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

Abstract

Provided herein are MAGEA1 immunogenic peptides, binding proteins that recognize MAGEA1 immunogenic peptides, and uses thereof. The present invention is based, at least in part, on the discovery of MAGEA1 immunogenic peptides and binding proteins that recognize such MAGEA1 immunogenic peptides based on an unbiased functional selection method used to discover antigens of TCR clonotypes identified from subjects with disorders 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).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 342,415, filed May 16, 2022, U.S. Provisional Application No. 63 / 317,337, filed March 7, 2022, and U.S. Provisional Application No. 63 / 277,924, filed November 10, 2021, the entire contents of each of which are incorporated herein by reference. [Background technology]

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

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

[0004] The present invention is based, at least in part, on the discovery of MAGEA1 immunogenic peptides and binding proteins that recognize such MAGEA1 immunogenic peptides based on an unbiased functional selection method used to discover antigens of TCR clonotypes identified from subjects with disorders 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). The identified TCRs bind to various HLA alleles (e.g., HLA-C, HLA-D, HLA-E, HLA-F ... * In the context of the MAGEA1 immunogenic peptides (e.g., those listed in Table 1) and the TCRs described herein, MAGEA1 immunogenic peptides were recognized, such as those listed in Table 1. It has been 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 described herein) to bind to MAGEA1 immunogenic peptides and elicit an immune response that kills MAGEA1-expressing cells (e.g., cancer cells) demonstrates the utility of such binding proteins in a wide variety of applications, including appropriate diagnostic, prognostic, therapeutic, and drug screening methods for disorders characterized by MAGEA1 expression.

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

[0006] In another embodiment, immunogenic peptides are provided that consist of peptide epitopes selected from the peptide sequences listed in Table 1.

[0007] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the immunogenic peptide is derived from the MAGEA1 protein, and optionally the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In another embodiment, the immunogenic peptide is capable of eliciting an immune response in a subject against MAGEA1 and / or MAGEA1-expressing cells, and optionally the immune response is selected from the group consisting of: i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell development (e.g., proliferation), cytokine release, and / or cytotoxic killing.

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

[0009] Numerous embodiments are further provided that may be applied to any aspect encompassed 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 MAGEA1-expressing cells, optionally the immune response being selected from the group consisting of: i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing.

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

[0011] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, and optionally, the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule is an HLA-A * 02. HLA-A * 03. HLA-A * 01. HLA-A * 11. HLA-A * 24. 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, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A * 0205, HLA-A * 0206, HLA-A * 0207, HLA-A * 0210, HLA-A * 0211, HLA-A * 0212, HLA-A * 0213, HLA-A* 0214、SONGS-A * 0216、SONGS-A * 0217、SONGS-A * 0219、SONGS-A * 0220、SONGS-A * 0222、SONGS-A * 0224、SONGS-A * 0230、SONGS-A * 0242、SONGS-A * 0253、SONGS-A * 0260、SONGS-A * 0274アレル、SONGS-A * 0301、SONGS-A * 0302、SONGS-A * 0305、SONGS-A * 0307、SONGS-A * 0101、SONGS-A * 0102、SONGS-A * 0103、SONGS-A * 0116アレル、SONGS-A * 1101、SONGS-A * 1102、SONGS-A * 1103、SONGS-A * 1104、SONGS-A * 1105、SONGS-A * 1119アレル、SONGS-A * 2402、SONGS-A * 2403、SONGS-A * 2405、SONGS-A * 2407、SONGS-A * 2408、SONGS-A * 2410、SONGS-A * 2414、SONGS-A * 2417、SONGS-A * 2420、SONGS-A * 2422、SONGS-A * 2425、SONGS-A * 2426、SONGS-A * 2458. HLA-B * 0702、SONG-B * 0704、SONG-B * 0705、SONG-B * 0709、SONG-B* 0710, HLA-B * 0715, HLA-B * 0721, HLA-C * 0702, HLA-C * 0701, HLA-C * 0401, HLA-C * 0602, HLA-C * 0304, HLA-C * 0501, HLA-C * 1601, HLA-C * 0202, HLA-C * 0303, HLA-C * 1203, HLA-C * 0802, HLA-C * 0102, HLA-C * 1701, HLA-C * 1502, HLA-C * 1402, HLA-C * 1202, HLA-C * 0704, HLA-C * 0801, HLA-C * 0302, HLA-C * 1801, HLA-C * 1505, HLA-C * 1602, HLA-C * 0804, HLA-C * 0305, and HLA-C * 1403 alleles.

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

[0013] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, and optionally, the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule is an HLA-A * 02. HLA-A *03、SONGS-A * 01、SONGS-A * 11、SONGS-A * 24. SONG-B * 07、SONGS-C * 07、SONGS-C * 01、SONGS-C * 02、SONGS-C * 03、SONGS-C * 04、SONGS-C * 05、SONGS-C * 06、SONGS-C * 08、SONGS-C * 12、SONGS-C * 14、SONGS-C * 15、SONGS-C * 16. HLA-C * 17 and HLA-C * 18. This is a very nice song by MHC On the other side of the spectrum is HLA-A * 0201、SONGS-A * 0202、SONGS-A * 0203、SONGS-A * 0204、SONGS-A * 0205、SONGS-A * 0206、SONGS-A * 0207、SONGS-A * 0210、SONGS-A * 0211、SONGS-A * 0212、SONGS-A * 0213、SONGS-A * 0214、SONGS-A * 0216、SONGS-A * 0217、SONGS-A * 0219、SONGS-A * 0220、SONGS-A * 0222、SONGS-A * 0224、SONGS-A * 0230、SONGS-A * 0242、SONGS-A * 0253、SONGS-A * 0260、SONGS-A * 0274アレル、SONGS-A * 0301、SONGS-A *0302、HLA-A * 0305、HLA-A * 0307、HLA-A * 0101、HLA-A * 0102、HLA-A * 0103、HLA-A * 0116アラル、HLA-A * 1101、HLA-A * 1102、HLA-A * 1103、HLA-A * 1104、HLA-A * 1105、HLA-A * 1119アラル、HLA-A * 2402、HLA-A * 2403、HLA-A * 2405、HLA-A * 2407、HLA-A * 2408、HLA-A * 2410、HLA-A * 2414、HLA-A * 2417、HLA-A * 2420、HLA-A * 2422、HLA-A * 2425、HLA-A * 2426、HLA-A * 2458アラル、HLA-B * 0702、HLA-B * 0704、HLA-B * 0705、HLA-B * 0709、HLA-B * 0710、HLA-B * 0715、HLA-B * 0721、HLA-C * 0702、HLA-C * 0701、HLA-C * 0401、HLA-C * 0602、HLA-C * 0304、HLA-C * 0501、HLA-C * 1601、HLA-C * 0202、HLA-C * 0303、HLA-C * 1203、HLA-C *0802, HLA-C * 0102, HLA-C * 1701, HLA-C * 1502, HLA-C * 1402, HLA-C * 1202, HLA-C * 0704, HLA-C * 0801, HLA-C * 0302, HLA-C * 1801, HLA-C * 1505, HLA-C * 1602, HLA-C * 0804, HLA-C * 0305, and HLA-C * 1403 allele. In yet another embodiment, the peptide epitope and the MHC molecule are covalently linked, and / or the alpha and beta chains of the MHC molecule are covalently linked. In another embodiment, the stable MHC-peptide complex comprises a detectable label, and optionally, the detectable label is a fluorophore.

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

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

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

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

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

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

[0020] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, the T cells are CD8+ T cells. In another embodiment, the detecting and / or determining is performed using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, Western blot, or intracellular flow assay. In yet another embodiment, the sample comprises T cells that have been in contact with, or are suspected of being in contact with, one or more MAGEA1 proteins or fragments thereof.

[0021] In yet another aspect, there is provided a method for determining whether T cells have been exposed to MAGEA1, comprising: a) incubating a cell population comprising T cells with an immunogenic peptide described herein or a stable MHC-peptide complex described herein; and b) detecting the presence or level of reactivity, wherein the presence or higher level of reactivity compared to a control level indicates that the T cells have been exposed to MAGEA1, and optionally the cell population comprising T cells is obtained from a subject.

[0022] In yet another aspect, there is provided a method for predicting clinical outcome in a subject suffering from a disorder characterized by MAGEA1 expression, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein; and b) comparing the presence or level of reactivity with that from a control obtained from a subject with a good clinical outcome, wherein the presence or higher level of reactivity in the subject sample compared to the control indicates that the subject will have a good clinical outcome.

[0023] In another aspect, a method for assessing the effectiveness of a therapy for a disorder characterized by MAGEA1 expression is provided, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein in a first sample obtained from the subject before 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 described herein or one or more stable MHC-peptide complexes described herein and T cells obtained from the subject present in a second sample obtained from the subject after providing the therapy to the subject, wherein the presence or higher level of reactivity in the second sample compared to the first sample is an indication that the therapy is effective in treating the disorder characterized by MAGEA1 expression in the subject.

[0024] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the level of responsiveness is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, where optionally, T cell activation or effector function is T cell proliferation, killing, or cytokine release. In another embodiment, the method further comprises repeating steps a) and b) at a subsequent time point, optionally, wherein between the first and subsequent time points, the subject has undergone treatment to ameliorate a disorder characterized by MAGEA1 expression. In yet 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), immunochemically, Western blot, or intracellular flow assay. In yet another embodiment, the control level is a reference number. In another embodiment, the control level is the level in a subject without a disorder characterized by MAGEA1 expression.

[0025] In yet another aspect, a method for 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 described herein.

[0026] In yet another aspect, methods are provided for identifying a peptide-binding molecule, or antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, the method comprising: a) providing a cell displaying a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule on its surface; 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.

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

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

[0029] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the plurality of candidate peptide-binding molecules comprises an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain. In another embodiment, the plurality of candidate peptide-binding molecules comprises at least 2, 5, 10, 100, 10 3 , 10 4 , 10 5 , 106 , 10 7 , 10 8 , 10 9 The plurality of candidate peptide-binding molecules comprises one or more species of different candidate peptide-binding molecules. In yet another embodiment, the plurality of candidate peptide-binding molecules comprises one or more candidate peptide-binding molecules obtained from a sample from a subject or a population of subjects; or the plurality of candidate peptide-binding molecules comprises one or more candidate peptide-binding molecules comprising mutations in a parent scaffold peptide-binding molecule obtained from a sample from a subject. In yet another embodiment, the subject or population of subjects a) is not afflicted with a disorder characterized by MAGEA1 expression and / or has recovered from a disorder characterized by MAGEA1 expression, or b) is afflicted with a disorder characterized by MAGEA1 expression. In another embodiment, the subject or population of subjects has been administered a composition described herein. In yet another embodiment, the subject is an animal model of a disorder characterized by MAGEA1 expression and / or a mammal, optionally, the mammal is a human, primate, or 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.

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

[0031] In yet another aspect, there is provided a method of treating a disorder characterized by MAGEA1 expression in a subject, the method comprising administering to the subject a therapeutically effective amount of engineered T cells that express a peptide-binding molecule or antigen-binding fragment thereof that i) binds to a peptide epitope selected from the sequences listed in Table 1, ii) is identified according to the methods described herein, and / or iii) binds to a stable MHC-peptide complex comprising a peptide epitope selected from the sequences listed in Table 1 in the context of an MHC molecule; optionally the peptide-binding molecule or antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain; and optionally the MHC or MHC-peptide complex is as described herein.

[0032] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, T cells are isolated from a) a subject, b) a donor not afflicted with a disorder characterized by MAGEA1 expression, or c) a donor who has recovered from a disorder characterized by MAGEA1 expression.

[0033] In another aspect, there is provided a method of treating a disorder characterized by MAGEA1 expression in a subject, comprising transfusing antigen-specific T cells into the subject, wherein the antigen-specific T cells are generated by: a) stimulating immune cells from the subject with a composition described herein; and b) expanding the antigen-specific T cells in vitro or ex vivo, optionally wherein i) the immune cells are isolated from the subject before stimulating the immune cells, and / or ii) the immune cells comprise PBMCs, T cells, CD8+ T cells, naive T cells, central memory T cells and / or effector memory T cells.

[0034] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the agent is contacted under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, immunogenic peptide, stable MHC-peptide complex, T cell receptor, and / or immune cell. In another embodiment, the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor are expressed by cells, and the cells are expanded and / or isolated during one or more steps. In yet another embodiment, the disorder characterized by MAGEA1 expression is cancer or its recurrence, optionally 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 of a disorder characterized by MAGEA1 expression and / or a mammal, optionally the mammal is a human, primate, or rodent.

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

[0036] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the binding protein comprises: a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence having at least about 80% identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, and optionally, the binding affinity is about 5×10 -4 K below M d In another embodiment, the binding protein has a) a TCR alpha chain variable (V) α ) TCR V domain sequences selected from the group consisting of α TCR V with at least about 80% identity to the domain sequence α and / or b) a TCR beta chain variable (V) domain sequence listed in Table 2. β ) TCR V domain sequences selected from the group consisting of β TCR V with at least about 80% identity to the domain sequence β domain sequence, and the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In yet another embodiment, the binding protein comprises a) a TCR alpha chain sequence having at least about 80% identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence having at least about 80% identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, and optionally, the binding affinity is about 5×10 -4K below M d In yet another embodiment, the binding protein comprises a) a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In yet another embodiment, the binding proteins provided have a) a TCR alpha chain variable (V) α ) TCR V domain sequences selected from the group consisting of α and / or b) a TCR beta chain variable (V) domain sequence listed in Table 2. β ) TCR V domain sequences selected from the group consisting of β domain sequence, and the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In another embodiment, a binding protein is provided that comprises a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5×10 -4 K below M d In another embodiment, the TCR comprises 1) a TCR alpha chain CDR, a TCR V α 1) the TCR beta chain CDRs, TCR V domain, and / or TCR alpha chain are encoded by a TRAV, TRAJ, and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2; and / or 2) the TCR beta chain CDRs, TCR V domain, and / or TCR alpha chain are encoded by a TRAV, TRAJ, and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2. β2) the domain, and / or the TCR beta chain are encoded by a TRBV, TRBJ, and / or TRBC gene or fragment 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 up to five amino acid substitutions, insertions, deletions, or a combination thereof compared to a cognate reference CDR sequence listed in Table 2. In yet another embodiment, the binding protein is chimeric, humanized, or human. In yet another embodiment, the binding protein comprises a binding domain having a transmembrane domain and an intracellular effector domain. In another embodiment, the TCR alpha chain and the TCR beta chain are covalently linked, optionally via a linker peptide. In yet another embodiment, the TCR alpha chain and / or the TCR beta chain are covalently linked to a substructure, optionally comprising an affinity tag or 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, Halo tag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or the label is a fluorescent protein. In another embodiment, the covalently linked moiety is selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof. In yet another embodiment, the binding protein binds to a pMHC complex on the cell surface. In yet another embodiment, the MHC or MHC-peptide complex is described herein. In another embodiment, binding of the binding protein to the MAGEA1 peptide-MHC (pMHC) complex elicits an immune response, optionally the immune response is selected from the group consisting of i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell proliferation, cytokine release, and / or cytotoxic killing. In yet another embodiment, the binding protein binds to a MAGEA1 immunogenic peptide-MHC (pMHC) complex at a concentration of about 1×10 -4M or less, about 5×10 -5 M or less, about 1×10 -5 M or less, about 5×10 -6 M or less, about 1×10 -6 M or less, about 5×10 -7 M or less, about 1×10 -7 M or less, about 5×10 -8 M or less, about 1×10 -8 M or less, about 5×10 -9 M or less, about 1×10 -9 M or less, about 5×10 -10 M or less, about 1×10 -10 M or less, about 5×10 -11 M or less, about 1×10 -11 M or less, about 5×10 -12 Below M, または approx. 1×10 -12 K below M dIn yet another embodiment, the binding protein has a higher binding affinity for peptide-MHC (pMHC) compared to a known T cell receptor, optionally where the higher binding affinity is at least 1.05-fold higher. In another embodiment, the binding protein induces a stronger T cell proliferation, cytokine release, and / or cytotoxic killing when contacted with target cells having heterozygous expression of MAGEA1 compared to a known T cell receptor, optionally where the induction is at least 1.05-fold stronger. As used herein, reference to fold change can, in some embodiments, be in comparison to any reference mode of interest, for example, a comparison to a different binding protein; a comparison to the same binding protein in a different context, such as expression of the same binding protein in different immune cells, at different levels, in combination with other agents described herein, and similar comparisons. In yet another embodiment, the cytotoxic killing is the cytotoxic killing of target cancer cells. In yet another embodiment, the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastric cancer, invasive breast cancer, and bladder urothelial carcinoma. In another embodiment, the binding protein does not bind to a peptide-MHC (pMHC) complex containing a LAMB4-IR, TLE3, SLC16A2, SLC6A13, BTBD2, RXFP1, and / or PIGO peptide epitope.These genes are well known and art recognized to be annotated according to the following NCBI gene ID numbers, each available on the World Wide Web at ncbi.nlm.nih.gov / gene: TLE3: gene ID 7090, as well as NM_005078.4 as a representative clone. NP_005069.2; SLC16A2: gene ID 6567, and NM_006517.5 and NP_006508.2 as representative clones; SLC6A13: gene ID 6540, and NM_016615.5 and NP_057699.2 as representative clones; BTBD2: gene ID 55643, and NM_017797.4 and NP_060267.2 as representative clones; RXFP1: gene ID 59350, and NM_021634.4 and NP_067647.2 as representative clones; and PIGO: gene ID 84720, and NM_032634.4 and NP_116023.2 as representative clones. LAMB4-IR is a bioinformatically predicted transcript that has not yet been identified in cells, whose coding sequence (CDS) and protein sequence are provided below in Table 3. LAMB4-IR has a chromosomal location of chr7:108043489-108123164, referenced according to the Genome Reference Consortium Human GRCh38.p13(GCA_000001405.28)(UCSC HG38) assembly (accession available on the World Wide Web at ncbi.nlm.nih.gov / assembly / GCA_000001405.28 / ).

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

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

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

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

[0041] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment 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), and a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In another embodiment, the nucleic acid sequences encoding CD8α, CD8β, DN-TGFβRII, and the selectable protein marker are operably linked to a nucleic acid encoding a tag. In yet another embodiment, the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding CD8α, 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 acids described herein, alone or in combination with a nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII and / or a selectable protein marker, are interconnected with an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide, hi yet another embodiment, the self-cleaving peptide is P2A, E2A, F2A, or T2A.

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

[0043] Numerous further embodiments are provided that may be applied to any aspect encompassed 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 group consisting of the α1 macroglobulin gene, the α2 macroglobulin gene, the α3 macroglobulin gene, the β1 microglobulin gene, the β2 microglobulin gene, and combinations thereof. In yet another embodiment, the host cell comprises a knockout of a TCR gene selected from the group consisting of a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and combinations thereof. In yet another embodiment, the host cell expresses CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally, the selectable protein marker is DHFR, and further optionally, the CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker fused to a CD34 enrichment tag. In another embodiment, the host cell is enriched using a CD34 enrichment tag. In yet another embodiment, the host cell is a hematopoietic progenitor cell, a peripheral blood mononuclear cell (PBMC), an umbilical cord blood cell, or an immune cell. In yet another embodiment, the immune cell is a T cell, a cytotoxic lymphocyte, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte progenitor cell, a cytotoxic lymphocyte stem cell, a CD4 + T cells, CD8 +The host cells may be T cells, CD4 / CD8 double-negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or a combination thereof. In yet another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or a combination thereof. In another embodiment, the T cells are primary T cells or cells of a T cell line. In yet another embodiment, the T cells do not express an endogenous TCR or have reduced surface expression thereof. In yet another embodiment, the host cells are capable of producing cytokines or cytotoxic molecules when contacted with target cells containing a peptide-MHC (pMHC) complex containing a MAGEA1 peptide epitope in the context of an MHC molecule. In another embodiment, the host cells are contacted with the target cells in vitro, ex vivo, or in vivo. In yet another embodiment, the cytokine is TNF-α, IL-2, and / or IFN-γ. In yet another embodiment, the cytotoxic molecule is perforin and / or granzyme, and optionally, the cytotoxic molecule is granzyme B. In another embodiment, the host cell is capable of producing higher levels of cytokines or cytotoxic molecules when contacted with target cells having heterozygous expression of MAGEA1. In yet another embodiment, the host cell is capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules. In yet another embodiment, the host cell is capable of killing target cells containing a peptide-MHC (pMHC) complex containing a MAGEA1 peptide epitope in the context of an MHC molecule. In another embodiment, killing is determined by a killing assay. In yet another embodiment, the ratio of host cells to target cells in the killing assay is 20:1 to 1:4. In yet another embodiment, the target cells are target cells pulsed with 1 μg / mL to 50 pg / mL of MAGEA1 peptide, and optionally, the target cells are monoallelic with respect to an MHC matched to the MAGEA1 peptide. In another embodiment, the host cell is capable of killing a greater number of target cells when contacted with target cells having heterozygous expression of MAGEA1, optionally with cell killing being at least 1.05-fold greater.In yet another embodiment, the target cells are cell lines or primary cells, and optionally, 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 yet another embodiment, the MAGEA1 immunogenic peptide is as described herein, and / or the MHC or MHC-peptide complex is as described herein. In another embodiment, the host cells do not induce T cell proliferation, cytokine release, or cytotoxic killing when contacted with target cells comprising a peptide-MHC (pMHC) complex comprising a LAMB4-IR, TLE3, SLC16A2, SLC6A13, BTBD2, RXFP1, and / or PIGO peptide epitope. In yet another embodiment, the host cells do not express the MAGEA1 antigen, a serotype HLA-C not recognized by the binding proteins described herein. * Not of 07 and / or HLA-C * Does not express the 07 allele.

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

[0045] In yet another aspect, a composition is provided that includes 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 host cells described herein, and a carrier.

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

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

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

[0049] In yet another aspect, there is provided a method for detecting the presence or absence of a MAGEA1 antigen and / or a cell expressing MAGEA1, optionally wherein the cell is a hyperproliferative cell, the method comprising detecting the presence or absence of said 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 host cells described herein, wherein detection of the MAGEA1 antigen indicates the presence of the MAGEA1 antigen and / or a cell expressing MAGEA1.

[0050] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, at least one binding protein or the at least one host cell forms a complex with a MAGEA1 peptide in association with an MHC molecule, and the complex is detected by fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, Western blot, or intracellular flow assay. In another embodiment, the method further comprises obtaining a sample from the subject.

[0051] In another aspect, a method is provided for detecting the level 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 the level of reactivity, wherein the presence or higher level of reactivity compared to a control level indicates the level of a disorder characterized by MAGEA1 expression in the subject.

[0052] Numerous embodiments are further provided that may be applied to any aspect encompassed 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 who does not have a disorder characterized by MAGEA1 expression.

[0053] In yet another aspect, there is provided a method for tracking and assessing the progression of a disorder characterized by MAGEA1 expression in a subject, comprising: a) detecting in a subject sample the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; b) repeating step a) at a subsequent time point; and c) comparing the levels of MAGEA1 or cells of interest expressing MAGEA1 detected in steps a) and b) to track and assess the progression of the disorder characterized by MAGEA1 expression in the subject, wherein the absence or decrease of the MAGEA1 level or cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates a suppression of the progression of the disorder characterized by MAGEA1 expression in the subject, and the presence or increase of the MAGEA1 level or cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates the progression of the disorder characterized by MAGEA1 expression in the subject.

[0054] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, between the first time point and the subsequent time point, the subject has undergone therapy to treat a disorder characterized by MAGEA1 expression.

[0055] In yet another aspect, there is provided a method for predicting clinical outcome in a subject suffering from a disorder characterized by MAGEA1 expression, comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) comparing the presence or level of reactivity with that from a control obtained from a subject with a good clinical outcome, wherein an absence or reduced level of reactivity in the subject sample compared to the control indicates that the subject will have a good clinical outcome.

[0056] In another aspect, a method for assessing the effectiveness of a therapy for a disorder characterized by MAGEA1 expression is provided, comprising: a) determining the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein in a first sample obtained from the subject before providing at least a portion of the therapy for the disorder characterized by MAGEA1 expression to the subject; and b) determining the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein in a second sample obtained from the subject after providing the therapy for the disorder characterized by MAGEA1 expression, wherein an absence or decreased level of reactivity in the second sample compared to the first sample is an indication that the therapy is effective in treating the disorder characterized by MAGEA1 expression in the subject, and a presence or increased level of reactivity in the second sample compared to the first sample is an indication that the therapy is not effective in treating the disorder characterized by MAGEA1 expression in the subject.

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

[0058] In yet another aspect, there is provided a method for preventing and / or treating a disorder characterized by MAGEA1 expression, comprising contacting target cells expressing MAGEA1 with a therapeutically effective amount of a composition comprising cells expressing at least one binding protein described herein, and optionally, the composition is administered to the subject.

[0059] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the cells are allogeneic, syngeneic, or autologous cells. In another embodiment, the cells are host cells described herein, or a population of host cells described herein. In yet 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 in a subject against target cells expressing MAGEA1. In yet another embodiment, the cell composition induces an antigen-specific T cell immune response in a subject against target cells expressing MAGEA1. In yet another embodiment, the antigen-specific T cell immune response is a CD4 + and at least one of a helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the method further comprises administering at least one additional treatment for a disorder characterized by 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 yet another embodiment, the disorder characterized by MAGEA1 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. In yet another embodiment, the subject is an animal model of a disorder characterized by MAGEA1 expression and / or a mammal, optionally wherein the mammal is a human, primate, or rodent. [Brief explanation of the drawings]

[0060] [Figure 1-1] We demonstrate nucleic acid gene expression of MAGEA1 in a broad panel of tumor types and normal tissues, and further demonstrate that MAGEA1 is frequently expressed in several target indications, enabling multiplexed TCR-T therapy. [Figure 1-2] We demonstrate nucleic acid gene expression of MAGEA1 in a broad panel of tumor types and normal tissues, and further demonstrate that MAGEA1 is frequently expressed in several target indications, enabling multiplexed TCR-T therapy. [Figure 2A-1] Figure 2A shows the identification and HLA restriction of MAGEA1 antigenic peptides. Overlapping peptide sequences from MAGEA1 were identified for predicted binding to HLA-C*07:02. The top predicted MHC-binding peptides were synthesized and used to pulse HLA-C*07:02-expressing HEK293T cells. The reactivity of pools of TCR-transduced T cells was measured using a granzyme-IFP reporter. Figure 2A shows the results and frequencies of pools of TCR-transduced T cells cocultured with HEK293T cells expressing a granzyme-activated fluorescent reporter, six HLA alleles: A*01:01, A*03:01, B*07:02, B*08:01, C*07:01, and C*07:02, and an overlapping library of approximately 600,000 90-mer peptides spanning the human proteosome. Tiles with identical overlapping peptide sequences and an enrichment factor >2.5 are highlighted with a dot corresponding to the underlying common gene. [Figure 2A-2]Figure 2A shows the identification and HLA restriction of MAGEA1 antigenic peptides. Overlapping peptide sequences from MAGEA1 were identified for predicted binding to HLA-C*07:02. The top predicted MHC-binding peptides were synthesized and used to pulse HLA-C*07:02-expressing HEK293T cells. The reactivity of pools of TCR-transduced T cells was measured using a granzyme-IFP reporter. Figure 2A shows the results and frequencies of pools of TCR-transduced T cells cocultured with HEK293T cells expressing a granzyme-activated fluorescent reporter, six HLA alleles: A*01:01, A*03:01, B*07:02, B*08:01, C*07:01, and C*07:02, and an overlapping library of approximately 600,000 90-mer peptides spanning the human proteosome. Tiles with identical overlapping peptide sequences and an enrichment factor >2.5 are highlighted with a dot corresponding to the underlying common gene. [Figure 2B] Figure 2B shows the identification and HLA restriction of MAGEA1 antigenic peptides. Overlapping peptide sequences from MAGEA1 were identified for predicted binding to HLA-C*07:02. The top predicted MHC-binding peptides were synthesized and used to pulse HLA-C*07:02-expressing HEK293T cells. The granzyme-IFP reporter was used to measure the reactivity of pools of TCR-transduced T cells. Figure 2B shows the HLA deconvolution of MAGEA1 to HLA-C*07:02. HEK293T cells expressing a granzyme-activated fluorescent reporter and the indicated individual HLA alleles, or all six HLA alleles (Pt.32 6Mer) as used in Figure 2A, were transduced with or without MAGEA1 and cocultured with pools of TCR-transduced T cells. Heatmaps representing the % IFP+ cells for each culture condition are shown, with triplicates. Each row represents a reporter cell line expressing the indicated HLA. The legend on the right indicates the range of IFP signals. [Figure 2C]Figure 2C shows the identification and HLA restriction of MAGEA1 antigenic peptides. Overlapping peptide sequences from MAGEA1 were identified for predicted binding to HLA-C*07:02. The top predicted MHC-binding peptides were synthesized and used to pulse HLA-C*07:02-expressing HEK293T cells. The granzyme-IFP reporter was used to measure the reactivity of pools of TCR-transduced T cells. Figure 2C shows representative sequences of MAGEA1 peptides identified in the screening data. The overlapping sequences (solid boxes) were analyzed for HLA-C*07:02 predicted binding, and a top predicted binder with the sequence VRFFFPSL was identified. [Figure 2D] Identification and HLA restriction of MAGEA1 antigenic peptides are shown. Overlapping peptide sequences from MAGEA1 were identified for predicted binding to HLA-C*07:02. The top predicted MHC-binding peptides were synthesized and used to pulse HLA-C*07:02-expressing HEK293T cells. The reactivity of pooled TCR-transduced T cells was measured using a granzyme-IFP reporter. Figures 2D and 2E (left panels) show the reactivity of TCR-transduced T cells cocultured with HLA-C*07:02 monoallelic HEK293T cells expressing a granzyme-activated fluorescent reporter pulsed with the indicated peptides, confirming that VRFFFPSL is the minimal MAGEA1 epitope. [Figure 2E]Identification and HLA restriction of MAGEA1 antigenic peptides are shown. Overlapping peptide sequences from MAGEA1 were identified for predicted binding to HLA-C*07:02. The top predicted MHC-binding peptides were synthesized and used to pulse HLA-C*07:02-expressing HEK293T cells. The reactivity of pooled TCR-transduced T cells was measured using a granzyme-IFP reporter. Figures 2D and 2E (left panels) show the reactivity of TCR-transduced T cells cocultured with HLA-C*07:02 monoallelic HEK293T cells expressing a granzyme-activated fluorescent reporter pulsed with the indicated peptides, confirming that VRFFFPSL is the minimal MAGEA1 epitope. Figure 2E (right panel) shows that both TCR39 and TCR41 exhibit similar affinity for the peptide VRFFFPSL pulsed into HEK293 cells in suspension. Each point represents the mean of 2-3 technical replicates measuring IFNg levels, along with the standard deviation. [Figure 3A] Figure 3A shows the identification of MAGEA1-reactive TCRs from a pool of TCR-transduced T cells. Target cells were transduced with a 90-mer from MAGEA1 and cocultured with a pool of TCR-transduced T cells. Reactive TCRs were identified by sequencing the foreign TCR from sorted CD137 / CD69 double-positive cells. Figure 3A shows the percentage of cells expressing the indicated TCRs in the input (left) and sorted cells (right) (TCR32-39 and TCR32-41 are labeled "39" and "41," respectively). [Figure 3B] Figure 3B shows the identification of MAGEA1-reactive TCRs from a pool of TCR-transduced T cells. Target cells were transduced with a 90-mer from MAGEA1 and co-cultured with a pool of TCR-transduced T cells. Reactive TCRs were identified by sequencing the foreign TCR from sorted CD137 / CD69 double-positive cells. Figure 3B shows the fold enrichment of selected TCRs reactive to MAGEA1-transduced cells over the input library. [Figure 4A]We show that TCR32-39 (also known as TCR39) and TCR32-41 (also known as TCR41) recognize cancer cells expressing MAGEA1. Figure 4A reports mRNA expression data in transcripts per million (TPM) from a publicly available dataset (CCLE Cancer Cell Line Encyclopedia). [Figure 4B] Figure 4B shows that TCR32-39 (also known as TCR39) and TCR32-41 (also known as TCR41) recognize MAGEA1-expressing cancer cells. Figure 4B demonstrates the reactivity of the TCRs to cancer cell lines as measured by IFNγ release. [Figure 4C] Figure 4C demonstrates that TCR32-39 (also known as TCR39) and TCR32-41 (also known as TCR41) recognize MAGEA1-expressing cancer cells. Figure 4C demonstrates TCR reactivity to cancer cell lines as measured by CD137 / CD69 double-positive staining. [Figure 5A] Figure 5A shows that TCR32-41 transduced into pan T cells (including both CD3+:CD4+ or CD8+ T cells) ("TSC-204-C7") recognizes cancer cells expressing MAGEA1. As described further below, the term "TSC-204-C7" refers to an engineered whole T cell population (including CD8+ and CD4+ T cells) expressing TCR32-41 in addition to other components, including CD8α, CD8β, and CD34 enrichment tags. In some embodiments, TSC-204-C7 may further express DN-TGFβRII. Figure 5A reports mRNA expression data in transcripts per million (TPM) from a publicly available dataset (CCLE Cancer Cell Line Encyclopedia). [Figure 5B]Figure 5B demonstrates that TCR32-41 transduced into pan T cells (including both CD3+, CD4+, and CD8+ T cells) ("TSC-204-C7") recognizes cancer cells expressing MAGEA1. As described further below, the term "TSC-204-C7" refers to an engineered whole T cell population (including CD8+ and CD4+ T cells) expressing TCR32-41 in addition to other components, including CD8α, CD8β, and CD34 enrichment tags. In some embodiments, TSC-204-C7 may further express DN-TGFβRII. Figure 5B demonstrates the reactivity of TCR transduced into either CD3+ or CD8+ T cells to cancer cell lines, as measured by secretion of the indicated cytokines (IFNg, TNFα, and granzyme B). Plotted are the mean ± standard deviation (n=3). [Figure 5C] Figure 5C shows that TCR32-41 transduced into pan T cells (including both CD3+:CD4+ or CD8+ T cells) ("TSC-204-C7") recognizes cancer cells expressing MAGEA1. As described further below, the term "TSC-204-C7" refers to an engineered whole T cell population (including CD8+ and CD4+ T cells) expressing TCR32-41 in addition to other components, including CD8α, CD8β, and CD34 enrichment tags. In some embodiments, TSC-204-C7 may further express DN-TGFβRII. Figure 5C provides a summary chart of the TCR41 CD3 data from Figure 5B. [Figure 6A] We demonstrate the cytotoxic activity of TCR32-39 or 32-41 against cell lines expressing variable levels of MAGEA1. Figure 6A shows that TCR32-39 and 32-41 kill cancer cell lines expressing high to moderate levels of MAGEA1 (TPM is shown for each cell line). [Figure 6B]Figure 6 demonstrates the cytotoxic activity of TCR32-39 or 32-41 against cell lines expressing variable levels of MAGEA1. Figure 6B shows that TCR32-41 does not kill cell lines with low to zero MAGEA1 expression. The indicated cell lines were transduced with Incucyte® NucLight™ Red and cocultured with either TCR32-39 (triangles), TCR32-41 (circles), untransduced T cells (diamonds), or no T cells (squares). T cells were added at the indicated E:T ratio at the start of the assay. Red fluorescence was measured over time using the Incucyte® instrument and displayed as total red integrated intensity per well normalized to time point 0. Summary data, presented as bar graphs below, are plots of the viability of each cell line, with error bars representing the mean level of cell viability at 72 hours (n=2), with error bars representing a range of 0.5. [Figure 6C] Figure 6C shows the cytotoxic activity of TCR32-39 or 32-41 against cell lines expressing variable levels of MAGEA1. Figure 6C simplifies the data from Figures 6A and 6B to show only the TCR41 data at an E:T ratio of 1:1. [Figure 7] This shows that TCR32-41 does not exhibit any alloreactivity to the 110 most common MHC alleles (HLA types). TCR32-41-expressing pan T cells or untransduced control T cells were cocultured for 48 hours with MHC-null HEK293T cells expressing one of the 110 most frequently occurring class I MHC alleles in the American population. A positive control consisting of HEK293T cells expressing both a fragment of MAGE-A1 containing the epitope (VRFFFPSL) and HLA-C*07:02 was included in the screen. After 48 hours of coculture, inhibition of target cell growth by TCR32-41-expressing pan T cells relative to that by untransduced control T cells was measured as a readout of TCR32-41 reactivity to allogeneic MHC molecules. The positive control is shown. [Figure 8]Results of a genome-wide SafetyScan screen to identify putative non-targets of TCR32-41 are shown. SafetyScan screening data for TCR32-41 is shown, identifying several potential non-targets in a screen of 600,000 protein fragments spanning all wild-type (wt) human proteins. The screen was designed to over-predict non-targets by overexpressing a 90-aa protein fragment, which is processed more efficiently than the full-length protein. Putative non-targets are identified by gene name. LAMB4-IR is a predicted cancer-specific intron-retaining transcript that has never been identified as expressed in normal cells. No expression of this variant was detected using RNA-seq analysis of normal tissue samples (Human Protein Atlas). [Figure 9] Figure 9A shows that putative non-targets of TCR32-41 are not recognized as full-length proteins. Figure 9A shows that co-culture of 32-41-expressing T cells with C*07:02 HEK reporter cells expressing the indicated full-length proteins, with the exception of LAMB4-IR, does not result in any reporter activation measured as IFP%. When challenged with dilutions of cross-reactive peptides from LAMB4-IR, Figure 9B shows poor reactivity of TCR32-41 to LAMB4-IR peptides below 2,000 ng / mL. A table summarizing the results obtained to evaluate predicted non-targets from genome-wide SafetyScan screening is shown. [Figure 10] This shows that TCR32-41 does not exhibit reactivity against normal human cells. 32-41-expressing pan T cells or untransduced (NTD) cells were tested for their reactivity against primary cells from healthy HLA-C*07:02+ human donors. Target cells were pulsed or unpulsed with MAGE-A1 peptide and co-cultured with TCR32-41 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR32-41 reactivity against target cells. Bars reflect the mean ± standard deviation of triplicates. [Figure 11] 1 provides an overview of the enhanced TCR-T cell products encompassed by the present invention.

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

[0062] The present invention is based, at least in part, on the discovery of MAGEA1 immunogenic peptides (e.g., comprising or consisting of the sequences listed in Table 1), binding proteins that recognize the MAGEA1 antigen (e.g., having the sequences listed in Table 2), and uses thereof. A systematic comprehensive search was performed to map the precise T cell targets recognized by the initial pool of T cells of interest.

[0063] Thus, the present invention relates, in part, to identified epitopes (immunodominant peptides) of the MAGEA1 protein of therapeutic importance, and related compositions (e.g., immunodominant peptides, vaccines, and the like), compositions comprising the immunogenic peptides alone or in combination with MHC molecules, stable MHC-peptide complexes, methods for diagnosing, prognosing, and tracking immune responses to disorders characterized by MAGEA1 expression, and methods for preventing and / or treating disorders characterized by MAGEA1 expression. The present invention also relates, in part, to identified binding proteins (e.g., TCRs), host cells expressing the binding proteins (e.g., TCRs), compositions comprising the binding proteins (e.g., TCRs) and host cells expressing the binding proteins (e.g., TCRs), methods for diagnosing, prognosing, and tracking T-cell responses to cells expressing MAGEA1, and methods for preventing and / or treating disorders characterized by MAGEA1 expression.

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

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

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

[0067] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, e.g., a protein, peptide, or hapten. The antigen may be a MAGEA1 antigen or a fragment thereof against which a protective or therapeutic immune response is desired. An "epitope" is the portion of an antigen that is bound by a natural or synthetic substance.

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

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

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

[0071] As used herein, the term "antigen-binding portion" of a binding protein (e.g., a TCR) refers to one or more portions of a TCR that retain the ability to bind (e.g., specifically and / or selectively) to an antigen (e.g., a MAGEA1 antigen). Such portions are, for example, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, preferably about 8 to about 300 amino acids in length, e.g., about 8 to about 200 amino acids in length, or about 10 amino acids up to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of a TCR can be performed by fragments of a full-length TCR. Examples of binding portions encompassed by the term "antigen-binding portion" of a TCR include: (i) the V α and V β (ii) an Fv fragment consisting of an isolated complementarity determining region (CDR), or (iii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. α and V β are encoded by separate genes, but V α and V β V as a single protein chain whose domains pair to form a monovalent molecule (known as a single-chain TCR (scTCR)). α and V β V is provided by a synthetic linker that allows α and V βSuch single-chain TCRs are also intended to be encompassed by the term "antigen-binding portion" of a TCR. These TCR fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same manner as the intact binding protein. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

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

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

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

[0075] The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region antiparallel to the first region when that residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region antiparallel to the first region when that residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid when the two regions are in an antiparallel arrangement and at least one nucleotide residue in the first region can base pair with a residue in the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, provided that when the first and second portions are in an antiparallel configuration, at least about 50% of the nucleotide residues in the first portion, 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, inclusive, e.g., at least about 80% to 100%, can base pair with nucleotide residues in the second portion. In some embodiments, all nucleotide residues in the first portion can base pair with nucleotide residues in the second portion.

[0076] As used herein, with respect to activated immune cells, the term "costimulate" includes that a costimulatory molecule can provide a second, non-stimulatory receptor-mediated signal (a "costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell receptor-mediated signal. Immune cells that have received a cell receptor-mediated signal, e.g., via an activating receptor, are referred to herein as "activated immune cells."

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

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

[0079] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein that has been engineered to contain two or more amino acid sequences linked together in a manner that does not occur naturally or in a host cell, and that is capable of functioning as a receptor when present on the surface of a cell. CARs encompassed by the present invention comprise an extracellular portion comprising an antigen-binding domain (i.e., obtained from or derived from an immunoglobulin or immunoglobulin-like molecule, e.g., a TCR specific for the MAGEA1 antigen, a single-chain TCR-derived binding protein, an scFv derived from an antibody, an antigen-binding domain derived from or derived from a killer immune receptor from an NK cell, etc.) linked to a transmembrane domain and one or more intracellular signaling domains (e.g., an effector domain optionally comprising costimulatory domain(s)). (See, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; see also, Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

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

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

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

[0083] The term "dominant-negative TGFβ receptor" or "DN-TGFβR" refers to a variant or mutant of a transforming growth factor (TGF) beta receptor that exhibits resistance to TGFβ signaling. There are five type II receptors (activating receptors) and seven type I receptors (receptors that propagate signaling). Activated TGFβ receptors are heterotetramers consisting of two TGFβ receptor I (TGFβRI) and two TGFβ receptor II (TGFβRII). In some embodiments, the DN-TGFβR is DN-TGFβRII (i.e., a variant or mutant of TGFβ receptor II). In some embodiments, the resistance is to the suppressive effects of TGFβ signaling on immune cells, e.g., T cells, where TGFβ may be produced by cancer cells or by other immune cells in the cellular environment, e.g., stromal cells, macrophages, myeloid cells, epithelial cells, natural killer cells, and the like. TGFβ signaling inhibitors are well known in the art and include, but are not limited to, mutant TGFβ, antibodies that bind to TGFβ and / or TGFβ receptors (e.g., lerdelimumab, metlimumab, fresolimumab, and the like) that sequester the receptors and thereby inhibit signaling, soluble TGFβ binding proteins, such as portions of the TGFβ receptor that sequester TGFβ (e.g., TGFβRII-Fc fusion protein), or other binding agents such as betaglycan. Any and all known TGFβ signaling inhibitors can be used in place of or in addition to the DN-TGFβR (e.g., DN-TGFβRII) described herein. In some embodiments, the DN-TGFβR lacks an intracellular portion required for TGFβ-mediated signaling, such as the entire intracellular domain, the kinase signaling domain, etc.DN-TGFβR constructs are well known in the art (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 No. WO2009 / 152610, PCT Publication No. WO2017 / 156484, Kloss et al. (See representative non-limiting embodiments in PCT Publication Nos. WO2019 / 089884, WO2020 / 042647, and WO2020 / 042648).

[0084] In some embodiments, immune cell products (e.g., engineered T cells) comprising one or more binding proteins (e.g., TCRs) described herein are resistant to β-mediated immunosuppression. As described above and below, TGFβ, produced by tumor cells and cells in the tumor microenvironment, is an immunosuppressive cytokine. TGFβ inhibits the function and development of cytotoxic and Th1 helper T cells, resulting in the suppression of tumor-specific T cell responses (Dahmani and Delisle (2018) Cancers 10:194). TGFβ signaling in T cells can be abrogated by expression of a dominant-negative TGFβ type II receptor (DN-TGFβRII) (Wieser et al. (1993) Mol. Cell. Biol. 13:7239-7247; Bollard et al. (2002) Blood 99:3179-3187). Once bound to TGFβ, wild-type TGFβRII phosphorylates and thereby activates TGFβRI, initiating intracellular signaling. In cells expressing a truncated form of TGFβRII (DN-TGFβRII) lacking the intracellular kinase domain, this signaling cascade is disrupted, rendering the cells resistant to inhibition by TGFβ. DN-TGFβRII blocks TGFβ signaling in engineered T cells (both CAR-T and TCR-T cells) (Bollard et al. (2002) Blood 99:3179-3187, Foster et al. (2008) J. Immunother. 31:500-505, Kloss et al. (2018) Mol. Ther. 26:1855-1866, Alabanza et al. (2022) Front. Immunol. 13:832645, Silk et al. (2022) J. Immunol. 208:169-180, Li et al. (2020) Front. Oncol. 10:1117).In a representative study, evaluation of DN-TGFβRII-loaded EBV-specific T cells for the treatment of Hodgkin lymphoma showed that DN-TGFβRII-engineered T cells were both safe and effective (Bollard et al. (2018) J. Clin. Oncol. 36:1128-1139).

[0085] As used herein, a "hematopoietic progenitor cell" is a cell that can be derived from hematopoietic stem cells or fetal tissue and can further differentiate into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include CD24 Lo Lin-CD117 + These include those with a phenotype similar to that of thymocytes, or those found in the thymus (called primitive thymocytes).

[0086] As used herein, "homology" refers to the similarity of nucleotide sequences between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. If a nucleotide residue position in both regions is occupied by the same nucleotide residue, the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the percentage of nucleotide residue positions in the two regions that are occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, provided that 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, inclusive, e.g., at least about 80%-100%, of the nucleic acid residue positions of each of the portions are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0087] The term "immune response" includes T cell and / or B cell immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses indirectly mediated by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0088] An increased ability to stimulate an immune response or the immune system may result from increased agonist activity of T cell costimulatory receptors and / or increased antagonist activity of inhibitory receptors. An increased ability to stimulate an immune response or the immune system may result from increased EC in assays that measure immune responses, such as assays that measure cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly via detecting CD107a or granzymes), and changes in proliferation. 50 The ability to stimulate an immune response or activity of the immune system may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500%, or more.

[0089] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate a host's immune system to generate an immune response to a viral infection in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0090] The term "immune cell" refers to any cell of the immune system that arises from hematopoietic stem cells in the bone marrow, which give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells, e.g., monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes), and lymphoid progenitor cells (which give rise to lymphoid cells, e.g., T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 +These include T cells, CD4 CD8 double negative T cells, gd T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells are sometimes called "antigen-presenting cells" or "APCs," which are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of the APC, complexed with peptide, interact with TCRs on the surface of the T cell.

[0091] "Isolated protein" refers to a protein that, when isolated from a cell or produced by recombinant DNA techniques, is substantially free of other proteins, cellular material, isolation and culture medium, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. An "isolated" or "purified" protein, or a biologically active portion thereof, is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the binding protein, antibody, polypeptide, peptide, or fusion protein is derived, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. The language "substantially free of cellular material" includes preparations of a biomarker polypeptide or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of biomarker proteins or fragments thereof having less than about 30% (by dry weight) non-biomarker protein (also referred to herein as "contaminating proteins"), or in some embodiments about 25%, 20%, 15%, 10%, 5%, 1%, or less, or any ranges therebetween, inclusive, e.g., about less than 1% to 5% non-biomarker protein. When a binding protein, antibody, polypeptide, peptide, or fusion protein, or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it may be substantially free of culture medium, i.e., culture medium may comprise about 20%, 15%, 10%, 5%, 1%, or less, or any ranges therebetween, inclusive, e.g., about less than 1% to 5%, of the volume of the protein preparation.

[0092] As used herein, the term "isotype" refers to the class of antibody (e.g., IgM, IgG1, IgG2C, etc.) encoded by heavy chain constant region genes.

[0093] As used herein, the term "K D" is intended to refer to the dissociation equilibrium constant of a particular binding protein-antigen interaction. The binding affinity of binding proteins encompassed by the present invention can be measured or determined by standard binding protein-target binding assays, e.g., competition assays, saturation assays, or standard immunoassays, e.g., ELISA or RIA. A relatively lower Kd value indicates a relatively higher binding affinity (e.g., about 5 x 10 -4 The Kd value for M (500uM) or less is 1×10 -4 M (100uM), where a Kd of 100uM indicates a relatively higher binding affinity compared to a Kd of 500uM).

[0094] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent (e.g., a probe or small molecule for detecting and / or affecting the expression of a marker encompassed by the invention). The kit may be advertised, distributed, or sold as a unit for practicing a method encompassed by the invention. The kit may include one or more reagents necessary to express a composition useful in a method encompassed by the invention. In some embodiments, the kit may further include a reference standard (e.g., a nucleic acid encoding a protein that does not affect or modulate a signaling pathway controlling cell proliferation, division, migration, survival, or apoptosis). One of skill in the art can envision many such control proteins, including, but not limited to, common molecular tags (e.g., green fluorescent protein and beta-galactosidase), proteins that do not fall into any of the pathways encompassing cell proliferation, division, migration, survival, or apoptosis according to Gene Ontology standards, or ubiquitous housekeeping proteins. The reagents in the kit may be provided in individual containers, or a mixture of two or more reagents in a single container. Additionally, instructional materials describing the use of the compositions within the kit may be included.

[0095] As used herein, the term "linked" refers to the association of two or more molecules. The association may be covalent or non-covalent. The association may also be genetic (i.e., recombinantly fused). Such association may be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0096] "Linker," in some embodiments, can refer to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs, and can provide a spacer function that accommodates the interaction of two sub-binding domains so that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., an scTCR) or signaling activity (e.g., a 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.

[0097] 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 forward strand. GRCh38:CM000685.2), also known as cancer / testis antigen 1.1 (CT1.1), melanoma-associated antigen 1, MAGE1, melanoma antigen family A,1 (induces 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. 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. 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 through interaction with SNW1, recruitment of the histone deacetylase HDAC1, and inhibition of Notch intracellular domain (NICD) transactivation. It is also thought to be involved in fetal development, several genetic disorders (e.g., dyskeratosis congenita), and / or tumor transformation or progression (e.g., gastric cancer, hepatocellular carcinoma, etc.). MAGEA1 is not highly expressed in normal tissues except in the testis, but is expressed in various histological types of tumors, including melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastric cancer, invasive breast cancer, and bladder urothelial carcinoma.

[0098] 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 publicly available from the National Center for Biotechnology Information (NCBI) (see, e.g., ncbi.nlm.nih.gov / gene / 4100). For example, human MAGEA1 (NP_004979.3) is encoded by transcript (NM_004988.5). Nucleic acid and polypeptide sequences of MAGEA1 orthologues from non-human organisms are well known, including chimpanzee MAGEA1 (XM_529226.2 and XP_529226.2) and mouse MAGEA1 (chromosome X:155088686-155089793; Ensembl Mus Musculus version 104.39 (GRCm39)). Representative MAGEA1 sequences are also presented in Table 3 below.

[0099] 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). Examples of antibodies that can be used to detect MAGEA1 expression include 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, among the commercial product lists of various companies, there are several types of siRNA, shRNA, and CRISPR constructs for regulating MAGEA1 expression, such as open reading frame (ORF) clones MG212171, MR212171, MR212171L3, MR212171L3V, MR212171L4, MR212171L4V, RC202134, RC202134L3, RC202134L3V, RC202134L4, RC202134L4V, and RG202134 (OriGene, Rockville, MD), CRISPR knockouts GA102785, GA202134L3V, and RG202134 (OriGene, Rockville, MD). 2555, KN202134, KN202134BN, KN202134LP, KN202134RB, KN402134, and KN509652 (OriGene, Rockville, MD), as well as RNA interference (RNAi) clones, such as siRNA and shRNA clones, for example, SR302776, TL311617, SR410578, TL311617V, TTL516288, TL516288V, TL704467, TL04467V, TR311617, TR516288, and TR704467 (OriGene, Rockville, MD). Note that the term may also be used to refer to any combination of the features described herein for the MAGEA1 molecule. For example, any combination of sequence composition, percent identity, sequence length, domain structure, functional activity, etc. may be used to describe MAGEA1 molecules encompassed by the present invention.

[0100] The terms "MAGEA1 antigen" or "MAGEA1 peptide antigen" or "MAGEA1-containing peptide antigen" or "MAGEA1 epitope" or "MAGEA1 peptide epitope" or "MAGEA1 peptide" refer to a naturally occurring or synthetically produced immunogenic portion of MAGEA1. In some embodiments, a MAGEA1 antigen protein can range in length from about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, or any range therebetween, inclusive of the endpoints, e.g., 8-15 amino acids. In some embodiments, a MAGEA1 antigen protein can form a complex with an MHC (e.g., HLA) molecule, such that a binding protein of the present disclosure that recognizes a MAGEA1 peptide:MHC (e.g., HLA) complex can bind (e.g., specifically and / or selectively) to such a complex. Representative MAGEA1 peptide antigen sequences are shown in Table 1.

[0101] 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 a chain (with three a domains) and a non-covalently associated b2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and b, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides from the cytosol to the cell surface, where the peptide antigen-MHC (pMHC) complex is expressed by CD8 + MHC class II molecules deliver peptides from the vesicle system to the cell surface, where they are recognized by CD4 T cells. + Recognized by T cells. Human MHC is called human leukocyte antigen (HLA).

[0102] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk of or susceptible to developing the disease, disorder, or condition.

[0103] The term "prognosis" includes a prediction of the likely course and outcome of a viral infection, or the likelihood of recovery from the disease. In some embodiments, a statistical algorithm is used to predict a prognosis of a viral infection in an individual. For example, the prognosis can be surgery, development of a clinical subtype of the viral infection, the occurrence of one or more clinical factors, or recovery from the disease.

[0104] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology," which may be determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This described algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the polynucleotides described herein. BLAST protein searches are performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0105] The phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating substance, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another organ or part of the body.

[0106] The term "ratio" refers to a relationship between two numbers (e.g., a score, a sum, etc.). While a ratio may be expressed in a particular order (e.g., a to b, or a:b), one skilled in the art will recognize that the underlying relationship between the numbers may be expressed in any order without losing the significance of the underlying relationship, although the appearance of trends and correlations based on the ratio may be reversed.

[0107] The term "recombinant host cell" (or simply "host cell") refers to a cell containing a nucleic acid that does not naturally occur in the cell, e.g., a cell into which a recombinant expression vector has been introduced. It should be understood that a cell of the invention is intended to encompass not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the term cell of the invention.

[0108] The terms "cancer response," "response to immunotherapy," or "response to T-cell-mediated cytotoxicity modulator / immunotherapy combination therapy" refer to any response of a hyperproliferative disorder (e.g., cancer) to a cancer drug, e.g., a T-cell-mediated cytotoxicity modulator, and immunotherapy, preferably to a change in tumor weight and / or volume after initiation of neoadjuvant or adjuvant therapy. The term "neoadjuvant therapy" refers to a treatment given before primary treatment. Examples of neoadjuvant therapy include chemotherapy, radiation therapy, and hormonal therapy. The response of a hyperproliferative disorder may be evaluated, for example, for efficacy or in the neoadjuvant or adjuvant setting, where the size of the tumor after systemic intervention may be measured by CT, PET, mammogram, ultrasound, or palpation to compare the size and dimensions of the tumor after systemic intervention with the initial size and dimensions. Response may also be evaluated by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Responses may be recorded in a quantitative manner, such as the percentage change in tumor volume, or in a qualitative manner, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Assessment of hyperproliferative disorder response may be performed early after the initiation of neoadjuvant or adjuvant therapy, e.g., hours, days, weeks, or preferably months. A typical endpoint for response assessment is the end of neoadjuvant chemotherapy or surgical resection of residual tumor cells and / or tumor bed. This is typically three months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). Clinical benefit rate is measured by determining the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the number of patients with stable disease (SD) at least 6 months after the end of therapy. Briefly, this formula is CBR = CR + PR + SD beyond 6 months.In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for assessing response to cancer therapy relate to "survival," including all of the following: survival to death, also known as overall survival (where death can be either cause-unrelated or tumor-related); "recurrence-free survival" (where the term recurrence includes both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease includes cancer and related diseases). The above survival lengths can be calculated based on defined starting points (e.g., time of diagnosis or initiation of treatment) and end points (e.g., death, recurrence, or metastasis). Furthermore, criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a population of subjects may be administered a particular cancer treatment regimen and the outcome may be correlated with biomarker measurements determined before any cancer treatment is administered. The outcome measurement may be a pathological response to a therapy administered in a neoadjuvant setting. Alternatively, outcome measurements, such as overall survival and disease-free survival, may be followed for a period of time after cancer therapy for which biomarker measurements are known. In certain embodiments, the administered dose is a standard dose known in the art for cancer therapeutic agents. The length of time for which subjects are followed may vary. For example, subjects may be followed for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds that correlate with cancer therapy outcomes may be determined using methods well known in the art, such as those described in the Examples section.

[0109] As indicated, these terms can also refer to improved prognosis, for example, as reflected by an increased time to recurrence (which is the period until first recurrence, censored by a second primary cancer as the first event or death without evidence of recurrence) or an increased overall survival (which is the period from treatment to death from any cause). Responding or having a response means that there is a beneficial endpoint that is achieved when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated by exposure to a stimulus. It will be understood that assessing the likelihood that a tumor or subject will respond favorably is equivalent to assessing the likelihood that a tumor or subject will not respond favorably (i.e., exhibit a lack of response or be non-responsive).

[0110] The term "resistance" refers to the acquired or natural resistance of a cancer sample or mammal to a cancer therapy (i.e., non-responsiveness to therapeutic treatment or a reduced or limited response to therapeutic treatment), for example, a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater decrease in response to therapeutic treatment, such as a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or greater decrease, or any range therebetween, inclusive of the endpoints. The reduced response can be measured by comparing the same cancer sample or mammal before resistance was acquired, or by comparing with a different cancer sample or mammal known not to have resistance to therapeutic treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance may be mediated by P-glycoprotein or other mechanisms, or may occur when a mammal is infected with a multidrug-resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of a skilled clinician, and may be measured, for example, by cell proliferation and cell death assays, as described herein as "sensitizing." In some embodiments, the term "reverse resistance" refers to the use of a second agent in conjunction with a primary cancer therapy (e.g., chemotherapy or radiation therapy) to significantly reduce tumor volume compared to that of an untreated tumor at a statistically significant level (e.g., p<0.05) in situations where the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone fails to reduce tumor volume statistically significantly compared to that of an untreated tumor. This typically applies to tumor volume measurements performed when untreated tumors are growing exponentially.

[0111] The term "sample" as used to detect or determine the absence or presence, or level, of at least one biomarker, typically refers to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., those described above under the definition of "body fluid"), or a tissue sample (e.g., biopsy material), such as skin, colon sample, or surgically resected tissue. In some embodiments, methods encompassed by the present invention further comprise the step of obtaining a sample from an individual before detecting or determining the absence or presence, or level, of at least one marker in the sample.

[0112] The term "sensitizing" refers to altering cancer or tumor cells in a way that allows the associated cancer to be more effectively treated with a cancer therapy (e.g., anti-immune checkpoint therapy, chemotherapy, and / or radiation therapy). In some embodiments, normal cells are not affected so much that they are unduly harmed by the therapy. Increased or decreased susceptibility to therapeutic treatment is measured according to methods known in the art for the particular treatments and methods described below in this application, including, but not limited to, cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164) and cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., In: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Press). Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90. Sensitivity or resistance can also be measured in animals by measuring the reduction in tumor size over a period of time (e.g., 6 months for humans, 4-6 weeks for mice). A composition or method sensitizes a response to a therapeutic treatment if the increase in therapeutic sensitivity or decrease in resistance compared to the therapeutic sensitivity or resistance in the absence of such composition or method is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any ranges therebetween, inclusive of the endpoints.Determining sensitivity or resistance to therapeutic treatment is a common practice in the art and is within the skill of a skilled clinician. It should be understood that any method described herein for enhancing the effectiveness of cancer therapy can be equally applied to methods for sensitizing hyperproliferative cells or other cancerous cells (e.g., resistant cells) to cancer therapy.

[0113] The term "small molecule" is a term of the art and includes molecules with a molecular weight of less than about 1000, or molecules with a molecular weight of less than about 500. In one embodiment, a small molecule is not composed exclusively of peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63-68), and natural product extract libraries. In another embodiment, the compound is a small organic non-peptide compound. In a further embodiment, the small molecule is not a biosynthetic product.

[0114] The term "specific binding" refers to the binding of a binding protein to a predetermined antigen. Typically, a binding protein binds to a specific antigen at a specific binding site on the order of about 5×10 6 as measured by a binding assay, for example, surface plasmon resonance (SPR) technology on a BIAcore™ assay instrument using the antigen of interest as the analyte and the binding protein as the ligand. -4 M or less, approximately 1×10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10-10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, approximately 1×10 -12 Affinity (K) of less than or equal to M or even less, or any range therebetween, inclusive of the endpoints, e.g., about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, etc. D ) In some embodiments, a binding protein binds to a predetermined antigen with an affinity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). As used herein, the phrases "binding protein that recognizes an antigen" and "binding protein specific for an antigen" are used interchangeably with the term "binding protein that specifically binds to an antigen." Selective binding is a relative term that refers to the ability of a binding protein to distinguish binding of one antigen from binding of another antigen, e.g., to distinguish binding of a particular family member or antigen target from binding of related family members or antigen targets. For example, the analytical data provided in the Examples section demonstrate that the binding proteins described herein specifically bind to MAGEA1 immunogenic epitopes and / or selectively bind to many related epitopes (e.g., MAGEA1 immunogenic epitopes and closely related sequences), thereby distinguishing such targets from the vast majority of other possible epitopes available in the human genome.

[0115] 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. The term "subject" is interchangeable with "patient."

[0116] The term "survival" includes all of the following: survival until death, also known as overall survival (where death can be either cause-unrelated or tumor-related); "recurrence-free survival" (where the term recurrence includes both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease includes cancer and related diseases). The length of survival may be calculated based on a defined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy may be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0117] The term "synergistic effect" refers to a combined effect of two or more agents (e.g., a MAGEA1-related agent described herein and another therapy for treating a disorder characterized by MAGEA1 expression) that is greater than the sum of the separate effects of the cancer agents / therapies alone.

[0118] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, e.g., effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0119] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analog of such RNA or cDNA) that is complementary to or homologous to all or a portion of the mature mRNA produced by transcription of a biomarker nucleic acid and by normal post-transcriptional processing of the RNA transcript, if any (e.g., splicing), and by reverse transcription of the RNA transcript.

[0120] "T cells" are immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells are naive (not exposed to antigen; T CM increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO compared to normal T cells), memory T cells (T M ) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). M are central memory T cells (T CM , which have increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA compared to naive T cells), and effector memory T cells (T EM , naive T cells or T CM These cells can be further divided into effector T cells (T) and effector T cells (T) (which have decreased expression of CD62L, CCR7, CD28, and CD45RA, and increased expression of CD127 compared to T cells). E ) is T CM CD4 T cells are antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, and CD28 compared to CD4 T cells, and are positive for granzymes and perforin. Other exemplary T cells include regulatory T cells, e.g., CD4 + CD25 + (Foxp3 + ) Regulatory T cells and Treg17 cells, as well as Tr1, Th3, and CD8 + CD28 and Qa-1 restricted T cells.

[0121] Conventional T cells (also known as Tconv or Teff) have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) and augment immune responses by expressing one or more T cell receptors. Tcon or Teff are generally defined as any T cell population that is not a Treg, including, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated toward, for example, a Th1 lineage or a Th2 lineage. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is a CD4+ Teff, or a CD8+ Teff, such as a CD4+ helper T lymphocyte (e.g., Th0, Th1, Tfh, or Th17), and a CD8+ cytotoxic T lymphocyte. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tcon" are CD4 T cells that have differentiated in the bone marrow and successfully undergone the processes of positive and negative central selection in the thymus, but have not yet been activated by exposure to antigen. + Naive T cells are generally characterized by the absence of surface expression of L-selectin (CD62L), activation markers such as CD25, CD44, or CD69, and memory markers such as CD45RO. Thus, naive T cells are considered quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see, at least, WO 2010 / 101870). The presence and activity of such cells is undesirable in the context of suppressing immune responses. Unlike Tregs, T cells are not anergic and can proliferate in response to antigen-driven T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637).

[0122] "T Effector" ("T eff " or "T E") cells refer to T cells (e.g., CD4+ and CD8+ T cells) and T helper (Th) cells with cytolytic activity that secrete cytokines to activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0123] "T cell receptor" or "TCR" refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al. (1997) Curr. Biol. Publ. 4:33) that can bind (e.g., specifically and / or selectively) to an antigenic peptide bound to an MHC receptor. TCRs can be found on the surface of cells or in soluble form and are generally composed of heterodimers having an alpha chain and a beta chain (also known as TCRα and TCRβ, respectively) or a gamma chain and a delta chain (also known as TCRγ and TCRδ, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portions of TCR chains (e.g., α and β chains) contain two immunoglobulin domains: a variable domain (e.g., an α chain variable domain or V chain) at the N-terminus; 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," U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 5 th ed.), and one constant domain at the C-terminus that is close 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 117-295 based on Kabat are used. Also similar to immunoglobulins, variable domains contain complementarity-determining regions ("CDRs," also called hypervariable regions or "HVRs") separated by framework regions ("FRs") (see, e.g., Fores et al. (1990) Proc. Natl. Acad Sci. USA 87:9138; Chothia et al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol. 27:55). In some embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and associate with the CD3 complex. The source of TCRs encompassed by the present invention may be from various animal species, e.g., human, mouse, rat, rabbit, or other mammals.

[0124] The term "T cell receptor" or "TCR" should be understood to encompass complete TCRs and antigen-binding portions or fragments thereof. In some embodiments, the TCR is a complete or full-length TCR, e.g., an αβ-type TCR or a γδ-type TCR. In some embodiments, the TCR is an antigen-binding portion that is shorter than the full-length TCR but binds to a specific peptide bound in an MHC molecule, e.g., an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR contains only a portion of the structural domain of a full-length or complete TCR but is still capable of binding to a peptide epitope (e.g., an MHC-peptide complex) bound by the full-length TCR. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, e.g., the variable α chain and variable β chain of the TCR, to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity-determining regions (CDRs) involved in recognizing peptides, MHC, and / or MHC-peptide complexes.

[0125] Nomenclature has been established by the International Immunogenetics Information System (IMGT) (Scaviner and Lefranc (2000) Exp. Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; see also "T Cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). IMGT provides unique sequences used to describe TCRs, and the sequences described in this application can be identified based on such unique sequences provided in this application. TCR sequences are publicly available in the IMGT database at imgt.org.

[0126] As described above, natural alpha / beta heterodimeric TCRs have an alpha chain and a beta chain. Generally, each chain contains a variable region, a joining region, and a constant region. The beta chain usually also contains a short diversity region between the variable and joining regions, although this diversity region is often considered part of the joining region. Each variable region contains three hypervariable CDRs (complementarity-determining regions) embedded in a framework sequence. CDR3 is well known to be the primary mediator of antigen recognition. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions, which are 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 with a unique framework, CDR1 and CDR2 sequences, and a CDR3 that is partially defined by amino acid sequences conserved across various TCRs but also contains amino acid sequences that vary from TCR to TCR. Similarly, "TRBV2" defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence. There are known to be 54 alpha variable genes (44 of which are functional) and 67 beta variable genes (42 of which are functional) within the alpha and beta loci, respectively.

[0127] Similarly, the joining regions of TCRs are defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions are defined by the IMGT TRAC and TRBC nomenclature. In the IMGT nomenclature, the diversity region of the beta chain is referred to by the abbreviation TRBD. As noted, the linked TRBD / TRBJ regions are often collectively referred to as the joining region.

[0128] The gene pools encoding the TCR alpha and beta chains are located on different chromosomes and contain distinct V, D, J, and C gene segments, which are brought together by rearrangement during T cell development. This results in a very high degree of diversity for T cell alpha and beta chains due to the numerous potential recombination events that occur between the 54 TCR alpha variable genes and the 61 alpha J genes, or between the 67 beta variable genes, the 2 beta D genes, and the 13 beta J genes. The recombination process is not strict, introducing additional diversity within the CDR3 region. Each alpha and beta variable gene can also contain allelic variants, designated TRAVxx*01 and *02 or TRBVx-x*01 and *02 in the IMGT nomenclature, respectively, thus further increasing the amount of diversity. Similarly, some TRBJ sequences have two known variants. (Note that the absence of the "*" qualifier means that only one allele is known for the relevant sequence.) The natural repertoire of human TCRs resulting from recombination and thymic selection is approximately 10 , determined by CDR3 diversity. 6 It is estimated that each beta chain contains a unique beta chain sequence for each species (Arstila et al. (1999) Science 286:958-961), and may even be higher (Robins et al. (2009) Blood 114:4099-4107). Each beta chain is estimated to pair with at least 25 different alpha chains, thus generating further diversity (Arstila et al. (1999) Science 286:958-961).

[0129] Thus, the term "TCR alpha variable domain" refers to the concatenated TRAV and TRAJ regions; the TRAV region alone; or the TRAV and a partial TRAJ region, and the term TCR alpha constant domain refers to the extracellular TRAC region, or a C-terminally truncated or full-length TRAC sequence. Similarly, the term "TCR beta variable domain" refers to the concatenated TRBV and TRBD / TRBJ regions; the TRBV and TRBD regions alone; the TRBV and TRBJ regions alone; or the TRBV and a partial TRBD and / or TRBJ region, and the term TCR beta constant domain refers to the extracellular TRBC region, or a C-terminally truncated or full-length TRBC sequence. These TCR alpha variable domain and TCR beta variable domain nomenclature equally apply to the variable domains of the TCR gamma chain and TCR delta chain, respectively, for gamma / delta TCRs. Those skilled in the art can obtain the gene sequences for TRAV, TRAJ, TRAC, TRBV, TRBJ and TRBC, for example, through the publicly available IMGT database.

[0130] The term "TCR complex" refers to a complex formed by the association of CD3 with a TCR. For example, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0131] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or in promoting desirable physical or mental development and conditions in animals or humans.

[0132] The terms "therapeutically effective amount" and "effective amount" refer to that amount of a substance that produces some desired effect (e.g., a desired local or systemic therapeutic effect) in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any treatment. In some embodiments, a therapeutically effective amount of a substance depends on the substance's therapeutic index, solubility, pharmacokinetics, half-life, etc. Toxicity and therapeutic efficacy of a subject compound can be determined, for example, by the LD 50 and ED 50 The LD can be determined by standard pharmaceutical methods in cell cultures or experimental animals to determine the therapeutic index. In some embodiments, compositions that exhibit large therapeutic indices are used. In some embodiments, the LD 50 The lethal dose (ED) may be measured, for example, for a drug, and may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to when the drug is not administered. 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) may be measured, and may, for example, for a drug, be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to when the drug is not administered. Similarly, the IC 50For example, for an agent, an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more may be measured compared to administration of no agent. In some embodiments, the T cell immune response in the assay may be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In other embodiments, a reduction in viral load of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% may be achieved.

[0133] The term "treating" refers to the therapeutic management or amelioration of a symptom of interest (e.g., a disease or disorder). Treatment can include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically performed to alter the course of a disease (this term is used to refer to any disease, disorder, syndrome, or undesirable condition that warrants or potentially warrants therapy) in a manner beneficial to the subject. The effect of treatment can include reversing, alleviating, reducing the severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of the occurrence or recurrence of a disease or one or more symptoms or signs of a disease. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improving prognosis. A therapeutic agent may be administered to a subject who has a disease or who is at increased risk of developing a disease compared to members of the general population. In some embodiments, a therapeutic agent may be administered to a subject who previously had a disease but no longer shows evidence of the disease. The agent may be administered, for example, to reduce the likelihood of overt disease recurrence. The therapeutic agent may be administered prophylactically, i.e., before any symptoms or signs of the disease develop. "Preventive treatment" refers to providing medical and / or surgical treatment to a subject who has not developed or shows no evidence of the disease, for example, to reduce the likelihood of the disease occurring or to reduce the severity of the disease if it does occur. The subject may have been identified as being at risk for developing the disease (e.g., at higher risk compared to the general population) or as having risk factors that increase the likelihood of developing the disease.

[0134] The term "unresponsiveness" includes the refractoriness of cancer cells to treatment or the unresponsiveness of therapeutic cells (e.g., immune cells) to stimulation (e.g., stimulation via an activating receptor or cytokine). Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigen. As used herein, the term "anergy" or "tolerance" includes unresponsiveness to activating receptor-mediated stimulation. Such unresponsiveness is generally antigen-specific and persists even after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by a lack of cytokine (e.g., IL-2) production. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (a T cell receptor- or CD3-mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, when the cells are re-exposed to the same antigen (even in the presence of costimulatory polypeptides), they are unable to produce cytokines and therefore unable to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells are unable to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by multimers of AP1 sequences that can be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0135] The term "vaccine" refers to a pharmaceutical composition that induces an immune response against an antigen of interest. A vaccine can also confer protective immunity in a subject.

[0136] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR) that is involved in binding the protein to an antigen (e.g., a TCR α or β chain (or the γ and δ chains of a γδ TCR)). The variable domains of the α and β chains of a native TCR (V α and V β ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. α The domains are encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ); β The domains are encoded by three separate DNA segments: the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). α or V β The V domain may be sufficient to confer antigen-binding specificity. Furthermore, a TCR that binds to a particular antigen may be identified by a V domain derived from the TCR that binds the antigen. α or V β The domains were isolated using the complementary V α or V β A library of domains may be screened.

[0137] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, a vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, a vector is capable of autonomous replication and / or expression of nucleic acids to which it is linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of "plasmids," which generally refer to circular double-stranded DNA loops that, in their vector form, are not bound to the chromosome. As used herein, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. However, as will be understood by those of skill in the art, the present invention is intended to include other forms of expression vectors subsequently known in the art which serve equivalent functions.

[0138] There is a known and definite correspondence, as defined by the genetic code (below), between the amino acid sequence of a particular protein and the nucleotide sequence capable of encoding that protein. Similarly, there is a known and definite correspondence, as defined by the genetic code, between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid. [Table 4]

[0139] An important and well-known feature of the genetic code is its redundancy, whereby more than one coding nucleotide triplet can be used for most of the amino acids used to make proteins (as exemplified above). Thus, multiple different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than others). Furthermore, methylation variants of purines or pyrimidines can occasionally be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and its corresponding amino acid.

[0140] In view of the above, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to obtain a polypeptide amino acid sequence using the genetic code to translate DNA or RNA into an amino acid sequence. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence that can encode the polypeptide can be inferred from the genetic code (which, due to its redundancy, will represent multiple nucleic acid sequences for any given amino acid sequence). Thus, any description and / or disclosure herein of a nucleotide sequence that encodes a polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, any description and / or disclosure herein of a polypeptide amino acid sequence should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0141] II. Peptides In certain aspects, provided herein are methods and compositions for the treatment and / or prevention of disorders associated with MAGEA1 expression by inducing an immune response against MAGEA1 or cells expressing MAGEA1, involving the administration of a MAGEA1 immunogenic peptide described herein, a nucleic acid encoding same, and / or a cell expressing same.

[0142] In certain embodiments, a MAGEA1 immunogenic peptide comprises (e.g., consists of) a peptide epitope selected from the peptide sequences listed in Table 1, e.g., Table 1A. The peptide epitopes described herein can be combined with MHC molecules, such as particular HLA molecules with particular HLA alpha chain alleles. For example, the peptides in Table 1A can be combined with MHC molecules, such as HLA-C, as further described in the Examples section. * HLA-C alpha chain, such as that encoded by the 0702 allele * In some embodiments, the MAGEA1 immunogenic peptides may be combined with an MHC molecule, the MHC molecule being associated with 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, and optionally the HLA allele is HLA-A* 0201、SONGS-A * 0202、SONGS-A * 0203、SONGS-A * 0204、SONGS-A * 0205、SONGS-A * 0206、SONGS-A * 0207、SONGS-A * 0210、SONGS-A * 0211、SONGS-A * 0212、SONGS-A * 0213、SONGS-A * 0214、SONGS-A * 0216、SONGS-A * 0217、SONGS-A * 0219、SONGS-A * 0220、SONGS-A * 0222、SONGS-A * 0224、SONGS-A * 0230、SONGS-A * 0242、SONGS-A * 0253、SONGS-A * 0260、SONGS-A * 0274アレル、SONGS-A * 0301、SONGS-A * 0302、SONGS-A * 0305、SONGS-A * 0307、SONGS-A * 0101、SONGS-A * 0102、SONGS-A * 0103、SONGS-A * 0116アレル、SONGS-A * 1101、SONGS-A * 1102、SONGS-A * 1103、SONGS-A * 1104、SONGS-A * 1105、SONGS-A * 1119アレル、SONGS-A * 2402、SONGS-A * 2403、SONGS-A * 2405、SONGS-A * 2407、SONGS-A * 2408、SONGS-A * 2410、SONGS-A* 2414、HLA-A * 2417、HLA-A * 2420、HLA-A * 2422、HLA-A * 2425、HLA-A * 2426、HLA-A * 2458アラル、HLA-B * 0702、HLA-B * 0704、HLA-B * 0705、HLA-B * 0709、HLA-B * 0710、HLA-B * 0715、HLA-B * 0721、HLA-C * 0702、HLA-C * 0701、HLA-C * 0401、HLA-C * 0602、HLA-C * 0304、HLA-C * 0501、HLA-C * 1601、HLA-C * 0202、HLA-C * 0303、HLA-C * 1203、HLA-C * 0802、HLA-C * 0102、HLA-C * 1701、HLA-C * 1502、HLA-C * 1402、HLA-C * 1202、HLA-C * 0704、HLA-C * 0801、HLA-C * 0302、HLA-C * 1801、HLA-C * 1505、HLA-C * 1602、HLA-C * 0804、HLA-C * 0305、&HLA-C *1403 allele. In some embodiments, the MAGEA1 immunogenic peptide is derived from a human MAGEA1 protein and / or a MAGEA1 protein shown in Table 3. In some embodiments, one or more MAGEA1 immunogenic peptides are administered alone or in combination with an adjuvant.

[0143] In certain aspects, provided herein are compositions comprising one or more MAGEA1 immunogenic peptides described herein and an adjuvant.

[0144] [Table 1]

[0145] In some embodiments, provided herein are 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 also 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.

[0146] In some embodiments, the MAGEA1 polypeptide comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 1 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, In some embodiments, the MAGEA1 polypeptide comprises, consists essentially of, or consists of 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 373, or more contiguous amino acids, or any range therebetween (e.g., 7-25, 8-22, 9-22, etc.), inclusive of 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, e.g., Table 1A.

[0147] As is well known to those skilled in the art, polypeptides that share significant sequence similarity can generate the same or a very similar immune response in a host animal. Thus, in some embodiments, derivatives, equivalents, variants, fragments, or variants of the MAGEA1 immunogenic peptides or fragments thereof described herein may also be suitable for the methods and compositions provided herein.

[0148] In some embodiments, variants or derivatives of MAGEA1 immunogenic polypeptides are provided herein. A modified polypeptide may have an amino acid sequence altered, for example, by conservative substitution, yet still elicit an immune response reactive to the unmodified protein antigen and is considered a functional equivalent. As used herein, the term "conservative substitution" refers to the replacement of an amino acid residue with another, biologically similar residue. It is well known in the art that amino acids within the same conservative group can typically be substituted for each other without substantially affecting protein function. According to certain embodiments, a derivative, equivalent, variant, or mutant of the ligand-binding domain of a MAGEA1 immunogenic peptide is a polypeptide that is at least 85% homologous to the sequence of a MAGEA1 immunogenic peptide or fragment thereof described herein. In some embodiments, the homology is at least 90%, at least 95%, at least 98%, or greater.

[0149] Immunogenic peptides encompassed by the present invention may include peptide epitopes derived from the MAGEA1 protein, such as those listed in Table 1, e.g., Table A. In some embodiments, the immunogenic peptides are 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide amino acid sequence is modified, which may include conservative or non-conservative mutations. The peptide may include up to 1, 2, 3, 4, or more mutations. In some embodiments, the peptide may include at least 1, 2, 3, 4, or more mutations.

[0150] In some embodiments, peptides may be chemically modified. For example, peptides may be mutated to alter peptide properties, such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is one example of methylation that may occur on peptides of the present disclosure. In some embodiments, peptides may be modified by methylation of free amines, for example, reductive methylation with formaldehyde and sodium cyanoborohydride.

[0151] Chemical modifications may include polymers, polyethers, polyethylene glycol, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains such as palmitate or myristoleate, or albumin. Chemical modifications of peptides with Fc regions may be fusion Fc-peptides. Polyamino acids may include, for example, polyamino acid sequences with repeating single amino acids (e.g., polyglycine) and polyamino acid sequences with mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the above. In some embodiments, peptides encompassed by the present disclosure may be modified to increase the stability and / or half-life of the peptide. In some embodiments, the half-life of peptides encompassed by the present disclosure can be extended using hydrophobic moieties, for example, at the N-terminus, C-terminus, or internal amino acids. In other embodiments, peptides may include post-translational modifications (e.g., methylation and / or amidation), which can affect, for example, serum half-life. In some embodiments, a simple carbon chain can be conjugated to the fusion protein or peptide (e.g., by myristoylation and / or palmitylation). In some embodiments, the simple carbon chain can allow the fusion protein or peptide to be easily separated from unconjugated material. For example, methods that can be used to separate the fusion protein or peptide from unconjugated material include, but are not limited to, solvent extraction and reverse-phase chromatography. The lipophilic moiety can extend half-life by reversible binding to serum albumin. The conjugated moiety can be a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative, such as cholestene, cholestane, cholestadiene, and oxysterols. In some embodiments, the peptide can be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptide can be coupled (e.g., conjugated) to a half-life modifier.Examples of half-life modifying agents 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, molecules that bind to Fc regions, fatty acids, palmitic acid, or albumin. In some embodiments, a spacer or linker, e.g., one, two, three, four, or more amino acid residues that act as a spacer or linker, may be coupled to the peptide to facilitate conjugation or fusion to another molecule and to facilitate cleavage of the peptide from such a conjugated or fused molecule. In some embodiments, a fusion protein or peptide may be conjugated to other moieties that, for example, can modify or alter the properties of the peptide.

[0152] In some embodiments, the peptide may be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or label. The affinity tag may be selected from the group consisting of glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, Halo tag, HA tag, Flag® tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of an inflammatory agent, an anti-inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody, e.g., a single-chain Fv.

[0153] Peptides may be conjugated to agents used in imaging, research, therapeutics, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiation therapy. In some embodiments, peptides may be conjugated or fused to detectable agents, such as fluorophores, near-infrared dyes, contrast agents, nanoparticles, metal-containing nanoparticles, metal chelates, X-ray contrast agents, PET agents, metals, radioisotopes, dyes, radionuclide chelators, or other suitable materials that can be used for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties may be linked to the peptide. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not easily quenched by biological tissues and fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation in the wavelength range of 650 nm to 4000 nm, and such radiation is used to detect such agents. Non-limiting examples of fluorescent dyes that can be used as conjugating molecules include DyLight®-680, DyLight®-750, VivoTag®-750, DyLight®-800, IRDye®-800, VivoTag®-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, near-infrared dyes are often cyanine dyes (e.g., Cy7, Cy5.5,and Cy5). Further non-limiting examples of fluorescent dyes for use as conjugating molecules in the present disclosure include acrazine orange or yellow, Alexa Fluors® (e.g., Alexa Fluor® 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dye and any derivatives thereof, auramine-rhodamine stain and any derivatives thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, DyLight Fluors and any derivatives thereof, epicocconone, ethidium bromide, FlAsH-EDT2, Fluo dyes and any derivatives thereof, FluoProbe and any derivatives thereof, fluorescein and any derivatives thereof, Fura and any derivatives thereof, GelGreen and any derivatives thereof, GelRed and any derivatives thereof, fluorescent proteins and any derivatives thereof, m isoform proteins and any derivatives thereof, such as mCherry, hetamethine dyes dye) and any derivatives thereof, Hoechst stain, iminocoumarin, Indian yellow, indo-1 and any derivatives thereof, laurdan, lucifer yellow and any derivatives thereof, luciferin and any derivatives thereof, luciferase and any derivatives thereof, mercocyanin and any derivatives thereof, Nile dye and any derivatives thereof, perylene, phloxine, phycodye and any derivatives thereof, propium iodide, pyranine, rhodamine and any derivatives thereof, ribogreen, RoGFP, rubrene, stilbene and any derivatives thereof, sulforhodamine and any derivatives thereof,Other suitable fluorescent dyes include, but are not limited to, SYBR™ and any derivatives thereof, synaptofluorin, tetraphenylbutadiene, tetrasodium tris, Texas Red, titanium yellow, TSQ, umbelliferone, violanthrone, yellow fluorescent protein, and YOYO-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 5G, rhodamine 6G, rhodamine 5 ... coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA)), Oregon Green® dyes (e.g., Oregon Green® 488, Oregon Green® 500, Oregon Green® 514), Texas Red, Texas Red-X, Spectrum Red, Spectrum Green, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5), ALEXA FLUOR® dyes (e.g., ALEXA FLUOR® 350, ALEXA FLUOR® 488, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 633, ALEXA FLUOR® 660, ALEXA FLUOR® 680, etc.), BODIPY® dyes (e.g., BODIPY® FL, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, BODIPY® 530 / 550,Suitable radioisotopes include BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, etc.), IRD dyes (e.g., IRD40, IRD700, IRD800, etc.). Additional suitable detectable agents are described in PCT / US14 / 56177. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.

[0154] The peptide may be conjugated to a radiosensitizer or photosensitizer. Radiosensitizers include, but are not limited to, ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleobase derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to, fluorescent molecules or beads that generate heat when irradiated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, etc. dyes), chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, Nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rose bengals), dimeric and oligomeric porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this approach allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent (e.g., a drug) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the peptide is fused to, or covalently or non-covalently linked to, an agent, e.g., directly or via a linker.

[0155] In some embodiments, binding proteins may be chemically modified. For example, binding proteins may be mutated to alter peptide properties, such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is one example of methylation that may occur on binding proteins encompassed by the present invention. In some embodiments, binding proteins may be modified by methylation of free amines, for example, reductive methylation with formaldehyde and sodium cyanoborohydride.

[0156] Chemical modifications can include polymers, polyethers, polyethylene glycol, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains such as palmitate or myristoleate, or albumin. Chemical modifications of binding proteins with Fc regions can be fusion Fc-peptides. Polyamino acids can include, for example, polyamino acid sequences with repeating single amino acids (e.g., polyglycine) and polyamino acid sequences with mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the above.

[0157] In some embodiments, binding proteins encompassed by the present invention may be modified. In some embodiments, the modifications produce functional variants that have substantial or significant sequence identity with the parent binding protein and that maintain one or more biophysical and / or biological activities of the parent binding protein (e.g., maintain pMHC-binding specificity). In some embodiments, the mutations are conservative amino acid substitutions.

[0158] In some embodiments, binding proteins encompassed by the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. β-hydroxyphenylalanine, phenylglycine, 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, oc-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and oc-tert-butylglycine.

[0159] The binding proteins encompassed by the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted to acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0160] In some embodiments, the half-life of peptides encompassed by the present invention may be extended using hydrophobic moieties, e.g., at the N-terminus, C-terminus, or internal amino acid. In other embodiments, the binding protein may contain post-translational modifications (e.g., methylation and / or amidation), which may affect, for example, serum half-life. In some embodiments, a simple carbon chain (e.g., by myristoylation and / or palmitylation) may be conjugated to the binding protein. In some embodiments, the simple carbon chain may allow the binding protein to be easily separated from unconjugated materials. For example, methods that may be used to separate the binding protein from unconjugated materials include, but are not limited to, solvent extraction and reverse-phase chromatography. Lipophilic moieties can extend half-life by reversible binding to serum albumin. The conjugated moiety may be a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative, such as cholestene, cholestane, cholestadiene, and oxysterol. In some embodiments, the binding protein may be conjugated to myristic acid (tetradecanoic acid) or its derivatives. In other embodiments, the binding protein may be coupled (e.g., conjugated) to a half-life modifying agent. Examples of half-life modifying agents include, but are not limited to, polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers containing glycine, glutamic acid, and serine, Fc regions, fatty acids, palmitic acid, or molecules that bind to albumin.In some embodiments, a spacer or linker, e.g., 1, 2, 3, 4, or more amino acid residues that act as a spacer or linker, may be coupled to the binding protein to facilitate conjugation or fusion to another molecule and to facilitate cleavage of the peptide from such a conjugated or fused molecule. In some embodiments, the binding protein may be conjugated to other moieties that, for example, can modify or alter the properties of the binding protein.

[0161] 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 performed by known synthetic methods, for example, using fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. Protein fragments can be joined together enzymatically or synthetically.

[0162] In aspects encompassed by the invention, provided herein are methods of producing the proteins described herein, the methods comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of the binding protein; and (ii) recovering the expressed binding protein.

[0163] Useful methods for isolating and purifying recombinantly produced binding proteins can include, for example, 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 suitable purification matrix or a series of suitable matrices, such as an affinity matrix or ion exchange resin. One or more reverse-phase HPLC steps may be utilized to further purify the recombinant polypeptide. These purification methods can also be employed when isolating immunogens from their natural environment. Methods for large-scale production of one or more of the binding proteins described herein include batch cell cultures, which are monitored and controlled to maintain appropriate culture conditions. Purification of the binding proteins can be carried out according to methods described herein and known in the art.

[0164] In some embodiments, provided herein are nucleic acids encoding a MAGEA1 immunogenic peptide or fragment thereof described herein, e.g., a DNA molecule encoding a MAGEA1 immunogenic peptide. In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a MAGEA1 immunogenic peptide or fragment thereof described herein. In some embodiments, the nucleic acid comprises regulatory elements required for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, an enhancer may be included. These elements may be operably linked to a sequence encoding a MAGEA1 immunogenic polypeptide or fragment thereof. Representative vectors, promoters, regulatory elements, etc. useful for expressing proteins, e.g., peptides, are further described below.

[0165] III. MHC-Peptide Complexes In certain aspects, provided herein are compositions comprising a MAGEA1 immunogenic peptide described herein and an MHC molecule. In some embodiments, the MAGEA1 immunogenic peptide forms a stable complex with the MHC molecule.

[0166] The MHC protein may be conjugated to an agent, such as a detection moiety, radiosensitizer, photosensitizer, etc., and / or may be chemically modified as described above for peptides.

[0167] The MHC proteins provided and used in the compositions and methods encompassed by the present disclosure can be any suitable MHC molecule known in the art. Generally, they have the formula (α-β-P) n where n is at least 2, e.g., 2-10, e.g., 4. α is the α chain of a class I or class II MHC protein. β is the β chain, defined herein as a class II MHC protein or β2-microglobulin of an MHC class I protein. P is a peptide antigen.

[0168] In some embodiments, the MHC protein is an MHC class I complex, such as an HLA I complex.

[0169] MHC proteins can be from any mammalian or avian species, such as primates, particularly humans; rodents, including mice, rats, and hamsters; rabbits; horses; cows; dogs; cats; etc. For example, MHC proteins can be derived from human HLA proteins or mouse H-2 proteins. HLA proteins include the class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, as well as the class I proteins HLA-A, HLA-B, HLA-C, and β2-microglobulin. H-2 proteins include the class I subunits H-2K, H-2D, and H-2L, as well as the class II subunits I-Aα, I-Aβ, I-Eα, and I-Eβ, as well as β2-microglobulin. Sequences of several representative MHC proteins can be found in Kabat et al., Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pp. 724-815. MHC protein subunits suitable for use in the present invention are soluble forms of normally membrane-bound proteins, prepared as known in the art, for example, by deletion of transmembrane and cytoplasmic domains.

[0170] For class I proteins, soluble forms may contain the α1, α2, and α3 domains. Soluble class II subunits may contain the α1 and α2 domains for the α subunits and the β1 and β2 domains for the β subunits.

[0171] The α and β subunits may be produced separately and associated in vitro to form a stable heteroduplex, or both subunits may be expressed in a single cell. Methods for producing MHC subunits are known in the art.

[0172] In certain embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1 and an MHC. In some embodiments, the MHC molecule is 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, and optionally the HLA allele is HLA-A * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0204, HLA-A * 0205, HLA-A * 0206, HLA-A * 0207, HLA-A * 0210, HLA-A * 0211, HLA-A * 0212, HLA-A * 0213, HLA-A * 0214, HLA-A * 0216, HLA-A * 0217, HLA-A * 0219, HLA-A * 0220, HLA-A * 0222, HLA-A * 0224, HLA-A * 0230, HLA-A *0242、SONGS-A * 0253、SONGS-A * 0260、SONGS-A * 0274アレル、SONGS-A * 0301、SONGS-A * 0302、SONGS-A * 0305、SONGS-A * 0307、SONGS-A * 0101、SONGS-A * 0102、SONGS-A * 0103、SONGS-A * 0116アレル、SONGS-A * 1101、SONGS-A * 1102、SONGS-A * 1103、SONGS-A * 1104、SONGS-A * 1105、SONGS-A * 1119アレル、SONGS-A * 2402、SONGS-A * 2403、SONGS-A * 2405、SONGS-A * 2407、SONGS-A * 2408、SONGS-A * 2410、SONGS-A * 2414、SONGS-A * 2417、SONGS-A * 2420、SONGS-A * 2422、SONGS-A * 2425、SONGS-A * 2426、SONGS-A * 2458. HLA-B * 0702、SONG-B * 0704、SONG-B * 0705、SONG-B * 0709、SONG-B * 0710、SONG-B * 0715、SONG-B * 0721、SONGS-C * 0702、SONGS-C * 0701、SONGS-C * 0401、SONGS-C * 0602、SONGS-C * 0304、SONGS-C *0501, HLA-C * 1601, HLA-C * 0202, HLA-C * 0303, HLA-C * 1203, HLA-C * 0802, HLA-C * 0102, HLA-C * 1701, HLA-C * 1502, HLA-C * 1402, HLA-C * 1202, HLA-C * 0704, HLA-C * 0801, HLA-C * 0302, HLA-C * 1801, HLA-C * 1505, HLA-C * 1602, HLA-C * 0804, HLA-C * 0305, and HLA-C * In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1A and an allele of HLA-C1403. * MHC with 07 serotype, e.g., HLA-C * 0702, HLA-C * 0701 and / or HLA-C * and the MHC encoded by the 0704 allele.

[0173] To prepare MHC-peptide complexes, subunits may be combined with antigenic peptides and folded in vitro to form stable heterodimeric complexes with intrachain disulfide-bonded domains. The peptide may be included in the initial folding reaction or added to the empty heterodimer in a later step. In the compositions and methods encompassed by the present invention, this is a MAGEA1 immunogenic peptide or a fragment thereof. Conditions allowing folding and association of subunits and peptides are known in the art. As an example, approximately equimolar amounts of solubilized α and β subunits may be mixed in a solution of urea. Refolding is initiated by dilution or dialysis with a buffer solution that does not contain urea. The peptide may be introduced into the empty class II heterodimer at a pH of about 5-5.5 for about 1-3 days, followed by neutralization, concentration, and buffer exchange. However, specific folding conditions are not essential for practicing the present invention.

[0174] The monomeric complex (α-β-P) (herein, monomer) may be multimerized, e.g., to form an MHC tetramer. The resulting multimer is stable over long periods of time. Preferably, the multimer may be formed by binding the monomer to a multivalent entity via specific binding sites on the α or β subunit, as known in the art (e.g., as described in U.S. Pat. No. 5,635,363). Alternatively, the MHC protein, in either monomeric or multimeric form, may be conjugated to beads or any other support.

[0175] The multimeric complexes may be labeled for direct detection when used in immunostaining or other art-known methods, or may be used in conjunction with a secondary labeled immunoreagent that specifically and / or selectively binds to the complex (e.g., binds to MHC protein subunits) as known to those skilled in the art. For example, the detectable label may be a fluorophore such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin (PE), allophycocyanin (APC), Brilliant Violet™ 421, Brilliant UV™ 395, Brilliant Violet™ 480, Brilliant Violet™ 421 (BV421), Brilliant Blue™ 515, APC-R700, or APC-Fire 750. In some embodiments, the multimeric complexes are labeled with a moiety that can specifically and / or selectively bind to another moiety. For example, the label may be biotin, streptavidin, an oligonucleotide, or a ligand. Other labels of interest may include fluorophores, dyes, enzymes, chemiluminescent agents, particles, radioisotopes, or other directly or indirectly detectable agents.

[0176] In some embodiments, cells presenting immunogenic peptides in the context of MHC molecules on their surface are generated by transfecting or transducing the cells with a vector (e.g., a viral vector) that contains a nucleic acid encoding a recombinant or heterologous antigen within the cell. In some embodiments, the vector is introduced into the cell under conditions such that one or more peptide antigens, e.g., optionally one or more peptide antigens of an expressed heterologous protein, are expressed, processed, and presented on the surface of the cell in the context of a major histocompatibility complex (MHC) molecule.

[0177] Generally, the cell contacted with the vector is a cell that expresses MHC, i.e., an MHC-expressing cell. The cell can normally express MHC on the cell surface, can be induced to express and / or upregulate MHC expression on the cell surface, or can be engineered to express MHC molecules on the cell surface. In some embodiments, the MHC contains a polymorphic peptide-binding site, or binding groove, that can optionally complex with a polypeptide peptide antigen, e.g., a peptide antigen that has been processed by the cellular machinery. In some cases, the MHC molecule may be presented or expressed on the cell surface, e.g., as a complex with a peptide, i.e., as an MHC-peptide complex, for presentation of the antigen in a conformation that can be recognized by a TCR on a T cell or by other peptide-binding molecules.

[0178] 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, for example, an endothelial cell line or a primary endothelial cell. In some embodiments, the cell is a fibroblast, for example, a fibroblast cell line or a primary fibroblast.

[0179] In some embodiments, the cells are artificial antigen-presenting cells (aAPCs). Typically, aAPCs include characteristics of natural APCs, such as expression of MHC molecules, stimulatory and costimulatory molecule(s), Fc receptors, adhesion molecule(s), and / or the ability to produce or secrete cytokines (e.g., IL-2). Typically, aAPCs are cell lines that lack expression of one or more of the above, including MHC molecules, low affinity Fc receptors (CD32), high affinity Fc receptors (CD64), costimulatory signals (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, 3 / TR6, or B7-H3 ligands; or CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor, or CD83 ligands). aAPCs are generated by introducing (e.g., by transfection or transduction) one or more missing elements of one or more of the following: an antibody that specifically binds to MHC molecules; a cell adhesion molecule (e.g., ICAM-1, or LFA-3); and / or a cytokine (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon alpha (IFN alpha), interferon beta (IFN beta), interferon gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), tumor necrosis factor beta (TNF beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (GCSF)). In some cases, aAPCs do not normally express MHC molecules but can be engineered to express MHC molecules, or in some cases, can be induced or induced to express MHC molecules, such as by stimulation with cytokines. In some cases, the aAPCs are loaded with a stimulatory ligand, such as an anti-CD3 antibody, an anti-CD28 antibody, or an anti-CD2 antibody.The exemplary cell line that can be used as a scaffold for producing aAPC is the K562 cell line, or a fibroblast cell line.Various aAPCs are known in the art, see, for example, U.S. Patent No. 8,722,400, published application US2014 / 0212446, Butler and Hirano (2014) Immunol Rev.257:10.1111 / imr.12129, Suhoshki et al. (2007) Mol.Ther.15:981-988).

[0180] Determining or identifying the specific MHC or allele expressed by a cell is well within the skill of one of ordinary skill in the art. In some embodiments, expression of a specific MHC molecule may be assessed or confirmed before contacting the cell with the vector, for example, by using an antibody specific for the specific MHC molecule. Antibodies to MHC molecules are known in the art, such as any of the antibodies described below.

[0181] In some embodiments, cells can be selected to express MHC alleles for the desired MHC restriction. In some embodiments, MHC typing of cells, e.g., cell lines, is well known in the art. In some embodiments, the MHC typing of cells, e.g., primary cells obtained from a subject, can be determined using procedures well known in the art, for example, by performing tissue typing using a molecular haplotype assay (BioTest ABC SSPtray, BioTest Diagnostics Corp., Denville, NJ; SeCore Kits, Life Technologies, Grand Island, NY). In some cases, it is well within the skill of one of ordinary skill in the art to perform standard typing of cells to determine their HLA genotype, for example, by using sequence-based typing (SBT) (Adams et al. (2004) J. Transl. Med., 2:30; Smith (2012) Methods Mol Biol., 882:67-86). In some cases, the HLA typing of cells, e.g., fibroblasts, is known. For example, the human fetal lung fibroblast cell line MRC-5 expresses HLA-A * 0201, A29, B13, B44 Cw7(C * 0702), human foreskin fibroblast cell line Hs68 is HLA-A1, A29, B8, B44, Cw7, Cw16, and WI-38 cell line is A * 6801, B * 0801 (Solache et al. (1999) J Immunol, 163:5512-5518; Ameres et al. (2013) PloS Pathog. 9:e1003383). The human transfectant fibroblast cell line M1DR1 / Ii / DM expresses HLA-DR and HLA-DM (Karakikes et al. (2012) FASEB J., 26:4886-96).

[0182] In some embodiments, the cells that are contacted or introduced with the vector are engineered or transfected to express MHC molecules. In some embodiments, the cell line can be prepared by genetically modifying a parent cell line. In some embodiments, the cells are engineered to express specific MHC molecules that are normally deficient in those molecules. In some embodiments, the cells are genetically engineered using recombinant DNA technology.

[0183] In some embodiments, the stable MHC-peptide complexes described herein are used to detect T cells that bind to the stable MHC-peptide complexes. In some embodiments, the stable MHC-peptide complexes described herein are used to track and assess T cell responses in a subject, for example, by detecting the amount and / or percentage of T cells (e.g., CD8+ T cells) that specifically and / or selectively bind to fluorescently labeled MHC-peptide complexes. Methods for generating, labeling, and using MHC-peptide complexes (e.g., MHC-peptide tetramers) to detect T cells specific for MHC-peptide complexes are well known in the art. Further description can be found, for example, in U.S. Pat. No. 7,776,562, U.S. Pat. No. 8,268,964, and U.S. Patent Publication No. 2019 / 0085048.

[0184] IV. Immunogenic composition In some aspects, provided herein are pharmaceutical compositions (e.g., vaccine compositions) comprising a MAGEA1 immunogenic peptide and / or a nucleic acid encoding a MAGEA1 immunogenic peptide, and an adjuvant. In some aspects, provided herein are pharmaceutical compositions (e.g., vaccine compositions) comprising 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 composition further comprises a pharmaceutically acceptable carrier.

[0185] The pharmaceutical compositions disclosed herein may be specially formulated for administration in solid or liquid form, e.g., in a form suitable for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those intended for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions, or sustained-release formulations.

[0186] Methods of preparing these formulations or compositions include the step of bringing into association the MAGEA1 immunogenic peptides and / or nucleic acids described herein with the adjuvant, carrier, and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the agents described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0187] Pharmaceutical compositions suitable for parenteral administration include the MAGEA1 immunogenic peptides and / or nucleic acids described herein in combination with 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, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0188] Examples of suitable aqueous and non-aqueous carriers that can be used in pharmaceutical compositions include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants.

[0189] Regardless of the route of administration selected, the agents provided herein, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions disclosed herein, are formulated into pharmaceutically acceptable dosage forms by routine methods known to those skilled in the art.

[0190] In some embodiments, the described pharmaceutical compositions, when administered to a subject, are capable of eliciting an immune response against cells infected with MAGEA1. Such pharmaceutical compositions may be useful as vaccine compositions for the prophylactic and / or therapeutic treatment of disorders characterized by MAGEA1 expression.

[0191] In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the adjuvant employed results in improved immunogenicity of the pharmaceutical composition. Such additional immune response-stimulating compounds or adjuvants can (i) be mixed with the pharmaceutical composition of the present invention after reconstitution of the peptide and optional emulsifier with an oil-based adjuvant as defined above, (ii) be part of the reconstituted composition of the present invention as defined above, (iii) be physically linked to the peptide(s) to be reconstituted, or (iv) be administered separately to the subject, mammal, or human to be treated. The adjuvant can be one that provides delayed release of the antigen (e.g., the adjuvant can be a liposome), or it can be an adjuvant that is immunogenic in its own right and thereby functions 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 substance that promotes antigen uptake, recruits immune system cells to the site of administration, or promotes immunostimulation of responding lymphoid cells. Adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, bacto-adjuvant, synthetic polymers such as polyamino acids and amino acid copolymers, saponin, paraffin oil, and muramyl dipeptide. In some embodiments, the adjuvant is adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptides, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, Quil A, trehalose dimycolate, or zymosan.

[0192] In some embodiments, the adjuvant is an immunomodulatory molecule. For example, the immunomodulatory molecule can be a recombinant protein cytokine, chemokine, or immunostimulatory agent, or a nucleic acid encoding a cytokine, chemokine, or immunostimulatory agent, designed to enhance an immunological response.

[0193] Examples of immunomodulatory cytokines include interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, and IL-20), tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1.alpha, MIP-1β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as functional fragments of any of the above.

[0194] In some embodiments, immunomodulatory chemokines that bind to chemokine receptors, i.e., CXC, CC, C, or CX3C chemokine receptors, may also be included in the compositions provided herein. Examples of chemokines include, but are not limited to, Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, McP-1, McP-2, McP-3, McP-4, McP-5, Eotaxin, Tarc, Elc, I309, IL-8, Gcp-2 Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), as well as functional fragments of any of the above.

[0195] In some embodiments, the composition comprises a nucleic acid encoding a MAGEA1 immunogenic polypeptide described herein, e.g., a DNA molecule encoding a MAGEA1 immunogenic peptide. In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a MAGEA1 immunogenic peptide.

[0196] When taken up by a cell (e.g., a host cell, an antigen-presenting cell (APC), e.g., a dendritic cell, a macrophage, etc.), the DNA molecule may be present in the cell as an extrachromosomal molecule and / or may be integrated into a chromosome. DNA may be introduced into the cell in the form of a plasmid, which may remain separate genetic material. Alternatively, linear DNA may be introduced into the cell, which may be integrated into a chromosome. Optionally, when DNA is introduced into a cell, a reagent may be added that promotes integration of the DNA into a chromosome.

[0197] V. Binding Proteins In some embodiments, binding moieties that bind to the peptides described herein and / or stable MHC-peptide complexes described herein are provided, e.g., specifically and / or selectively bind to the peptides and / or stable MHC-peptide complexes, e.g., at about 10 -4 M or less (e.g., about 10 -4 , 10 -5 , 10 -6 , 10 -7 , about 10 -8 , about 10 -9 , about 10 10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 etc.) d Binding proteins, such as T cell receptors (TCRs), antibodies and the like, that bind at

[0198] In aspects encompassed by the invention, provided herein are binding proteins that bind (e.g., specifically and / or selectively) to peptide-MHC (pMHC) complexes that include a MAGEA1 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the binding protein binds to about 5×10 -4 M or less, approximately 1×10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, approximately 1×10 -12 K less than or equal to M, or any range therebetween, including the endpoints, e.g., about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, etc. d In some embodiments, the MHC molecule is capable of binding (e.g., specifically and / or selectively) to a MAGEA1 peptide-MHC (pMHC) complex. * 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、SONGS-C * 12、SONGS-C * 14、SONGS-C * 15、SONGS-C * 16. HLA-C * 17 and HLA-C * 18. This is a very nice song by MHC On the other side of the spectrum is HLA-A * 0201、SONGS-A * 0202、SONGS-A * 0203、SONGS-A * 0204、SONGS-A * 0205、SONGS-A * 0206、SONGS-A * 0207、SONGS-A * 0210、SONGS-A * 0211、SONGS-A * 0212、SONGS-A * 0213、SONGS-A * 0214、SONGS-A * 0216、SONGS-A * 0217、SONGS-A * 0219、SONGS-A * 0220、SONGS-A * 0222、SONGS-A * 0224、SONGS-A * 0230、SONGS-A * 0242、SONGS-A * 0253、SONGS-A * 0260、SONGS-A * 0274アレル、SONGS-A * 0301、SONGS-A * 0302、SONGS-A * 0305、SONGS-A * 0307、SONGS-A * 0101、SONGS-A * 0102、SONGS-A * 0103、SONGS-A * 0116アレル、SONGS-A * 1101、SONGS-A * 1102、SONGS-A * 1103、SONGS-A * 1104、SONGS-A* 1105、HLA-A * 1119アラル、HLA-A * 2402、HLA-A * 2403、HLA-A * 2405、HLA-A * 2407、HLA-A * 2408、HLA-A * 2410、HLA-A * 2414、HLA-A * 2417、HLA-A * 2420、HLA-A * 2422、HLA-A * 2425、HLA-A * 2426、HLA-A * 2458アラル、HLA-B * 0702、HLA-B * 0704、HLA-B * 0705、HLA-B * 0709、HLA-B * 0710、HLA-B * 0715、HLA-B * 0721、HLA-C * 0702、HLA-C * 0701、HLA-C * 0401、HLA-C * 0602、HLA-C * 0304、HLA-C * 0501、HLA-C * 1601、HLA-C * 0202、HLA-C * 0303、HLA-C * 1203、HLA-C * 0802、HLA-C * 0102、HLA-C * 1701、HLA-C * 1502、HLA-C * 1402、HLA-C * 1202、HLA-C * 0704、HLA-C * 0801、HLA-C * 0302、HLA-C * 1801、HLA-C * 1505、HLA-C* 1602, HLA-C * 0804, HLA-C * 0305, and HLA-C * In some embodiments, the HLA serotype is selected from the group consisting of HLA-C 1403 alleles. * 07, and / or the HLA allele is HLA-C * 0702, HLA-C * 0701 and HLA-C * In certain embodiments, the HLA allele is selected from the group consisting of HLA-C 0704 alleles. * 0702. In some embodiments, the binding proteins provided herein are genetically engineered, isolated and / or purified.

[0199] In some embodiments, the binding protein binds to MAGEA1 peptide-MHC (pMHC) with higher binding affinity compared to a known T cell receptor (e.g., a TCR from van Kunert et al. (2016) J. Immunol. 197:2541-2552, or others described herein). For example, the binding protein binds to MAGEA1 peptide-MHC with at least 1.2-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold It may have a binding affinity for MAGEA1 peptide-MHC (pMHC) that is 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 5000-fold, 10000-fold, 50000-fold, 100000-fold, 500000-fold, 1000000-fold or more, or any range therebetween inclusive of the endpoints, for example, 1.2-fold to 2-fold higher.

[0200] In some embodiments, compared to known T cell receptors, the binding protein induces stronger T cell proliferation, cytokine release, and / or cytotoxic killing when contacted with target cells that express MAGEA1 at or below a certain level (e.g., see the Examples section for representative cell lines expressing MAGEA1 at various levels). For example, in some embodiments of any aspect described herein, MAGEA1 levels are expressed at or below a certain level, such as transcripts per million. million), for example, about 1,000 transcripts per million transcripts (about 1,000 TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM 1,000 TPM or less, or any range therebetween inclusive of the endpoints, e.g., from about 1,000 TPM or less to about 73 TPM or less). As further described herein, TPM is measured according to well-known techniques, e.g., RNA-Seq, and gene expression TPM data is well known in the art for various cell lines, tissue types, etc. (see, e.g., the Broad Institute Cancer Cell Line Encyclopedia (CCLE) at portals.broadinstitute.org on the World Wide Web).In some embodiments, the binding protein increases T cell proliferation, cytokine release and / or cytotoxic killing by at least 1.2 fold, 1.5 fold, 1.8 fold, 2.0 fold, 2.2 fold, 2.5 fold, 2.8 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold when contacted with target cells having heterozygous expression of MAGEA1 compared to a known T cell receptor. , 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x or more, or any range therebetween inclusive of the endpoints, e.g., 1.2x to 2x increase.

[0201] In some embodiments, MAGEA1 expression is detected using RNA sequencing (RNA-seq). RNA-seq generally involves the following steps: obtaining a sample containing genetic material, isolating total RNA from the obtained sample, preparing an amplified cDNA library from the total RNA, sequencing the amplified cDNA library, and analyzing and profiling the amplified cDNA to assess the expression levels of various transcripts. The sample may be a population of cells, a tissue sample, a biopsy sample, a cell culture, or a single cell. Total RNA can be isolated from biological samples using any method known in the art. In certain embodiments, total RNA is extracted from plasma. Extraction of plasma RNA is described in Enders et al., "The Concentration of Circulating Corticotropin-Releasing Homer mRNA in Material Plasma Is Inclined in Preclampsia," Clin. 1999, 10:101–104. 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 into an RNeasy mini-column (Qiagen) and processed according to the manufacturer's recommendations.

[0202] In some embodiments, the RNA-seq method described herein involves preparing amplified cDNA from total RNA. For example, cDNA is prepared and the isolated RNA sample is randomly amplified without dilution, or a mixture containing genetic material in the isolated RNA is dispersed into individual reaction samples. In certain embodiments, amplification is initiated randomly from the 3' end of the transcriptome in the sample to amplify both mRNA and non-polyadenylated transcripts. In this manner, the double-stranded cDNA amplification product is optimized to generate a sequencing library for next-generation sequencing platforms. Kits suitable for amplifying cDNA using the methods encompassed by the present invention include, for example, the Ovation® RNA-Seq System.

[0203] In some embodiments, the RNA-seq described herein comprises sequencing the amplified cDNA.Any known sequencing method can be used to sequence the amplified cDNA mixture, for example, including single molecule sequencing.In certain embodiments, the amplified cDNA is sequenced by whole transcriptome shotgun sequencing.Whole transcriptome shotgun sequencing can be carried out using various next-generation sequencing platforms, for example, Illumina Genome Analyzer platform, ABI SOLiD™ sequencing platform or Life Science's 454 sequencing platform.

[0204] In some embodiments, the RNA-seq method described herein further comprises digitally counting and analyzing the cDNA. The number of amplified sequences for each transcript in the amplified sample can be quantified by sequence reads (one read per amplified strand). In some embodiments, transcripts per million (TPM) are used to quantify the expression level of a particular transcript. TPM can be calculated as shown in Wagner et al. (2012) Theory in Biosciences 131:281-285, the entire contents of which are incorporated herein by reference.

[0205] In certain embodiments, the binding proteins recognize MAGEA1 immunogenic peptides in complex with MHC molecules, such as specific HLA molecules with specific HLA alpha chain alleles. For example, the binding proteins listed in Table 2A recognize MAGEA1 immunogenic peptides in complex with MHC molecules, such as specific HLA molecules with specific HLA alpha chain alleles, as further described in the Examples section. * MHC with 07 serotype, e.g., HLA-B * 0702, HLA-C * 0701 and / or HLA-C * The binding protein has been identified as a binder of a MAGEA1 immunogenic peptide in association with an MHC molecule encoded by the 0704 allele. In some embodiments, the binding protein recognizes a complex of a MAGEA1 immunogenic peptide and an MHC molecule, and the MHC molecule is 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、SONGS-C * 14、SONGS-C * 15、SONGS-C * 16. HLA-C * 17 and HLA-C * 18. This is a very nice song by MHC On the other side of the spectrum is HLA-A * 0201、SONGS-A * 0202、SONGS-A * 0203、SONGS-A * 0204、SONGS-A * 0205、SONGS-A * 0206、SONGS-A * 0207、SONGS-A * 0210、SONGS-A * 0211、SONGS-A * 0212、SONGS-A * 0213、SONGS-A * 0214、SONGS-A * 0216、SONGS-A * 0217、SONGS-A * 0219、SONGS-A * 0220、SONGS-A * 0222、SONGS-A * 0224、SONGS-A * 0230、SONGS-A * 0242、SONGS-A * 0253、SONGS-A * 0260、SONGS-A * 0274アレル、SONGS-A * 0301、SONGS-A * 0302、SONGS-A * 0305、SONGS-A * 0307、SONGS-A * 0101、SONGS-A * 0102、SONGS-A * 0103、SONGS-A * 0116アレル、SONGS-A * 1101、SONGS-A * 1102、SONGS-A * 1103、SONGS-A * 1104、SONGS-A *1105、HLA-A * 1119アラル、HLA-A * 2402、HLA-A * 2403、HLA-A * 2405、HLA-A * 2407、HLA-A * 2408、HLA-A * 2410、HLA-A * 2414、HLA-A * 2417、HLA-A * 2420、HLA-A * 2422、HLA-A * 2425、HLA-A * 2426、HLA-A * 2458アラル、HLA-B * 0702、HLA-B * 0704、HLA-B * 0705、HLA-B * 0709、HLA-B * 0710、HLA-B * 0715、HLA-B * 0721、HLA-C * 0702、HLA-C * 0701、HLA-C * 0401、HLA-C * 0602、HLA-C * 0304、HLA-C * 0501、HLA-C * 1601、HLA-C * 0202、HLA-C * 0303、HLA-C * 1203、HLA-C * 0802、HLA-C * 0102、HLA-C * 1701、HLA-C * 1502、HLA-C * 1402、HLA-C * 1202、HLA-C * 0704、HLA-C * 0801、HLA-C * 0302、HLA-C * 1801、HLA-C * 1505、HLA-C *1602, HLA-C * 0804, HLA-C * 0305, and HLA-C * 1403 allele. In some embodiments, the MAGEA1 immunogenic peptide is derived from a human MAGEA1 protein and / or a MAGEA1 protein shown in Table 3. In some embodiments, one or more MAGEA1 immunogenic peptides are administered alone or in combination with an adjuvant.

[0206] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% affinity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha sequences listed in Table 2. , 99% or more identity to a TCR alpha chain sequence; and / or b) a TCR beta chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chains listed in Table 2.

[0207] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2.

[0208] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain variable (V) antibody listed in Table 2 α) TCR V domain sequences selected from the group consisting of α a TCR alpha chain variable (V) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the V domain sequence; α ) domain sequence; and / or b) a TCR beta chain variable (V) domain sequence listed in Table 2 β ) TCR V domain sequences selected from the group consisting of β a TCR beta chain variable (V) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the V β ) domain array.

[0209] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain variable (V) antibody listed in Table 2 α ) TCR V domain sequences selected from the group consisting of α and / or b) a TCR beta chain variable (V) domain sequence listed in Table 2. β ) TCR V domain sequences selected from the group consisting of β Domain array.

[0210] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of) at least one (e.g., one, two, or three, e.g., CDR3 alone or in combination with CDR1 and CDR2) TCR alpha chain complementarity determining region (CDR) sequences having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2. CDR3 is believed to be the primary CDR responsible for recognizing processed antigen, while CDR1 and CDR2 primarily interact with MHC; therefore, in some embodiments, binding proteins are provided that comprise a CDR3 from a TCR alpha chain alone and / or a CDR3 from a TCR beta chain alone as listed in Table 2, wherein each CDR3 has a sequence homology as listed in this paragraph.

[0211] In some embodiments, the binding proteins provided herein may also comprise (e.g., comprise, consist essentially of, or consist of) at least one (e.g., one, two, or three, e.g., CDR3 alone or in combination with CDR1 and CDR2) TCR beta chain complementarity determining region (CDR) sequences having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2. As noted above, CDR3 is believed to be the primary CDR responsible for recognizing processed antigen, while CDR1 and CDR2 primarily interact with MHC; therefore, in some embodiments, binding proteins are provided that comprise a CDR3 alone from a TCR beta chain and / or a CDR3 alone from a TCR alpha chain listed in Table 2, wherein each CDR3 has a sequence homology as listed in this paragraph.

[0212] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of at least one (e.g., one, two, or three) complementarity determining region (CDR) of a TCR alpha chain listed in Table 2).

[0213] In some embodiments, the binding proteins provided herein may also comprise (e.g., may comprise, consist essentially of, or consist of at least one (1, 2, or 3) of) the complementarity determining regions (CDRs) of the TCR beta chain listed in Table 2.

[0214] In some embodiments, the binding proteins provided herein comprise a TCR alpha chain constant region (C) listed in Table 2. α96%, 97%, 98%, 99% or more identity to a TCR Cα sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, ...

[0215] In some embodiments, the binding proteins provided herein also contain a TCR beta chain constant region (C) listed in Table 2. β ) TCR C 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 with the sequence β It may comprise (e.g., may comprise, consist essentially of, or consist of) a sequence.

[0216] In some embodiments, the binding proteins provided herein comprise a TCR alpha chain constant region (C) listed in Table 2. α ) TCR C sequences selected from the group consisting of α Comprises (e.g., comprises, consists essentially of, or consists of) a sequence.

[0217] In some embodiments, the binding proteins provided herein also contain a TCR beta chain constant region (C) listed in Table 2. β ) TCR C sequences selected from the group consisting of β It may comprise (e.g., may comprise, consist essentially of, or consist of) a sequence.

[0218] [Table 2-1] [Table 2-2] [Table 2-3]

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

[0219] In some embodiments, the binding proteins provided herein comprise a constant region that is chimeric, humanized, human, primate, or rodent (e.g., rat or mouse). For example, a human variable region may be chimerized with a mouse constant region, or a mouse variable region may be humanized with a human constant region and / or human framework regions. In some embodiments, the constant region may be mutated to alter functionality (e.g., introducing non-naturally occurring cysteine ​​substitutions at opposing residue positions in the TCR alpha and beta chains to create disulfide bonds useful for improving affinity between the TCR alpha and beta chains). Similarly, mutations may be made in the transmembrane domain of the constant region to alter functionality (e.g., increasing hydrophobicity by introducing non-naturally occurring substitutions of residues with hydrophobic amino acids). In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof compared to a reference CDR sequence. In some embodiments, mutations may be made in the constant region to increase cell surface expression.

[0220] In some embodiments, the binding proteins disclosed herein may be engineered protein scaffolds, antibodies or antigen-binding fragments thereof, TCR mimetic antibodies, and the like.Such binding moieties can be designed and / or generated against the peptides and / or MHC-peptide complexes described herein using conventional immunological methods, such as immunizing a host, obtaining antibody-producing cells and / or antibodies thereto, and generating hybridomas useful for producing monoclonal antibodies (see, e.g., Watt et al. (2006) Nat. Biotechnol. 24:177-183; Gebauer and Skerra (2009) Curr. Opin. Chem Biol. 13:245-255; Skerra et al. (2008) FEBS J. 275:2677-2683; Nygren et al. (2008) FEBS J. 275:2668-2676; Dana et al. (2012) Exp. Rev. Mol. Med. 14:e6; Sergeva et al. (2011) Blood 117:4262-4272, PCT Publication Nos. WO2007 / 143104, PCT / US86 / 02269 and WO86 / 01533, U.S. Patent No. 4,816,567, Better et al. (1988) Science 240:1041-1043, Liu et al. al.(1987) Proc.Natl.Acad.Sci.USA84:3439-3443, Liu et al.(1987) J.Immunol.139:3521-3526, Sun et al.(1987) Proc.Natl.Acad.Sci.84:214-218, Nishimura et al.(1987) Cancer Res.47:999-1005, Wood et al. al. (1985) Nature 314:446-449, Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559), Morrison, SL (1985) Science 229:1202-1207, Oi et al. (1986) Biotechniques 4:214, U.S. Patent No. 5,225,539, Jones et al. (1986) Nature 321:552-525, Verhoeyan et al. (1988) Science 239:1534, and Beidler et al. (1988) J. Immunol. 141:4053-4060.If desired, the binding moiety may be isolated or purified using conventional procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, and high performance liquid chromatography (HPLC) (e.g., Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY).

[0221] The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE), as well as recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, and fragments and derivatives of all of the above, which fragments and derivatives retain at least an antigen-binding site. Antibody derivatives may include proteins or chemical moieties conjugated to the antibody.

[0222] Additionally, intrabodies are well-known antigen-binding molecules that have the characteristics of antibodies but can be expressed intracellularly to bind to and / or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods for adapting antibodies to target (e.g., inhibit) intracellular portions are well known in the art, including, for example, the use of single-chain antibodies (scFv), engineering immunoglobulin VL domains for hyperstability, engineering antibodies to tolerate reducing intracellular environments, and creating fusion proteins that improve intracellular stability and / or modulate intracellular localization. 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, see Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).

[0223] As used herein, the term "antibody" also includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen-binding portion," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically and / or selectively bind to an antigen (e.g., a peptide and / or MHC-peptide complex described herein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be recombinantly joined by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426, Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a particular scFv can be ligated to human immunoglobulin constant region cDNA or genomic sequences to create expression vectors encoding the entire IgG polypeptide or other isotype.VH and VL can also be used to create Fab, Fv, or other immunoglobulin fragments using either protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short 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. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0224] Furthermore, an antibody, or antigen-binding portion thereof, may be part of a larger immunoadhesion polypeptide formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include the use of a streptavidin core region to generate tetrameric scFv polypeptides (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, protein subunit peptides, and a C-terminal polyhistidine tag to generate bivalent biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

[0225] 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, antibodies of the present invention bind specifically and / or selectively, or substantially specifically and / or selectively, to the peptides and / or MHC-peptide complexes described herein. The terms "monoclonal antibody" and "monoclonal antibody composition," as used herein, refer to a population of antibody polypeptides that contain only one 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. A monoclonal antibody composition typically exhibits a single binding affinity for the particular antigen with which it immunoreacts.

[0226] Like other binding moieties described herein, antibodies may be "humanized," which is intended to include antibodies made by non-human cells having variable and constant regions that have been modified to more closely resemble antibodies made by human cells. For example, modifying the amino acid sequence of a non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies of the invention may include amino acid residues, e.g., in the CDRs, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro / ex vivo or by somatic mutation in vivo). The term "humanized antibody," as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences.

[0227] In some embodiments, the binding proteins disclosed herein may comprise a T cell receptor (TCR), an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In some embodiments, the binding proteins disclosed herein may comprise two polypeptide chains, each comprising a variable region comprising the CDR3 of the TCR alpha chain and the CDR3 of the TCR beta chain, or comprising the CDR1, CDR2, and CDR3 of both the TCR alpha chain and the TCR beta chain. In some embodiments, the binding protein comprises a single-chain TCR (scTCR), which comprises a TCR V α and TCR V β domains but only one TCR constant domain (C α or C β The term "chimeric antigen receptor" (CAR) refers to a fusion protein engineered to contain two or more naturally occurring amino acid sequences linked to each other in a manner that does not occur naturally or in a host cell, and that can function as a receptor when present on the surface of a cell. CARs encompassed by the present invention can include an extracellular portion comprising an antigen-binding domain (i.e., an antigen-binding domain derived from or derived from an immunoglobulin or immunoglobulin-like molecule, e.g., an antibody or TCR, or an antigen-binding domain derived from or derived from a killer immune receptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing costimulatory domain(s)) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; and Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0228] In some embodiments, 1) the TCR alpha chain CDRs, TCR V αand / or 2) the TCR beta chain CDRs, TCR V domains and / or TCR alpha chain CDRs are encoded by a TRAV, TRAJ and / or TRAC gene selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2, or fragments thereof. β The domain and / or TCR beta chain is encoded by a TRBV, TRBJ and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof compared to the cognate reference CDR sequence listed in Table 2.

[0229] In some embodiments, the binding proteins disclosed in the present application (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs)) are chimeric (e.g., contain amino acid residues or motifs from more than one donor or species), humanized (e.g., contain residues from a non-human organism that have been modified or substituted to reduce the risk of immunogenicity in humans), or human.

[0230] Methods for producing engineered binding proteins, such as TCRs, CARs, and antigen-binding fragments thereof, are well known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 5:3000, U.S. Patent No. 6,410,319, U.S. Patent No. 7,446,191, U.S. Patent Publication No. 2010 / 065818, U.S. Patent No. 8,822,647, PCT Publication No. WO2014 / 031687, U.S. Patent No. 7,514,537, and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0231] In some embodiments, the binding proteins described herein are TCRs or antigen-binding fragments thereof expressed on the cell surface, wherein the TCR expressed on the cell surface can associate with CD3 protein more efficiently than the endogenous TCR. When expressed on the surface of a cell, such as a T cell, the binding proteins encompassed by the present invention, e.g., TCRs, may have stronger surface expression on the cell than endogenous binding proteins, e.g., endogenous TCRs. In some embodiments, provided herein are CARs in which the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0232] Further provided are the VGAs disclosed herein as starting materials for engineering modified binding proteins that may have altered properties from the starting binding protein. α and / or V β The modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) can be prepared according to well-known methods using binding proteins having one or more of the following sequences: α and / or V β ), for example, by modifying one or more residues in one or more CDR regions and / or in one or more framework regions. Additionally, or alternatively, binding proteins may be engineered by modifying residues in the constant region(s).

[0233] Another type of variable region modification is the V α and / or V βThe goal of this study is to mutate amino acid residues in the CDR1, CDR2, and / or CDR3 regions of a CDR1 protein to thereby improve one or more binding characteristics (e.g., affinity) of a binding protein of interest. Site-directed mutagenesis or PCR-mediated mutagenesis may be performed to introduce the mutation(s), and the effect on protein binding or other functional properties of interest may be assessed in in vitro, ex vivo, or in vivo assays such as those described herein and provided in the Examples. In some embodiments, conservative modifications (as described above) may be introduced. The mutations may be amino acid substitutions, additions, or deletions. In some embodiments, the mutations are substitutions. Furthermore, typically, no more than 1, 2, 3, 4, or 5 residues within the CDR regions are modified.

[0234] In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) described herein may have one or more amino acid substitutions, deletions, or additions compared to naturally occurring TCRs. In some embodiments, each CDR of a binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof compared to the cognate reference CDR sequence listed in Table 2. Conservative amino acid substitutions are well known and may occur naturally, or may be introduced when the binding protein is recombinantly produced. Amino acid substitutions, deletions, and additions can be introduced into proteins using mutagenesis methods known in the art (e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide-site-directed (or segment-directed) mutagenesis procedures may be employed to result in modified polynucleotides with specific codons altered according to the desired substitution, deletion, or insertion. Alternatively, immunogenic polypeptide variants may be prepared using random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis (see, e.g., Sambrook et al., supra).

[0235] Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid substitution or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids can be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, although considered more difficult to classify, shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, substitution of glutamine for glutamic acid or asparagine for aspartic acid can be considered similar substitutions in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of a polypeptide and its conserved amino acid substitutes to the sequence of a second polypeptide (e.g., using the GENEWORKS™, Align, BLAST algorithms, or other algorithms described herein and practiced in the art).

[0236] In some embodiments, the encoded binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) may include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). The signal peptide directs a newly synthesized polypeptide to the appropriate location inside or outside of a cell. The signal peptide may be removed from the polypeptide during localization or secretion, or once localization or secretion is complete. A polypeptide having a signal peptide is referred to herein as a "preprotein," and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In some embodiments, the binding proteins (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) described herein include a mature V α Domain, mature V β In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) described herein comprise a mature TCR beta chain, a mature TCR alpha chain, or both.

[0237] 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 a MAGEA1 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule).

[0238] As used herein, an "effector domain" or "immune effector domain" is an intracellular portion or domain of a fusion protein or receptor that can directly or indirectly promote an immune response in a cell upon receiving an appropriate signal. In some embodiments, the effector domain is derived from an immune cell protein or portion thereof or immune cell protein complex that receives a signal upon binding (e.g., CD3ζ) or when the immune cell protein or portion thereof or immune cell protein complex directly binds to a target molecule to trigger signaling from the effector domain in the immune cell.

[0239] An effector domain can directly promote a cellular response if it contains one or more signaling domains or motifs, such as intracellular tyrosine-dependent activation motifs (ITAMs), such as those found in costimulatory molecules. Without wishing to be bound by theory, ITAMs are thought to serve for T cell activation following ligand engagement by the T cell receptor or by a fusion protein containing a T cell effector domain. In some embodiments, the intracellular component or functional portion thereof comprises an ITAM. Exemplary immune effector domains include, but are not limited to, immune effector domains derived from CD3ε, CD3δ, CD3ζ, CD25, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the effector domain comprises a lymphocyte receptor signaling domain (e.g., CD3ζ, or a functional portion or variant thereof).

[0240] In further embodiments, the intracellular component of the fusion protein comprises a costimulatory domain or functional portion thereof selected from a ligand that binds (e.g., specifically and / or selectively) to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-1 (CD54), LFA-1 (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, or a functional variant thereof, or any combination thereof. In some embodiments, the intracellular component comprises a CD28 costimulatory domain or a functional portion or variant thereof (which may optionally comprise an LL-GG mutation at positions 186-187 of the native CD28 protein (e.g., Nguyen et al. (2003) Blood 702:4320), a 4-1BB costimulatory domain or a functional portion or variant thereof, or both.

[0241] In some embodiments, the effector domain comprises a CD3ε endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD27 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD28 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a 4-1BB endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an OX40 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD2 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD5 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICAM-1 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an LFA-1 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICOS endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof.

[0242] The extracellular and intracellular components encompassed by the present invention are linked by a transmembrane domain. As used herein, a "transmembrane domain" is a portion of a transmembrane protein that can be inserted into or span a cell membrane. A transmembrane domain has a three-dimensional structure that is thermodynamically stable in the cell membrane and generally ranges in length from about 15 to about 30 amino acids. The structure of a transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof. In some embodiments, the transmembrane domain comprises or is derived from a known transmembrane protein (e.g., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, or any combination thereof).

[0243] In some embodiments, the extracellular component of the fusion protein further comprises a linker positioned between the binding domain and the transmembrane domain. As used herein, a "linker," when referring to a component of the fusion protein linking the binding domain and the transmembrane domain, can be an amino acid sequence having from about 2 amino acids to about 500 amino acids that can provide flexibility and room for conformational movement between the two regions, domains, motifs, fragments, or modules connected by the linker. For example, linkers encompassed by the present invention can position the binding domain away from the surface of the host cell expressing the fusion protein, allowing for proper contact, antigen binding, and activation between the host cell and the target cell (Patel et al. (1999) Gene Therapy 6:412-419). Linker length may be varied to maximize antigen recognition based on the seize and affinity of the selected target molecule, selected binding epitope, or antigen-binding domain (see, e.g., Guest et al. (2005) Immunother. 28:203-11, and PCT Publication No. WO2014 / 031687). Exemplary linkers include Gly x Ser yIn the formula, x and y are each independently an integer of 0 to 10, provided that both x and y are not 0 (for example, (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or a combination thereof, such as ((Gly3Ser)2Gly2Ser)).

[0244] The binding proteins may be conjugated to an agent, such as a detection moiety, radiosensitizer, photosensitizer, etc., and / or may be chemically modified as described above for peptides.

[0245] In any of the embodiments disclosed herein, the encoded binding protein is capable of binding to a peptide-MHC (pMHC) complex comprising a MAGEA1 immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule).

[0246] A variety of assays are known for assessing binding affinity and / or for determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand (e.g., a peptide antigen-MHC complex). It is within the level of ordinary skill in the art to determine the binding affinity of a binding protein for a target, e.g., a T-cell peptide epitope of a target polypeptide, for example, by using any of several binding assays known in the art. For example, in some embodiments, a Biacore™ instrument may be used to determine the binding constant of a complex between two proteins. The dissociation constant (K D) can be determined by monitoring the change in refractive index with time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs), or measuring binding by monitoring changes in the spectroscopic or optical properties of the proteins by fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore™) analysis (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660, Wilson (2002) Science 295:2103, Wolff et al. (1993) Cancer Res. 53:2560, and U.S. Pat. Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, apparent affinity for a target is measured by assessing binding to various concentrations of tetramer, e.g., by flow cytometry using labeled multimers, such as MHC-antigen tetramers. In one representative example, the apparent K of a binding protein is measured. D is measured using a range of two-fold dilutions of the labeled tetramer, followed by determination of the binding curve by nonlinear regression, whereas the apparent K D is determined as the concentration of ligand that results in half-maximal binding.

[0247] VI. Nucleic Acids and Vectors In aspects encompassed by the present invention, provided herein are nucleic acid molecules encoding the proteins described herein, such as MAGEA1 immunogenic peptides and fragments thereof, MHC molecules, binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, CARs, etc.).

[0248] In some embodiments, a nucleic acid molecule hybridizes under stringent conditions to the complement of a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, e.g., over the entire length, to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3.

[0249] 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.

[0250] In some embodiments, the nucleic acid molecule comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence that encodes a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-3.

[0251] In some embodiments, the nucleic acid sequence encodes a MAGEA1 immunogenic peptide described herein.

[0252] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α chain CDR shown in Table 2. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α chain CDR shown in Table 2. α TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. αIn some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR alpha chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR alpha chain sequence shown in Table 2.

[0253] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β chain CDR shown in Table 2. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β chain CDR shown in Table 2. β TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. β In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR β chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β chain sequence shown in Table 2.

[0254] The term "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, which may be single- or double-stranded, synthetic or obtained from natural sources (e.g., isolated and / or purified), and which may contain natural, non-natural, or modified nucleotides, and which may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides. In one embodiment, nucleic acid includes complementary DNA (cDNA).

[0255] 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 a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule resulting from replication of the molecule described in (i) above. For purposes herein, replication can be in vitro, ex vivo, or in vivo replication.

[0256] 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. For example, nucleic acids may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to improve the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that may be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids encompassed by the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA).

[0257] In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound by any particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence improves the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but that can be translated by a more readily available tRNA in the cell, thereby increasing translation efficiency. Optimizing a nucleotide sequence may also reduce secondary structures in the mRNA that would otherwise interfere with translation, thereby increasing translation efficiency. In some embodiments, the nucleotide sequences described herein are codon-optimized for expression in a host cell (e.g., an immune cell such as a T cell).

[0258] The present invention also provides nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0259] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" refers to nucleotide sequences that specifically and / or selectively hybridize to a target sequence (any of the nucleotide sequences of the nucleic acids described herein) with a detectable degree of hybridization greater than that observed in nonspecific hybridization. High stringency conditions include conditions that will distinguish polynucleotides with exact complementary sequences or polynucleotides containing only minor interspersed mismatches from random sequences that coincidentally contain small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity melt more easily than full-length complements of 14-17 bases or more, making them readily distinguishable by high stringency hybridization. Relatively high stringency conditions may include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions are particularly suitable for detecting expression of any of the TCRs of the present invention, as they tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand. It is widely recognized that conditions can be made more stringent by adding increasing amounts of formamide.

[0260] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids described herein.

[0261] Typically, the nucleic acid is a DNA or RNA molecule that may be comprised in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0262] The terms "vector," "cloning vector," and "expression vector" refer to delivery vehicles capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell, thereby transforming the host and promoting expression (e.g., transcription and translation) of the introduced sequences. Accordingly, a further object encompassed by the present invention relates to vectors comprising the nucleic acids encompassed by the present invention.

[0263] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to bring about or direct the expression of the above-mentioned polypeptides when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and the immunoglobulin H chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983).

[0264] Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1 beta d2-4- (Miyaji H et al. 1990). Other representative examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids, such as pUC, pcDNA, and pBR. Representative examples of viral vectors include adenoviral, retroviral, lentiviral, herpesviral, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses are well known in the art and can be found, for example, in PCT Publication WO95 / 14785, PCT Publication WO96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and PCT Publication WO94 / 19478.

[0265] In some embodiments, the expression vector is a nanoplasmid. The term "nanoplasmid" is used herein to refer to a circular DNA sequence with reduced bacterial sequences, providing a smaller plasmid with a desired cargo insert. The reduced size of the vector is associated with limited toxicity induced by the DNA sequence upon transfection, potentially longer persistence in cells, potentially better viability after transfection, and potentially higher transposition efficiency. In some embodiments, the nanoplasmid is an antibiotic resistance marker-free nanoplasmid. In some embodiments, the nanoplasmid contains a selectable marker and / or a nonsense suppressor marker. Due to the small backbone size, e.g., less than 500 bp, such nanoplasmids maximize the size of the desired cargo insert. The desired cargo insert (e.g., eukaryotic transgene) can be of any size that can be delivered to a target cell, such as 50 kb, 45 kb, 40 kb, 35 kb, 30 kb, 25 kb, 20 kb, 18 kb, 15 kb, 12 kb, 10 kb, 5.0 kb, 4.5 kb, 4.0 kb, 3.5 kb, 3.0 kb, 2.8 kb, 2.5 kb, 2.2 kb, 2 kb, 1.8 kb, 1.5 kb, 1.2 kb, 1 kb or less, or any range therebetween, including the endpoints, such as 8-12 kb. They also provide high transgene expression and reduce transgene silencing, allowing for tunable cargo integration efficiency relative to random integration of the cargo in the genome of the engineered cell.

[0266] In some embodiments, a Nanoplasmid may contain elements shown in the vectors provided in Table 3 (e.g., R6K, and RNA-OUT). For example, in some embodiments, a Nanoplasmid contains a minimized bacterial ColE1 or R6K origin of replication (which allows such Nanoplasmids to replicate in a bacterial host strain), a selectable marker (e.g., a bacterial RNA selectable marker), and a eukaryotic gene region. An RNA selectable marker is a non-translated RNA expressed from the vector that regulates a target gene expressed from the chromosome, allowing for selection of the vector. This is in contrast to a plasmid-borne nonsense marker that regulates a suppressible, selectable nonsense chromosomal target. Repressor tRNAs may be, for example, those described in U.S. Patent No. 6,977,174, which is incorporated herein by reference. They may also include antisense repressor RNAs carried on plasmids, RNA-OUT genes that repress targets regulated by RNA-IN, pMB1 plasmid origin-encoded RNAI that represses targets regulated by RNAII, IncB plasmid pMU720 origin-encoded RNAI that represses targets regulated by RNAII, ParB locus Sok of plasmid RI that represses targets regulated by Hok, Flm locus FlmB of F plasmid that represses targets regulated by flmA, and naturally occurring antisense repressor RNAs, for example, Wagner (2002) Adv. Genet. 46:361, and Franch and Gerdes (2000) Current Opin. Microbiol. 3:159, or engineered repressor RNAs, e.g., small synthetic RNAs such as SgrS, MicC, or MicF scaffolds, as described in Park et al. (2013) Nature Biotechnology 31:170-174.

[0267] Exemplary nanoplasmids generated by the antibiotic-free RNA-OUT selection system and methods for making such nanoplasmids are described, for example, in PCT Publication No. WO2008153733, U.S. Patent No. 9,737,620, U.S. Patent Publication No. 2010 / 0303859, and U.S. Patent No. 9,109,012, each of which is incorporated by reference in its entirety. Further exemplary nanoplasmids are described, for example, in PCT Application Nos. PCT / US2013 / 000259, PCT / US2013 / 00067, and PCT / US2013 / 00068, and U.S. Patent Publication No. 2015 / 0275221, each of which is incorporated by reference in its entirety.

[0268] Nanoplasmids are commercially available. For example, Nature Technology Corporation, a subsidiary of Aldevron, offers Nanoplasmid vectors that combine an RNA selectable marker with an R6K, ColE2, or ColE2-related origin of replication. These nanoplasmid vectors include, for example, NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors, and modified forms thereof, such as those described in PCT Application No. PCT / US2013 / 00068; NTC9385R-BE, NTC9385Ra-O1, and NTC9385Ra-O2 vectors, such as those described in U.S. Pat. No. 10,144,935; and NTC9385C2, NTC9385C2a, NTC9385R2, NTC9385R2a, NTC9385R2b, NTC9385Ra, NTC9385RaF, and NTC9385RbF replicating minicircle vectors, such as those described in U.S. Patent Publication No. 2021 / 0189407, which are incorporated by reference in their entireties.

[0269] In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a binding protein or polypeptide, or fragment thereof, described herein. In some embodiments, the nucleic acid comprises regulatory elements required for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. Additionally, enhancers may be included. These elements may be operably linked to the sequence encoding the binding protein, polypeptide, or fragment thereof.

[0270] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, a dominant-negative TGFβ receptor (e.g., DN-TGFβRII), a selectable protein marker, optionally the selectable protein marker is dihydrofolate reductase (DHFR). In certain embodiments, 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 (e.g., a CD34 enrichment tag). In certain embodiments, the nucleic acid sequences described herein, e.g., the nucleic acid sequences encoding TCRα, TCRβ, CD8α, CD8β, DN-TGFβR and / or the selectable protein marker, are interconnected to a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide such as P2A, E2A, F2A, or T2A.

[0271] In some embodiments, the expression vectors provided herein comprise a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids set forth in Tables 1-3.

[0272] As described above, representative examples of promoters include, but are not limited to, promoters from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), e.g., the HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV), e.g., the CMV immediate early promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Examples of suitable polyadenylation signals include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal.

[0273] In addition to the regulatory elements required for expression, other elements may be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include promoters described herein. In some embodiments, enhancers / promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers, such as those from CMV, RSV, and EBV.

[0274] In some embodiments, the nucleic acid may be operably incorporated into a carrier or delivery vector, as described further below. Useful delivery vectors include, but are not limited to, biodegradable microcapsules, immunostimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live carriers, such as viruses or bacteria.

[0275] In some embodiments, the vector is a viral vector, such as a lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, fowlpox, AVpox, modified vaccinia Ankara (MVA), and other recombinant viruses. For example, a lentiviral vector may be used to infect T cells.

[0276] In some embodiments, the recombinant expression vector is capable of delivering the polynucleotide to a suitable host cell, e.g., a T cell or an antigen-presenting cell, i.e., a cell that presents peptide / MHC complexes on its cell surface (e.g., a dendritic cell) and lacks CD8. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. For example, the immune system cell is a CD4 + T cells, CD8 + The host may be a T cell, a CD4 / CD8 double-negative T cell, a gd T cell, a natural killer cell, a dendritic cell, or any combination thereof. In some embodiments, a T cell is the host, and the T cell may be a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. Thus, the recombinant expression vector may also include a lymphoid tissue-specific transcriptional regulatory element (TRE), for example, a TRE specific for B lymphocytes, T lymphocytes, or dendritic cells. 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).

[0277] In some embodiments, the recombinant expression vector comprises nucleotide sequences encoding a TCR alpha chain, a TCR beta chain, and / or a linker peptide. For example, in some embodiments, the recombinant expression vector comprises nucleotide sequences encoding the full-length TCR alpha and TCR beta chains of a binding protein with a linker therebetween, wherein the nucleotide sequence encoding the beta chain is located 5' to the nucleotide sequence encoding the alpha chain. In some embodiments, the nucleotide sequences encode the full-length TCR alpha and TCR beta chains with a linker therebetween, wherein the nucleotide sequence encoding the TCR beta chain is located 3' to the nucleotide sequence encoding the TCR alpha chain. In some embodiments, the full-length TCR alpha and / or TCR beta chains are replaced with fragments thereof.

[0278] As described further below, another aspect encompassed by the present invention relates to cells transfected, infected, or transformed with nucleic acids and / or vectors according to the present invention. A host cell includes any individual cell or cell culture capable of receiving a vector or the incorporation of nucleic acids and / or proteins, as well as any progeny. The term also encompasses the progeny of a host cell, whether genetically or phenotypically identical or different. Suitable host cells will depend on the vector and may include mammalian, animal, human, simian, insect, yeast, and bacterial cells. These cells can be induced to take up vectors or other materials by transformation via viral vectors, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring Harbor Laboratory)). The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0279] For example, in some embodiments, engineered immune cells (e.g., T cells) are pan-T cells (CD4 T cells) engineered by transposon / transposase-mediated gene delivery to express genetic cargo encoding elements such as the alpha and beta chains of a recombinant T cell receptor (TCR) that is specific for a given target antigen presented on a particular MHC (e.g., class I HLA). + and CD8 + and T cells). +The vector can express one or more elements selected from the group consisting of: a) CD8α and CD8β co-receptors to enable T cell engagement; b) a CD34-derived QBEND / 10 epitope tag fused to the amino terminus of CD8α to enable in vitro and in vivo tracking of engineered cells; c) a dominant-negative type II TGFβ receptor (DN-TGFβRII) to overcome tumor-mediated immunosuppression; and d) a selectable marker, e.g., a mutant form of dihydrofolate reductase (DHFRdm), to facilitate enrichment of engineered cells during the manufacturing process. The α and β chains of the foreign TCR and the α and β chains of CD8 can be encoded on a single mRNA molecule under the control of a single promoter, e.g., the murine stem cell virus (MSCV) promoter. Post-translational processing at a self-cleaving peptide element, e.g., the P2A site, can result in independent polypeptides, e.g., four individual polypeptides corresponding to each element in the vector. Similarly, DN-TGFβRII and DHFRdm may be encoded by a single mRNA molecule driven by a single promoter, such as the human elongation factor 1α (EF1α) promoter. Post-translational processing at a self-cleaving peptide element, such as the P2A site, can result in independent polypeptides corresponding to each individual element in the vector.

[0280] Nucleic acids encompassed by the invention may be used in a suitable expression system to produce recombinant polypeptides encompassed by the invention. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for expression of a protein encoded by foreign DNA, e.g., carried by a vector and introduced into the host cell.

[0281] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al. (1980)), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, YB2 / 0 cells are used because the ADCC activity of chimeric or humanized binding proteins is enhanced when expressed in YB2 / 0 cells.

[0282] The present invention also encompasses methods for producing recombinant host cells expressing binding proteins, peptides, and fragments thereof, comprising the steps of (i) introducing, in vitro or ex vivo, a recombinant nucleic acid or vector as described above into a competent host cell, (ii) culturing, in vitro or ex vivo, the resulting recombinant host cell, and (iii) optionally selecting cells expressing the binding protein, peptide, and fragments thereof. Such recombinant host cells may be used in the diagnostic, prognostic, and / or therapeutic methods encompassed by the present invention.

[0283] In another aspect, as described above, the present invention provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, polynucleotides encompassed by the present invention can be used to identify, isolate, or amplify partial or full-length clones in a stored library. In some embodiments, the polynucleotides are genomic or cDNA sequences that are complementary to cDNAs isolated from or derived from a human or mammalian nucleic acid library. In some embodiments, the cDNA library contains at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more full-length sequences, or any range therebetween, inclusive, e.g., at least about 80%-100%. The cDNA library may be normalized to enhance the representation of rare sequences. Low or medium stringency hybridization conditions are typically, but not exclusively, employed for sequences with low sequence identity to complementary sequences. Medium and high stringency conditions may optionally be employed for sequences with higher identity. Low stringency conditions allow for selective hybridization of sequences with about 70% sequence identity and may be used to identify orthologous or paralogous sequences. Optionally, polynucleotides encompassed by the present invention will encode at least a portion of a binding protein encoded by a polynucleotide described herein. Polynucleotides encompassed by the present invention include nucleic acid sequences that can be employed for selective hybridization to polynucleotides encoding binding proteins encompassed by the present invention (see, e.g., Ausubel, supra, and Colligan, supra).

[0284] VII. Manipulated cells In aspects encompassed by the present invention, provided herein are host cells that express a protein described herein, e.g., a MAGEA1 immunogenic peptide, a MAGEA1 immunogenic peptide-MHC (pMHC) complex, a MAGEA1 binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, a CAR, or a fusion protein comprising a TCR and an effector domain), etc. In some embodiments, the host cell comprises a nucleic acid or vector described herein.

[0285] In some embodiments, polynucleotides encoding the binding proteins are used to transform, transfect, or transduce host cells (e.g., T cells) for use in adoptive transfer therapy. Advances in nucleic acid sequencing and specific TCR sequencing have been described (e.g., Robins et al. (2009) Blood 114:4099; Robins et al. (2010) Sci. Translat. Med. 2:47ra64; Robins et al. (2011) J. Imm. Meth.; and Warren et al. (2011) Genome Res. 21:790), which may be used in the course of practicing embodiments encompassed by the present invention. Similarly, methods for transfecting or transducing T cells with a desired nucleic acid are well known in the art (e.g., U.S. Patent Application Publication No. 2004 / 0087025), as are adoptive transfer procedures using T cells with a desired antigen specificity (e.g., Schmitt et al. (2009) Hum. Gen. 20:1240; Dossett et al. (2009) Mol. Ther. 77:742; Till et al. (2008) Blood 772:2261; Wang et al. (2007) Hum. Gene Ther. 18:112; Kuball et al. (2007) Blood 709:2331; U.S. Patent Publication No. 2011 / 0243972; U.S. Publication No. 2011 / 0189141; and Leen et al. al.(2007)Ann.Rev.Immunol.25:243).

[0286] Any suitable immune cell, e.g., a T cell, an NK cell, or an NK-T cell, can be modified to contain a heterologous polynucleotide encompassed by the present invention. In some embodiments, the cell can be a primary cell or a cell line. In some embodiments, the modified immune cell is a CD4 + T cells, CD8 + T cells, or both. For purposes herein, T cells can be any T cell, e.g., cultured T cells, e.g., primary T cells, or cells from a cultured T cell line, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. If obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are T cells isolated from a human. T cells can be any type of T cell and at any developmental stage, including, but not limited to, cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), and naive T cells.

[0287] Any suitable method can be used to transfect or transduce cells (e.g., T cells) or administer the nucleotide sequences or compositions encompassed by the methods described herein. Methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercially available products, such as in vivo-jetPEI®. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, biolistics, transposon-mediated transfer, and the like. Additional methods for transfecting or transducing host cells employ vectors, which are described in more detail in this application.

[0288] The modified immune cells described herein can be functionally characterized using methodologies to assess T cell activity, including determining T cell binding, activation, or induction, and also determining antigen-specific T cell responses. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., from pre-loaded target cells), and T cell proliferation. 51 These include determining T cell function (by detecting Cr release), changes in T cell phenotypic marker expression, and other measures of T cell function.

[0289] Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: A Comprehensive Sourcebook of Techniques, 1998), and Current Protocols in Immunology, Weir, (1986) Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA, Mishell and Shigii (eds.) (1979) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA, Green and Reed (1998) Science 281:1309, and references cited therein.

[0290] In some embodiments, the apparent affinity for a binding protein, e.g., a TCR or its antigen-binding portion, can be measured by assessing binding to various concentrations of MHC multimers. "MHC-peptide multimer staining" refers to an assay used to detect antigen-specific T cells, which in some embodiments focuses on tetramers of MHC molecules, each containing an identical peptide with a homologous (e.g., identical or related) amino acid sequence to at least one antigen (e.g., a MAGEA1 immunogenic peptide), and the complex can bind to a binding protein that recognizes the cognate antigen, e.g., a TCR or its antigen-binding portion. Each MHC molecule may be tagged with a biotin molecule. The biotinylated MHC / peptide may be multimerized (e.g., tetramerized) by the addition of streptavidin (which may be fluorescently labeled).

[0291] Multimers can be detected by flow cytometry via fluorescent labeling. In some embodiments, the affinity-enhanced binding proteins encompassed by the present invention, e.g., TCRs or antigen-binding portions thereof, are detected or selected using pMHC multimer assays. In some examples, the apparent K D is measured using a range of two-fold dilutions of the labeled tetramer, followed by determination of the binding curve by nonlinear regression, whereas the apparent K D is determined as the concentration of ligand that results in half-maximal binding.

[0292] Cytokine levels can be determined using methods described herein, such as ELISA, ELISPOT, intracellular cytokine staining and flow cytometry, and combinations thereof (e.g., intracellular cytokine staining and flow cytometry).

[0293] Immune cell proliferation and clonal expansion resulting from antigen-specific induction or stimulation of an immune response can be determined by isolating lymphocytes, e.g., circulating lymphocytes in a sample of peripheral blood cells or lymph node-derived cells, stimulating the cells with an antigen, and measuring cytokine production, cell proliferation, and / or cell viability, e.g., by tritiated thymidine incorporation or non-radioactive assays, such as the MTT assay. The effect of the immunogens described herein on the balance between Th1 and Th2 immune responses can be examined, for example, by determining the levels of Th1 cytokines, e.g., IFN-γ, IL-12, IL-2, and TNF-β, and type 2 cytokines, e.g., IL-4, IL-5, IL-9, IL-10, and IL-13.

[0294] Host cells encompassed by the present invention may contain a single polynucleotide encoding a binding protein described herein, or the binding protein may be encoded by more than one polynucleotide. In other words, components or portions of a binding protein may be encoded by more than one polynucleotide, and these polynucleotides may be contained on a single nucleic acid molecule or on more than one nucleic acid molecule.

[0295] Moreover, as further described below and in the working examples, host cells encompassed by the present invention may encode and / or express, in addition to the binding proteins described herein, useful accessory proteins, either on the same polynucleotide as the binding protein or components thereof or on different polynucleotides. For example, the host cells may encode and / or express CD8α, CD8β, DN-TGFβR (e.g., DN-TGFβRII), and / or a selectable protein marker, optionally the selectable protein marker is DHFR.

[0296] In some embodiments, polynucleotides encoding two or more components or portions of a binding protein encompassed by the invention comprise two or more coding sequences operably linked in a single open reading frame. Advantageously, such an arrangement can allow for coordinated expression of the desired gene products, e.g., simultaneous expression of the alpha and beta chains of a TCR, such that they are produced in a ratio of about 1:1. In some embodiments, two or more alternative gene products of a binding protein encompassed by the invention, e.g., a TCR (e.g., an alpha and beta chain) or a CAR, are expressed as separate molecules and post-translationally associate. In further embodiments, two or more alternative gene products of a binding protein encompassed by the invention are expressed as a single peptide with portions separated by a cleavable or removable segment. For example, self-cleaving peptides useful for expressing separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, the porcine teschovirus-1 2A (P2A) peptide, the zosea asignavirus 2A (T2A) peptide, the equine rhinitis A virus (ERAV) 2A (E2A) peptide, and the foot-and-mouth disease vims 2A (F2A) peptide.

[0297] In some embodiments, binding proteins encompassed by the invention comprise one or more junction amino acids. A "junction amino acid" or "junction amino acid residue" refers to one or more (e.g., 2 to about 10) amino acid residues located between two adjacent motifs, regions, or domains of a polypeptide, e.g., between a binding domain and an adjacent constant domain, or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can result from the design of a construct encoding a fusion protein (e.g., amino acid residues resulting from the use of restriction enzyme sites during construction of a nucleic acid molecule encoding a fusion protein), or can result from cleavage of a self-cleaving peptide adjacent to one or more domains of an encoded binding protein encompassed by the invention (e.g., a P2A peptide located between the TCR a chain and the TCR β chain that, upon self-cleavage, leaves one or more junction amino acids in the a chain, the TCR β chain, or both).

[0298] The engineered immune cells encompassed by the present invention can be administered, for example, as a treatment for disorders characterized by MAGEA1 expression. In some situations, it may be desirable to reduce or stop activity associated with cellular immunotherapy. Thus, in some embodiments, the engineered immune cells encompassed by the present invention comprise heterologous polynucleotides encoding binding proteins and accessory proteins, e.g., safety switch proteins, that can be targeted with the same drugs or other compounds to selectively modulate (e.g., reduce or eliminate) the activity of such cells, if desired. Safety switch proteins used in this regard include, for example, truncated EGF receptor polypeptides (huEGFRt) that lack the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retain the native amino acid sequence, type I transmembrane cell surface localization, and conformationally intact binding epitopes for pharmaceutical-grade anti-EGFR monoclonal antibodies, such as cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al. (2011) Blood 118:1255-1263), caspase polypeptides (e.g., iCasp9; Straathof et al. (2005) Blood 105:4247-4254, Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Zhou and Brenner (2016) Hematol. pii:S0301-472X:30513-30516), RQR8 (Philip et al. (2014) Blood 124:1277-1287), and the human c-myc protein tag (Kieback et al. (2008) Proc. Natl. Acad. Sci. USA 105:623-628).

[0299] Other useful ancillary components for therapeutic cells include tags or selection markers (e.g., CD34 enrichment tags) that allow cells to be identified, sorted, isolated, enriched, or tracked. For example, marked immune cells with desired characteristics (e.g., antigen-specific TCR and safety switch protein) can be sorted from marker-free cells in a sample and more efficiently activated and expanded for inclusion in a therapeutic product of desired purity.

[0300] As used herein, the term "selection marker" includes a nucleic acid construct that confers an identifiable change to a cell, allowing for the detection and positive selection of immune cells transduced with a polynucleotide containing the selection marker. For example, RQR is a selection marker that contains the large extracellular loop of CD20 and two minimal binding sites of CD34. In some embodiments, the polynucleotide encoding RQR contains a polynucleotide encoding a minimal 16-amino acid epitope of CD34. In some embodiments, such as certain embodiments provided in the Examples herein, the minimal CD34 epitope is incorporated into the amino-terminal position of the CD8 stalk domain (Q8). In further embodiments, the minimal binding site sequence of CD34 may be combined with a target epitope of CD20 to form a small marker / suicide gene for T cells (RQR8) (Philip et al. 2014). This construct allows for the selection of immune cells expressing the construct using, for example, a CD34-specific antibody (Miltenyi) coupled to magnetic beads, and the selective elimination of engineered T cells expressing the transgene using the clinically approved pharmaceutical antibody rituximab (e.g., Philip et al. (2014) Blood 124:1277-1287; U.S. Patent Publication No. 2015-0093401; and U.S. Patent Publication No. 2018-0051089).

[0301] Further exemplary selection markers include several truncated type I transmembrane proteins not normally expressed on T cells: truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (e.g., Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Mavilio et al. (1994) Blood 83:1988-1997, and Fehse et al. (2000) Mol. Ther. 7:448-456). A particularly attractive feature of CD19 and CD34 is the availability of the Miltenyi CliniMACs™ selection system, which can target these markers for clinical-grade selection. However, CD19 and CD34 are relatively large surface proteins, which may impair the packaging capacity of vectors and the transcription efficiency of integrated vectors. Surface markers containing extracellular, non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR, etc.) may also be employed. Any selection marker may be employed, provided that it is acceptable under good manufacturing practice. In some embodiments, the selection marker is expressed together with a polynucleotide encoding a gene product of interest (e.g., a binding protein encompassed by the present invention, such as a TCR or CAR, or an antigen-binding fragment thereof). Further examples of selection markers include, for example, reporters such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In some embodiments, a selection marker such as CD34 is expressed by the cells, and CD34 can be used to select, enrich, or isolate transduced cells of interest for use in the methods described herein (e.g., by immunomagnetic selection). As used herein, the CD34 marker is distinct from anti-CD34 antibodies, or, for example, scFvs, TCRs, or other antigen-recognition moieties that bind to CD34.

[0302] In some embodiments, the selectable marker comprises an RQR polypeptide, a truncated low affinity nerve growth factor (tNGFR), a truncated CD19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.

[0303] As background, CD4 + Inclusion of T cells in immunotherapy cell products can result in antigen-induced IL-2 secretion and transfer of cytotoxic CD8 + T cell persistence and function can be enhanced (e.g., Kennedy et al. (2008) Immunol. Rev. 222:129, and Nakanishi et al. Nature (2009) 52:510). In some embodiments, CD4 + Class I-restricted TCRs in T cells may require the transfer of a CD8 coreceptor to enhance the sensitivity of the TCR to class I HLA-peptide complexes. The CD4 coreceptor is structurally distinct from CD8 and cannot effectively be used as a substitute for the CD8 coreceptor (e.g., Stone & Kranz (2013) Front. Immunol. 4:244, and Cole et al. (2012) Immunology 737:139). Therefore, another auxiliary protein for use in the compositions and methods encompassed by the present invention includes the CD8 coreceptor or a component thereof. Engineered immune cells comprising a heterologous polynucleotide encoding a binding protein encompassed by the present invention may, in some embodiments, further comprise a heterologous polynucleotide encoding the CD8 coreceptor protein or a beta or alpha chain component thereof.

[0304] Host cells can be efficiently transduced to contain and efficiently express a single polynucleotide encoding the binding protein, the safety switch protein, the selectable marker and the CD8 co-receptor protein.

[0305] In one embodiment, a host cell encompassed by the invention further comprises a nucleic acid encoding a costimulatory molecule, such that the modified T cell expresses the costimulatory molecule. In some embodiments, the costimulatory domain is selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L.

[0306] In any of the foregoing embodiments, the host cell expressing the binding protein described herein may be a universal immune cell. A "universal immune cell" includes an immune cell that has been modified to reduce or eliminate expression of one or more endogenous genes encoding polypeptide products selected from PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA molecule, a TCR molecule, or any combination thereof. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, TIGIT) or may interfere with the binding activity of heterologously expressed binding proteins encompassed by the invention (e.g., endogenous TCRs that bind to non-MAGEA1 antigens and interfere with binding of modified immune cells to target cells that express MAGEA1 antigens, such as MAGEA1 immunogenic peptides, in the context of MHC molecules. Furthermore, endogenous proteins (e.g., immune cell proteins, e.g., HLA alleles) expressed on donor immune cells may be recognized as foreign by the allogeneic host, which may lead to the elimination or suppression of the modified donor immune cells by the allogeneic host.

[0307] Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerance, or persistence of engineered immune cells in an autologous or allogeneic host environment and allow the cells to be administered universally (e.g., to any recipient regardless of HLA type). In some embodiments, the cells of the present invention are syngeneic, meaning genetically identical or sufficiently identical and immunologically compatible to permit transplantation. In some embodiments, the universal immune cells are donor cells (e.g., allogeneic) or autologous cells. In some embodiments, modified immune cells (e.g., universal immune cells) encompassed by the invention comprise one or more chromosomal gene knockouts of genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g., a gene encoding alpha 1 macroglobulin, alpha 2 macroglobulin, alpha 3 macroglobulin, beta 1 microglobulin, or beta 2 microglobulin), or a TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai et al. (2016) Nature Sci. Rep. 6:21757, Torikai et al. (2012) Blood 179:5697, and Torikai et al. (2013) Blood 722:1341, which also provide representative exemplary gene editing techniques, compositions, and adoptive cell therapies that are useful according to the invention).

[0308] As used herein, the term "chromosomal gene knockout" refers to a genetic modification or inhibitor introduced into a host cell that prevents (e.g., reduces, delays, suppresses, or eliminates) the production by the host cell of a functionally active endogenous polypeptide product. Modifications that result in a chromosomal gene knockout can include, for example, the introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0309] In some embodiments, chromosomal gene knockouts or gene knock-ins can be created by chromosomal editing of a host cell. Chromosomal editing can be performed, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of phosphodiester bonds within a polynucleotide chain. In some embodiments, the endonuclease can cleave a target gene, thereby inactivating or "knockout" the target gene. The endonuclease can be a natural, recombinant, genetically modified, or fusion endonuclease. Nucleic acid strand breaks caused by endonucleases are generally repaired through distinct mechanisms: homologous recombination or non-homologous end joining (NHEJ). During the homologous recombination process, a donor nucleic acid molecule can be used for donor gene "knock-in," for target gene "knock-out," and, optionally, for target gene inactivation via a donor gene knock-in or target gene knock-out event. NHEJ is an error-prone repair process that often results in changes in the DNA sequence at the cut site (for example, substitution, deletion, or addition of at least one nucleotide). NHEJ can be used to "knock out" target genes. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0310] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein containing a zinc finger DNA-binding domain fused to a nonspecific DNA cleavage domain, e.g., Fok1 endonuclease. Each zinc finger motif of approximately 30 amino acids binds to approximately three base pairs of DNA, and amino acids at certain residues may be altered to alter the triplet sequence specificity (e.g., Desjarlais et al. (1993) Proc. Natl. Acad. Sci. 90:2256-2260, and Wolfe et al. (1999) J. Mol. Biol. 255:1917-1934). Multiple zinc finger motifs may be linked in tandem to create binding specificity for a desired DNA sequence, e.g., a region having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and the directed integration of transgenes containing adjacent sequences homologous to the genome at the DSB site is promoted by homology-directed repair. Alternatively, DSBs generated by ZFNs can result in knockout of the target gene through repair by non-homologous end joining (NHEJ), an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the break site. In some embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, created using ZFN molecules.

[0311] As used herein, "transcription activator-like effector nuclease" (TALEN) refers to a fusion protein comprising a TALE DNA-binding domain and a DNA-cleavage domain, e.g., Fok1 endonuclease. A "TALE DNA-binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each of which generally has a highly conserved 33-35 amino acid sequence with variable 12th and 13th amino acids. The TALE repeat domain is responsible for TALE binding to target DNA sequences. The variable amino acid residues, referred to as repeat variable dinucleotides (RVDs), correlate with specific nucleotide recognition. The natural (canonical) codes for DNA recognition of these TALEs have been determined: the HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE results in the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) binds to an A, NN (asparagine-asparagine) binds to a G or A nucleotide, NG (asparagine-glycine) binds to a T nucleotide, etc. Non-canonical (atypical) RVDs are also well known in the art (e.g., U.S. Patent Publication No. 2011 / 0301073, the entire disclosure of which is incorporated herein by reference). TALENs can be used to introduce site-specific double-strand breaks (DSBs) into the genome of T cells. Non-homologous end joining (NHEJ) ligates the DNA on both sides of double-strand breaks that have little or no overlapping sequence to anneal, thereby introducing errors that knock out gene expression.Alternatively, homology-directed repair can introduce transgene into DSB site if there is a homologous flanking sequence in the transgene.In some embodiments, gene knockout includes insertion, deletion, mutation or a combination thereof, and is made by using TALEN molecule.

[0312] As used herein, the term "clustered regularly interspaced short palindromic repeats / Cas" (CRISPR / Cas) nuclease system refers to a system that uses a CRISPR RNA (crRNA)-dependent Cas nuclease to recognize target sites (known as protospacers) within the genome by base-pairing complementarity and then cleave DNA when the complementary target sequence is immediately 3' followed by a short, conserved protospacer-associated motif (PAM). CRISPR / Cas systems are classified into three types (i.e., Type I, Type II, and Type III) based on the sequence and structure of the Cas nuclease. In Types I and III, the crRNA-dependent surveillance complex requires multiple Cas subunits. Type II systems, the most studied, contain at least three components: an RNA-dependent Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA contains a duplex-forming region. The crRNA and tracrRNA form a duplex that interacts with the Cas9 nuclease and guides the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA by Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA upstream of the PAM. The Cas9 nuclease cleaves the double-stranded break within the region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions that disrupt expression of the target locus. Alternatively, transgenes with homologous flanking sequences can be introduced at the site of the DSB via homology-directed repair. The crRNA and tracrRNA can be engineered into single-stranded guide RNAs (sgRNAs or gRNAs) (e.g., Jinek et al. (2012) Science 337:816-821).Additionally, the region of the guide RNA that is complementary to the target site may be modified or programmed to target a desired sequence (Xie et al. (2014) PLOS One 9:el00448, U.S. Patent Publication Nos. US2014 / 0068797, US2014 / 0186843, U.S. Patent No. 8,697,359, and PCT Publication No. WO2015 / 071474). In some embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, and is made using a CRISPR / Cas nuclease system.

[0313] Exemplary gRNA sequences and exemplary methods of using gRNA sequences to knock out endogenous genes encoding immune cell proteins include those described in Ren et al. (2017) Clin. Cancer Res. 23:2255-2266, which provides representative exemplary gRNAs, CAS9 DNA, vectors, and gene knockout techniques.

[0314] As used herein, "meganuclease," also known as "homing endonuclease," refers to an endonuclease characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, RI-Sce, I-ScelV, I-Csml, I-Panl, I-Scell, I-Ppol, I-SceIII, I-Crel, I-Tevl, I-TevII, and I-TevIII, and their recognition sequences are known (e.g., U.S. Pat. Nos. 5,420,032 and 6,833,252; Belfort et al. (1997) Nucl. Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 52:115-118; Perler et al. (1994) Nucl. Acids Res. 22:1125-1127; Jasin (1996) Trends Genet.72:224-228, Gimble et al. (1996) J.Mol.Biol.263:163-180, and Argast et al. (1998) J.Mol.Biol.280:345-353).

[0315] In some embodiments, naturally occurring meganucleases may be used to facilitate site-specific genomic modification of targets of interest, such as immune checkpoints, HLA-encoding genes, or TCR component-encoding genes.

[0316] In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genome modification (e.g., Porteus et al. (2005) Nat. Biotechnol. 23:967-73; Sussman et al. (2004) J. Mol. Biol. 342:31-41; Epinat et al. (2003) Nucl. Acids Res. 37:2952-2962; Chevalier et al. (2002) Mol. Cell 70:895-905; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Curr. Gene Ther. 7:49-66, and U.S. Patent Publication Nos. US2007 / 0117128, US2006 / 0206949, US2006 / 0153826, US2006 / 0078552, and US2004 / 0002092. In a further embodiment, homing endonucleases engineered with the modular DNA-binding domain of TALENs to create fusion proteins known as megaTALs are used to generate chromosomal gene knockouts. megaTALs can be utilized not only to knock out one or more target genes, but also to introduce (knock in) heterologous or foreign polynucleotides when used in conjunction with an exogenous donor template encoding a polypeptide of interest.

[0317] In some embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that binds (e.g., specifically and / or selectively) to a MAGEA1 antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor, and the encoded target-specific inhibitor inhibits expression of an endogenous gene (i.e., PD-1, TIM3, LAG3, CTLA4, TIGIT, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.

[0318] Chromosomal gene knockout can be confirmed directly by DNA sequencing of host immune cells after the knockout procedure or drug application.

[0319] A chromosomal gene knockout can also be inferred from the absence of gene expression following the knockout (eg, the absence of mRNA or polypeptide product encoded by the gene).

[0320] In some embodiments, host cells encompassed by the present invention are capable of specifically and / or selectively killing 50% or more of target cells that contain a peptide-MHC (pMHC) complex that includes a MAGEA1 immunogenic peptide in the context of an MHC molecule.

[0321] In some embodiments, the modified immune cells are capable of producing cytokines when contacted with target cells that contain a peptide-MHC (pMHC) complex that includes a MAGEA1 immunogenic peptide in the context of an MHC molecule.

[0322] In some embodiments, the cytokine comprises IFN-γ or IL2. In some embodiments, the cytokine comprises TNF-α.

[0323] In some embodiments, the host cell has expression of MAGEA1 at about 1,000 transcrip...

Claims

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

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

3. The immunogenic peptide of claim 1 or claim 2, wherein the immunogenic peptide is derived from the MAGEA1 protein, and optionally the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

4. the immunogenic peptide is capable of inducing an immune response against MAGEA1 and / or MAGEA1-expressing cells in a subject; Optionally, the immune response comprises: i) a T cell response and / or a CD8+ T cell response; and / or ii) selected from the group consisting of T cell proliferation, cytokine release and / or cytotoxic killing; The immunogenic peptide according to any one of claims 1 to 3.

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

6. The immunogenic composition of claim 5 further comprising an adjuvant.

7. the immunogenic composition is capable of inducing an immune response against MAGEA1 and / or MAGEA1-expressing cells in the subject; Optionally, the immune response comprises: i) a T cell response and / or a CD8+ T cell response; and / or ii) selected from the group consisting of T cell proliferation, cytokine release and / or cytotoxic killing; The immunogenic composition according to claim 5 or claim 6.

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

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

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

11. The MHC molecule is \LA-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、-HLA-C ＊ 18 and optionally, the HLA allele comprises an MHC alpha chain that is an HLA serotype selected from the group consisting of: EXPOSED ＊ 0201、HYS ＊ 202、HYS ＊ 203、HYS ＊ 0204、HYS ＊ 2005、DYS ＊ 206、HYS ＊ 2007、HYS ＊ 210、HYS ＊ 211. CHRISTIAN ＊ 212、HYS ＊ 213、HYS ＊ 214、HAR ＊ 216、HYS ＊ 217、HYS ＊ 219、HYS ＊ 220、HYS ＊ 222、HYS ＊ 2224、HAR ＊ 230、HYS ＊ 242、HAR ＊ 253、HYS ＊ 260、HYS ＊ 274アレル ＊ 301、HYS ＊ 302、HYS ＊ 0305、HYS ＊ 307、HYS ＊ 1101、HYS ＊ 102、HYS ＊ 1103、HYS ＊ 116アレル ＊ 1101、HARSH ＊ 1102、HYS ＊ 1103、HARSH ＊ 1104、HAR ＊ 1105、HARSH ＊ 1119アレル ＊ 2402、HAR ＊ 2403、HYS ＊ 2405、DYS ＊ 2407、HAR ＊ 2408、HAR ＊ 2410、HLA-A ＊ 2414、HLA-A ＊ 2417、HLA-A ＊ 2420、HLA-A ＊ 2422、HLA-A ＊ 2425、HLA-A ＊ 2426、HLA-A ＊ 2458アリール、HLA-B ＊ 0702、HLA-B ＊ 0704、HLA-B ＊ 0705、HLA-B ＊ 0709、HLA-B ＊ 0710、HLA-B ＊ 0715、HLA-B ＊ 0721, HLA-C ＊ 0702, HLA-C ＊ 0701, HLA-C ＊ 0401、HLA-C ＊ 0602、HLA-C ＊ 0304、HLA-C ＊ 0501、HLA-C ＊ 1601、HLA-C ＊ 0202、HLA-C ＊ 0303、HLA-C ＊ 1203、HLA-C ＊ 0802、HLA-C ＊ 0102、HLA-C ＊ 1701, HLA-C ＊ 1502、HLA-C ＊ 1402、HLA-C ＊ 1202、HLA-C ＊ 0704、HLA-C ＊ 0801、HLA-C ＊ 0302、HLA-C ＊ 1801、HLA-C ＊ 1505、HLA-C ＊ 1602、HLA-C ＊ 0804、HLA-C ＊ 0305、ZHLA-C ＊ 1403アラル The composition according to any one of claims 9 to 11, selected from the group consisting of:

12. A stable MHC-peptide complex comprising an immunogenic peptide according to any one of claims 1 to 4 in association with an MHC molecule.

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

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

15. The MHC molecule is \LA-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、-HLA-C ＊ 18 and optionally, the HLA allele comprises an MHC alpha chain that is an HLA serotype selected from the group consisting of: EXPOSED ＊ 0201、HYS ＊ 202、HYS ＊ 203、HYS ＊ 0204、HYS ＊ 2005、DYS ＊ 206、HYS ＊ 2007、HYS ＊ 210、HYS ＊ 211. CHRISTIAN ＊ 212、HYS ＊ 213、HYS ＊ 214、HAR ＊ 216、HYS ＊ 217、HYS ＊ 219、HYS ＊ 220、HYS ＊ 222、HYS ＊ 2224、HAR ＊ 230、HYS ＊ 242、HAR ＊ 253、HYS ＊ 260、HYS ＊ 274アレル ＊ 301、HYS ＊ 302、HYS ＊ 0305、HYS ＊ 307、HYS ＊ 1101、HYS ＊ 102、HYS ＊ 1103、HYS ＊ 116アレル ＊ 1101、HARSH ＊ 1102、HYS ＊ 1103、HARSH ＊ 1104、HAR ＊ 1105、HARSH ＊ 1119アレル ＊ 2402、HAR ＊ 2403、HYS ＊ 2405、DYS ＊ 2407、HAR ＊ 2408、HAR ＊ 2410、HLA-A ＊ 2414、HLA-A ＊ 2417、HLA-A ＊ 2420、HLA-A ＊ 2422、HLA-A ＊ 2425、HLA-A ＊ 2426、HLA-A ＊ 2458アリール、HLA-B ＊ 0702、HLA-B ＊ 0704、HLA-B ＊ 0705、HLA-B ＊ 0709、HLA-B ＊ 0710、HLA-B ＊ 0715、HLA-B ＊ 0721, HLA-C ＊ 0702, HLA-C ＊ 0701, HLA-C ＊ 0401、HLA-C ＊ 0602、HLA-C ＊ 0304、HLA-C ＊ 0501、HLA-C ＊ 1601、HLA-C ＊ 0202、HLA-C ＊ 0303、HLA-C ＊ 1203、HLA-C ＊ 0802、HLA-C ＊ 0102、HLA-C ＊ 1701, HLA-C ＊ 1502、HLA-C ＊ 1402、HLA-C ＊ 1202、HLA-C ＊ 0704、HLA-C ＊ 0801、HLA-C ＊ 0302、HLA-C ＊ 1801、HLA-C ＊ 1505、HLA-C ＊ 1602、HLA-C ＊ 0804、HLA-C ＊ 0305、ZHLA-C ＊ 1403アラル The stable MHC-peptide complex of any one of claims 12 to 14, selected from the group consisting of:

16. 16. The stable MHC-peptide complex of any one of claims 12 to 15, wherein the peptide epitope and the MHC molecule are covalently linked and / or the alpha and beta chains of the MHC molecule are covalently linked.

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

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

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

20. A vector comprising the isolated nucleic acid of claim 19.

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

22. 1. An apparatus or kit comprising: a) one or more immunogenic peptides according to any one of claims 1 to 4, and / or b) one or more stable MHC-peptide complexes according to any one of claims 12 to 17. Including, Optionally, a reagent for detecting binding of a) and / or b) to the binding protein; Optionally, the binding protein comprises: An antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain That is, The device or kit.

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

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

25. The method according to any one of claims 22 to 24, wherein said detecting and / or determining is carried out by means of fluorescence activated cell sorting (FACS), by means of an enzyme-linked immunosorbent assay (ELISA), by means of a radioimmunoassay (RIA), immunochemically, by means of a Western blot or by means of an intracellular flow assay.

26. The sample is T cells in contact with or suspected of being in contact with one or more MAGEA1 proteins or fragments thereof The method according to any one of claims 22 to 25, comprising:

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

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

29. 1. A method for assessing the efficacy of a therapy for a disorder characterized by MAGEA1 expression, comprising: a) determining in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides according to any one of claims 1 to 4 or one or more stable MHC-peptide complexes according to any one of claims 12 to 17; and b) determining the presence or level of reactivity between said immunogenic peptide according to any one of claims 1 to 4 or said stable MHC-peptide complex according to any one of claims 12 to 17 and T cells obtained from said subject present in a second sample obtained from said subject after provision of said therapy to said subject. Including, The presence or higher level of said reactivity in said second sample compared to said first sample is an indication that said therapy is effective in treating said disorder characterized by MAGEA1 expression in said subject. The method.

30. The level of reactivity is a) the presence of a bond, and / or b) T cell activation and / or effector function and optionally said T cell activation or effector function is T cell proliferation, killing or cytokine release.

31. repeating steps a) and b) at a subsequent time point; Optionally, between the first time point and the subsequent time point, the subject has undergone a treatment to ameliorate the disorder characterized by MAGEA1 expression. The method according to any one of claims 27 to 30.

32. 32. The method of any one of claims 27 to 31, wherein the T cell binding, activation and / or effector function is detected by means of fluorescence activated cell sorting (FACS), by means of enzyme-linked immunosorbent assay (ELISA), by means of radioimmunoassay (RIA), immunochemically, by means of Western blot, or by means of an intracellular flow assay.

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

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

35. A method for 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 described in any one of claims 1 to 22.

36. 1. A method for identifying a peptide binding molecule, or an antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, comprising: a) providing a cell presenting a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule on the surface of said cell; b) determining the binding of a plurality of candidate peptide binding molecules or antigen-binding fragments thereof to the peptide epitope in the context of the MHC molecule on the cell; and c) identifying one or more peptide binding molecules or antigen-binding fragments thereof that bind to said peptide epitope in the context of said MHC molecule; The method comprising:

37. The step a) contacting said MHC molecule on said surface of said cell with a peptide epitope selected from the peptide sequences listed in Table 1.

37. The method of claim 36, comprising:

38. The step a) expressing said peptide epitope selected from the peptide sequences listed in Table 1 in said cell using a vector comprising a heterologous sequence encoding said peptide epitope.

37. The method of claim 36, comprising:

39. 1. A method for identifying a peptide binding molecule, or an antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, comprising: a) providing a peptide epitope, alone or in the context of an MHC molecule, selected from the peptide sequences listed in Table 1, either alone or in a stable MHC-peptide complex; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to said peptide or said stable MHC-peptide complex; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to said peptide epitope or said stable MHC-peptide complex. Including, Optionally, the MHC or the MHC-peptide complex is as described in any one of claims 8 to 17. The method.

40. 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 40. The method of claim 39, comprising:

41. The plurality of candidate peptide-binding molecules may comprise at least 2, 5, 10, 100, 10 ３ , 10 ４ , 10 ５ , 10 ６ , 10 ７ , 10 ８ , 10 ９ 41. The method of claim 39 or claim 40, comprising a species or more of different candidate peptide binding molecules.

42. the plurality of candidate peptide-binding molecules comprises one or more candidate peptide-binding molecules obtained from a sample from a subject or a population of subjects; or The plurality of candidate peptide-binding molecules includes one or more candidate peptide-binding molecules that contain a mutation in a parent scaffold peptide-binding molecule obtained from a sample from a subject.

42. The method according to any one of claims 39 to 41.

43. The subject or population of subjects a) is not afflicted with a disorder characterized by MAGEA1 expression and / or has recovered from a disorder characterized by MAGEA1 expression, or b) suffering from a disorder characterized by MAGEA1 expression; 43. The method of claim 42.

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

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

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

48. The peptide-binding molecule or antigen-binding fragment thereof identified according to any one of claims 39 to 48, optionally comprising: An antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain The peptide-binding molecule or antigen-binding fragment thereof,

49. 1. A method of treating a disorder characterized by MAGEA1 expression in a subject, comprising: A peptide-binding molecule or antigen-binding fragment thereof, which i) binds to a peptide epitope selected from the sequences listed in Table 1, ii) is identified according to the method of any one of claims 39 to 48, 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. administering to the subject a therapeutically effective amount of engineered T cells expressing: An antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain and optionally the MHC or the MHC-peptide complex is according to any one of claims 8 to 17.

50. The T cell is a) the subject, b) a donor not suffering from said disorder characterized by MAGEA1 expression, or c) Donors who have recovered from a disorder characterized by MAGEA1 expression.

50. The method of claim 49, wherein the compound is isolated from

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

52. 52. The method of claim 51, wherein the agent is contacted under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, the immunogenic peptide, the stable MHC-peptide complex, the T cell receptor and / or the immune cell.

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

54. 54. The method of any one of claims 23 to 53, wherein the disorder characterized by MAGEA1 expression is cancer or its recurrence, and optionally 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. A method according to any one of claims 23 to 54, wherein the subject is an animal model of a disorder characterized by MAGEA1 expression, and / or a mammal, 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, to an immunogenic peptide according to any one of claims 1 to 4, and / or to a stable MHC-peptide complex according to any one of claims 12 to 17, optionally comprising An antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain The binding protein,

57. a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity with a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence having at least about 80% identity with a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2. Including, the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex; Optionally, the binding affinity is about 5×10 －４ K below M ｄ having 57. The binding protein of claim 56.

58. a) TCR alpha chain variable (V α ) TCR V domain sequences selected from the group consisting of α A TCR V domain sequence having at least about 80% identity with α Domain sequences; and / or b) TCR beta chain variable (V β ) TCR V domain sequences selected from the group consisting of β A TCR V domain sequence having at least about 80% identity with β Domain Array Including, the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex; Optionally, the binding affinity is about 5×10 －４ K below M ｄ having 57. The binding protein of claim 56.

59. a) a TCR alpha chain sequence having at least about 80% identity with a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence having at least about 80% identity with a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2. Including, the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex; Optionally, the binding affinity is about 5×10 －４ K below M ｄ having 57. The binding protein of claim 56.

60. a) a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2; and / or b) a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2 Including, the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex; Optionally, the binding affinity is about 5×10 －４ K below M ｄ having 57. The binding protein of claim 56.

61. a) TCR alpha chain variable (V α ) TCR V domain sequences selected from the group consisting of α Domain sequences; and / or b) TCR beta chain variable (V β ) TCR V domain sequences selected from the group consisting of β Domain Array Including, the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex; Optionally, the binding affinity is about 5×10 －４ K below M ｄ having 57. The binding protein of claim 56.

62. a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2 Including, the binding protein is capable of binding to a MAGEA1 immunogenic peptide-MHC (pMHC) complex; Optionally, the binding affinity is about 5×10 －４ K below M ｄ having 57. The binding protein of claim 56.

63. 1) The TCR alpha chain CDR, TCR V α and / or the TCR alpha chain is encoded by a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ and TRAC genes listed in Table 2; and / or 2) The TCR beta chain CDR, TCR V β and / or the TCR beta chain is encoded by a TRBV, TRBJ and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ and TRBC genes listed in Table 2; and / or 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof, compared to the cognate reference CDR sequence listed in Table 2; 63. The binding protein according to any one of claims 56 to 62.

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

65. The binding protein is a transmembrane domain, and Intracellular effector domains 65. The binding protein of any one of claims 56 to 64, comprising a binding domain having the following structure:

66. 66. The binding protein of any one of claims 56-65, wherein the TCR alpha chain and the TCR beta chain are covalently linked, and optionally, the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide.

67. 67. The binding protein of any one of claims 56 to 66, wherein the TCR alpha chain and / or the TCR beta chain are covalently linked to a substructure, optionally wherein the covalently linked substructure comprises an affinity tag or label.

68. the affinity tag is selected from the group consisting of a CD34 enrichment tag, a glutathione-S-transferase (GST), a calmodulin-binding protein (CBP), a protein C tag, a Myc tag, a Halo tag, a HA tag, a Flag tag, a His tag, a biotin tag and a V5 tag; and / or The label is a fluorescent protein.

68. The binding protein of claim 67.

69. 69. The binding protein of any one of claims 56-68, wherein the covalently linked moiety is selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof.

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

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

72. Binding of the binding protein to the MAGEA1 peptide-MHC (pMHC) complex elicits an immune response, and optionally, the immune response comprises: i) a T cell response and / or a CD8+ T cell response; and / or ii) selected from the group consisting of T cell proliferation, cytokine release and / or cytotoxic killing; A binding protein according to any one of claims 56 to 71.

73. The binding protein binds to the MAGEA1 immunogenic peptide-MHC (pMHC) complex at a concentration of about 1×10 －４ M or less, approximately 5 x 10 －５ M or less, approximately 1×10 －５ M or less, approximately 5 x 10 －６ M or less, approximately 1×10 －６ M or less, approximately 5 x 10 －７ M or less, approximately 1×10 －７ M or less, approximately 5 x 10 －８ M or less, approximately 1×10 －８ M or less, approximately 5 x 10 －９ M or less, approximately 1×10 －９ M or less, approximately 5 x 10 －１０ M or less, approximately 1×10 －１０ M or less, approximately 5 x 10 －１１ M or less, approximately 1×10 －１１ M or less, approximately 5 x 10 －１２ M or less, or about 1 x 10 －１２ K below M ｄ 73. The binding protein of any one of claims 56 to 72, which is capable of specifically and / or selectively binding to

74. 74. The binding protein of any one of claims 56-73, wherein the binding protein has a higher binding affinity to the peptide-MHC (pMHC) compared to a known T cell receptor, optionally the higher binding affinity is at least 1.05 fold higher.

75. 75. The binding protein of any one of claims 56 to 74, wherein the binding protein induces a stronger T cell proliferation, cytokine release and / or cytotoxic killing when contacted with a target cell having heterozygous expression of MAGEA1 compared to a known T cell receptor, optionally wherein the induction is at least 1.05 fold stronger.

76. 76. The binding protein of claim 75, wherein the cytotoxic kill is a target cancer cell.

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

78. The binding protein is ALKDVEERV / HLA-A ＊ 02, LLFGLALIEV / HLA-A ＊ 02, SESIKKKVL / HLA-B ＊ 44, ASSTLYLVF / HLA-B ＊ 57, and pMHC complexes comprising LAMB4-IR, TLE3, SLC16A2, SLC6A13, BTBD2, RXFP1 and / or PIGO peptide epitopes.

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

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

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

82. 82. A vector comprising the isolated nucleic acid of claim 80 or claim 81, optionally comprising: 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; The vector.

83. 83. The vector of claim 82, wherein 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).

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

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

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

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

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

89. 82. A host cell comprising the isolated nucleic acid of claim 80 or claim 81, and / or comprising a vector according to any one of claims 82 to 88; Expressing the binding protein according to any one of claims 56 to 78, Optionally, the cells are genetically engineered. The host cell.

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

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

92. The host cell comprises TCR alpha variable region gene, TCR beta variable region gene, TCR constant region gene, and combinations thereof The host cell according to any one of claims 89 to 91, comprising a knockout of a TCR gene selected from:

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

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

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

96. The immune cells include T cells, cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 ＋ T cells, CD8 ＋ 96. The host cell of claim 95, which is a T cell, a CD4 / CD8 double negative T cell, a gamma delta (γδ) T cell, a natural killer (NK) cell, an NK-T cell, a dendritic cell, or a combination thereof.

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

99. 99. The host cell of any one of claims 89 to 98, wherein the T cell does not express or has reduced surface expression of an endogenous TCR.

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

101. The host cell of claim 100, wherein the host cell is contacted with the target cell in vitro, ex vivo or in vivo.

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

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

104. A host cell according to any one of claims 89 to 103, which is capable of producing higher levels of cytokines or cytotoxic molecules when contacted with a target cell having heterozygous expression of MAGEA1.

105. The host cell of claim 104, wherein the host cell is capable of producing at least 1.05-fold higher levels of a cytokine or cytotoxic molecule.

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

107. The host cell of claim 106, wherein the killing is determined by a killing assay.

108. 108. The host cell of claim 106 or claim 107, wherein the ratio of said host cells to said target cells in said killing assay is between 20:1 and 1:

4.

109. The host cell according to any one of claims 106 to 108, wherein the target cell is a target cell pulsed with 1 μg / mL to 50 pg / mL of MAGEA1 peptide, and optionally the target cell is a monoallelic cell with respect to MHC matching the MAGEA1 peptide.

110. The host cell of any one of claims 106 to 109, wherein the host cell is capable of killing a greater number of target cells when contacted with target cells having heterozygous expression of MAGEA1, and optionally the cell killing is at least 1.05-fold greater.

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

113. The host cell comprises LAMB4-IR, TLE3, SLC16A2, SLC6A13, BTBD2, RXFP1 and / or PIGO peptide epitopes 113. The host cell of any one of claims 89 to 112, which does not induce T cell proliferation, cytokine release or cytotoxic killing when contacted with a target cell comprising a peptide-MHC (pMHC) complex comprising:

114. The host cell comprises does not express the MAGEA1 antigen; Not recognized by the binding protein according to any one of claims 56 to 78, Serotype HLA-C ＊ Not of 07, and / or HLA-C ＊ does not express the 07 allele, A host cell according to any one of claims 89 to 113.

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

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

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

118. A method for producing a binding protein according to any one of claims 56 to 77, comprising the steps of: (i) culturing a transformed host cell, transformed with a nucleic acid comprising a sequence encoding a binding protein according to any one of claims 56 to 77, under suitable conditions allowing expression of said binding protein; and (ii) recovering the expressed binding protein. The method comprising:

119. A method for producing a host cell expressing a binding protein according to any one of claims 56 to 77, comprising the steps of: (i) introducing into said host cell a nucleic acid comprising a sequence encoding a binding protein according to any one of claims 56 to 77; and (ii) culturing the transformed host cell under suitable conditions to allow expression of the binding protein. The method comprising:

120. 1. A method for detecting the presence or absence of a MAGEA1 antigen and / or a cell expressing MAGEA1, comprising: Optionally, the cell is a hyperproliferative cell; The method comprises: At least one binding protein according to any one of claims 56 to 77, At least one host cell according to any one of claims 89 to 114, or The population of host cells of claim 115. detecting the presence or absence of said MAGEA1 antigen in a sample by using The detection of the MAGEA1 antigen indicates the presence of the MAGEA1 antigen and / or cells expressing MAGEA1; The method.

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

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

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

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

125. The method of claim 123 or claim 124, wherein the control level is a level from a subject who does not have the disorder characterized by MAGEA1 expression.

126. 1. A method for tracking and assessing the progression of a disorder characterized by MAGEA1 expression in a subject, comprising: a) detecting in a subject sample the presence or level of reactivity between a sample obtained from said subject and at least one binding protein according to any one of claims 56 to 77, at least one host cell according to any one of claims 89 to 114, or a population of host cells according to claim 115; b) repeating step a) at a subsequent time point; and c) comparing the levels of MAGEA1 or of cells of interest expressing MAGEA1 detected in steps a) and b) to follow and assess the progression of said disorder characterized by MAGEA1 expression in said subject. Including, The absence or reduction of the MAGEA1 level or the cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates an inhibition of the progression of the disorder characterized by MAGEA1 expression in the subject, The presence or increase of MAGEA1 levels or of the cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates the progression of the disorder characterized by MAGEA1 expression in the subject. The method.

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

128. 1. A method for predicting a clinical outcome in a subject suffering from a disorder characterized by MAGEA1 expression, comprising: a) determining the presence or level of reactivity between a sample obtained from said subject and at least one binding protein according to any one of claims 56 to 77, at least one host cell according to any one of claims 89 to 114, or a population of host cells according to claim 115; and b) comparing the presence or level of said reactivity with that from a control. Including, The control is obtained from a subject with a good clinical outcome, An absence or reduced level of the reactivity in the subject sample compared to the control indicates that the subject will have a favorable clinical outcome. The method.

129. 1. A method for assessing the efficacy of a therapy for a disorder characterized by MAGEA1 expression, comprising: a) determining in a first sample obtained from the subject prior to providing to the subject at least a portion of said therapy for said disorder characterized by MAGEA1 expression, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein according to any one of claims 56 to 77, at least one host cell according to any one of claims 89 to 114, or a population of host cells according to claim 115; and b) determining in a second sample obtained from the subject after the provision of said therapy for said disorder characterized by MAGEA1 expression the presence or level of reactivity between the sample obtained from the subject and at least one binding protein according to any one of claims 56 to 77, at least one host cell according to any one of claims 89 to 114 or a population of host cells according to claim 115. Including, an absence or decreased level of said responsiveness in said second sample compared to said first sample is an indication that said therapy is effective in treating said disorder characterized by MAGEA1 expression in said subject; The presence or elevated level of said reactivity in said second sample compared to said first sample is an indication that said therapy is ineffective in treating said disorder characterized by MAGEA1 expression in said subject. The method.

130. The level of reactivity is a) the presence of a bond, and / or b) T cell activation and / or effector function is shown by Optionally, the T cell activation or effector function is T cell proliferation, killing, or cytokine release.

130. The method of any one of claims 120 to 129.

131. 131. The method of any one of claims 120 to 130, wherein the T cell binding, activation and / or effector function is detected by means of fluorescence activated cell sorting (FACS), by means of enzyme-linked immunosorbent assay (ELISA), by means of radioimmunoassay (RIA), immunochemically, by means of Western blot, or by means of an intracellular flow assay.

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

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

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

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

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

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

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

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

140. and further comprising administering at least one additional treatment for said disorder characterized by MAGEA1 expression; Optionally, the at least one additional treatment for the disorder characterized by MAGEA1 expression is administered simultaneously or sequentially with the composition. A method according to any one of claims 49 to 55 and claims 132 to 139.

141. The method of any one of claims 132 to 140, wherein the disorder characterized by MAGEA1 expression is cancer or its recurrence, and optionally, 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. A method according to any one of claims 132 to 141, wherein the subject is an animal model of a disorder characterized by MAGEA1 expression, and / or a mammal, optionally wherein the mammal is a human, a primate or a rodent.

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