MAGEA1 immunogenic peptides, binding proteins recognizing MAGEA1 immunogenic peptides, and uses thereof

Abstract

Provided herein are MAGEA1 immunogenic peptides, binding proteins that recognize the MAGEA1 immunogenic peptides, and uses thereof.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application Serial No. 63 / 413,560, filed on October 5, 2022, and U.S. Provisional Application Serial No. 63 / 468,842, filed on May 25, 2023; the entire contents of each of said applications are incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION

[0004] Adoptive cell transfer (ACT) using engineered T cells has proven highly efficacious in treating certain types of liquid tumors and holds promise for treating solid tumors. T - cell receptor - engineered T cells (TCR - T) are T cells that express an exogenous TCR capable of recognizing antigens present in cancer cells. The TCR - antigen interaction is a core component of the targeting mechanism by which TCR - T cells kill cancer cells. One of the challenges facing the widespread testing and adoption of TCR - T therapies is the lack of TCR - antigen pairs applicable to a wide range of patients and indications.

[0005] In addition, since it is difficult to discover new TCR - antigen pairs, which typically requires predicting MHC - presented epitopes, the number of antigens being pursued is limited. However, such epitopes may not be immunogenic, making it difficult to identify reactive TCRs, or the epitopes may not be physiologically processed and presented by cancer cells. Thus, there is a great need in the art to identify TCR - antigen pairs in the context of a variety of widely applicable HLA alleles in order to develop useful reagents for diagnosing, prognosticating, treating disorders characterized by expression of said antigens, and screening agents related to said disorders. SUMMARY OF THE INVENTION

[0006] The present invention is at least in part based on the discovery of MAGEA1 immunogenic peptides and binding proteins that recognize such MAGEA1 immunogenic peptides, which discovery is based on an unbiased functional screen for antigens of TCR clonotypes identified from subjects suffering from a disorder associated with MAGEA1 expression (e.g., subjects suffering from melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, or bladder urothelial carcinoma). In the case of a variety of HLA alleles (e.g., HLA-A*02:01), the identified TCRs recognize MAGEA1 immunogenic peptides, such as those listed in Table 1. It is demonstrated herein that MAGEA1 is selectively expressed in cancer and testicular tissues, but not in normal somatic tissues, making it an ideal target for ACT. The ability of MAGEA1 binding proteins (e.g., TCRs as described herein) to bind MAGEA1 immunogenic peptides and initiate an immune response that kills cells expressing MAGEA1 (e.g., cancer cells) demonstrates the utility of such binding proteins in a variety of uses, including methods of diagnosis, prognosis, treatment of disorders characterized by MAGEA1 expression, and screening for agents associated with such disorders.

[0007] In one aspect, there is provided an immunogenic peptide comprising a peptide epitope selected from the peptide sequences listed in Table 1.

[0008] In another aspect, there is provided an immunogenic peptide consisting of a peptide epitope selected from the peptide sequences listed in Table 1.

[0009] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. By way of example, in one embodiment, the immunogenic peptide is derived from the MAGEA1 protein, optionally wherein the immunogenic peptide has a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In another embodiment, the immunogenic peptide is capable of eliciting an immune response in a subject against MAGEA1 and / or cells expressing MAGEA1, 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 expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing.

[0010] In another aspect, there is provided an immunogenic composition comprising at least one immunogenic peptide described herein.

[0011] Also provided are multiple embodiments, which can be applied to any aspect covered by the present invention and / or 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 in a subject against MAGEA1 and / or cells expressing MAGEA1, 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 expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing.

[0012] In yet another aspect, a composition is provided that comprises a peptide epitope selected from the peptide sequences listed in Table 1, and an MHC molecule.

[0013] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally wherein the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In another embodiment, the MHC molecule comprises an MHC α 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, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04,HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In yet another embodiment, the HLA serotype is HLA-A*02, such as HLA-A*02:01.,

[0014] In another aspect, there is provided a stabilized MHC-peptide complex comprising the immunogenic peptide described herein in the context of an MHC molecule.

[0015] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally wherein the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In another embodiment, the MHC molecule comprises an MHC α chain, which is an HLA serotype selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02,HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05, and HLA-C*14:03 alleles. In yet another embodiment, the peptide epitope and the MHC molecule are covalently linked and / or the α-chain and β-chain of the MHC molecule are covalently linked. In another embodiment, the stable MHC-peptide complex comprises a detectable label, optionally wherein the detectable label is a fluorophore.,

[0016] In another aspect, there is provided an immunogenic composition comprising the stable MHC-peptide complex described herein and an adjuvant.

[0017] In yet another aspect, there is provided an isolated nucleic acid encoding the immunogenic peptide described herein, or its complement.

[0018] In another aspect, there is provided a vector comprising the isolated nucleic acid described herein.

[0019] In another aspect, there is provided a cell that: a) comprises the isolated nucleic acid described herein, b) comprises the vector described herein, and / or c) produces one or more of the immunogenic peptides described herein and / or presents one or more of the stable MHC-peptide complexes described herein on the cell surface, optionally wherein the cell is genetically engineered.

[0020] In yet another aspect, there is provided an apparatus or kit comprising: a) one or more of the immunogenic peptides described herein and / or b) one or more of the stable MHC-peptide complexes described herein, the apparatus or kit optionally comprising a reagent for detecting the 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.

[0021] In another aspect, there is provided a method for detecting T cells that bind to a stable MHC-peptide complex, the method comprising: a) contacting a sample comprising T cells with the stable MHC-peptide complex described herein; and b) detecting the 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 (PBMC).

[0022] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the T cell is a CD8+ T cell. In another embodiment, the detection and / or determination is performed using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow cytometry. In another embodiment, the sample comprises T cells that have been contacted with or are suspected of having been contacted with one or more MAGEA1 proteins or fragments thereof.

[0023] In another aspect, provided is a method for determining whether a T cell has been exposed to MAGEA1, the method comprising: a) incubating a cell population comprising T cells with an immunogenic peptide as described herein or a stable MHC-peptide complex as described herein; and b) detecting the presence or level of reactivity, wherein the presence of reactivity or a higher level of reactivity compared to a control level indicates that the T cell has been exposed to MAGEA1, optionally wherein the cell population comprising T cells is obtained from a subject.

[0024] In yet another aspect, provided is a method for predicting the clinical outcome of a subject suffering from a disorder characterized by MAGEA1 expression, the method comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides as described herein or one or more stable MHC-peptide complexes as described herein; and b) comparing the presence or level of reactivity with the reactivity from a control, wherein the control is obtained from a subject with a favorable clinical outcome, wherein the presence of reactivity or a higher level of reactivity in the subject sample compared to the control indicates that the subject has a favorable clinical outcome.

[0025] In another aspect, provided is a method for assessing the efficacy of a therapy for a disorder characterized by MAGEA1 expression, the method comprising: a) determining the presence or level of reactivity between T cells obtained from a subject and one or more immunogenic peptides as described herein or one or more stable MHC-peptide complexes as 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 immunogenic peptides as described herein or one or more stable MHC-peptide complexes as described herein and T cells obtained from the subject, wherein the T cells are present in a second sample obtained from the subject after providing the therapy to the subject, wherein the presence of reactivity or a higher level of reactivity in the second sample compared to the first sample indicates that the therapy is effective in treating the subject's disorder characterized by MAGEA1 expression.

[0026] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or 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 wherein the T cell activation or effector function is T cell proliferation, killing, or cytokine release. In another embodiment, the method further includes repeating steps a) and b) at a subsequent time point, optionally wherein the subject has been treated between the first time point and the subsequent time point to improve a condition characterized by MAGEA1 expression. In another embodiment, T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, western blotting, or intracellular flow cytometry. In yet another embodiment, the control level is a reference number. In another embodiment, the control level is the level of a subject not suffering from a condition characterized by MAGEA1 expression.

[0027] In another aspect, a method of preventing and / or treating a condition in a subject characterized by MAGEA1 expression, the method comprising administering to the subject a therapeutically effective amount of the composition described herein.

[0028] In yet another aspect, a method of 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 is provided, the method comprising: a) providing a cell that presents on its cell surface a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule; 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 the peptide epitope in the context of the MHC molecule.

[0029] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, step a) comprises contacting an MHC molecule on the cell surface 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 the cell using a vector comprising a heterologous sequence encoding the peptide epitope.

[0030] In another aspect, a method of 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 is provided, the method comprising: a) providing a peptide epitope alone or as a stable MHC-peptide complex, which comprises a peptide epitope selected from the peptide sequences listed in Table 1 alone or in the context of an MHC molecule; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to the peptide or the 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 the stable MHC-peptide complex, optionally wherein the MHC or MHC-peptide complex is as described herein.

[0031] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the plurality of candidate peptide-binding molecules comprise antibodies, antigen-binding fragments of antibodies, TCRs, antigen-binding fragments of TCRs, single-chain TCRs (scTCRs), chimeric antigen receptors (CARs), or fusion proteins comprising a TCR and an effector domain. In another embodiment, the plurality of candidate peptide-binding molecules comprise at least 2, 5, 10, 100, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species, 10 9 species or more different candidate peptide-binding molecules. In another embodiment, the plurality of candidate peptide-binding molecules comprise one or more candidate peptide-binding molecules obtained from a sample from a subject or a group of subjects; or the plurality of candidate peptide-binding molecules comprise one or more candidate peptide-binding molecules comprising mutations in a parental scaffold peptide-binding molecule obtained from a sample from a subject. In yet another embodiment, the subject or group of subjects: a) does not have a disorder characterized by MAGEA1 expression and / or has recovered from a disorder characterized by MAGEA1 expression, or b) has a disorder characterized by MAGEA1 expression. In another embodiment, the composition described herein has been administered to the subject or group of subjects. In another embodiment, the subject is an animal model and / or a mammal of a disorder characterized by MAGEA1 expression, optionally wherein 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 MAGEA1 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.

[0032] In another aspect, there is provided a peptide-binding molecule or an antigen-binding fragment thereof identified according to the methods described herein, optionally wherein the peptide-binding molecule or an 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.

[0033] In yet another aspect, there is provided a method of treating a subject having a disorder characterized by MAGEA1 expression, the method comprising administering to the subject a therapeutically effective amount of genetically engineered T cells that express a peptide-binding molecule or an antigen-binding fragment thereof, wherein the peptide-binding molecule or an antigen-binding fragment thereof 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 an 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, optionally wherein the MHC or MHC-peptide complex is as described herein.

[0034] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. By way of example, in one embodiment, the T cells are isolated from: a) a subject, b) a donor not having a disorder characterized by MAGEA1 expression, or c) a donor recovered from a disorder characterized by MAGEA1 expression.

[0035] In another aspect, there is provided a method of treating a subject having a disorder characterized by MAGEA1 expression, the method comprising infusing 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 i) isolating the immune cells from the subject prior to stimulating the immune cells and / or ii) wherein the immune cells comprise PBMCs, T cells, CD8+ T cells, naive T cells, central memory T cells, and / or effector memory T cells.

[0036] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the agent is contacted and placed under conditions and for a time suitable for forming at least one immune complex between a peptide epitope, an immunogenic peptide, a stable MHC-peptide complex, a T cell receptor, and / or an immune cell. In another embodiment, the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor are expressed by a cell and the cell is expanded and / or isolated during one or more steps. In another embodiment, the disorder characterized by MAGEA1 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, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma. In yet another embodiment, the subject is an animal model and / or a mammal of a disorder characterized by MAGEA1 expression, optionally wherein the mammal is a human, a primate, or a rodent.

[0037] In another aspect, there is provided 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, 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.

[0038] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the binding protein comprises: a) a T cell receptor (TCR) α-chain CDR sequence that has at least about 80% identity with a TCR α-chain CDR sequence selected from the group consisting of the TCR α-chain CDR sequences listed in Table 2; and / or b) a TCR β-chain CDR sequence that has at least about 80% identity with a TCR β-chain CDR sequence selected from the group consisting of the TCR β-chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K -4 of less than or equal to about 5 × 10 d M. In another embodiment, the binding protein comprises: a) a TCR α-chain variable (V α ) domain sequence that has 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) a TCR β-chain variable (V β) A domain sequence that has at least about 80% identity with a TCR V domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 β A TCR V domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2 β and has a K of less than or equal to about 5×10 -4 M d . In another embodiment, the binding protein comprises: a) a TCRα chain sequence that has at least about 80% identity with a TCRα chain sequence selected from the group consisting of the TCRα chain sequences listed in Table 2; and / or b) a TCRβ chain sequence that has at least about 80% identity with a TCRβ chain sequence selected from the group consisting of the TCRβ chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d . In yet another embodiment, the binding protein comprises: a) a TCRα chain CDR sequence selected from the group consisting of the TCRα chain CDR sequences listed in Table 2; and / or b) a TCRβ chain CDR sequence selected from the group consisting of the TCRβ chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d . In yet another embodiment, there is provided a binding protein that comprises: a) a TCRα chain variable (V α ) domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2; and / or b) a TCRβ chain variable (V α ) domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 β and has a K of less than or equal to about 5×10 β M -4 M d . In another embodiment, there is provided a binding protein that comprises: a) a TCRα chain sequence selected from the group consisting of the TCRα chain sequences listed in Table 2; and / or b) a TCRβ chain sequence selected from the group consisting of the TCRβ chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 Md 。In another embodiment, 1) the TCR α-chain CDR, TCR V α domain and / or the TCR α-chain are encoded by TRAV, TRAJ, and / or TRAC genes or fragments thereof selected from the group consisting of the TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) the TCR β-chain CDR, TCR V β domain and / or the TCR β-chain are encoded by TRBV, TRBJ, and / or TRBC genes or fragments thereof selected from the group consisting of the TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) compared to the homologous reference CDR sequences listed in Table 2, each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions, or combinations thereof. In 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 intracellular effector domain. In another embodiment, the TCR α-chain and the TCR β-chain are covalently linked, optionally wherein the TCR α-chain and the TCR β-chain are covalently linked via a linker peptide. In another embodiment, the TCR α-chain and / or the TCR β-chain are covalently linked to a moiety, optionally wherein the covalently linked moiety comprises an affinity tag or a label. In yet another embodiment, the affinity tag is selected from the group consisting of: a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin-binding protein (CBP), Protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or wherein the label is a fluorescent protein. In another embodiment, the covalently linked moiety is selected from the group consisting of: a pro-inflammatory factor, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or an antigen-binding fragment thereof. In 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, the binding of the binding protein to the MAGEA1 peptide-MHC (pMHC) complex elicits an immune response, 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 expansion, cytokine release, and / or cytotoxic killing. In another embodiment, the binding protein is capable of binding with a dissociation constant less than or equal to about 1×10 -4 M, less than or equal to about 5×10 -5 M, less than or equal to about 1×10 -5 M, less than or equal to about 5×10 -6 M, less than or equal to about 1×10 -6 M, less than or equal to about 5×10 -7 M, less than or equal to about 1×10 -7 M, less than or equal to about 5×10-8 M, less than or equal to about 1×10 -8 M, less than or equal to about 5×10 - 9 M, less than or equal to about 1×10 -9 M, less than or equal to about 5×10 -10 M, less than or equal to about 1×10 -10 M, less than or equal to about 5×10 -11 M, less than or equal to about 1×10 -11 M, less than or equal to about 5×10 -12 M or less than or equal to about 1×10 -12 K of M dSpecifically and / or selectively binds to a MAGEA1 immunogenic peptide-MHC (pMHC) complex. In another embodiment, the binding protein has a higher binding affinity for the peptide-MHC (pMHC) compared to a known T cell receptor, optionally wherein the higher binding affinity is at least 1.05-fold higher. In another embodiment, when contacting a target cell with MAGEA1 heterozygous expression, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing compared to a known T cell receptor, optionally wherein the induction is at least 1.05-fold higher. As used herein, in some embodiments, references to fold changes can be compared to any reference pattern of interest, such as comparing to different binding proteins; comparing the same binding protein in different contexts, such as in combination with other agents described herein, where the same binding protein is expressed at different levels in different immune cells; and the like. In another embodiment, the cytotoxic killing is directed against target cancer cells. In another embodiment, the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma. In another embodiment, the binding protein does not bind to a peptide-MHC (pMHC) complex comprising a PIEZO1, NBEAL1, NBEAL2, and / or EPN2 peptide epitope. These genes are well-known and are recognized in the art to be annotated according to the following NCBI gene ID numbers, each of which is available on the world wide web at ncbi.nlm.nih.gov / gene: PIEZO1: gene ID 9780 and NM_001142864.4 and NP_001136336.2 as representative clones; NBEAL1: gene ID 65065 and NM_001114132.2 and NP_001107604.1, and NM_001378026.1 and NP_001364955.1 as representative clones; NBEAL2: gene ID 23218 and NM_001365116.2 and NP_001352045.1 and NM_015175.3 and NP_055990.1 as representative clones; EPN2: gene ID 22905 and NM_001102664.2 and NP_001096134.1, NM_014964.5 and NP_055779.2, and NM_148921.4 and NP_683723.2 as representative clones.

[0039] In yet another aspect, provided is a TCR α-chain and / or β-chain, the TCR α-chain and / or β-chain being selected from the group consisting of the TCR α-chain and β-chain sequences listed in Table 2.

[0040] In another aspect, a isolated nucleic acid molecule is provided, the isolated nucleic acid molecule: 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 that is at least about 80% homologous 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 that is at least about 80% homologous to a nucleic acid encoding the nucleic acid listed in Table 2, optionally wherein the isolated nucleic acid molecule comprises 1) a TRAV, TRAJ, and / or TRAC gene selected from the group consisting of the TRAV, TRAJ, and TRAC genes listed in Table 2 or a fragment thereof and / or 2) a TRBV, TRBJ, and / or TRBC gene selected from the group consisting of the TRBV, TRBJ, and TRBC genes listed in Table 2 or a fragment thereof.

[0041] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the nucleic acid is codon-optimized for expression in a host cell.

[0042] In another aspect, a vector is provided, the 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.

[0043] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, a dominant negative TGFβ receptor II (DN-TGFβRII), a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In another embodiment, the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag. In another embodiment, the nucleic acid encoding the tag is at the 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein, such that the tag is fused to the N-terminus of CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker. In yet another embodiment, the tag is a CD34 enrichment tag. In another embodiment, the isolated nucleic acid described herein alone or in combination with the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker is ligated to an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide. In another embodiment, the self-cleaving peptide is P2A, E2A, F2A, or T2A.

[0044] In yet another aspect, provided is a host cell comprising the isolated nucleic acid described herein, comprising the vector described herein, and / or expressing the binding protein described herein, optionally wherein the cell is genetically engineered.

[0045] Also provided are multiple embodiments that can be applied to any aspect covered by the present invention and / or 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 the following: alpha-1 macroglobulin gene, alpha-2 macroglobulin gene, alpha-3 macroglobulin gene, beta-1 microglobulin gene, beta-2 microglobulin gene, and combinations thereof. In another embodiment, the host cell comprises a knockout of a TCR gene selected from the following: TCR alpha variable region gene, TCR beta variable region gene, TCR constant region gene, and combinations thereof. In yet another embodiment, the host cell expresses CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR, and further optionally wherein CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker are fused to a CD34 enrichment tag. In another embodiment, the host cell is enriched using a CD34 enrichment tag. In another embodiment, the host cell is a hematopoietic progenitor cell, peripheral blood mononuclear cell (PBMC), cord blood cell, or immune cell. In yet another embodiment, the immune cell is a T cell, cytotoxic lymphocyte, cytotoxic lymphocyte precursor cell, cytotoxic lymphocyte progenitor cell, cytotoxic lymphocyte stem cell, CD4 + T cell, CD8 +T cells, CD4 / CD8 double-negative T cells, γδ (gammadelta) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or combinations thereof. In another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or combinations thereof. In another embodiment, the T cells are primary T cells or cells of a T cell line. In another embodiment, the T cells do not express endogenous TCR or have low surface expression of endogenous TCR. In another embodiment, the host cell is capable of producing cytokines or cytotoxic molecules upon contact with a target cell comprising a peptide-MHC (pMHC) complex comprising a MAGEA1 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 another embodiment, the cytokines are TNF-α, IL-2, and / or IFN-γ. In another embodiment, the cytotoxic molecules are perforin and / or granzyme, optionally wherein the cytotoxic molecule is granzyme B. In another embodiment, the host cell is capable of producing higher levels of cytokines or cytotoxic molecules upon contact with a target cell having heterozygous expression of MAGEA1. In another embodiment, the host cell is capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules. In another embodiment, the host cell is capable of killing a target cell comprising a peptide-MHC (pMHC) complex comprising a MAGEA1 peptide epitope in the context of an MHC molecule. In another embodiment, the killing is measured by a killing assay. In another embodiment, the ratio of host cells to target cells in the killing assay is from 20:1 to 1:4. In another embodiment, the target cells are target cells pulsed with 1 μg / mL to 50 pg / mL of MAGEA1 peptide, optionally wherein the target cells are single-allelic cells of an MHC that matches the MAGEA1 peptide. In another embodiment, the host cell is capable of killing a higher number of target cells upon contact with a target cell having heterozygous expression of MAGEA1, optionally wherein the cell killing is at least 1.05-fold higher. In another embodiment, the target cells are cell lines or primary cells, optionally wherein the target cells are selected from the group consisting of HEK293-derived cell lines, cancer cell lines, primary cancer cells, transformed cell lines, and immortalized cell lines. In another embodiment, the MAGEA1 immunogenic peptide is as described herein and / or wherein the MHC or MHC-peptide complex is as described herein. In another embodiment, the host cell does not induce T cell expansion, cytokine release, or cytotoxic killing upon contact with a target cell comprising a peptide-MHC (pMHC) complex comprising a PIEZO1, NBEAL1, NBEAL2, and / or EPN2 peptide epitope.In another embodiment, the host cell does not express the MAGEA1 antigen, is not recognized by the binding proteins described herein, does not belong to the HLA-A*02 serotype, and / or does not express the HLA-A*02 allele.

[0046] In another aspect, a population of the host cells described herein is provided.

[0047] In another aspect, a composition is provided, the composition comprising a) a binding protein described herein, b) an isolated nucleic acid described herein, c) a vector described herein, d) a host cell described herein, and / or e) a population of the host cells described herein, and a carrier.

[0048] In yet another aspect, an apparatus or a kit is provided, the apparatus or the kit comprising a) a binding protein described herein, b) an isolated nucleic acid described herein, c) a vector described herein, d) a host cell described herein, and / or e) a population of the host cells described herein, and the apparatus or the kit optionally comprises a reagent for detecting the binding of a), d), and / or e) to the pMHC complex.

[0049] In another aspect, a method for producing the binding protein described herein is provided, wherein the method comprises the steps of: (i) culturing a transformed host cell under conditions suitable to permit the expression of the binding protein, the host cell having been transformed with a nucleic acid comprising a sequence encoding the binding protein described herein; and (ii) recovering the expressed binding protein.

[0050] In another aspect, a method for producing a host cell expressing the binding protein described herein is provided, wherein the method comprises the steps of: (i) introducing into a host cell a nucleic acid comprising a sequence encoding the binding protein described herein; and (ii) culturing the transformed host cell under conditions suitable to permit the expression of the binding protein.

[0051] In yet another aspect, a method for detecting the presence or absence of the MAGEA1 antigen and / or cells expressing MAGEA1 is provided, optionally wherein the cells are hyperproliferative cells, the method comprising detecting the presence or absence of the MAGEA1 antigen in a sample by using at least one binding protein described herein, at least one host cell described herein, or a population of the host cells described herein, wherein detection of the MAGEA1 antigen indicates the presence of the MAGEA1 antigen and / or cells expressing MAGEA1.

[0052] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, at least one binding protein or at least one host cell forms a complex with a MAGEA1 peptide in the context of an MHC molecule, and the complex is detected in the form of fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting, or intracellular flow cytometry. In another embodiment, the method further comprises obtaining a sample from a subject.

[0053] In another aspect, provided is a method for detecting the extent of a disorder characterized by MAGEA1 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 a level of reactivity, wherein the presence or a higher level of reactivity compared to a control level indicates the extent of the disorder characterized by MAGEA1 expression in the subject.

[0054] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or 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 not suffering from a disorder characterized by MAGEA1 expression.

[0055] In another aspect, provided is a method for monitoring the progression of a disorder characterized by MAGEA1 expression in a subject, the method comprising: a) detecting 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 in a sample from the subject; b) repeating step a) at a subsequent time point; and c) comparing the MAGEA1 levels or the cells of interest expressing MAGEA1 detected in steps a) and b) to monitor the progression of the disorder characterized by MAGEA1 expression in the subject, wherein the absence or decrease of the MAGEA1 levels or the cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates that the progression of the disorder characterized by MAGEA1 expression in the subject is inhibited, and the presence or increase of the MAGEA1 levels or the cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates that the disorder characterized by MAGEA1 expression in the subject is progressing.

[0056] Multiple embodiments are also provided, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, a subject has been treated between a first time point and a subsequent time point to treat a disorder characterized by MAGEA1 expression.

[0057] In yet another aspect, a method for predicting the clinical outcome of a subject suffering from a disorder characterized by MAGEA1 expression is provided, the method 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 the reactivity from a control, wherein the control is obtained from a subject with a favorable clinical outcome; wherein the absence of reactivity or a decrease in the level of reactivity in the subject sample compared to the control indicates that the subject has a favorable clinical outcome.

[0058] In another aspect, a method for assessing the efficacy of a therapy for a disorder characterized by MAGEA1 expression is provided, the method 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 in a first sample obtained from the subject prior to providing at least a portion of the therapy for a disorder characterized by MAGEA1 expression, and b) 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 in a second sample obtained from the subject after providing the therapy for a disorder characterized by MAGEA1 expression, wherein the absence of reactivity or a decrease in the level of reactivity in the second sample compared to the first sample indicates that the therapy effectively treats the subject's disorder characterized by MAGEA1 expression, and wherein the presence of reactivity or an increase in the level of reactivity in the second sample compared to the first sample indicates that the therapy does not effectively treat the subject's disorder characterized by MAGEA1 expression.

[0059] A number of embodiments are also provided, which can be applied to any aspect covered by the present invention and / or combined with any other embodiments 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 wherein the T cell activation or effector function is 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), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, western blotting, or intracellular flow cytometry.

[0060] In another aspect, a method of preventing and / or treating a disorder characterized by MAGEA1 expression is provided, the method comprising contacting a target cell expressing MAGEA1 with a therapeutically effective amount of a composition comprising cells expressing at least one of the binding proteins described herein, optionally wherein the composition is administered to a subject.

[0061] A number of embodiments are also provided, which can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the cells are allogeneic cells, syngeneic cells, or autologous cells. In another embodiment, the cells are the host cells described herein or a population of the host cells described herein. In another embodiment, the target cells are cancer cells expressing MAGEA1. In yet another embodiment, the cell composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the cell composition induces an immune response against the target cells expressing MAGEA1 in a subject. In another embodiment, the cell composition induces an antigen-specific T cell immune response against the target cells expressing MAGEA1 in a subject. In yet another embodiment, the antigen-specific T cell immune response comprises CD4 + helper T lymphocyte (Th) response and at least one of CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the method further comprises administering at least one additional treatment for a disorder characterized by MAGEA1 expression, optionally wherein the at least one additional treatment for a disorder characterized by MAGEA1 expression is administered simultaneously or sequentially with the composition. In another embodiment, the disorder characterized by MAGEA1 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, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma. In yet another embodiment, the subject is an animal model and / or a mammal of a disorder characterized by MAGEA1 expression, optionally wherein the mammal is a human, a primate, or a rodent. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Certain working examples and figures refer to certain control TCRs, such as a) "Comparator", also known as "Comparator 1", which corresponds to the Immatics-based TCR further described herein, for example in Table 4, and b) "Comparator 2", which corresponds to the T-Knife-based TCR further described herein, for example in Table 4.

[0063] Figure 1A and 1B The identification of 1676 MAGEA1 278-286 specific TCRs is shown. Figure 1A A co-culture system is shown. Briefly, on day -4, CD14 + monocytes were isolated from PBMCs of HLA-A*02:01 healthy donors and differentiated into mature DCs. On day -1, naive CD8 T cells were isolated from autologous PBMCs and rested overnight. As part of the multiplex screening, after 3-hour pulsed delivery of 1 μg / mL MAGEA1 278-286 peptide to DCs, the naive CD8 T cells were co-cultured with DCs, followed by an 11-day cell expansion period. Figure 1B A screening process is shown. Dextramer staining was performed with HLA-A*02:01-restricted MAGEA1 278-286 (KVLEYVIKV)dextramer to identify clones and sort MAGEA1 278-286 specific cells. The isolated T cells were sequenced using the 10X Genomics platform and the TCR α and β chains were paired.

[0064] Figure 2A and 2B The selection of 30 TCRs from 500 TCRs by multiple rounds of VAYG screening for functional assessment is shown. Figure 2A The T cell cytotoxicity against NCIH1703 (HLA-A*02:01 + MAGEA1) targets at an E:T of 5:1 is shown. Figure 2B The selection of 30 TCRs from 500 TCRs by multiple rounds of VAYG screening for functional assessment is shown.

[0065] Figures 3A - 3E The results of selecting MAGEA1 278-286 TCRs based on expression and cytotoxic function are shown. Figure 3A The expression of MAGEA1 TCR on the surface of engineered T cells is shown. Figure 3B The T cell cytotoxicity against NCIH1703 (HLA-A*02:01 + MAGEA1) targets at an effector cell to T cell (E:T) ratio of 4:1 is shown.Figure 3C Shows the T cell cytotoxicity of the Hs936T (HLA-A*02:01 + MAGEA1) target at an E:T of 4:1. Figure 3D Shows the T cell cytotoxicity of the A375 (HLA-A*02:01 + MAGEA1) target at an E:T of 4:1. Figure 3E Shows the T cell cytotoxicity of the HEK293T (HLA-A*02:01 - MAGEA1) target at an E:T of 4:1.

[0066] Figures 4A - 4F Shows the 278-286 functional evaluation of the MAGEA1 Figure 4A TCR. Shows the expression of MAGEA1 278-286 TCR 1134 and TCR 1479 on the surface of engineered T cells. Figure 4B Shows the cytokine production of TCR 1134 in response to the HLA-A*02:01 + MAGEA1 + / - target. Figure 4C Shows the cytokine production of TCR 1479 in response to the HLA-A*02:01 + MAGEA1 + / - target. Figure 4D Shows the T cell cytotoxicity of the NCIH1703 (HLA-A*02:01 + MAGEA1) target at an E:T of 4:1. Figure 4E Shows the T cell cytotoxicity of the Hs936T (HLA-A*02:01 + MAGEA1) target at an E:T of 4:1. Figure 4F Shows the T cell cytotoxicity of the HEK293T (HLA-A*02:01 - MAGEA1) target at an E:T of 4:1.

[0067] Figure 5 Shows the peptide dilution curve of TCR MAGE-A1-1479.

[0068] Figure 6 Shows the putative off-targets of TCR MAGE-A1-1479 identified by proprietary whole-genome screening.

[0069] Figure 7 Shows that TCR MAGE-A1-1479 did not show alloreactivity to 109 out of 110 MHCs tested.

[0070] Figures 8A - 8D Shows that MAGE-A1-1479 did not show reactivity to healthy human primary cells.

[0071] Figure 9Shows the pMHC dose-dependent function of the process representative TSC-204-A0201 TCR-T cells. T2 cells were pulsed with various concentrations of MAGE-A1 peptide and co-cultured with three batches of process representative TSC-204-A0201 TCR-T cells. The figure shows IFN-γ secretion as a readout of TCR-T cell reactivity to various homologous peptide doses. Note: IFN-γ was normalized, where 0% was based on the minimum mean in each dataset (n = 3) and 100% was based on the maximum mean in each dataset. Results are presented as percentages. A non-linear regression fit was used to represent the "normalized response" model.

[0072] Figure 10 A-10E shows that TSC-204-A0201 TCR-T cells secrete granzyme B and the inflammatory cytokines IFN-γ, TNF-α, and IL-2 in a target-dependent manner. Granzyme B and inflammatory cytokines in the supernatants of co-cultures (E:T 1:1) of TSC-204-A0201 TCR-T cells (orange (i.e., each pair of left columns)) or untransfected (UTF) control T cells from matched donors (gray) with the indicated cell lines were quantified using automated ELISA (ELLA). The dashed line indicates the cytokine and granzyme B levels of unstimulated TCR-T cells, i.e., TCR-T cells not co-cultured with cancer cell lines. Note that for some conditions, the baseline was too low to be shown on the graph. Additionally, for the UTF controls, some values were below the detection level (indicated by asterisks). Note that different Y-axis scales were used to depict strong ( Figure 10 A-10C) and weak or absent ( Figure 10 D and 10E) cytokine and granzyme B responses.

[0073] Figure 11 A-11E shows that both helper (CD4 + ) T cells and cytotoxic (CD4 - ) T cells in TSC-204-A0201 proliferate in a target-dependent manner. TSC-204-A0201 TCR-T cells were labeled with CTV dye and co-cultured with the indicated cancer cell lines for 3.5 days. Subsequently, the transduced fraction of TSC-204-A0201 TCR-T cells (i.e., CD34 + ) and helper T cells (CD34 + CD4 + ) and cytotoxic T cells (CD34 + CD4 -(CTV dye dilution (indicating proliferation) within (orange (darkest shade)). Percentage of TSC-204-A0201 T cells indicating one, two, or three or more cycles. Proliferation in untransfected (UTF) control T cells (gray) from a matched donor was also assessed. For the UTF control, CD34 - CD4 + and CD34 - CD4 - proliferation in the population. The three dashed lines represent the baseline proliferation of UTF controls matched from three donors: PD269 (low), PD272 (medium), and PD274 (high).

[0074] Figure 12A and 12B show that TSC-204-A0201 TCR-T cells display potent and selective killing activity. Figure 12A shows the analysis of the cytotoxic potential of three batches of process representative TSC-204-A0201 TCR-T cells (orange (shaded), circles) and untransfected (UTF) control T cells (gray, circles) from a matched donor against the indicated target cell lines in a -based cytotoxicity assay. Effector TCR-T cells and target cells were co-cultured across a range of effector cell to target cell ratios (E:T ranging from 10:1 to 0.3:1), and the growth of target cells was measured over a 72-hour period. The data presented were obtained from TSC-204-A0201 TCR-T cells and UTF from batch PD272 and are representative of data obtained from all 3 batches of process representative material tested. Target cells cultured alone are shown as a negative control (black, triangles). Figure 14 Panel B shows the cytotoxic activity of three batches of process representative TSC-204-A0201 TCR-T cells over a 72-hour period and is summarized as normalized target cell growth, which is calculated as the ratio of the area under the curve (AUC) of target cell growth co-cultured with TSC-204-A0201 of the indicated batch at an E:T of 5:1 for 72 hours compared to the area under the curve of target cell growth under the same conditions co-cultured with a matched UTF control T cell.

[0075] Figure 13A-13D shows that the expression of DN-TGFβRII confers resistance to TGFβ-mediated inhibition of target-induced cytokine and granzyme B secretion. After pre-incubating with 0 or 5 ng / mL TGFβ for 20 hours, TSC-204-A0201 TCR-T cells were incubated continuously with target cells in two rounds: to deplete pre-formed cytokine mRNA and granzyme B protein. First, the TCR-T cells were incubated with HLA-A*02:01-positive and MAGE-A1-positive U266B1 cells for 20 hours. Then the TCR-T cells were briefly centrifuged, the supernatant was discarded, and the TCR-T cells were re-incubated with the second-round target cells U266B1 (HLA-A*02:01-positive and MAGE-A1-positive) or LOUCY (HLA-A*02:01-positive, MAGE-A1-negative) indicated in the figure for another 20 hours. The TGFβ concentration was maintained at 0 or 5 ng / mL throughout the two-round co-culture. At the end of the second-round co-culture, cytokines (IFN-γ, TNF-α, and IL-2) and granzyme B secretion were evaluated using automated ELISA (ELLA). The DN-TGFβRII-negative TSC-204-A0201 TCR-T cells (D5662) shown in black (i.e., the left two columns) correspond to the similarly processed TCR-T cells generated in Example 15 and are included here as a control for TGFβ inhibition. The cytokine and granzyme B secretion of three batches of process-representative TCR-T cells (batches PD269, PD272, and PD274) are shown in orange (i.e., the right two columns).

[0076] Figure 14 Shows the vaccination, dosing, and analysis schedules for the animals in Groups 1-5.

[0077] Figure 15 Shows the vaccination, dosing, and sampling schedules for the animals in Groups 6-7.

[0078] Figure 16A and 16B Shows the in vivo efficacy of TSC-204-A0201 TCR-T cells. NCG mice were subcutaneously inoculated with U266B1. Once tumor transplantation was successful (21 days after inoculation, the tumors reached an average of 100 mm 3 ), the animals were randomly assigned to different treatment groups. Then the animals received two intravenous injections of process-representative TSC-204-A0201 TCR-T cells (2 batches tested, PD269 and PD272) or untransfected (UTF) control T cells from a matched donor or vehicle (PBS) on Day 1 and Day 8 of the study (arrows). For each batch, the total number of cells injected corresponded to 2E7 CD34 + . Figure 16ARepresents the average tumor volume of different groups over time. Figure 16B Shows the tumor growth of individual mice in each group over time, separating the two batches of TSC-204-A0201.

[0079] Figure 17 Shows the average body weight development over time across different groups. NCG mice were subcutaneously inoculated with U266B1. Once tumor transplantation was successful (21 days after inoculation, the average tumor reached 100 mm 3 ), the animals were randomly assigned to different treatment groups. Then the animals received two intravenous injections of the process representative TSC-204-A0201 TCR-T cells (two batches tested, PD269 and PD272) or untransfected (UTF) control T cells or vehicle (PBS) from a matched donor on day 1 and day 8 (arrows) of the study. The body weight (BW) of the animals was measured every 3 days after the start of treatment. The average body weight of each treatment group is shown.

[0080] Figure 18A and 18B Shows T cell persistence in peripheral blood. Blood samples were taken on the indicated days after the start of treatment and analyzed by flow cytometry to identify murine (mCD45) and human (hCD45) cells. The CD34 positivity of human cells was further analyzed. The figure shows the percentage of human CD45 Figure 18A immune cells and ( + ) human immune cells positive for CD34 ( Figure 21B ) in the blood of mice given TSC-204-A0201 TCR-T cells from batches PD269 and PD272. Figure 18B )

[0081] Figure 19 Shows the steps and timeline of the cytokine assay to test the off-tumor reactivity of TSC-204-A0201 TCR-T cells.

[0082] Figure 20 Shows the expression of MAGE-A1 and putative off-targets of the therapeutic TCR used in TSC-204-A0201 TCR-T cells in cancer cell lines. NA was extracted from the cancer cell lines and sequenced. The heatmap shows TPM (transcripts per million) calculated based on the counts. The scale used in the RNAseq heatmap sets zero TPM values to white, and values above zero follow a continuous color density scale up to 100 TPM.

[0083] Figures 21A - 21CIt was shown that TSC-204-A0201 TCR-T cells did not show reactivity against HLA-A*02:01+ cancer cell lines expressing TCR off-targets. TSC-204-A0201 TCR-T cells and donor-matched UTF cells were co-cultured with a panel of cancer cell lines, and the IFN-γ levels in the supernatants were evaluated as a measure of T cell reactivity.

[0084] Figure 22 It shows the expression of MAGE-A1 and off-targets of the therapeutic TCR used in TSC-204-A0201 TCR-T cells in primary cells and iPSC-derived cells. NA was extracted from the cells and sequenced. TPM (transcripts per million) was calculated based on the counts, and the average TPM of replicates of the same cell type was calculated. The color scale used in the RNAseq heatmap sets zero TPM values to white, and values above zero follow a continuous color density scale up to 100 TPM.

[0085] Figures 23A - 23C Representative graphs indicating that TSC-204-A0201 TCR-T cells did not show reactivity against HLA-A*02:01+ primary cells are provided. TSC-204-A0201 TCR-T cells and donor-matched UTF cells were co-cultured with a panel of primary cells, and the IFN-γ levels in the supernatants were evaluated as a measure of T cell reactivity.

[0086] Figure 24 It shows the procedure and timeline of the oncogenicity assay used to evaluate cytokine dependence of proliferating T cells.

[0087] Figure 25 It shows the results of T cell viability analysis. The data show the normalized (using Count Bright beads) numbers of live (eFlour660 negative) UTF-T cells (gray bars; right bar of each pair) and TSC-204-A0201 TCR-T cells (orange bars; left bar of each pair) from donor PD268, donor PD269, and donor PD272 after 5 days of in vitro culture in the absence (-) or presence (+) of cytokines and ImmunoCult. The dotted line represents the initial cell number (80,000) used in the assay. ****p≤0.0001; ***p≤0.001; **p≤0.01; *p≤0.05; 'ns' means not significant, p>0.05.

[0088] Figure 26Shows the results of T cell proliferation analysis. The data show the normalized (using Count Bright beads) numbers of proliferating (dividing) UTF-T cells (gray bars; right bars of each pair) and TSC-204-A0201 TCR-T cells (orange bars; left bars of each pair) from donors PD268, donor PD269, and donor PD272 after 5 days of in vitro culture in the absence (-) or presence (+) of cytokines and ImmunoCult. ****p≤0.0001; ***p≤0.001; **p≤0.01; *p≤0.05; 'ns' means not significant, p>0.05.

[0089] Figure 27 Shows the percentage of dividing cells from the proliferation assay. The data show the percentage (%) of proliferating (dividing) UTF-T (gray bars; right bars of each pair) and TSC-204-A0201 TCR-T (orange bars; left bars of each pair) gated live cells from donors PD268, donor PD269, and donor PD272 after 5 days of culture in the absence (-) or presence (+) of cytokines and ImmunoCult. ****p≤0.0001; ***p≤0.001; **p≤0.01; *p≤0.05; 'ns' means not significant, p>0.05.

[0090] Figure 28 Shows MAGE-A1 expression in 48 normal human organs.

[0091] Figure 29 Shows MAGE-A1 expression in 24 different brain tissues.

[0092] Figure 30 Shows a schematic diagram of the expression vector used for TSC-204-A0201 TCR-T cell engineering.

[0093] Figure 31 A and 31B show that the expression of DN-TGFβRII confers resistance to TGFβ-mediated inhibition of target-induced cytokine and granzyme B secretion. After pre-incubating with 0 or 5 ng / mL TGFβ for 20 hours, TCR-T cells were continuously incubated with target cells in two rounds: to deplete pre-formed cytokine mRNA and granzyme B protein, TCR-T cells were first incubated with MAGE-A1-positive U266B1 cells for 20 hours. Then the TCR-T cells were briefly centrifuged, the supernatant was discarded, and the TCR-T cells were incubated with a second round of target cells, MAGE-A1-positive U266B1 cells ( Figure 31 A) or MAGE-A1-negative LOUCY cells ( Figure 31B) Incubate for another 20 hours. The TGFβ concentration was maintained at 0 or 5 ng / mL throughout the two rounds of co-culture. At the end of the second round of co-culture, cytokines (IFN-γ, TNF-α, and IL-2) and granzyme B secretion were evaluated using automated ELISA (ELLA). TCR-T cells engineered from two donors (D5662 and D6418) were evaluated. In addition, for each donor, TCR-T cells expressing DN-TGFβRII were compared with TCR-T cells lacking DN-TGFβRII expression (see the legend below the figure). The cytokine and granzyme B responses of TSC-204-A0201 TCR-T cells were depicted.

[0094] Figure 32 A-32F shows prevention of TGFβ-mediated inhibition of T cell expansion and proliferation in TSC-204-A0201 TCR-T cells expressing DN-TGFβRII. T cells from two donors (D5662 and D6418) were engineered with the clinical TSC-204-A0201 vector or a vector identical to the clinical vector but lacking the DN-TGFβRII gene. Then, DN-TGFβRII-positive and DN-TGFβRII-negative TSC-204-A0201 TCR-T cells were labeled with Cell Trace Violet dye (CTV) and co-cultured with cancer cell lines expressing HLA-A*02:01 and MAGE-A1 ( Figure 32 A and 32B, SW1271; Figure 32 C and 32D, HS936T) or cell lines expressing HLA-A*02:01 but negative for MAGE-A1 ( Figure 32 E and 32F, LOUCY) for 3.5 days. TGFβ was added to the co-cultures at a final concentration of 0 or 5 ng / mL. At the end of the co-culture, cell counts (left panel) and proliferation (right panel) were assessed by flow cytometry. Based on the CTV dye dilution, the total percentage of proliferating cells, as well as the percentage of cells that had undergone one, two, or three or more cell cycles, were quantified as indicated in the legend below the proliferation plot.

[0095] Figure 33A and 33B shows the co-culture of TSC-204-A0201 TCR-T cells with U266B1 cells. T cells from two donors (D5662 and D6418) were engineered with DN-TGFβRII-positive or DN-TGFβRII-negative TSC-204-A0201 TCR-T cells. Then, DN-TGFβRII-positive and DN-TGFβRII-negative TCR-T cells were Violet dye (CTV) was used to label and co-cultured with the MAGE-A1, HLA-A*02:01 positive cell line U266B1 for 3.5 days. TGFβ was added to the co-culture at a final concentration of 0 or 5 ng / mL. At the end of the co-culture, the cell count (left panel) and proliferation (right panel) of TSC-204-A0201 TCR-T cells were assessed by flow cytometry. Based on the CTV dye dilution, the total percentage of proliferating cells, as well as the percentage of cells that had undergone one, two, or three or more cell cycles, were quantified as indicated in the legend below the proliferation plot.

[0096] Figure 34 It is shown that addition of DN-TGFbRII to TCR-T cells enables target-dependent proliferation in the presence of TGFb.

[0097] Figure 35 It is shown that expression of DN-TGFβRII has little effect on the cytotoxic activity of TSC-204-A0201 TCR-T cells. Cytotoxicity assays were performed with TSC-204-A0201 TCR-T cells expressing DN-TGFβRII or not (orange circles (light shade), DN-TGFβRII positive; black circles, DN-TGFβRII negative) based on The MAGE-A1 positive target cells (SW1271, HS936T or AU565) were co-cultured with TCR-T cells at variable effector to target cell ratios (0.04 - 20). TGFβ was added at a final concentration of 0 or 5 ng / mL and target cell growth was measured at 72 hours. The figure depicts the area under the curve (AUC) plotted against the E:T ratio.

[0098] Figure 36 It is shown that DN-TGFβRII enhances the in vivo activity duration.

[0099] Figure 37 The figure of the pNVVD 136 (i.e., pNVVD136_TSC-204-A02_TCR-1479_MSCV-TCR-1479-CD8-EF1α-dnTGFbRII-DHFR) vector is shown. CD: Cluster of differentiation. RNA-OUT: Antisense RNA against the bacterial levansucrase encoded by sacB. SV: Simian virus. TCR: T cell receptor, TIR: Terminal inverted repeat sequence, QBend: Mouse anti-human CD34 antibody, dnTGFbRII: Dominant negative TGFβ receptor II, DHFR: Dihydrofolate reductase selection marker.

[0100] Figure 38Shows a diagram of the pNVVD 166 (i.e., pNVVD166_TSC-204-A02_TCR-1479_MSCV-TCR-1479-CD8-EF1a-DHFR) vector. CD: Cluster of Differentiation. RNA-OUT: Antisense RNA against bacterial levansucrase encoded by sacB. SV: Simian virus. TCR: T cell receptor, TIR: Terminal Inverted Repeat, QBend: Mouse anti-human CD34 antibody, DHFR: Dihydrofolate reductase selection marker.

[0101] Unless otherwise indicated, for any figure showing a bar graph, curve, or other data related to a legend, each bar, curve, or other data presented from left to right directly and sequentially corresponds to the boxes in the legend from top to bottom or from left to right. Detailed Description

[0102] The present invention is at least partially based on the discovery of MAGEA1 immunogenic peptides (e.g., peptides comprising or consisting of the sequences listed in Table 1), binding proteins that recognize the MAGEA1 antigen (e.g., binding proteins having the sequences listed in Table 2), and their uses. A systematic comprehensive investigation was conducted to determine the precise T cell targets recognized by the initial T cell pool of interest.

[0103] Accordingly, the present invention is in part directed to the identified epitopes (immunodominant peptides) of the MAGEA1 protein relevant to therapy and related compositions (e.g., immunodominant peptides, vaccines, etc.); compositions comprising immunogenic peptides alone or together with MHC molecules; stabilized MHC-peptide complexes; methods for diagnosing, prognosticating, and monitoring the immune response to disorders characterized by MAGEA1 expression; and methods for preventing and / or treating disorders characterized by MAGEA1 expression. The present invention is also in part directed to the 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, prognosticating, and monitoring the response of T cells to cells expressing MAGEA1; and methods for preventing and / or treating disorders characterized by MAGEA1 expression.

[0104] I. Definitions

[0105] For convenience, certain terms used in this specification, examples, and the appended claims are collected herein.

[0106] The article "a / an" is used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "an element" means one element or more than one element. In addition, unless otherwise specified, references to the tables provided herein cover all sub-tables of the table.

[0107] The term "administer" means to provide an agent or composition to a subject and includes, but is not limited to, administration by a medical professional and self-administration. This involves physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. In some embodiments, the administration routes of the binding proteins described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, intraspinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the binding proteins described herein may be administered via a non-parenteral route, such as a topical, epidermal, or mucosal administration route, such as intranasal, oral, vaginal, rectal, sublingual, or topical. Administration may also be carried out, for example, once, multiple times, and / or over one or more extended time periods.

[0108] 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 the MAGEA1 antigen or a fragment thereof for which a protective or therapeutic immune response is desired. An "epitope" is the part of the antigen to which a natural or synthetic substance binds.

[0109] As used herein, the term "adjuvant" refers to a substance that promotes, prolongs, and / or enhances the quality and / or intensity of an immune response to an antigen when administered before, simultaneously with, or after the administration of the antigen, as compared to the administration of the antigen alone. Adjuvants can increase the magnitude and duration of the immune response induced by vaccination.

[0110] The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H) and a heavy chain constant region. In some naturally occurring antibodies, the heavy chain constant region consists of three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region consists of one domain, CL. V H and V L regions can also be further divided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). V H and V L Each consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: 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 can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0111] 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).

[0112] As used herein, the term "antigen-binding portion" of a binding protein such as a TCR refers to one or more portions of the TCR that retain the ability to bind (e.g., specifically and / or selectively) an antigen (e.g., the MAGEA1 antigen). The length of such portions is, for example, from about 8 to about 1,500 amino acids, suitably from about 8 to about 745 amino acids, suitably from about 8 to about 300, for example from about 8 to about 200 amino acids, or from about 10 to about 50 or 100 amino acids. It has been shown that the antigen-binding function of the TCR can be performed by fragments of the full-length TCR. Examples of binding portions encompassed by the term "antigen-binding portion" of the TCR include: (i) an Fv fragment consisting of the V α and V β domains of the TCR; (ii) isolated complementarity-determining regions (CDRs); or (iii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. In addition, although V α and V β are encoded by separate genes, they can be joined by recombinant methods using a synthetic linker such that they are able to form a single protein chain, in which V α and V βThe regions pair to form a single-chain molecule (referred to as a single-chain TCR (scTCR)). Such single-chain TCRs are also intended to be encompassed within the "antigen-binding portion" of the term TCR. These TCR fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as for full-length binding proteins. The antigen-binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0113] The terms "complementary determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to certain contiguous amino acid sequences within, for example, the variable regions of TCRs, which confer antigen specificity and / or binding affinity. For TCRs, generally, there are 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 the major CDR responsible for recognition of processed antigen. CDR1 and CDR2 interact mainly with MHC.

[0114] 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 fluid or pre-ejaculatory semen, chyle, chyme, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubricant, vitreous humor, vomitus). In some embodiments, the body fluid contains immune cells, optionally wherein the immune cells are cytotoxic lymphocytes, such as cytotoxic T cells and / or NK cells, CD4+ T cells, etc.

[0115] 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 region of a nucleotide sequence that is not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0116] 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 in a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region when the residue in the second nucleic acid region is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand is capable of base pairing with a residue in a second nucleic acid strand that is antiparallel to the first strand when the residue in the second nucleic acid strand is guanine. Two regions are complementary if at least one nucleotide residue in the first region is capable of base pairing with a residue in the second region when the first region of the nucleic acid is arranged antiparallel to the second region of the same or a different nucleic acid. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, wherein when the first portion and the second portion are arranged antiparallel to each other, at least about 50% and 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), such as at least about 80%-100% of the nucleotide residues in the first portion are capable of base pairing with the nucleotide residues in the second portion. In some embodiments, all of the nucleotide residues in the first portion are capable of base pairing with the nucleotide residues in the second portion.

[0117] As used herein, the term "costimulation" with respect to an activated immune cell includes the ability of a costimulatory molecule to provide a second signal ("costimulatory signal") that is non-activating receptor-mediated and can induce proliferation or effector function. For example, a costimulatory signal may cause cytokine secretion, for example, in a T cell that has received a T cell receptor-mediated signal. An immune cell that has received a signal mediated by a cell receptor, such as via an activating receptor, is referred to herein as an "activated immune cell".

[0118] "CD3" is known in the art as a multi - protein complex having six chains (see Abbas and Lichtman, Cellular and Molecular Immunology (9th Edition) (2018); Janeway et al. (Immunobiology) (9th Edition) (2016)). In mammals, the complex comprises one CD3γ chain, one CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. The CD3γ chain, CD3δ chain, and CD3ε chain are related cell - surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ chain, CD3δ chain, and CD3ε chain are negatively charged, and this feature is thought to allow these chains to associate with the positively charged regions or residues of the T - cell receptor chains. The intracellular tails of the CD3γ chain, CD3δ chain, and CD3ε chain each contain a single conserved motif called an immunoreceptor tyrosine - based 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 according to the present invention can be from various animal species, including human, mouse, rat, or other mammals.

[0119] As used herein, "components of the TCR complex" refers to TCR chains (i.e., TCRα, TCRβ, TCRγ, or TCRδ), CD3 chains (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or complexes 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).

[0120] "Comparative T cell receptor" refers to the current state-of-the-art, e.g., at least one benchmark T cell receptor (e.g., Immatics-based or T-Knife-based) reported in U.S. Patent No. 10,874,731 (Immatics) and Obenaus et al. (2014) Nat. Biotechnol. 33:402-407. In some embodiments, "Comparator 1", also abbreviated as "Comparator," is a TCR based on the Immatics R37P1C9 TCR from U.S. Patent No. 10,874,731. Engineered forms of such parental sequences were used in the working examples and the sequences of such engineered forms are set forth in Table 4. In some embodiments, "Comparator 2" refers to a TCR based on the T-Knife-T1367 TCR from Obenaus et al. (2014) Nat. Biotechnol. 33:402-407. Engineered forms of such parental sequences were used in the working examples and the sequences of such engineered forms are set forth in Table 4. In some embodiments, the comparative T cell receptor has the sequences set forth in Table 4.

[0121] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein engineered to contain two or more amino acid sequences joined in a manner that does not occur naturally or does not occur naturally in a host cell, which fusion protein functions as a receptor when present on the cell surface. CARs encompassed by the present invention include an extracellular portion that contains an antigen-binding domain (i.e., obtained from or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR specific for the MAGEA1 antigen, a binding protein derived from a single-chain TCR, an scFv derived from an antibody, an antigen-binding domain derived from or obtained from a killer immunoglobulin receptor from an NK cell, etc.), which antigen-binding domain is linked to a transmembrane domain and one or more intracellular signaling domains (e.g., effector domains, optionally containing co-stimulatory domains) (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).

[0122] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. The CTL response is primarily mediated by CD8 + T cells.

[0123] The term "consisting essentially of" is not equivalent to "comprising", and means the specific materials or steps of a claim, or those materials or steps that do not materially affect the basic characteristics of the claimed subject matter. For example, a protein domain, region or module (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 specific amino acid sequence" when the amino acid sequence of the domain, region, module or protein includes, in combination, at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain, region, module or protein and does not significantly affect (i.e., does not reduce the activity by more than 50%, e.g., no more than 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) the activity of the domain, region, module or protein (e.g., the target binding affinity of a binding protein), such as an extension, deletion, mutation or combination thereof (e.g., an amino acid or an amino acid between domains at the amino or carboxyl terminus).

[0124] The term "determining a treatment regimen suitable for a subject" means determining a treatment regimen for a subject (i.e., a single therapy or a combination of different therapies for preventing and / or treating a viral infection in a subject), the treatment regimen being initiated, modified and / or ended based on or essentially based on or at least partially based on the results of the analysis according to the present invention. One example is starting adjuvant therapy after surgery, the aim of which is to reduce the risk of recurrence, and another example would be modifying the dose of a particular chemotherapy. In addition to the results of the analysis according to the present invention, the determination may also be based on the personal characteristics of the subject to be treated. In most cases, the actual treatment regimen suitable for a subject will be determined by the attending physician or doctor.

[0125] The term "dominant negative TGFβ receptor" or "DN-TGFβR" refers to a transforming growth factor (TGF) β receptor variant or mutant that resists TGFβ signaling. There are five type II receptors (activating receptors) and seven type I receptors (signal-propagating receptors). The active TGFβ receptor is a heterotetramer composed of two TGFβ receptor I (TGFβRI) and two TGFβ receptor II (TGFβRII). In some embodiments, the DN-TGFβR is DN-TGFβRII (i.e., a TGFβ receptor II variant or mutant). In some embodiments, the inhibition of TGFβ signaling on immune cells such as, for example, T cells is resisted, and the TGFβ can be produced by cancer cells or by other immune cells within the cellular environment, such as by stromal cells, macrophages, myeloid cells, epithelial cells, natural killer cells, etc. TGFβ signaling inhibitors are well known in the art and include, without limitation, mutant TGFβ that chelates the receptor and thus inhibits signaling; antibodies that bind to TGFβ and / or TGFβ receptors (such as lerdelimumab, metlimumab, fressolimumab, etc.); soluble TGFβ-binding proteins, such as portions of TGFβ receptors that chelate TGFβ (such as TGFβRII-Fc fusion proteins); or other binding agents, such as betaglycan. Instead of or in addition to the DN-TGFβR described herein (such as DN-TGFβRII), any and all known TGFβ signaling inhibitors can be used. In some embodiments, the DN-TGFβR lacks the intracellular portion required for TGFβ-mediated signaling, such as the entire intracellular domain, kinase signaling domain, etc. DN-TGFβR constructs are well known in the art. (See, for representative non-limiting embodiments, Brand et al. (1993) J. Biol. Chem. 268:11500-11503; Weiser et al. (1993) Mol. Cell Biol. 13:7239-7247; Bollard et al. (2002) Blood 99::3179-3187; PCT Publication WO 2009 / 152610; PCT Publication WO 2017 / 156484; Kloss et al. (2018) Mol. Ther. 26:1855-1866; PCT Publication WO.2019 / 089884; PCT Publication WO 2020 / 042647; and PCT Publication WO 2020 / 042648.)

[0126] As used herein, a "hematopoietic progenitor cell" is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiating into mature cell types (such as cells of the immune system). Exemplary hematopoietic progenitor cells include those having CD24Lo Lin-CD117 + Phenotypic hematopoietic progenitor cells or hematopoietic progenitor cells found in the thymus (referred to as thymic progenitor cells).

[0127] As used herein, "homologous" refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When the nucleotide residue positions in two regions are occupied by the same nucleotide residue, the regions are homologous at that position. If at least one nucleotide residue position in each region is occupied by the same residue, the first region is homologous to the second region. The homology between two regions is expressed as the proportion 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, wherein at least about 50% and 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), such as at least about 80%-100% of the nucleotide residue positions in each portion are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions in each portion are occupied by the same nucleotide residue.

[0128] The term "hyperproliferative disorder characterized by MAGEA1 antigen expression" can be any hyperproliferative disorder in which the MAGEA1 antigen is present in an MHC (e.g., HLA) complex expressed by at least some hyperproliferating cells in a subject. Examples of hyperproliferative disorders characterized by the MAGEA1:HLA complex include solid malignancies, such as the solid malignancies described in detail below.

[0129] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0130] Enhanced agonistic activity of T cell co-stimulatory receptors and / or enhanced antagonistic activity of inhibitory receptors may result in enhanced ability to stimulate an immune response or the immune system. Enhanced ability to stimulate an immune response or the immune system can be determined by measuring the EC in an assay of the immune response 50or the increase multiple of the maximum activity level, and the assay is, for example, an assay that measures changes in cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly via detection of CD107a or granzyme), and proliferation. The ability to stimulate an immune response or immune system activity can be enhanced 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.

[0131] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate an immune response in a subject's host immune system against cancer cells. A variety of immunotherapeutic agents can be used in the compositions and methods described herein.

[0132] The term "immune cell" refers to any immune system cell derived from hematopoietic stem cells in the bone marrow, which gives rise to two main lineages: myeloid progenitors (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes); and lymphoid progenitors (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 CD8 double-negative T cells, gd T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells can be referred to as "antigen-presenting cells" or "APCs", which are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor complexed with a peptide on the surface of the APC interacts with the TCR on the surface of the T cell.

[0133] "Isolated protein" refers to a protein that is isolated from cells or produced by recombinant DNA techniques and is substantially free of other proteins, cellular material, isolation media, and culture media, or a protein that is substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or a bioactive 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 produced, or is substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a biomarker polypeptide or a fragment thereof, wherein the protein is isolated from the cellular components of the cell from which the protein is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of a biomarker protein or a fragment thereof that have less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as "contaminating protein"), or in some embodiments, less than about 25%, 20%, 15%, 10%, 5%, 1% or less, or any range therebetween (including the endpoints), such as less than about 1% to 5% of non-biomarker protein. When a binding protein, antibody, polypeptide, peptide, or fusion protein or a fragment thereof (e.g., a bioactive fragment thereof) is recombinantly produced, it may be substantially free of culture media, i.e., the culture media accounts for less than about 20%, 15%, 10%, 5%, 1% or less, or any range therebetween (including the endpoints), such as less than about 1% to 5% of the volume of the protein preparation.

[0134] As used herein, the term "isotype" refers to the class of antibody encoded by the heavy chain constant region gene (e.g., IgM, IgG1, IgG2C, etc.).

[0135] As used herein, the term "K" D " refers to the dissociation equilibrium constant of a particular binding protein-antigen interaction. The binding affinity of a binding protein 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 low Kd value indicates a relatively high binding affinity (e.g., a Kd value of less than or equal to about 5 × 10 -4 M (500 uM) includes a Kd value of 1 × 10 -4 M (100 uM) and a 100 uM Kd indicates a relatively higher binding affinity compared to a 500 uM Kd).

[0136] A "kit" is any article (e.g., a package or container) that contains at least one reagent, such as a probe or a small molecule, which is used for specifically detecting and / or affecting the expression of a biomarker covered by the present invention. The kit can be marketed, resold, or sold as a unit for performing the methods covered by the present invention. The kit can contain one or more reagents necessary for expressing the compositions useful in the methods covered by the present invention. In some embodiments, the kit can also contain reference standards, such as nucleic acids encoding proteins that do not affect or regulate signal transduction pathways controlling cell growth, division, migration, survival, or apoptosis. Those skilled in the art can envision many such control proteins, including but not limited to common molecular tags (e.g., green fluorescent protein and β-galactosidase), proteins not classified by GeneOntology reference into any pathway covering cell growth, division, migration, survival, or apoptosis, or ubiquitous housekeeping proteins. The reagents in the kit can be provided in separate containers or in a mixture of two or more reagents in a single container. In addition, instructional materials describing the use of the compositions in the kit can be included.

[0137] As used herein, the term "linkage" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinant fusion). Such linkages can be achieved using a variety of well-known techniques, such as chemical conjugation and recombinant protein production.

[0138] In some embodiments, a "linker" can refer to an amino acid sequence that links two proteins, polypeptides, peptides, domains, regions, or motifs and can provide a spacer function compatible with the interaction of two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., scTCR) or retains signal transduction 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.

[0139] The term "MAGEA1" refers to a specific member of the melanoma antigen gene family clustered on human chromosome Xq28 (e.g., chromosome X: 153,179,284-153,183,880 positive strand. GRCh38: CM000685.2), also known as cancer / testis antigen 1.1 (CT1.1); melanoma-associated antigen 1; MAGE1; melanoma antigen family A1 (directing the expression of antigen MZ2-E); cancer / testis antigen family 1 member 1; melanoma-associated antigen MZ2-E; melanoma antigen family A1; cancer / testis antigen 1.1; melanoma antigen MAGE-1; MAGE-1 antigen; antigen Z2-E, MGC9326; and MAGE1A (Mao et al. (2019) J. Hematol. Oncol. 12:106; Fanipakdel et al. (2019) J. Cell Physiol. 234:12080-12086; Gu et al. (2018) Thorac. Cancer 9:431-438; Mecklenburg et al. (2017) Clin. Cancer Res. 23:1213-1219; Wang et al. (2016) Biochem. Biophys. Res. Commun. 473:959-965; Kozakova et al. (2015) Cell Cycle 14:920-930; Cannuyer et al. (2013) PLoS One 8:e5874; Pereira et al. (2012) Oncol. Rep. 27:1843-1848; Ogata et al. (2011) Ann. Surg. Oncol. 18:1195-1203; Roch et al. (2010) Anticancer Res. 30:1617-1623; Dango et al. (2010) Lung Cancer 67:290-295; van der Bruggen et al. (1991) Science 254:1643-1647). MAGEA1 is a melanoma antigen recognized by cytolytic T lymphocytes and is thought to be involved in transcriptional regulation via interaction with SNW1 and recruitment of histone deacetylase HDAC1, and inhibits the transactivation of the Notch intracellular domain (NICD), embryonic development, causes some genetic disorders (such as dyskeratosis congenita) and / or tumor transformation or tumor progression (such as gastric cancer, hepatocellular carcinoma, etc.). MAGEA1 is not highly expressed in normal tissues except the testis and is expressed in tumors of various histological types, such as melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma.

[0140] The term "MAGEA1" is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human MAGEA1 cDNA and human MAGEA1 protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI) (see, e.g., ncbi.nlm.nih.gov / gene / 4100). By way of example, human MAGEA1 (NP_004979.3) can be encoded by the transcript (NM_004988.5). Nucleic acid and polypeptide sequences of MAGEA1 orthologs in organisms other than humans are well known and include, for example, chimpanzee MAGEA1 (XM_529226.2 and XP_529226.2) and mouse MAGEA1 (chromosome X: 155088686-155089793; Ensembl Mus musculus release 104.39 (GRCm39)). Representative sequences of the MAGEA1 sequences are presented in Table 3 below.

[0141] Anti-MAGEA1 antibodies suitable for detecting MAGEA1 protein are well-known in the art and include, for example, antibodies AM32863PU, AM50138PU, AP06212PU, AP13128PU-TA312178, TA39275, TA339275, TA339276, and TA347677 (OriGene, Rockville, MD); antibodies orb167376 and orb11016 (Biorbyt, Cambridge, United Kingdom); antibodies A03570 and AO3570-1 (Boster Bio, Pleasanton, CA); antibodies E22-11B2-E9 and N1C3 (GeneTex, Irvince, CA); antibodies AFLGC-MAGEA1, MA5-37821, and MA1-91067 (Invitrogen, Waltham, MA); antibodies ABIN2782493 and ABIN2782494 (Antibodies-online, Limerick, PA); and antibodies MA454 and 6C1 (Santa Cruz Biotechnology, Dallas, TX). In addition, reagents for detecting MAGEA1 expression are well-known. Furthermore, various siRNAs, shRNAs, and CRISPR constructs for modulating MAGEA1 expression can be found in the commercial product listings of multiple companies, such as open reading frame (ORF) clones MG212171, MR212171, MR212171L3, MR212171L3V, MR212171L4, MR212171L4V, RC202134, RC202134L3, RC202134L3V, RC202134L4, RC202134L4V, and RG202134 (OriGene, Rockville, MD); CRISPR knockout GA102785, GA202555, KN202134, KN202134BN, KN202134LP, KN202134RB, KN402134, and KN509652 (OriGene, Rockville, MD); and RNA interference (RNAi) clones, such as siRNA and shRNA clones, including SR302776, TL311617, SR410578, TL311617V, TTL516288, TL516288V, TL704467, TL04467V, TR311617, TR516288, and TR704467 (OriGene, Rockville, MD).It should be noted that this term can also be used to refer to any combination of the characteristics of the MAGEA1 molecule described herein. For example, any combination of sequence composition, percentage identity, sequence length, domain structure, functional activity, etc. can be used to describe the MAGEA1 molecules encompassed by the present invention.

[0142] The term "MAGEA1 antigen" or "MAGEA1 peptide antigen" or "peptide antigen containing MAGEA1" or "MAGEA1 epitope" or "MAGEA1 peptide epitope" or "MAGEA1 peptide" refers to the immunogenic portion of MAGEA1, which can be naturally occurring or synthetically produced. In some embodiments, the length of the MAGEA1 antigen protein can range 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 in between, such as 8 - 15 amino acids. In some embodiments, the MAGEA1 antigen protein can form a complex with an MHC (e.g., HLA) molecule such that the binding proteins of the present disclosure that recognize the MAGEA1 peptide:MHC (e.g., HLA) complex can bind to such complexes (e.g., specifically and / or selectively). Representative MAGEA1 peptide antigen sequences are shown in Table 1.

[0143] 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 transmembrane chain (with three α domains) and non-covalently associated β2-microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, 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 recognized by CD8 + T cells. MHC class II molecules deliver peptides derived from the vesicular system to the cell surface, where they are recognized by CD4 + T cells. The human MHC is called human leukocyte antigen (HLA).

[0144] The terms "prevent / preventing / prevention", "preventive treatment", etc. refer to reducing the probability that a subject who does not have a disease, disorder or condition but is at risk or susceptible to developing the disease, disorder or condition will develop the disease, disorder or condition.

[0145] The term "prognosis" includes a prediction of the likely course and outcome of cancer or the likelihood of recovery from a disease. In some embodiments, statistical algorithms are used to provide a prognosis for an individual's cancer. By way of example, the prognosis can be the outcome of surgery, the development of a cancer clinical subtype, the development of one or more clinical factors, or recovery from the disease.

[0146] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology" and can be determined using the Karlin and Altschul algorithms (1990 Proc. Natl. Acad. Sci. USA 87:2264-2268) modified by Karlin and Altschul (1993 Proc. Natl. Acad. Sci. USA 90:5873-5877). The described algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990 J. Mol. Biol. 215:403-410). The BLAST nucleotide searches are performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the polynucleotides described herein. The BLAST protein searches are performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the reference polypeptides. To obtain gapped alignments for comparison purposes, gapped BLAST is used as described in Altschul et al. (1997 Nuc. Acids Res. 25:3389-3402). When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used.

[0147] The phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting a compound of the invention from one organ or part of the body to another organ or part of the body.

[0148] The term "ratio" refers to the relationship between two numbers (e.g., fractions, sums, 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 can be expressed in any order and that the underlying relationship does not lose meaning, although the observations and correlations of trends based on the ratio may be reversed.

[0149] The term "recombinant host cell" (or simply "host cell") refers to a cell that contains nucleic acid not naturally present in the cell, such as a cell that has been introduced with a recombinant expression vector. It should be understood that the cells according to the invention refer not only to the specific subject cells but also to the progeny of such cells. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parental cells, but are still included within the scope of the term cells according to the invention.

[0150] The terms "cancer response", "response to immunotherapy", or "response to a combination therapy of a T cell-mediated cytotoxicity modulator / immunotherapy" refer to any response of a hyperproliferative disease (such as cancer) to a cancer agent (such as a T cell-mediated cytotoxicity modulator) and immunotherapy, preferably a change in tumor mass and / or volume after the initiation of neoadjuvant or adjuvant therapy. The term "neoadjuvant therapy" refers to a therapy given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiotherapy, and hormone therapy. The response of a hyperproliferative disorder can be evaluated, for example, for efficacy or in a neoadjuvant or adjuvant setting, where the tumor size after a systemic intervention can be compared to the initial size and dimensions by CT, PET, mammogram, ultrasound, or palpation. The response can also be evaluated by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. The response can be recorded quantitatively, such as the percentage change in tumor volume, or qualitatively, such as "pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. The evaluation of the response of a hyperproliferative disorder can be performed early after the initiation of neoadjuvant or adjuvant therapy, for example, after a few hours, days, weeks, or preferably months. The typical endpoint of response evaluation is when neoadjuvant chemotherapy is terminated or when residual tumor cells and / or the tumor bed are surgically resected. This is typically three months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatment described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the following: the percentage of patients with complete remission (CR) at a time point at least 6 months from the end of therapy, the number of patients with partial remission (PR), and the number of patients with stable disease (SD). The shorthand for this formula is CBR = CR + PR + SD within 6 months. In some embodiments, the CBR for a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or higher. Other criteria for evaluating the response to cancer therapy are related to "survival" and include all of the following: survival to death, also known as overall survival (where the death can be regardless of cause or related to the tumor); "recurrence-free survival" (where the term recurrence should include local recurrence and distant recurrence); metastasis-free survival; disease-free survival (where the term disease should include cancer and related diseases). The length of the survival can be calculated by reference to a defined starting point (such as the time of diagnosis or the start of treatment) and endpoint (such as death, recurrence, or metastasis). In addition, the criteria for treatment efficacy can be extended to include the response to chemotherapy, the probability of survival, the probability of metastasis within a given time period, and the probability of tumor recurrence.For example, to determine an appropriate threshold, a particular cancer treatment regimen may be administered to a population of subjects and the results may be correlated with biomarker measurements determined prior to the administration of any cancer therapy. The outcome measure may be the pathologic response to the therapy administered in the neoadjuvant setting. Alternatively, outcome metrics such as overall survival and disease-free survival may be monitored in subjects with known biomarker measurements for a period of time following the cancer therapy. In certain embodiments, the dose administered is a standard dose of a cancer therapeutic agent known in the art. The period of time for which the subjects are monitored may vary. For example, the subjects may be monitored for at least 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 14 months, 16 months, 18 months, 20 months, 25 months, 30 months, 35 months, 40 months, 45 months, 50 months, 55 months, or 60 months. Well-known methods in the art, such as those described in the Examples section, may be used to determine the biomarker measurement thresholds associated with cancer therapy outcomes.

[0151] As noted, the term may also refer to an improved prognosis, such as reflected in an increase in time to recurrence, which is the period of time to the first recurrence review of a second primary cancer as a first event or death with no evidence of recurrence; or an increase in overall survival, which is the period of time from treatment to death from any cause. Respond or response means achieving a beneficial endpoint when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated when exposed to the stimulus. It should be understood that assessing the likelihood that a tumor or subject will exhibit a favorable response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a favorable response (i.e., will exhibit a lack of response or non-response).

[0152] The term "resistance" refers to acquired or natural resistance of a cancer sample or a mammal to a cancer therapy (i.e., non-responsiveness or reduced or limited responsiveness to a therapeutic treatment), such as a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more reduction in response to a therapeutic treatment, such as a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more reduction, or any range therebetween (including the endpoints). The reduction in response can be measured by comparing with the same cancer sample or mammal prior to acquisition of resistance, or by comparing with a different cancer sample or mammal known to be non-resistant to the therapeutic treatment. A typical acquired resistance to chemotherapy is referred to as "multi-drug resistance". Multi-drug resistance may be mediated by P-glycoprotein, or may be mediated by other mechanisms, or may occur when a mammal is infected with multi-drug resistant microorganisms or combinations of microorganisms. The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinary clinician, e.g., it can be measured by cell proliferation assays and cell death assays described herein as "sensitization". In some embodiments, the term "reversing drug resistance" means that in a situation where a single primary cancer therapy (e.g., chemotherapy or radiotherapy) alone does not produce a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor, a second agent in combination with the primary cancer therapy (e.g., chemotherapy or radiotherapy) is capable of producing a significant reduction in tumor volume (e.g., p<0.05) to a degree of statistical significance compared to the tumor volume of the untreated tumor. This generally applies to tumor volume measurements made when an untreated tumor is growing logarithmically.

[0153] The term "sample" for detecting or assaying the absence, presence or level of at least one biomarker is generally a brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, feces (e.g., stool), tears and any other body fluid (e.g., as 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 include obtaining a sample from an individual prior to detecting or assaying the absence, presence or level of at least one biomarker in the sample.

[0154] The term "sensitize" means to alter cancer cells or tumor cells in a manner that permits more effective treatment of the associated cancer with a cancer therapy (e.g., anti-immune checkpoint, chemotherapy, and / or radiotherapy). In some embodiments, normal cells are not affected to an extent that results in excessive damage to the normal cells due to the treatment. The increase or decrease in sensitivity to a therapeutic treatment is measured according to methods known in the art for a particular treatment and the methods described below, 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., Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P editors, Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90). The sensitivity or resistance of an animal can also be measured by measuring the reduction in tumor size over a period of time, e.g., 6 months in humans and 4-6 weeks in mice. If the treatment sensitivity is increased or the resistance is decreased by 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 (including the endpoints), compared to the treatment sensitivity or resistance in the absence of the composition or method, then such a composition or method sensitizes the response to the therapeutic treatment. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinary clinician. It should be understood that any method described herein for enhancing the efficacy of a cancer therapy can equally be applied to methods for sensitizing hyperproliferative or other cancer cells (e.g., resistant cells) to a cancer therapy.

[0155] The term "small molecule" is a term in the art and includes molecules having a molecular weight of less than about 1000 or a molecular weight of less than about 500. In one embodiment, the small molecule does not only contain peptide bonds. In another embodiment, the small molecule is not an oligomer. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (such as polyketides) (Cane et al. (1998) Science 282:63-68), and libraries of natural product extracts. In another embodiment, the compound is a small organic non-peptide compound. In another embodiment, the small molecule is not biosynthetic.

[0156] The term "specifically binds" refers to the binding of a binding protein to a predetermined antigen. Generally, when measured in a binding assay, such as surface plasmon resonance (SPR) technology, using the antigen of interest as the analyte and the binding protein as the ligand in a BIAcore TM assay instrument, the binding protein has an affinity (K -4 of less than or equal to about 5×10 -4 M, less than or equal to about 1×10 -5 M, less than or equal to about 5×10 -5 M, less than or equal to about 1×10 -6 M, less than or equal to about 5×10 - 6 M, less than or equal to about 5×10 -7 M, less than or equal to about 1×10 -7 M, less than or equal to about 5×10 -8 M, less than or equal to about 1×10 -8 M, less than or equal to about 5×10 -9 M, less than or equal to about 1×10 -9 M, less than or equal to about 5×10 -10 M, less than or equal to about 1×10 -10 M, less than or equal to about 5×10 -11 M, less than or equal to about 1×10 -11 M, less than or equal to about 5×10 -12 M, less than or equal to about 1×10 -12 M or lower, or any range in between (including the endpoints), such as an affinity of about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, etc. (K D) Binding. In some embodiments, the binding protein binds to the predetermined antigen with an affinity that is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold or 10.0-fold greater than its affinity for non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). The phrases "binding protein that recognizes an antigen" and "binding protein that is specific for an antigen" are used interchangeably herein with the term "binding protein that specifically binds to an antigen". Selective binding is a relative term and refers to the ability of a binding protein to distinguish the binding of one antigen from the binding of another antigen, e.g., the ability to distinguish the binding of a particular family member or antigen target from the binding of a related family member or antigen target. By way of example, the assay data provided in the Examples section demonstrate that the binding proteins described herein specifically bind to the MAGEA1 immunogenic epitope and / or selectively bind to a number of related epitopes (e.g., the MAGEA1 immunogenic epitope and closely related sequences), thereby distinguishing such targets from the numerous other possible epitopes available in the human genome.

[0157] The term "subject" refers to any healthy animal, mammal or human, or any animal, mammal or human suffering from a disorder characterized by MAGEA1 expression (e.g., a non-malignant disorder, a hyperproliferative disorder or a recurrence of a hyperproliferative disorder characterized by MAGEA1 expression). The term "subject" may be used interchangeably with "patient".

[0158] The term "survival" includes all of the following: survival to death, also known as overall survival (wherein the death may be irrespective of cause or related to the tumor); "recurrence-free survival" (wherein the term recurrence should include local recurrence and distant recurrence); metastasis-free survival; disease-free survival (wherein the term disease should include cancer and diseases associated therewith). The length of survival can be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and an end point (e.g., death, recurrence or metastasis). In addition, the criteria for therapeutic efficacy can be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time period and probability of tumor recurrence.

[0159] The term "synergy" refers to the combined action of two or more agents (e.g., the MAGE A1-related agents described herein and another therapy for treating a disorder characterized by MAGEA1 expression, such as another MAGEA1-targeted TCR, an anti-cancer therapy, an immunotherapy, etc.) being greater than the sum of the individual actions of the individual cancer agents / therapies.

[0160] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + cells). T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0161] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of such RNA or cDNA) that is complementary or homologous to all or a portion of a mature mRNA, which is produced by transcription of a biomarker nucleic acid and (if present) normal post-transcriptional processing (e.g., splicing) of the RNA transcript and reverse transcription of the RNA transcript.

[0162] A "T cell" is a cell of the immune system that matures in the thymus and produces a T cell receptor (TCR). T cells can be naive T cells (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA and decreased expression of CD45RO compared to T CM ), memory T cells (T M ) (which have experienced antigen and have a long lifespan), and effector cells (which have experienced antigen and are cytotoxic). T M can also be divided into central memory T cells (T CM , 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 , decreased expression of CD62L, CCR7, CD28, and CD45RA and increased expression of CD127 compared to naive T cells or T CM ). Effector T cells (T E ) refer to CD8+ cytotoxic T lymphocytes that have experienced antigen, with decreased expression of CD62L, CCR7, and CD28 compared to T CM and being positive for granzyme and perforin. Other exemplary T cells include regulatory T cells, such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Tregl7 cells, as well as Trl, Th3, CD8 + CD28, and Qa-1 restricted T cells.

[0163] Conventional T cells (also known as Tconv or Teff) have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) to enhance the immune response by virtue of their expression of one or more T cell receptors. Tcon or Teff is generally defined as any T cell population that is not Treg and includes, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon that have differentiated into, for example, Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is CD4+ Teff or CD8+ Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T lymphocytes. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. A "naive Tcon" is a CD4 + T cell that has differentiated in the bone marrow and successfully undergone positive and negative central selection processes in the thymus but has not been activated by exposure to antigen. Naive Tcon are typically characterized by surface expression of L-selectin (CD62L), lack of activation markers (e.g., CD25, CD44, or CD69), and lack of memory markers (e.g., CD45RO). Thus, naive Tcon are believed to be quiescent and non-dividing and require interleukin 7 (IL-7) and interleukin 15 (IL-15) to maintain homeostatic survival (see, at least, WO 2010 / 101870). In the context of suppressing the immune response, the presence and activity of such cells are not desired. Unlike Treg, Tcon are not anergic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:625-637).

[0164] "T effector" ("T eff " or "T E ") cells refer to T cells (e.g., CD4+ and CD8+ T cells) with cytolytic activity, as well as T helper (Th) cells that secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0165] "T cell receptor" or "TCR" refers to a member of the immunoglobulin superfamily that is capable of binding (e.g., specifically and / or selectively) to an antigenic peptide that binds to an MHC receptor (having variable binding domains, constant domains, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al. (1997) Curr. Biol. Publ. 4:33). TCRs can be present on the cell surface or in a soluble form and are typically composed of a heterodimer having α and β chains (also referred to as TCRα and TCRβ, respectively) or γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portions of TCR chains (e.g., the α and β chains) contain two immunoglobulin domains: a variable domain at the N-terminus (e.g., the α-chain variable domain or V α and the β-chain variable domain or V β ; typically amino acids 1 to 116 based on 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, 5th ed.), and a constant domain at the C-terminus adjacent to the cell membrane (e.g., the α-chain constant domain or C α , typically amino acids 117 to 259 based on Kabat; the β-chain constant domain or C β , typically amino acids 117 to 295 based on Kabat). Additionally, like immunoglobulins, the variable domains contain complementarity-determining regions ("CDRs", also referred to as 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 present on the surface of T cells (or T lymphocytes) and are associated with the CD3 complex. The sources of TCRs encompassed by the present invention can be from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0166] The term "T cell receptor" or "TCR" is to be understood to encompass the full TCR as well as its antigen-binding portion or antigen-binding fragment. In some embodiments, the TCR is a full or full-length TCR, including TCRs in the αβ form or the γδ form. In some embodiments, the TCR is less than full-length but binds to a specific peptide bound in an MHC molecule, such as an antigen-binding portion that binds to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR may contain only partial domains of the full-length or full TCR but is still capable of binding to the peptide epitope bound by the full TCR, such as an MHC-peptide complex. In some cases, the antigen-binding portion contains the variable domains of the TCR, such as the variable α-chain and variable β-chain of the TCR, sufficient to form a binding site that binds to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain complementarity-determining regions (CDRs) involved in the recognition of the peptide, MHC, and / or MHC-peptide complex.

[0167] Nomenclature is established by the International Immunogenetics Information System (IMGT) (see also Scaviner and Lefranc (2000) Exp. Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; "T Cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). IMGT provides unique sequences for describing TCRs, and the sequences described herein can be identified by reference to such unique sequences provided herein. TCR sequences are publicly available in the IMGT database at imgt.org.

[0168] As described above, a native α / β heterodimeric TCR has an α chain and a β chain. Broadly speaking, each chain contains a variable region, a joining region, and a constant region, and the β chain usually also contains a short diversity region between the variable and joining regions, although this diversity region is typically considered part of the joining region. Each variable region contains three hypervariable CDRs (complementary determining regions) embedded within framework sequences. It is well known that CDR3 is the primary mediator of antigen recognition. There are several types of α chain variable (Vα) regions and several types of β chain variable (Vβ) regions, which are distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. In the IMGT nomenclature, Vα types are designated by unique TRAV numbers. For example, "TRAV4" defines a TCR Vα region that has a unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence that is partially defined by an amino acid sequence conserved between TCRs but also includes amino acid sequences that vary between TCRs. Similarly, "TRBV2" defines a TCR Vβ region that has a unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence. It is known that there are 54 α variable genes within the α locus, 44 of which are functional, and 67 β variable genes within the β locus, 42 of which are functional.

[0169] Similarly, the joining regions of TCRs are defined by the unique IMGT TRAJ and TRBJ nomenclatures, and the constant regions are defined by the IMGT TRAC and TRBC nomenclatures. In the IMGT nomenclature, the β chain diversity region is abbreviated as TRBD, and as mentioned previously, the tandem TRBD / TRBJ regions are generally considered together as the joining region.

[0170] The gene pools encoding the TCR α and β chains are located on different chromosomes and contain separate V, (D), J, and C gene segments that are brought together by rearrangement during T cell development. Due to the large number of possible recombination events between the 54 TCR α variable genes and 61 α J genes, or between the 67 β variable genes, two β D genes, and 13 β J genes, this gives rise to extremely high diversity in the T cell α and β chains. The recombination process is not precise and introduces further diversity within the CDR3 region. Each α and β variable gene can also contain allelic variants, designated as TRAVxx*01 and *02, or TRBVx-x*01 and *02, respectively, in the IMGT nomenclature, thus further increasing the amount of variation. Similarly, some TRBJ sequences have two known variants. (Note that the absence of the "*" qualifier means that only one allele of the relevant sequence is known). The natural repertoire of human TCRs generated by recombination and thymic selection is estimated to contain approximately 10 6A unique β-chain sequence, determined by CDR3 diversity (Arstila et al. (1999) Science 286:958-961), and potentially even higher (Robins et al. (2009) Blood 114:4099-4107). It is estimated that each β-chain pairs with at least 25 different α-chains, generating further diversity (Arstila et al. (1999) Science 286:958-961).

[0171] Thus, the term "TCRα variable domain" refers to the tandem of TRAV and TRAJ regions; the TRAV region only; or the TRAV and part of the TRAJ region, and the term TCRα constant domain refers to the extracellular TRAC region, or the C-terminally truncated or full-length TRAC sequence. Similarly, the term "TCRβ variable domain" refers to the tandem of TRBV and TRBD / TRBJ regions; the TRBV and TRBD regions only; the TRBV and TRBJ regions only; or the TRBV region and part of the TRBD and / or TRBJ regions, and the term TCRβ constant domain refers to the extracellular TRBC region, or the C-terminally truncated or full-length TRBC sequence. These nomenclatures for TCRα and TCRβ variable domains similarly apply to the variable domains of TCRγ and TCRδ chains of γ / δ TCRs. A person of ordinary skill in the art can obtain TRAV, TRAJ, TRAC, TRBV, TRBJ, and TRBC gene sequences, for example, through the publicly available IMGT database.

[0172] The term "TCR complex" refers to the complex formed by the association of CD3 with TCR. For example, the TCR complex can be composed of one CD3γ chain, one CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, one TCRα chain, and one TCRβ chain. Alternatively, the TCR complex can be composed of one CD3γ chain, one CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, one TCRγ chain, and one TCRδ chain.

[0173] The term "therapeutic effect" refers to the local or systemic effect caused by a pharmacologically active substance in animals, particularly mammals, and more particularly in humans. Thus, the term means any substance intended for the diagnosis, cure, mitigation, treatment, or prevention of a disease in animals or humans or for enhancing their desired physical or mental development and condition.

[0174] The terms "therapeutically effective amount" and "effective amount" mean an amount of a substance that produces some desired effect, such as a desired local or systemic therapeutic effect, in at least one cell subset in an animal at a reasonable benefit / risk ratio suitable for any therapy. In some embodiments, the therapeutically effective amount of a substance will depend on the therapeutic index, solubility, pharmacokinetics, half-life, etc. of the substance. It can be determined in cell cultures or experimental animals by standard pharmaceutical procedures such as those used to determine LD 50 and ED 50 to determine the toxicity and therapeutic efficacy of the subject compounds. In some embodiments, compositions with a large therapeutic index are used. In some embodiments, LD 50 (lethal dose) can be measured, and relative to not administering the agent, it can be reduced, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more when the agent is administered. Similarly, ED 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured, and relative to not administering the agent, it can be increased, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more when the agent is administered. Similarly, IC 50 can also be measured, and relative to not administering the agent, it can be increased, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more when the agent is administered. In some embodiments, in one assay, the T cell immune response can 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 another embodiment, 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% can be achieved.

[0175] The term "treatment" refers to the therapeutic management or amelioration of a disorder of interest (e.g., a disease or condition). Treatment can include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically carried out in an effort to alter, in a manner beneficial to the subject, the course of a disease (the term is used to denote any disease, condition, syndrome, or adverse condition that requires or may require therapy). Treatment effects can include reversing, alleviating one or more symptoms or manifestations of the disease, reducing its severity, delaying its onset, curing it, inhibiting its progression, and / or reducing the likelihood of its occurrence or recurrence. Desirable treatment effects include, but are not limited to: preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. A therapeutic agent can be administered to a subject having a disease or having an increased risk of disease development relative to a member of the general population. In some embodiments, a therapeutic agent can be administered to a subject who has had a disease but no longer shows signs of the disease. An agent can be administered, for example, to reduce the likelihood of overt disease recurrence. A therapeutic agent can be administered prophylactically, i.e., before the appearance of any symptoms or manifestations of the disease. "Prophylactic treatment" refers to providing medical and / or surgical treatment to a subject who has not yet developed a disease or shown signs of a disease, for example, to reduce the likelihood of the disease occurring or to reduce the severity when the disease occurs. The subject may have been identified as being at risk of developing the disease (e.g., having an increased risk relative to the general population or having a risk factor that increases the likelihood of developing the disease).

[0176] The term "anergy" includes the refractoriness of cancer cells to therapy, or of therapeutic cells, such as immune cells, to stimulation, such as stimulation via activation receptors or cytokines. Anergy can occur, for example, due to exposure to immunosuppressants or to high doses of antigen. As used herein, the terms "incompetence" or "tolerance" include refractoriness to activation receptor-mediated stimulation. This refractoriness is generally antigen-specific and persists after cessation of exposure to the tolerizing antigen. For example, T cell anergy (as opposed to unresponsiveness) is characterized by a lack of production of cytokines such as IL-2. T cell anergy occurs when T cells are exposed to antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of co-stimulatory polypeptides) results in an inability to produce cytokines and thus an inability to proliferate. However, anergic T cells may proliferate if cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by measuring the lack of production of IL-2 by T lymphocytes using ELISA or a proliferation assay using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells are unable to initiate IL-2 gene transcription controlled by the 5' IL-2 gene enhancer or induced by multimers of AP1 sequences that may be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0177] The term "vaccine" refers to a pharmaceutical composition that elicits an immune response against an antigen of interest. A vaccine can also confer protective immunity to a subject.

[0178] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., TCR) that is involved in the binding of the immunoglobulin superfamily binding protein to antigen (e.g., TCR α-chain or β-chain (or γ-chain and δ-chain for γδ TCR)). The variable domains of the α-chain and β-chain of a native TCR (V α and V β ) generally have a similar structure, each domain containing four conserved framework regions (FR) and three CDRs. The V α domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J); the V β domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J). A single V α or V β domain may be sufficient to confer antigen-binding specificity. In addition, V α or V βThe domain separates the TCR that binds a specific antigen from the TCR that binds the antigen, to separately screen a library of complementary V α or V β domains.

[0179] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, the vector is those vectors capable of autonomous replication and / or expression of the nucleic acid to which it is linked. A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector". Generally, the expression vectors used in recombinant DNA technology are usually in the form of "plasmids", and plasmids generally refer to circular double-stranded DNA rings, the vector form of which is not combined with chromosomes. In this specification, "plasmid" and "vector" may be used interchangeably because plasmids are the most commonly used vector forms. However, as those skilled in the art will appreciate, the present invention is intended to include other forms of such expression vectors that provide equivalent functions and are subsequently known in the art.

[0180] There is a known and definite correspondence between the amino acid sequence of a specific protein and the nucleotide sequence capable of encoding the protein, as defined by the genetic code (shown below). Similarly, there is a known and definite correspondence between the nucleotide sequence of a specific nucleic acid and the amino acid sequence encoded by the nucleic acid, as defined by the genetic code.

[0181] Genetic code Alanine (Ala, A) GCA, GCC, GCG, GCT

[0182] Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT

[0183] Aspartic acid (Asp, D) GAC, GAT

[0184] Cysteine (Cys, C) TGC, TGT

[0185] Glutamic acid (Glu, E) GAA, GAG

[0186] Glutamine (Gln, Q) CAA, CAG

[0187] Glycine (Gly, G) GGA, GGC, GGG, GGT

[0188] Histidine (His, H) CAC, CAT

[0189] Isoleucine (Ile, I) ATA, ATC, ATT

[0190] Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

[0191] Lysine (Lys, K) AAA, AAG

[0192] Methionine (Met, M) ATG

[0193] Phenylalanine (Phe, F) TTC, TTT

[0194] Proline (Pro, P) CCA, CCC, CCG, CCT

[0195] Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

[0196] Threonine (Thr, T) ACA, ACC, ACG, ACT

[0197] Tryptophan (Trp, W) TGG

[0198] Tyrosine (Tyr, Y) TAC, TAT

[0199] Valine (Val, V) GTA, GTC, GTG, GTT

[0200] Stop signal (end) TAA, TAG, TGA

[0201] An important and well-known feature of the genetic code is its redundancy, i.e., for most of the amino acids used to make proteins, more than one coding nucleotide triplet (as shown above) may be employed. Thus, many different nucleotide sequences may code for a given amino acid sequence. These nucleotide sequences are considered to be functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although some organisms may translate some sequences more efficiently than others). In addition, occasional methylated variants of purines or pyrimidines may be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0202] Given the above, a nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any part thereof) can be used to derive a polypeptide amino acid sequence by translating the DNA or RNA into an amino acid sequence using the genetic code. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence that can encode the polypeptide can be inferred from the genetic code (due to its redundancy, multiple nucleic acid sequences will result for any given amino acid sequence). Thus, a description and / or disclosure of a nucleotide sequence encoding a polypeptide herein should be considered to also include a description and / or disclosure of the amino acid sequence encoded by that nucleotide sequence. Similarly, a description and / or disclosure of a polypeptide amino acid sequence herein should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode that amino acid sequence.

[0203] II. Peptide

[0204] In certain aspects, the present disclosure provides methods and compositions for treating and / or preventing a disorder associated with MAGEA1 expression by inducing an immune response against MAGEA1 or cells expressing MAGEA1, which involve administering an MAGEA1 immunogenic peptide, a nucleic acid encoding an MAGEA1 immunogenic peptide, and / or a cell expressing an MAGEA1 immunogenic peptide as described herein.

[0205] In certain embodiments, the MAGEA1 immunogenic peptide comprises a peptide epitope (e.g., consists of) selected from the peptide sequences listed in Table 1, such as those listed in Table 1A. The peptide epitopes described herein can be combined with MHC molecules, such as specific HLA molecules having a particular HLA α-chain allele. For example, it was identified that the peptides of Table 1A associate with MHC having an HLA-A*02 serotype for the α-chain, such as the MHC encoded by the HLA-A*02:01 allele, as further described in the Examples section. In some embodiments, the MAGEA1 immunogenic peptide can be combined with an MHC molecule, wherein the MHC molecule comprises an MHC α-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, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLThe alleles HLA-A*11:04, HLA-A*11:05, HLA-A*11:19, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03. In some embodiments, the MAGEA1 immunogenic peptides are derived from the human MAGEA1 protein and / or the MAGEA1 proteins shown in Table 3. In some embodiments, one or more MAGEA1 immunogenic peptides are administered alone or in combination with an adjuvant.

[0206] In certain aspects, there are provided compositions comprising one or more of the MAGEA1 immunogenic peptides described herein and an adjuvant.

[0207] Table 1: MAGEA1 Epitopes

[0208] Table 1A

[0209] MAGEA1 Epitopes Presented by HLA Serotype HLA-A*02

[0210] Peptide epitope KVLEYVIKV VLEYVIKV KVLEYVIK

[0211] *Table 1, for example Table 1A, includes peptide epitopes, and polypeptide molecules having an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity to the amino acid sequence of any of the sequences listed in Table 1, for example Table 1A, or a portion thereof, over its full length. Such polypeptides can have the functions of the full-length peptides or polypeptides further described herein.

[0212] In some embodiments, the present disclosure provides MAGEA1 polypeptides and / or nucleic acids encoding MAGEA1 polypeptides. In some embodiments, the MAGEA1 polypeptide is a polypeptide comprising an amino acid sequence of sufficient length to elicit a MAGEA1-specific immune response. In certain embodiments, the MAGEA1 polypeptide further comprises amino acids that do not correspond to the amino acid sequence (e.g., a fusion protein comprising a MAGEA1 amino acid sequence and an amino acid sequence corresponding to a non-MAGEA1 protein or polypeptide). In some embodiments, the MAGEA1 polypeptide comprises only the amino acid sequence corresponding to the MAGEA1 protein or a fragment thereof.

[0213] In some embodiments, the amino acid sequence of the MAGEA1 polypeptide comprises, consists essentially of, or consists of: the amino acid sequence of the MAGEA1 protein, such as 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, 110, 120, 130, 140, 150, 160, 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 (e.g., 7 - 25, 8 - 22, 9 - 22, etc.) (including the endpoints). In some embodiments, the contiguous amino acids are identical to the amino acid sequence of MAGEA1 shown in Table 3. In some embodiments, the MAGEA1 polypeptide comprises, consists essentially of, or consists of one or more peptide epitopes selected from the group consisting of the MAGEA1 peptide epitopes listed in Table 1, such as Table 1A.

[0214] As is well known to those skilled in the art, polypeptides with significant sequence similarity can elicit the same or very similar immune responses in a host animal. Thus, in some embodiments, derivatives, equivalents, variants, fragments, or mutants of the MAGEA1 immunogenic peptides or fragments thereof described herein may also be suitable for the methods and compositions provided herein.

[0215] In some embodiments, provided herein are variants or derivatives of MAGEA1 immunogenic polypeptides. The altered polypeptides can have, for example, an amino acid sequence altered by conservative substitutions but still elicit an immune response reactive with the unaltered protein antigen and are considered functional equivalents. As used herein, the term "conservative substitution" means that an amino acid residue is replaced by another biologically similar residue. It is well known in the art that amino acids within the same conservative group can generally be substituted for one another with little or no effect on the function of the protein. According to certain embodiments, derivatives, equivalents, variants, or mutants of the ligand-binding domain of the MAGEA1 immunogenic peptide are polypeptides that are at least 85% homologous to the sequence of the MAGEA1 immunogenic peptide or a fragment thereof described herein. In some embodiments, the homology is at least 90%, at least 95%, at least 98% or higher.

[0216] The immunogenic peptides encompassed by the present invention can comprise peptide epitopes derived from the MAGEA1 protein, such as those listed in Table 1, for example, those listed in Table 1A. In some embodiments, the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide amino acid sequence is modified, which can include conservative or non-conservative mutations. The peptide can comprise up to 1, 2, 3, 4, or more mutations. In some embodiments, the peptide can comprise at least 1, 2, 3, 4, or more mutations.

[0217] In some embodiments, the peptide can be chemically modified. By way of example, the peptide can be mutated to modify peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is an example of methylation that can occur in the peptides of the present disclosure. In some embodiments, the peptide can be modified by methylation of the free amine, for example, by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0218] Chemical modifications can include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains (such as palmitate or myristate), or albumin. Chemical modification of a peptide having an Fc region can be a fusion Fc - peptide. Polyamino acids can include, for example, polyamino acid sequences having repeating single amino acids (such as polyglycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the foregoing. In some embodiments, the peptides encompassed by the present disclosure can be modified such that the modification increases the stability and / or half - life of the peptide. In some embodiments, attachment of a hydrophobic moiety (such as attachment to the N - terminus, C - terminus, or internal amino acids) can be used to extend the half - life of the peptides encompassed by the present disclosure. In other embodiments, the peptide can include post - translational modifications (such as, methylation and / or amidation) that affect, for example, the serum half - life. In some embodiments, simple carbon chains (such as, by myristoylation and / or palmitoylation) can be conjugated to a fusion protein or peptide. In some embodiments, simple carbon chains can facilitate separation of the fusion protein or peptide from unconjugated materials. For example, methods that can be used to separate a fusion protein or peptide from unconjugated materials include, but are not limited to, solvent extraction and reverse - phase chromatography. The lipophilic moiety can extend the 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, including cholestene, cholestane, cholestadiene, and oxysterol. In some embodiments, the peptide can be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptide can be coupled (such as, 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, glutamate, and serine, Fc regions, fatty acids, palmitic acid, or a molecule that binds to albumin. In some embodiments, a spacer or linker can be coupled to the peptide, such as 1, 2, 3, 4, or more amino acid residues used as a spacer or linker, in order to facilitate conjugation or fusion with another molecule, and to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, a fusion protein or peptide can be conjugated to other moieties that can modify or effect a change in the properties of the peptide.

[0219] In some embodiments, the peptide can be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or label. The affinity tag can be selected from the group consisting of: glutathione - S - transferase (GST), calmodulin - binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, tags, His tags, biotin tags, and V5 tags. The label can be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of pro-inflammatory factors, anti-inflammatory agents, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies, such as single-chain Fvs.

[0220] The peptide can be conjugated to agents for imaging, research, therapeutics, theranostics, pharmacy, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, the peptide can be conjugated or fused to a detectable agent, such as a fluorophore, near-infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radiometal chelator, or another suitable material for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties can be attached 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 body fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation with a wavelength 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 conjugated molecules include Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, near-infrared dyes generally include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Additional non-limiting examples of fluorescent dyes used as conjugated molecules in the present disclosure include acridine orange or acridine yellow, Alexa (e.g., Alexa 790, 750, 700, 680, 660, and 647) and any of its derivatives, 7-Actinomycin D, 8-Anilino-1-naphthalenesulfonic acid, ATTO dyes and any of its derivatives, Auramine-Rhodamine stain and any of its derivatives, Bensanthrone, Bimane, 9,10-Bis(phenylethynyl)anthracene, 5,12-Bis(phenylethynyl)naphthacene, Bisbenzimide, Brainbow, Calcein, Carboxyfluorescein and any of its derivatives, 1-Chloro-9,10-bis(phenylethynyl)anthracene and any of its derivatives, DAPI, DiOC6, DyLight Fluors and any of its derivatives, Epicocconone, Ethidium bromide, FlAsH-EDT2, Fluo dyes and any of its derivatives, FluoProbe and any of its derivatives, Fluorescein and any of its derivatives, Fura and any of its derivatives, GelGreen and any of its derivatives, GelRed and any of its derivatives, Fluorescent proteins and any of its derivatives, m isotype proteins and any of its derivatives (e.g., mCherry), Hetamethine dyes and any of its derivatives, Hoeschst stains, Imidocoumarin, Indian Yellow, Indo-1 and any of its derivatives, Laurdan, Lucifer Yellow and any of its derivatives, Fluorescein and any of its derivatives, Luciferase and any of its derivatives, Merocyanine and any of its derivatives, Nile dyes and any of its derivatives, Perylene, Phloxin e, Phycobiliprotein dyes and any of its derivatives, Propidium iodide, Pyranine, Rhodamine and any of its derivatives, Ribogreen, RoGFP, Rubrene, Stilbene and any of its derivatives, Sulfonylrhodamine and any of its derivatives, SYBR TMand any of its derivatives, synapto-pHluorin, tetraphenylbutadiene, tris tetrasodium, Texas Red, Titan Yellow, TSQ, umbelliferone, perylene, yellow fluorescent protein, and YOYO-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), cyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon dye (e.g., Oregon 488, Oregon 500, Oregon 514, etc.), Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA dye (e.g., ALEXA 350, ALEXA 488, ALEXA 532, ALEXA 546, ALEXA 568, ALEXA 594, ALEXA 633, ALEXA 660, ALEXA FL 680, etc.), dye (e.g., FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRDye (e.g., IRD40, IRD 700, IRD 800, etc.), 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.

[0221] The peptide can be conjugated to a radiosensitizer or a photosensitizer. Examples of radiosensitizers include, but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to: fluorescent molecules or beads, nanoparticles, porphyrins, and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metal porphyrins, metal phthalocyanines, angelicin, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins, and related compounds (e.g., lumazines and riboflavins), fullerenes, pheophorbide, pyropheophorbide, cyanines (e.g., merocyanine 540), pheophytin, sapphyrin, texaphyrin, purpurin, porphyrinogens, phenothiaziniums, methylene blue derivatives, naphthalimides, nile blue derivatives, quinones, perylenequinones (e.g., hypericin, hypocrellin, and cercosporin), psoralen, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosin, erythrosin, rose bengal), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, the method allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent (e.g., a drug) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the peptide is fused to the agent, or covalently or non-covalently linked to the agent, e.g., directly or via a linker.

[0222] In some embodiments, the binding protein can be chemically modified. For example, the binding protein can be mutated to modify peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is an example of methylation that can occur in the binding proteins encompassed by the present invention. In some embodiments, the binding protein can be modified by methylating free amines, for example, by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0223] The chemical modification can include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains (such as palmitate or myristate), or albumin. The chemical modification of a binding protein having an Fc region can be a fusion Fc-protein. The polyamino acids can include, for example, polyamino acid sequences having repeated single amino acids (such as polyglycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the foregoing.

[0224] In some embodiments, the binding proteins encompassed by the present invention can be modified. In some embodiments, the modification has substantial or significant sequence identity with the parental binding protein to produce a functional variant that maintains one or more biophysical and / or biological activities of the parental binding protein (e.g., maintaining pMHC binding specificity). In some embodiments, the mutation is a conservative amino acid substitution.

[0225] In some embodiments, the binding proteins encompassed by the present invention can contain synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxy phenylalanine, phenylglycine, α-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, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0226] The binding proteins encompassed by the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0227] In some embodiments, the half-life of the peptides encompassed by the present invention can be extended by attachment of a hydrophobic moiety (e.g., attachment to the N-terminus, C-terminus, or internal amino acids). In other embodiments, the binding proteins can include post-translational modifications (e.g., methylation and / or amidation) that can affect, for example, the serum half-life. In some embodiments, simple carbon chains (e.g., by myristoylation and / or palmitoylation) can be conjugated to the binding proteins. In some embodiments, the simple carbon chains can facilitate the separation of the binding proteins from unconjugated materials. For example, methods that can be used to separate the binding proteins from unconjugated materials include, but are not limited to, solvent extraction and reverse phase chromatography. The lipophilic moiety can extend the 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, including cholestene, cholestane, cholestadiene, and oxysterol. In some embodiments, the binding protein can be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding protein can 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 region, fatty acids, palmitic acid, or molecules that bind to albumin. In some embodiments, a spacer or linker can be coupled to the binding protein, such as 1, 2, 3, 4, or more amino acid residues that serve as a spacer or linker, to facilitate conjugation or fusion with another molecule, and to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, the binding protein can be conjugated to other moieties that can modify or effect a change in the properties of the binding protein.

[0228] Proteins, such as peptides, can be produced recombinantly or synthetically, for example, by solid-phase peptide synthesis or solution-phase peptide synthesis. Protein synthesis can be carried out by known synthetic methods, such as using fluorenylmethoxycarbonyl (Fmoc) chemistry or by tert-butyloxycarbonyl (Boc) chemistry. Protein fragments can be joined together enzymatically or synthetically.

[0229] In one aspect covered by the present invention, provided herein are methods for generating the proteins described herein, the methods comprising the steps of: (i) culturing transformed host cells under conditions suitable to permit expression of the binding protein described herein, the host cells having been transformed with a nucleic acid comprising a sequence encoding the binding protein; and (ii) recovering the expressed binding protein.

[0230] For example, methods useful for isolating and purifying recombinantly produced binding proteins can include obtaining a supernatant from a suitable host cell / vector system that secretes the binding protein into the culture medium, followed by concentrating the culture medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps can be employed to further purify the recombinant polypeptide. These purification methods can also be used when isolating an immunogen from its natural environment. Methods for large-scale manufacture of one or more binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. The binding proteins can be purified according to methods described herein and known in the art.

[0231] In some embodiments, provided herein is a nucleic acid encoding the MAGEA1 immunogenic polypeptide or a fragment thereof described herein, such as a DNA molecule encoding a MAGEA1 immunogenic peptide. In some embodiments, the composition comprises an expression vector that contains an open reading frame encoding the MAGEA1 immunogenic peptide or a fragment thereof described herein. In some embodiments, the nucleic acid includes regulatory elements necessary for expressing the open reading frame. Such elements can include, for example, a promoter, a start codon, a stop codon, and a polyadenylation signal. Additionally, enhancers can be included. These elements are operably linked to the sequence encoding the MAGEA1 immunogenic polypeptide or a fragment thereof. Representative vectors, promoters, regulatory elements, etc. that can be used for expressing proteins such as peptides are further described below.

[0232] III. MHC-Peptide Complexes

[0233] In certain aspects, provided are compositions comprising the MAGEA1 immunogenic peptide and an MHC molecule described herein. In some embodiments, the MAGEA1 immunogenic peptide forms a stable complex with the MHC molecule.

[0234] The MHC protein can be conjugated to an agent such as a detection moiety, a radiosensitizer, a photosensitizer, etc., and / or can be chemically modified as described above for the peptide.

[0235] The MHC proteins provided and used in the compositions and methods covered by the present disclosure can be any suitable MHC molecule known in the art. Generally, it has the formula (α-β-P)n , where n is at least 2, for example between 2 and 10, such as 4. α is the α-chain of a class I or class II MHC protein. β is the β-chain, defined herein as the β-chain of a class II MHC protein or β2-microglobulin of a MHC class I protein. P is a peptide antigen.

[0236] In some embodiments, the MHC protein is a MHC class I complex, such as a HLA I complex.

[0237] The MHC protein can be from any mammalian or avian species, such as a primate species, especially human; a rodent, including mouse, rat, and hamster; rabbit; horse, cow, dog, cat, etc. For example, the MHC protein can be derived from a human HLA protein or a murine H-2 protein. HLA proteins include class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, and 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, and class II subunits I-Aα, I-Aβ, I-Eα, and I-Eβ, and β2-microglobulin. Sequences of some representative MHC proteins can be found in Kabat et al., Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pages 724-815. The MHC protein subunits suitable for use in the present invention are soluble forms of normal membrane-bound proteins, which are prepared as known in the art, for example, by deletion of the transmembrane domain and cytoplasmic domain.

[0238] For class I proteins, the soluble form can include the α1, α2, and α3 domains. Soluble class II subunits can include the α1 and α2 domains of the α-subunit, and the β1 and β2 domains of the β-subunit.

[0239] The α and β subunits can be produced separately and allowed to associate in vitro to form a stable heteroduplex complex, or the two subunits can be expressed in a single cell. Methods for producing MHC subunits are known in the art.

[0240] In certain embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1 and an MHC. In some embodiments, the MHC molecule comprises an MHC α chain, which 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, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01,HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1A and an MHC having an α-chain with an HLA-A*02 serotype, such as an MHC encoded by the HLA-A*02:01 allele.

[0241] To prepare the MHC-peptide complex, the subunits can be combined with the antigenic peptide and allowed to fold in vitro to form a stable heterodimeric complex having an intrastrand disulfide-bonded domain. The peptide can be included in the initial folding reaction or can be added to the empty heterodimer in a subsequent step. In the compositions and methods encompassed by the present invention, the peptide is a MAGEA1 immunogenic peptide or a fragment thereof. Conditions for allowing the subunits and peptide to fold and associate are known in the art. As an example, approximately equimolar amounts of dissolved α and β subunits can be mixed in a urea solution. Refolding is initiated by dilution or dialysis into a urea-free buffer solution. The peptide can be loaded into the empty class II heterodimer at about pH 5 to 5.5 for about 1 to 3 days, followed by neutralization, concentration, and buffer exchange. However, the specific folding conditions are not critical to the practice of the present invention.

[0242] The monomeric complex (α-β-P) (monomer herein) can be multimerized, such as into an MHC tetramer. The resulting multimer is stable over a long period of time. Preferably, the multimer can be formed by binding the monomer to a multivalent entity through specific attachment sites on the α or β subunit, as is known in the art (e.g., as described in U.S. Patent No. 5,635,363). Whether in monomeric or multimeric form, the MHC protein can also be conjugated to beads or any other support.

[0243] The multimeric complex can be labeled so that it can be directly detected when used for immunostaining or other methods known in the art, or it can be used in conjunction with a secondary labeled immunological reagent that specifically and / or selectively binds to the complex (e.g., binds to an MHC protein subunit), as known in the art. For example, the detectable label can be a fluorophore such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin (PE), allophycocyanin (APC), Brilliant Violet TM 421, Brilliant UV TM 395, Brilliant Violet TM 480, BrilliantViolet TM 421 (BV421), Brilliant Blue TM 515, APC-R700 or APC-Fire750. In some embodiments, the multimeric complex is labeled with a moiety capable of specifically and / or selectively binding to another moiety. For example, the label can be biotin, streptavidin, an oligonucleotide, or a ligand. Other labels of interest can include fluorescent dyes, dyes, enzymes, chemiluminescent agents, particles, radioisotopes, or other directly or indirectly detectable agents.

[0244] In some embodiments, cells presenting an immunogenic peptide in the context of an MHC molecule on the cell surface are produced by transfecting or transducing cells with a vector (e.g., a viral vector) that introduces a nucleic acid encoding a recombinant or heterologous antigen into the cells. In some embodiments, the vector is introduced into the cells under conditions where one or more peptide antigens (in some cases, including one or more peptide antigens of the expressed heterologous protein) are expressed, processed, and presented on the cell surface by the cells in the context of major histocompatibility complex (MHC) molecules.

[0245] Generally, the cells contacted by the vector are cells that express MHC, i.e., MHC-expressing cells. The cells can be cells that normally express MHC on the cell surface, cells that are induced to express MHC and / or upregulate MHC expression on the cell surface, or cells that are engineered to express MHC molecules on the cell surface. In some embodiments, the MHC contains polymorphic peptide-binding sites or binding grooves that can, in some cases, complex with peptide antigens of a polypeptide, including peptide antigens processed by cellular mechanisms. In some cases, MHC molecules can be presented or expressed on the cell surface, including in the form of a complex with a peptide, i.e., an MHC-peptide complex, for presenting an antigen in a conformation recognizable by a TCR or other peptide-binding molecule on a T cell.

[0246] 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, dendritic cell, B cell, endothelial cell, or fibroblast. In some embodiments, the cell is an endothelial cell, such as an endothelial cell line or primary endothelial cell. In some embodiments, the cell is a fibroblast, such as a fibroblast cell line or primary fibroblast.

[0247] In some embodiments, the cell is an artificial antigen-presenting cell (aAPC). Generally, aAPCs include features of natural APCs, including the expression of MHC molecules, stimulatory and co-stimulatory molecules, Fc receptors, adhesion molecules, and / or the ability to produce or secrete cytokines (e.g., IL-2). Generally, aAPCs are cell lines that lack the expression of one or more of the above and are generated by introducing (e.g., by transfection or transduction) one or more of the following: elements missing in MHC molecules, low-affinity Fc receptors (CD32), high-affinity Fc receptors (CD64), one or more co-stimulatory 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 β receptor, ILT3, ILT4, 3 / TR6 or B7-H3 ligand; or antibodies that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor or CD83 ligand), cell adhesion molecules (e.g., ICAM-1 or LFA-3), and / or cytokines (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon-α (IFNα), interferon-β (IFNβ), interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), tumor necrosis factor-β (TNFβ), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (GCSF)). In some cases, aAPCs generally do not express MHC molecules but can be engineered to express MHC molecules or, in some cases, be induced or be inducible to express MHC molecules, e.g., by stimulation with cytokines. In some cases, aAPCs can also be loaded with stimulatory ligands, which can include, for example, anti-CD3 antibodies, anti-CD28 antibodies, or anti-CD2 antibodies. Exemplary cell lines that can be used as a backbone for generating aAPCs are the K562 cell line or fibroblast cell lines. A variety of aAPCs are known in the art, see, e.g., U.S. Patent No. 8,722,400; Published Application No. US2014 / 0212446; Butler and Hirano (2014) Immunol Rev. 257:10.1111 / imr.12129; Suhoshki et al. (2007) Mol. Ther. 15:981-988).

[0248] Determining or identifying specific MHCs or alleles expressed by a cell is well within the level of a skilled artisan. In some embodiments, prior to contacting the cell with a vector, expression of a specific MHC molecule can be evaluated or confirmed, for example, by using an antibody specific for the particular MHC molecule. Antibodies directed against MHC molecules are known in the art, such as any of the antibodies described below.

[0249] In some embodiments, cells can be selected to express a desired MHC-restricted MHC allele. In some embodiments, MHC typing of cells (e.g., cell lines) is known in the art. In some embodiments, well-known procedures in the art can be used, such as performing tissue typing by using molecular haplotyping (BioTest ABC SSPtray, BioTest Diagnostics Corporation, Denville, N.J.; SeCore Kits, Life Technologies, Grand Island, N.Y.) to determine the MHC typing of cells (e.g., primary cells obtained from a subject). In some cases, determining HLA genotypes by performing standard cell typing by using sequence-based typing (SBT) is well within the level of a skilled artisan (Adams et al. (2004) J. Transl. Med., 2:30; Smith (2012) Methods MolBiol., 882:67-86). In some cases, the HLA typing of cells (e.g., fibroblasts) is known. By way of example, the human fetal lung fibroblast cell line MRC-5 is HLA-A*02:01, A29, B13, B44 Cw7 (C*0702); the human foreskin fibroblast cell line Hs68 is HLA-A1, A29, B8, B44, Cw7, Cw16; and the WI-38 cell line is A*68:01, B*08:01 (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).

[0250] In some embodiments, the cells contacted with or into which the vector is introduced are cells that have been engineered or transfected to express an MHC molecule. In some embodiments, a cell line can be prepared by genetically modifying a parental cell line. In some embodiments, the cells typically lack a specific MHC molecule and are engineered to express such a specific MHC molecule. In some embodiments, the cells are genetically engineered using recombinant DNA technology.

[0251] 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 monitor T cell responses in a subject, such as 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 MHC-peptide complex-specific T cells are well known in the art. Additional descriptions can be found, for example, in U.S. Patent No. 7,776,562; U.S. Patent No. 8,268,964; and U.S. Patent Publication 2019 / 0085048.

[0252] IV. Immunogenic Compositions

[0253] In some aspects, the present disclosure provides pharmaceutical compositions (e.g., vaccine compositions) that comprise a MAGEA1 immunogenic peptide and / or a nucleic acid encoding a MAGEA1 immunogenic peptide and an adjuvant. In some aspects, the present disclosure provides pharmaceutical compositions (e.g., vaccine compositions) that include a stable MHC-peptide complex comprising a MAGEA1 immunogenic peptide in the context of an MHC molecule and an adjuvant. In some embodiments, the composition comprises a combination of multiple (e.g., two or more) MAGEA1 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 MAGEA1 immunogenic peptide in the context of an MHC molecule and an adjuvant. In some embodiments, the above-described compositions further comprise a pharmaceutically acceptable carrier.

[0254] The pharmaceutical compositions disclosed herein can be specifically formulated for administration in solid or liquid form, including those forms suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), lozenges applied to the tongue, powders, granules, pastes; or (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection in the form of, for example, a sterile solution or suspension or a sustained release formulation.

[0255] Methods for preparing these formulations or compositions include the step of combining a MAGEA1 immunogenic peptide and / or nucleic acid described herein with an adjuvant, a carrier, and optionally one or more accessory ingredients selected as appropriate. Generally, the formulations are prepared by uniformly and intimately bringing the agents described herein into association with a liquid carrier or a finely divided solid carrier or both, and then shaping the product, if necessary.

[0256] A pharmaceutical composition suitable for parenteral administration comprises a combination of a MAGEA1 immunogenic peptide and / or nucleic acid as described herein and an adjuvant, and one or more pharmaceutically 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, bacteriostatic agents, solutes that render the preparation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0257] Examples of suitable aqueous and non-aqueous carriers for use in pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). For example, appropriate fluidity can be maintained by using coating materials such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0258] Regardless of the selected route of administration, the agents provided herein and / or the pharmaceutical compositions disclosed herein, when presented in a suitable hydrated form, can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0259] In some embodiments, when administered to a subject, the pharmaceutical composition can elicit an immune response against cells infected with MAGEA1. Such pharmaceutical compositions can be used as prophylactic and / or therapeutic vaccines for treating conditions characterized by MAGEA1 expression.

[0260] In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the adjuvant used increases the immunogenicity of the pharmaceutical composition. Such compounds or adjuvants that stimulate further immune responses can: (i) be mixed into the pharmaceutical composition according to the invention after the peptide is reconstituted and optionally emulsified with an oil-based adjuvant as defined above, (ii) can be part of the reconstituted composition of the invention as defined above, (iii) can be physically linked to the peptide to be reconstituted, or (iv) can be administered separately to the subject, mammal or human to be treated. The adjuvant can be an adjuvant that provides slow release of the antigen (e.g., the adjuvant can be a liposome), or it can be an adjuvant that is immunogenic per se and thus acts synergistically with the antigen (i.e., the antigen present in the MAGEA1 immunogenic peptide). For example, the adjuvant can be a known adjuvant, or other substances that promote antigen uptake, recruit immune system cells to the site of administration, or promote the immune activation of responding lymphoid cells. Adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, dextran, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, synthetic polymers (e.g., polyamino acids and amino acid copolymers), saponins, paraffin oil, and muramyl dipeptide. In some embodiments, the adjuvant is Adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, 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.

[0261] In some embodiments, the adjuvant is an immunomodulatory molecule. For example, the immunomodulatory molecule can be a recombinant protein cytokine, chemokine or immunostimulant designed to enhance the immune response, or a nucleic acid encoding a cytokine, chemokine or immunostimulant.

[0262] 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-1α, MIP-1β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and functional fragments of any of the foregoing.

[0263] 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), and functional fragments of any of the foregoing.

[0264] In some embodiments, the composition comprises a nucleic acid encoding a MAGEA1 immunogenic polypeptide as described herein, such as a DNA molecule encoding a MAGEA1 immunogenic peptide. In some embodiments, the composition comprises an expression vector that comprises an open reading frame encoding a MAGEA1 immunogenic peptide.

[0265] When taken up by a cell (e.g., a host cell, an antigen-presenting cell (APC), such as a dendritic cell, macrophage, etc.), the DNA molecule can exist as an extrachromosomal molecule in the cell and / or can integrate into the chromosome. The DNA can be introduced into the cell in plasmid form, which can remain as an independent genetic material. Alternatively, linear DNA that can integrate into the chromosome can also be introduced into the cell. Optionally, when introducing the DNA into the cell, a reagent that promotes integration of the DNA into the chromosome can be added.

[0266] V. Binding Proteins

[0267] In some aspects, a binding moiety is provided that combines a peptide described herein and / or a stabilized MHC-peptide complex described herein. For example, binding proteins such as T cell receptors (TCRs), antibodies, etc. are provided that bind, for example, with a K of less than or equal to about 10 -4 M (e.g., about 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 10 10 -11 10 -12 10 -13 10 -14 10 d etc.) specifically and / or selectively to the peptide and / or the stabilized MHC-peptide complex.

[0268] In one aspect encompassed by the present invention, binding proteins are provided herein that bind (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex comprising an immunogenic peptide of MAGEA1 in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the binding protein is capable of binding (e.g., specifically and / or selectively) to the MAGEA1 peptide-MHC (pMHC) complex with a K d less than or equal to about 5×10 -4 M, less than or equal to about 1×10 -4 M, less than or equal to about 5×10 - 5 M, less than or equal to about 1×10 -5 M, less than or equal to about 5×10 -6 M, less than or equal to about 1×10 -6 M, less than or equal to about 5×10 -7 M, less than or equal to about 1×10 -7 M, less than or equal to about 5×10 -8 M, less than or equal to about 1×10 -8 M, less than or equal to about 5×10 -9 M, less than or equal to about 1×10 -9 M, less than or equal to about 5×10 -10 M, less than or equal to about 1×10 -10 M, less than or equal to about 5×10 -11 M, less than or equal to about 1×10 -11 M, less than or equal to about 5×10 -12 M, less than or equal to about 1×10 -12M, or any range in between (including endpoints), such as about 1 - 50 micromoles, 1 - 100 micromoles, 0.1 - 500 micromoles, etc. In some embodiments, the MHC molecule comprises an MHC α-chain, which 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, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21,HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In some embodiments, the HLA serotype is HLA-A*02 and / or the HLA allele is the HLA-A*02:01 allele. In some embodiments, the binding proteins provided herein are genetically engineered, isolated, and / or purified.

[0269] In some embodiments, the binding protein has a higher binding affinity for the MAGEA1 peptide-MHC (pMHC) than known T cell receptors (e.g., the TCR from van Kunert et al. (2016) J. Immunol. 197:2541-2552 or other TCRs described herein). For example, the binding protein may have a binding affinity for the MAGEA1 peptide-MHC (pMHC) that is 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, 5000-fold, 10000-fold, 50000-fold, 100000-fold, 500000-fold, 1000000-fold or more higher than known T cell receptors, or any range therebetween (including the endpoints), such as 1.2-fold to 2-fold.

[0270] In some embodiments, when contacting target cells expressing MAGEA1 at a certain level or lower, compared to known T cell receptors, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing (see, for example, the Examples section for representative cell lines expressing MAGEA1 at different levels). For example, in some embodiments of any aspect described herein, the MAGEA1 level can be expressed per million transcripts and can be, for example, less than or equal to about 1,000 transcripts per million transcripts (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 any range therebetween (including the endpoints), such as less than or equal to about 1,000 TPM to less than or equal to about 35 TPM). In some embodiments, a low MAGEA1 expression level is referred to as "heterozygous expression", meaning between about 1 TPM and about 35 TPM, or any range therebetween (including the endpoints), such as 32 TPM or 1 - 32 TPM. Higher expression is 36 TPM and higher. As further described herein, TPM is measured according to well-known techniques, such as RNA-Seq, and gene expression TPM data for a variety of cell lines, tissue types, etc. are well-known in the art (see, for example, the Broad Institute Cancer Cell Line Encyclopedia (CCLE) at portals.broadinstitute.org on the World Wide Web).In some embodiments, when contacting target cells that express a MAGEA1 peptide epitope, such as heterologously expressing a MAGEA1 peptide epitope, the binding protein induces 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 or more, or any range therebetween (including the endpoints), such as 1.2-fold to 2-fold, increase in T cell expansion, cytokine release, and / or cytotoxic killing compared to a known T cell receptor (e.g., a comparator TCR described herein).

[0271] In some embodiments, expression of MAGEA1 is detected using RNA sequencing (RNA-seq). RNA-seq generally includes the steps of: 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 assess the expression levels of different transcripts. The sample can be a cell population, a tissue sample, a biopsy sample, a cell culture, or a single cell. Any method known in the art can be used to isolate total RNA from a biological sample. 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 HomermRNA in Material Plasma Is Inclined in Preclampsia”, Clinr. As described therein, 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 placed in an RNeasy MinElute column (Qiagen) and processed according to the manufacturer's recommendations.

[0272] In some embodiments, the RNA-seq described herein includes the step of preparing amplified cDNA from total RNA. For example, cDNA is prepared and the isolated RNA sample is randomly amplified without dilution, or the mixture of genetic material in the isolated RNA is dispersed into individual reaction samples. In certain embodiments, the amplification randomly starts at the 3' end and proceeds throughout the entire transcriptome in the sample to amplify 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 covered by the present invention include, for example RNA-Seq systems.

[0273] In some embodiments, the RNA-seq described herein includes the step of sequencing the amplified cDNA. Any known sequencing method can be used to sequence the amplified cDNA mixture, including single-molecule sequencing methods. In certain embodiments, the amplified cDNA is sequenced by whole transcriptome shotgun sequencing. Whole transcriptome shotgun sequencing can be performed using various next-generation sequencing platforms, such as genome analysis platforms, ABI SOLiD TM sequencing platforms or the 454 sequencing platform of LifeScience.

[0274] In some embodiments, the RNA-seq described herein further includes digital counting and analysis of the cDNA. The number of amplified sequences of 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 specific transcript. TPM can be calculated as shown in Wagner et al. (2012) Theory in Biosciences 131:281-285, the content of which is incorporated herein by reference in its entirety.

[0275] In certain embodiments, the binding protein recognizes an immunogenic peptide of MAGEA1 in complex with an MHC molecule, such as a particular HLA molecule having a specific HLA α-chain allele. For example, the binding proteins listed in Table 2A were identified as binders of an immunogenic peptide of MAGEA1 associated with an MHC having the HLA-A*02 serotype of the α-chain, such as the MHC encoded by the HLA-A*02:01 allele, as further described in the Examples section. In some embodiments, the binding protein recognizes a complex of an immunogenic peptide of MAGEA1 and an MHC molecule, wherein the MHC molecule comprises an MHC α-chain that is selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18 HLA serotypes, optionally wherein the HLA allele is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20,HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In some embodiments, the MAGEA1 immunogenic peptides are derived from the human MAGEA1 protein and / or the MAGEA1 proteins shown in Table 3. In some embodiments, one or more MAGEA1 immunogenic peptides are administered alone or in combination with an adjuvant.

[0276] In some embodiments, the binding protein does not bind to a peptide-MHC (pMHC) complex, optionally wherein the peptide is derived from an "off-target" as described herein.

[0277] In some embodiments, the binding protein does not bind to an "off-target" as described herein that is complexed with an MHC peptide-MHC (pMHC) complex.

[0278] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or consist of the following): a) a TCR α-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 α-chain sequence selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) a TCR β-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 β-chain sequence selected from the group consisting of the TCR β-chain sequences listed in Table 2.

[0279] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or consist of the following): a) a TCR α-chain sequence selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) a TCR β-chain sequence selected from the group consisting of the TCR β-chain sequences listed in Table 2.

[0280] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or consist of the following): a) a TCR α-chain variable (V α ) 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 a TCR α-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) a TCR β-chain variable (V β ) 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 a TCR β-chain variable (V β ) domain sequence selected from the group consisting of the TCR V β ) domain sequences listed in Table 2.

[0281] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or consist of the following): a) a TCR α-chain variable (V α) Domain sequences selected from the group consisting of TCR V domain sequences listed in Table 2; and / or b) TCR β-chain variable (V) domain sequences selected from the group consisting of TCR V domain sequences listed in Table 2. α In some embodiments, the binding proteins provided herein include (e.g., comprise at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2), consist essentially of, or consist of) TCR α-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 the group consisting of TCR α-chain CDR sequences listed in Table 2. CDR3 is considered the primary CDR responsible for recognizing processed antigen, and CDR1 and CDR2 primarily interact with MHC. Thus, in some embodiments, binding proteins are provided that include CDR3 alone from the TCR α-chain listed in Table 2 and / or CDR3 alone from the TCR β-chain listed in Table 2, each CDR3 having sequence homology as described in this paragraph. β ) Domain sequences selected from the group consisting of TCR V domain sequences listed in Table 2; and / or b) TCR β-chain variable (V) domain sequences selected from the group consisting of TCR V domain sequences listed in Table 2. β In some embodiments, the binding proteins provided herein include (e.g., comprise at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2), consist essentially of, or consist of) TCR β-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 the group consisting of TCR β-chain CDR sequences listed in Table 2. As described above, CDR3 is considered the primary CDR responsible for recognizing processed antigen, and CDR1 and CDR2 primarily interact with MHC. Thus, in some embodiments, binding proteins are provided that include CDR3 alone from the TCR β-chain listed in Table 2 and / or CDR3 alone from the TCR α-chain listed in Table 2, each CDR3 having sequence homology as described in this paragraph.

[0282] In some embodiments, the binding proteins provided herein include (e.g., comprise at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2), consist essentially of, or consist of) TCR α-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 the group consisting of TCR α-chain CDR sequences listed in Table 2. CDR3 is considered the primary CDR responsible for recognizing processed antigen, and CDR1 and CDR2 primarily interact with MHC. Thus, in some embodiments, binding proteins are provided that include CDR3 alone from the TCR α-chain listed in Table 2 and / or CDR3 alone from the TCR β-chain listed in Table 2, each CDR3 having sequence homology as described in this paragraph.

[0283] In some embodiments, the binding proteins provided herein further include (e.g., comprise at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2), consist essentially of, or consist of) TCR β-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 the group consisting of TCR β-chain CDR sequences listed in Table 2. As described above, CDR3 is considered the primary CDR responsible for recognizing processed antigen, and CDR1 and CDR2 primarily interact with MHC. Thus, in some embodiments, binding proteins are provided that include CDR3 alone from the TCR β-chain listed in Table 2 and / or CDR3 alone from the TCR α-chain listed in Table 2, each CDR3 having sequence homology as described in this paragraph.

[0284] In some embodiments, the binding proteins provided herein include (e.g., comprise at least one (e.g., one, two, or three), consist essentially of, or consist of) TCR α-chain complementarity determining region (CDR) listed in Table 2.

[0285] In some embodiments, the binding proteins provided herein may also include (e.g., comprise at least one (e.g., one, two, or three), consist essentially of, or consist of) the TCRβ chain complementarity determining regions (CDRs) listed in Table 2.

[0286] In some embodiments, the binding proteins provided herein include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCRα chain constant region (C α ) 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 TCR Cα sequences listed in Table 2.

[0287] In some embodiments, the binding proteins provided herein may also include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCR C β 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 TCR C β ) sequence of the TCRβ chain constant region.

[0288] In some embodiments, the binding proteins provided herein include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCRα chain constant region (C α sequence selected from the group consisting of the TCR C α ) sequences listed in Table 2.

[0289] In some embodiments, the binding proteins provided herein may also include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCR C β sequence selected from the group consisting of the TCR C β ) sequences of the TCRβ chain constant region listed in Table 2.

[0290] Table 2: TCR Sequences Recognizing MAGEA1 Antigen

[0291] Table 2A

[0292] TCR Sequences Recognizing MAGEA1 Antigen Presented by HLA Serotype HLA-A*02

[0293] MAGEA1-278-1479WT Sequence

[0294] Alpha chain: TRAV20 / TRAJ26 / TRAC alpha chain DNA sequence

[0295]

[0296] Alpha chain protein sequence

[0297]

[0298] Beta chain:

[0299] TRBV5-8 / TRBJ1-1 / TRBC1 beta chain DNA sequence

[0300]

[0301] Beta chain protein sequence

[0302]

[0303]

[0304] MAGEA1-278-1479 MGTM codon-optimized sequence (also known as clone "MAGE-A1-1479", "TCR1479", "1479", TCR expressed by "TSC-204-A02", and TCR expressed by "TSC-204-A0201")

[0305] Alpha chain:

[0306] TRAV20 / TRAJ26 / MGTM-modified TRAC

[0307] Alpha chain DNA sequence

[0308]

[0309] Alpha chain protein sequence

[0310]

[0311]

[0312] Beta chain:

[0313] TRBV5-8 / TRBJ1-1 / MGTM-modified TRBC beta chain DNA sequence

[0314]

[0315] Beta chain protein sequence

[0316]

[0317] Full β and α ORF DNA sequences (the underlined italicized region in the "Furin - P2A" site encodes a sequence that allows the expression of two polypeptide chains in a single cassette)

[0318]

[0319]

[0320] Full β and α ORF protein sequences (the underlined italicized region in the "Furin - P2A" site allows the expression of two polypeptide chains in a single cassette)

[0321]

[0322] *Table 2 part provides representative TCR sequences, which are grouped according to MHC serotype presentation and different peptide subgroups presented by MHC serotype and bound by subgrouped TCRs. Describe and claim individual TCRs, such as those in the representative examples in the table, and classes of binding proteins that bind to the peptide epitope sequences described herein, either alone or in complex with MHC, such as those grouped in the tables provided herein. In addition, the TRAV, TRAJ, and TRAC genes of each TCR α - chain described herein, and the TRBV, TRBJ, and TRBC genes of each TCR β - chain described herein are provided. The sequences of each TCR described herein are provided as pairs of homologous α - chain and β - chain for each named TCR. Annotate the TCR sequences described herein. Variable domain sequences are in uppercase. Constant domain sequences are in lowercase. CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined text. CDR1, CDR2, and CDR3 are shown in the standard order of occurrence from left (N - terminus) to right (C - terminus). The TRAV, TRAJ, and TRAC genes of each TCR α - chain described herein, and the TRBV, TRBJ, and TRBC genes of each TCR β - chain described herein are annotated according to the well - known IMGT nomenclature described herein. Similarly, CDR1 and CDR2 of TRAV and TRBV are well - known in the art as they are based on well - known and annotated TRAV and TRBV sequences (e.g., as annotated in the databases of IMGT available at imt.org and IEDB available at iedb.org).

[0323] Table 3

[0324] Representative human MAGEA1 cDNA sequence

[0325] atgtctcttgagcagaggagtctgcactgcaagcctgaggaagcccttgaggcccaacaagaggccctgggcctggtgtgtgtgcaggctgccacctcctcctcctctcctctggtcctgggcaccctggaggaggtgcccactgctgggtcaacagatcctccccagagtcctcagggagcctccgcctttcccactaccatcaacttcactcgacagaggcaacccagtgagggttccagcagccgtgaagaggaggggccaagcacctcttgtatcctggagtccttgttccgagcagtaatcactaagaaggtggctgatttggttggttttctgctcctcaaatatcgagccagggagccagtcacaaaggcagaaatgctggagagtgtcatcaaaaattacaagcactgttttcctgagatcttcggcaaagcctctgagtccttgcagctggtctttggcattgacgtgaaggaagcagaccccaccggccactcctatgtccttgtcacctgcctaggtctctcctatgatggcctgctgggtgataatcagatcatgcccaagacaggcttcctgataattgtcctggtcatgattgcaatggagggcggccatgctcctgaggaggaaatctgggaggagctgagtgtgatggaggtgtatgatgggagggagcacagtgcctatggggagcccaggaagctgctcacccaagatttggtgcaggaaaagtacctggagtaccggcaggtgccggacagtgatcccgcacgctatgagttcctgtggggtccaagggccctcgctgaaaccagctatgtg aaagtccttgagtatgtgatcaaggtc agtgcaagagttcgctttttcttcccatccctgcgtgaagcagctttgagagaggaggaagagggagtctga

[0326] Representative human MAGEA1 protein sequence

[0327] MSLEQRSLHCKPEEALEAQQEALGLVCVQAATSSSSPLVLGTLEEVPTAGSTDPPQSPQGASAFPTTINFTRQRQPSEGSSSREEEGPSTSCILESLFRAVITKKVADLVGFLLLKYRAREPVTKAEMLESVIKNYKHCFPEIFGKASESLQLVFGIDVKEADPTGHSYVLVTCLGLSYDGLLGDNQIMPKTGFLIIVLVMIAMEGGHAPEEEIWEELSVMEVYDGREHSAYGEPRKLLTQDLVQEKYLEYRQVPDSDPARYEFLWGPRALAETSYV KVLEYVIKV SARVRFFFPSLREA

[0328] ALREEEEGV*

[0329] Representative HLA-A*02:01 DNA sequence

[0330]

[0331] Representative HLA-A*02:01 protein sequence

[0332] MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV*

[0333] Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pTSLV102-MSCV-HA1-10-30-MGTM-Q-CD8

[0334] tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacg

[0335]

[0336]

[0337]

[0338]

[0339]

[0340] tagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcct

[0341] atggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttc

[0342] ctgcgttatccCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT

[0343] GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCG

[0344] AGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAAC

[0345] CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCAAGCT

[0346] CATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGC

[0347] CTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTT

[0348] GGAGGCCTAGGCTTTTGCAAAAAGCTCCCCGTGGCACGACAGG

[0349] TTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG

[0350] TGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATG

[0351] CTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAAT

[0352] TTCACACAGGAAACAGCTATGACATGATTACGAATTTCACAAA

[0353] TAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAAC

[0354] TCATCAATGTATCTTATCATGTCTGGATCAACTGGATAACTCAA

[0355] GCTAACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTA

[0356] CCACTGCCAATTACCTGTGGTTTCATTTACTCTAAACCTGTGAT

[0357] TCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTTGTTAAAT

[0358] ATGTACTACAAACTtagtagt

[0359] Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pHAGE-MSCV-HN-P32-41-P2A-dnTGFbRII (dnTGFbRII is highlighted in bold)

[0360] tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgag

[0361]

[0362]

[0363]

[0364]

[0365] atttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccctta

[0366] acgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt

[0367] ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaag

[0368] agctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgt

[0369] agccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttac

[0370] cagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataa

[0371] ggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacac

[0372] cgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac

[0373] aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc

[0374] ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggg

[0375] ggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgct

[0376] cacatgttctttcctgcgttatccCCTGATTCTGTGGATAACCGTATTACCGCC

[0377] TTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC

[0378] GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATAC

[0379] GCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAG

[0380] CAAGCTCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGA

[0381] GGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGC

[0382] TTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCCGTGGCAC

[0383] GACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCA

[0384] ATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC

[0385] ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGA

[0386] TAACAATTTCACACAGGAAACAGCTATGACATGATTACGAATT

[0387] TCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTG

[0388] TCCAAACTCATCAATGTATCTTATCATGTCTGGATCAACTGGAT

[0389] AACTCAAGCTAACCAAAATCATCCCAAACTTCCCACCCCATAC

[0390] CCTATTACCACTGCCAATTACCTGTGGTTTCATTTACTCTAAAC

[0391] CTGTGATTCCTCTGAATTATTTTCATTTTAAAGAAATTGTATTT

[0392] GTTAAATATGTACTACAAACTtagtagt

[0393] Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pNVVD136_TSC-204-A02_TCR-1479_MSCV-TCR-1479-CD8-EF1α-dnTGFbRII-DHFR

[0394]

[0395]

[0396]

[0397]

[0398]

[0399] TCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACAGGTTACCTCAGTCTCCTAGGTACGTCTTATATCTATGAAAAAACATTCAAAAGCACAACATCTAGAAGAACTTACCTTTTTTCACCACTCTATTGCAAAGATATGTACCGATTTCTCTCGAAGTACAAAAAACCGCTAGTTTTCAAATTCACCTCAAGACTTTGAAAAAAAATTGAATCTGTCAATGTCAAATAAAATCAGAAACAAATGTCATAATGTTACGTTAATGTTGTCAGGTCGAAAAATAAAATTGCAAATAGAAATTTTGTTCCTTTTTTATTGGTTTTTATTGGTGGGAAAAATATTCCCTCTAACTGCAAAAGGGTTAATTATGTTAGAGGTAGAGTCGACAAGCTT

[0400] Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pNVVD166_TSC-204-A02_TCR-1479_MSCV-TCR-1479-CD8-EF1a-DHFR

[0401] GCTAGCTGGCTTGTTGTCCACAACCATTAAACCTTAAAAGCTTTAAAAGCCTTATATATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTGCTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACGTGAAACATGAGAGCTTAGTACATTAGCCATGAGAGCTTAGTACATTAGCCATGAGGGTTTAGTTCATTAAACATGAGAGCTTAGTACATTAAACATGAGAGCTTAGTACATACTATCAACAGGTTGAACTGCTGATCTGTACAGTAGAATTGGTAAAGAGAGTTGTGTAAAATATTGAGTTCGCACATCTTGTTGTCTGATTATTGATTTTTGGCGAAACCATTTGATCATATGACAAGATGTGTATCTACCTTAACTTAATGATTTTGATAAAAATCATTAGGTACCAATTACATTGCTTGCAATTAACCCTTTAACGGTTATAAGGATCTAGATGAGATAGAAAGATTTGGTTTTCGGATTTGTGTTACATAAGATGCCTAAAATAAAAATTGAGATTCAATTTTTTTTAAACTTTTTTTTAATTGGTGGTAAGAATATTCCCTCTACCTGTTTGAGAGTAATGAAATTGTAGTATGATTTTTCAACAAACTAAAA

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408] *For certain depicted vectors, the MSCV promoter is in bold. The β-chain is annotated with bold and italic text. The α-chain is annotated with bold and underlined text. The CD34 enrichment tag (Q-tag) is annotated with italic and underlined text. CD8-α is italic. CD8-β is underlined.

[0409] Table 4

[0410] T-Knife-based T1367 TCR MGTM codon-optimized sequence

[0411] α-chain:

[0412] TRAV5 / TRAJ41 / MGTM-modified TRAC

[0413] α-chain DNA sequence

[0414]

[0415]

[0416] α-chain protein sequence

[0417]

[0418] β-chain:

[0419] TRBV28 / TRBJ2-7 / MGTM-modified TRBC β-chain DNA sequence

[0420]

[0421]

[0422] β-chain protein sequence

[0423]

[0424] Full β and α ORF DNA sequence (the italicized and underlined region in the "Furin-P2A" site encodes the sequence allowing the expression of two polypeptide chains in a single cassette)

[0425]

[0426]

[0427] Full β and α ORF protein sequence (the italicized and underlined region in the "Furin-P2A" site allows the expression of two polypeptide chains in a single cassette)

[0428]

[0429]

[0430] Immatics-based R37P1C9 TCR MGTM codon-optimized sequence alpha chain:

[0431] TRAV26-2 / TRAJ21 / MGTM-modified TRAC alpha chain DNA sequence

[0432]

[0433]

[0434] Alpha chain protein sequence

[0435]

[0436] Beta chain:

[0437] TRBV15 / TRBJ1-4 / MGTM-modified TRBC beta chain DNA sequence

[0438]

[0439]

[0440] Beta chain protein sequence

[0441]

[0442] Complete beta and alpha ORF DNA sequence (the underlined italicized region in the "Furin-P2A" site encodes the sequence allowing the expression of two polypeptide chains in a single cassette)

[0443]

[0444]

[0445] Complete beta and alpha ORF protein sequence (the underlined italicized region in the "Furin-P2A" site allows the expression of two polypeptide chains in a single cassette)

[0446]

[0447] EPVHLPCNHS TISGTDY IHWYRQLPSQGPEYVIH GLTSN VNNRM

[0448] ASLAIAEDRKSSTLILHRATLRDAAVYY CILFNFNKFYF GSGTKL

[0449] NVKPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksn

[0450] savawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfn

[0451] llmtlrlws

[0452] * For certain depicted vectors, the MSCV promoter is in bold. The β chain is annotated with bold and italic text. The α chain is annotated with bold and underlined text. The CD34 enrichment tag (Q tag) is annotated with italic and underlined text. CD8-α is italic. CD8-β is underlined.

[0453] * Tables 1-4 include peptide epitopes, and polypeptide molecules, or portions thereof, having an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity to the amino acid sequence of any sequence listed in Table 1 over its full length. Such polypeptides can have the functions of the full-length peptides or polypeptides further described herein.

[0454] * Tables 1-4 include RNA nucleic acid molecules (e.g., thymine replaced with uracil), nucleic acid molecules encoding orthologs of the encoded proteins, and DNA or RNA nucleic acid sequences, or portions thereof, having a nucleic acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity to the nucleic acid sequence of any sequence listed in Tables 1-4 over its full length. Such nucleic acid molecules can have the functions of the full-length nucleic acids further described herein.

[0455] In some embodiments, the binding proteins provided herein comprise chimeric, humanized, human, primate, or rodent (e.g., rat or mouse) constant regions. For example, a human variable region can be chimerized with a murine constant region, or a murine variable region can be humanized with a human constant region and / or human framework regions. In some embodiments, the constant region can be mutated to modify functionality (e.g., introducing a non-naturally occurring cysteine substitution in the relative residue positions in the TCR α and β chains to provide a disulfide bond that can be used to increase the affinity between the TCR α and β chains). Similarly, mutations can be made in the transmembrane domain of the constant region to modify functionality (e.g., increasing hydrophobicity by introducing a non-naturally occurring residue substitution with a hydrophobic amino acid). In some embodiments, each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions, or combinations thereof as compared to a reference CDR sequence. In some embodiments, the constant region can be mutated to increase cell surface expression.

[0456] In some embodiments, the binding proteins disclosed herein can be engineered protein scaffolds, antibodies or antigen-binding fragments thereof, TCR-mimicking antibodies, etc. Such binding moieties can be designed and / or generated using conventional immunological methods against the peptides and / or MHC-peptide complexes described herein, such as immunizing a host, obtaining antibody-producing cells and / or their antibodies, and generating hybridomas that can be used to produce monoclonal antibodies (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 No. WO 2007 / 143104, PCT / US86 / 02269 and WO 86 / 01533; U.S. Patent No. 4,816,567; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:3439-3443; 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. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S.L. (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).When necessary, conventional procedures can be used to isolate or purify the binding moiety, such procedures as protein A-agarose, 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, N.Y.).

[0457] The term “antibody / antibodies” broadly encompasses naturally occurring antibody forms (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, as well as fragments and derivatives of all of the foregoing antibodies that have at least one antigen binding site. Antibody derivatives can comprise a protein or chemical moiety conjugated to the antibody.

[0458] In addition, intracellular antibodies are well-known antigen-binding molecules that have antibody characteristics but are capable of being 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 for targeting (e.g., inhibiting) intracellular compartments are well-known in the art, such as using single-chain antibodies (scFv), modifying immunoglobulin VL domains to obtain hyperstability, modifying antibodies to resist the reducing intracellular environment, generating fusion proteins that increase intracellular stability and / or regulate intracellular localization, and the like. 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 Lett. 508:407-412; Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).

[0459] As used herein, the term "antibody" also includes an "antigen-binding portion" (or simply "antibody portion") of an antibody. As used herein, the term "antigen-binding portion" 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 within the term "antigen-binding portion" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are divalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (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 (Ward et al., (1989) Nature 341:544-546), which consist of a VH domain; and (vi) isolated complementarity-determining regions (CDRs). In addition, although the two domains, VL and VH, of an Fv fragment are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single protein chain, where the VL region pairs with the VH region to form a monovalent polypeptide (referred to 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). It is also intended that such single-chain antibodies be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of a particular scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate an expression vector encoding a complete IgG polypeptide or other isotype. VH and VL can also be used to generate Fab, Fv, or other immunoglobulin fragments using protein chemistry or recombinant DNA techniques. Also encompassed are other forms of single-chain antibodies such as, for example, bispecific antibodies. Bispecific antibodies are bivalent bispecific antibodies in which the VH domain and the VL domain are expressed on a single polypeptide chain but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0460] Alternatively, an antibody or antigen-binding portion thereof can be part of a larger immunoadhesion polypeptide formed by the covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include a tetrameric scFv polypeptide made using a streptavidin core region (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and a bivalent and biotinylated scFv polypeptide made using cysteine residues, protein subunit peptides, and a C-terminal polyhistidine tag (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions such as Fab and F(ab')2 fragments can be prepared from intact antibodies using conventional techniques, such as papain digestion or pepsin digestion of intact antibodies, respectively. In addition, as described herein, antibodies, antibody portions, and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques.

[0461] 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 invention bind specifically and / or selectively or substantially specifically and / or selectively to the peptides and / or MHC-peptide complexes described herein. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one antigen-binding site capable of immunoreacting with a particular epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple antigen-binding sites capable of interacting with a particular antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the particular antigen with which they immunoreact.

[0462] Similar to other binding portions described herein, antibodies can also be "humanized," which is intended to include antibodies made by non-human cells having variable and constant regions that have been altered to be more closely similar to antibodies that would be made by human cells. By way of example, the amino acid sequence of a non-human antibody is altered to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention can, for example, include amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro / in vivo random or site-specific mutagenesis or by in vivo somatic mutation). As used herein, the term "humanized antibody" also includes antibodies in which the CDR sequences from the germline of another mammalian species have been grafted onto human framework sequences.

[0463] In some embodiments, the binding proteins disclosed herein can 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 can comprise two polypeptide chains, each of which comprises a variable region that comprises CDR3 of the TCRα chain and CDR3 of the TCRβ chain, or CDR1, CDR2, and CDR3 of both the TCRα chain and the TCRβ chain. In some embodiments, the binding protein comprises a single-chain TCR (scTCR) that comprises both TCR V α and TCR V β domains, but comprises only a single TCR constant domain (C α or C β ). The term "chimeric antigen receptor" (CAR) refers to a fusion protein that has been engineered to contain two or more naturally occurring amino acid sequences that are linked together in a non-naturally occurring manner or in a manner that is not naturally occurring in a host cell, and that functions as a receptor when present on the cell surface. CARs encompassed by the present invention can include an extracellular portion that comprises an antigen-binding domain (i.e., obtained from or derived from an immunoglobulin or immunoglobulin-like molecule, such as an antibody or TCR, or an antigen-binding domain derived from or obtained from a killer immunoglobulin receptor from an NK cell), which antigen-binding domain is linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing a co-stimulatory domain) (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).

[0464] In some embodiments, 1) the TCRα chain CDRs, TCR V α domain, and / or the TCRα chain are encoded by TRAV, TRAJ, and / or TRAC genes or fragments thereof selected from the group consisting of the TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) the TCRβ chain CDRs, TCR V β domain, and / or the TCR β chain are encoded by TRBV, TRBJ, and / or TRBC genes or fragments thereof selected from the group consisting of the TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions, or combinations thereof compared to the homologous reference CDR sequences listed in Table 2.

[0465] In some embodiments, the binding proteins disclosed herein (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs)) are chimeric (e.g., comprising amino acid residues or motifs from more than one donor or species), humanized (e.g., comprising residues from a non-human organism that have been altered or replaced to reduce the risk of human immunogenicity), or human.

[0466] Methods for generating engineered binding proteins (e.g., 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).

[0467] In some embodiments, the binding proteins described herein are TCRs or antigen-binding fragments thereof expressed on the cell surface, wherein the cell surface-expressed TCR is capable of associating more effectively with CD3 protein compared to the endogenous TCR. When expressed on the surface of a cell such as a T cell, the binding proteins (e.g., TCRs) encompassed by the present invention may also have higher surface expression on the cell compared to an endogenous binding protein (e.g., endogenous TCR). In some embodiments, a CAR is provided herein, wherein the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0468] Also provided are modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs), which can be engineered according to well-known methods using the binding proteins disclosed herein having one or more V α and / or V β sequences as starting materials, and the modified binding proteins may have properties that are altered compared to the starting binding protein. The binding protein can be engineered by modifying one or more residues within one or two variable regions (i.e., V α and / or V β ), e.g., within one or more CDR regions and / or within one or more framework regions. Alternatively or additionally, the binding protein can be engineered by modifying residues within the constant region.

[0469] Another type of variable region modification is to mutate the amino acid residues in the V α and / or V β CDR1, CDR2, and / or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the binding protein of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effects on protein binding or other functional properties of interest can be evaluated in in vitro, ex vivo, or in vivo assays as described herein and provided in the examples. In some embodiments, conservative modifications (as discussed above) can be introduced. The mutations can be amino acid substitutions, additions, or deletions. In some embodiments, the mutations are substitutions. Additionally, typically no more than one, two, three, four, or five residues in the CDR regions are modified.

[0470] In some embodiments, the binding proteins described herein (e.g., TCR, antigen-binding fragments of TCR, or CAR) can have one or more amino acid substitutions, deletions, or additions relative to a naturally occurring TCR. In some embodiments, each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions, or combinations thereof compared to the homologous reference CDR sequences listed in Table 2. Conservative substitutions of amino acids are well known and can occur naturally or can be introduced during the recombinant production of the binding protein. Amino acid substitutions, deletions, and additions can be introduced into the protein using mutagenesis methods known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide site-specific (or segment-specific) mutagenesis procedures can be employed to provide an altered polynucleotide having specific codons that are changed according to the desired substitution, deletion, or insertion. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis, can be used to prepare immunogenic polypeptide variants (see, e.g., Sambrook et al., supra).

[0471] A variety of criteria known to those of ordinary skill 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 or conservative amino acid substitution is a substitution 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 having basic side chains (e.g., lysine, arginine, histidine); amino acids having acidic side chains (e.g., aspartic acid, glutamic acid); amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline is considered more difficult to classify and shares characteristics with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, substituting glutamine for glutamic acid or asparagine for aspartic acid can be considered a similar substitution since glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As would be understood in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence of the polypeptide and its conservative amino acid substitutions with the sequence of a second polypeptide (e.g., using GENEWORKS TM , Align, BLAST algorithms or other algorithms described herein and practiced in the art).

[0472] In some embodiments, the encoded binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) can include a "signal peptide" (also referred to as a leader sequence, leader peptide, or transit peptide). The signal peptide targets the newly synthesized polypeptide to its appropriate location inside or outside the cell. The signal peptide can be removed from the polypeptide during 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 described herein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) include a mature V α domain, a mature V β domain, or both. In some embodiments, the binding proteins described herein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) include a mature TCR β-chain, a mature TCR α-chain, or both.

[0473] 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 comprising an immunogenic MAGEA1 peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the MHC molecule comprises an MHC α-chain that is of the HLA serotype HLA-A*02. In some embodiments, the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, and HLA-A*02:07 alleles. In certain embodiments, the HLA allele is HLA-A*02:01.

[0474] As used herein, an "effector domain" or "immune effector domain" is the intracellular portion or domain of a fusion protein or receptor that, upon receipt of an appropriate signal, can directly or indirectly promote an immune response in a cell. In some embodiments, the effector domain is from an immune cell protein or a portion thereof or an immune cell protein complex that receives a signal when binding occurs (e.g., CD3ζ), or when the immune cell protein or a portion thereof or an immune cell protein complex directly binds to a target molecule and triggers signal transduction of the effector domain in an immune cell.

[0475] When the effector domain contains one or more signaling domains or motifs, such as intracellular tyrosine activation motifs (ITAMs), such as those found in co-stimulatory molecules, it can directly promote a cellular response. Without wishing to be bound by theory, it is believed that ITAMs can be used for T cell activation following ligation of a T cell receptor or a fusion protein comprising a T cell effector domain to a ligand. In some embodiments, the intracellular component or a functional portion thereof comprises an ITAM. Exemplary immune effector domains include, but are not limited to, those 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).

[0476] In other embodiments, the intracellular component of the fusion protein comprises a co-stimulatory domain or a functional portion thereof selected from: CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-l (CD54), LFA-l (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that binds to (e.g., specifically and / or selectively) CD83 or a functional variant thereof, or any combination thereof. In some embodiments, the intracellular component comprises a CD28 co-stimulatory domain or a functional portion or variant thereof (which may optionally include the LL-GG mutation at positions 186-187 of the native CD28 protein (e.g., Nguyen et al. (2003) Blood 702:4320), a 4-1BB co-stimulatory domain or a functional portion or variant thereof, or both.

[0477] In some embodiments, the effector domain comprises the intracellular domain of CD3ε or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD27 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD28 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of 4-1BB or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of OX40 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD2 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD5 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of ICAM-1 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of LFA-1 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of ICOS or a functional (e.g., signaling) portion thereof, or a functional variant thereof.

[0478] The extracellular and intracellular components encompassed by the present invention are linked by a transmembrane domain. As used herein, a "transmembrane domain" is the portion of a transmembrane protein that can be inserted into or span the 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 amino acids to about 30 amino acids. The structure of a transmembrane domain can comprise an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof. In some embodiments, the transmembrane domain comprises or is derived from a known transmembrane protein (e.g., the transmembrane domain of CD4, the transmembrane domain of CD8, the transmembrane domain of CD27, the transmembrane domain of CD28, or any combination thereof).

[0479] In some embodiments, the extracellular component of the fusion protein further comprises a linker disposed between the binding domain and the transmembrane domain. As used herein when referring to the component that links the binding domain and the transmembrane domain in the fusion protein, a "linker" can be an amino acid sequence having from about two amino acids to about 500 amino acids, which can provide flexibility and space for conformational movement between the two regions, domains, motifs, fragments, or modules linked by the linker. For example, the linker encompassed by the present invention can position the binding domain away from the surface of the host cell expressing the fusion protein such that proper contact, antigen binding, and activation between the host cell and the target cell can be achieved (Patel et al. (1999) Gene Therapy 6:412-419). The linker length can vary based on the selected target molecule, the selected binding epitope, or the capture and affinity of the antigen-binding domain to maximize antigen recognition (see, e.g., Guest et al. (2005) Immunother. 28:203-11, and PCT Publication No. WO 2014 / 031687). Exemplary linkers include those having a glycine-serine amino acid chain having one to about ten Gly x Ser y repeat sequences, where x and y are each independently an integer from 0 to 10, with the proviso that x and y are not both 0 (e.g., (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or combinations thereof, such as ((Gly3Ser)2Gly2Ser)).

[0480] The binding protein can be conjugated to an agent such as a detection moiety, radiosensitizer, photosensitizer, etc., and / or can be chemically modified as described above for the peptide.

[0481] In some embodiments, the binding proteins encompassed by the present invention can be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or label. The affinity tag can be selected from the group consisting of glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label can be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of pro-inflammatory factors, anti-inflammatory agents, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies, such as single-chain Fv.

[0482] The binding protein can be conjugated to agents for imaging, research, therapeutics, theranostics, pharmacy, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, the binding protein can be conjugated or fused to a detectable agent such as a fluorophore, near-infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radionuclide chelator, or another suitable material for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties can be attached to the binding protein. 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 readily quenched by biological tissues and body fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation having a wavelength between 650 nm and 4000 nm, such emission being used to detect such agents. Non-limiting examples of fluorescent dyes that can be used as conjugated molecules include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, near-infrared dyes generally include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Additional non-limiting examples of fluorescent dyes used as conjugated molecules according to the present invention include acridine orange or acridine yellow, Alexa (e.g., Alexa 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilino-1-naphthalenesulfonic acid, dye and any derivatives thereof, auramine-rhodamine stain and any derivatives thereof, bensanthrone, bimane, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene, bisbenzimide, brainbow, calcein, carboxyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC 6, and any derivatives thereof, epicocconone, ethidium bromide, Fluo dye and any derivatives thereof, and any of its derivatives, fluorescein and any of its derivatives, and any of its derivatives, GelG and any of its derivatives, and any of its derivatives, fluorescent proteins and any of their derivatives, m isotype proteins and any of their derivatives (such as mCherry), heta methine dyes and any of their derivatives, hoeschst stains, iminocoumarins, Indian yellow, indo-1 and any of its derivatives, laurdan, fluorescein yellow and any of its derivatives, fluorescein and any of its derivatives, luciferase and any of its derivatives, merocyanine and any of its derivatives, nile dyes and any of their derivatives, perylene, phloxine, phycobiliprotein dyes and any of their derivatives, propidium iodide, pyranine, rhodamine and any of its derivatives, ribogreen, RoGFP, rubrene, stilbene and any of its derivatives, sulforhodamine and any of its derivatives, SYBR and any of its derivatives, synapto-pHluorin, tetraphenylbutadiene, trisodium tetraborate, Texas Red, Titan 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 isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green TM dyes (e.g., Oregon Green TM 488, Oregon Green TM 500, OregonGreen TM 514, etc.), Texas Texas -X, SPECTRUM SPECTRUM Cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), Alexa dyes (e.g., Alexa 350, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 633, Alexa 660, Alexa 680, etc.), dyes (e.g., FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRD dyes (e.g., IRD40 TM , IRD700 TM , IRD800 TM , etc.). Other suitable detectable agents are well known in the art (e.g., PCT Publication No. PCT / US14 / 56177). Non-limiting examples of radioisotopes include α emitters, β emitters, positron emitters, and γ 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.

[0483] The binding protein can be conjugated with a radiosensitizer or a photosensitizer. Examples of radiosensitizers include, but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to: fluorescent molecules or beads, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metal porphyrins, metal phthalocyanines, angelicin, chalcogenopyrylium dyes, chlorophylls, coumarins, flavins, and related compounds (e.g., lumazines and riboflavins), fullerenes, pheophorbide a, pyropheophorbide a, cyanines (e.g., merocyanine 540), pheophytin, thiaflavin, tespaflavin, purpurin, porphycene, phenothiazinium, methylene blue derivatives, naphthalimides, nile blue derivatives, quinones, perylenequinones (e.g., hypericin, hypocrellin, and cercosporin), psoralens, quinones, retinoids, rhodamines, thiophenes, wilkinsons, xanthene dyes (e.g., eosin, erythrosin, rose bengal), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, the method allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent (e.g., a drug) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the binding protein is fused to the agent, or covalently or non-covalently linked to the agent, e.g., directly or via a linker.

[0484] In some embodiments, the binding protein can be chemically modified. For example, the binding protein can be mutated to modify peptide properties, such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is an example of methylation that can occur in the binding proteins encompassed by the present invention. In some embodiments, the binding protein can be modified by methylating free amines, e.g., by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0485] The chemical modification can comprise a polymer, a polyether, a polyethylene glycol, a biopolymer, a zwitterionic polymer, a polyamino acid, a fatty acid, a dendrimer, an Fc region, a simple saturated carbon chain (e.g., palmitate or myristate), or albumin. The chemical modification of a binding protein having an Fc region can be a fusion Fc-protein. The polyamino acid can include, for example, a polyamino acid sequence having a repeating single amino acid (e.g., polyglycine), and a polyamino acid sequence having a mixed polyamino acid sequence that may or may not follow a pattern, or any combination of the foregoing.

[0486] In some embodiments, the binding proteins encompassed by the present invention can be modified. In some embodiments, the modification has substantial or significant sequence identity with the parental binding protein to produce a functional variant that maintains one or more biophysical and / or biological activities of the parental binding protein (e.g., maintaining pMHC binding specificity). In some embodiments, the mutation is a conservative amino acid substitution.

[0487] In some embodiments, the binding proteins encompassed by the present invention can contain synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxy phenylalanine, phenylglycine, α-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, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0488] The binding proteins encompassed by the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via a disulfide bridge), or converted to an acid addition salt, and / or optionally dimerized or polymerized, or conjugated.

[0489] In some embodiments, attachment of a hydrophobic moiety (e.g., attachment to the N-terminus, C-terminus, or an internal amino acid) can be used to extend the half-life of the peptides encompassed by the present invention. In other embodiments, the binding protein can include post-translational modifications (e.g., methylation and / or amidation) that can affect, for example, the serum half-life. In some embodiments, a simple carbon chain (e.g., by myristoylation and / or palmitoylation) can be conjugated to the binding protein. In some embodiments, the simple carbon chain can facilitate separation of the binding protein from unconjugated material. For example, methods that can be used to separate the binding protein from unconjugated material include, but are not limited to, solvent extraction and reverse-phase chromatography. The lipophilic moiety can extend the 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, including cholestene, cholestane, cholestadiene, and oxysterol. In some embodiments, the binding protein can be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding protein can 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, glutamate, and serine, Fc region, fatty acids, palmitic acid, or molecules that bind to albumin. In some embodiments, a spacer or linker can be coupled to the binding protein, such as 1, 2, 3, 4, or more amino acid residues that serve as a spacer or linker, to facilitate conjugation or fusion with another molecule and to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, the binding protein can be conjugated to other moieties that can modify or effect a change in the properties of the binding protein, for example.

[0490] The binding protein can be produced recombinantly or synthetically, for example, by solid-phase peptide synthesis or solution-phase peptide synthesis. Polypeptide synthesis can be carried out by known synthetic methods, such as using fluorenylmethoxycarbonyl (Fmoc) chemistry or by tert-butyloxycarbonyl (Boc) chemistry. The polypeptide fragments can be joined together enzymatically or synthetically.

[0491] In one aspect encompassed by the present invention, provided herein is a method for producing the binding protein described herein, the method comprising the steps of: (i) culturing a transformed host cell under conditions suitable to permit expression of the binding protein described herein, the host cell having been transformed with a nucleic acid comprising a sequence encoding the binding protein; and (ii) recovering the expressed binding protein.

[0492] For example, methods for separating and purifying recombinantly produced binding proteins can include obtaining a supernatant from a suitable host cell / vector system that secretes the binding protein into the culture medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse-phase HPLC steps can be employed to further purify the recombinant polypeptide. These purification methods can also be used when isolating immunogens from a natural environment. Methods for large-scale manufacturing of one or more binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. The binding proteins can be purified according to methods described herein and known in the art. In any of the embodiments disclosed herein, the encoded binding protein is capable of binding to a peptide-MHC (pMHC) complex comprising an MAGEA1 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the MHC molecule comprises an MHC α-chain that is of the HLA serotype HLA-A*02. In some embodiments, the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, and HLA-A*02:07 alleles. In a particular embodiment, the HLA allele is HLA-A*02:01.

[0493] There are a variety of well-known assays for assessing binding affinity and / or determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand (e.g., a peptide antigen-MHC complex). For example, determining the binding affinity of a binding protein for a target (e.g., a T cell peptide epitope of a target polypeptide) is within the level of skill of a person skilled in the art by using any of a variety of binding assays well known in the art. For example, in some embodiments, a Biacore TM machine can be used to determine the binding constant of a complex between two proteins. The dissociation constant (K D ) of the complex can be determined by monitoring the change in refractive index over time as buffer 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 assay (ELISA) and radioimmunoassay (RIA), or measuring binding by monitoring changes in the spectral 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 blotting, ELISA, analytical ultracentrifugation, spectroscopic analysis, and surface plasmon resonance (Biacore TM)Analysis (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; Wilson (2002) Science 295:2103; Wolff et al. (1993) Cancer Res. 53:2560; and U.S. Patent Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, the apparent affinity for a target is measured by assessing binding to various concentrations of a labeled multimer, such as an MHC-antigen tetramer, e.g., by flow cytometry. In a representative example, a two-fold dilution series of a labeled tetramer at a series of concentrations is used to measure the apparent K D , and then the binding curve is determined by non-linear regression to determine the apparent K D which is the ligand concentration that produces half-maximal binding.

[0494] VI. Nucleic Acids and Vectors

[0495] In one aspect encompassed by the present invention, nucleic acid molecules are provided herein that encode the proteins described herein, such as MAGEA1 immunogenic peptides and fragments thereof, MHC molecules, binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, CARs, etc.), and the like.

[0496] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to a complement of a sequence that has at least about 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 nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3 over the full length.

[0497] 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.

[0498] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3 (e.g., comprising it, consisting essentially of it, or consisting of it).

[0499] In some embodiments, the nucleic acid sequence encodes the MAGEA1 immunogenic peptide described herein.

[0500] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one (e.g., one, two, or three) of the TCRα chain CDRs described in Table 2 (e.g., comprising it, consisting essentially of it, or consisting of it). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCR V α domain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCR V α domain has 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 higher identical to the TCR V α domain sequence described in Table 2. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCRα chain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCRα chain has 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 higher identical to the TCRα chain sequence described in Table 2.

[0501] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one (e.g., one, two, or three) of the TCRβ chain CDRs described in Table 2 (e.g., comprising it, consisting essentially of it, or consisting of it). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCR V β domain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCR V β domain has 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 higher identical to the TCR V β domain sequence described in Table 2. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCRβ chain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCRβ chain has 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 higher identical to the TCRβ chain sequence described in Table 2.

[0502] The term "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally refers to a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources (e.g., isolated and / or purified), which can contain natural, unnatural, or modified nucleotides, and can contain natural, unnatural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, rather than the phosphodiester found between nucleotides of unmodified oligonucleotides. In one embodiment, the nucleic acid comprises complementary DNA (cDNA).

[0503] In some embodiments, the nucleic acids encompassed by the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside of a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in a living cell, or (ii) a molecule produced by the replication of those molecules described in (i) above. For the purposes herein, replication can be in vitro, ex vivo, or in vivo replication.

[0504] Nucleic acids can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. See, e.g., Green and Sambrook et al., supra. By way of example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides that are designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can 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-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-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, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, scyllo-inosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetate, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more nucleic acids encompassed by the present invention may be purchased from companies such as Integrated DNA Technologies (Coralville, IA).

[0505] In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound by a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence can improve the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but can be translated by a more readily available tRNA within the cell, thereby improving translation efficiency. Optimization of the nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, thereby improving 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).

[0506] The present invention also provides a nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or that hybridizes to the nucleotide sequence of any nucleic acid described herein under stringent conditions.

[0507] Nucleotide sequences that hybridize under stringent conditions may hybridize under highly stringent conditions. "Highly stringent conditions" mean that a nucleotide sequence hybridizes specifically and / or selectively to a target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. Highly stringent conditions include conditions that distinguish a polynucleotide with an exact complementary sequence or a polynucleotide containing only a few scattered mismatches from a random sequence having only a few small regions (e.g., 3-10 bases) that match the nucleotides exactly. Such small regions of complementarity are more readily melted compared to a full-length complement of 14-17 or more bases, and highly stringent hybridization makes it easy to distinguish. Relatively high stringency conditions will include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or equivalents at a temperature of about 50-70 °C. Such highly stringent conditions allow little or no mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the expression of any TCR of the present invention. It is generally understood that the conditions can be made more stringent by adding incremental amounts of formamide.

[0508] The present invention also provides a nucleic acid, which comprises a nucleotide 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 higher identity to any of the nucleic acids described herein.

[0509] Generally, the nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0510] The terms "vector", "cloning vector" and "expression vector" mean a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and facilitate the expression (e.g., transcription and translation) of the introduced sequence. Accordingly, another object encompassed by the present invention relates to a vector comprising the nucleic acid encompassed by the present invention.

[0511] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the polypeptide after administration to a subject. Examples of promoters and enhancers for use in animal cell expression vectors include the early promoter and enhancer of SV40 (Mizukami T et al. 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al. 1987), the promoter of immunoglobulin H chain (Mason J O et al. 1985) and enhancer (Gillies SD et al. 1983), etc.

[0512] Any animal cell expression vector 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), pSG1βd2-4- (Miyaji H et al. 1990), etc. Other representative examples of plasmids include replicating plasmids containing an origin of replication, or integrating 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, such as by transfection of packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of viral 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 WO 95 / 14785, PCT publication WO 96 / 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 WO 94 / 19478.

[0513] In some embodiments, the composition comprises an expression vector that contains an open reading frame encoding the binding protein or polypeptide or fragment thereof described herein. In some embodiments, the nucleic acid includes regulatory elements necessary for expressing the open reading frame. Such elements can include, for example, a promoter, start codon, stop codon, and polyadenylation signal. Additionally, enhancers can be included. These elements are operably linked to the sequence encoding the binding protein, polypeptide, or fragment thereof.

[0514] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, a dominant negative TGFβ receptor (such as DN-TGFβRII), a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In certain embodiments, the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβR, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag (such as a CD34 enrichment tag). In specific embodiments, the nucleic acid sequences described herein, such as those encoding TCRα, TCRβ, CD8α, CD8β, DN-TGFβR, and / or the selectable protein marker, are linked to an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, E2A, F2A, or T2A, etc.).

[0515] In some embodiments, the expression vectors provided herein comprise nucleotide 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 higher identity to any of the nucleic acids shown in Tables 1-3.

[0516] As described above, representative examples of promoters include, but are not limited to, the promoter from Simian virus 40 (SV40); the Mouse mammary tumor virus (MMTV) promoter; promoters from the Human immunodeficiency virus (HIV), such as the HIV long terminal repeat (LTR) promoter; the promoter from Moloney virus; the promoter from Cytomegalovirus (CMV), such as the CMV immediate early promoter; the promoter from Epstein Barr Virus (EBV); the promoter from 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.

[0517] In addition to the regulatory elements required for expression, other elements may also be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include the promoters described above. In some embodiments, the enhancer / promoter includes, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as those from CMV, RSV, and EBV.

[0518] In some embodiments, the nucleic acid is operably incorporated into a carrier or delivery vehicle as further described below. Suitable delivery vehicles include, but are not limited to, biodegradable microcapsules, immunostimulating complexes (ISCOMs), or liposomes, as well as genetically engineered attenuated live carriers such as viruses or bacteria.

[0519] In some embodiments, the vector is a viral vector, such as a lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, Fowl pox virus, AV-pox virus, Modified vaccinia virus Ankara (MVA), and other recombinant viruses. For example, lentiviral vectors can be used to infect T cells.

[0520] In some embodiments, the recombinant expression vector is capable of delivering the polynucleotide to a suitable host cell, such as a T cell or an antigen-presenting cell, i.e., a cell that displays a peptide / MHC complex on its cell surface and lacks CD8 (e.g., a dendritic cell). In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. For example, the immune system cell can be a CD4 + T cell, a CD8 + T cell, a CD4 / CD8 double-negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof. In some embodiments, where the T cell is the host, the T cell can be a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. Thus, the recombinant expression vector can also include, for example, a lymphoid tissue-specific transcriptional regulatory element (TRE), such as a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue-specific TREs are known in the art (see, e.g., 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).

[0521] In some embodiments, the recombinant expression vector contains a nucleotide sequence encoding a TCR α-chain, a TCR β-chain, and / or a linker peptide. For example, in some embodiments, the recombinant expression vector contains a nucleotide sequence encoding full-length TCR α- and TCR β-chains of a binding protein (with a linker therebetween), wherein the nucleotide sequence encoding the β-chain is 5' to the nucleotide sequence encoding the α-chain. In some embodiments, the nucleotide sequence encodes full-length TCR α- and TCR β-chains (with a linker therebetween), wherein the nucleotide sequence encoding the TCR β-chain is 3' to the nucleotide sequence encoding the TCR α-chain. In some embodiments, the full-length TCR α- and / or TCR β-chains are replaced by fragments thereof.

[0522] As further described below, another aspect covered by the present invention relates to a cell that has been transfected, infected, or transformed with a nucleic acid and / or vector according to the present invention. A host cell can include any individual cell or cell culture that can accept a vector or incorporate nucleic acids and / or proteins, as well as any progeny cells. The term also encompasses the progeny of the host cell, whether genetically or phenotypically identical or different. Suitable host cells can depend on the vector and can include mammalian cells, animal cells, human cells, ape cells, insect cells, yeast cells, and bacterial cells. These cells can be induced to incorporate the vector or other materials by using viral vectors, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory)). The term "transformation" means introducing an "exogenous" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence, thereby producing the desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. A host cell that accepts and expresses the introduced DNA or RNA has been "transformed."

[0523] The nucleic acids covered by the present invention can be used to produce recombinant polypeptides covered by the present invention in a suitable expression system. The term "expression system" means a host cell and a compatible vector that express a protein encoded by exogenous DNA carried by the vector and introduced into the host cell under suitable conditions.

[0524] Common expression systems include Escherichia 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 Escherichia coli; yeast of the genus Kluyveromyces or Saccharomyces; mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.); and primary or established mammalian cell cultures (e.g., generated from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0...

Claims

1. An immunogenic peptide, the immunogenic peptide comprising a peptide epitope selected from the peptide sequences listed in Table 1.

2. An immunogenic peptide, the immunogenic peptide consisting of a peptide epitope selected from the peptide sequences listed in Table 1.

3. The immunogenic peptide according to claim 1 or 2, wherein the immunogenic peptide is derived from the MAGEA1 protein, optionally wherein the length of 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.

4. The immunogenic peptide according to any one of claims 1-3, wherein the immunogenic peptide is capable of eliciting an immune response against MAGEA1 and / or cells expressing MAGEA1 in a subject, 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 expansion, cytokine release and / or cytotoxic killing.

5. An immunogenic composition, the immunogenic composition comprising at least one immunogenic peptide according to any one of claims 1-4.

6. The immunogenic composition according to claim 5, the immunogenic composition further comprising an adjuvant.

7. The immunogenic composition according to claim 5 or 6, wherein the immunogenic composition is capable of eliciting an immune response against MAGEA1 and / or cells expressing MAGEA1 in a subject, 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 expansion, cytokine release and / or cytotoxic killing.

8. A composition, the composition comprising a peptide epitope selected from the peptide sequences listed in Table 1, and an MHC molecule.

9. The composition according to claim 8, wherein the MHC molecule is an MHC multimer, optionally wherein the MHC multimer is a tetramer.

10. The composition according to claim 8 or 9, wherein the MHC molecule is an MHC class I molecule.

11. The composition according to any one of claims 9 - 11, wherein the MHC molecule comprises an MHC α-chain, the chain being an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01,HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles., 12. A stable MHC-peptide complex, the stable MHC-peptide complex comprising the immunogenic peptide according to any one of claims 1-4 in the context of an MHC molecule.

13. The stable MHC-peptide complex according to claim 12, wherein the MHC molecule is an MHC multimer, optionally wherein the MHC multimer is a tetramer.

14. The stable MHC-peptide complex according to claim 12 or 13, wherein the MHC molecule is an MHC class I molecule.

15. The stable MHC-peptide complex according to any one of claims 12-14, wherein the MHC molecule comprises an MHC α chain, and the chain 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, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01,HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles; optionally, wherein the HLA serotype is HLA-A*02; and further optionally, wherein the HLA-A*02 is HLA-A*02:01., 16. The stable MHC-peptide complex according to any one of claims 12-15, wherein the peptide epitope and the MHC molecule are covalently linked and / or wherein the α-chain and β-chain of the MHC molecule are covalently linked.

17. The stable MHC-peptide complex according to any one of claims 12-16, wherein the stable MHC-peptide complex comprises a detectable label, optionally wherein the detectable label is a fluorophore.

18. An immunogenic composition comprising a stabilized MHC-peptide complex according to any one of claims 12-17 and an adjuvant.

19. An isolated nucleic acid encoding an immunogenic peptide according to any one of claims 1-4, or a complement thereof.

20. A vector comprising the isolated nucleic acid according to claim 19.

21. A cell that: a) comprises the isolated nucleic acid according to claim 19, b) comprises the vector according to claim 20, and / or c) produces one or more immunogenic peptides according to any one of claims 1-4 and / or presents one or more stabilized MHC-peptide complexes according to any one of claims 12-17 on the cell surface, optionally wherein the cell is genetically engineered.

22. A device or kit comprising: a) one or more immunogenic peptides according to any one of claims 1-4 and / or b) one or more stabilized MHC-peptide complexes according to any one of claims 12-17, the device or kit optionally comprising reagents for detecting the 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.

23. A method of detecting T cells that bind to a stabilized MHC-peptide complex, the method comprising: a) contacting a sample comprising T cells with a stabilized MHC-peptide complex according to any one of claims 12-17; and b) detecting the binding of the T cells to the stabilized MHC-peptide complex, optionally further determining the percentage of stabilized MHC-peptide-specific T cells that bind to the stabilized MHC-peptide complex, optionally wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

24. The method according to claim 23, wherein the T cells are CD8+ T cells.

25. The method according to any one of claims 22-24, wherein the detection and / or the determination is performed using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, western blotting, or intracellular flow cytometry.

26. The method according to any one of claims 22-25, wherein the sample comprises T cells that have been exposed to or are suspected of having been exposed to one or more MAGEA1 proteins or fragments thereof.

27. A method of determining whether a T cell has been exposed to MAGEA1, the method comprising: a) incubating a population of cells comprising T cells with an immunogenic peptide according to any one of claims 1-4 or a stabilized MHC-peptide complex according to any one of claims 12-17; and b) detecting the presence or level of reactivity. The presence of reactivity or a higher level of reactivity compared to a control level indicates that the T cells have been exposed to MAGEA1, optionally wherein the cell population comprising the T cells is obtained from a subject.

28. A method for predicting the clinical outcome of a subject suffering from a disorder characterized by MAGEA1 expression, the method comprising: a) determining the presence or level of reactivity of T cells obtained from the subject with one or more immunogenic peptides according to any one of claims 1-4 or one or more stable MHC-peptide complexes according to any one of claims 12-17; and b) comparing the presence or level of reactivity with that of a control obtained from a subject with a favorable clinical outcome, wherein the presence of reactivity or a higher level of reactivity in the subject sample compared to the control indicates that the subject has a favorable clinical outcome.

29. A method for assessing the efficacy of a therapy for a disorder characterized by MAGEA1 expression, the method comprising: a) determining the presence or level of reactivity of T cells obtained from the subject with one or more immunogenic peptides according to any one of claims 1-4 or one or more stable MHC-peptide complexes according to any one of claims 12-17 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 of one or more immunogenic peptides according to any one of claims 1-4 or the one or more stable MHC-peptide complexes according to any one of claims 12-17 with T cells obtained from the subject, the T cells being present in a second sample obtained from the subject after providing the therapy to the subject, wherein the presence of reactivity or a higher level of reactivity in the second sample compared to the first sample indicates that the therapy effectively treats the disorder characterized by MAGEA1 expression in the subject.

30. The method according to any one of claims 27-29, wherein the level of reactivity is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, optionally wherein the T cell activation or effector function is T cell proliferation, killing, or cytokine release.

31. The method according to any one of claims 27-30, the method further comprising repeating steps a) and b) at a subsequent time point, optionally wherein the subject has been treated between the first time point and the subsequent time point to improve the disorder characterized by MAGEA1 expression.

32. The method according to any one of claims 27-31, wherein the T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, western blotting, or intracellular flow cytometry.

33. The method according to any one of claims 27 - 32, wherein the control level is a reference number.

34. The method according to any one of claims 27 - 33, wherein the control level is the level of a subject not suffering from the disorder characterized by MAGEA1 expression.

35. A method of preventing and / or treating a disorder characterized by MAGEA1 expression in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 1 - 22.

36. A method of 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, the method comprising: a) providing a cell that presents on the surface of the cell a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule; 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 the peptide epitope in the context of the MHC molecule.

37. The method according to claim 36, wherein step a) comprises contacting the MHC molecule on the surface of the cell with a peptide epitope selected from the peptide sequences listed in Table 1.

38. The method according to claim 36, wherein step a) comprises expressing, in the cell, a peptide epitope selected from the peptide sequences listed in Table 1 using a vector comprising a heterologous sequence encoding the peptide epitope.

39. A method of 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, the method comprising: a) providing a peptide epitope alone or as a stable MHC - peptide complex, which comprises a peptide epitope selected from the peptide sequences listed in Table 1 alone or in the context of an MHC molecule; b) determining the binding of a plurality of candidate peptide - binding molecules or antigen - binding fragments thereof to the peptide or the 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 the stable MHC - peptide complex, optionally wherein the MHC or the MHC - peptide complex is as defined in any one of claims 8 - 17.

40. The method according to claim 39, wherein 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.

41. The method according to claim 39 or 40, wherein the plurality of candidate peptide binding molecules comprises at least 2, 5, 10, 100, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species, 10 9 species or more different candidate peptide binding molecules.

42. The method according to any one of claims 39 - 41, wherein the plurality of candidate peptide - binding molecules comprises one or more candidate peptide - binding molecules obtained from a sample from a subject or a group of subjects; or the plurality of candidate peptide - binding molecules comprises one or more candidate peptide - binding molecules comprising mutations in a parental scaffold peptide - binding molecule obtained from a sample from a subject.

43. The method according to claim 42, wherein the subject or the group of subjects: a) does not have a disorder characterized by MAGEA1 expression and / or has recovered from a disorder characterized by MAGEA1 expression, or b) has a disorder characterized by MAGEA1 expression.

44. The method according to claim 42 or 43, wherein the composition according to any one of claims 1-22 has been administered to the subject or the group of subjects.

45. The method according to any one of claims 42-44, wherein the subject is an animal model and / or a mammal of a disorder characterized by MAGEA1 expression, optionally wherein the mammal is a human, a primate or a rodent.

46. The method according to any one of claims 42-45, wherein the subject is an animal model of a disorder characterized by MAGEA1 expression, an HLA transgenic mouse and / or a human TCR transgenic mouse.

47. The method according to any one of claims 42-46, wherein the sample comprises peripheral blood mononuclear cells (PBMCs), T cells and / or CD8+ memory T cells.

48. A peptide-binding molecule or an antigen-binding fragment thereof identified according to any one of claims 39-48, optionally wherein the peptide-binding molecule or the 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.

49. A method of treating a disorder characterized by MAGEA1 expression in a subject, the method comprising administering to the subject a therapeutically effective amount of genetically engineered T cells that express a peptide-binding molecule or an antigen-binding fragment thereof, the peptide-binding molecule or the antigen-binding fragment thereof: i) binds to a peptide epitope selected from the sequences listed in Table 1, ii) is identified according to the method according to any one of claims 39-48, and / or iii) binds to a stable MHC-peptide complex of 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 the 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, optionally wherein the MHC or the MHC-peptide complex is as according to any one of claims 8-17.

50. The method according to claim 49, wherein the T cells are isolated from: a) the subject, b) a donor who does not have the disorder characterized by MAGEA1 expression, or c) a donor who has recovered from a disorder characterized by MAGEA1 expression.

51. A method of treating a subject having a disorder characterized by MAGEA1 expression, the method comprising infusing 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 according to any one of claims 1-22; and b) expanding antigen-specific T cells in vitro or ex vivo, optionally i) isolating immune cells from the subject prior to stimulating the immune cells and / or ii) wherein the immune cells comprise PBMC, T cells, CD8+ T cells, naive T cells, central memory T cells, and / or effector memory T cells.

52. The method according to claim 51, wherein the agent is contacted and placed under conditions and for a time suitable for forming 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 cells.

53. The method according to claim 51 or 52, wherein the peptide epitope, the immunogenic peptide, the stable MHC-peptide complex, and / or the T cell receptor are expressed by cells and the cells are expanded and / or isolated during one or more steps.

54. The method according to any one of claims 23-53, wherein the disorder characterized by MAGE A1 expression is cancer or a recurrence thereof, optionally wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma.

55. The method according to any one of claims 23-54, wherein the subject is an animal model and / or a mammal having a disorder characterized by MAGEA1 expression, optionally wherein the mammal is a human, a primate, or a rodent.

56. A binding protein that binds to a polypeptide comprising an immunogenic peptide sequence according to any one of claims 1 to 4, the immunogenic peptide according to any one of claims 1-4, and / or the stable MHC-peptide complex according to any one of claims 12-17, 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.

57. The binding protein according to claim 56, the binding protein comprising: a) a T cell receptor (TCR) α-chain CDR sequence that has at least about 80% identity with a TCR α-chain CDR sequence selected from the group consisting of the TCR α-chain CDR sequences listed in Table 2; and / or b) A TCRβ chain CDR sequence that has at least about 80% identity with a TCRβ chain CDR sequence selected from the group consisting of the TCRβ chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d .

58. The binding protein according to claim 56, the binding protein comprising: a) TCR α-chain variable (V α ) domain sequence that has 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) The variable (V) domain sequence of the TCR β chain, which has at least about 80% identity with a TCR V domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 β ) domain sequence, which has at least about 80% identity with a TCR V domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2 β domain sequences, and the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex β domain sequence, and optionally the binding affinity has a K of less than or equal to about 5×10 -4 M d .

59. The binding protein according to claim 56, the binding protein comprising: a) a TCR α-chain sequence that has at least about 80% identity with a TCR α-chain sequence selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) A TCRβ chain sequence that has at least about 80% identity with a TCRβ chain sequence selected from the group consisting of the TCRβ chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d .

60. The binding protein according to claim 56, wherein the binding protein comprises: a) a TCRα chain CDR sequence selected from the group consisting of the TCRα chain CDR sequences listed in Table 2; and / or b) A TCRβ chain CDR sequence selected from the group consisting of the TCRβ chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d .

61. The binding protein according to claim 56, wherein the binding protein comprises: a) The variable (V α ) domain sequence of the TCRα chain, which is selected from the group consisting of the TCR V α domain sequences listed in Table 2; and / or b) The variable (V β ) domain sequence of the TCRβ chain, which is selected from the group consisting of the TCR V β domain sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K -4 of less than or equal to about 5×10 d M 62. The binding protein according to claim 56, wherein the binding protein comprises: a) a TCRα chain sequence selected from the group consisting of the TCRα chain sequences listed in Table 2; and / or b) A TCRβ chain sequence selected from the group consisting of the TCRβ chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d .

63. The binding protein according to any one of claims 56-62, wherein 1) the TCRα chain CDR, the TCR V α domain and / or the TCRα chain is encoded by a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group consisting of the TRAV, TRAJ and TRAC genes listed in Table 2, and / or 2) the TCRβ chain CDR, the TCR V β domain and / or the TCRβ chain is encoded by a TRBV, TRBJ and / or TRBC gene or a fragment thereof selected from the group consisting of the TRBV, TRBJ and TRBC genes listed in Table 2, and / or 3) compared to the homologous reference CDR sequences listed in Table 2, each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions or combinations thereof.

64. The binding protein according to any one of claims 56 - 63, wherein the binding protein is chimeric, humanized or human.

65. The binding protein according to any one of claims 56 - 64, wherein the binding protein comprises a binding domain having a transmembrane domain and an intracellular effector domain.

66. The binding protein according to any one of claims 56 - 65, wherein the TCRα chain and the TCRβ chain are covalently linked, optionally wherein the TCRα chain and the TCRβ chain are covalently linked through a linker peptide.

67. The binding protein according to any one of claims 56 - 66, wherein the TCRα chain and / or the TCRβ chain are covalently linked to a moiety, optionally wherein the covalently linked moiety comprises an affinity tag or a label.

68. The binding protein according to claim 67, 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, HaloTag, HA tag, Flag tag, His tag, biotin tag and V5 tag, and / or wherein the label is a fluorescent protein.

69. The binding protein according to any one of claims 56 - 68, wherein the covalently linked moiety is selected from the group consisting of: pro - inflammatory factors, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies or antigen - binding fragments thereof.

70. The binding protein according to any one of claims 56 - 69, wherein the binding protein binds to the pMHC complex on the cell surface.

71. The binding protein according to any one of claims 56 - 70, wherein the MHC or the MHC - peptide complex is as described in any one of claims 8 - 17.

72. The binding protein according to any one of claims 56 - 71, wherein the binding of the binding protein to the MAGEA1 peptide - MHC (pMHC) complex elicits an immune response, 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 expansion, cytokine release and / or cytotoxic killing.

73. The binding protein according to any one of claims 56-72, wherein the binding protein is capable of binding specifically and / or selectively to the MAGEA1 immunogenic peptide-MHC (pMHC) complex at a K of less than or equal to about 1×10 -4 M, less than or equal to about 5×10 -5 M, less than or equal to about 1×10 -5 M, less than or equal to about 5×10 -6 M, less than or equal to about 1×10 -6 M, less than or equal to about 5×10 -7 M, less than or equal to about 1×10 -7 M, less than or equal to about 5×10 -8 M, less than or equal to about 1×10 -8 M, less than or equal to about 5×10 -9 M, less than or equal to about 1×10 -9 M, less than or equal to about 5×10 -10 M, less than or equal to about 1×10 -10 M, less than or equal to about 5×10 -11 M, less than or equal to about 1×10 -11 M, less than or equal to about 5×10 -12 M, or less than or equal to about 1×10 -12 M d ​ 74. The binding protein according to any one of claims 56 - 73, wherein the binding protein has a higher binding affinity for the peptide - MHC (pMHC) compared to a known T - cell receptor, optionally wherein the higher binding affinity is at least 1.05 - fold higher.

75. A binding protein according to any one of claims 56 - 74, wherein when contacting a target cell with heterozygous expression of MAGEA1, compared with a known T cell receptor, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing, optionally wherein the induction is at least 1.05 - fold higher.

76. The binding protein according to claim 75, wherein the cytotoxic killing is against target cancer cells.

77. The binding protein according to claim 76, wherein the cancer is selected from the group consisting of: melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma.

78. The binding protein according to any one of claims 56 - 77, wherein the binding protein does not bind to a pMHC complex comprising a PIEZO1, NBEAL1, NBEAL2, and / or EPN2 peptide epitope.

79. A TCRα chain and / or β chain, wherein the TCRα chain and / or β chain is selected from the group consisting of the TCRα chain and β chain sequences listed in Table 2.

80. An isolated nucleic acid molecule, the isolated nucleic acid molecule: 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 with 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 with at least about 80% homology to the nucleic acid listed in Table 2, optionally wherein the isolated nucleic acid molecule comprises: 1) a TRAV, TRAJ, and / or TRAC gene or a fragment thereof selected from the group consisting of the 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 consisting of the TRBV, TRBJ, and TRBC genes listed in Table 2.

81. The isolated nucleic acid according to claim 80, wherein the nucleic acid is codon - optimized for expression in a host cell.

82. A vector, the vector comprising the isolated nucleic acid according to claim 80 or 81, optionally wherein i) the vector is a cloning vector, an expression vector, or a viral vector and / or ii) the vector comprises the vector sequences listed in Table 3.

83. The vector according to claim 82, wherein the vector further comprises nucleic acid sequences encoding CD8α, CD8β, dominant - negative TGFβ receptor II (DN - TGFβRII), a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR).

84. The vector according to claim 83, wherein the nucleic acid sequences encoding CD8α, CD8β, the DN - TGFβRII, and / or the selectable protein marker are operably linked to a nucleic acid encoding a tag.

85. The vector according to claim 83 or 84, wherein the nucleic acid encoding the tag is at the 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII, and / or the selective protein marker, such that the tag is fused to the N-terminus of CD8α, CD8β, the DN-TGFβRII, and / or the selective protein marker.

86. The vector according to claim 84 or 85, wherein the tag is a CD34 enrichment tag.

87. The nucleic acid or vector according to any one of claims 80-86, wherein the nucleic acid sequence encoding TCRα, TCRβ, CD8α, CD8β, the DN-TGFβRII, and / or the selective protein marker is linked to an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide.

88. The nucleic acid or vector according to claim 87, wherein the self-cleaving peptide is P2A, E2A, F2A, or T2A.

89. A host cell comprising the isolated nucleic acid according to claim 80 or 81, comprising the vector according to any one of claims 82-88, and / or expressing the binding protein according to any one of claims 56-78, optionally wherein the cell is genetically engineered.

90. The host cell according to claim 89, wherein the host cell comprises a chromosomal gene knockout of the TCR gene, the HLA gene, or both.

91. The host cell according to claim 89 or 90, wherein the host cell comprises a knockout of an HLA gene selected from the following: α1-macroglobulin gene, α2-macroglobulin gene, α3-macroglobulin gene, β1-microglobulin gene, β2-microglobulin gene, and combinations thereof.

92. The host cell according to any one of claims 89-91, wherein the host cell comprises a knockout of a TCR gene selected from the following: TCRα variable region gene, TCRβ variable region gene, TCR constant region gene, and combinations thereof.

93. The host cell according to any one of claims 89-92, wherein the host cell expresses CD8α, CD8β, DN-TGFβRII, and / or a selective protein marker, optionally wherein the selective protein marker is DHFR, and further optionally wherein CD8α, CD8β, the DN-TGFβRII, and / or the selective protein marker is fused to a CD34 enrichment tag.

94. The host cell according to claim 93, wherein the host cell is enriched using the CD34 enrichment tag.

95. The host cell according to any one of claims 89-94, wherein the host cell is a hematopoietic progenitor cell, a peripheral blood mononuclear cell (PBMC), a cord blood cell, or an immune cell.

96. The host cell according to claim 95, wherein the immune cell is a T cell, cytotoxic lymphocyte, cytotoxic lymphocyte precursor cell, cytotoxic lymphocyte progenitor cell, cytotoxic lymphocyte stem cell, CD4 + T cell, CD8 + T cell, CD4 / CD8 double-negative T cell, γδ (gamma delta) T cell, natural killer (NK) cell, NK-T cell, dendritic cell, or a combination thereof.

97. The host cell according to any one of claims 89-96, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof.

98. A host cell according to any one of claims 89 - 97, wherein the T cell is a primary T cell or a cell of a T cell line.

99. A host cell according to any one of claims 89 - 98, wherein the T cell does not express an endogenous TCR or has a lower surface expression of the endogenous TCR.

100. A host cell according to any one of claims 89 - 99, wherein the host cell is capable of producing cytokines or cytotoxic molecules when contacting a target cell comprising a peptide - MHC (pMHC) complex, the peptide - MHC (pMHC) complex comprising a MAGEA1 peptide epitope in the context of an MHC molecule.

101. The host cell according to claim 100, wherein the host cell contacts the target cell in vitro, ex vivo or in vivo.

102. The host cell according to claim 100 or 101, wherein the cytokine is TNF - α, IL - 2 and / or IFN - γ.

103. A host cell according to any one of claims 89 - 102, wherein the cytotoxic molecule is perforin and / or granzyme, optionally wherein the cytotoxic molecule is granzyme B.

104. A host cell according to any one of claims 89 - 103, wherein the host cell is capable of producing a higher level of cytokines or cytotoxic molecules when contacting a target cell with heterozygous expression of MAGEA1.

105. The host cell according to claim 104, wherein the host cell is capable of producing a level of cytokines or cytotoxic molecules that is at least 1.05 - fold higher.

106. A host cell according to any one of claims 89 - 103, wherein the host cell is capable of killing a target cell comprising a peptide - MHC (pMHC) complex comprising the MAGEA1 peptide epitope in the context of an MHC molecule.

107. The host cell according to claim 106, wherein the killing is measured by a killing assay.

108. The host cell according to claim 106 or 107, wherein the ratio of the host cell to the target cell in the killing assay is from 20:1 to 1:

4.

109. A host cell according to any one of claims 106 - 108, wherein the target cell is a target cell pulsed with 1 μg / mL to 50 pg / mL of MAGEA1 peptide, optionally wherein the target cell is a single - allele cell of an MHC that matches the MAGEA1 peptide.

110. A host cell according to any one of claims 106 - 109, wherein the host cell is capable of killing a higher number of target cells when contacting a target cell with heterozygous expression of MAGEA1, optionally wherein the cell killing is at least 1.05 - fold higher.

111. A host cell according to any one of claims 89 - 110, wherein the target cell is a cell line or a primary cell, optionally wherein 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.

112. A host cell according to any one of claims 89 - 111, wherein the MAGEA1 immunogenic peptide is as defined in any one of claims 1 to 4 and / or wherein the MHC or the MHC - peptide complex is as defined in any one of claims 8 - 17.

113. A host cell according to any one of claims 89 - 112, wherein the host cell does not induce T cell expansion, cytokine release or cytotoxic killing when contacting a target cell comprising a peptide - MHC (pMHC) complex, the peptide - MHC (pMHC) complex comprising a PIEZO1, NBEAL1, NBEAL2 and / or EPN2 peptide epitope.

114. A host cell according to any one of claims 89 - 113, wherein the host cell does not express the MAGEA1 antigen, is not recognized by a binding protein as defined in any one of claims 56 - 78, does not belong to the serotype HLA - A*02, and / or does not express the HLA - A*02 allele.

115. A population of host cells according to any one of claims 89 - 114.

116. A composition comprising: a) a binding protein according to any one of claims 56 - 77, b) an isolated nucleic acid according to claim 80 or 81, c) a vector according to any one of claims 82 to 88, d) a host cell according to any one of claims 89 - 114, and / or e) a population of host cells according to claim 115, and a carrier.

117. A device or kit comprising: a) a binding protein according to any one of claims 56 - 77, b) an isolated nucleic acid according to claim 80 or 81, c) a vector according to any one of claims 82 to 88, d) a host cell according to any one of claims 89 - 114, and / or e) a population of host cells according to claim 115, the device or kit optionally comprising a reagent for detecting the binding of a), d) and / or e) to the pMHC complex.

118. A method of producing an associated protein according to any one of claims 56 - 77, wherein the method comprises the following steps: (i) Culturing a transformed host cell under conditions suitable to permit expression of the binding protein, the host cell having been transformed with a nucleic acid comprising a sequence encoding a binding protein according to any one of claims 56 - 77; and (ii) recovering the expressed binding protein.

119. A method for generating a host cell expressing a binding protein according to any one of claims 56 - 77, wherein the method comprises the following steps: (i) Introducing a nucleic acid into the host cell, the nucleic acid comprising a sequence encoding a binding protein according to any one of claims 56 - 77; and (ii) culturing the transformed host cell under conditions suitable to permit expression of the binding protein.

120. A method for detecting the presence or absence of MAGEA1 antigen and / or cells expressing MAGEA1, optionally wherein the cells are hyperproliferative cells, the method comprising detecting the presence or absence of the MAGEA1 antigen in a sample by using at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114 or a population of host cells according to claim 115, wherein detection of the MAGEA1 antigen indicates the presence of MAGEA1 antigen and / or cells expressing MAGEA1.

121. The method according to claim 120, wherein the at least one binding protein or the at least one host cell forms a complex with the MAGEA1 peptide in the context of an MHC molecule, and the complex is detected in the form of fluorescence - activated cell sorting (FACS), enzyme - linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting or intracellular flow cytometry.

122. The method according to claim 120 or 121, the method further comprising obtaining the sample from a subject.

123. A method for detecting the degree of a disorder characterized by MAGEA1 expression in a subject, the method comprising: a) contacting a sample obtained from the subject with at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114 or a population of host cells according to claim 115; and b) detecting the level of reactivity, wherein the presence of reactivity or a higher level of reactivity compared to a control level indicates the degree of the disorder characterized by MAGEA1 expression in the subject.

124. The method according to claim 123, wherein the control level is a reference number.

125. The method according to claim 123 or 124, wherein the control level is a level from a subject not suffering from the disorder characterized by MAGEA1 expression.

126. A method for monitoring the progression of a disorder characterized by MAGEA1 expression in a subject, the method comprising: a) detecting the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114 or a population of host cells according to claim 115; b) repeating step a) at a subsequent time point; and c) Comparing the MAGEA1 levels detected in steps a) and b) or the cells of interest expressing MAGE A1 to monitor the progression of the disorder characterized by MAGEA1 expression in the subject, wherein the absence or decrease of the MAGEA1 level detected in step b) or the cells of interest expressing MAGEA1 as compared to step a) indicates that the progression of the disorder characterized by MAGEA1 expression in the subject is inhibited, and the presence or increase of the MAGEA1 level detected in step b) or the cells of interest expressing MAGEA1 as compared to step a) indicates that the disorder characterized by MAGEA1 expression in the subject has progressed.

127. The method of claim 126, wherein between the first time point and the subsequent time point, the subject has been treated to treat the disorder characterized by MAGEA1 expression.

128. A method for predicting the clinical outcome of a subject suffering from a disorder characterized by MAGEA1 expression, the method comprising: a) Determining the presence or level of reactivity between a sample obtained from the subject and a population of at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114, or the host cell population according to claim 115; and b) Comparing the presence or level of reactivity with the reactivity from a control, wherein the control is obtained from a subject with a favorable clinical outcome; wherein the absence of reactivity or a decrease in the level of reactivity in the subject sample as compared to the control indicates that the subject has a favorable clinical outcome.

129. A method for assessing the efficacy of a therapy on a disorder characterized by MAGEA1 expression, the method comprising: a) Determining the presence or level of reactivity between a sample obtained from the subject and a population of at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114, or the host cell population according to claim 115 in a first sample obtained from the subject prior to providing at least a portion of the therapy for the disorder characterized by MAGEA1 expression, and b) Determining the presence or level of reactivity between a sample obtained from the subject and a population of at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114, or the host cell population according to claim 115 in a second sample obtained from the subject after providing the therapy for the disorder characterized by MAGEA1 expression, Absence of reactivity or reduced reactivity levels in the second sample relative to the first sample indicates that the therapy is effective in treating the disorder characterized by MAGEA1 expression in the subject, and presence of reactivity or increased reactivity levels in the second sample relative to the first sample indicates that the therapy is not effective in treating the disorder characterized by MAGEA1 expression in the subject.

130. The method according to any one of claims 120-129, wherein the reactivity level is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, optionally wherein the T cell activation or effector function is T cell proliferation, killing, or cytokine release.

131. The method according to any one of claims 120-130, wherein the T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, western blotting, or intracellular flow cytometry.

132. A method of preventing and / or treating a disorder characterized by MAGEA1 expression, the method comprising contacting a target cell expressing MAGEA1 with a therapeutically effective amount of a composition comprising cells expressing at least one binding protein according to any one of claims 56-77, optionally wherein the composition is administered to a subject.

133. The method according to any one of claims 49-55 and 132, wherein the cells are allogeneic cells, syngeneic cells, or autologous cells.

134. The method according to any one of claims 49-55, 132, and 133, wherein the cells are host cells according to any one of claims 89-114 or a population of host cells according to claim 115.

135. The method according to any one of claims 49-55 and 132-134, wherein the target cell is a cancer cell expressing MAGEA1.

136. The method according to any one of claims 49-55 and 132-135, wherein the composition further comprises a pharmaceutically acceptable carrier.

137. The method according to any one of claims 49-55 and 132-136, wherein the composition induces an immune response against the target cell expressing MAGEA1 in the subject.

138. The method according to any one of claims 49-55 and 132-137, wherein the composition induces an antigen-specific T cell immune response against the target cell expressing MAGEA1 in the subject.

139. The method according to any one of claims 49-55 and 132-138, wherein the antigen-specific T cell immune response comprises CD4 + helper T lymphocyte (Th) response and at least one of CD8+ cytotoxic T lymphocyte (CTL) response.

140. The method according to any one of claims 49-55 and 132-139, the method further comprising administering at least one additional therapy for the disorder characterized by MAGEA1 expression, optionally wherein the at least one additional therapy for the disorder characterized by MAGEA1 expression is administered concurrently or sequentially with the composition.

141. The method according to any one of claims 132-140, wherein the disorder characterized by MAGEA1 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, hepatocellular carcinoma, invasive breast cancer, and bladder urothelial carcinoma.

142. The method according to any one of claims 132-141, wherein the subject is an animal model and / or a mammal of a disorder characterized by MAGEA1 expression, optionally wherein the mammal is a human, a primate, or a rodent.

Images