet another embodiment, the T cell does not express an endogenous TCR, or has less surface expression thereof. In yet another embodiment, the host cell is capable of producing a cytokine or a cytotoxic molecule when contacted with a target cell comprising a peptide-MHC (pMHC) complex comprising a MAGEC2 peptide epitope in the context of an MHC molecule. In another embodiment, the host cell contacts the target cell in vitro, ex vivo, or in vivo. In yet another embodiment, the cytokine is TNF-α, IL-2, and / or IFN-γ. In yet another embodiment, the cytotoxic molecule is perforin and / or granzyme, optionally the cytotoxic molecule is granzyme B. In another embodiment, the host cell is capable of producing higher levels of cytokines or cytotoxic molecules when contacted with a target cell having heterozygous expression of MAGEC2. In yet another embodiment, the host cell is capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules. In yet another embodiment, the host cell is capable of killing a target cell comprising a peptide-MHC (pMHC) complex comprising a MAGEC2 peptide epitope in the context of an MHC molecule. In another embodiment, the killing is determined by a killing assay. In yet another embodiment, the ratio of host cells to target cells in the killing assay is 20:1 to 1:4. In yet another embodiment, the target cell is a target cell pulsed with 1 μg / mL to 50 pg / mL of MAGEC2 peptide, optionally the target cell is monoallelic for MHC matched to the MAGEC2 peptide. In another embodiment, the host cell is capable of killing a greater number of target cells when contacted with target cells having heterozygous expression of MAGEC2, optionally, cell killing is at least 1.05-fold greater.In yet another embodiment, the target cell is a cell line or a primary cell, optionally the target cell is selected from the group consisting of a HEK293 derived cell line, a cancer cell line, a primary cancer cell, a transformed cell line, and an immortalized cell line. In yet another embodiment, the MAGEC2 immunogenic peptide is as described herein, and / or the MHC or MHC-peptide complex is as described herein. In another embodiment, the host cell is ALKDVEERV / HLA-A. * 02. LLFGLALIEV / HLA-A * 02, SESIKKKVL / HLA-B * 44, and ASSTLYLVF / HLA-B * 57. In yet another embodiment, the host cell does not induce T cell proliferation, cytokine release, or cytotoxic killing when contacted with a target cell comprising a peptide-MHC (pMHC) complex selected from the group consisting of 57. In yet another embodiment, the host cell does not express the MAGEC2 antigen, is not recognized by the binding proteins described herein, and is of the serotype HLA-B * HLA-B, not 07 * Does not express 07 allele, serotype HLA-A * 24 and / or HLA-A * The 24 allele is not expressed.

[0044] In another aspect, a population of the host cells described herein is provided.

[0045] In yet another aspect, there is provided a composition comprising a) a binding protein as described herein, b) an isolated nucleic acid as described herein, c) a vector as described herein, d) a host cell as described herein, and / or e) a population of host cells as described herein, and a carrier.

[0046] In yet another aspect there is provided a device or kit comprising a) a binding protein as described herein, b) an isolated nucleic acid as described herein, c) a vector as described herein, d) a host cell as described herein, and / or e) a population of host cells as described herein, and optionally reagents for detecting binding of a), d) and / or e) to a pMHC complex.

[0047] In another aspect, there is provided a method of producing a binding protein as described herein, the method comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein as described herein under suitable conditions to allow expression of said binding protein; and (ii) recovering the expressed binding protein.

[0048] In yet another aspect, there is provided a method of producing a host cell expressing a binding protein described herein, the method comprising the steps of: (i) introducing into a host cell a nucleic acid comprising a sequence encoding a binding protein described herein; and (ii) culturing the transformed host cell under suitable conditions to allow expression of the binding protein.

[0049] In yet another aspect, there is provided a method for detecting the presence or absence of a MAGEC2 antigen and / or a cell expressing MAGEC2, optionally wherein the cell is a hyperproliferative cell, the method comprising detecting the presence or absence of said MAGEC2 antigen in a sample by using at least one binding protein as described herein, at least one host cell as described herein, or a population of host cells as described herein, wherein detection of the MAGEC2 antigen indicates the presence of the MAGEC2 antigen and / or a cell expressing MAGEC2.

[0050] Numerous embodiments are further provided which may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein.For example, in one embodiment, at least one binding protein or said at least one host cell forms a complex with MAGEC2 peptide in association with an MHC molecule, and the complex is detected by fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, by Western blot, or by intracellular flow assay.In another embodiment, the method further comprises obtaining a sample from the subject.

[0051] In another aspect, there is provided a method for detecting the level of a disorder characterized by MAGEC2 expression in a subject, the method comprising: a) contacting a sample obtained from the subject with at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) detecting the level of reactivity, wherein the presence or higher level of reactivity compared to a control level indicates the level of a disorder characterized by MAGEC2 expression in the subject.

[0052] Numerous embodiments are further provided which may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the control level is a reference number. In another embodiment, the control level is a level from a subject without a disorder characterized by MAGEC2 expression.

[0053] In yet another aspect, there is provided a method for tracking and assessing the progression of a disorder characterized by MAGEC2 expression in a subject, comprising: a) detecting in a subject sample the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; b) repeating step a) at a subsequent time point; and c) comparing the levels of MAGEC2, or of a cell of interest expressing MAGEC2, detected in steps a) and b) to track and assess the progression of the disorder characterized by MAGEC2 expression in the subject, wherein an absence or decrease in the level of MAGEC2 or of a cell of interest expressing MAGEC2 detected in step b) compared to step a) indicates an inhibition of the progression of the disorder characterized by MAGEC2 expression in the subject, and a presence or increase in the level of MAGEC2 or of a cell of interest expressing MAGEC2 detected in step b) compared to step a) indicates the progression of the disorder characterized by MAGEC2 expression in the subject.

[0054] Numerous embodiments are further provided which may be applied to any aspect encompassed by the invention and / or may be combined with any other embodiment described herein For example, in one embodiment, between the first time point and the subsequent time point the subject has undergone therapy to treat a disorder characterized by MAGEC2 expression.

[0055] In yet another aspect, there is provided a method of predicting clinical outcome in a subject suffering from a disorder characterized by MAGEC2 expression, comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) comparing the presence or level of reactivity with that from a control, wherein the control is obtained from a subject having a good clinical outcome, and wherein an absence or reduced level of reactivity in the subject sample compared to the control indicates that the subject will have a good clinical outcome.

[0056] In another aspect, there is provided a method of assessing the effectiveness of a therapy for a disorder characterized by MAGEC2 expression, comprising: a) determining in a first sample obtained from the subject prior to providing at least a portion of a therapy for a disorder characterized by MAGEC2 expression to the subject, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) determining in a second sample obtained from the subject after providing the therapy for a disorder characterized by MAGEC2 expression, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, wherein an absence or decreased level of reactivity in the second sample compared to the first sample is an indication that the therapy is effective in treating the disorder characterized by MAGEC2 expression in the subject, and a presence or increased level of reactivity in the second sample compared to the first sample is an indication that the therapy is not effective in treating the disorder characterized by MAGEC2 expression in the subject.

[0057] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the level of reactivity is indicated by a) the presence of binding, and / or b) T cell activation and / or effector function, optionally T cell activation or effector function being T cell proliferation, killing, or cytokine release. In another embodiment, T cell binding, activation, and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using intracellular flow assay.

[0058] In yet another aspect, there is provided a method for preventing and / or treating a disorder characterized by MAGEC2 expression, comprising contacting a target cell expressing MAGEC2 with a therapeutically effective amount of a composition comprising a cell expressing at least one binding protein described herein, and optionally, the composition is administered to the subject.

[0059] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the cells are allogeneic, syngeneic or autologous cells. In another embodiment, the cells are host cells as described herein, or a population of host cells as described herein. In yet another embodiment, the target cells are cancer cells expressing MAGEC2. In yet another embodiment, the cell composition further comprises a pharma- ceutically acceptable carrier. In another embodiment, the cell composition induces an immune response in the subject against target cells expressing MAGEC2. In yet another embodiment, the cell composition induces an antigen-specific T cell immune response in the subject against target cells expressing MAGEC2. In yet another embodiment, the antigen-specific T cell immune response is a CD4 + The response includes at least one of a helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the method further comprises administering at least one additional treatment for a disorder characterized by MAGEC2 expression, optionally administered simultaneously or sequentially with the composition. In yet another embodiment, the disorder characterized by MAGEC2 expression is cancer or a recurrence thereof, optionally the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma. In yet another embodiment, the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally the mammal is a human, a primate or a rodent. [Brief description of the drawings]

[0060] [Figure 1A] Figures 1A and 1B show nucleic acid gene expression of MAGEC2 in a broad panel of tumor types and normal tissues (Figure 1A), and compared to tumor-associated biomarkers MAGEA4 and NY-ESO-1 across a representative set of tumor types (Figure 1B). [Figure 1B] Figures 1A and 1B show nucleic acid gene expression of MAGEC2 in a broad panel of tumor types and normal tissues (Figure 1A), and compared to tumor-associated biomarkers MAGEA4 and NY-ESO-1 across a representative set of tumor types (Figure 1B). [Figure 2A] Figure 2 shows the identification of MAGEC2 antigenic peptides. Overlapping peptide sequences from MAGEC2 were identified for predicted binding to HLA-B*07:02. The top predicted MHC binding peptides were synthesized and used to pulse HLA-B*07:02 expressing HEK293T cells. The activation markers CD137 and CD69 were used to measure the reactivity of pools of TCR-transduced T cells. Figure 2A shows the results of pools of TCR-transduced T cells co-cultured with HLA-B*07:02 monoallelic HEK293T cells expressing a granzyme-activated fluorescent reporter and an overlapping library of 60mer peptides across 1,670 cancer-testis antigens. Tiles with identical overlapping peptide sequences and enrichment factors >4 are highlighted with dots corresponding to the underlying common genes. [Figure 2B]Identification of MAGEC2 antigenic peptides. Overlapping peptide sequences from MAGEC2 were identified for predicted binding to HLA-B*07:02. The top predicted MHC binding peptides were synthesized and used to pulse HLA-B*07:02 expressing HEK293T cells. The activation markers CD137 and CD69 were used to measure the reactivity of the pool of TCR transduced T cells. Figure 2B shows representative sequences of MAGEC2 peptides identified in the screening data. The overlapping sequences (solid boxes) were analyzed for HLA-B*07:02 predicted binding to identify a top predicted binder with the sequence RAREFMELL (referred to as the "RAR" peptide, dotted box). [Figure 2C] Figure 2 shows the identification of MAGEC2 antigenic peptides. Overlapping peptide sequences from MAGEC2 were identified for predicted binding to HLA-B*07:02. The top predicted MHC binding peptides were synthesized and used to pulse HLA-B*07:02 expressing HEK293T cells. The reactivity of the pool of TCR-transduced T cells was measured using activation markers CD137 and CD69. Figure 2C shows the reactivity of the TCR-transduced T cell pool to HLA-B*07:02 binding peptides identified for the top two enriched genes in the screening data. [Diagram 3] A and B show the identification of MAGEC2-reactive TCRs from a pool of TCR-transduced T cells. Target cells were pulsed with a peptide from MAGEC2 (RAR) and co-cultured with a pool of TCR-transduced T cells. Reactive TCRs were identified by sequencing the foreign TCR from sorted CD137 / CD69 double positive cells. A shows the fold enrichment difference of sorted TCRs reactive to RAR- and SPQ-pulsed cells over the input library. B shows the percentage of sorted cells expressing the indicated TCRs (magenta: TCR8-3, purple: TCR4-1, light blue: TCR11-2). [Figure 4A]We show that TCR8-3 recognizes and kills cells pulsed with RAR peptide. Figure 4A shows the results of wild-type HEK293T cells pulsed with serial dilutions of RAR peptide and co-cultured with TCR8-3-transduced T cells. TCR reactivity was measured by IFNγ release. [Figure 4B] Figure 4B shows that TCR8-3 recognizes and kills cells pulsed with RAR peptides. Figure 4B shows the results of HLA-B*07:02 monoallelic HEK293T cells expressing a granzyme-activated fluorescent reporter pulsed with the indicated concentrations of RAR peptides and co-cultured with TCR8-3-transduced T cells. [Figure 5A] Figure 5 shows that TCR8-3 recognizes melanoma cells expressing MAGEC2. Figure 5A shows a Western blot illustrating MAGEC2 protein levels in the indicated melanoma cell lines. [Figure 5B] Figure 5B shows that TCR8-3 recognizes melanoma cells expressing MAGEC2. Figure 5B shows quantification of MAGEC2 protein levels relative to GAPDH. [Figure 5C] Figure 5C shows that TCR8-3 recognizes melanoma cells expressing MAGEC2. Figure 5C shows the correspondence between MAGEC2 protein levels and MAGEC2 mRNA expression data from a public database (TRON cell line portal). [Figure 5D] Figure 5D shows that TCR8-3 recognizes melanoma cells expressing MAGEC2. Figure 5D shows the reactivity of 8-3 TCR against melanoma cell lines as measured by IFNγ release. [Figure 5E] Figure 5E shows that TCR8-3 recognizes melanoma cells expressing MAGEC2. Figure 5E shows that 8-3 TCR reactivity corresponds to MAGEC2 expression. [Figure 6]Figure 1 shows that TCR8-3 kills MAGEC2-expressing melanoma cell lines. The indicated cell lines were transduced with Incucyte® NucLight™ Red and either co-cultured with TCR8-3 (magenta), MHC-mismatched control TCR (blue) or without T cells (yellow). T cells were added at the beginning of the assay at an E:T ratio of 2:1. Red fluorescence was measured over time on an Incucyte® instrument and displayed as total red object counts normalized to time point 0. [Figure 7A] Figure 7 shows the identification of MAGEC2 antigenic peptides. Overlapping peptide sequences from MAGEC2 were identified for predicted binding to HLA-A*24:02. The top predicted MHC binding peptides were synthesized and used to pulse HLA-A*24:02 expressing HEK293T cells. The reactivity of pools of TCR transduced T cells was measured using activation-induced markers (AIMs), CD137 and CD69 (i.e., percentage of AIM double positive cells; see Figure 2 for further assay details). Figure 7A shows the results of pools of TCR transduced T cells co-cultured with HLA-A*24:02 monoallelic HEK293T cells expressing a granzyme-activated fluorescent reporter and an overlapping library of 60mer peptides across 1,670 cancer-testis antigens. Tiles with identical overlapping peptide sequences and enrichment factors >4 are highlighted with dots corresponding to the underlying common genes. [Figure 7B] Identification of MAGEC2 antigenic peptides. Overlapping peptide sequences from MAGEC2 were identified for predicted binding to HLA-A*24:02. The top predicted MHC binding peptides were synthesized and used to pulse HLA-A*24:02 expressing HEK293T cells. The reactivity of the pool of TCR transduced T cells was measured using activation-induced markers (AIMs), CD137 and CD69 (i.e., percentage of AIM double positive cells; see FIG. 2 for further assay details). FIG. 7B shows representative sequences of MAGEC2 peptides identified in the screening data. Overlapping sequences were analyzed for HLA-A*24:02 predicted binding to identify the top predicted binder sequences. [Figure 7C] Figure 7 shows the identification of MAGEC2 antigenic peptides. Overlapping peptide sequences from MAGEC2 were identified for predicted binding to HLA-A*24:02. The top predicted MHC binding peptides were synthesized and used to pulse HLA-A*24:02 expressing HEK293T cells. The reactivity of the pool of TCR transduced T cells was measured using activation-induced markers (AIMs), CD137 and CD69 (i.e., percentage of AIM double positive cells; see Figure 2 for further assay details). Figure 7C shows the reactivity of the TCR transduced T cell pool to HLA-A*24:02 binding peptides identified for peptides identified in the screening data, including a peptide with the sequence VGPDHFCVF (referred to as the "VGP" peptide) (see also Table 1B). [Figure 8] A-C show the identification of MAGEC2-reactive TCRs from a pool of TCR-transduced T cells. Target cells were pulsed with peptide from MAGEC2 (VGP) and co-cultured with a pool of TCR-transduced T cells. Reactive TCRs were identified by sequencing the exogenous TCR from sorted CD137 / CD69 double positive cells. A shows the fold enrichment difference of sorted TCRs reactive to VGO-pulsed and non-pulsed cells on the input library. B shows the percentage of sorted cells expressing the indicated TCRs. C shows the reactivity of TCR4-58-transduced T cells measured using activation-induced markers (AIMs), CD137 and CD69 (i.e., percentage of AIM double positive cells; see FIG. 2 for further assay details). [Figure 9]A shows that TCR4-58 recognizes and kills cells pulsed with VGP peptide. A shows the results of wild-type HEK293T cells pulsed with serial dilutions of VGP peptide and co-cultured with TCR4-58 transduced T cells. TCR reactivity was measured by IFP+ reporter-based cell killing. B shows the results of TCR4-58 MAGEC2 expressing cell lines, e.g. melanoma cell lines. The indicated cell lines were transduced with Incucyte® NucLight™ Red and co-cultured with TCR4-58 (magenta), non-transduced control T cells (purple), or without T cells (blue). T cells were added at the beginning of the assay at an E:T ratio of 4:1. Red fluorescence was measured over time with an Incucyte® instrument and displayed as total red object counts normalized to time point 0.

[0061] In any figure showing bar histograms, curves, or other data associated with a legend, the bars, curves or other data presented from left to right in each display correspond sequentially to the boxes shown in the legend from top to bottom or left to right. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The present invention is based, at least in part, on the discovery of MAGEC2 immunogenic peptides (e.g., those comprising or consisting of sequences listed in Table 1), binding proteins that recognize the MAGEC2 antigen (e.g., those having sequences listed in Table 2), and uses thereof. A systematic comprehensive search was performed to map the precise T cell targets recognized by the initial pool of T cells of interest.

[0063] Thus, the present invention relates, in part, to identified epitopes (immunodominant peptides) of the MAGEC2 protein of therapeutic importance, and related compositions (e.g., immunodominant peptides, vaccines and the like), compositions comprising the immunogenic peptides alone or in combination with MHC molecules, stable MHC-peptide complexes, methods for diagnosing, prognosing and tracking immune responses to disorders characterized by MAGEC2 expression, and methods for preventing and / or treating disorders characterized by MAGEC2 expression. The present invention also relates, in part, to identified binding proteins (e.g., TCRs), host cells expressing the binding proteins (e.g., TCRs), compositions comprising the binding proteins (e.g., TCRs) and host cells expressing the binding proteins (e.g., TCRs), methods for diagnosing, prognosing and tracking T cell responses to cells expressing MAGEC2, and methods for preventing and / or treating disorders characterized by MAGEC2 expression.

[0064] I. Definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0065] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0066] The term "administering" refers to providing a pharmaceutical agent or composition to a subject, including, but not limited to, administration by a medical professional and self-administration. This includes physically introducing a composition containing a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those skilled in the art. In some embodiments, routes of administration for the binding proteins described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. The term "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the binding proteins described herein may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically, and administration may be performed, e.g., once, multiple times, and / or over one or more extended periods of time.

[0067] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be a MAGEC2 antigen or a fragment thereof against which it is desired to elicit a protective or therapeutic immune response. An "epitope" is the portion of an antigen that is bound by a natural or synthetic substance.

[0068] As used herein, the term "adjuvant" refers to a substance that, when administered before, together with, or after the administration of an antigen, accelerates, prolongs, and / or enhances the quality and / or strength of the immune response to an antigen compared to administration of the antigen alone. Adjuvants can increase the magnitude and duration of the immune response induced by vaccination.

[0069] As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion"), or single chains thereof. An "antibody" refers, in one embodiment, to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding site thereof. Each heavy chain comprises a heavy chain variable region (referred to herein as V H In certain naturally occurring antibodies, the heavy chain constant region is composed of three domains, CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain is composed of a light chain variable region (abbreviated herein as V L The light chain constant region is composed of one domain, CL. H and V L The regions may be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) separated from each other by more conserved regions called framework regions (FRs). H And each V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0070] The term "antigen-presenting cell" or "APC" includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0071] As used herein, the term "antigen-binding portion" of a binding protein (e.g., a TCR) refers to one or more portions of a TCR that retain the ability to bind (e.g., specifically and / or selectively) to an antigen (e.g., a MAGEC2 antigen). Such portions are, for example, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, preferably about 8 to about 300 amino acids in length, such as about 8 to about 200 amino acids in length, or from about 10 amino acids to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of a TCR can be performed by fragments of a full-length TCR. Examples of binding portions encompassed by the term "antigen-binding portion" of a TCR include (i) the V α and V β The Fv fragments include (i) an Fv fragment consisting of a V domain, (ii) an isolated complementarity determining region (CDR), or (iii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. α and V β are encoded by separate genes, but using recombinant methods, α and V β V is expressed as a single protein chain whose domains pair to form a monovalent molecule, known as a single-chain TCR (scTCR). α and V β V is provided by a synthetic linker that allows α and V β Such single chain TCRs are also intended to be encompassed by the term "antigen-binding portion" of a TCR. These TCR fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for usefulness in the same manner as the complete binding protein. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of the complete immunoglobulin.

[0072] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to a sequence of non-contiguous amino acids that confer antigen specificity and / or binding affinity in a particular binding protein, e.g., TCR variable region. In TCRs, there are generally three CDRs in each α chain variable region (αCDR1, αCDR2, and αCDR3) and three CDRs in each β chain variable region (βCDR1, βCDR2, and βCDR3). CDR3 is considered to be the main CDR responsible for recognizing processed antigen. CDR1 and CDR2 primarily interact with MHC.

[0073] The term "body fluid" refers to fluids excreted or secreted from the body, as well as fluids that are not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's or pre-ejaculate fluid, chyle, chyme, feces, Skene's fluid, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal fluid, vitreous humor, vomit). In some embodiments, the body fluid comprises immune cells, and optionally the immune cells are cytotoxic lymphocytes, e.g., cytotoxic T cells, and / or NK cells, CD4+ T cells, and the like.

[0074] The term "coding region" refers to the region of a nucleotide sequence that contains codons that are translated into amino acid residues, while the term "non-coding region" refers to the regions of a nucleotide sequence that are not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0075] The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region that is antiparallel to the first region if that residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand can form specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region that is antiparallel to the first region if that residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if the two regions are in an antiparallel arrangement and at least one nucleotide residue of the first region can base pair with a residue of the second region. In some embodiments, the first region includes a first portion and the second region includes a second portion, whereby when the first portion and the second portion are arranged in an antiparallel manner, at least about 50% of the nucleotide residues of the first portion, in other embodiments at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range therebetween, including the endpoints, e.g., at least about 80% to 100%, can base pair with nucleotide residues of the second portion. In some embodiments, all nucleotide residues of the first portion can base pair with nucleotide residues in the second portion.

[0076] As used herein, the term "costimulate" in reference to activated immune cells includes that a costimulatory molecule can provide a second, non-stimulatory receptor-mediated signal ("costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion, for example in a T cell that has received a T cell receptor-mediated signal. An immune cell that has received a cell receptor-mediated signal, for example through an activating receptor, is referred to herein as an "activated immune cell."

[0077] "CD3" is known in the art as a six-chain multiprotein complex (Abbas and Lichtman, Cellular and Molecular Immunology (9 th Edition)(2018), Janeway et al.(Immunobiology)(9 th (see, e.g., 1999 American Association of Clinical Oncology (ACM) (2016)). In mammals, the complex comprises a homodimer of CD3γ, CD3δ, two CD3ε, and CD3ζ chains. The CD3γ, CD3δ, and CD3ε chains are related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that is believed to allow these chains to associate with positively charged regions or residues of the T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-dependent activation motif, or ITAM, while each CD3ζ chain has three ITAMs. Without wishing to be bound by theory, it is believed that the ITAMs are important for the signaling capacity of the TCR complex. The CD3 used in accordance with the present invention may be derived from a variety of animal species, including human, mouse, rat, or other mammals.

[0078] As used herein, a "component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ and two CD3ε chains).

[0079] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein that has been engineered to contain two or more amino acid sequences linked together in a manner that does not occur naturally or in a host cell, and that can function as a receptor when present on the surface of a cell. CARs encompassed by the present invention comprise an extracellular portion that comprises an antigen-binding domain (i.e., derived from or derived from an immunoglobulin or immunoglobulin-like molecule, e.g., a TCR specific for the MAGEC2 antigen, a single chain TCR-derived binding protein, an scFv derived from an antibody, an antigen-binding domain derived from or derived from a killer immune receptor from a NK cell, etc.) linked to a transmembrane domain and one or more intracellular signaling domains (e.g., an effector domain optionally including a costimulatory domain(s)). (See, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; see also, Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0080] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily CD8 + Mediated by T cells.

[0081] The term "consisting essentially of" is not equivalent to "comprising" and refers to materials or steps specified in a claim or to materials or steps that do not materially affect the basic characteristics of the claimed subject matter. For example, a domain, region, or module of a protein (e.g., a binding domain, hinge region, linker module), or a protein (which may have one or more domains, regions, or modules) "consists essentially of" a particular amino acid sequence if the amino acid sequence of the domain, region, module, or protein contains extensions, deletions, mutations, or combinations thereof (e.g., amino acids at the amino or carboxy terminus or between domains) that, in combination, account for up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein, and do not substantially affect the activity (e.g., target binding affinity of a binding protein) of the domain(s), region(s), module(s), or protein (i.e., activity is reduced by no more than 50%, e.g., no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%).

[0082] The term "determining a suitable therapeutic regimen for a subject" is understood to mean determining a therapeutic regimen for a subject (i.e., a single therapy or a combination of different therapies used to prevent and / or treat a viral infection in a subject) that is initiated, modified and / or terminated based on, or essentially based on, or at least partially based on, the results of the analysis according to the present invention. One example would be to initiate an adjuvant therapy after surgery to reduce the risk of recurrence, and another example would be to change the dose of a specific chemotherapy. The decision may be based on the results of the analysis according to the present invention as well as on the personal characteristics of the subject to be treated. In most cases, the actual decision of the therapeutic regimen suitable for the subject will be made by the attending physician or doctor.

[0083] As used herein, a "hematopoietic progenitor cell" is a cell that can be derived from hematopoietic stem cells or fetal tissue and can further differentiate into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include CD24 Lo Lin-CD117 + These include those that have a phenotype similar to that of primary thymocytes or those found in the thymus (called primitive thymocytes).

[0084] "Homology" as used herein refers to the similarity of nucleotide sequences between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. A nucleotide residue position in both regions is homologous at that position if the same nucleotide residue is occupied by the same nucleotide residue. A first region is homologous to a second region if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the percentage of nucleotide residue positions in the two regions that are occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, with the proviso that at least about 50%, in other embodiments at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range therebetween, inclusive of the endpoints, e.g., at least about 80%-100%, of the nucleic acid residue positions of each of the portions are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions of each of the sites are occupied by the same nucleotide residue.

[0085] The term "immune response" includes T cell and / or B cell immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses that are indirectly mediated by T cell activation, such as antibody production (humoral response), and activation of cytokine-responsive cells (e.g., macrophages).

[0086] An increased immune response or ability to stimulate the immune system may result from increased agonist activity of T cell costimulatory receptors and / or increased antagonist activity of inhibitory receptors. An increased immune response or ability to stimulate the immune system may result from increased EC in assays that measure immune responses, such as assays that measure cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly via detecting CD107a or granzymes) and changes in proliferation. 50 The ability to stimulate an immune response or activity of the immune system may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500%, or more.

[0087] The term "immunotherapeutic agent" can include any molecule, peptide, antibody or other agent capable of stimulating the host's immune system to generate an immune response to a viral infection in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0088] The term "immune cell" refers to any cell of the immune system that arises from hematopoietic stem cells in the bone marrow that give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells, e.g., monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes), and lymphoid progenitor cells (which give rise to lymphoid cells, e.g., T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + These include T cells, CD4 CD8 double negative T cells, gd T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells are sometimes referred to as "antigen-presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC, complexed with a peptide, interacts with a TCR on the surface of the T cell.

[0089] "Isolated protein" refers to a protein that is substantially free of other proteins, cellular material, isolation and culture media, if isolated from a cell or produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals, if chemically synthesized. An "isolated" or "purified" protein, or a biologically active portion thereof, is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the binding protein, antibody, polypeptide, peptide, or fusion protein is derived, or, if chemically synthesized, is substantially free of chemical precursors or other chemicals. The language "substantially free of cellular material" includes preparations of a biomarker polypeptide or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of biomarker proteins or fragments thereof having less than about 30% (by dry weight) non-biomarker proteins (also referred to herein as "contaminating proteins"), or in some embodiments about 25%, 20%, 15%, 10%, 5%, 1%, or less, or any ranges therebetween inclusive of the endpoints, e.g., about less than 1% to 5% non-biomarker proteins. When a binding protein, antibody, polypeptide, peptide, or fusion protein, or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it may be substantially free of culture medium, i.e., culture medium may comprise about 20%, 15%, 10%, 5%, 1%, or less, or any ranges therebetween inclusive of the endpoints, e.g., about less than 1% to 5% of the volume of the protein preparation.

[0090] As used herein, the term "isotype" refers to the class of antibody (e.g., IgM, IgG1, IgG2C, etc.) that is encoded by heavy chain constant region genes.

[0091] As used herein, the term "K D" is intended to refer to the dissociation equilibrium constant of a particular binding protein-antigen interaction. The binding affinity of the binding proteins encompassed by the present invention can be measured or determined by standard binding protein-target binding assays, such as competitive assays, saturation assays, or standard immunoassays, such as ELISA or RIA. A relatively lower Kd value indicates a relatively higher binding affinity (e.g., about 5×10 -4 The Kd value for M(500uM) or less is 1×10 -4 M (100 uM), where a Kd of 100 uM indicates a relatively higher binding affinity compared to a Kd of 500 uM).

[0092] A "kit" is any article of manufacture (e.g., package, or container) that includes at least one reagent (e.g., a probe or small molecule for detecting and / or affecting the expression of a marker encompassed by the present invention). The kit may be advertised, distributed, or sold as a unit for carrying out a method encompassed by the present invention. The kit may include one or more reagents necessary to express a composition useful in a method encompassed by the present invention. In some embodiments, the kit may further include a reference standard (e.g., a nucleic acid encoding a protein that does not affect or modulate a signaling pathway controlling cell proliferation, division, migration, survival, or apoptosis). One of skill in the art can envision many such control proteins, including, for example, but not limited to, common molecular tags (e.g., green fluorescent protein and beta-galactosidase), proteins that are not classified according to the GeneOntology standards into any of the pathways encompassing cell proliferation, division, migration, survival, or apoptosis, or ubiquitous housekeeping proteins). The reagents in the kit may be provided in individual containers or a mixture of two or more reagents in a single container. Additionally, instructional materials describing the use of the compositions within the kit may be included.

[0093] As used herein, the term "linked" refers to the association of two or more molecules. The association may be covalent or non-covalent. The association may also be genetic (i.e., recombinantly fused). Such association may be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0094] "Linker" may, in some embodiments, refer to an amino acid sequence that links two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function that accommodates the interaction of two sub-binding domains such that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., scTCR) or signaling activity (e.g., TCR complex). In some embodiments, the linker is composed of, for example, about 2 to about 35 amino acids, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids.

[0095] The term "MAGEC2" refers to a specific member of the melanoma antigen gene family clustered on human chromosome Xq26-q27, also known as cancer / testis antigen 10 (CT10), hepatocellular carcinoma antigen 587 (HCA587) and melanoma antigen, family E, 1, cancer / testis specific (MAGEE1) (Gure et al. (2000) Int. J. Cancer 85:726-732; Li et al. (2003) Lab Invest. 83:1185-1192; Ma et al. (2004) Int. J. Cancer 109:698-702; Godelaine et al. (2007) Cancer Immunol. Immunother. 56:753-759; Reiner et al. (2009) Int. J. Cancer 124:352-357; Doyle et al. (2009) Int. J. Cancer 124:352-357). al.(2010)Mol.Cell 39:93-974, von Boehmer et al.(2011)PLoS One 6,e21366, Wen et al.(2011)Cancer Sci.102:1455-1461, de Carvalho et al.(2013)Cancer Immunol.Immunother.62:191-195, Bhatia et al. (2013) J.Invest.Dermatol.133:759-767), Yang et al. (2014) Breast Cancer Res.Treat.145:23-32, and Kunert et al. al. (2016) J. Immunol. 197:2541-2552). MAGEC2 is believed to enhance the ubiquitin ligase activity of RING-type zinc finger-containing E3 ubiquitin-protein ligases, for example by recruiting and / or stabilizing Ub1-conjugating enzymes (E2s) in the E3:substrate complex. MAGEC2 enhances the ubiquitin ligase activity of TRIM28 in vitro and stimulates p53 / TP53 ubiquitination in the presence of the Ub1-conjugating enzyme UBE2H, leading to p53 / TP53 degradation.MAGEC2 is not expressed in normal tissues except for testis, but is expressed in tumors of various histological types, including melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer, and bladder urothelial carcinoma.

[0096] The term "MAGEC2" is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof. Representative human MAGEC2 cDNA and human MAGEC2 protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI) (see, e.g., ncbi.nlm.nih.gov / gene / 51438). For example, human MAGEC2 (NP_057333.1) is encoded by the transcript (NM_016249.4). Nucleic acid and polypeptide sequences of MAGEC2 orthologues from non-human organisms are well known, including chimpanzee MAGEC2 (NM_001302428.1, NP_001289357.1, XM_016942653.1, and XP_016798142.1) and rhesus macaque MAGEC2 (NM_001265825.1, NP_001252754.1, XM_028841693.1, and XP_028697526.1). Representative sequences of MAGEC2 sequences are also provided in Table 3 below.

[0097] Suitable anti-MAGEC2 antibodies for detecting MAGEC2 protein are well known in the art, including, for example, antibodies TA315476 and TA342769 (OriGene, Rockville, MD), antibodies orb353181 and orb125944 (Biorbyt, Cambridge, United Kingdom), antibodies A05335 and AO5335-1 (Boster Bio, Pleasanton, CA), and antibodies ABIN2788251 and ABIN2706502 (Antibodies-online, Limerick, PA). In addition, reagents for detecting MAGEC2 expression are well known. Moreover, in the commercial product listings of various companies, several kinds of siRNA, shRNA, CRISPR constructs for regulating MAGEC2 expression can be found, such as open reading frame (ORF) clones SC07208 and RN211555 (OriGene, Rockville, MD), and CRISPR knockouts GA109805 and KN403064 (OriGene, Rockville, MD). It should be noted that the term may also be used to refer to any combination of the characteristics described herein for MAGEC2 molecules. For example, any combination of sequence composition, identity percentage, sequence length, domain structure, functional activity, etc. may be used to describe the MAGEC2 molecules encompassed by the present invention.

[0098] The term "MAGEC2 antigen" or "MAGEC2 peptide antigen" or "MAGEC2-containing peptide antigen" or "MAGEC2 epitope" or "MAGEC2 peptide epitope" or "MAGEC2 peptide" refers to a naturally or synthetically produced immunogenic portion of MAGEC2. In some embodiments, the MAGEC2 antigen protein can range in length from about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids, or any range therebetween, including the endpoints, e.g., 8-15 amino acids. In some embodiments, the MAGEC2 antigen protein can form a complex with an MHC (e.g., HLA) molecule such that a binding protein of the disclosure that recognizes a MAGEC2 peptide:MHC (e.g., HLA) complex can bind (e.g., specifically and / or selectively) to such a complex. Representative MAGEC2 peptide antigen sequences are shown in Table 1.

[0099] The term "major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a membrane-spanning a chain (with three a domains) and a non-covalently associated b2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and b, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides derived from the cytosol to the cell surface, where the peptide antigen-MHC (pMHC) complex is expressed by CD8 + MHC class II molecules deliver peptides from the vesicle system to the cell surface, where they are recognized by CD4 + Recognized by T cells. Human MHC is called human leukocyte antigen (HLA).

[0100] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk of developing it or is susceptible to developing it.

[0101] The term "prognosis" includes a prediction of the likely course and outcome of a viral infection, or the likelihood of recovery from a disease. In some embodiments, a statistical algorithm is used to predict the prognosis of a viral infection in an individual. For example, the prognosis may be surgery, the development of a clinical subtype of a viral infection, the occurrence of one or more clinical factors, or recovery from the disease.

[0102] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology," which may be determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This described algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the polynucleotides described herein. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0103] The phrase "pharmacologically acceptable carrier" means a pharma- ceutically acceptable substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating substance, involved in carrying or transporting a compound of interest from one organ or part of the body to another organ or part of the body.

[0104] The term "ratio" refers to a relationship between two numbers (e.g., a score, a sum, etc.). Although a ratio may be expressed in a particular order (e.g., a to b, or a:b), one of ordinary skill in the art will recognize that the underlying relationship between the numbers may be expressed in any order without losing the significance of the underlying relationship, even though the appearance of trends and correlations based on the ratio may be reversed.

[0105] The term "recombinant host cell" (or simply "host cell") refers to a cell that contains a nucleic acid that does not naturally occur in the cell, e.g., a cell into which a recombinant expression vector has been introduced. It should be understood that the cells of the invention are intended to mean to encompass not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in subsequent generations, either by mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the term cell of the invention.

[0106] The term "cancer response", "response to immunotherapy", or "response to modulator of T-cell mediated cytotoxicity / immunotherapy combination therapy" refers to any response of a hyperproliferative disorder (e.g., cancer) to a cancer drug, e.g., a modulator of T-cell mediated cytotoxicity, and immunotherapy, preferably to the change in tumor weight and / or volume after starting neoadjuvant or adjuvant therapy. The term "neoadjuvant therapy" refers to a treatment given before the primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormonal therapy. The response of a hyperproliferative disorder can be evaluated, for example, for efficacy or in the neoadjuvant or adjuvant setting, where the size of the tumor after the systemic intervention can be compared to the initial size and dimensions, measured by CT, PET, mammogram, ultrasound, or palpation. Response can also be evaluated by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Responses may be recorded in a quantitative manner, such as percentage change in tumor volume, or in a qualitative manner, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Assessment of hyperproliferative disorder response may be performed early, for example, hours, days, weeks, or preferably months, after initiation of neoadjuvant or adjuvant therapy. A typical endpoint for response assessment is the end of neoadjuvant chemotherapy or surgical removal of residual tumor cells and / or tumor bed. This is typically 3 months after initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). Clinical benefit rate is measured by determining the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the total number of patients with stable disease (SD) at least 6 months after the end of therapy. In simple terms, this formula is CBR=CR+PR+SD beyond 6 months.In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating response to cancer therapy relate to "survival time", including all of the following: survival time to death, also known as overall survival (where death can be either cause-unrelated or tumor-related); "recurrence-free survival" (wherein the term recurrence includes both local and distant recurrence); metastasis-free survival; disease-free survival (wherein the term disease includes cancer and its associated diseases). The above survival time length can be calculated based on a defined starting point (e.g., at the time of diagnosis or at the start of treatment) and an end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for efficacy of treatment may be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a population of subjects may be administered a particular cancer treatment regimen and the outcome may be correlated with biomarker measurements determined before any cancer treatment is administered. The outcome measurements may be pathological responses to the therapy administered in a neoadjuvant setting. Alternatively, outcome measurements, such as overall survival and disease-free survival, may be followed up over a period of time for subjects following cancer therapy for which biomarker measurements are known. In certain embodiments, the dose administered is a standard dose known in the art for cancer therapeutic agents. The duration of time for which subjects are followed up may vary. For example, subjects may be followed up for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds that correlate with cancer therapy outcomes may be determined using methods well known in the art, such as those described in the Examples section.

[0107] As indicated, these terms may also refer to improved prognosis, for example, as reflected by increased time to recurrence (which is the time to first recurrence, censored by a second primary cancer as the first event, or death without evidence of recurrence) or increased overall survival (which is the time from treatment to death from any cause). To respond or to be responsive means that there is a beneficial endpoint that is achieved when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated by exposure to a stimulus. It will be understood that assessing the likelihood that a tumor or subject will respond favorably is equivalent to assessing the likelihood that a tumor or subject will not respond favorably (i.e., show a lack of response or be non-responsive).

[0108] The term "resistance" refers to the acquired or natural resistance of a cancer sample or mammal to a cancer therapy (i.e., unresponsiveness to a therapeutic treatment or reduced or limited response to a therapeutic treatment), for example, a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater decrease in response to a therapeutic treatment, such as a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or greater decrease, or any range therebetween, including the endpoints. The decrease in response can be measured by comparing to the same cancer sample or mammal before resistance is acquired, or by comparing to a different cancer sample or mammal known not to have resistance to a therapeutic treatment. A typical acquired resistance to chemotherapy is called "multidrug resistance". Multidrug resistance may be mediated by P-glycoprotein or by other mechanisms, or may occur when a mammal is infected with a multidrug resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of the skilled clinician, and may be measured, for example, by cell proliferation and cell death assays, as described herein as "sensitizing". In some embodiments, the term "reverse resistance" refers to the use of a second agent in conjunction with a primary cancer therapy (e.g., chemotherapy or radiation therapy) in situations where the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone is unable to reduce the tumor volume statistically significantly compared to that of an untreated tumor, thereby significantly reducing the tumor volume at a statistically significant level (e.g., p<0.05) compared to that of an untreated tumor. This is usually applied to tumor volume measurements taken when untreated tumors are growing exponentially.

[0109] The term "sample" used to detect or determine the absence or presence, or level, of at least one biomarker, typically refers to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., those described above under the definition of "body fluid"), or a tissue sample (e.g., a biopsy), such as a skin, colon sample, or surgically resected tissue. In some embodiments, the methods encompassed by the present invention further comprise the step of obtaining a sample from an individual before detecting or determining the absence or presence, or level, of at least one marker in the sample.

[0110] The term "sensitizing" refers to altering cancer or tumor cells in a way that allows the associated cancer to be more effectively treated with a cancer therapy (e.g., anti-immune checkpoint, chemotherapy, and / or radiation therapy). In some embodiments, normal cells are not affected so much that they are unduly harmed by the therapy. Increased susceptibility or decreased susceptibility to therapeutic treatment is measured according to methods known in the art for the particular treatments and methods described below in this application, including, but not limited to, cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164), and cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., In: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Press, 2006). Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90. Sensitivity or resistance can also be measured in animals by measuring the reduction in tumor size over a period of time (e.g., 6 months for humans, 4-6 weeks for mice). A composition or method sensitizes a response to a therapeutic treatment if the increase in therapeutic sensitivity or decrease in resistance compared to the therapeutic sensitivity or resistance in the absence of such composition or method is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range therebetween inclusive of the endpoints.Determining sensitivity or resistance to therapeutic treatment is a common practice in the art and is within the skill of a skilled clinician.It should be understood that any method described herein for enhancing the effectiveness of cancer therapy can be equally applied to the method for sensitizing hyperproliferative cells or other cancerous cells (e.g., resistant cells) to cancer therapy.

[0111] The term "small molecule" is a term of the art and includes molecules with a molecular weight of less than about 1000, or molecules with a molecular weight of less than about 500. In one embodiment, a small molecule is not composed solely of peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63-68), and natural product extract libraries. In another embodiment, the compound is a small organic non-peptide compound. In a further embodiment, the small molecule is not a biosynthetic product.

[0112] The term "specific binding" refers to the binding of a binding protein to a given antigen. Typically, a binding protein binds to a given antigen with a specific binding activity of approximately 5×10 , as measured by a binding assay, e.g., surface plasmon resonance (SPR) technology on a BIAcore™ assay instrument, using the antigen of interest as the analyte and the binding protein as the ligand. -4 M or less, approximately 1×10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10-10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, approximately 1×10 -12 Affinity (K) of less than or equal to M, or even less, or any range therebetween, including the endpoints, e.g., about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, etc. D In some embodiments, a binding protein binds to a given antigen with an affinity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times greater than its affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). As used herein, the phrases "binding protein that recognizes an antigen" and "binding protein specific for an antigen" are used interchangeably with the term "binding protein that specifically binds to an antigen." Selective binding is a relative term that refers to the ability of a binding protein to distinguish binding of one antigen from binding of another antigen, e.g., to distinguish binding of a particular family member or antigen target from binding of a related family member or antigen target. For example, the analytical data provided in the Examples section demonstrates that the binding proteins described herein specifically bind to the MAGEC2 immunogenic epitope and / or selectively bind to many related epitopes (e.g., the MAGEC2 immunogenic epitope and closely related sequences), thereby distinguishing such targets from the vast majority of other possible epitopes available in the human genome.

[0113] The term "subject" refers to any healthy animal, mammal or human, or any animal, mammal or human suffering from a disorder characterized by MAGEC2 expression. The term "subject" is interchangeable with "patient."

[0114] The term "survival" includes all of the following: survival to death, also known as overall survival (where death can be either cause-free or tumor-related); "recurrence-free survival" (where the term recurrence includes both local and distant recurrence); metastasis-free survival; disease-free survival (where the term disease includes cancer and related diseases). The above survival lengths can be calculated based on a defined starting point (e.g., time of diagnosis or start of treatment) and an end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for the efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0115] The term "synergistic effect" refers to a combined effect of two or more agents (e.g., a MAGEC2-related agent described herein and another therapy for treating a disorder characterized by MAGEC2 expression) that is greater than the sum of the separate effects of the cancer agents / therapies alone.

[0116] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, e.g., effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0117] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., an mRNA, hnRNA, cDNA, or analog of such an RNA or cDNA) that is complementary to or homologous to all or a portion of the mature mRNA produced by transcription of a biomarker nucleic acid and, if any, by normal post-transcriptional processing of the RNA transcript (e.g., splicing), and by reverse transcription of the RNA transcript.

[0118] "T cells" are immune system cells that mature in the thymus and produce a T cell receptor (TCR). T cells are naive (not exposed to antigen; T CM expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO, compared with normal T cells), memory T cells (T M ) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). M are central memory T cells (T CM , which have increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA, compared to naive T cells), and effector memory T cells (T EM , naive T cells or T CM These cells can be further divided into effector T cells (T E ) is T CM CD8+ T lymphocytes are antigen-experienced cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzymes and perforin compared to CD4+ T lymphocytes. Other exemplary T cells include regulatory T cells, e.g., CD4+ T lymphocytes. + CD25 + (Foxp3 + ) Regulatory T cells and Treg17 cells, as well as Tr1, Th3, and CD8 + CD28 and Qa-1 restricted T cells.

[0119] Conventional T cells (also known as Tconv or Teff) have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) and augment immune responses by expressing one or more T cell receptors. Tcon or Teff is generally defined as any T cell population that is not a Treg, including, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated, for example, toward a Th1 lineage or a Th2 lineage. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is a CD4+ Teff, or a CD8+ Teff, such as a CD4+ helper T lymphocyte (e.g., Th0, Th1, Tfh, or Th17), and a CD8+ cytotoxic T lymphocyte. As further described in this application, the cytotoxic T cell is a CD8+ T lymphocyte. "Naive Tcon" are CD4 T cells that have differentiated in the bone marrow and successfully undergone the processes of positive and negative central selection in the thymus, but have not yet been activated by exposure to antigen. + T cells. Naive Tcons are generally characterized by the absence of surface expression of L-selectin (CD62L), activation markers such as CD25, CD44 or CD69, and memory markers such as CD45RO. Naive Tcons are therefore considered to be quiescent and non-dividing, and require interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see at least WO2010 / 101870). The presence and activity of such cells is undesirable in the context of suppressing immune responses. Unlike Tregs, Tcons are not anergic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637).

[0120] "T Effector" ("T eff " or "T E") cells refer to T cells (e.g., CD4+ and CD8+ T cells) and T helper (Th) cells with cytolytic activity, which secrete cytokines to activate and signal other immune cells, but do not include regulatory T cells (Treg cells).

[0121] "T cell receptor" or "TCR" refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al. (1997) Curr. Biol. Publ. 4:33) that can bind (e.g., specifically and / or selectively) to an antigenic peptide bound to an MHC receptor. TCRs can be found on the surface of cells or in a soluble form and are generally composed of a heterodimer having an alpha and a beta chain (also known as TCRα and TCRβ, respectively) or a gamma and a delta chain (also known as TCRγ and TCRδ, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portions of the TCR chains (e.g., α and β chains) contain two immunoglobulin domains: a variable domain (e.g., α chain variable domain or V chain variable domain) at the N-terminus; α、 and the β chain variable domain or V β Typically, amino acids 1 to 116 based on the Kabat numbering (Kabat et al. (1991) "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 5 th ed.), and one constant domain at the C-terminus that is adjacent to the cell membrane (e.g., the α chain constant domain or C α Typically, amino acids 117 to 259 based on Kabat, a β chain constant domain or C β、Typically, amino acids 117-295 based on Kabat). Also similar to immunoglobulins, variable domains contain complementarity determining regions ("CDRs", also called hypervariable regions or "HVRs") separated by framework regions ("FRs") (see, e.g., Fores et al. (1990) Proc. Natl. Acad Sci. US.A. 87:9138; Chothia et al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol. 27:55). In some embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and are associated with the CD3 complex. Sources of TCRs encompassed by the present invention may be derived from various animal species, e.g., humans, mice, rats, rabbits, or other mammals.

[0122] The term "T cell receptor" or "TCR" should be understood to encompass complete TCRs and antigen-binding portions or fragments thereof. In some embodiments, the TCR is a complete or full-length TCR, e.g., an αβ-type TCR or a γδ-type TCR. In some embodiments, the TCR is an antigen-binding portion that is shorter than the full-length TCR but binds to a specific peptide bound in an MHC molecule, e.g., an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR contains only a portion of the structural domain of the full-length or complete TCR, but can still bind to a peptide epitope (e.g., an MHC-peptide complex) that the full-length TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, e.g., the variable α chain and the variable β chain of the TCR, to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity determining regions (CDRs) involved in recognition of peptides, MHC and / or MHC-peptide complexes.

[0123] The nomenclature has been established by the International Immunogenetics Information System (IMGT) (Scaviner and Lefranc (2000) Exp. Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; see also T Cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). IMGT provides unique sequences used to describe TCRs, and the sequences described in this application can be identified based on such unique sequences provided in this application. TCR sequences are publicly available in the IMGT database at imgt.org.

[0124] As mentioned above, natural alpha / beta heterodimeric TCRs have an alpha chain and a beta chain. In general, each chain contains a variable region, a joining region and a constant region, and the beta chain usually also contains a short diversity region between the variable region and the joining region, but this diversity region is often considered as part of the joining region. Each variable region contains three hypervariable CDRs (complementarity determining regions) embedded in a framework sequence. It is well known that CDR3 is the main mediator of antigen recognition. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions, which are differentiated by their framework, CDR1 and CDR2 sequences, and by a partially defined CDR3 sequence. In the IMGT nomenclature, the Vα types are referred to by a unique TRAV number. For example, "TRAV4" defines a TCR Vα region with a unique framework, and CDR1 and CDR2 sequences, and a CDR3 that is partially defined by amino acid sequences that are conserved across various TCRs, but also contains amino acid sequences that differ from one TCR to another. Similarly, "TRBV2" defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence. There are 54 alpha variable genes (of which 44 are functional) and 67 beta variable genes (of which 42 are functional) known to exist within the alpha and beta loci, respectively.

[0125] Similarly, the joining regions of the TCR are defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions are defined by the IMGT TRAC and TRBC nomenclature. In the IMGT nomenclature, the diversity region of the beta chain is referred to by the abbreviation TRBD. As noted, the linked TRBD / TRBJ regions are often collectively referred to as the joining region.

[0126] The gene pools encoding the TCR alpha and beta chains are located on different chromosomes and contain distinct V, (D), J and C gene segments, which are brought together by rearrangement during T cell development. This results in a very high degree of diversity for the T cell alpha and beta chains due to the large number of potential recombination events that occur between the 54 TCR alpha variable genes and the 61 alpha J genes, or between the 67 beta variable genes and the 2 beta D and 13 beta J genes. The recombination process is not stringent, introducing further diversity within the CDR3 region. Each alpha and beta variable gene may also contain allelic variants, designated TRAVxx*01 and *02, or TRBVx-x*01 and *02, respectively, in the IMGT nomenclature, thus further increasing the amount of diversity. Similarly, some of the TRBJ sequences have two known varieties. (Note that the absence of the "*" modifier means that only one allele is known for the relevant sequence). The natural repertoire of human TCRs resulting from recombination and thymic selection consists of approximately 10 6 It is estimated that each beta chain contains a unique beta chain sequence for each species (Arstila et al. (1999) Science 286:958-961) and may even be higher (Robins et al. (2009) Blood 114:4099-4107). Each beta chain is estimated to pair with at least 25 different alpha chains, thus generating further diversity (Arstila et al. (1999) Science 286:958-961).

[0127] Thus, the term "TCR alpha variable domain" refers to the TRAV and TRAJ regions together; the TRAV region alone; or the TRAV and a portion of the TRAJ region, and the term TCR alpha constant domain refers to the extracellular TRAC region, or the C-terminally truncated or full-length TRAC sequence. Similarly, the term "TCR beta variable domain" refers to the TRBV and TRBD / TRBJ regions together; the TRBV and TRBD regions alone; the TRBV and TRBJ regions alone; or the TRBV and a portion of the TRBD and / or TRBJ regions, and the term TCR beta constant domain refers to the extracellular TRBC region, or the C-terminally truncated or full-length TRBC sequence. These TCR alpha variable domain and TCR beta variable domain nomenclature apply equally to the variable domains of the TCR gamma chain and the TCR delta chain, respectively, for gamma / delta TCRs. One skilled in the art can obtain the gene sequences for TRAV, TRAJ, TRAC, TRBV, TRBJ and TRBC, for example, via the publicly available IMGT database.

[0128] The term "TCR complex" refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of a CD3 zeta chain, a TCR alpha chain, and a TCR beta chain. Alternatively, a TCR complex may be composed of a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, a homodimer of a CD3 zeta chain, a TCR gamma chain, and a TCR delta chain.

[0129] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease, or to enhance desirable physical or mental development and conditions in animals or humans.

[0130] The terms "therapeutically effective amount" and "effective amount" refer to an amount of a substance that produces some desired effect (e.g., a desired local or systemic therapeutic effect) in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any treatment. In some embodiments, a therapeutically effective amount of a substance depends on the substance's therapeutic index, solubility, pharmacokinetics, half-life, etc. Toxicity and therapeutic efficacy of a subject compound can be determined, for example, by the LD 50 and ED 50 The LD can be determined by standard pharmaceutical methods in cell cultures or experimental animals to determine the therapeutic index. In some embodiments, compositions are used that exhibit large therapeutic indices. In some embodiments, the LD 50 The lethal dose (ED) may be measured, for example, for a drug, and may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to when the drug is not administered. 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) may be measured, and may, for example, for a drug, be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to when the drug is not administered. Similarly, the IC 50For example, for an agent, an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more may be measured compared to when the agent is not administered. In some embodiments, the T cell immune response in the assay may be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In other embodiments, a reduction in viral load of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% may be achieved.

[0131] The term "treating" refers to the therapeutic management or amelioration of a symptom (e.g., a disease or disorder) of interest. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically performed to alter the course of a disease (the term is used to indicate any disease, disorder, syndrome, or undesirable symptoms that warrant or potentially warrant therapy) in a manner that is beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing the severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of the occurrence or recurrence of a disease or one or more symptoms or signs of a disease. Desirable effects of treatment include, but are not limited to, prevention of onset or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastasis, reduction in the rate of disease progression, improvement or amelioration of a disease state, and remission or improvement of prognosis. Therapeutic agents may be administered to subjects who have a disease or who are at high risk of developing a disease compared to members of the general population. In some embodiments, the therapeutic agent may be administered to a subject who had a disease but no longer shows evidence of the disease. The agent may be administered, for example, to reduce the likelihood of overt disease recurrence. The therapeutic agent may be administered prophylactically, i.e., before any symptoms or signs of disease develop. "Preventive treatment" refers to providing medical and / or surgical treatment to a subject who does not develop or shows no evidence of disease, for example, to reduce the likelihood of disease developing or to reduce the severity of disease if disease does develop. The subject may have been identified as being at risk of developing a disease (e.g., at increased risk compared to the general population) or as having a risk factor that increases the likelihood of developing a disease.

[0132] The term "unresponsiveness" includes refraactivity of cancer cells to treatment or unresponsiveness of therapeutic cells (e.g., immune cells) to stimulation (e.g., stimulation via activating receptors or cytokines). Unresponsiveness can occur, for example, due to exposure to immunosuppressants or exposure to high doses of antigen. As used herein, the term "anergy" or "tolerance" includes unresponsiveness to activating receptor-mediated stimulation. Such unresponsiveness is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by a lack of cytokine (e.g., IL-2) production. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, when the cells are re-exposed to the same antigen (even if the re-exposure occurs in the presence of a costimulatory polypeptide), they are unable to produce cytokines and therefore cannot proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or by a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct can be used. For example, anergic T cells cannot initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by a multimer of AP1 sequences that can be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0133] The term "vaccine" refers to a pharmaceutical composition that induces an immune response against an antigen of interest. A vaccine can also confer protective immunity in a subject.

[0134] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR) that is involved in binding the protein to an antigen (e.g., a TCR α or β chain (or the γ and δ chains of a γδ TCR). The variable domains of the α and β chains of a native TCR (V α and V β ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. α The domains are encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ); β The domains are encoded by three separate DNA segments, the variable gene segment, the diversity gene segment and the joining gene segment (VDJ). α or V β The domain may be sufficient to confer antigen-binding specificity. Furthermore, a TCR that binds a particular antigen may be identified by a V domain derived from the TCR that binds the antigen. α or V β The complementary V domains were isolated using α or V β A library of domains may be screened.

[0135] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid that is linked to it. In some embodiments, a vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, a vector is a vector capable of autonomously replicating and / or expressing a nucleic acid that is linked to it. A vector capable of directing the expression of a gene that is operably linked to it is referred to herein as an "expression vector." Generally, expression vectors useful in recombinant DNA techniques are often in the form of a "plasmid," which generally refers to a circular double-stranded DNA loop that, in its vector form, is not bound to a chromosome. In this specification, "plasmid" and "vector" are used interchangeably, since the plasmid is the most commonly used form of vector. However, as will be understood by those skilled in the art, the present invention is intended to include other forms of expression vectors that subsequently become known in the art, which serve equivalent functions.

[0136] There is a known and definite correspondence, as defined by the genetic code (below), between the amino acid sequence of a particular protein and the nucleotide sequence capable of encoding that protein. Similarly, there is a known and definite correspondence, as defined by the genetic code, between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid. [Table 4]

[0137] An important and well-known feature of the genetic code is its redundancy, whereby more than one coding nucleotide triplet can be used for most of the amino acids used to make proteins (as exemplified above). Thus, several different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered to be functionally equivalent, since they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than others). Furthermore, occasionally methylation variants of purines or pyrimidines can be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and its corresponding amino acid.

[0138] In view of the above, the nucleotide sequence of the DNA or RNA encoding the biomarker nucleic acid (or any portion thereof) can be used to obtain a polypeptide amino acid sequence using the genetic code to translate the DNA or RNA into an amino acid sequence. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence that can encode the polypeptide can be inferred from the genetic code (which, due to its redundancy, will provide multiple nucleic acid sequences for any given amino acid sequence). Thus, any description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, any description and / or disclosure herein of a polypeptide amino acid sequence should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0139] II. Peptides In certain aspects, provided herein are methods and compositions for the treatment and / or prevention of disorders associated with MAGEC2 expression by inducing an immune response against MAGEC2 or cells expressing MAGEC2, involving administration of a MAGEC2 immunogenic peptide described herein, a nucleic acid encoding same, and / or a cell expressing same.

[0140] In certain embodiments, the MAGEC2 immunogenic peptide comprises (e.g., consists of) a peptide epitope selected from the peptide sequences listed in Table 1, e.g., Table 1A and Table 1B. The peptide epitopes described herein may be combined with MHC molecules, such as particular HLA molecules with particular HLA alpha chain alleles. For example, the peptides of Table 1A may be combined with MHC molecules, such as HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H ... * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and / or HLA-B * HLA-B alpha chain, as encoded by the 0721 allele * The peptides in Table 1B were identified in association with MHC having the HLA-A serotype 07, as further described in the Examples section. * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * HLA-A alpha chain, as encoded by the 2458 allele *In some embodiments, the MAGEC2 immunogenic peptides may be combined with MHC molecules, the MHC molecules being HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-H ... * 02. HLA-A * 03. HLA-A * 01. HLA-A * 11. HLA-A * 24, and / or HLA-B * 07, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A * 0205, HLA-A * 0206, HLA-A * 0207, HLA-A * 0210, HLA-A * 0211, HLA-A * 0212, HLA-A * 0213, HLA-A * 0214, HLA-A * 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A * 0242, HLA-A * 0253, HLA-A * 0260, HLA-A * 0274 allele, HLA-A * 0301, HLA-A * 0302, HLA-A * 0305, HLA-A * 0307, ​​HLA-A * 0101, HLA-A * 0102, HLA-A * 0103, HLA-A * 0116 allele, HLA-A *1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104, HLA-A * 1105, HLA-A * 1119 allele, HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and HLA-B * 0721 allele. In some embodiments, the MAGEC2 immunogenic peptide is derived from a human MAGEC2 protein and / or a MAGEC2 protein shown in Table 3. In some embodiments, one or more MAGEC2 immunogenic peptides are administered alone or in combination with an adjuvant.

[0141] In certain aspects, provided herein are compositions comprising one or more MAGEC2 immunogenic peptides described herein and an adjuvant.

[0142] [Table 1]

[0143] In some embodiments, provided herein are MAGEC2 polypeptides and / or nucleic acids encoding MAGEC2 polypeptides. In some embodiments, the MAGEC2 polypeptide is a polypeptide comprising an amino acid sequence of sufficient length to elicit a MAGEC2-specific immune response. In certain embodiments, the MAGEC2 polypeptide also comprises amino acids that do not correspond to the amino acid sequence (e.g., a fusion protein comprising a MAGEC2 amino acid sequence and an amino acid sequence that corresponds to a non-MAGEC2 protein or polypeptide). In some embodiments, the MAGEC2 polypeptide comprises only an amino acid sequence that corresponds to a MAGEC2 protein or fragment thereof.

[0144] In some embodiments, the MAGEC2 polypeptide comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 121, 122, 123, 1 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, In some embodiments, the MAGEC2 polypeptide comprises, consists essentially of, or consists of 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 373 or more contiguous amino acids, or any range therebetween, inclusive of the endpoints (e.g., 7-25, 8-22, 9-22, etc.). In some embodiments, the contiguous amino acids are identical to the amino acid sequence of MAGEC2 shown in Table 3. In some embodiments, the MAGEC2 polypeptide comprises, consists essentially of, or consists of one or more peptide epitopes selected from the group consisting of the MAGEC2 peptide epitopes listed in Table 1, e.g., Tables 1A and 1B.

[0145] As is well known to those skilled in the art, polypeptides that have significant sequence similarity can generate the same or a very similar immune response in a host animal. Thus, in some embodiments, derivatives, equivalents, variants, fragments or mutants of the MAGEC2 immunogenic peptides or fragments thereof described herein may also be suitable for the methods and compositions provided herein.

[0146] In some embodiments, variants or derivatives of MAGEC2 immunogenic polypeptides are provided herein. Modified polypeptides may have an amino acid sequence that is modified, for example, by conservative substitution, but still elicit an immune response that reacts with unmodified protein antigens and are considered functional equivalents. As used herein, the term "conservative substitution" refers to the replacement of an amino acid residue with another biologically similar residue. It is well known in the art that amino acids within the same conservative group can typically be used in place of each other without substantially affecting the function of the protein. According to certain embodiments, derivatives, equivalents, variants or mutants of the ligand-binding domain of MAGEC2 immunogenic peptides are polypeptides that are at least 85% homologous to the sequence of the MAGEC2 immunogenic peptides or fragments thereof described herein. In some embodiments, the homology is at least 90%, at least 95%, at least 98%, or more.

[0147] Immunogenic peptides encompassed by the present invention may include peptide epitopes derived from MAGEC2 protein, such as those listed in Table 1, e.g., Table 1A and Table 1B. In some embodiments, the immunogenic peptides are 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide amino acid sequence is modified, which may include conservative or non-conservative mutations. The peptides may include up to 1, 2, 3, 4, or more mutations. In some embodiments, the peptides may include at least 1, 2, 3, 4, or more mutations.

[0148] In some embodiments, peptides may be chemically modified. For example, peptides may be mutated to alter peptide properties, such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is one example of methylation that may occur on peptides of the present disclosure. In some embodiments, peptides may be modified by methylation on free amines, for example, reductive methylation with formaldehyde and sodium cyanoborohydride.

[0149] Chemical modifications may include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains, such as palmitate or myristole, or albumin. Chemical modifications of peptides with Fc regions may be fusion Fc-peptides. Polyamino acids may include, for example, polyamino acid sequences with repeating single amino acids (e.g., polyglycines) and polyamino acid sequences with mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the above. In some embodiments, peptides encompassed by the present disclosure may be modified to increase the stability and / or half-life of the peptides by modification. In some embodiments, the half-life of peptides encompassed by the present disclosure may be extended using attachment of hydrophobic moieties, for example, to the N-terminus, C-terminus, or internal amino acids. In other embodiments, peptides may include post-translational modifications (e.g., methylation and / or amidation), which may affect, for example, serum half-life. In some embodiments, a simple carbon chain can be conjugated to the fusion protein or peptide (e.g., by myristoylation and / or palmitylation). In some embodiments, the simple carbon chain can make the fusion protein or peptide easily separable from unconjugated material. For example, methods that can be used to separate the fusion protein or peptide from unconjugated material include, but are not limited to, solvent extraction and reverse phase chromatography. The lipophilic moiety can extend half-life by reversible binding to serum albumin. The conjugated moiety can be a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol, or a cholesterol derivative, such as cholestene, cholestane, cholestadiene, and oxysterols. In some embodiments, the peptide may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptide may be coupled (e.g., conjugated) to a half-life modifier.Examples of half-life modifying agents include, but are not limited to, molecules that bind to polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine and serine, water-soluble polymers containing glycine, glutamic acid and serine, Fc regions, fatty acids, palmitic acid, or albumin. In some embodiments, a spacer or linker may be coupled to the peptide to facilitate conjugation or fusion to another molecule and to facilitate cleavage of the peptide from such a conjugated or fused molecule, e.g., one, two, three, four, or more amino acid residues that act as a spacer or linker. In some embodiments, the fusion protein or peptide may be conjugated to other moieties that can, for example, modify or effect changes to the properties of the peptide.

[0150] The peptide may, in some embodiments, be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or label. The affinity tag may be selected from the group consisting of glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, Halo tag, HA tag, Flag® tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of an inflammatory agent, an anti-inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody, such as a single chain Fv.

[0151] The peptides may be conjugated to agents used in imaging, research, therapeutics, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiation therapy. In some embodiments, the peptides may be conjugated or fused to detectable agents, such as fluorophores, near-infrared dyes, contrast agents, nanoparticles, metal-containing nanoparticles, metal chelates, X-ray contrast agents, PET agents, metals, radioisotopes, dyes, radionuclide chelators, or other suitable materials that may be used for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties may be linked to the peptide. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225, or lead-212. In some embodiments, the near-infrared dye is not easily quenched by biological tissues and fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation at wavelengths between 650 nm and 4000 nm, and such emission is used to detect such agents. Non-limiting examples of fluorescent dyes that can be used as conjugating molecules include DyLight®-680, DyLight®-750, VivoTag®-750, DyLight®-800, IRDye®-800, VivoTag®-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, the near-infrared dye is often a cyanine dye (e.g., Cy7, Cy5.5,Further non-limiting examples of fluorescent dyes for use as conjugating molecules in the present disclosure include acridine orange or yellow, Alexa Fluors® (e.g., Alexa Fluor® 790, 750, 700, 680, 660 and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dyes and any derivatives thereof, auramine-rhodamine stains and any derivatives thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, DyLight Fluors and any derivatives thereof, epicocconone, ethidium bromide, FlAsH-EDT2, Fluo dyes and any derivatives thereof, FluoProbe and any derivatives thereof, fluorescein and any derivatives thereof, Fura and any derivatives thereof, GelGreen and any derivatives thereof, GelRed and any derivatives thereof, fluorescent proteins and any derivatives thereof, m-isoform proteins and any derivatives thereof, such as mCherry, hetamethine dyes dye) and any derivatives thereof, Hoechst stain, iminocoumarin, Indian yellow, indo-1 and any derivatives thereof, laurdan, lucifer yellow and any derivatives thereof, luciferin and any derivatives thereof, luciferase and any derivatives thereof, mercocyanin and any derivatives thereof, Nile dye and any derivatives thereof, perylene, phloxine, phycodyes and any derivatives thereof, propium iodide, pyranine, rhodamine and any derivatives thereof, ribogreen, RoGFP, rubrene, stilbene and any derivatives thereof, sulforhodamine and any derivatives thereof,SYBR™ and any derivatives thereof, synaptofluorin, tetraphenylbutadiene, tetrasodium tris, Texas Red, titanium yellow, TSQ, umbelliferone, violanthrone, yellow fluorescent protein, and YOYO-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 5G, rhodamine 6H, rhodamine 7H, rhodamine 8H, rhodamine 9H, rhodamine 10H, rhodamine 11H, rhodamine 12H, rhodamine 13H, rhodamine 14H, rhodamine 15H, rhodamine 16H, rhodamine 17H, rhodamine 18H, rhodamine 19H, rhodamine 20H, rhodamine 21H, rhodamine 22H, rhodamine 23H, rhodamine 24H, rhodamine 25H, rhodamine 26H, rhodamine 27H, rhodamine 28H, rhodamine 29H, rhodamine 30H, rhodamine 31H, rhodamine 32H, rhodamine coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA) and the like), Oregon Green® dyes (e.g., Oregon Green® 488, Oregon Green® 500, Oregon Green® 514 and the like), Texas Red, Texas Red-X, Spectrum Red, Spectrum Green, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5 and the like), ALEXA FLUOR® dyes (e.g., ALEXA FLUOR® 350, ALEXA FLUOR® 488, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 633, ALEXA FLUOR® 660, ALEXA FLUOR® 680, etc.), BODIPY® dyes (e.g., BODIPY® FL, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, BODIPY® 530 / 550,Suitable detectable agents include BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, etc., IRD dyes (e.g., IRD40, IRD700, IRD800, etc.). Additional suitable detectable agents are described in PCT / US14 / 56177. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225, or lead-212.

[0152] The peptide may be conjugated to a radiosensitizer or photosensitizer. Radiosensitizers include, but are not limited to, ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleobase derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to, fluorescent molecules or beads that generate heat when irradiated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, and the like. dyes), chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, Nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdines, xanthene dyes (e.g., eosins, erythrosins, rose bengals), dimeric and oligomeric porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this approach allows for highly specific targeting of cells of interest (e.g., immune cells) using both therapeutic agents (e.g., drugs) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the peptide is fused to or covalently or non-covalently linked to an agent, e.g., directly or via a linker.

[0153] In some embodiments, the binding protein may be chemically modified. For example, the binding protein may be mutated to alter peptide properties, such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is one example of methylation that may occur on the binding protein encompassed by the present invention. In some embodiments, the binding protein may be modified by methylation of free amines, for example, reductive methylation with formaldehyde and sodium cyanoborohydride.

[0154] Chemical modifications may include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains such as palmitate or myristole, or albumin. Chemical modifications of binding proteins with Fc regions may be fusion Fc-peptides. Polyamino acids may include, for example, polyamino acid sequences with repeating single amino acids (e.g., polyglycine) and polyamino acid sequences with mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the above.

[0155] In some embodiments, binding proteins encompassed by the invention may be modified. In some embodiments, the modifications generate functional variants that have substantial or significant sequence identity with the parent binding protein and that maintain one or more biophysical and / or biological activities of the parent binding protein (e.g., maintain pMHC binding specificity). In some embodiments, the mutations are conservative amino acid substitutions.

[0156] In some embodiments, the binding proteins encompassed by the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. β-hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentanecarboxylic acid, oc-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and oc-tert-butylglycine.

[0157] The binding proteins encompassed by the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted to an acid addition salt, and / or optionally dimerized or polymerized, or conjugated.

[0158] In some embodiments, the attachment of hydrophobic moieties, for example to the N-terminus, C-terminus, or internal amino acids, may be used to extend the half-life of peptides encompassed by the present invention. In other embodiments, the binding protein may include post-translational modifications (e.g., methylation and / or amidation), which may affect, for example, serum half-life. In some embodiments, a simple carbon chain (e.g., by myristoylation and / or palmitylation) may be conjugated to the binding protein. In some embodiments, a simple carbon chain may make the binding protein easily separable from unconjugated material. For example, methods that may be used to separate the binding protein from unconjugated material include, but are not limited to, solvent extraction and reverse phase chromatography. Lipophilic moieties may extend half-life by reversible binding to serum albumin. The conjugated moiety may be a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety may be cholesterol, or a cholesterol derivative, such as cholestene, cholestane, cholestadiene, and oxysterols. In some embodiments, the binding protein may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding protein may be coupled (e.g., conjugated) to a half-life modifier. Examples of half-life modifiers include, but are not limited to, polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water soluble polymers, zwitterionic water soluble polymers, water soluble poly(amino acids), water soluble polymers of proline, alanine, and serine, water soluble polymers containing glycine, glutamic acid, and serine, Fc regions, fatty acids, palmitic acid, or molecules that bind to albumin.In some embodiments, a spacer or linker, e.g., one, two, three, four, or more amino acid residues that act as a spacer or linker, may be coupled to the binding protein to facilitate conjugation or fusion to another molecule and to facilitate cleavage of the peptide from such a conjugated or fused molecule. In some embodiments, the binding protein may be conjugated to other moieties that, for example, can modify or effect changes in the properties of the binding protein.

[0159] Proteins, such as peptides, can be produced recombinantly or synthetically, for example, by solid-phase or solution-phase peptide synthesis. Protein synthesis can be performed by known synthetic methods, for example, using fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. Protein fragments can be joined together enzymatically or synthetically.

[0160] In an aspect encompassed by the invention, provided herein is a method of producing a protein described herein, comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of said binding protein; and (ii) recovering the expressed binding protein.

[0161] Methods useful for isolating and purifying recombinantly produced binding proteins may include, for example, obtaining a supernatant from a suitable host cell / vector system that secretes the binding protein into the culture medium, and then concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a suitable purification matrix or a series of suitable matrices, such as affinity matrices or ion exchange resins. One or more reverse-phase HPLC steps may be utilized to further purify the recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large-scale production of one or more of the binding proteins described herein include batch cell cultures, which are monitored and controlled to maintain appropriate culture conditions. Purification of the binding proteins may be performed according to methods described herein and known in the art.

[0162] In some embodiments, provided herein is a nucleic acid encoding a MAGEC2 immunogenic peptide or fragment thereof as described herein, e.g., a DNA molecule encoding a MAGEC2 immunogenic peptide. In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a MAGEC2 immunogenic peptide or fragment thereof as described herein. In some embodiments, the nucleic acid comprises regulatory elements required for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers may be included. These elements may be operably linked to a sequence encoding a MAGEC2 immunogenic polypeptide or fragment thereof. Representative vectors, promoters, regulatory elements, etc. useful for expressing proteins, e.g., peptides, are further described below.

[0163] III. MHC-Peptide Complexes In certain aspects, provided herein is a composition comprising a MAGEC2 immunogenic peptide described herein and an MHC molecule, in some embodiments, the MAGEC2 immunogenic peptide forms a stable complex with the MHC molecule.

[0164] The MHC protein may be conjugated to an agent, such as a detection moiety, a radiosensitizer, a photosensitizer, etc., and / or may be chemically modified as described above for the peptides.

[0165] The MHC proteins provided and used in the compositions and methods encompassed by this disclosure can be any suitable MHC molecule known in the art. Generally, they have the formula (α-β-P): n where n is at least 2, such as 2 to 10, e.g., 4. α is the α chain of a class I or class II MHC protein. β is herein defined as the β chain of a class II MHC protein or the β chain of a class I MHC protein. 2 The beta chain is defined as microglobulin. P is a peptide antigen.

[0166] In some embodiments, the MHC protein is an MHC class I complex, such as an HLA I complex.

[0167] The MHC protein can be from any mammalian or avian species, such as primate species, particularly humans; rodents, including mice, rats and hamsters; rabbits; horses; cows; dogs; cats, etc. For example, the MHC protein can be derived from human HLA proteins or mouse H-2 proteins. HLA proteins include class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, as well as class I proteins HLA-A, HLA-B, HLA-C, and β2-microglobulin. H-2 proteins include class I subunits H-2K, H-2D, H-2L, as well as class II subunits I-Aα, I-Aβ, I-Eα, and I-Eβ, as well as β2-microglobulin. Sequences of several representative MHC proteins can be found in Kabat et al. Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pp724-815. MHC protein subunits suitable for use in the present invention are soluble forms of normally membrane-bound proteins, which may be prepared as known in the art, e.g., by deletion of transmembrane and cytoplasmic domains.

[0168] In the case of class I proteins, the soluble forms may contain the α1, α2, and α3 domains. Soluble class II subunits may contain the α1 and α2 domains for the α subunits, and the β1 and β2 domains for the β subunits.

[0169] The α and β subunits may be produced separately and associated in vitro to form a stable heteroduplex complex, or both subunits may be expressed in a single cell. Methods for producing MHC subunits are known in the art.

[0170] In certain embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1 (e.g., Table 1A and Table 1B) and MHC. In some embodiments, the MHC molecule is HLA-A * 02. HLA-A * 03. HLA-A * 01. HLA-A * 11. HLA-A * 24, and / or HLA-B * 07, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A * 0205, HLA-A * 0206, HLA-A * 0207, HLA-A * 0210, HLA-A * 0211, HLA-A * 0212, HLA-A * 0213, HLA-A * 0214, HLA-A * 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A * 0242, HLA-A * 0253, HLA-A * 0260, HLA-A * 0274 allele, HLA-A * 0301, HLA-A * 0302, HLA-A * 0305, HLA-A * 0307, ​​HLA-A * 0101, HLA-A * 0102, HLA-A * 0103, HLA-A * 0116 allele, HLA-A *1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104, HLA-A * 1105, HLA-A * 1119 allele, HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and HLA-B * In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1A and an allele in which the alpha chain is selected from the group consisting of HLA-B, 0721, 0722, 0723, 0724, 0725, 0726, 0727, 0728, 0729, 0730, 0731, 0732, 0733, 07 * 07 Serotype-bearing MHC, e.g., HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and / or HLA-B * In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1B and an MHC encoded by the alpha chain of HLA-A. * MHC with 24 serotypes, e.g., HLA-A * 2402, HLA-A* 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426 and / or HLA-A * and the MHC encoded by the 2458 allele.

[0171] To prepare MHC-peptide complexes, the subunits may be combined with antigenic peptides and folded in vitro to form stable heterodimeric complexes with intrachain disulfide-bonding domains. The peptide may be included in the initial folding reaction or may be added to the empty heterodimer in a later step. In the compositions and methods encompassed by the present invention, this is a MAGEC2 immunogenic peptide or a fragment thereof. Conditions that allow folding and association of the subunits and peptides are known in the art. As an example, approximately equimolar amounts of solubilized α and β subunits may be mixed in a solution of urea. Refolding is initiated by dilution or dialysis against a buffer solution that does not contain urea. The peptide may be introduced into the empty class II heterodimer at a pH of about 5-5.5 for about 1-3 days, followed by neutralization, concentration and buffer exchange. However, no particular folding conditions are essential for the practice of the present invention.

[0172] The monomeric complex (α-β-P) (herein, monomer) may be multimerized, for example, for MHC tetramers. The resulting multimers are stable for long periods of time. Preferably, the multimers may be formed by binding the monomers to a multivalent entity via specific binding sites on the α or β subunits, as known in the art (e.g., as described in U.S. Pat. No. 5,635,363). The MHC protein in monomeric or multimeric form may also be conjugated to beads or any other support.

[0173] The multimeric complexes may be labeled so that they can be detected directly when used for immunostaining or other methods known in the art, or may be used in conjunction with a secondary labeled immunoreagent that specifically and / or selectively binds to the complex (e.g., binds to MHC protein subunits) as known to those skilled in the art. For example, the detectable label may be a fluorophore, such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin (PE), allophycocyanin (APC), Brilliant Violet™ 421, Brilliant UV™ 395, Brilliant Violet™ 480, Brilliant Violet™ 421 (BV421), Brilliant Blue™ 515, APC-R700, or APC-Fire750. In some embodiments, the multimeric complexes are labeled with a moiety that can specifically and / or selectively bind to another moiety. For example, the label may be biotin, streptavidin, an oligonucleotide, or a ligand. Other labels of interest may include fluorophores, dyes, enzymes, chemiluminescent agents, particles, radioisotopes, or other directly or indirectly detectable agents.

[0174] In some embodiments, cells presenting immunogenic peptides in the context of MHC molecules on the cell surface are created by transfecting or transducing the cells with a vector (e.g., a viral vector) that contains a nucleic acid encoding a recombinant or heterologous antigen within the cell. In some embodiments, the vector is introduced into the cell under conditions such that one or more peptide antigens, e.g., one or more peptide antigens of an expressed heterologous protein, in some cases, are expressed, processed, and presented on the surface of the cell in the context of a major histocompatibility complex (MHC) molecule.

[0175] Generally, the cell contacted with the vector is a cell that expresses MHC, i.e., an MHC-expressing cell. The cell may normally express MHC on the cell surface, may be induced to express and / or upregulate its expression on the cell surface, or may be engineered to express MHC molecules on the cell surface. In some embodiments, the MHC contains a polymorphic peptide binding site, or binding groove, which can optionally complex with a peptide antigen of the polypeptide, e.g., a peptide antigen processed by the cellular machinery. In some cases, the MHC molecule may be presented or expressed on the cell surface, e.g., as a complex with a peptide, i.e., as an MHC-peptide complex, for presentation of the antigen in a conformation that can be recognized by the TCR on a T cell or by other peptide-binding molecules.

[0176] In some embodiments, the cell is a nucleated cell. In some embodiments, the cell is an antigen-presenting cell. In some embodiments, the cell is a macrophage, a dendritic cell, a B cell, an endothelial cell, or a fibroblast. In some embodiments, the cell is an endothelial cell, for example, an endothelial cell line or a primary endothelial cell. In some embodiments, the cell is a fibroblast, for example, a fibroblast cell line or a primary fibroblast.

[0177] In some embodiments, the cell is an artificial antigen presenting cell (aAPC). Typically, an aAPC comprises characteristics of a natural APC, such as expression of MHC molecules, stimulatory and costimulatory molecule(s), Fc receptors, adhesion molecule(s), and / or the ability to produce or secrete cytokines (e.g., IL-2). Typically, aAPCs are cell lines that lack expression of one or more of the above, including but not limited to, MHC molecules, low affinity Fc receptors (CD32), high affinity Fc receptors (CD64), costimulatory signals (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, 3 / TR6, or ligand for B7-H3; or CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor, or ligand for CD83. The aAPCs are generated by introduction (e.g., by transfection or transduction) of one or more of the missing elements of one or more of the following: an antibody that specifically binds to MHC; a cell adhesion molecule (e.g., ICAM-1, or LFA-3); and / or a cytokine (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon alpha (IFN.alpha.), interferon beta (IFN.beta.), interferon gamma (IFN.gamma.), tumor necrosis factor alpha (TNF.alpha.), tumor necrosis factor beta (TNF.beta.), granulocyte macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (GCSF)). In some cases, aAPCs do not normally express MHC molecules but may be engineered to express MHC molecules, or in some cases, may be induced or induced to express MHC molecules, such as by stimulation with cytokines. Optionally, the aAPCs are loaded with a stimulatory ligand, such as, for example, an anti-CD3 antibody, an anti-CD28 antibody, or an anti-CD2 antibody.An exemplary cell line that may be used as a scaffold for generating aAPC is the K562 cell line, or a fibroblast cell line.Various aAPCs are known in the art, see, for example, U.S. Patent No. 8,722,400, published application US2014 / 0212446, Butler and Hirano (2014) Immunol Rev.257:10.1111 / imr.12129, Suhoshki et al. (2007) Mol.Ther.15:981-988).

[0178] It is well within the skill of the art to determine or identify the specific MHC or allele expressed by a cell. In some embodiments, before contacting the cell with the vector, the expression of a specific MHC molecule may be evaluated or confirmed, for example, by using an antibody specific for a specific MHC molecule. Antibodies for MHC molecules are known in the art, such as any of the antibodies described below.

[0179] In some embodiments, the cells can be selected to express the desired MHC-restricted MHC alleles. In some embodiments, MHC typing of cells, e.g., cell lines, is well known in the art. In some embodiments, MHC typing of cells, e.g., primary cells obtained from a subject, can be determined using procedures well known in the art, e.g., by performing tissue typing using molecular haplotype assays (BioTest ABC SSPray, BioTest Diagnostics Corp., Denville, NJ; SeCore Kits, Life Technologies, Grand Island, NY). In some cases, it is well within the skill of the art to perform standard typing of cells to determine HLA genotype, e.g., by using sequence-based typing (SBT) (Adams et al. (2004) J. Transl. Med., 2:30; Smith (2012) Methods Mol Biol., 882:67-86). In some cases, HLA typing of cells, e.g., fibroblasts, is known. For example, the human fetal lung fibroblast cell line MRC-5 expresses HLA-A * 0201, A29, B13, B44 Cw7(C * 0702), human foreskin fibroblast cell line Hs68 is HLA-A1, A29, B8, B44, Cw7, Cw16, and WI-38 cell line is A * 6801, B * 0801 (Solache et al. (1999) J Immunol, 163:5512-5518; Ameres et al. (2013) PloS Pathog. 9:e1003383). The human transfectant fibroblast cell line M1DR1 / Ii / DM expresses HLA-DR and HLA-DM (Karakikes et al. (2012) FASEB J., 26:4886-96).

[0180] In some embodiments, the cell that the vector is contacted or introduced into is a cell that is engineered or transfected to express MHC molecules. In some embodiments, the cell line can be prepared by genetically modifying a parent cell line. In some embodiments, the cell is engineered to express a specific MHC molecule that is normally deficient in such a specific MHC molecule. In some embodiments, the cell is genetically engineered using recombinant DNA technology.

[0181] In some embodiments, the stable MHC-peptide complexes described herein are used to detect T cells that bind to the stable MHC-peptide complexes. In some embodiments, the stable MHC-peptide complexes described herein are used to track and evaluate T cell responses in a subject, for example, by detecting the amount and / or percentage of T cells (e.g., CD8+ T cells) that specifically and / or selectively bind to a fluorescently labeled MHC-peptide complex. Methods for generating, labeling, and using MHC-peptide complexes (e.g., MHC-peptide tetramers) to detect T cells specific for MHC-peptide complexes are well known in the art. Further description can be found, for example, in U.S. Pat. No. 7,776,562, U.S. Pat. No. 8,268,964, and U.S. Patent Publication No. 2019 / 0085048.

[0182] IV. Immunogenic composition In some aspects, provided herein is a pharmaceutical composition (e.g., a vaccine composition) comprising a MAGEC2 immunogenic peptide and / or a nucleic acid encoding a MAGEC2 immunogenic peptide, and an adjuvant. In some aspects, provided herein is a pharmaceutical composition (e.g., a vaccine composition) comprising a stable MHC-peptide complex comprising a MAGEC2 immunogenic peptide in association with an MHC molecule, and an adjuvant. In some embodiments, the composition comprises a combination of multiple (e.g., two or more) MAGEC2 immunogenic peptides or nucleic acids, and an adjuvant. In some embodiments, the composition comprises a combination of multiple (e.g., two or more) stable MHC-peptide complexes comprising a MAGEC2 immunogenic peptide in association with an MHC molecule, and an adjuvant. In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier.

[0183] The pharmaceutical compositions disclosed herein may be specially formulated for administration in solid or liquid form, e.g., in a form suitable for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., intended for buccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous or epidural injection, e.g., a sterile solution or suspension, or a sustained release formulation.

[0184] Methods of preparing these formulations or compositions include the step of bringing into association the MAGEC2 immunogenic peptides and / or nucleic acids described herein with the adjuvants, carriers, and optionally one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the agents described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0185] Pharmaceutical compositions suitable for parenteral administration include the MAGEC2 immunogenic peptides and / or nucleic acids described herein in combination with an adjuvant, and one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0186] Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0187] Regardless of the route of administration selected, the agents provided herein, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions disclosed herein, are formulated into pharma- ceutically acceptable dosage forms by routine methods known to those of skill in the art.

[0188] In some embodiments, the described pharmaceutical compositions, when administered to a subject, are capable of eliciting an immune response against cells infected with MAGEC2. Such pharmaceutical compositions may be useful as vaccine compositions for the prophylactic and / or therapeutic treatment of disorders characterized by MAGEC2 expression.

[0189] In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the adjuvant employed provides for improved immunogenicity of the pharmaceutical composition. Such additional immune response stimulating compounds or adjuvants can (i) be mixed with the pharmaceutical composition of the present invention after reconstitution of the peptide and optional emulsifier with an oil-based adjuvant as defined above, (ii) be part of the reconstituted composition of the present invention as defined above, (iii) be physically linked to the peptide(s) to be reconstituted, or (iv) be administered separately to the subject, mammal or human to be treated. The adjuvant can be one that provides delayed release of the antigen (e.g., the adjuvant can be a liposome), or it can be an adjuvant that is immunogenic in itself and thereby functions synergistically with the antigen (i.e., the antigen present in the MAGEC2 immunogenic peptide). For example, the adjuvant can be a known adjuvant or other substance that promotes antigen uptake, recruits immune system cells to the site of administration, or promotes immunostimulation of responsive lymphoid cells. Adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, bacto-adjuvant, synthetic polymers such as polyamino acids and copolymers of amino acids, saponin, paraffin oil, and muramyl dipeptide. In some embodiments, the adjuvant is adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptides, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalose dimycolate, or zymosan.

[0190] In some embodiments, the adjuvant is an immunomodulatory molecule. For example, the immunomodulatory molecule can be a recombinant protein cytokine, chemokine, or immunostimulant, or a nucleic acid encoding a cytokine, chemokine, or immunostimulant, designed to enhance an immunological response.

[0191] Examples of immunomodulatory cytokines include interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, and IL-20), tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1.alpha, MIP-1β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as functional fragments of any of the above.

[0192] In some embodiments, immunomodulatory chemokines that bind to chemokine receptors, i.e., CXC, CC, C, or CX3C chemokine receptors, may also be included in the compositions provided herein. Examples of chemokines include, but are not limited to, Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tarc, Elc, I309, IL-8, Gcp-2 Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), as well as functional fragments of any of the above.

[0193] In some embodiments, the composition comprises a nucleic acid encoding a MAGEC2 immunogenic polypeptide as described herein, e.g., a DNA molecule encoding a MAGEC2 immunogenic peptide. In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a MAGEC2 immunogenic peptide.

[0194] When taken up by a cell (e.g., a host cell, an antigen presenting cell (APC), e.g., a dendritic cell, a macrophage, etc.), the DNA molecule may be present in the cell as an extrachromosomal molecule and / or may be integrated into a chromosome. DNA may be introduced into the cell in the form of a plasmid, which may remain separate genetic material. Alternatively, linear DNA may be introduced into the cell, which may be integrated into a chromosome. Optionally, when DNA is introduced into a cell, a reagent may be added that promotes integration of the DNA into a chromosome.

[0195] V. Binding Proteins In some embodiments, binding moieties that bind to the peptides described herein and / or stable MHC-peptide complexes described herein are provided, e.g., specific and / or selective to the peptides and / or stable MHC-peptide complexes, e.g., at about 10 -4 M or less (for example, about 10 -4 , 10 -5 , 10 -6 , 10 -7 , about 10 -8 , about 10 -9 , about 10 10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 etc.) d Binding proteins, such as T cell receptors (TCRs), antibodies and the like, that bind at .

[0196] In aspects encompassed by the invention, provided herein are binding proteins that bind (e.g., specifically and / or selectively) to peptide-MHC (pMHC) complexes that include a MAGEC2 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the binding protein binds to about 5×10 -4 M or less, approximately 1×10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, approximately 1×10 -12 K less than or equal to M, or any range therebetween, including the endpoints, e.g., about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, etc. d In some embodiments, the MHC molecule is capable of binding (e.g., specifically and / or selectively) to a MAGEC2 peptide-MHC (pMHC) complex. * 02. HLA-A * 03. HLA-A * 01. HLA-A * 11. HLA-A * 24, and / or HLA-B * 07, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A *0205、SONGS-A * 0206、SONGS-A * 0207、SONGS-A * 0210、SONGS-A * 0211、SONGS-A * 0212、SONGS-A * 0213、SONGS-A * 0214、SONGS-A * 0216、SONGS-A * 0217、SONGS-A * 0219、SONGS-A * 0220、SONGS-A * 0222、SONGS-A * 0224、SONGS-A * 0230、SONGS-A * 0242、SONGS-A * 0253、SONGS-A * 0260、SONGS-A * 0274アレル、SONGS-A * 0301、SONGS-A * 0302、SONGS-A * 0305、SONGS-A * 0307、SONGS-A * 0101、SONGS-A * 0102、SONGS-A * 0103、SONGS-A * 0116アレル、SONGS-A * 1101、SONGS-A * 1102、SONGS-A * 1103、SONGS-A * 1104、SONGS-A * 1105、SONGS-A * 1119アレル、SONGS-A * 2402、SONGS-A * 2403、SONGS-A * 2405、SONGS-A * 2407、SONGS-A * 2408、SONGS-A * 2410、SONGS-A * 2414、SONGS-A * 2417、SONGS-A * 2420、SONGS-A * 2422、SONGS-A *2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and HLA-B * In some embodiments, the HLA serotype is selected from the group consisting of HLA-B, 0721, 0722, 0723, 0724, 0725, 0726, 0727, 0728, 0729, 0730, 0731, 0732, 0733, 0734, 0735, 0736, 0737, 0 * 07 and / or the HLA allele is HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and HLA-B * 0721 allele. In certain embodiments, the HLA allele is selected from the group consisting of HLA-B * 0702. In some embodiments, the HLA serotype is HLA-A * 24, and / or the HLA allele is HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426 and HLA-A * 2458 allele. In certain embodiments, the HLA allele is selected from the group consisting of HLA-A * 2402. In some embodiments, the binding proteins provided herein are engineered, isolated and / or purified.

[0197] In some embodiments, the binding protein binds to MAGEC2 peptide-MHC (pMHC) with a higher binding affinity compared to a known T cell receptor (e.g., a TCR from van Kunert et al. (2016) J. Immunol. 197:2541-2552, or others described herein). For example, the binding protein binds to MAGEC2 peptide-MHC with at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 110 fold, 120 fold, 130 fold, 140 fold, 150 fold, 160 fold, 170 fold, 180 fold, 190 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 It may have a binding affinity for MAGEC2 peptide-MHC (pMHC) that is 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1000 fold, 5000 fold, 100000 fold, 500000 fold, 1000000 fold, or more, or any range therebetween inclusive of the endpoints, for example 1.2-2 fold higher.

[0198] In some embodiments, the binding protein induces greater T cell proliferation, cytokine release and / or cytotoxic killing when contacted with target cells that have a certain or lower level of MAGEC2 expression, relative to a known T cell receptor (e.g., see the Examples section for representative cell lines expressing MAGEC2 at various levels). For example, in some embodiments of any aspect described herein, MAGEC2 levels are expressed as transcripts per million. million), for example, about 1,000 transcripts per million transcripts (about 1,000 TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM, M, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM or less, or any range therebetween, inclusive of the endpoints, for example, from about 1,000 TPM or less to about 73 TPM or less. As further described herein, TPM is measured according to well-known techniques, e.g., RNA-Seq, and gene expression TPM data is well known in the art for various cell lines, tissue types, etc. (see, e.g., the Broad Institute Cancer Cell Line Encyclopedia (CCLE), available on the world wide web at portals.broadinstitute.org).In some embodiments, the binding protein enhances T cell proliferation, cytokine release and / or cytotoxic killing by at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, or more when contacted with a target cell having heterozygous expression of MAGEC2 compared to a known T cell receptor. , 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or more, or any range therebetween inclusive of the endpoints, for example, an increase of 1.2x to 2x.

[0199] In some embodiments, the expression of MAGEC2 is detected using RNA-sequencing (RNA-seq). RNA-seq generally includes the following steps: obtaining a sample containing genetic material, isolating total RNA from the obtained sample, preparing an amplified cDNA library from the total RNA, sequencing the amplified cDNA library, and analyzing and profiling the amplified cDNA to evaluate the expression levels of various transcripts. The sample may be a population of cells, a tissue sample, a biopsy sample, a cell culture, or a single cell. Total RNA may be isolated from a biological sample using any method known in the art. In certain embodiments, total RNA is extracted from plasma. Extraction of plasma RNA is described in Enders et al., "The Concentration of Circulating Corticotropin-Releasing Homer mRNA in Material Plasma Is Inclined in Preclampsia," Clinr. As described therein, the plasma collected after a centrifugation step is mixed with Trizol LS reagent (Invitrogen) and chloroform. The mixture is centrifuged and the aqueous layer is transferred to a new tube. Ethanol is added to the aqueous layer. The mixture is then loaded into an RNeasy mini column (Qiagen) and processed according to the manufacturer's recommendations.

[0200] In some embodiments, the RNA-seq described herein includes preparing amplified cDNA from total RNA. For example, cDNA is prepared and the isolated RNA sample is randomly amplified without dilution, or the mixture containing genetic material in the isolated RNA is dispersed into individual reaction samples. In certain embodiments, amplification is randomly initiated from the 3' end of the transcriptome in the sample to amplify both mRNA and non-polyadenylated transcripts. In this way, the double-stranded cDNA amplification products are optimized to generate a sequencing library for next-generation sequencing platforms. Kits suitable for amplifying cDNA by the methods encompassed by the present invention include, for example, the Ovation® RNA-Seq system.

[0201] In some embodiments, the RNA-seq described herein comprises sequencing the amplified cDNA.Any known sequencing method can be used to sequence the amplified cDNA mixture, for example, single molecule sequencing.In certain embodiments, the amplified cDNA is sequenced by whole transcriptome shotgun sequencing.Whole transcriptome shotgun sequencing can be carried out using various next-generation sequencing platforms, for example, Illumina Genome Analyzer platform, ABI SOLiD™ sequencing platform, or Life Science's 454 sequencing platform.

[0202] In some embodiments, RNA-seq as described herein further comprises digitally counting and analyzing the cDNA. The number of amplified sequences for each transcript in the amplified sample can be quantified by sequence reads (one read per amplified strand). In some embodiments, transcripts per million (TPM) are used to quantify the expression level of a particular transcript. TPM can be calculated as shown in Wagner et al. (2012) Theory in Biosciences 131:281-285, the contents of which are incorporated herein by reference in their entirety.

[0203] In certain embodiments, the binding proteins recognize MAGEC2 immunogenic peptides in complex with MHC molecules, such as specific HLA molecules with specific HLA alpha chain alleles. For example, the binding proteins listed in Table 2A recognize MAGEC2 immunogenic peptides in complex with MHC molecules, such as specific HLA molecules with specific HLA alpha chain alleles, as further described in the Examples section. * 07 Serotype-bearing MHC, e.g., HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and / or HLA-B * The binding proteins listed in Table 2B were identified as binders of MAGEC2 immunogenic peptides associated with MHC encoded by the 0721 allele. Similarly, the binding proteins listed in Table 2B were identified as binders of MAGEC2 immunogenic peptides associated with MHC encoded by the 0721 allele, as further described in the Examples section. * MHC with 24 serotypes, e.g., HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A* 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426 and / or HLA-A * The binding protein has been identified as a binder of MAGEC2 immunogenic peptides associated with MHC encoded by the 2458 allele. In some embodiments, the binding protein recognizes a complex of the MAGEC2 immunogenic peptide and an MHC molecule, the MHC molecule being associated with HLA-A * 02. HLA-A * 03. HLA-A * 01. HLA-A * 11. HLA-A * 24, and / or HLA-B * 07, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A * 0205, HLA-A * 0206, HLA-A * 0207, HLA-A * 0210, HLA-A * 0211, HLA-A * 0212, HLA-A * 0213, HLA-A * 0214, HLA-A * 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A * 0242, HLA-A * 0253, HLA-A * 0260, HLA-A * 0274 allele, HLA-A * 0301, HLA-A * 0302, HLA-A *0305, HLA-A * 0307, ​​HLA-A * 0101, HLA-A * 0102, HLA-A * 0103, HLA-A * 0116 allele, HLA-A * 1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104, HLA-A * 1105, HLA-A * 1119 allele, HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 and HLA-B * 0721 allele. In some embodiments, the MAGEC2 immunogenic peptide is derived from a human MAGEC2 protein and / or a MAGEC2 protein shown in Table 3. In some embodiments, one or more MAGEC2 immunogenic peptides are administered alone or in combination with an adjuvant.

[0204] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% affinity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha sequences listed in Table 2. , 99% or more identity to a TCR alpha chain sequence; and / or b) a TCR beta chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chains listed in Table 2.

[0205] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2.

[0206] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain variable (V α ) TCR V domain sequences selected from the group consisting of α A TCR alpha chain variable (V) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the V α ) domain sequence; and / or b) a TCR beta chain variable (V β ) TCR V domain sequences selected from the group consisting of βA TCR beta chain variable (V) having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the domain sequence. β ) Domain array.

[0207] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain variable (V α ) TCR V domain sequences selected from the group consisting of α and / or b) a TCR beta chain variable (V β ) TCR V domain sequences selected from the group consisting of β Domain array.

[0208] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of) at least one (e.g., one, two or three, e.g., CDR3 alone or in combination with CDR1 and CDR2) TCR alpha chain complementarity determining region (CDR) sequences having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2. CDR3 is believed to be the primary CDR responsible for recognizing processed antigen, while CDR1 and CDR2 primarily interact with MHC, and therefore in some embodiments binding proteins are provided that comprise only a CDR3 from the TCR alpha chain and / or only a CDR3 from the TCR beta chain listed in Table 2, wherein each CDR3 has a sequence homology as listed in this paragraph.

[0209] In some embodiments, the binding proteins provided herein may also comprise (e.g., may comprise, consist essentially of, or consist of) at least one (e.g., one, two or three, e.g., CDR3 alone or in combination with CDR1 and CDR2) TCR beta chain complementarity determining region (CDR) sequences having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2. As noted above, CDR3 is believed to be the primary CDR responsible for recognizing processed antigen, with CDR1 and CDR2 primarily interacting with MHC, and therefore in some embodiments there is provided a binding protein comprising only a CDR3 from a TCR beta chain and / or only a CDR3 from a TCR alpha chain as listed in Table 2, wherein each CDR3 has a sequence homology as listed in this paragraph.

[0210] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of at least one (e.g., one, two, or three) of the complementarity determining regions (CDRs) of the TCR alpha chain listed in Table 2.

[0211] In some embodiments, the binding proteins provided herein may also comprise (e.g., may comprise, consist essentially of, or consist of at least one (1, 2, or 3) of a complementarity determining region (CDR) of a TCR beta chain listed in Table 2).

[0212] In some embodiments, the binding proteins provided herein comprise a TCR alpha chain constant region (C α) TCR C sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence α Comprises (e.g., comprises, consists essentially of, or consists of) the sequence.

[0213] In some embodiments, the binding proteins provided herein also comprise a TCR beta chain constant region (C β ) TCR C sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence β It may comprise (eg, it may comprise, consist essentially of, or consist of) the sequence.

[0214] In some embodiments, the binding proteins provided herein comprise a TCR alpha chain constant region (TCRC) or a TCR alpha chain constant region (TCC) as listed in Table 2. α ) TCR C sequences selected from the group consisting of α Comprises (e.g., comprises, consists essentially of, or consists of) the sequence.

[0215] In some embodiments, the binding proteins provided herein also comprise a TCR beta chain constant region (C β ) TCR C sequences selected from the group consisting of β It may comprise (eg, it may comprise, consist essentially of, or consist of) the sequence.

[0216] [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 3-1

Table 3-2

Table 3-3

[0217] In some embodiments, the binding proteins provided herein include constant regions that are chimeric, humanized, human, primate, or rodent (e.g., rat or mouse). For example, a human variable region may be chimerized with a mouse constant region, or a mouse variable region may be humanized with a human constant region and / or human framework regions. In some embodiments, the constant region may be mutated to modify functionality (e.g., introducing non-naturally occurring cysteine ​​substitutions at opposing residue positions in the TCR alpha and beta chains to provide disulfide bonds useful for improving affinity between the TCR alpha and beta chains). Similarly, mutations may be made in the transmembrane domains of the constant region to modify functionality (e.g., improving hydrophobicity by introducing non-naturally occurring substitutions of residues with hydrophobic amino acids). In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof, compared to a reference CDR sequence. In some embodiments, mutations may be made in the constant region to increase cell surface expression.

[0218] In some embodiments, the binding proteins disclosed herein may be engineered protein scaffolds, antibodies or antigen-binding fragments thereof, TCR mimetic antibodies, and the like.Such binding moieties can be designed and / or generated against the peptides and / or MHC-peptide complexes described herein using conventional immunological methods, such as immunizing a host, obtaining antibody-producing cells and / or their antibodies, and generating hybridomas useful for producing monoclonal antibodies (see, e.g., Watt et al. (2006) Nat. Biotechnol. 24:177-183; Gebauer and Skerra (2009) Curr. Opin. Chem Biol. 13:245-255; Skerra et al. (2008) FEBS J. 275:2677-2683; Nygren et al. (2008) FEBS J. 275:2668-2676; Dana et al. (2012) Exp. Rev. Mol. Med. 14:e6; Sergeva et al. (2011) Blood 117:4262-4272, PCT Publication Nos. WO2007 / 143104, PCT / US86 / 02269 and WO86 / 01533, U.S. Patent No. 4,816,567, Better et al. (1988) Science 240:1041-1043, Liu et al. al.(1987) Proc.Natl.Acad.Sci.USA84:3439-3443, Liu et al.(1987) J.Immunol.139:3521-3526, Sun et al.(1987) Proc.Natl.Acad.Sci.84:214-218, Nishimura et al.(1987) Cancer Res.47:999-1005, Wood et al. al. (1985) Nature 314:446-449, Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559), Morrison, SL (1985) Science 229:1202-1207, Oi et al. (1986) Biotechniques 4:214, U.S. Patent No. 5,225,539, Jones et al. (1986) Nature 321:552-525, Verhoeyan et al. (1988) Science 239:1534, and Beidler et al. (1988) J. Immunol. 141:4053-4060.If desired, the binding moiety may be isolated or purified using conventional procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, and high performance liquid chromatography (HPLC) (e.g., Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY).

[0219] The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE), as well as recombinant antibodies, such as single chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, and fragments and derivatives of all of the above, which fragments and derivatives contain at least an antigen-binding site. Antibody derivatives may include proteins or chemical moieties conjugated to the antibody.

[0220] In addition, intrabodies are well-known antigen-binding molecules that have the characteristics of antibodies but can be expressed intracellularly to bind and / or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods for adapting antibodies to target (e.g., inhibit) intracellular moieties are well known in the art and include, for example, the use of single-chain antibodies (scFv), engineering immunoglobulin VL domains for hyperstability, engineering antibodies to tolerate reducing intracellular environments, creating fusion proteins to improve intracellular stability and / or modulate intracellular localization, etc. Intracellular antibodies can also be introduced into and expressed in one or more cells, tissues, or organs of a multicellular organism, e.g., for prophylactic and / or therapeutic purposes (e.g., as gene therapy) (see at least PCT Publication Nos. WO 08 / 020079, WO 94 / 02610, WO 95 / 22618, and WO 03 / 014960; U.S. Patent No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.); Kontermann (2004) Methods 34:163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS 11:162-163; Lett. 508:407-412, see Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).

[0221] The term "antibody" as used herein also includes an "antigen-binding portion" (or simply "antibody portion") of an antibody. The term "antigen-binding portion" as used herein refers to one or more fragments of an antibody that retain the ability to specifically and / or selectively bind to an antigen (e.g., a peptide and / or MHC-peptide complex as described herein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab') fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; 2(iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments, which consist of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining regions (CDRs). Moreover, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant techniques that allows them to be made as a single protein chain (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, and Osbourn et al. 1998, Nature Biotechnology 16: 778) in which the VL and VH regions pair to form a monovalent polypeptide. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a particular scFv can be ligated to human immunoglobulin constant region cDNA or genomic sequences to create an expression vector encoding a complete IgG polypeptide or other isotype. VH and VL can also be used to generate Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain to generate two antigen-binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0222] Additionally, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesin polypeptide formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesin polypeptides include the use of a streptavidin core region to generate tetrameric scFv polypeptides (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, protein subunit peptides and a C-terminal polyhistidine tag to generate bivalent biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab') 2 Fragments can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions, and immunoadherence polypeptides can be obtained using standard recombinant DNA techniques as described herein.

[0223] Antibodies can be polyclonal or monoclonal, xenogeneic, allogeneic or syngeneic, or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies can also be fully human. Preferably, the antibodies of the present invention bind specifically and / or selectively, or substantially specifically and / or selectively, to the peptides and / or MHC-peptide complexes described herein. The terms "monoclonal antibody" and "monoclonal antibody composition" as used herein refer to a population of antibody polypeptides that contain only one type of antigen-binding site capable of immunoreacting with a particular epitope of an antigen, whereas the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple types of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically exhibits a single binding affinity for the particular antigen with which it immunoreacts.

[0224] As with other binding moieties described herein, antibodies may be "humanized," which is intended to include antibodies made by non-human cells with variable and constant regions that have been modified to more closely resemble antibodies made by human cells. For example, by modifying a non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies of the invention may contain amino acid residues, for example in the CDRs, that are not encoded in human germline immunoglobulin sequences (e.g., mutations introduced by in vitro / ex vivo random or site-specific mutagenesis or by in vivo somatic mutation). The term "humanized antibody," as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species are joined to human framework sequences.

[0225] In some embodiments, the binding proteins disclosed herein may comprise a T cell receptor (TCR), an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In some embodiments, the binding proteins disclosed herein may comprise two polypeptide chains, each of which comprises a variable region comprising the CDR3 of the TCR alpha chain and the CDR3 of the TCR beta chain, or the CDR1, CDR2, and CDR3 of both the TCR alpha chain and the TCR beta chain. In some embodiments, the binding protein comprises a single chain TCR (scTCR), which comprises a TCR V α and TCR V β Contains both domains but only one TCR constant domain (C α or C β). The term "chimeric antigen receptor" (CAR) refers to a fusion protein engineered to contain two or more naturally occurring amino acid sequences linked together in a manner that does not occur naturally or in a host cell, and which can function as a receptor when present on the surface of a cell. CARs encompassed by the present invention can include an extracellular portion that includes an antigen-binding domain (i.e., an antigen-binding domain derived from or derived from an immunoglobulin or immunoglobulin-like molecule, e.g., an antibody or TCR, or an antigen-binding domain derived from or derived from a killer immune receptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing a costimulatory domain(s)) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388, Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220, and Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0226] In some embodiments, 1) the TCR alpha chain CDRs, TCR V α and / or 2) the TCR beta chain CDRs, TCR V domain and / or the TCR alpha chain are encoded by a TRAV, TRAJ and / or TRAC gene selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2, or a fragment thereof; and / or β The domain and / or TCR beta chain is encoded by a TRBV, TRBJ and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has up to 5 amino acid substitutions, insertions, deletions or combinations thereof compared to the cognate reference CDR sequence listed in Table 2.

[0227] In some embodiments, the binding proteins disclosed in the present application (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs) are chimeric (e.g., containing amino acid residues or motifs from more than one donor or species), humanized (e.g., containing residues from a non-human organism that have been modified or substituted to reduce the risk of immunogenicity in humans), or human.

[0228] Methods for producing engineered binding proteins, such as TCRs, CARs and antigen-binding fragments thereof, are well known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 5:3000, U.S. Patent No. 6,410,319, U.S. Patent No. 7,446,191, U.S. Patent Publication No. 2010 / 065818, U.S. Patent No. 8,822,647, PCT Publication No. WO2014 / 031687, U.S. Patent No. 7,514,537, and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0229] In some embodiments, the binding proteins described herein are TCRs or antigen-binding fragments thereof expressed on the cell surface, where the TCR expressed on the cell surface can associate with CD3 protein more efficiently than the endogenous TCR. The binding proteins encompassed by the present invention, e.g., TCRs, when expressed on the surface of a cell, such as a T cell, may have higher surface expression on the cell compared to the endogenous binding protein, e.g., the endogenous TCR. In some embodiments, provided herein are CARs, in which the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0230] Further provided are the VGAs disclosed herein as starting materials for engineering modified binding proteins that may have altered properties from the starting binding protein. α and / or Vβ The modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) can be prepared according to well-known methods using binding proteins having one or more of the following sequences: α and / or V β ), for example in one or more CDR regions and / or in one or more framework regions. Additionally or alternatively, a binding protein may be engineered by modifying residues in the constant region(s).

[0231] Another type of variable region modification is α and / or V β The present invention relates to a method for modifying an amino acid residue in the CDR1, CDR2 and / or CDR3 region of a nucleotide sequence ...

[0232] In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) described herein may have one or more amino acid substitutions, deletions, or additions compared to naturally occurring TCRs. In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof compared to the cognate reference CDR sequence listed in Table 2. Conservative substitutions of amino acids are well known and may occur naturally, or may be introduced when the binding protein is produced recombinantly. Amino acid substitutions, deletions, and additions may be introduced into the protein using mutagenesis methods known in the art (e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide-site-directed (or segment-directed) mutagenesis procedures may be employed to result in modified polynucleotides with specific codons modified according to the desired substitutions, deletions, or insertions. Alternatively, immunogenic polypeptide variants may be prepared using random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis (see, e.g., Sambrook et al., supra).

[0233] Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid substitution or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids can be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline is considered more difficult to classify, but shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, the substitution of glutamine for glutamic acid or asparagine for aspartic acid can be considered similar substitutions in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of a polypeptide and its conserved amino acid substitutes to the sequence of a second polypeptide (e.g., using the GENEWORKS™, Align, BLAST algorithms, or other algorithms described herein and practiced in the art).

[0234] In some embodiments, the encoded binding protein (e.g., TCR, antigen-binding fragment of a TCR, or CAR) may include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). The signal peptide directs a newly synthesized polypeptide to an appropriate location inside or outside the cell. The signal peptide may be removed from the polypeptide during localization or secretion, or once localization or secretion is complete. A polypeptide having a signal peptide is referred to herein as a "preprotein," and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In some embodiments, the binding proteins (e.g., TCR, antigen-binding fragment of a TCR, or CAR) described herein are mature V proteins. α Domain, mature V β In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) described herein comprise a mature TCR β chain, a mature TCR α chain, or both.

[0235] In some embodiments, the binding protein is a fusion protein comprising: (a) an extracellular component comprising a TCR or an antigen-binding fragment thereof; (b) an intracellular component comprising an effector domain or a functional portion thereof; and (c) a transmembrane domain linking the extracellular and intracellular components. In some embodiments, the fusion protein is capable of binding (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex that comprises a MAGEC2 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule).

[0236] As used herein, an "effector domain" or "immune effector domain" is an intracellular portion or domain of a fusion protein or receptor that can directly or indirectly promote an immune response in a cell upon receiving an appropriate signal. In some embodiments, the effector domain is derived from an immune cell protein or portion thereof or an immune cell protein complex that receives a signal when bound (e.g., CD3ζ) or when the immune cell protein or portion thereof or immune cell protein complex directly binds to a target molecule to trigger signaling from the effector domain in the immune cell.

[0237] An effector domain may directly promote a cellular response if it contains one or more signaling domains or motifs, such as intracellular tyrosine-dependent activation motifs (ITAMs), such as those found in costimulatory molecules. Without wishing to be bound by theory, it is believed that ITAMs serve for T cell activation following ligand engagement by the T cell receptor or by a fusion protein that includes a T cell effector domain. In some embodiments, the intracellular component or functional portion thereof includes an ITAM. Exemplary immune effector domains include, but are not limited to, immune effector domains derived from CD3ε, CD3δ, CD3ζ, CD25, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the effector domain comprises a lymphocyte receptor signaling domain (e.g., CD3ζ, or a functional portion or variant thereof).

[0238] In further embodiments, the intracellular component of the fusion protein comprises a costimulatory domain or functional portion thereof selected from a ligand that binds (e.g., specifically and / or selectively) to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-1 (CD54), LFA-1 (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, or a functional variant thereof, or any combination thereof. In some embodiments, the intracellular component comprises a CD28 costimulatory domain or a functional portion or variant thereof (which may optionally comprise a LL-GG mutation at positions 186-187 of the native CD28 protein (e.g., Nguyen et al. (2003) Blood 702:4320), a 4-1BB costimulatory domain or a functional portion or variant thereof, or both.

[0239] In some embodiments, the effector domain comprises a CD3ε endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD27 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD28 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a 4-1BB endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an OX40 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD2 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD5 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICAM-1 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an LFA-1 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICOS endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof.

[0240] The extracellular and intracellular components encompassed by the present invention are linked by a transmembrane domain. A "transmembrane domain," as used herein, is a portion of a transmembrane protein that can be inserted into or span a cell membrane. A transmembrane domain has a three-dimensional structure that is thermodynamically stable in the cell membrane and generally ranges in length from about 15 to about 30 amino acids. The structure of a transmembrane domain may include an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof. In some embodiments, the transmembrane domain comprises or is derived from a known transmembrane protein (e.g., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, or any combination thereof).

[0241] In some embodiments, the extracellular component of the fusion protein further comprises a linker disposed between the binding domain and the transmembrane domain. When referring to the component of the fusion protein linking the binding domain and the transmembrane domain, a "linker," as used herein, can be an amino acid sequence having from about 2 amino acids to about 500 amino acids that can provide flexibility and room for conformational movement between the two regions, domains, motifs, fragments, or modules linked by the linker. For example, linkers encompassed by the present invention can position the binding domain away from the surface of the host cell expressing the fusion protein to allow proper contact, antigen binding, and activation between the host cell and the target cell (Patel et al. (1999) Gene Therapy 6:412-419). Linker length may be varied to maximize antigen recognition based on the seize and affinity of a selected target molecule, a selected binding epitope, or an antigen-binding domain (see, e.g., Guest et al. (2005) Immunother. 28:203-11, and PCT Publication No. WO2014 / 031687). Exemplary linkers include Gly, x Ser yIn the formula, x and y are each independently an integer of 0 to 10, provided that both x and y are not 0 (for example, (Gly 4 Ser) 2 , (Gly 3 Ser) 2 , Gly 2 Ser, or combinations thereof, for example, ((Gly 3 Ser) 2 Gly 2 Ser)).

[0242] The binding proteins may be conjugated to an agent, such as a detection moiety, a radiosensitizer, a photosensitizer, etc., and / or may be chemically modified as described above for the peptides.

[0243] In any of the embodiments disclosed herein, the encoded binding protein is capable of binding to a peptide-MHC (pMHC) complex that comprises a MAGEC2 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule).

[0244] A variety of assays are known for assessing binding affinity and / or for determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand (e.g., a peptide antigen-MHC complex). It is within the level of ordinary skill in the art to determine the binding affinity of a binding protein to a target, e.g., a T cell peptide epitope of a target polypeptide, for example, by using any of a number of binding assays known in the art. For example, in some embodiments, a Biacore™ instrument may be used to determine the binding constant of a complex between two proteins. The dissociation constant (K D) can be determined by monitoring the change in refractive index with respect to time as a buffer solution passes over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs), or measuring binding by monitoring changes in the spectroscopic or optical properties of the proteins by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blots, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore™) analysis (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660, Wilson (2002) Science 295:2103, Wolff et al. (1993) Cancer Res. 53:2560, and U.S. Pat. Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, apparent affinity for a target is measured by assessing binding to various concentrations of tetramers, e.g., by flow cytometry using labeled multimers, such as MHC-antigen tetramers. In one representative example, the apparent K of a binding protein is determined by measuring the binding affinity of the target protein to various concentrations of tetramers, e.g., ... D is measured using a range of two-fold dilutions of the labeled tetramer, followed by determination of the binding curve by nonlinear regression, whereas the apparent K D is determined as the concentration of ligand that results in half-maximal binding.

[0245] VI. Nucleic Acids and Vectors In aspects encompassed by the present invention, provided herein are nucleic acid molecules encoding the proteins described herein, such as MAGEC2 immunogenic peptides and fragments thereof, MHC molecules, binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, CARs, etc.).

[0246] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to the complement of a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, e.g., over the entire length, to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3.

[0247] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to the complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3.

[0248] In some embodiments, the nucleic acid molecule comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3.

[0249] In some embodiments, the nucleic acid sequence encodes a MAGEC2 immunogenic peptide as described herein.

[0250] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α chain CDR as shown in Table 2. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α chain CDR as shown in Table 2. α TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. αIn some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR alpha chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the TCR alpha chain sequences shown in Table 2.

[0251] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β chain CDR as shown in Table 2. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β chain CDR as shown in Table 2. β TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. β In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR β chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β chain sequence shown in Table 2.

[0252] The term "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, which may be single- or double-stranded, synthetic or obtained from natural sources (e.g., isolated and / or purified), and which may contain natural, non-natural, or modified nucleotides, and which may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages in place of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides. In one embodiment, a nucleic acid comprises complementary DNA (cDNA).

[0253] In some embodiments, the nucleic acid encompassed by the present invention is recombinant.As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule that can be replicated in the living cell, or (ii) a molecule resulting from replication of the molecule described in (i) above.For purposes herein, replication can be in vitro, ex vivo or in vivo replication.

[0254] Nucleic acids can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. See, for example, Green and Sambrook et al., supra. For example, nucleic acids may be chemically synthesized using naturally occurring nucleotides or using various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to improve the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that may be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids encompassed by the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA).

[0255] In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound to a particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence improves the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence may include replacing a natural codon with another codon that codes for the same amino acid but can be translated by a more readily available tRNA in the cell, thus increasing translation efficiency. Optimization of a nucleotide sequence may also reduce secondary structures in the mRNA that would interfere with translation, thus increasing translation efficiency. In some embodiments, the nucleotide sequences described herein are codon-optimized for expression in a host cell (e.g., an immune cell such as a T cell).

[0256] The present invention also provides a nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0257] Nucleotide sequences that hybridize under stringent conditions may hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence specifically and / or selectively hybridizes to a target sequence (any nucleotide sequence of the nucleic acids described herein) in a detectably stronger amount than would be the case for non-specific hybridization. High stringency conditions include conditions that would distinguish polynucleotides with exact complementary sequences or polynucleotides containing only scattered minor mismatches from random sequences that happen to have small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small complementary regions melt more easily than full-length complements of 14-17 bases or more, making them easily distinguishable by high stringency hybridization. Relatively high stringency conditions would include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions are particularly suitable for detecting expression of any of the TCRs of the present invention, as they tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand, It is widely recognized that conditions can be made more stringent by the addition of increasing amounts of formamide.

[0258] The present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids described herein.

[0259] Typically, the nucleic acid is a DNA or RNA molecule that may be comprised in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0260] The terms "vector", "cloning vector" and "expression vector" refer to a delivery vehicle capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell, thereby transforming the host and promoting expression (e.g., transcription and translation) of the introduced sequences. Thus, a further object encompassed by the present invention relates to vectors comprising the nucleic acids encompassed by the present invention.

[0261] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., for effecting or directing the expression of the above-mentioned polypeptides when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), immunoglobulin H chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), etc.

[0262] Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al.1990), pAGE103 (Mizukami T et al.1987), pHSG274 (Brady G et al.1984), pKCR (O'Hare K et al.1981), pSG1 beta d2-4- (Miyaji H et al.1990), etc. Other representative examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids, such as pUC, pcDNA, pBR, etc. Representative examples of viral vectors include adenovirus, retrovirus, lentivirus, herpesvirus and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses are well known in the art and can be found, for example, in PCT Publication WO95 / 14785, PCT Publication WO96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and PCT Publication WO94 / 19478.

[0263] In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a binding protein or polypeptide, or a fragment thereof, as described herein. In some embodiments, the nucleic acid comprises regulatory elements required for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers may be included. These elements may be operably linked to the sequence encoding the binding protein, polypeptide, or a fragment thereof.

[0264] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8α and / or CD8β. In certain embodiments, the nucleic acid sequence encoding CD8α or CD8β is operably linked to a nucleic acid encoding a tag (e.g., a CD34 enrichment tag). In certain embodiments, the nucleic acid sequence encoding CD8α and / or CD8β is interconnected to a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide, such as P2A, E2A, F2A, or T2A.

[0265] In some embodiments, the expression vectors provided herein comprise a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids set forth in Tables 1-3.

[0266] As described above, representative examples of promoters include, but are not limited to, promoters from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), such as the HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV), such as the CMV immediate early promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Examples of suitable polyadenylation signals include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal.

[0267] In addition to the regulatory elements required for expression, other elements may be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include promoters as described herein. In some embodiments, enhancers / promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers, such as those from CMV, RSV, and EBV.

[0268] In some embodiments, the nucleic acid may be functionally incorporated into a carrier or delivery vector, as described further below. Useful delivery vectors include, but are not limited to, biodegradable microcapsules, immune stimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live carriers, such as viruses or bacteria.

[0269] In some embodiments, the vector is a viral vector, such as a lentivirus, retrovirus, herpes virus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, fowlpox, AV pox, modified vaccinia Ankara (MVA), and other recombinant viruses. For example, a lentivirus vector may be used to infect T cells.

[0270] In some embodiments, the recombinant expression vector is capable of delivering the polynucleotide to a suitable host cell, e.g., a T cell or an antigen-presenting cell, i.e., a cell that presents peptide / MHC complexes on its cell surface (e.g., a dendritic cell) and lacks CD8. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. For example, the immune system cell is a CD4 + T cells, CD8 + The host may be a T cell, a CD4 / CD8 double negative T cell, a gd T cell, a natural killer cell, a dendritic cell, or any combination thereof. In some embodiments, a T cell is the host, and the T cell may be a naive, a central memory T cell, an effector memory T cell, or any combination thereof. Thus, the recombinant expression vector may also include a lymphoid tissue-specific transcriptional regulatory element (TRE), such as a TRE specific for B lymphocytes, for T lymphocytes, or for dendritic cells. Lymphoid tissue-specific TREs are known in the art (see, for example, Thompson et al. (1992) Mol. Cell. Biol. 72:1043, Todd et al. (1993) J. Exp. Med. 777:1663, and Penix et al. (1993) J. Exp. Med. 775:1483).

[0271] In some embodiments, the recombinant expression vector comprises a nucleotide sequence encoding a TCR alpha chain, a TCR beta chain, and / or a linker peptide. For example, in some embodiments, the recombinant expression vector comprises a nucleotide sequence encoding the full length TCR alpha and TCR beta chains of the binding protein with a linker therebetween, where the nucleotide sequence encoding the beta chain is located 5' to the nucleotide sequence encoding the alpha chain. In some embodiments, the nucleotide sequence encodes the full length TCR alpha and TCR beta chains with a linker therebetween, where the nucleotide sequence encoding the TCR beta chain is located 3' to the nucleotide sequence encoding the TCR alpha chain. In some embodiments, the full length TCR alpha and / or TCR beta chains are replaced with fragments thereof.

[0272] As described further below, another aspect encompassed by the present invention relates to cells transfected, infected or transformed by the nucleic acid and / or vector according to the present invention. A host cell can include any individual cell or cell culture that can accept the vector or the incorporation of the nucleic acid and / or protein, as well as any progeny cells. The term also includes the progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells can depend on the vector and include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells. These cells can be induced to take up vectors or other materials by transformation via the use of viral vectors, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection or other methods (see, for example, Sambrook el al. (1989) Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory)). The term "transformation" means the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed."

[0273] The nucleic acids encompassed by the invention may be used in a suitable expression system to produce a recombinant polypeptide encompassed by the invention. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for the expression of a protein encoded by foreign DNA, e.g., carried by a vector and introduced into the host cell.

[0274] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al. (1980)), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, YB2 / 0 cells are used because the ADCC activity of the chimeric or humanized binding protein is enhanced when expressed in the YB2 / 0 cells.

[0275] The invention also encompasses methods for producing recombinant host cells expressing the binding proteins, peptides and fragments thereof, comprising the steps of (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell, in vitro or ex vivo, (ii) culturing the resulting recombinant host cell, in vitro or ex vivo, and (iii) optionally selecting cells expressing the binding proteins, peptides and fragments thereof. Such recombinant host cells may be used in the diagnostic, prognostic and / or therapeutic methods encompassed by the invention.

[0276] In another aspect, as described above, the present invention provides an isolated nucleic acid that hybridizes to the polynucleotide disclosed herein under selective hybridization conditions.Thus, the polynucleotide of this embodiment can be used to isolate, detect and / or quantify the nucleic acid that contains such a polynucleotide.For example, the polynucleotide encompassed by the present invention can be used to identify, isolate or amplify partial or full-length clones in a storage library.In some embodiments, the polynucleotide is a genomic or cDNA sequence that is complementary to a cDNA isolated from or derived from a human or mammalian nucleic acid library. In some embodiments, the cDNA library contains at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or any range therebetween, including the endpoints, for example, at least about 80% to 100% of full-length sequences. The cDNA library may be normalized to enhance the representation of rare sequences. Low or medium stringency hybridization conditions are typically, but not exclusively, employed for sequences with low sequence identity to complementary sequences. Medium and high stringency conditions may optionally be employed for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity and may be used to identify orthologous or paralogous sequences. Optionally, the polynucleotides encompassed by the present invention will encode at least a portion of the binding proteins encoded by the polynucleotides described herein. The polynucleotides encompassed by the present invention include nucleic acid sequences that can be employed for selective hybridization to polynucleotides encoding the binding proteins encompassed by the present invention (see, e.g., Ausubel, supra, and Colligan, supra).

[0277] VII. Manipulated cells In aspects encompassed by the invention, provided herein are host cells that express a protein described herein, such as a MAGEC2 immunogenic peptide described herein, a MAGEC2 immunogenic peptide-MHC (pMHC) complex, a MAGEC2 binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, a CAR, or a fusion protein comprising a TCR and an effector domain), etc. In some embodiments, the host cell comprises a nucleic acid or vector described herein.

[0278] In some embodiments, polynucleotides encoding the binding proteins are used to transform, transfect or transduce host cells (e.g., T cells) for use in adoptive transfer therapy. Advances in nucleic acid sequencing and specific TCR sequencing have been described (e.g., Robins et al. (2009) Blood 114:4099; Robins et al. (2010) Sci. Translat. Med. 2:47ra64; Robins et al. (2011) J. Imm. Meth.; and Warren et al. (2011) Genome Res. 21:790), which may be used in the course of carrying out embodiments encompassed by the present invention. Similarly, methods for transfecting or transducing T cells with a desired nucleic acid are well known in the art (e.g., U.S. Patent Application Publication No. 2004 / 0087025), as are adoptive transfer procedures using T cells with a desired antigen specificity (e.g., Schmitt et al. (2009) Hum. Gen. 20:1240, Dossett et al. (2009) Mol. Ther. 77:742, Till et al. (2008) Blood 772:2261, Wang et al. (2007) Hum. Gene Ther. 18:112, Kuball et al. (2007) Blood 709:2331, U.S. Patent Publication No. 2011 / 0243972, U.S. Publication No. 2011 / 0189141, and Leen et al. al.(2007)Ann.Rev.Immunol.25:243).

[0279] Any suitable immune cell, for example, a T cell, a NK cell, or a NK-T cell, can be modified to contain a heterologous polynucleotide encompassed by the present invention. In some embodiments, the cell can be a primary cell or a cell line cell. In some embodiments, the modified immune cell is a CD4 + T cells, CD8 + For purposes herein, a T cell may be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a number of sources, e.g., but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may also be enriched or purified. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are T cells isolated from a human. The T cells may be any type of T cell and may be at any stage of development, e.g., but not limited to, cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells, naive T cells, etc.

[0280] Any suitable method may be used to transfect or transduce cells (e.g., T cells) or to administer the nucleotide sequences or compositions encompassed by the methods described herein. Methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercial products, such as in vivo-jetPEI®. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, particle bombardment, transposon-mediated transfer, and the like. Additional methods of transfecting or transducing host cells employ vectors, which are described in more detail in this application.

[0281] The modified immune cells described herein may be functionally characterized using methodologies to assess T cell activity, including determining T cell binding, activation or induction, and also determining antigen-specific T cell responses. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., from pre-loaded target cells). 51 These include determining whether T cell function is related to T cell proliferation or proliferation (e.g., by detecting Cr release), changes in T cell phenotypic marker expression, and other measures of T cell function.

[0282] Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: Hie Comprehensive Sourcebook of Techniques, 1998), and Current Protocols in Immunology, Weir, (1986) Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA, Mishell and Shigii (eds.) (1979) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA, Green and Reed (1998) Science 281:1309, and references cited therein.

[0283] In some embodiments, the apparent affinity for a binding protein, e.g., a TCR or antigen-binding portion thereof, can be measured by assessing binding to various concentrations of MHC multimers. "MHC-peptide multimer staining" refers to an assay used to detect antigen-specific T cells, which in some embodiments focuses on tetramers of MHC molecules, each containing an identical peptide with a homologous (e.g., identical or related) amino acid sequence to at least one antigen (e.g., a MAGEC2 immunogenic peptide), and the complex can bind to a binding protein, e.g., a TCR or antigen-binding portion thereof, that recognizes the cognate antigen. Each MHC molecule may be tagged with a biotin molecule. The biotinylated MHC / peptide may be multimerized (e.g., tetramerized) by the addition of streptavidin, which may be fluorescently labeled.

[0284] Multimers can be detected by flow cytometry via fluorescent labeling. In some embodiments, the affinity-enhanced binding proteins encompassed by the present invention, such as TCRs or antigen-binding portions thereof, are detected or selected using pMHC multimer assays. In some examples, the apparent K D is measured using a range of two-fold dilutions of the labeled tetramer, followed by determination of the binding curve by nonlinear regression, whereas the apparent K D is determined as the concentration of ligand that results in half-maximal binding.

[0285] Cytokine levels can be determined using methods described herein, such as ELISA, ELISPOT, intracellular cytokine staining and flow cytometry, and combinations thereof (eg, intracellular cytokine staining and flow cytometry).

[0286] Immune cell proliferation and clonal expansion resulting from antigen-specific induction or stimulation of an immune response can be determined by isolating lymphocytes, e.g., circulating lymphocytes in a sample of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and / or cell viability, e.g., by tritiated thymidine incorporation or non-radioactive assays, e.g., MTT assays, etc. The effect of the immunogens described herein on the balance between Th1 and Th2 immune responses can be examined, for example, by determining the levels of Th1 cytokines, e.g., IFN-g, IL-12, IL-2, and TNF-b, and type 2 cytokines, e.g., IL-4, IL-5, IL-9, IL-10, and IL-13.

[0287] Host cells encompassed by the present invention may contain a single polynucleotide encoding a binding protein as described herein, or the binding protein may be encoded by more than one polynucleotide. In other words, components or portions of a binding protein may be encoded by more than one polynucleotide, and these polynucleotides may be contained on a single nucleic acid molecule or may be contained on more than one nucleic acid molecule.

[0288] In some embodiments, a polynucleotide encoding two or more components or portions of a binding protein encompassed by the present invention comprises two or more coding sequences functionally associated in a single open reading frame. Advantageously, such an arrangement can allow for coordinated expression of the desired gene products, for example, simultaneous expression of the alpha and beta chains of a TCR, so that they are produced in a ratio of about 1:1. In some embodiments, two or more alternative gene products of a binding protein encompassed by the present invention, for example, a TCR (e.g., an alpha and beta chain) or a CAR, are expressed as separate molecules and post-translationally associated. In further embodiments, two or more alternative gene products of a binding protein encompassed by the present invention are expressed as a single peptide with portions separated by a cleavable or removable segment. For example, self-cleaving peptides useful for expressing separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, the porcine teschovirus-1 2A (P2A) peptide, the zosea signavirus 2A (T2A) peptide, the equine rhinitis A virus (ERAV) 2A (E2A) peptide, and the foot and mouth disease vims 2A (F2A) peptide.

[0289] In some embodiments, a binding protein encompassed by the invention comprises one or more junction amino acids. A "junction amino acid" or "junction amino acid residue" refers to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, e.g., between a binding domain and an adjacent constant domain, or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can result from the design of a construct encoding a fusion protein (e.g., amino acid residues resulting from the use of restriction enzyme sites during the construction of a nucleic acid molecule encoding a fusion protein), or can result from cleavage of an auto-cleaving peptide adjacent to, for example, one or more domains of an encoded binding protein encompassed by the invention (e.g., a P2A peptide located between the TCRa and TCR β chains that self-cleaves to leave one or more junction amino acids in the a chain, the TCR β chain, or both).

[0290] The engineered immune cells encompassed by the present invention can be administered, for example, as a therapeutic agent for disorders characterized by MAGEC2 expression. In some circumstances, it may be desirable to reduce or stop the activity associated with cellular immunotherapy. Thus, in some embodiments, the engineered immune cells encompassed by the present invention comprise heterologous polynucleotides that encode binding proteins and auxiliary proteins, such as safety switch proteins, that can be targeted using the same drugs or other compounds to selectively modulate (e.g., reduce or eliminate) the activity of such cells when desired. Safety switch proteins for use in this regard include, for example, truncated EGF receptor polypeptides (huEGFRt) that lack the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retain the native amino acid sequence, type I transmembrane cell surface localization, and conformationally intact binding epitopes for pharmaceutical grade anti-EGFR monoclonal antibodies, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al. (2011) Blood 118:1255-1263), caspase polypeptides (e.g., iCasp9; Straathof et al. (2005) Blood 105:4247-4254, Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Zhou and Brenner (2016) Hematol. pii:S0301-472X:30513-30516), RQR8 (Philip et al. (2014) Blood 124:1277-1287), and the human c-myc protein tag (Kieback et al. (2008) Proc. Natl. Acad. Sci. USA 105:623-628).

[0291] Other ancillary components useful for therapeutic cells include tags or selection markers (e.g., CD34 enrichment tags) that allow the cells to be identified, sorted, isolated, enriched, or tracked. For example, marked immune cells with desired characteristics (e.g., antigen-specific TCR and safety switch protein) can be sorted from marker-free cells in a sample and more efficiently activated and expanded for inclusion in a therapeutic product of desired purity.

[0292] As used herein, the term "selection marker" includes a nucleic acid construct that confers an identifiable change to a cell to allow for detection and positive selection of immune cells transduced with a polynucleotide comprising the selection marker. For example, RQR is a selection marker that comprises the large extracellular loop of CD20 and two minimal binding sites of CD34. In some embodiments, the polynucleotide encoding RQR comprises a polynucleotide encoding a minimal epitope of 16 amino acids of CD34. In some embodiments, such as certain embodiments provided in the Examples herein, the minimal epitope of CD34 is incorporated at the amino-terminal position of the CD8 stalk domain (Q8). In further embodiments, the minimal binding site sequence of CD34 may be combined with a target epitope of CD20 to form a small marker / suicide gene for T cells (RQR8) (Philip et al. 2014). This construct allows for the selection of immune cells expressing the construct using, for example, a CD34-specific antibody (Miltenyi) coupled to magnetic beads, and the selective elimination of engineered T cells expressing the transgene using the clinically approved pharmaceutical antibody, rituximab (e.g., Philip et al. (2014) Blood 124:1277-1287; U.S. Patent Publication No. 2015-0093401; and U.S. Patent Publication No. 2018-0051089).

[0293] Further exemplary selection markers include several truncated type I transmembrane proteins that are not normally expressed on T cells: truncated low affinity nerve growth factor, truncated CD19, and truncated CD34 (e.g., Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Mavilio et al. (1994) Blood 83:1988-1997, and Fehse et al. (2000) Mol. Ther. 7:448-456). A particularly attractive feature of CD19 and CD34 is the availability of ready-made Miltenyi CliniMACs™ selection systems that can target these markers for clinical grade sorting. However, CD19 and CD34 are relatively large surface proteins that may impair vector packaging capacity and transcription efficiency of integrated vectors. Surface markers containing extracellular, non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR, etc.) may be employed. Any selection marker may be employed and should be acceptable in good manufacturing practice. In some embodiments, the selection marker is expressed together with the polynucleotide encoding the gene product of interest (e.g., the binding protein encompassed by the present invention, such as TCR or CAR, or antigen-binding fragments thereof). Further examples of selection markers include, for example, reporters such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In some embodiments, a selection marker such as CD34 is expressed by the cells, and CD34 can be used to select, enrich, or isolate transduced cells of interest for use in the methods described herein (e.g., by immunomagnetic selection). As used herein, the CD34 marker is distinct from anti-CD34 antibodies, or, for example, scFvs, TCRs, or other antigen-recognition moieties that bind to CD34.

[0294] In some embodiments, the selection marker comprises an RQR polypeptide, a truncated low affinity nerve growth factor (tNGFR), a truncated CD19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.

[0295] As background, CD4 + Inclusion of T cells in immunotherapy cell products can result in antigen-induced IL-2 secretion, and the transferred cytotoxic CD8 + T cell persistence and function can be enhanced (e.g., Kennedy et al. (2008) Immunol. Rev. 222:129, and Nakanishi et al. Nature (2009) 52:510). In some embodiments, CD4 + Class I-restricted TCRs in T cells may require the transfer of CD8 co-receptors to enhance the sensitivity of TCRs to class I HLA-peptide complexes. The CD4 co-receptor is structurally distinct from CD8 and cannot effectively be substituted for the CD8 co-receptor (e.g., Stone & Kranz (2013) Front. Immunol. 4:244, and Cole et al. (2012) Immunology 737:139). Therefore, another auxiliary protein for use in the compositions and methods encompassed by the present invention includes the CD8 co-receptor or a component thereof. The engineered immune cells comprising a heterologous polynucleotide encoding a binding protein encompassed by the present invention may, in some embodiments, further comprise a heterologous polynucleotide encoding a CD8 co-receptor protein or a beta or alpha chain component thereof.

[0296] The host cell can be efficiently transduced to contain and efficiently express a single polynucleotide encoding the binding protein, the safety switch protein, the selection marker and the CD8 co-receptor protein.

[0297] In one embodiment, a host cell encompassed by the invention further comprises a nucleic acid encoding a costimulatory molecule, such that the engineered T cell expresses the costimulatory molecule. In some embodiments, the costimulatory domain is selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L.

[0298] In any of the foregoing embodiments, the host cell expressing a binding protein described herein may be a universal immune cell. A "universal immune cell" includes an immune cell that has been modified to reduce or eliminate expression of one or more endogenous genes encoding a polypeptide product selected from PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA molecule, a TCR molecule, or any combination thereof. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, TIGIT) or may interfere with the binding activity of the heterologously expressed binding proteins encompassed by the invention (e.g., endogenous TCRs that bind non-MAGEC2 antigens and interfere with binding of modified immune cells to target cells expressing MAGEC2 antigens, such as MAGEC2 immunogenic peptides in the context of MHC molecules. Furthermore, endogenous proteins expressed on donor immune cells (e.g., immune cell proteins, e.g., HLA alleles) may be recognized as foreign by the allogeneic host, which may lead to the elimination or suppression of the modified donor immune cells by the allogeneic host.

[0299] Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve activity, tolerance or persistence of the engineered immune cells in an autologous or allogeneic host environment and allow the cells to be universally administered (e.g., to any recipient regardless of HLA type). In some embodiments, the cells of the invention are syngeneic, meaning genetically identical or sufficiently identical and immunologically compatible to allow for transplantation. In some embodiments, the universal immune cells are donor cells (e.g., allogeneic) or autologous cells. In some embodiments, modified immune cells (e.g., universal immune cells) encompassed by the invention comprise one or more chromosomal gene knockouts of genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, HLA components (e.g., genes encoding alpha 1 macroglobulin, alpha 2 macroglobulin, alpha 3 macroglobulin, beta 1 microglobulin, or beta 2 microglobulin), or TCR components (e.g., genes encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai el al. (2016) Nature Sci. Rep. 6:21757, Torikai et al. (2012) Blood 179:5697, and Torikai et al. (2013) Blood 722:1341, which also provide representative exemplary gene editing techniques, compositions, and adoptive cell therapies that are useful according to the invention).

[0300] As used herein, the term "chromosomal gene knockout" refers to a genetic modification or inhibitor introduced into a host cell that prevents (e.g., reduces, delays, suppresses, or eliminates) the production by the host cell of a functionally active endogenous polypeptide product. Modifications that result in a chromosomal gene knockout can include, for example, the introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0301] In some embodiments, chromosomal gene knockout or gene knock-in can be created by chromosomal editing of a host cell. Chromosomal editing can be performed, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of phosphodiester bonds in a polynucleotide chain. In some embodiments, the endonuclease can cleave a target gene, thereby inactivating or "knocking out" the target gene. The endonuclease can be a natural, recombinant, genetically modified, or fusion endonuclease. Nucleic acid strand breaks caused by endonucleases are generally repaired through separate mechanisms: homologous recombination or non-homologous end joining (NHEJ). During the process of homologous recombination, a donor nucleic acid molecule can be used for donor gene "knock-in," for target gene "knock-out," and, optionally, for inactivating a target gene via a donor gene knock-in or target gene knock-out event. NHEJ is an error-prone repair process that often results in changes in DNA sequence at the cut site (e.g., substitution, deletion or addition of at least one nucleotide). NHEJ can be used to "knock out" target genes. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0302] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein that includes a zinc finger DNA binding domain fused to a non-specific DNA cleavage domain, e.g., Fok1 endonuclease. Each zinc finger motif of about 30 amino acids binds about three base pairs of DNA, and amino acids at certain residues may be altered to modify the triple sequence specificity (e.g., Desjarlais et al. (1993) Proc. Natl. Acad. Sci. 90:2256-2260, and Wolfe et al. (1999) J. Mol. Biol. 255:1917-1934). Multiple zinc finger motifs may be linked in tandem to create binding specificity for a desired DNA sequence, e.g., a region having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and the directional integration of transgenes containing flanking sequences homologous to the genome at the DSB site is promoted by homology-directed repair. Alternatively, the DSBs generated by ZFNs can result in knockout of the target gene via repair by non-homologous end joining (NHEJ), an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the break site. In some embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof made using a ZFN molecule.

[0303] As used herein, "Transcription Activator-Like Effector Nuclease" (TALEN) refers to a fusion protein that includes a TALE DNA-binding domain and a DNA cleavage domain, e.g., Fok1 endonuclease. A "TALE DNA-binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each of which generally has a highly conserved 33-35 amino acid sequence with variable 12th and 13th amino acids. The TALE repeat domain is responsible for the binding of the TALE to the target DNA sequence. The variable amino acid residues, referred to as repeat variable dipeptides (RVDs), correlate with specific nucleotide recognition. The natural (canonical) codes for DNA recognition of these TALEs have been determined, where the HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE results in the binding of the TALE to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) binds to an A, NN (asparagine-asparagine) binds to a G or A nucleotide, NG (asparagine-glycine) binds to a T nucleotide, and so on. Non-canonical (atypical) RVDs are also well known in the art (e.g., U.S. Patent Publication No. 2011 / 0301073, all of which atypical RVDs are incorporated herein by reference). TALENs can be used to introduce site-specific double-strand breaks (DSBs) into the genome of T cells. Non-homologous end joining (NHEJ) ligates the DNA on both sides of the double-strand break with little or no sequence overlap to anneal, thereby introducing errors that knock out gene expression.Alternatively, homologous recombination repair can introduce transgenes into DSB sites if there are homologous flanking sequences in the transgene.In some embodiments, gene knockouts include insertions, deletions, mutations or combinations thereof, and are made using TALEN molecules.

[0304] As used herein, the "clustered regularly interspaced short palindromic repeats / Cas" (CRISPR / Cas) nuclease system refers to a system that uses a CRISPR RNA (crRNA)-dependent Cas nuclease to recognize target sites (known as protospacers) in the genome by base-pairing complementarity and then cleave DNA if the complementary target sequence is followed immediately 3' by a short conserved protospacer-associated motif (PAM). CRISPR / Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nuclease. In types I and III, the crRNA-dependent surveillance complex requires multiple Cas subunits. Type II systems, which are the most studied, contain at least three components: an RNA-dependent Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA contains a double-strand forming region. The crRNA and tracrRNA form a duplex that can interact with Cas9 nuclease and guide the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA by Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA upstream of the PAM. The Cas9 nuclease cleaves the double-stranded break within the region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions that disrupt expression of the target locus. Alternatively, transgenes with homologous flanking sequences can be introduced at the site of the DSB via homology-directed repair. The crRNA and tracrRNA can be engineered into single-stranded guide RNAs (sgRNAs or gRNAs) (e.g., Jinek et al. (2012) Science 337:816-821).Additionally, the region of the guide RNA that is complementary to the target site may be modified or programmed to target a desired sequence (Xie et al. (2014) PLOS One 9:el00448, U.S. Patent Publication No. US2014 / 0068797, U.S. Patent Publication No. US2014 / 0186843, U.S. Patent No. 8,697,359, and PCT Publication No. WO2015 / 071474). In some embodiments, gene knockouts include insertions, deletions, mutations, or combinations thereof, and are made using the CRISPR / Cas nuclease system.

[0305] Exemplary gRNA sequences and exemplary methods of using gRNA sequences to knock out endogenous genes encoding immune cell proteins include those described in Ren et al. (2017) Clin. Cancer Res. 23:2255-2266, which provides representative exemplary gRNAs, CAS9 DNA, vectors and gene knockout techniques.

[0306] As used herein, "meganucleases," also called "homing endonucleases," refer to endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, RI-Sce, I-ScelV, I-Csml, I-Panl, I-Scell, I-Ppol, I-SceIII, I-Crel, I-Tevl, I-TevII, and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Pat. Nos. 5,420,032 and 6,833,252; Belfort et al. (1997) Nucl. Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 52:115-118; Perler et al. (1994) Nucl. Acids Res. 22:1125-1127; Jasin (1996) Trends Genet.72:224-228, Gimble et al. (1996) J.Mol.Biol.263:163-180, and Argast et al. (1998) J.Mol.Biol.280:345-353).

[0307] In some embodiments, naturally occurring meganucleases may be used to facilitate site-specific genomic modification of targets of interest, such as immune checkpoints, HLA-encoding genes, or TCR component-encoding genes.

[0308] In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genome modification (see, e.g., Porteus et al. (2005) Nat. Biotechnol. 23:967-73; Sussman et al. (2004) J. Mol. Biol. 342:31-41; Epinat et al. (2003) Nucl. Acids Res. 37:2952-2962; Chevalier et al. (2002) Mol. Cell 70:895-905; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Curr. Genet. 10:111-112; (See, Ther. 7:49-66, and US Patent Publication Nos. US2007 / 0117128, US2006 / 0206949, US2006 / 0153826, US2006 / 0078552, and US2004 / 0002092). In a further embodiment, a homing endonuclease modified with a modular DNA-binding domain of TALEN is used to create a fusion protein known as a megaTAL to generate a chromosomal gene knockout. MegaTALs can be utilized not only to knock out one or more target genes, but also to introduce (knock in) a heterologous or foreign polynucleotide when used in conjunction with an exogenous donor template that encodes a polypeptide of interest.

[0309] In some embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that binds (e.g., specifically and / or selectively) to a MAGEC2 antigen, where the inhibitory nucleic acid molecule encodes a target-specific inhibitor, and the encoded target-specific inhibitor inhibits expression of an endogenous gene (i.e., PD-1, TIM3, LAG3, CTLA4, TIGIT, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.

[0310] Chromosomal gene knockout can be confirmed directly by DNA sequencing of host immune cells following the knockout procedure or drug application.

[0311] A chromosomal gene knockout can also be inferred from the absence of gene expression following the knockout (eg, the absence of mRNA or polypeptide product encoded by the gene).

[0312] In some embodiments, host cells encompassed by the present invention are capable of specifically and / or selectively killing 50% or more of target cells that contain a peptide-MHC (pMHC) complex that comprises a MAGEC2 immunogenic peptide in the context of an MHC molecule.

[0313] In some embodiments, the modified immune cells are capable of producing cytokines when contacted with a target cell that contains a peptide-MHC (pMHC) complex that includes a MAGEC2 immunogenic peptide in the context of an MHC molecule.

[0314] In some embodiments, the cytokine comprises IFN-γ or IL2. In some embodiments, the cytokine comprises TNF-α.

[0315] In some embodiments, the host cell has expression of MAGEC2 at about 1,000 transcripts per million transcripts (about 1,000 TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 32 TPM, 33 TPM, 34 ... In one embodiment, the IL-16 receptor agonist (IL-16 receptor agonist) can produce higher levels of cytokines or cytotoxic molecules when contacted with target cells having levels of IL-16 receptor agonist (IL-16 receptor agonist), such as IL-16 receptor agonist (IL-16 receptor agonist), ... In some embodiments, low MAGEC2 expression levels are referred to as "heterozygous expression", which means from about 1 TPM to about 35 TPM, or any range therebetween, inclusive of the endpoints, for example, 1 to 32 TPM. For example, the host cell may produce levels of a cytokine or cytotoxic molecule that are at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1000 fold higher or more, or any range therebetween inclusive of the endpoints, e.g., 1.2 fold to 2 fold higher.

[0316] In some embodiments, the host cell is capable of specifically and / or selectively killing target cells expressing MAGEC2 (e.g., hyperproliferative cells expressing MAGEC2). In certain embodiments, the target cells express a MAGEC2 immunogenic peptide in the context of an MHC molecule (e.g., a matching MHC molecule).

[0317] In some embodiments, the host cell does not express the MAGEC2 antigen, which is not recognized by the binding proteins described herein, and is of the serotype HLA-B. * Not 07 and / or HLA-B * 07 allele, e.g., HLA-B * 0702, HLA-B * 0704, HLA-B * 0705, HLA-B * 0709, HLA-B * 0710, HLA-B * 0715 or HLA-B * For example, the patient may be MAGEC2 negative or MHC negative (e.g., HLA-B) that presents the MAGEC2 immunogenic peptides described herein and does not express the 0721 allele. * 07:02 Negative and / or HLA-A *The patient may receive host cells from a healthy donor who is MAGEC2 negative (HLA-B 24:02 negative) or even selected and / or engineered autologous cells. The cells isolated from the donor may be used as a source of transplant material. In particular, the T cells isolated from the donor may be genetically engineered to recognize MAGEC2, for example by expressing a MAGEC2 binding protein as described herein. The donor cells may be used to engraft a cell population in the patient (e.g., hematopoietic stem cells used to reconstitute the immune system) and the host cells may be infused into the patient to induce a highly specific anti-tumor effect. The engineered donor T cells may be designed to recognize and eliminate MAGEC2 positives in the patient, thereby preventing recurrence of the disorder characterized by MAGEC2 expression and promoting a complete cure. The transplanted cells are derived from the donor and therefore MAGEC2 negative, seronegative for MHC presenting the MAGEC2 immunogenic peptides as described herein (e.g., HLA-B * 07 Seronegative and / or HLA-A * 24:02 serotype negative), and / or negative for MHC presenting MAGEC2 immunogenic peptides described herein (e.g., HLA-B * 07:02 Negative and / or HLA-A * 24:02 negative), the engineered cells described herein may have minimal adverse side effects. Such host cells and treatment methods that are compatible with the patient may be used according to the treatment methods described further below.

[0318] In some embodiments, killing is determined by a killing assay. In some embodiments, the killing assay is performed by co-culturing host cells and target cells at a ratio of 20:1 to 0.625:1, such as 15:1 to 1.25:1, 10:1 to 1.5:1, 8:1 to 3:1, 6:1 to 5:1, 20:1 to 5:1, 10:1 to 2.5:1, etc. In some embodiments, the target cells are pulsed with 1 μg / mL to 50 pg / mL of MAGEC2 peptide, such as 1 ug / mL to 10 ng / mL, 500 ng / mL to 0.5 ng / mL, 10 ng / mL to 10 pg / mL, 250 ng / mL to 1 ng / mL, 50 ng / mL to 5 ng / mL, 20 ng / mL to 10 ng / mL, etc.

[0319] In some embodiments, the host cell is adapted to express MAGEC2 at levels of about 1,000 transcripts per million transcripts (about 1,000 TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM and 1 TPM, or less, or any range therebetween inclusive of the endpoints, for example, about 1,000 TPM or less to about 73 TPM or less, can kill a greater number of target cells. For example, a host cell may be capable of killing at least 1.2x, 1.5x, 1.8x, 2.0x, 2.2x, 2.5x, 2.8x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x more, or more, or any range therebetween inclusive of the endpoints, e.g., 1.2x to 2x more, number of target cells.

[0320] The invention further provides a population of cells comprising at least one host cell as described herein. The population of cells may be a heterogeneous population, comprising host cells comprising any of the recombinant expression vectors described, as well as at least one other cell not comprising any of the recombinant expression vectors, such as a host cell (e.g., a T cell), or a cell other than a T cell, such as a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, and the like. Alternatively, the population of cells may be a substantially homogeneous population that comprises primarily host cells that comprise (e.g., consist essentially of) a recombinant expression vector. The population may also be a clonal population of cells, where all cells of the population are clones of a single host cell that comprises a recombinant expression vector, such that all cells of the population comprise a recombinant expression vector. In one embodiment encompassed by the invention, the population of cells is a clonal population that comprises host cells that comprise a recombinant expression vector as described herein.

[0321] In one embodiment encompassed by the present invention, the number of cells in the population may be rapidly expanded. Expanding the number of T cells can be achieved by any of several methods well known in the art (e.g., U.S. Patent Nos. 8,034,334 and 8,383,099, U.S. Patent Publication No. 2012 / 0244133, Dudley et al. (2003) J. Immunother. 26:332-242, and Riddell et al. (1990) J. Immunol. Methods 128:189-201). For example, expanding the number of T cells can be achieved by culturing T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0322] VIII. Pharmaceutical Compositions In another aspect encompassed by the present invention, provided herein is a pharmaceutical composition comprising a composition described herein (e.g., a binding protein, a nucleic acid, a cell, etc.) and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0323] The term "pharmacologically acceptable" refers to agents, materials, compositions and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0324] The medicaments and other compositions encompassed by the present invention may be specially formulated for administration in solid or liquid form, for example, in a form suitable for various routes of administration, such as (1) a form suitable for oral administration, for example, drenches (aqueous or non-aqueous liquids or suspensions), tablets, boli, powders, granules, pastes; (2) a form suitable for parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection, for example, as a sterile solution or suspension; (3) a form suitable for topical administration, for example, as a cream, plaster or spray applied to the skin; (4) a form suitable for vaginal or rectal administration, for example, as a vaginal suppository, cream or foam; or (5) an aerosol, for example, an aqueous aerosol, liposomal formulation or solid particles containing the compound. Any suitable form factor for the medicaments or compositions described herein is contemplated, for example, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories and enemas.

[0325] Pharmaceutical compositions encompassed by the present invention may be presented as separate dosage forms, such as capsules, pre-sachets or tablets, or liquids or aerosol sprays, each containing a predetermined amount of the active ingredient as a powder, or in granules, liquids, or suspensions made with aqueous or non-aqueous liquids, oil-in-water emulsions, water-in-oil liquid emulsions, powders for reconstitution, powders for oral ingestion, in bottles (e.g., powders or liquids in bottles), in mouth-dissolving films, lozenges, pastes, tubes, gums, and packs. Such dosage forms may be prepared by any of the methods of pharmacy.

[0326] Suitable excipients include water, saline, dextrose, glycerol or the like, and combinations thereof. In some embodiments, the compositions comprising the host cells, binding proteins or fusion proteins disclosed herein further comprise a suitable infusion medium. The suitable infusion medium may be any isotonic medium formulation, typically utilizing normal saline, Normosol™-R (Abbott) or Plasma-Lyte™ A (Baxter), 5% dextrose in water, lactated Ringer's solution. The infusion medium may be supplemented with human serum albumin or other human serum components. A unit dose comprising an effective amount of the host cells or composition is also contemplated.

[0327] Also provided herein is a unit dose comprising an effective amount of a host cell, or an effective amount of a composition comprising a host cell. As described herein, host cells include immune cells, T cells (CD4 + T cells and / or CD8+ T cells), cytotoxic lymphocytes (e.g., cytotoxic T cells and / or natural killer (NK) cells), and the like. For example, in some embodiments, the unit dose comprises a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%), at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the engineered cells, alone or in combination with other cells, e.g., at least about 30%, at least about 40%, at least about 50%, at least about 60%), at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the other cells. In some embodiments, undesirable cells are present in reduced amounts or are substantially absent, e.g., less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the population of cells in the composition.

[0328] The amount of cells in a composition or unit dose may include at least one cell (e.g., at least one modified CD8 +T cells, modified CD4 + T cells, and / or NK cells), or more typically, 10 2 More than 10 cells, e.g. 6 , up to 10 7 , up to 10 8 cells, up to 10 9 cells, or 10 10 In some embodiments, the cells are from about 10 to about 10 10 cells / m 2 range, for example, about 10 5 ~about 10 9 cells / m 2 The number of cells will depend on the intended end use of the composition as well as the type of cells contained therein. For example, cells modified to contain a binding protein specific for a particular antigen will include a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. In the applications provided herein, the cells are generally in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In embodiments, the desired cell density is typically 10 4 cells / ml, typically 10 7 cells / ml, typically 10 8 The cells can be administered as a single infusion or in multiple infusions over a range of time periods. Clinically relevant numbers of immune cells can be administered in a cumulative total of 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 In some embodiments, a unit dose of the engineered immune cells may be co-administered (e.g., simultaneously or concomitantly) with hematopoietic stem cells from an allogeneic donor.

[0329] Pharmaceutical compositions can be administered in a manner appropriate to the disease or condition to be treated (or prevented) and determined by those skilled in the medical arts. The appropriate dose of the composition, as well as the suitable duration and frequency of its administration, will be determined by factors such as the patient's health status, the patient's size (i.e., weight, mass, or body surface area), the type and severity of the patient's condition, the specific form of the active ingredient, and the method of administration. In general, an appropriate dosage regimen and treatment regimen will provide the composition(s) in an amount sufficient to provide a therapeutic and / or prophylactic benefit (e.g., as described herein, including improved clinical outcomes such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or reduced symptom severity).

[0330] An effective amount of a pharmaceutical composition refers to an amount sufficient to bring about the desired clinical results or beneficial treatment described herein at the required dosage and for the required period of time.An effective amount can be delivered in one or more administrations.When administration is performed on a subject who is already known or confirmed to have a disease or disease state, the term "therapeutically effective amount" may be used in relation to treatment, while "prophylactically effective amount" is used to refer to administering an effective amount as a prophylactic process to a subject who is susceptible to or at risk of developing a disease or disease state (e.g., recurrence).

[0331] The pharmaceutical compositions described herein may be provided in unit-dose or multi-dose containers (e.g., sealed ampoules or vials). Such containers may be frozen to maintain the stability of the formulation until the time of infusion into the patient. In some embodiments, a unit dose comprises about 10 host cells described herein. 7 cells / m 2 ~about 10 11 cells / m 2 Development of suitable dosing and treatment regimens (e.g., parenteral or intravenous administration or formulations) for use with certain compositions described herein in various treatment regimens.

[0332] When the subject composition is administered parenterally, the composition may also include a sterile aqueous or oily solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol mixed with water, ethanol, propylene glycol or polyethylene glycol. Aqueous solutions or suspensions may further include one or more buffers, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharma- ceutically pure and substantially non-toxic in the amounts employed. Furthermore, active compounds may be incorporated into sustained release formulations and preparations. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated; each unit may contain a predetermined amount of engineered immune cells or active compound calculated to produce the desired effect in association with a suitable pharmaceutical carrier.

[0333] In some embodiments, the pharmaceutical compositions described herein and described above for the immunogenic compositions exemplified with respect to peptides, when administered to a subject, are capable of eliciting an immune response against cells of interest expressing MAGEC2. Such pharmaceutical compositions may be useful as vaccines for the prophylactic and / or therapeutic treatment of disorders characterized by MAGEC2 expression.

[0334] IX. Uses and Methods The compositions described herein may be used in a variety of diagnostic, prognostic, and therapeutic applications. In any method described in this application, e.g., diagnostic, prognostic, therapeutic, or combination thereof, all steps of the method may be performed by a single actor or by more than one actor. For example, the actor providing the therapeutic treatment may directly perform the diagnosis. Alternatively, the person providing the therapeutic agent may request that a diagnostic assay be performed. The diagnostician and / or interventionist may interpret the diagnostic assay results to determine a treatment strategy. Similarly, such alternative processes may be applied to other assays, e.g., prognostic assays.

[0335] Some uses and methods encompassed by the present invention utilize a subject or a subject sample. In some embodiments, the subject is an animal. The animal may be an animal of either sex and at any stage of development. In some embodiments, the animal is a vertebrate, such as a mammal. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a domestic animal, such as a dog, cat, cow, pig, horse, sheep or goat. In some embodiments, the subject is a companion animal, such as a dog or cat. In some embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep or goat. In some embodiments, the subject is a zoo animal. In som...

Claims

1. An immunogenic peptide having the amino acid sequence RAREFMEL (SEQ ID NO: 7), or comprising or consisting of a peptide epitope selected from the peptide sequences listed in Table 1, or an immunogenic composition comprising at least one of said immunogenic peptides, optionally comprising: 1) the immunogenic peptide is derived from a MAGEC2 protein, and optionally the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length; 2) the immunogenic peptide comprises or consists of the amino acid sequence RAREFMELL (SEQ ID NO:5); 3) the immunogenic peptide is capable of eliciting an immune response in a subject against MAGEC2 and / or MAGEC2 expressing cells, optionally wherein the immune response is i) a T cell response and / or a CD8+ T cell response, and / or ii) selected from the group consisting of T cell proliferation, cytokine release and / or cytotoxic killing; 4) the immunogenic composition further comprises an adjuvant; and / or 5) An immunogenic peptide or immunogenic composition capable of inducing an immune response in a subject against MAGEC2 and / or MAGEC2 expressing cells, optionally wherein the immune response is i) a T cell response and / or a CD8+ T cell response, and / or ii) selected from the group consisting of T cell proliferation, cytokine release and / or cytotoxic killing.

2. A composition comprising a peptide epitope having the amino acid sequence RAREFMEL (SEQ ID NO: 7), or selected from the peptide sequences listed in Table 1, and an MHC molecule, optionally wherein the MHC molecule is 1) an MHC multimer, optionally wherein the MHC multimer is a tetramer; 2) is an MHC class I molecule; and / or 3) comprises an MHC alpha chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and / or HLA-B*07, and optionally the HLA allele is selected from the group consisting of HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0215, HLA-A*0216, HLA-A*0217, HLA-A*0218, HLA-A*0219, HLA-A*0300, HLA-A*0301, HLA-A*0302, HLA-A*0303, HLA-A*0304, HLA-A*0305, HLA-A*0306, HLA-A*0307, ​​HLA-A*0308, HLA-A*0309, HLA-A*0310, HLA-A*0311, HLA-A*0312, HLA-A*0313, HLA-A*0314, HLA-A*0315, HLA-A*0316, HLA-A*0317, HLA-A*0318, HLA-A*0319, HLA-A*0320, HLA-A*0321, HLA-A*0322, HLA-A*0323, HLA-A*0324, H 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A * 0242, HLA-A * 0253, HLA-A * 0260, HLA-A * 0274 allele, HLA-A * 0301, HLA-A * 0302, HLA-A * 0305, HLA-A * 0307, ​​HLA-A * 0101, HLA-A * 0102, HLA-A * 0103, HLA-A *0116 allele, HLA-A *1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104, HLA-A * 1105, HLA-A * 1119 allele, HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B*0702, HLA-B*0704, HLA-B* 0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles.

3. A stable MHC-peptide complex comprising an immunogenic peptide according to claim 1 in association with an MHC molecule, or an immunogenic composition comprising said stable MHC-peptide complex and an adjuvant, optionally comprising 1) The MHC molecule is a) an MHC multimer, optionally wherein the MHC multimer is a tetramer; b) is an MHC class I molecule; and / or c) comprises an MHC alpha chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and / or HLA-B*07, and optionally the HLA allele is selected from the group consisting of HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0215, HLA-A*0216, HLA-A*0217, HLA-A*0218, HLA-A*0219, HLA-A*0300, HLA-A*0301, HLA-A*0302, HLA-A*0303, HLA-A*0304, HLA-A*0305, HLA-A*0306, HLA-A*0307, ​​HLA-A*0308, HLA-A*0309, HLA-A*0310, HLA-A*0311, HLA-A*0312, HLA-A*0313, HLA-A*0314, HLA-A*0315, HLA-A*0316, HLA-A*0317, HLA-A*0318, HLA-A*0319, HLA-A*0320, HLA-A*0321, HLA-A*0322, HLA-A*0323, HLA-A*0324, HLA 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A * 0242, HLA-A * 0253, HLA-A * 0260, HLA-A * 0274 allele, HLA-A * 0301, HLA-A * 0302, HLA-A * 0305, HLA-A * 0307, ​​HLA-A * 0101, HLA-A * 0102, HLA-A * 0103, HLA-A *0116 allele, HLA-A *1101, HLA-A * 1102, HLA-A * 1103, HLA-A * 1104, HLA-A * 1105, HLA-A * 1119 allele, HLA-A * 2402, HLA-A * 2403, HLA-A * 2405, HLA-A * 2407, HLA-A * 2408, HLA-A * 2410, HLA-A * 2414, HLA-A * 2417, HLA-A * 2420, HLA-A * 2422, HLA-A * 2425, HLA-A * 2426, HLA-A * 2458 allele, HLA-B*0702, HLA-B*0704, HLA-B* 0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles; 2) the peptide epitope and the MHC molecule are covalently linked, and / or the alpha and beta chains of the MHC molecule are covalently linked; and / or 3) A stable MHC-peptide complex or an immunogenic composition, wherein said stable MHC-peptide complex comprises a detectable label, optionally wherein said detectable label is a fluorophore.

4. 2. An isolated nucleic acid encoding the immunogenic peptide of claim 1, or its complement, or a vector comprising said isolated nucleic acid.

5. a) comprising the isolated nucleic acid of claim 4; b) comprising the vector according to claim 4; and / or c) producing one or more immunogenic peptides according to claim 1 and / or presenting one or more stable MHC-peptide complexes according to claim 3 on the cell surface. A cell, optionally wherein the cell is genetically engineered.

6. 1. An apparatus or kit comprising: a) one or more immunogenic peptides according to claim 1, and / or b) one or more stable MHC-peptide complexes according to claim 3; the device or kit optionally comprises a reagent for detecting binding of a) and / or b) to the binding protein; Optionally, the binding protein comprises: An antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain That is, The device or kit.

7. 1. A method for detecting T cells that bind to stable MHC-peptide complexes, comprising: a) contacting a sample containing T cells with the stable MHC-peptide complex of claim 3; and b) detecting binding of T cells to said stable MHC-peptide complexes, and optionally further determining the percentage of stable MHC-peptide specific T cells that bind to said stable MHC-peptide complexes. Including, Optionally, the sample comprises peripheral blood mononuclear cells (PBMCs), and optionally, 1) the T cell is a CD8+ T cell; 2) said detecting and / or determining is performed using fluorescence activated cell sorting (FACS), using an enzyme-linked immunosorbent assay (ELISA), using a radioimmunoassay (RIA), immunochemically, using a Western blot, or using an intracellular flow assay; and / or 3) The above method, wherein the sample comprises T cells that have been in contact or are suspected of being in contact with one or more types of MAGEC2 protein or fragments thereof.

8. 1. A method for determining whether a T cell has been exposed to MAGEC2, comprising: a) incubating a cell population comprising T cells with an immunogenic peptide of claim 1 or a stable MHC-peptide complex of claim 3; and b) detecting the presence or level of reactivity Including, The presence or higher level of reactivity as compared to a control level indicates that the T cells have been exposed to MAGEC2, and optionally, 1) the cell population comprising T cells is obtained from a subject; 2) the level of reactivity is: a) the presence of a bond, and / or b) T cell activation and / or effector function and optionally, the T cell activation or effector function is T cell proliferation, killing, or cytokine release; 3) the method further comprises repeating steps a) and b) at a subsequent time point, optionally wherein between the first time point and the subsequent time point, the subject has received a treatment to ameliorate the disorder characterized by MAGEC2 expression; 4) the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay; 5) The control level is a) is a reference number; and / or b) the level in subjects not having said disorder characterized by MAGEC2 expression; 6) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 7) The method, wherein the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent.

9. 1. A method for predicting a clinical outcome in a subject suffering from a disorder characterized by MAGEC2 expression, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides of claim 1 or one or more stable MHC-peptide complexes of claim 3; and b) comparing the presence or level of said reactivity with that from a control. Including, The control is obtained from a subject with a good clinical outcome, a presence or a higher level of said reactivity in the subject sample compared to the control is indicative of said subject having a favorable clinical outcome; and optionally 1) The level of reactivity is: a) the presence of a bond, and / or b) T cell activation and / or effector function and optionally, the T cell activation or effector function is T cell proliferation, killing, or cytokine release; 2) the method further comprises repeating steps a) and b) at a subsequent time point, optionally wherein between the first time point and the subsequent time point, the subject has received a treatment to ameliorate the disorder characterized by MAGEC2 expression; 3) the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay; 4) The control level is: a) is a reference number; and / or b) the level in subjects not having said disorder characterized by MAGEC2 expression; 5) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 6) The method, wherein the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent.

10. 1. A method for assessing the efficacy of a therapy for a disorder characterized by MAGEC2 expression, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides of claim 1 or one or more stable MHC-peptide complexes of claim 3 in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject; and b) determining the presence or level of reactivity between said immunogenic peptides according to claim 1 or said stable MHC-peptide complexes according to claim 3 and T cells obtained from said subject present in a second sample obtained from said subject after providing said therapy to said subject. Including, the presence or higher level of said reactivity in said second sample compared to said first sample is an indication that said therapy is effective in treating said disorder characterized by MAGEC2 expression in said subject; and optionally 1) The level of reactivity is: a) the presence of a bond, and / or b) T cell activation and / or effector function and optionally, the T cell activation or effector function is T cell proliferation, killing, or cytokine release; 2) the method further comprises repeating steps a) and b) at a subsequent time point, optionally wherein between the first time point and the subsequent time point, the subject has received a treatment to ameliorate the disorder characterized by MAGEC2 expression; 3) the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay; 4) The control level is: a) is a reference number; and / or b) the level in subjects not having said disorder characterized by MAGEC2 expression; 5) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 6) The method, wherein the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent.

11. The composition according to any one of claims 1 to 6 for use in a method for preventing and / or treating a disorder characterised by MAGEC2 expression in a subject.

12. 1. A method for identifying a peptide binding molecule, or an antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, comprising: a) providing a cell presenting a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule on the surface of said cell; b) determining the binding of a plurality of candidate peptide binding molecules or antigen-binding fragments thereof to the peptide epitope in the context of the MHC molecule on the cell; and c) identifying one or more peptide binding molecules or antigen-binding fragments thereof that bind to said peptide epitope in the context of said MHC molecule; and optionally 1) said step a) comprises contacting said MHC molecule on said surface of said cell with a peptide epitope selected from the peptide sequences listed in Table 1; and / or 2) the method, wherein step a) comprises expressing in the cell the peptide epitope selected from the peptide sequences listed in Table 1 using a vector comprising a heterologous sequence encoding the peptide epitope.

13. 1. A method for identifying a peptide binding molecule, or an antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, comprising: a) providing a peptide epitope, alone or in the context of an MHC molecule, selected from the peptide sequences listed in Table 1, either alone or in a stable MHC-peptide complex; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to said peptide or said stable MHC-peptide complex; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to said peptide epitope or said stable MHC-peptide complex. and optionally 1) The MHC or the MHC-peptide complex is as defined in claim 2 or 3; 2) the plurality of candidate peptide-binding molecules are a) an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain; b) at least 2, 5, 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or more different candidate peptide-binding molecules; and / or c) one or more candidate peptide-binding molecules obtained from a sample from a subject or a population of subjects, or one or more candidate peptide-binding molecules comprising a mutation in a parent scaffold peptide-binding molecule obtained from a sample from a subject. and optionally i) said subject or population of subjects a) is not afflicted with and / or has recovered from a disorder characterized by MAGEC2 expression, or b) is afflicted with a disorder characterized by MAGEC2 expression; ii) the subject or population of subjects has been administered the composition of claim 1; iii) the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent; iv) the subject is an animal model of a disorder characterized by MAGEC2 expression, an HLA transgenic mouse, and / or a human TCR transgenic mouse; and / or v) the sample comprises peripheral blood mononuclear cells (PBMCs), T cells and / or CD8+ memory T cells; 3) the peptide-binding molecule or antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain; 4) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 5) The method, wherein the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent.

14. 1. A composition for use in a method of treating a disorder characterized by MAGEC2 expression in a subject, said composition comprising engineered T cells, said engineered T cells comprising: i) binds to a peptide epitope selected from the sequences listed in Table 1; ii) is identified according to the method of claim 13; and / or iii) binds to a stable MHC-peptide complex comprising a peptide epitope selected from the sequences listed in Table 1 in the context of an MHC molecule. expressing a peptide binding molecule or an antigen-binding fragment thereof, and optionally 1) the peptide-binding molecule or antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain; 2) The MHC or the MHC-peptide complex is as defined in claim 2 or 3; 3) the T cells are isolated from a) the subject, b) a donor not afflicted with the disorder characterized by MAGEC2 expression, or c) a donor who has recovered from a disorder characterized by MAGEC2 expression; 4) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 5) the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent; 6) the cells are allogeneic, syngeneic or autologous; 7) The cell is a host cell according to claim 5, or a population of said host cells; 8) The target cell is a cancer cell expressing MAGEC2; 9) the composition further comprises a pharma- ceutically acceptable carrier; 10) the composition induces an immune response against the target cells expressing MAGEC2 in the subject, optionally wherein the composition induces an antigen-specific T cell immune response against the target cells expressing MAGEC2 in the subject, optionally wherein the antigen-specific T cell immune response comprises at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response; 11) the method further comprises administering at least one additional treatment for said disorder characterized by MAGEC2 expression, optionally wherein said at least one additional treatment for said disorder characterized by MAGEC2 expression is administered simultaneously or sequentially with said composition; 12) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 13) The composition, wherein the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent.

15. 1. A composition for use in a method of treating a disorder characterized by MAGEC2 expression in a subject, said composition comprising antigen-specific T cells, said antigen-specific T cells comprising: a) stimulating immune cells from the subject with a composition according to any one of claims 1 to 6; and b) expanding said antigen-specific T cells in vitro or ex vivo. and optionally i) isolating said immune cells from said subject prior to stimulating said immune cells; and / or ii) the immune cells comprise PBMCs, T cells, CD8+ T cells, naive T cells, central memory T cells and / or effector memory T cells, and optionally 1) the agent is contacted under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, the immunogenic peptide, the stable MHC-peptide complex, the T cell receptor, and / or the immune cell; 2) the peptide epitopes, immunogenic peptides, stable MHC-peptide complexes, and / or T cell receptors are expressed by cells, and the cells are expanded and / or isolated during one or more steps; 3) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 4) the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent; 5) the cells are allogeneic, syngeneic or autologous; 6) The cell is a host cell according to claim 5, or a population of said host cells; 7) The target cell is a cancer cell expressing MAGEC2; 8) the composition further comprises a pharma- ceutically acceptable carrier; 9) the composition induces an immune response against the target cells expressing MAGEC2 in the subject, optionally wherein the composition induces an antigen-specific T cell immune response against the target cells expressing MAGEC2 in the subject, optionally wherein the antigen-specific T cell immune response comprises at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response; 10) The method further comprises administering at least one additional treatment for the disorder characterized by MAGEC2 expression, optionally wherein the at least one additional treatment for the disorder characterized by MAGEC2 expression is administered simultaneously or sequentially with the composition.

16. A binding protein that binds to a polypeptide comprising the immunogenic peptide of claim 1 and / or the stable MHC-peptide complex of claim 3, optionally the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain, optionally the binding protein is 1) a) a T cell receptor (TCR) alpha chain variable domain comprising a CDR1 having residues 49-54 of SEQ ID NO:33, a CDR2 having residues 72-78 of SEQ ID NO:33, and a CDR3 having residues 113-124 of SEQ ID NO:33; and b) a TCR beta chain variable domain comprising a CDR1 having residues 46-50 of SEQ ID NO:35, a CDR2 having residues 68-73 of SEQ ID NO:35, and a CDR3 having residues 112-125 of SEQ ID NO:35; 2) a) a TCR alpha chain variable (V α ) domain comprising residues 1-136 of SEQ ID NO:33; and b) comprises a TCR beta chain variable (Vβ) domain comprising residues 1-136 of SEQ ID NO:35; 3) a) a TCR alpha chain comprising the sequence of SEQ ID NO: 33; and b) comprises a TCR beta chain comprising the sequence of SEQ ID NO: 35; 4) a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity with a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2; and / or b) comprising a TCR beta chain CDR sequence having at least about 80% identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein said binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity of about 5×10 −4 M or less, K d ; 5) a) a TCR alpha chain variable (V α ) domain sequence having at least about 80% identity with a TCR V α domain sequence selected from the group consisting of the TCR V α domain sequences listed in Table 2; and / or b) comprising a TCR beta chain variable (V β ) domain sequence having at least about 80% identity to a TCR V β domain sequence selected from the group consisting of the TCR V β ) domain sequences listed in Table 2, wherein said binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity with a K d of about 5×10 −4 M or less; 6) a) a TCR alpha chain sequence having at least about 80% identity with a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) comprising a TCR beta chain sequence having at least about 80% identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein said binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity with a K d of about 5×10 −4 M or less; 7) a) a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2; and / or b) comprising a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein said binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity with a K d of about 5×10 −4 M or less; 8) a) a TCR alpha chain variable (V α ) domain sequence selected from the group consisting of the TCR V α domain sequences listed in Table 2; and / or b) comprising a TCR beta chain variable (V β ) domain sequence selected from the group consisting of the TCR V β ) domain sequences listed in Table 2, wherein said binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity with a K d of about 5×10 −4 M or less; or 9) a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a binding protein comprising a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein said binding protein is capable of binding to a MAGEC2 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity with a K d of about 5×10 −4 M or less.

17. 1) The TCR alpha chain CDR, TCR V α the domain, and / or the TCR alpha chain is encoded by a TRAV, TRAJ and / or TRAC gene, or a fragment thereof, selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2; 2) The TCR beta chain CDR, TCR V β the domain, and / or the TCR beta chain is encoded by a TRBV, TRBJ and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ and TRBC genes listed in Table 2; 3) each CDR of said binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof, compared to the cognate reference CDR sequence listed in Table 2; 4) the binding protein is chimeric, humanized or human; 5) the binding protein comprises a binding domain having a transmembrane domain and an effector domain that is intracellular; 6) the TCR alpha chain and the TCR beta chain are covalently linked, and optionally, the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide; 7) The TCR alpha chain and / or the TCR beta chain are covalently linked to a substructure, and optionally the covalently linked substructure is a) comprising an affinity tag or label, optionally wherein the affinity tag is selected from the group consisting of CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin binding protein (CBP), protein C tag, Myc tag, Halo tag, HA tag, Flag tag, His tag, biotin tag and V5 tag, and / or the label is a fluorescent protein; and / or b) is selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof; 8) the binding protein binds to the pMHC complex on the cell surface; 9) The MHC or MHC-peptide complex is as defined in claim 2; 10) binding of said binding protein to said MAGEC2 peptide-MHC (pMHC) complex elicits an immune response, optionally said immune response being selected from the group consisting of: i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell proliferation, cytokine release and / or cytotoxic killing; and / or said binding protein binds to said MAGEC2 immunogenic peptide-MHC (pMHC) complex at about 1x10 -4 M or less, about 5x10 -5 M or less, about 1x10 -5 M or less, about 5x10 -6 M or less, about 1x10 -6 M or less, about 5x10 -7 M or less, about 1x10 -7 M or less, about 5x10 -8 M or less, about 1x10 -8 M or less, about 5x10 -9 M or less, about 1 ... specifically and / or selectively bind with a K d of about 1×10 −10 M or less, about 5×10 −10 M or less, about 1×10 −10 M or less, about 5×10 −11 M or less, about 1×10 −11 M or less, about 5×10 −12 M or less, or about 1×10 −12 M or less; 11) the binding protein has a higher binding affinity for the peptide-MHC (pMHC) compared to a known T cell receptor, optionally the higher binding affinity is at least 1.05-fold higher; 12) compared to a known T cell receptor, the binding protein induces greater T cell proliferation, cytokine release and / or cytotoxic killing when contacted with a target cell having heterozygous expression of MAGEC2, optionally said induction being at least 1.05-fold greater, and further optionally said cytotoxic killing targets cancer cells, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 13) The binding protein of claim 16, wherein the binding protein does not bind to a peptide-MHC (pMHC) complex selected from the group consisting of ALKDVEERV / HLA-A*02, LLFGLALIEV / HLA-A*02, SESIKKKVL / HLA-B*44, and ASSTLYLVF / HLA-B*57.

18. A TCR alpha and / or beta chain selected from the group consisting of the TCR alpha and beta chain sequences listed in Table 2.

19. 1. An isolated nucleic acid molecule comprising: i) hybridizes under stringent conditions to the complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2; ii) a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2; and / or iii) ii) a sequence having at least about 80% homology with a nucleic acid encoding one listed in Table 2 and optionally 1) a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2, and / or 2) A TRBV, TRBJ and / or TRBC gene selected from the group of TRBV, TRBJ and TRBC genes listed in Table 2, or a fragment thereof. Including, Optionally, the nucleic acid is codon optimized for expression in a host cell.

20. 20. A vector comprising the isolated nucleic acid of claim 19, optionally comprising: i) the vector is a cloning vector, an expression vector or a viral vector; ii) the vector comprises a vector sequence listed in Table 3; iii) the vector further comprises a nucleic acid sequence encoding CD8α and / or CD8β, optionally wherein the nucleic acid sequence encoding CD8α or CD8β is operably linked to a nucleic acid encoding a tag, optionally wherein the nucleic acid encoding the tag is 5′ upstream of the nucleic acid sequence encoding CD8α or CD8β such that the tag is fused to the N-terminus of CD8α or CD8β, and further optionally wherein the tag is a CD34 enrichment tag; and / or iv) the vector, wherein the isolated nucleic acid and the nucleic acid sequence encoding CD8α and / or CD8β are interconnected with an internal ribosome entry site or with a nucleic acid sequence encoding a self-cleaving peptide, optionally wherein the self-cleaving peptide is P2A, E2A, F2A or T2A.

21. 20. A host cell comprising the isolated nucleic acid of claim 19, and / or comprising the vector of claim 20 18. A host cell, or a population of said host cells, expressing the binding protein of claim 16 or 17, optionally comprising: 1) the cells are genetically engineered; 2) Includes chromosomal gene knockouts of TCR genes, HLA genes, or both; 3) knockout of an HLA gene selected from the group consisting of the α1 macroglobulin gene, the α2 macroglobulin gene, the α3 macroglobulin gene, the β1 microglobulin gene, the β2 microglobulin gene, and combinations thereof; 4) knockout of a TCR gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and a combination thereof; 5) expressing CD8α and / or CD8β, optionally fused to a CD34 enrichment tag, and further optionally, the host cells are enriched using the CD34 enrichment tag; 6) Hematopoietic progenitor cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells, or immune cells; Optionally, the immune cells are cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + T cells, CD8 + T cells, CD4 / CD8 double negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or combinations thereof; Optionally, the T cells are: a) naive T cells, central memory T cells, effector memory T cells, or a combination thereof; b) is a primary T cell, or a cell of a T cell line; and / or c) does not express an endogenous TCR or has reduced surface expression of the TCR; and / or 7) the host cell is capable of producing a cytokine or cytotoxic molecule when contacted with a target cell comprising a peptide-MHC (pMHC) complex that comprises a MAGEC2 peptide epitope in the context of an MHC molecule; and optionally a) said host cell is contacted with said target cell in vitro, ex vivo or in vivo; b) the cytokine is TNF-α, IL-2 and / or IFN-γ; c) the cytotoxic molecule is perforin and / or a granzyme, optionally, the cytotoxic molecule is granzyme B; d) the host cell is capable of producing higher levels of cytokines or cytotoxic molecules when contacted with a target cell having heterozygous expression of MAGEC2, optionally the host cell is capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules; e) said host cell is capable of killing target cells that contain a peptide-MHC (pMHC) complex that includes said MAGEC2 peptide epitope in the context of an MHC molecule, and optionally i) the killing is determined by a killing assay; ii) the ratio of host cells to target cells in the killing assay is between 20:1 and 1:4; iii) the target cells are target cells pulsed with 1 μg / mL to 50 pg / mL of a MAGEC2 peptide, and optionally the target cells are monoallelic for an MHC matched to the MAGEC2 peptide; iv) the host cell is capable of killing a greater number of target cells when contacted with target cells having heterozygous expression of MAGEC2, and optionally, the cell killing is at least 1.05-fold greater; and / or v) the target cell is a cell line or a primary cell, optionally the target cell is selected from the group consisting of a HEK293 derived cell line, a cancer cell line, a primary cancer cell, a transformed cell line, and an immortalized cell line; f) the MAGEC2 immunogenic peptide is that of claim 1; g) the MHC or MHC-peptide complex is as defined in claim 2; h) i) The host cell or population of host cells, wherein the host cell does not induce T cell proliferation, cytokine release or cytotoxic killing when contacted with a target cell comprising a peptide-MHC (pMHC) complex selected from the group consisting of ALKDVEERV / HLA-A*02, LLFGLALIEV / HLA-A*02, SESIKKKVL / HLA-B*44, and ASSTLYLVF / HLA-B*57, and / or ii) the host cell does not express the MAGEC2 antigen, is not recognized by the binding protein of claim 16, is not of serotype HLA-B*07, does not express an HLA-B*07 allele, is not of serotype HLA-A*24, and / or does not express an HLA-A*24 allele.

22. a) a binding protein according to claim 16 or 17, b) an isolated nucleic acid according to claim 19; c) a vector according to any one of claims 20, d) a host cell according to claim 21, and / or e) a population of host cells according to claim 21, and Carrier A composition comprising:

23. 1. An apparatus or kit comprising: a) a binding protein according to claim 16 or 17, b) an isolated nucleic acid according to claim 19; c) the vector according to claim 20 , d) a host cell according to claim 21, and / or e) A population of host cells according to claim 21. Including, Optionally, reagents for detecting binding of a), d) and / or e) to a pMHC complex. The device or kit.

24. A method for producing a binding protein according to claim 16 or 17, comprising the steps of: (i) culturing a transformed host cell, transformed with a nucleic acid comprising a sequence encoding a binding protein according to claim 16 or 17, under suitable conditions allowing expression of said binding protein; and (ii) recovering the expressed binding protein. The method comprising:

25. 18. A method for producing a host cell expressing a binding protein according to claim 16 or 17, comprising: (i) introducing into said host cell a nucleic acid comprising a sequence encoding a binding protein according to claim 16 or 17; and (ii) culturing the transformed host cell under suitable conditions to allow expression of the binding protein. The method comprising:

26. 1. A method for detecting the presence or absence of a MAGEC2 antigen and / or a cell expressing MAGEC2, comprising: Optionally, the cell is a hyperproliferative cell; The method comprises: At least one binding protein according to claim 16 or 17, At least one host cell according to claim 21, or The population of host cells of claim 21. detecting the presence or absence of said MAGEC2 antigen in a sample by using The detection of the MAGEC2 antigen indicates the presence of the MAGEC2 antigen and / or cells expressing MAGEC2, and optionally 1) the at least one binding protein or the at least one host cell forms a complex with the MAGEC2 peptide in association with an MHC molecule, and the complex is detected by fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, by Western blot, or by intracellular flow assay; 2) the method further comprises obtaining the sample from a subject; 3) the level of reactivity is: A) the presence of a bond; and / or B) T cell activation and / or effector function and optionally i) the T cell activation or effector function is T cell proliferation, killing or cytokine release; and / or ii) the T cell binding, activation and / or effector function is detected by means of fluorescence activated cell sorting (FACS), by means of enzyme-linked immunosorbent assay (ELISA), by means of radioimmunoassay (RIA), immunochemically, by means of Western blot, or by means of an intracellular flow assay.

27. 1. A method for detecting a level of a disorder characterized by MAGEC2 expression in a subject, comprising: a) contacting a sample obtained from the subject with at least one binding protein of claim 16 or 17, at least one host cell of claim 21, or a population of host cells of claim 21; and b) Detecting the level of reactivity Including, The presence or higher level of said reactivity as compared to a control level indicates the level of said disorder characterized by MAGEC2 expression in said subject, and optionally i) the control level is A) is a reference number; or B) levels from subjects not having the disorder characterized by MAGEC2 expression; ii) the level of reactivity is A) the presence of a bond; and / or B) T cell activation and / or effector function and optionally 1) the T cell activation or effector function is T cell proliferation, killing or cytokine release; and / or 2) The method, wherein the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay.

28. 1. A method for tracking and assessing the progression of a disorder characterized by MAGEC2 expression in a subject, comprising: a) detecting in a subject sample the presence or level of reactivity between a sample obtained from said subject and at least one binding protein of claim 16 or 17, at least one host cell of claim 21, or a population of host cells of claim 21; b) repeating step a) at a subsequent time point; and c) comparing the levels of MAGEC2 or of cells of interest expressing MAGEC2 detected in steps a) and b) to follow and assess the progression of said disorder characterized by MAGEC2 expression in said subject. Including, The absence or reduction of MAGEC2 levels or the cells of interest expressing MAGEC2 detected in step b) compared to step a) indicates an inhibition of progression of the disorder characterized by MAGEC2 expression in the subject, The presence or increase of MAGEC2 levels or the cells of interest expressing MAGEC2 detected in step b) compared to step a) indicates the progression of the disorder characterized by MAGEC2 expression in the subject, and optionally i) between the first time point and said subsequent time point, said subject has undergone a therapy for treating said disorder characterized by MAGEC2 expression; ii) the level of reactivity is A) the presence of a bond; and / or B) T cell activation and / or effector function and optionally 1) the T cell activation or effector function is T cell proliferation, killing or cytokine release; and / or 2) The method, wherein the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay.

29. 1. A method for predicting a clinical outcome in a subject suffering from a disorder characterized by MAGEC2 expression, comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein of claim 16 or 17, at least one host cell of claim 21, or a population of host cells of claim 21; and b) comparing the presence or level of said reactivity with that from a control. Including, The control is obtained from a subject with a good clinical outcome, an absence or reduced level of the reactivity in the subject sample compared to the control indicates that the subject will have a good clinical outcome; and optionally, i) the level of reactivity is A) the presence of a bond; and / or B) T cell activation and / or effector function and optionally 1) the T cell activation or effector function is T cell proliferation, killing or cytokine release; and / or 2) The method, wherein the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay.

30. 1. A method for assessing the efficacy of a therapy for a disorder characterized by MAGEC2 expression, comprising: a) determining in a first sample obtained from the subject prior to providing to the subject at least a portion of said therapy for said disorder characterized by MAGEC2 expression, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein of claim 16 or 17, at least one host cell of claim 21, or a population of host cells of claim 21; and b) determining in a second sample obtained from the subject after the provision of said therapy for said disorder characterized by MAGEC2 expression the presence or level of reactivity between the sample obtained from the subject and at least one binding protein of claim 16 or 17, at least one host cell of claim 21, or a population of host cells of claim 21. Including, an absence or decreased level of said responsiveness in said second sample compared to said first sample is an indication that said therapy is effective in treating said disorder characterized by MAGEC2 expression in said subject; the presence or elevated level of said reactivity in said second sample compared to said first sample is an indication that said therapy is ineffective in treating said disorder characterized by MAGEC2 expression in said subject; and optionally i) the level of reactivity is A) the presence of a bond; and / or B) T cell activation and / or effector function and optionally 1) the T cell activation or effector function is T cell proliferation, killing or cytokine release; and / or 2) The method, wherein the T cell binding, activation and / or effector function is detected using fluorescence activated cell sorting (FACS), using enzyme-linked immunosorbent assay (ELISA), using radioimmunoassay (RIA), immunochemically, using Western blot, or using an intracellular flow assay.

31. A composition for use in a method for preventing and / or treating a disorder characterized by MAGEC2 expression, said composition comprising a cell expressing at least one binding protein according to claim 16 or 17, and optionally 1) The composition is administered to a subject; 2) the cells are allogeneic, syngeneic or autologous; 3) the cell is a host cell according to claim 21, or a population of said host cells; 4) the target cell is a cancer cell expressing MAGEC2; 5) the composition further comprises a pharma- ceutically acceptable carrier; 6) the composition induces an immune response against the target cells expressing MAGEC2 in the subject, optionally wherein the composition induces an antigen-specific T cell immune response against the target cells expressing MAGEC2 in the subject, optionally wherein the antigen-specific T cell immune response comprises at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response; 7) the method further comprises administering at least one additional treatment for said disorder characterized by MAGEC2 expression, optionally wherein said at least one additional treatment for said disorder characterized by MAGEC2 expression is administered simultaneously or sequentially with said composition; 8) the disorder characterized by MAGEC2 expression is cancer or its recurrence, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, prostate cancer, multiple myeloma, hepatocellular carcinoma, invasive breast cancer and bladder urothelial carcinoma; and / or 9) The composition, wherein the subject is an animal model of a disorder characterized by MAGEC2 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent.

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