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.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 546,105, filed October 27, 2023, and U.S. Provisional Application Serial No. 63 / 554,734, filed February 16, 2024; the entire contents of each of these applications are incorporated herein by reference in their entirety. Background Technology

[0002] Adoptive cell transfer (ACT) using engineered T cells has demonstrated significant efficacy in treating certain types of liquid tumors and holds promise for treating solid tumors. T-cell receptor-engineered T cells (TCR-T) are T cells that express exogenous TCRs capable of recognizing antigens present in 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 to the widespread testing and adoption of TCR-T therapy is the lack of TCR-antigen pairs suitable for a broad range of patients and indications.

[0003] Furthermore, the discovery of novel TCR-antigen pairs is challenging, often requiring prediction of MHC-presented epitopes, thus limiting the number of antigens that can be tracked. However, such epitopes may lack immunogenicity, making it difficult to identify reactive TCRs, or they may fail to be physiologically processed and presented by cancer cells. Therefore, there is a strong need in the art to identify TCR-antigen pairs within a broadly applicable HLA allele spectrum in order to develop useful agents for diagnosing, prognosing, and treating conditions characterized by said antigen expression, and to screen agents associated with said conditions. Summary of the Invention

[0004] This 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 unbiased functional screening to identify antigens of TCR clones identified in subjects with conditions associated with MAGEA1 expression (e.g., 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 recognize MAGEA1 immunogenic peptides in a background of multiple HLA alleles (e.g., HLA-A*01:01, expressed in approximately 24% of the US population), such as those listed in Table 1. This paper demonstrates that MAGEA1 is selectively expressed in cancerous and testicular tissues but not in normal somatic tissues, thus making it an ideal target for ACT. The ability of MAGEA1-binding proteins (such as TCR as described herein) to bind to MAGEA1 immunogenic peptides and elicit an immune response that kills MAGEA1-expressing cells (such as cancer cells) demonstrates the utility of such binding proteins in a variety of applications, including methods for diagnosing, prognosticating, treating diseases characterized by MAGEA1 expression, and screening agents associated with said diseases.

[0005] MAGEA1 presented on HLA A01:01 was found 161-169 Epitope-specific TCRs are particularly challenging, and the results are often unexpected. Specifically, because the HLA-A*01:01 allele is expressed in only about 24% of the US population, the number of available peripheral blood mononuclear cells (PBMCs) for identifying HLA-A*01:01-specific TCRs is limited. Experience also shows that the T cell clonal frequency of HLA-A*01:01 (e.g., 1 clone per 5E6 naïve T cells) is significantly lower than that of other HLA alleles, such as HLA-A*02:01, which has a clonal frequency of over 20 clones per 5E6 naïve T cells. Therefore, more extensive screening of donors and T cells is required compared to other HLA alleles to identify candidate HLA-A*01:01-specific TCRs. Even among HLA-A*01:01-specific TCRs, empirical analysis further confirms that very few TCRs possess cytotoxic function. For example, as further described below, the expression frequency of the HLA-A*01:01 allele is significantly lower than that of other HLA alleles, such as HLA-A*02:01, which has a clonal frequency of over 200 clones per 5E6 naïve T cells. 161-169Functional screening of TCRs specific to the HLA*01:01-restricted epitope revealed 0 cytotoxic TCRs, and further screening of 1181 TCRs was required to identify 5 highly cytotoxic TCRs. In contrast, functional screening of 200 candidate TCRs specific to peptides restricted to other HLAs typically yielded a much higher yield of cytotoxic TCRs (e.g., functional screening of 200 candidate TCRs targeting the HLA-A*02:01 epitope identified 30 highly cytotoxic TCRs). Furthermore, less MAGEA1 was observed in peptide serial dilution assays, for example. 161-169 TCR showed favorable affinity. See also Chakraborty et al. (2004) Hum. Immunol. 65:794-802; Podaza et al. (2020) Front. Immunol. 11:1147; and Traversari et al. (1992) J. Exp. Med. 176:1453-1457.

[0006] In one aspect, an immunogenic peptide is provided, the immunogenic peptide comprising a peptide epitope selected from the peptide sequences listed in Table 1.

[0007] On the other hand, an immunogenic peptide is provided, which is composed of peptide epitopes selected from the peptide sequences listed in Table 1.

[0008] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the immunogenic peptide is derived from the MAGEA1 protein, optionally wherein the length of said 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 another embodiment, the immunogenic peptide is capable of eliciting an immune response against MAGEA1 and / or cells expressing MAGEA1 in a subject, optionally wherein said immune response is i) a T cell response and / or a CD8+ T cell response and / or ii) selected from the group consisting of T cell expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing.

[0009] In another aspect, an immunogenic composition is provided comprising at least one immunogenic peptide described herein.

[0010] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the immunogenic composition further comprises an adjuvant. In another embodiment, the immunogenic composition is capable of inducing an immune response against MAGEA1 and / or cells expressing MAGEA1 in a subject, optionally said immune response being i) a T cell response and / or a CD8+ T cell response and / or ii) selected from the group consisting of T cell expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing.

[0011] In another aspect, a composition is provided comprising a peptide epitope selected from the peptide sequences listed in Table 1, and an MHC molecule.

[0012] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally wherein said MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule comprises an MHC α chain, said chain being the HLA serotype HLA-A*01, optionally wherein the HLA allele is HLA-A*01:01.

[0013] On the other hand, a stable MHC-peptide complex is provided, the stable MHC-peptide complex comprising the immunogenic peptides described herein in an MHC molecular background.

[0014] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally wherein said MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule comprises an MHC α chain, said chain being the HLA serotype HLA-A*01, optionally wherein the HLA allele is HLA-A*01:01. In yet another embodiment, the peptide epitope and the MHC molecule are covalently linked and / or wherein the α and β chains of the MHC molecule are covalently linked. In another embodiment, the stable MHC-peptide complex comprises a detectable label, optionally wherein said detectable label is a fluorophore.

[0015] In another aspect, an immunogenic composition is provided comprising the stable MHC-peptide complex and adjuvant described herein.

[0016] In another aspect, an isolated nucleic acid is provided that encodes the immunogenic peptide described herein, or its complement.

[0017] On the other hand, a vector is provided that contains the isolated nucleic acids described herein.

[0018] In another aspect, a cell is provided that: a) contains isolated nucleic acids as described herein, b) contains a vector as described herein, and / or c) produces one or more immunogenic peptides as described herein and / or presents one or more stable MHC-peptide complexes as described herein on its cell surface, optionally wherein the cell is genetically engineered.

[0019] In another aspect, an apparatus or kit is provided comprising: a) one or more immunogenic peptides described herein and / or b) one or more stable MHC-peptide complexes described herein, wherein the apparatus or kit optionally comprises reagents for detecting the 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.

[0020] In another aspect, a method for detecting T cells bound to a stable MHC-peptide complex is provided, the method comprising: a) contacting a sample containing T cells with the stable MHC-peptide complex described herein; and b) detecting the binding of T cells to the stable MHC-peptide complex, optionally further determining the percentage of stable MHC-peptide-specific T cells bound to the stable MHC-peptide complex, optionally wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

[0021] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the T cells are CD8+ T cells. In another embodiment, detection and / or assay are performed using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow cytometry. In yet another embodiment, the sample comprises T cells that have been exposed to or are suspected of being exposed to one or more MAGEA1 proteins or fragments thereof.

[0022] In another aspect, a method is provided for determining whether T cells have been exposed to MAGEA1, the method comprising: a) incubating a cell population containing T cells with an immunogenic peptide or a stable MHC-peptide complex as described herein; and b) detecting the presence or level of reactivity, wherein the presence of reactivity or a higher level of reactivity compared to a control level indicates that the T cells have been exposed to MAGEA1, optionally wherein the cell population containing T cells is obtained from a subject.

[0023] In another aspect, a method is provided for predicting clinical outcomes in subjects suffering from a condition characterized by MAGEA1 expression, the method comprising: a) determining the presence or level of responsiveness 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 said responsiveness with responsiveness from a control obtained from a subject with good clinical outcomes, wherein the presence of responsiveness or a higher level of responsiveness in the subject's sample compared to the control indicates that the subject has good clinical outcomes.

[0024] On the other hand, a method is provided for evaluating the efficacy of a therapy for a condition characterized by MAGEA1 expression, the method comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein in a first sample obtained from the subject before administering at least a portion of the therapy; 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, the T cells being present in a second sample obtained from the subject after administering the therapy, wherein the presence or higher level of reactivity in the second sample relative to the first sample indicates that the therapy is effective in treating the subject's condition characterized by MAGEA1 expression.

[0025] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the level of reactivity is indicated by the presence of a) binding and / or b) T cell activation and / or effector function, optionally wherein said T cell activation or effector function is T cell proliferation, killing, or cytokine release. In another embodiment, the method further includes repeating steps a) and b) at subsequent time points, optionally wherein said subject has been treated between the first and subsequent time points to improve the condition characterized by MAGEA1 expression. In another embodiment, T cell binding, activation, and / or effector function are detected using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting, or intracellular flow cytometry. In yet another embodiment, the control level is a reference figure. In yet another embodiment, the control level is the level in subjects who do not have the condition characterized by MAGEA1 expression.

[0026] In another aspect, a method for preventing and / or treating a condition characterized by MAGEA1 expression in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition described herein.

[0027] In another aspect, a method is provided for identifying peptide-binding molecules or antigen-binding fragments thereof that bind to peptide epitopes selected from peptide sequences listed in Table 1, the method comprising: a) providing cells that present peptide epitopes selected from peptide sequences listed in Table 1 in an MHC molecular background on their cell surface; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof on the cells to peptide epitopes in the MHC molecular background; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to peptide epitopes in the MHC molecular background.

[0028] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, step a) includes contacting an MHC molecule on the cell surface with a peptide epitope selected from the peptide sequences listed in Table 1. In another embodiment, step a) includes expressing a peptide epitope selected from the peptide sequences listed in Table 1 in the cell using a vector containing a heterologous sequence encoding the peptide epitope.

[0029] In another aspect, a method is provided for identifying peptide-binding molecules or antigen-binding fragments thereof that bind to peptide epitopes selected from peptide sequences listed in Table 1, the method comprising: a) providing a peptide epitope, alone or in the form of a stable MHC-peptide complex, comprising a peptide epitope, alone or in an MHC molecule background, selected from peptide sequences listed in Table 1; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to a peptide or a stable MHC-peptide complex; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to a peptide epitope or a stable MHC-peptide complex, optionally wherein the MHC or MHC-peptide complex is as described herein.

[0030] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments 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, or 10 3 10 species 4 10 species 5 10 species 6 10 species 7 10 species 8 10 species 9 One or more different candidate peptide-binding molecules. In another embodiment, the multiple candidate peptide-binding molecules comprise one or more candidate peptide-binding molecules obtained from a sample from a subject or subject population; or the multiple candidate peptide-binding molecules comprise one or more candidate peptide-binding molecules containing a mutation in a parental scaffold peptide-binding molecule obtained from a sample from a subject. In yet another embodiment, the subject or subject population: a) does not have the condition characterized by MAGEA1 expression and / or has recovered from the condition characterized by MAGEA1 expression, or b) has the condition characterized by MAGEA1 expression. In another embodiment, the composition described herein has been administered to the subject or subject population. In another embodiment, the subject is an animal model and / or mammal of the condition characterized by MAGEA1 expression, optionally said mammal being a human, primate, or rodent. In yet another embodiment, the subject is an animal model of the condition 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.

[0031] In another aspect, peptide-binding molecules or antigen-binding fragments thereof identified according to the methods described herein are provided, 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.

[0032] In another aspect, a method is provided for treating a subject with a condition characterized by MAGEA1 expression, the method comprising administering to the subject a therapeutically effective amount of genetically engineered T cells expressing a peptide-binding molecule or an antigen-binding fragment thereof, the peptide-binding molecule or the antigen-binding fragment thereof i) binding to a peptide epitope selected from sequences listed in Table 1, ii) being identified according to the method described herein, and / or iii) binding to a stable MHC-peptide complex containing a peptide epitope selected from sequences listed in Table 1 in a background of MHC molecules, optionally wherein the peptide-binding molecule or the antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain, optionally wherein the MHC or MHC-peptide complex is as described herein.

[0033] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, T cells are isolated from: a) a subject, b) a donor not suffering from a disease characterized by MAGEA1 expression, or c) a donor who has recovered from a disease characterized by MAGEA1 expression.

[0034] On the other hand, a method is provided for treating a subject with a condition characterized by MAGEA1 expression, the method comprising infusing the subject with antigen-specific T cells, wherein the antigen-specific T cells are generated by: a) stimulating immune cells from the subject with the composition described herein; and b) expanding the antigen-specific T cells in vitro or ex vivo, optionally i) isolating immune cells from the subject prior to stimulating the immune cells and / or ii) wherein the immune cells comprise PBMCs, T cells, CD8+ T cells, naive T cells, central memory T cells, and / or effector memory T cells.

[0035] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the agent is placed in contact with the peptide epitope, immunogenic peptide, stable MHC-peptide complex, T-cell receptor, and / or immune cell under conditions and for a time suitable for the formation of at least one immune complex. 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 another embodiment, the condition characterized by MAGEA1 expression is cancer or a recurrence thereof, optionally said cancer being selected from the group consisting of melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma. In yet another embodiment, the subject is an animal model and / or mammal of a condition characterized by MAGEA1 expression, optionally said mammal being a human, primate, or rodent.

[0036] In another aspect, a binding protein is provided that binds a polypeptide comprising the immunogenic peptide sequence described herein, the immunogenic peptide described herein, and / or the stable MHC-peptide complex described herein, optionally wherein the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0037] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the binding protein comprises: a) a T-cell receptor (TCR) α-chain CDR sequence having at least about 80% identity with a TCR α-chain CDR sequence selected from the group of TCR α-chain CDR sequences listed in Table 2; and / or b) a TCR β-chain CDR sequence having at least about 80% identity with a TCR β-chain CDR sequence selected from the group of TCR β-chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a value less than or equal to about 5 × 10⁻⁶. -4 M of K d In another embodiment, the binding protein comprises: a) a variable TCR α chain (V α The sequence of structural domains, which are selected from the TCR V listed in Table 2 α TCR V of the group composed of domain sequences α The domain sequences have at least approximately 80% identity; and / or b) the TCR β chain is variable (Vβ The sequence of structural domains, which are selected from the TCR V listed in Table 2 β TCR V of the group composed of domain sequences β The domain sequences have at least about 80% identity, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has less than or equal to about 5 × 10⁻⁶. -4 M of K d In another embodiment, the binding protein comprises: a) a TCR α chain sequence having at least about 80% identity with a TCR α chain sequence selected from the group consisting of TCR α chain sequences listed in Table 2; and / or b) a TCR β chain sequence having at least about 80% identity with a TCR β chain sequence selected from the group consisting of TCR β chain sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has less than or equal to about 5 × 10⁻⁶. -4 M of K d In yet another embodiment, the binding protein comprises: a) a TCR α chain CDR sequence selected from the group consisting of TCR α chain CDR sequences listed in Table 2; and / or b) a TCR β chain CDR sequence selected from the group consisting of TCR β chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a binding affinity of less than or equal to about 5 × 10⁻⁶. -4 M of K d In yet another embodiment, a binding protein is provided, the binding protein comprising: a) a TCR α chain variable (V α The domain sequence is selected from the TCR V listed in Table 2. α Groups consisting of domain sequences; and / or b) TCR β-chain variable (V β The domain sequence is selected from the TCR V listed in Table 2. β A group of domain sequences, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a value of less than or equal to about 5 × 10⁻⁶. -4 M of K dIn another embodiment, a binding protein is provided, the binding protein comprising: a) a TCR α chain sequence selected from the group consisting of TCR α chain sequences listed in Table 2; and / or b) a TCR β chain sequence selected from the group consisting of TCR β chain sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a binding affinity of less than or equal to about 5 × 10⁻⁶. -4 M of K d In another embodiment, 1) TCR α chain CDR, TCR V α The domain and / or TCR α chain is encoded by the TRAV, TRAJ, and / or TRAC genes or fragments thereof selected from the groups of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) TCR β chain CDR, TCRV βThe domain and / or TCR β chain are encoded by TRBV, TRBJ, and / or TRBC genes or fragments 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 at most five amino acid substitutions, insertions, deletions, or combinations thereof compared to the homologous reference CDR sequences listed in Table 2. In another embodiment, the binding protein is chimeric, humanized, or human. In yet another embodiment, the binding protein comprises a binding domain having a transmembrane domain and an intracellular effector domain. In another embodiment, the TCR α chain and TCR β chain are covalently linked, optionally wherein the TCR α chain and TCR β chain are covalently linked through a linker peptide. In another embodiment, the TCR α chain and / or TCR β chain are covalently linked to a portion, optionally wherein the covalently linked portion comprises an affinity tag or label. In another embodiment, the affinity tag is selected from the group consisting of: CD34 enrichment tags, glutathione S-transferase (GST), calmodulin-binding protein (CBP), protein C tags, Myc tags, HaloTag, HA tags, Flag tags, His tags, biotin tags, and V5 tags, and / or those labeled as fluorescent proteins. In another embodiment, the covalently linked portion is selected from the group consisting of: pro-inflammatory factors, cytokines, toxins, cytotoxic molecules, radioactive isotopes, or antibodies or their antigen-binding fragments. In another embodiment, the binding protein binds to a pMHC complex on the cell surface. In yet another embodiment, MHC or MHC-peptide complexes are as described herein. In another embodiment, the binding protein binding to the MAGEA1 peptide-MHC (pMHC) complex triggers an immune response, optionally wherein said immune response is i) a T cell response and / or a CD8+ T cell response and / or ii) selected from the group consisting of T cell expansion, cytokine release, and / or cytotoxic killing. In another embodiment, the binding protein is capable of binding at a concentration of less than or equal to about 1 × 10⁻⁶. -4 M, less than or equal to approximately 5 × 10 -5 M, less than or equal to approximately 1 × 10 -5 M, less than or equal to approximately 5 × 10 -6 M, less than or equal to approximately 1 × 10 -6 M, less than or equal to approximately 5 × 10 -7 M, less than or equal to approximately 1 × 10 -7 M, less than or equal to approximately 5 × 10 -8 M, less than or equal to approximately 1 × 10 -8 M, less than or equal to approximately 5 × 10 -9 M, less than or equal to approximately 1 × 10 -9 M, less than or equal to approximately 5 × 10 -10 M, less than or equal to approximately 1 × 10 -10M, less than or equal to approximately 5 × 10 -11 M, less than or equal to approximately 1 × 10 -11 M, less than or equal to approximately 5 × 10 -12 M is less than or equal to approximately 1 × 10 -12 M of K dThe protein specifically and / or selectively binds to the MAGEA1 immunogenic peptide-MHC (pMHC) complex. In another embodiment, the binding protein has a higher binding affinity for peptide-MHC (pMHC) compared to known T cell receptors, optionally wherein the higher binding affinity is at least 1.05-fold higher. In another embodiment, upon contact with target cells exhibiting MAGEA1 heterozygous expression, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing compared to known T cell receptors, optionally wherein the induction is at least 1.05-fold higher. As used herein, in some embodiments, references to fold changes may be compared with any reference mode of interest, such as with different binding proteins; with the same binding protein in different backgrounds, such as in combination with other agents described herein, where the same binding protein is expressed at different levels in different immune cells; and so on. In another embodiment, cytotoxic killing targets cancer cells. In another implementation scheme, the cancers are 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 urothelial carcinoma of the bladder. In another embodiment, the binding protein does not bind to peptide-MHC (pMHC) complexes comprising the following: MAGEA10, MAGEA11, RPS6KA2, RPS6KA3, RPS6KA6, STIL, TECPR1, WDR45, CCDC168, SIRPB1, TENM1, ADAMTS20, CPO, SPATA22, and / or ZNF202 peptide epitopes (e.g., EVDPTGHSF MAGEA10 epitope, EVDPTSHSYMAGEA11 epitope, KEDIGVGSYSVCKRC RPS6KA2 epitope, KEDIGVGSYSVCKRC RPS6KA3 epitope, KEDIGVGSYSVCKRC RPS6KA6 epitope, QVQGTYKYGYLTMDETRKLLLL STIL epitope, CTKAGTKPPSLQWAWVSDWFVDFSVPGGTDQEGWQYASDFPASYHGSKTMKDFVRRRCWARKCKLVT). TECPR1 epitope, GTSSAPFTINAHQSDIACVSLNQPGTVVASASQKGTLIRLFDTQSKEKLVELRRGTDPATLYCINFS WDR45 epitope, MWQGENVADTFPNTTSFTPDSSDIKRQSGFQTEIDMRISGLSHTQPTQIESLAEGIARYSDPIDKRRTSNLVKGAKLHDRESGEEKQEHLCCDC268 epitope, TVSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDHVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDIT SIRPB1 epitope, PPTHTQFDFVKLMDGKQLVKQDSKGSDDTQHSPRNLILTSLQETGFIEYMDQGPWYLAFYNDGKKMEQVFVLTTAIEIMDDCSTNCNG TENM1 epitope, VFSKIRIDLTSMQIKTTDLLFSKTIFGNAVPFATAGDCYSAFRCPQGQFSINLSGTGMKISSTAKWLTQGSYTSVSIRRSEDGTRFFGKC ADAMTS20 epitope, TWTTDRLWRKSRSPHNNGTCFGTDLNRNFNASWCSIGASRNCQDQTFCGTGPVSEPETKAVASFIESKKDDILCFLTMHSYGQLILTPYG The CPO epitope, MKRSLNENSARSTADWAWEAVNPELAPVMKTVDTGQIPHSVSRPLRSQDSVFNSIQSNTGRSQGGWSYRDGNKNTSLKTWNKNDFKPQCK SPATA22 epitope and / or VALLTALSQGLVTFKDVAVCFSQDQWSDLDPTQKEFYGEYVLEED ZNF202 epitope.

[0038] These genes are well-known and are recognized for annotation according to the following NCBI gene IDs, each of which is available on the World Wide Web at ncbi.nlm.nih.gov / gene: ZNF202: Gene ID 7753 and NM_001301779.2 and NP_001288708.1, NM_001301780.2 and NP_001288709.1, NM_001301819.1 and NP_001288748.1, and NM_003455.4 and NP_003446.2 are used as representative clones; RPS6KA2: Gene ID 6196 and NM_001006932.3 and NP_001006933.3, NM_001318936.2 and NP_001305865.2, NM_001318937.2 and NP_001305866.1, NM_001318937.2 and NP_001305866.1, and NM_021135.6 and NP_066958.2 are used as representative clones; RPS6KA3: Gene ID 6197, NM_004586.3, and NP_004577.1 are used as representative clones; RPS6KA6: Gene ID 27330 and NM_001330512.1 and NP_001317441.1; and NM_014496.5 and NP_055311.1 are used as representative clones; MAGEA11: Gene ID 4110, NM_014496.5, and NP_055311.1; and NM_014496.5 and NP_055311.1 as representative clones; MAGEA10: Gene ID 4109 and NM_001011543.3 and NP_001011543.3; NM_001251828.2 and NP_001238757.2; and NM_021048.5 and NP_066386.3 are used as representative clones; WDR45: Gene ID 11152 and NM_001029896.2 and NP_001025067.1; and NM_001029896.2 and NP_001025067.1 as representative clones; TECPR1: Gene ID 25851, NM_015395.3, and NP_056210.1 are used as representative clones; CCDC168: Gene ID 643677, NM_001146197.3, and NP_001139669.1 are used as representative clones; SIRPB1: Gene ID 10326 and NM_001083910.4 and NP_001077379.1; NM_001135844.4 and NP_001129316.1; NM_001329157.2 and NP_001316086.1; NM_001330639.2 and NP_001317568.1; NM_006065.5 and NP_006056.2 are used as representative clones; TENM1: Gene ID 10178 and NM_001163278.2 and NP_001156750.1; NM_001163279.1 and NP_001156751.1; and NM_014253.3 and NP_055068.2 are used as representative clones; ADAMTS20: Gene ID 80070, NM_025003.5, and NP_079279.3 are used as representative clones; CPO: Gene ID 130749, NM_173077.3, and NP_775100.1 are used as representative clones; SPATA22: Gene ID 84690 and NM_001170695.2 and NP_001164166.1; NM_001170696.2 and NP_001164167.1; NM_001170697.2 and NP_001164168.1; NM_001170698.2 and NP_001164169.1; NM_001170699.2 and NP_001164170.1; NM_001321336.2 and NP_001308265.1; NM_001321337.2 and NP_001308266.1; and NM_032598.5 and NP_115987.2 as representative clones; and STIL: Gene ID 6491 and NM_001048166.1 and NP_001041631.1; NM_001282936.1 and NP_001269865.1; NM_001282937.1 and NP_001269866.1; NM_001282938.1 and NP_001269867.1; NM_001282939.1 and NP_001269868.1; NM_001377417.1 and NP_001364346.1; and NM_003035.2 and NP_003026.2 are used as representative clones.

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

[0040] On the other hand, an isolated nucleic acid molecule is provided, said isolated nucleic acid molecule: i) undergoes complement hybridization under stringent conditions with a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2; ii) has at least about 80% homology with a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2; and / or iii) has at least about 80% homology with a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2, optionally said isolated nucleic acid molecule comprising 1) TRAV, TRAJ and / or TRAC genes or fragments thereof selected from the group consisting of TRAV, TRAJ and TRAC genes listed in Table 2 and / or 2) TRBV, TRBJ and / or TRBC genes or fragments thereof selected from the group consisting of TRBV, TRBJ and TRBC genes listed in Table 2.

[0041] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, nucleic acids are codon-optimized for expression in host cells.

[0042] On the other hand, a vector is provided that contains 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 contains the vector sequences listed in Table 3.

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

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

[0045] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments 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: α1 macroglobulin gene, α2 macroglobulin gene, α3 macroglobulin gene, β1 microglobulin gene, β2 microglobulin gene, and combinations thereof. In another embodiment, the host cell comprises a knockout of a TCR gene selected from: TCR α variable region gene, TCR β variable region gene, TCR constant region gene, and combinations thereof. In yet another embodiment, the host cell expresses CD8α, CD8β, DN-TGFβRII, and / or a selective protein marker, optionally wherein the selective protein marker is DHFR, and further optionally wherein CD8α, CD8β, DN-TGFβRII, and / or the selective protein marker are fused with a CD34 enrichment tag. In another embodiment, the host cell is enriched using a CD34 enrichment tag. In another embodiment, the host cell is a hematopoietic progenitor cell, peripheral blood mononuclear cell (PBMC), umbilical cord blood cell, or immune cell. In yet another embodiment, the immune cell is a T cell, cytotoxic lymphocyte, cytotoxic lymphocyte precursor cell, cytotoxic lymphocyte progenitor cell, cytotoxic lymphocyte stem cell, or CD4+. + T cells, CD8 +T cells, CD4 / CD8 double-negative T cells, gamma-delta T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or combinations thereof. In another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or combinations thereof. In another embodiment, the T cells are primary T cells or cells of a T cell line. In another embodiment, the T cells do not express endogenous TCR or have low surface expression of endogenous TCR. In yet another embodiment, the host cells are capable of producing cytokines or cytotoxic molecules upon contact with target cells containing a peptide-MHC (pMHC) complex, said peptide-MHC (pMHC) complex comprising the MAGEA1 peptide epitope in an MHC molecular background. In another embodiment, the host cells contact the target cells in vitro, ex vivo, or in vivo. In another embodiment, the cytokines are TNF-α, IL-2, and / or IFN-γ. In yet another embodiment, the cytotoxic molecules are perforin and / or granzymes, optionally wherein the cytotoxic molecule is granzyme B. In another embodiment, the host cells are able to produce higher levels of cytokines or cytotoxic molecules upon contact with target cells exhibiting MAGEA1 heterozygous expression. In another embodiment, the host cells are able to produce at least 1.05-fold higher levels of cytokines or cytotoxic molecules. In yet another embodiment, the host cells are able to kill target cells containing a peptide-MHC (pMHC) complex, said peptide-MHC (pMHC) complex comprising a MAGEA1 peptide epitope in an MHC molecular background. In another embodiment, killing is determined by a killing assay. In another embodiment, the ratio of host cells to target cells in the killing assay is from 20:1 to 1:4. In yet another embodiment, the target cells are target cells pulsed with a MAGEA1 peptide from 1 µg / mL to 50 pg / mL, optionally wherein the target cells are monoallelic cells with MAGEA1 peptide-matched MHC. In another embodiment, the host cells are able to kill a higher number of target cells upon contact with target cells exhibiting MAGEA1 heterozygous expression, optionally wherein cell killing is at least 1.05-fold higher. In another embodiment, the target cells are cell lines or primary cells, optionally 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, host cells do not induce T cell expansion, cytokine release, or cytotoxic killing upon contact with target cells containing a peptide-MHC (pMHC) complex comprising the peptide epitopes MAGEA10, MAGEA11, RPS6KA2, RPS6KA3, RPS6KA6, STIL, TECPR1, WDR45, CCDC168, SIRPB1, TENM1, ADAMTS20, CPO, SPATA22, and / or ZNF202. In another embodiment, the host cells do not express the MAGEA1 antigen, are not recognized by the binding proteins described herein, do not belong to the serotype HLA-A*01, and / or do not express the HLA-A*01 allele.

[0046] On the other hand, a population of host cells as described in this article is provided.

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

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

[0049] In another aspect, a method for generating the binding protein described herein is provided, wherein the method comprises the steps of: (i) culturing transformed host cells under conditions suitable for allowing expression of the binding protein, said host cells having been transformed with nucleic acids containing a sequence encoding the binding protein described herein; and (ii) recovering the expressed binding protein.

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

[0051] In another aspect, a method is provided for detecting the presence or absence of MAGEA1 antigen and / or cells expressing MAGEA1, optionally wherein said cells are overproliferating cells, 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 said host cells described herein, wherein detection of MAGEA1 antigen indicates the presence of MAGEA1 antigen and / or cells expressing MAGEA1.

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

[0053] On the other hand, a method is provided for detecting the degree of a condition characterized by MAGEA1 expression in a subject, the method comprising: a) contacting a sample obtained from the subject with at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) detecting a reactivity level, wherein the presence of reactivity or a higher reactivity level compared to a control level indicates the degree of a condition characterized by MAGEA1 expression in the subject.

[0054] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the control level is a reference figure. In another embodiment, the control level is the level from subjects who do not have a condition characterized by MAGEA1 expression.

[0055] On the other hand, a method is provided for monitoring the progression of a disease characterized by MAGEA1 expression in a subject, the method comprising: a) detecting the presence or level of reactivity in a subject sample 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 MAGEA1 level or cells of interest expressing MAGEA1 detected in steps a) and b) to monitor the progression of a disease characterized by MAGEA1 expression in a subject, wherein the absence or reduction of the MAGEA1 level or cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates that the progression of the disease characterized by MAGEA1 expression in the subject is suppressed, and the presence or increase of the MAGEA1 level or cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates that the disease characterized by MAGEA1 expression in the subject has progressed.

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

[0057] In another aspect, a method is provided for predicting clinical outcomes in subjects suffering from a condition characterized by MAGEA1 expression, the method comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) comparing the presence or level of reactivity with reactivity from a control obtained from a subject with good clinical outcomes; wherein the absence of reactivity or a reduced level of reactivity in the subject's sample compared to the control indicates that the subject has good clinical outcomes.

[0058] On the other hand, a method is provided for evaluating the efficacy of a therapy for a condition characterized by MAGEA1 expression, the method comprising: a) determining, in a first sample obtained from the subject before administering at least a portion of the therapy to the subject for the condition characterized by MAGEA1 expression, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) determining, in a second sample obtained from the subject after administering the therapy to the subject for the condition characterized by MAGEA1 expression, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, wherein the absence of reactivity or a decreased level of reactivity in the second sample relative to the first sample indicates that the therapy effectively treats the subject's condition characterized by MAGEA1 expression, and wherein the presence of reactivity or an increased level of reactivity in the second sample relative to the first sample indicates that the therapy is not effectively treating the subject's condition characterized by MAGEA1 expression.

[0059] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the level of reactivity is indicated by the presence of a) binding and / or b) T cell activation and / or effector function, optionally wherein said T cell activation or effector function is T cell proliferation, killing, or cytokine release. In another embodiment, T cell binding, activation, and / or effector function is detected using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting, or intracellular flow cytometry.

[0060] On the other hand, a method for preventing and / or treating a condition characterized by MAGEA1 expression is provided, the method comprising contacting target cells expressing MAGEA1 with a therapeutically effective amount of a composition comprising cells expressing at least one binding protein described herein, optionally wherein the composition is administered to a subject.

[0061] Several embodiments are also provided, which can be applied to any aspect covered by the invention and / or combined with any other embodiments described herein. For example, in one embodiment, the cells are allogeneic cells, genotyped cells, or autologous cells. In another embodiment, the cells are host cells or a population of host cells described herein. In another embodiment, the target cells are cancer cells expressing MAGEA1. In yet another embodiment, the cell composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the cell composition induces an immune response in a subject against target cells expressing MAGEA1. In 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 comprises CD4+. + At least one of an assist T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the method further includes administering at least one additional treatment for a condition characterized by MAGEA1 expression, optionally said additional treatment for a condition characterized by MAGEA1 expression is administered concurrently or sequentially with the composition. In another embodiment, the condition characterized by MAGEA1 expression is cancer or a recurrence thereof, optionally said cancer is selected from the group consisting of: melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and bladder urothelial carcinoma. In yet another embodiment, the subject is an animal model and / or mammal of a condition characterized by MAGEA1 expression, optionally said mammal is a human, primate, or rodent. Attached Figure Description

[0062] Certain working embodiments and figures refer to certain comparative TCRs, such as a) “Comparative 1”, which corresponds to the T-Knife-based TCR described herein, for example, further described in Table 4; b) “Comparative 2”, also referred to as “Comparative”, which corresponds to the Immatics-based TCR described herein, for example, further described in Table 4; and c) “Comparative 3”, which corresponds to HN-32-41 from U.S. Patent Publication 2023 / 0270832 (which is incorporated herein by reference in its entirety) (i.e., TCR 32-41HM in Table 2A).

[0063] Figure 1A-1D A schematic diagram of the ReceptorScan platform used to identify 1181 candidate MAGEA1-specific TCRs is shown. Figure 1A The co-cultivation system was demonstrated. Figure 1AThe results of expanding MAGE-A1-specific T cells from an initial CD8 T cell population of A*01:01-positive healthy donors are presented. The initial CD8 T cells were co-cultured and expanded with autologous homologous mature dendritic cells (DCs) pulsed with A*01:01-restricted epitopes derived from MAGE-A1. Figure 1B This diagram illustrates a co-culture of antigen-specific CD8 T cells isolated using DNA-barcoded A*01:01 MAGE-A1 specific dextramer staining and sorting. Single-cell sequencing of dextramer-positive CD8 T cells was performed using the 10X Genomics platform. Figure 1C This demonstrates 1181 candidate MAGEA1s under an E:T ratio of 5:1. 161-169 Screening of 200 T-cell cytotoxicity targets targeting NCIH1703 (HLA-A*01:01 + MAGEA1+) in TCR. Figure 1D The comparison of cytotoxicity based on comparative TCRs (e.g., Comparative 1 is the T-Knife TCR; Comparative 2 is the Immatics TCR; sequence details are shown in Table 4) is presented. In this screening, 0 TCRs were selected from 200 for additional functional assessment. Further screening using ActivScan assays with a set of 1181 MAGE-A1-specific TCRs yielded 59 TCRs, which were prioritized for reproducible functional screening (see below). Figure 2A and 2B ).

[0064] Figure 2A and 2B The diagram illustrates the ActivScan strategy and the corresponding data, demonstrating how this strategy identified 14 MAGEA1 variants with high expression and high affinity. 161-169 TCR. Synthesized using TScan's proprietary PISTACHIO cloning method. Figure 1A and 1B The MAGE-A1-specific TCR library identified by the ReceptorScan platform described in [the document] Figure 2ATCRs with high expression were selected by transducing whole T cells with a virus encoding a TCR library of the PISTACHIO clone, followed by isolation of dextramer-bound cells. High-affinity TCRs were identified by sorting cells that responded to peptide titration using ActivScan. Fifty-nine (59) TCRs were prioritized for functional assessment. Fourteen (14) of the 59 TCRs showed cytotoxicity against endogenously MAGE-A1-expressing cell lines, similar to the comparison TCRs (i.e., the comparison TCRs were Immatics TCRs; sequence details are shown in Table 4). Figure 2B This study presents the results of transducing whole T cells to individually express 59 MAGEA1-specific TCRs. Engineered T cells were co-cultured with target cells labeled with Incucyte® NucLight Red, such as NCIH1703 cells. Target cell survival was quantified using time-dependent imaging as readout data for T cell cytotoxicity. The 72-hour (endpoint) survival of each TCR is shown. Figure 2B Untransduced cells (NTD) and two comparative TCRs (e.g., Comparative 1 was T-Knife TCR; Comparative 2 was Immatics TCR; sequence details are shown in Table 4) were used as controls. Figure 2B Fourteen (14) of the 59 TCRs shown were selected for further evaluation.

[0065] Figure 3A-3G MAGEA1 was showcased 161-169 Functional assessment results of specific TCRs. Whole T cells were isolated from healthy donor PBMCs and transduced to express the MAGE-A1161-169 specific TCR or a “comparative TCR” (e.g., Comparative 2 or the Immatics TCR with sequence details shown in Table 4; Comparative 3 was HN-32-41 from U.S. Patent Publication 2023 / 0270832 (in its entirety incorporated herein by reference) (i.e., TCR 32-41 HM in Table 2A), and their functional response to target cells positive and negative for MAGEA1 and HLA-A*01:01 was assessed. The functional response of T cells to target cells was assessed. The first 14 types are shown. Figure 3A and Figure 3B ) and the first 5 of the first 14 ( Figures 3C-3E Results of candidate TCRs. Figure 3A and Figure 3B 14 MAGEA1 varieties were showcased. 161-169 Specific TCR against HLA-A*01:01 + MAGEA1 + Target cell lines NCIH1703 and A101D ( Figure 3AA375 Figure 3B ) and HLA-A*01:01 + MAGEA1 negative control cell line HEK293T ( Figure 3B () functional response. Figure 3C MAGEA1 was showcased 161-169 Specific TCR against HLA-A*01:01 + MAGE-A1 + Target cell lines NCIH1703, A101D, and A375 (TPM values ​​for each cell line can be found here). Figure 3A and Figure 3B ) and HLA-A*01:01 + Results of cytotoxicity assays for the HEK293T MAGEA1 negative control cell line (E:T 5:1). Figure 3D This demonstrates the use of a second healthy donor (i.e., different from...) Figure 2A and Figure 2B Whole T cells isolated from PBMCs were transduced to express the first five MAGEA1 proteins. 161-169 The study evaluated specific TCRs and assessed their functional responses in target cells that were positive and negative for MAGEA1 and HLA-A*01:01. Five MAGEA1 variants were presented. 161-169 Specific TCR against HLA-A*01:01 + MAGEA1 + Target cell lines NCIH1703, A101D, and A375, and HLA-A*01:01 + Functional response of the MAGEA1 negative control cell line HEK293T. Engineered T cells were co-cultured with Incucyte® NucLight Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified on IncuCyte® as readout data of T cell cytotoxicity. Figure 3E The results of the measurement of IFN-γ production in the co-culture supernatant after 24 hours (E:T 1:1) are presented. Figure 3F The results showed that TCR 458 exhibited the strongest performance in peptide titration assays. Whole T cells isolated from healthy donor PBMCs were transduced to individually express the top five MAGEA1 proteins. 161-169 Specific TCRs were assessed, and cytokine secretion in response to co-culture with target cells was evaluated. HLA-A*01:01+ MAGEA1 negative control HEK293T cells were pulsed with 7 serially diluted MAGE-A1 peptide EADPTGHSY. Engineered T cells were co-cultured with pulsed-delivered and washed target cells at a 1:1 E:T ratio, and cytokine secretion in the cell supernatant was measured 24 hours after co-culture. Figure 3GA dot plot depicting TCR expression, constrained by MAGE-A1 at A*01:01, is shown. 161-169 Dextramer staining assessment. The comparative TCR identified MAGE-A1 on different HLA lines. Results showed strong cytotoxicity against cancer cell lines expressing various levels of MAGE-A1 and HLA-A*01:01 (see example...). Figure 3A and Figure 3B The TPM value for each cell line listed in the document is [value].

[0066] Figure 4 The in vivo efficacy of TSC-204-A0101 TCR-T cells was demonstrated. In vivo experiments were performed using research-grade materials to ensure that the T cells were prepared and characterized identically to those used in in vitro experiments (and the expression vector did not express TGFBRII). 2 × 10⁻⁶ cells were used. 6 NCI H1703 cells were subcutaneously implanted into the right ventral region of immunodeficient NCG mice (n = 7 mice / group). Tumor volume was monitored from day 7 to day 38 post-implantation. Mice were injected with 2 × 10⁻⁶ NCI H1703 cells on days 9 and 16. 7 One TSC-204-A0101 TCR-T cell or untransduced donor control T cells (NTD). The mean tumor volume over time is shown for different groups.

[0067] Figure 5A and 5B This demonstrates the putative off-target effect of TCR MAGE-A1-458 identified by screening with the proprietary whole-genome SafetyScan. Figure 5A A schematic diagram illustrates the SafetyScan screening strategy. TCRs are screened from >500,000 protein fragments for each protein in the entire human proteome to identify potential reactivity, including reactivity with low sequence homology to natural targets. Figure 5B The SafetyScan analysis results of TCR MAGE-A1-458 are presented, which identified nine proteins other than MAGEA1. When these proteins were overexpressed as fragments of 90 amino acids in length, they were presumed to be recognized by TCR on multiple tiles.

[0068] Figure 6A and 6BThis study demonstrates that TCR MAGE-A1-458 showed no reactivity to putative off-target effects, including MAGE-A10 and MAGE-A11. The physiological relevance of potential off-target effects can be assessed in detail by co-culturing TCR-T cells with primary cells that naturally express full-length proteins at normal levels. In a representative, non-restrictive analysis, TCR MAGE-A1-458 was activated against potential off-target peptides or potentially off-target full-length protein sequences, such as MAGE-A10 or MAGE-A11 peptides and / or full-length protein sequences. Figure 6A This demonstrates the pulsed delivery of peptides indicated in A*01:01 HEK293T reporter cells at sequential dilutions, and Figure 6B Results of full-length MAGEA10 ORF expressed in A*01:01 HEK293T cells are presented. For Figure 6A and Figure 6B After co-culturing with T cells expressing MAGE-A1-458 TCR for 24 hours, the IFN-γ in the supernatant was measured.

[0069] Figure 7 The study demonstrated that MAGE-A1-458 showed no reactivity in primary cells from healthy individuals. The reactivity of TCR MAGE-A1-458 to primary cells derived from healthy HLA-A*01:01+ tissues was tested; these primary cells naturally expressed the putative off-target effects identified in SafetyScreen (see above). Figure 5B ) Target cells were treated with MAGE-A1 161-169 (EADPTGHSY) peptide was administered via pulsed delivery or not, and co-cultured with whole T cells expressing MAGE-A1-458 TCR or untransduced (NTD). IFN-γ (E:T approx. 2:1) in the co-culture supernatant was measured after 24 hours. NCI-H1703 and A*01:01HEK cells were used as positive and negative controls, respectively.

[0070] Figure 8 The results showed that TCR MAGE-A1-458 did not exhibit allogeneic reactivity to any of the 110 MHCs.

[0071] T cells expressing the MAGE-A1-458 TCR or untransduced T cells were co-cultured with MHC-free HEK293T cells engineered to express the 110 most common class I MHCs in the US population. Inhibition of target cell growth was measured as readout data for allogeneic reactivity.

[0072] Figures 9A-9BThis demonstrates the pMHC-dependent function of process-representative TSC-204-A0101 TCR-T cells. NCI-H1395 cells (transduced with Nuclight Red) were pulsed with the indicated concentration of MAGE-A1 peptide and co-cultured with three batches of process-representative TSC-204-A0101 TCR-T cells (purple), donor-matched UTF control T cells at the highest peptide concentration (20,000 ng / mL) (blue), or cultured alone in the absence of peptide or effector T cells (black). Unpulsed NCI-H1395 cells (0.0 ng / mL) were co-cultured with TSC-204-A0101 TCR-T cells (grey) and UTF control T cells (red). Figure 9A The relative growth curves of NCI-H1395 cells and effector T cells co-cultured for 96 hours at each peptide concentration are shown, normalized relative to t = 0 hours, at an E:T ratio of 5:1. For each donor, three copies were co-cultured (n = 3). The error bars for each data point represent the standard error of the mean (SEM). Figure 9B The area under the curve (AUC) of growth induced by co-culturing NCI-H1395 cells with TSC-204-A0101 for 96 hours is shown, varying with peptide concentration. Data are normalized relative to cell growth (grey) under conditions without pulse delivery (0.0 ng / mL) and expressed as target cell “survival” over 96 hours. Data are normalized, with 0% representing the minimum value in each dataset and 100% representing the maximum value in each dataset. The IC50 of NCI-H1395 cell growth inhibition is then calculated using a nonlinear regression curve (using the equation sigmoid 4PL, where X is concentration). 50 .

[0073] Figures 10A-10H This demonstrates that TSC-204-A0101 TCR-T cells secrete granzyme B and pro-inflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-204-A0101 TCR-T cells from three batches (PD353, PD354, and PD355) were used in this study. Figure 10A-10D ) or donor-matched UTF control T cells ( Figure 10E-10HCells were cultured in the absence of target cells (black bars) or co-cultured at a 1:1 E:T ratio with HLA-A*01:01 positive MAGE-A1 negative target cells NCI-H1395 or three different HLA-A*01:01 positive MAGE-A1 positive cell lines (NCI-H1703; A375; A101D). After approximately 24 hours of co-culture, the supernatant was collected, and the levels of pro-inflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated 4-channel ELISA assay (ELLA from ProteinSimple). Co-culture was performed in triplicate, and error bars represent the standard error of the mean (SEM).

[0074] Figure 11A-11D This study demonstrates that TSC-204-A0101 TCR-T cells proliferate in a target-dependent manner. TSC-204-A0101 TCR-T cells were labeled with CTV dye and co-cultured with the indicated cancer cell line (E:T 1:1) for approximately 4 days. Figure 11A Demonstrated in total T cells (TCR) + CD34 + ), cytotoxic T cells (TCR) + CD34 + CD8 + CD4 - ) and helper T cells (TCR) + CD34 + CD8 + CD4 + CTV dilution was assessed within the population. The CTV peak of undivided T cells relative to dividing T cells was established using T cells cultured in the absence of the target ('T cells only'). Representative histograms of PD355 from TSC-204-A0101 batch co-cultured with the indicated target cell line are shown. Figure 11B-11D This graph displays the number of proliferating (dividing) TCR-T cells from batches PD353, PD354, and PD355 (TSC-204-A0101). The bars represent the total number of dividing T cells (i.e., TCR). + CD34 + ) ( Figure 11B ), helper T cells (TCR) + CD34 + CD8 + CD4 + ) ( Figure 11C ) and cytotoxic T cells (TCR) + CD34 + CD8 + CD4 - ) ( Figure 11DThe number of samples was determined. Triples were co-cultured, and the mean standard error (SEM) of each condition / donor was displayed.

[0075] Figure 12A-12B The TSC-204-A0101 TCR-T cells were shown to exhibit effective and selective cytotoxicity. Figure 12A This study demonstrates the cytotoxic potential of three batches of process-representative TSC-204-A0101 TCR-T cells (purple growth curves) and untransfected (UTF) control T cells from matched donors (grey growth curves) against an HLA-A*01:01-positive MAGE-A1-negative control cell line (NCI-H1395) or three different HLA-A*01:01-positive MAGE-A1-positive target cell lines (A375, NCI-H1703, and A101D) in an Incucyte-based cytotoxicity assay. Effector TCR-T cells and target cells were co-cultured across a range of effector cell to target cell ratios (E:T ranging from 5:1 to 0.625:1), and target cell growth was measured over 96 hours. Representative data from batch PD355 of TSC-204-A0101 TCR-T cells (left panel) and UTF control T cells (right panel) over 72 hours are presented. The target cells cultured separately were used as a negative control (black growth curve). Triples were co-cultured. The mean standard error (SEM) is less than the sign. Figure 12B The cytotoxic activity of three batches of process-representative TSC-204-A0101 TCR-T cells over 72 hours is shown, summarized as the area under the curve (AUC) of the growth curves of target cells co-cultured with TSC-204-A0101 at an E:T ratio of 2.5:1, normalized relative to the growth curves of target cells co-cultured with the corresponding UTF control T cells.

[0076] Figure 13Phosphorylation flow cytometry analysis of phosphorylated-SMAD2 or total SMAD2 in TSC-204-A0101 TCR-T or UTF cells is presented. Data are presented from: TSC-204-A0101 process-representative TCR-T cells (batch PD353, PD354, and PD355) untreated or treated with 10 ng / mL TGFβ-1 and process-similar UTF cells (PD353, PD354) from the same donor at laboratory scale and a third unrelated batch (D6446). Live single cells were characterized as CD34 positive (TSC-204-A0101 TCR-T cells) or CD34 negative (UTF control), and these subsets were further characterized by histogram analysis against phosphorylated-SMAD2 or total SMAD2. The histogram analysis overlay consists of the following: untreated CD34-positive TCR-T cells (dark purple, solid), TGFβ-1-treated CD34-positive TCR-T cells (dark purple, hollow), untreated CD34-negative UTF (black, solid), and TGFβ-1-treated CD34-negative UTF (black, hollow) containing phosphorylated SMAD2 or total SMAD2.

[0077] Figure 14 The schedule for vaccination, administration, and analysis is presented.

[0078] Figures 15A-15E The in vivo efficacy of TSC-204-A0101 was demonstrated. NCG mice were subcutaneously inoculated with NCI-H1703 tumor cells. Once tumor transplantation was successful (13 days post-inoculation), animals were randomly assigned to seven different treatment groups and injected with representative TSC-204-A0101 TCR-T cells (three batches tested, PD353, PD354, and PD355) on days 1 and 8 of the study; or untransfected (UTF) control T cells from matched donors; or PBS (triangular head). The mean tumor volume over time in each treatment group (n=12) is shown. Figure 15A The area under the curve (AUC) values ​​of the tumor growth curves for individual mice in each treatment group between day 0 and day 40 are shown. Figure 15B The tumor volume of individual mice over time is shown. Figure 15C-15E Compared with the corresponding UTF control group, ***P=0.0008, *P=0.0421. Error bars represent SEM.

[0079] Figure 16The percentage change in body weight over time is shown across different groups. NCG mice were subcutaneously inoculated with NCI-H1703 tumor cells. Once tumor transplantation was successful (6 days post-inoculation), animals were randomly assigned to 7 different treatment groups and injected on days 1 and 8 of the study with representative TSC-204-A0101 TCR-T cells (3 batches tested, PD353, PD354, and PD355); or untransfected (UTF) control T cells from matched donors; or mediator (PBS) (triangular heads). The percentage change in body weight for all treatment groups (n = 12) is shown. Error bars represent the mean SEM.

[0080] Figure 17 This study presents an allogeneic reactivity profile of mechanorepresentative TSC-204-A0101 TCR-T cells. The co-culture of mechanorepresentative TSC-204-A0101 TCR-T cells with MHC-free HEK293T cells, which re-express one of the 110 most common class I HLA types in the US population, was performed for the indicated time period. Screening included a positive control (red) consisting of HEK293T cells expressing the MAGE-A1 fragment containing the HLA-A*01:01-restricted epitope and HLA-A*01:01; and a negative control (blue) consisting of MHC-free HEK293T cells. The inhibition of target cell growth by TCR-T cells during the 48-hour co-culture period was measured relative to UTD control T cells, providing readout data on the responsiveness of the mechanorepresentative therapeutic TCR to allogeneic HLA proteins.

[0081] Figure 18 A flowchart is shown, which describes the steps and timeline of cytokine assays for testing the extratumor reactivity of TSC-204-A0101 TCR-T cells.

[0082] Figure 19 This study demonstrates the putative off-target expression of therapeutic TCRs in primary cells. RNA was extracted and sequenced from primary cells and iPSC-derived cells. The heatmap shows TPM (transcripts per million) calculated based on counts. The color scale used in the RNA-seq heatmap sets zero TPM values ​​to white, and values ​​above zero follow a continuous color scale up to 100 TPM. In the heatmap, monocytes are labeled as CD14, and human neurons are labeled as neurons.

[0083] Figures 20A-20C This demonstrates the effect of TSC-204-A0101 TCR-T cells on HLA-A*01:01. +Primary cells showed no reactivity. TSC-204-A0101 TCR-T cells and donor-matched UTF cells were co-cultured with the primary cell group, and the IFN-γ level in the supernatant was assessed as a measure of T cell reactivity.

[0084] Figure 21 The putative off-target expression of the therapeutic TCR used in TSC-204-A0101 TCR-T cells is shown in the cancer cell line. RNA was extracted from the cancer cell line and sequenced. The heatmap shows the TPM (transcripts per million) calculated based on counts. The color scale used in the RNA-seq heatmap sets zero TPM values ​​to white, and values ​​above zero follow a continuous color scale up to 100 TPM.

[0085] Figures 22A-22C This demonstrates the effect of TSC-204-A0101 TCR-T cells on HLA-A*01:01. + Cancer cells did not show reactivity. TSC-204-A0101 TCR-T cells and donor-matched UTF cells were co-cultured with cancer cell groups, and the IFN-γ level in the supernatant was assessed as a measure of T cell reactivity.

[0086] Figures 23A-23C This demonstrates the effect of TSC-204-A0101 TCR-T cells on HLA-A*01:01 cells overexpressing CPO. + HEK cells showed no reactivity. TSC-204-A0101 TCR-T cells and donor-matched UTF cells were co-cultured with a single-cell clone group of HEKA*01:01 cells overexpressing CPO, and the IFN-γ level in the supernatant was assessed as a measure of T cell reactivity.

[0087] Figure 24 A diagram showing the pNVVD236 vector (i.e., pNVVD236_TSC-204-A01_TCR-458_MSCV-TCR-458-CD8-EF1α-dnTGFbRII-DHFR). CD: differentiation cluster, RNA-OUT: antisense RNA against bacterial fructan sucrase encoded by sacB, SV40: simian virus 40, TCR: T cell receptor, ITR: terminal inverted repeat sequence, QBend: mouse anti-human CD34 antibody, dnTGFbRII: dominant-negative TGFβ receptor II, DHFR: dihydrofolate reductase selection marker.

[0088] Unless otherwise indicated, for any graph displaying bar histograms, curves or other data associated with a legend, each indication of the bars, curves or other data presented from left to right directly and sequentially corresponds to the boxes in the legend from top to bottom or from left to right. Detailed Implementation

[0089] This invention is based, in at least part, on the discovery of MAGEA1 immunogenic peptides (e.g., peptides comprising or composed of the sequences listed in Table 1), binding proteins that recognize the MAGEA1 antigen (e.g., binding proteins having the sequences listed in Table 2), and their uses. A comprehensive systematic investigation was conducted to determine the precise T-cell targets recognized by the initial pool of T cells of interest.

[0090] Therefore, this invention relates in part to identified epitopes (immunogenic dominance peptides) of the treatment-related MAGEA1 protein and related compositions (e.g., immunodominance peptides, vaccines, etc.); compositions comprising immunogenic peptides alone or together with MHC molecules; stabilizing MHC-peptide complexes; methods for diagnosing, prognosing, and monitoring immune responses to conditions characterized by MAGEA1 expression; and methods for preventing and / or treating conditions characterized by MAGEA1 expression. This invention also relates in part to identified binding proteins (e.g., TCRs); host cells expressing binding proteins (e.g., TCRs); compositions comprising binding proteins (e.g., TCRs) and host cells expressing binding proteins (e.g., TCRs); methods for diagnosing, prognosing, and monitoring T cell responses to cells expressing MAGEA1; and methods for preventing and / or treating conditions characterized by MAGEA1 expression.

[0091] I. Definition For convenience, certain terms used in this specification, embodiments, and appended claims are collected herein.

[0092] The article “a / an” is used in this document to refer to one or more (i.e., at least one) grammatical object. For example, “an element” means one element or more elements. Furthermore, unless otherwise stated, references to the tables provided herein cover all subtables of that table.

[0093] The term “administration” means providing a drug or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration. This involves physically introducing a composition containing a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. In some embodiments, the routes of administration of the binding protein described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” means a mode of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrasheath, intralymphatic, intralesional, intracapsular, intracardiac, intradermal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the binding protein described herein may be administered via a non-parenteral route, such as a local, epidermal, or mucosal administration route, such as intranasal, oral, vaginal, rectal, sublingual, or topical. Administration may also be performed, for example, once, multiple times, and / or over one or more extended time periods.

[0094] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. An antigen can be the MAGEA1 antigen or a fragment thereof, against which a protective or therapeutic immune response is required. An "epitope" is the portion of an antigen that binds to a natural or synthetic substance.

[0095] As used herein, the term "adjuvant" refers to a substance that, when administered before, simultaneously with, or after the administration of an antigen, promotes, prolongs, and / or enhances the quality and / or intensity of an immune response to an antigen, compared to administration of the antigen alone. Adjuvants can increase the magnitude and duration of the immune response induced by vaccination.

[0096] As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding moiety") or single chain thereof. In one embodiment, "antibody" refers to a glycoprotein, or its antigen-binding moiety, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V). H The heavy chain consists of a heavy chain constant region and a heavy chain constant region. In some naturally occurring antibodies, the heavy chain constant region consists of three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated as V in this article). L It consists of a light chain constant region and a structural domain CL. H and V L The region can be further subdivided into high-variability regions, called complementary determinant regions (CDRs), within which are scattered more conservative regions, called framing regions (FRs).H and V L Each antibody consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0097] 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).

[0098] As used herein, the term "antigen-binding moiety" in the context of TCR-binding proteins, for example, refers to one or more portions of the TCR that retain the ability to bind (e.g., specifically and / or selectively) antigens (e.g., MAGEA1 antigen). Such moieties are, for example, about 8 to about 1,500 amino acids in length, preferably about 8 to about 745 amino acids in length, preferably about 8 to about 300 amino acids in length, for example about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of the TCR can be performed by fragments of the full-length TCR. Examples of binding moieties encompassed within the term "antigen-binding moiety" of the TCR include: (i) those formed by the V of the TCR. α and V β Fv segments composed of structural domains; (ii) separate complementarity-determining regions (CDRs); or (iii) combinations of two or more separate CDRs, which may optionally be joined by a synthetic joint. Furthermore, although V α and V β Encoded by separate genes, but which can be joined together using recombination methods via synthetic adapters to form a single protein chain, where V α and V β Regions pair to form monovalent molecules (called single-stranded TCRs (scTCRs)). These single-stranded TCRs are also intended to be encompassed within the term "antigen-binding moiety" of TCR. These TCR fragments can be obtained using conventional techniques known to those skilled in the art, and fragments are screened for utility in the same manner as fully binding proteins. The antigen-binding moiety can be produced via recombinant DNA technology or through enzymatic or chemical cleavage of intact immunoglobulins.

[0099] The terms “complementarity-determining region” and “CDR” are synonymous with “hypervariant region” or “HVR” and are known in the art to refer to certain binding proteins, such as the discontinuous amino acid sequence within the variable region of the TCR, which confers antigen specificity and / or binding affinity. For the TCR, generally, there are three CDRs (αCDR1, αCDR2, and αCDR3) in each α-chain variable region and three CDRs (βCDR1, βCDR2, and βCDR3) in each β-chain variable region. CDR3 is considered the major CDR responsible for recognizing processed antigens. CDR1 and CDR2 primarily interact with the MHC.

[0100] The term "body fluid" refers to fluids excreted or secreted from the body, as well as fluids not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculate, chyle, chyme, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menstrual blood, breast milk, mucus, pleural effusion, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubricant, vitreous humor, vomitus). In some embodiments, body fluids contain immune cells, optionally wherein the immune cells are cytotoxic lymphocytes, such as cytotoxic T cells and / or NK cells, CD4+ T cells, etc.

[0101] The term "coding region" refers to the region of a nucleotide sequence that contains a codon that is translated into an amino acid residue, while the term "non-coding region" refers to the region of a nucleotide sequence that is not translated into an amino acid (e.g., the 5' and 3' untranslated regions).

[0102] The term "complementarity" is a broad concept referring to sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that adenine residues in the first nucleic acid region can form specific hydrogen bonds ("base pairing") with residues in the second nucleic acid region that are antiparallel to the first region, provided that residues in the second nucleic acid region are thymine or uracil. Similarly, it is known that cytosine residues in the first nucleic acid strand can base pair with residues in the second nucleic acid strand that are antiparallel to the first strand, provided that residues in the second nucleic acid strand are guanine. If, when the first region of a nucleic acid is arranged antiparallel to the second region of the same or different nucleic acids, at least one nucleotide residue in the first region can base pair with a residue in the second region, then the two regions are complementary. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, wherein when the first and second portions are arranged in an antiparallel manner, 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 between the two (including endpoints), such as at least about 80%-100%, are capable of base pairing with the nucleotide residues in the second portion. In some embodiments, all the nucleotide residues in the first portion are capable of base pairing with the nucleotide residues in the second portion.

[0103] As used herein, the term "co-stimulation" in relation to activated immune cells includes the ability of co-stimulatory molecules to provide a non-activated receptor-mediated second signal ("co-stimulatory signal") that can induce proliferation or effector function. For example, a co-stimulatory signal may, for instance, induce cytokine secretion in T cells that have already received a signal mediated by a T cell receptor. Immune cells that have received a signal mediated by a cell receptor, for example, via an activating receptor, are referred to herein as "activated immune cells".

[0104] "CD3" is known in the art as a six-chain multiprotein complex (see Abbas and Lichtman, Cellular and Molecular Immunology (9th edition) (2018); Janeway et al. (Immunobiology) (9th edition) (2016)). In mammals, the complex comprises a homodimer of one CD3γ chain, one CD3δ chain, two CD3ε chains, and one CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are associated cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature believed to allow these chains to associate with positively charged regions or residues of the T-cell receptor chain. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif called an immunoreceptor tyrosine activation motif, or ITAM, while each CD3ζ chain has three ITAMs. Not wanting to be bound by theory, it is believed that IT AM is important for the signal transduction ability of the TCR complex. The CD3 used in this invention can be derived from various animal species, including humans, mice, rats, or other mammals.

[0105] As used herein, “components 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).

[0106] "Comparative T-cell receptor" refers to at least one benchmark T-cell receptor (e.g., based on Immatics or T-Knife) reported in state-of-the-art technology, such as U.S. Patent No. 10,874,731 (Immatics) and Obenaus et al. (2014) Nat. Biotechnol. 33:402-407. In some embodiments, "Comparative 1" refers to a TCR based on the T-Knife-T1367 TCR from Obenaus et al. (2014) Nat. Biotechnol. 33:402-407. In some embodiments, "Comparative 2," also simply "Comparative," is a TCR based on the ImmaticsR37P1C9 TCR from U.S. Patent No. 10,874,731. Engineered forms of these parental sequences are used in the working examples, and the sequences of these engineered forms are illustrated in Table 4. In some embodiments, the comparative T-cell receptor has the sequences illustrated in Table 4. In some implementations, “Comparative Item 3” is HN-32-41 (i.e., TCR 32-41 HM in Table 2A) from U.S. Patent Publication 2023 / 0270832 (which is incorporated herein by reference in its entirety).

[0107] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein that is engineered to contain two or more amino acid sequences linked together in a manner not naturally present or in host cells, which can function as a receptor when present on the cell surface. The CARs covered by this invention include an extracellular portion comprising an antigen-binding domain (i.e., derived from or derived from immunoglobulins or immunoglobulin-like molecules, such as TCRs specific to the MAGEA1 antigen, binding proteins derived from single-chain TCRs, scFvs derived from antibodies, antigen-binding domains derived from or derived from cytotoxic immune receptors from NK cells, etc.), said antigen-binding domain being connected to a transmembrane domain and one or more intracellular signal transduction domains (e.g., effector domains, optionally containing co-stimulatory domains) (see, for example, 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).

[0108] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily mediated by CD8+ cells. + T cell mediated.

[0109] The term "substantially constitutes" is not equivalent to "comprising" and refers to the specific materials or steps of the claim, or those materials or steps that do not substantially affect the essential characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, or modules) is "substantially constituted" when the amino acid sequence of the domain, region, module, or protein includes, in combination, at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and does not significantly affect (i.e., does not reduce activity by more than 50%, e.g., no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain, region, module, or protein (e.g., the target binding affinity of the binding protein) by extension, deletion, mutation, or combination thereof (e.g., amino acids at the amino or carboxyl terminus or between domains).

[0110] The term "determining a suitable treatment regimen for a subject" means determining a treatment regimen for the subject (i.e., a single therapy or a combination of different therapies for the prevention and / or treatment of viral infection in the subject), said treatment regimen being initiated, modified, and / or terminated based on or substantially based on or at least partially based on the analytical results according to the invention. One example is initiating adjuvant therapy after surgery with the aim of reducing the risk of recurrence; another example would be modifying the dosage of a specific chemotherapy therapy. In addition to the analytical results according to the invention, the determination may also be based on the individual characteristics of the subject to be treated. In most cases, the actual treatment regimen suitable for the subject will be determined by the attending physician or doctor.

[0111] The term "dominant-negative TGFβ receptor" or "DN-TGFβR" refers to a variant or mutant of the transforming growth factor (TGF) β receptor that resists TGFβ signaling. There are five type II receptors (activating receptors) and seven type I receptors (signaling receptors). The active TGFβ receptor is a heterotetramer, composed of two TGFβ receptor I (TGFβRI) and two TGFβ receptor II (TGFβRII). In some embodiments, DN-TGFβR is DN-TGFβRII (i.e., a TGFβ receptor II variant or mutant). In some embodiments, resistance to TGFβ signaling is achieved on immune cells such as T cells, where the TGFβ may be produced by cancer cells or other immune cells in the cellular environment, such as stromal cells, macrophages, bone marrow cells, epithelial cells, natural killer cells, etc. TGFβ signaling inhibitors are well known in the art and include, but are not limited to, mutant TGFβ that chelates the receptor to inhibit signaling; antibodies that bind to TGFβ and / or the TGFβ receptor (e.g., lerdelimumab, metlimumab, fressolimumab, etc.); soluble TGFβ-binding proteins, such as portions of the TGFβ receptor that chelate TGFβ (e.g., TGFβRII-Fc fusion protein); or other binding agents, such as β-glycans. Any and all known TGFβ signaling inhibitors may be used instead of DN-TGFβR (e.g., DN-TGFβRII) or others. In some embodiments, DN-TGFβR lacks the intracellular portions required for TGFβ-mediated signaling, such as whole-cell intracellular domains, kinase signaling domains, etc. DN-TGFβR constructs are well known in the art. (For representative non-restrictive embodiments, see 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 WO 2009 / 152610; PCT WO 2017 / 156484; Kloss et al. (2018) Mol. Ther. 26:1855-1866; PCT WO. 2019 / 089884; PCT WO 2020 / 042647; and PCT WO 2020 / 042648.)

[0112] As used herein, a "hematopoietic progenitor cell" is a cell that can originate from hematopoietic stem cells or fetal tissue and is capable of further differentiating into mature cell types (such as immune system cells). Exemplary hematopoietic progenitor cells include those with CD24 Lo Lin-CD117 + Phenotypic hematopoietic progenitor cells or hematopoietic progenitor cells found in the thymus (called thymic progenitor cells).

[0113] As used herein, “homology” refers to the nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. Regions are homologous at that position when nucleotide residue positions in two regions are occupied by the same nucleotide residue. A first region is homologous to a second region if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the proportion of nucleotide residue positions in the two regions occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, wherein at least about 50% of the nucleotide residue positions of each 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 between the two (including endpoints), for example, at least about 80% to 100% are occupied by the same nucleotide residues. In some embodiments, all nucleotide residue positions of each portion are occupied by the same nucleotide residues.

[0114] The term "hyperplastic syndrome characterized by MAGEA1 antigen expression" can refer to any hyperplastic syndrome in which the MAGEA1 antigen is present in an MHC (e.g., HLA) complex expressed by at least some hyperproliferating cells in the subject. Examples of hyperplastic syndromes characterized by the MAGEA1:HLA complex include solid malignancies, such as those described in detail below.

[0115] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. Furthermore, the term "immune response" includes immune responses indirectly influenced by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0116] Enhanced agonistic activity of T cell co-stimulatory receptors and / or enhanced antagonistic activity of inhibitory receptors may lead to an increased ability to stimulate an immune response or the immune system. This increased ability to stimulate an immune response or the immune system can be measured by measuring ECGs in an immune response assay. 50 The assay may reflect an increase in the maximum activity level by a factor of 1, or, for example, by measuring changes in cytokine or chemokine release, cell lysis activity (measured directly on target cells or indirectly via detection of CD107a or granzyme), and proliferation. The ability to stimulate an immune response or immune system activity may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500%, or more.

[0117] The term "immunotherapy agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host's immune system in a subject to produce an immune response against cancer cells. Various immunotherapy agents can be used in the compositions and methods described herein.

[0118] The term "immune cell" refers to any immune system cell derived from hematopoietic stem cells in the bone marrow, which produces two main lineages: myeloid progenitor cells (producing myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes); and lymphoid progenitor cells (producing lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 cells. + T cells, CD8 + T cells, CD4 / CD8 double-negative T cells, gd T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells, also known as antigen-presenting cells or "APCs," are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor on the surface of APCs interacts with the TCR on the surface of T cells.

[0119] "Isolated protein" refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals during chemical synthesis. "Isolated" or "purified" proteins, or their biologically active portions, are substantially free of cellular material or other contaminating proteins from the cells or tissues from which binding proteins, antibodies, polypeptides, peptides, or fusion proteins are produced, or substantially free of chemical precursors or other chemicals during chemical synthesis. The term "substantially free of cellular material" includes formulations of biomarker polypeptides or fragments thereof, wherein the protein is isolated from cellular components of the cells from which the protein is recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes formulations of biomarker proteins or fragments thereof having less than about 30% (on a dry weight basis) of non-biomarker proteins (also referred to herein as "contaminating proteins"), or in some embodiments, less than about 25%, 20%, 15%, 10%, 5%, 1%, or less, or any range between the two (including endpoints), such as less than about 1% to 5% of non-biomarker proteins. When recombinant protein, antibody, polypeptide, peptide or fusion protein or fragment thereof (e.g. its bioactive fragment) is produced, it may be substantially free of culture medium, i.e., the culture medium accounts for less than about 20%, 15%, 10%, 5%, 1% or less or any range in between (including the endpoints) of the protein preparation volume, such as less than about 1% to 5%.

[0120] As used in this article, the term "isotype" refers to an antibody class (e.g., IgM, IgG1, IgG2C, etc.) encoded by a heavy chain constant region gene.

[0121] As used in this article, the term "K" D "Kd" refers to the dissociation equilibrium constant of a specific binding protein-antigen interaction. The binding affinity of the binding proteins covered by this invention can be measured or determined by standard binding protein-target binding assays, such as competitive assays, saturation assays, or standard immunoassays, such as ELISA or RIA. A relatively low Kd value indicates a relatively high binding affinity (e.g., less than or equal to about 5 × 10⁻⁶). -4 The Kd value of M (500 uM) includes 1×10 -4 The Kd value of M (100 uM) and 100 uM Kd indicates relatively high binding affinity compared to 500 uM Kd).

[0122] A “kit” is any article (e.g., package or container) containing at least one reagent, such as a probe or small molecule, for the specific detection and / or influencing of the expression of markers covered by this invention. Kits may be marketed, distributed, or sold as a unit for performing the methods covered by this invention. Kits may contain one or more reagents necessary for expressing compositions useful in the methods covered by this invention. In some embodiments, kits may also contain reference standards, such as nucleic acids encoding proteins that do not affect or regulate signaling pathways controlling cell growth, division, migration, survival, or apoptosis. Many such control proteins are contemplated by those skilled in the art, including but not limited to common molecular tags (e.g., green fluorescent protein and β-galactosidase), proteins not classified by GeneOntology references into any pathway covering cell growth, division, migration, survival, or apoptosis, or ubiquitous housekeeping proteins. Reagents in the kit may be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, explanatory material describing the use of the compositions within the kit may be included.

[0123] As used herein, the term "link" refers to the association of two or more molecules. Links can be covalent or non-covalent. Links can also be genetic (i.e., recombination fusion). Such links can be achieved using a variety of recognized techniques, such as chemical conjugation and recombinant protein production.

[0124] In some embodiments, a "connector" may refer to an amino acid sequence that links two proteins, polypeptides, peptides, domains, regions, or motifs, and may provide a spacer function compatible with the interaction of the two sub-binding domains, thereby resulting in a polypeptide that retains specific binding affinity for a target molecule (e.g., scTCR) or retains signal transduction activity (e.g., TCR complex). In some embodiments, the connector is composed of, for example, about 2 to about 35 amino acids, about 4 to about 20 amino acids, about 8 to about 15 amino acids, or about 15 to about 25 amino acids.

[0125] The term "MAGEA1" refers to a specific member of the melanoma antigen gene family clustered on human chromosome Xq28 (e.g., chromosome X: 153,179,284-153,183,880 positive strand. GRCh38: CM000685.2), also known as cancer / testis antigen 1.1 (CT1.1); melanoma-associated antigen 1; MAGE1; melanoma antigen family A1 (guiding the expression of antigen MZ2-E); cancer / testis antigen family 1 member 1; melanoma-associated antigen MZ2-E; melanoma antigen family A1; cancer / testis antigen 1.1; melanoma antigen MAGE-1; MAGE-1 antigen; antigen Z2-E, MGC9326; and MAGE1A (Mao et al. (2019) J. Hematol. Oncol. 12:106; Fanipakdel et al. (2019) J. Cell). Physiol. 234:12080-12086; Gu et al. (2018) Thorac. Cancer 9:431-438; Mecklenburg et al. (2017) Clin. Cancer Res. 23:1213-1219; Wang et al. (2016) Biochem. Biophys. Res. Commun. 473:959-965; Kozakova et al. (2015) Cell Cycle 14:920-930; Cannuyer et al. (2013) PLoS One 8:e5874; Pereira et al. (2012) Oncol. Rep. 27:1843-1848; Ogata et al. (2011) Ann. Surg. Oncol. 18:1195-1203; Roch et al. (2010) Anticancer Res. 30:1617-1623; Dango et al. (2010) Lung Cancer 67:290-295; van der Bruggen et al. (1991) Science 254:1643-1647). MAGEA1 is a melanoma antigen recognized by cytolytic T lymphocytes and is thought to be involved in transcriptional regulation through interaction with SNW1 and recruitment of histone deacetylase HDAC1, and to inhibit transactivation of the intracellular notch domain (NICD), embryonic development, and the development of some genetic disorders (e.g., congenital dyskeratosis) and / or tumor transformation or progression (e.g., gastric cancer, hepatocellular carcinoma, etc.). MAGEA1 is not highly expressed in normal tissues except the testes, but is expressed in various histological types of tumors, such as melanoma, head and neck cancer, lung cancer, cervical cancer, hepatocellular carcinoma, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and urothelial carcinoma of the bladder.

[0126] The term “MAGEA1” is intended to include its fragments, variants (e.g., allelic variants), and derivatives. Representative human MAGEA1 cDNA and human MAGEA1 protein sequences are well-known in the art and are publicly available from the National Center for Biotechnology Information (NCBI) (see, for example, ncbi.nlm.nih.gov / gene / 4100). For instance, human MAGEA1 (NP_004979.3) is encoded by the transcript (NM_004988.5). Nucleic acid and polypeptide sequences of MAGEA1 orthologs in organisms other than humans are well-known and include, for example, chimpanzee MAGEA1 (XM_529226.2 and XP_529226.2) and mouse MAGEA1 (chromosome X: 155088686-155089793; Ensembl mouse (mus musculus) type 104.39 (GRCm39)). Representative sequences of the MAGEA1 sequence are presented in Table 3 below.

[0127] Anti-MAGEA1 antibodies suitable for detecting MAGEA1 protein are well known in the art and include, for example, antibodies AM32863PU, AM50138PU, AP06212PU, AP13128PU, TA312178, TA39275, TA339275, TA339276, and TA347677 (OriGene, Rockville, MD); antibodies orb167376 and orb11016 (Biorbyt, Cambridge, United Kingdom); antibodies A03570 and AO3570-1 (Boster Bio, Pleasanton, CA); antibodies E22-11B2-E9 and N1C3 (GeneTex, Irvince, CA); and 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). Furthermore, reagents for detecting MAGEA1 expression are well-known. In addition, various siRNA, shRNA, and CRISPR constructs for regulating MAGEA1 expression can be found in the commercial product lists of multiple companies, such as open reading frame (ORF) clones MG212171, MR212171, MR212171L3, MR212171L3V, MR212171L4, MR212171L4V, RC202134, RC202134L3, RC202134L3V, RC202134L4, RC202134L4V, and RG202134 (OriGene, Rockville, MD); and CRISPR knockouts GA102785, GA202555, KN202134, KN202134BN, KN202134LP, KN202134RB, KN402134, and KN509652. (OriGene, Rockville, MD); and RNA interference (RNAi) clones, such as siRNA and shRNA clones, including SR302776, TL311617, SR410578, TL311617V, TTL516288, TL516288V, TL704467, TL04467V, TR311617, TR516288, and TR704467 (OriGene, Rockville, MD).It should be noted that this term can also be used to refer to any combination of characteristics of the MAGEA1 molecule described herein. For example, any combination of sequence composition, identity percentage, sequence length, domain structure, functional activity, etc., can be used to describe the MAGEA1 molecule covered by this invention.

[0128] The terms “MAGEA1 antigen,” “MAGEA1 peptide antigen,” “MAGEA1-containing peptide antigen,” “MAGEA1 epitope,” “MAGEA1 peptide epitope,” or “MAGEA1 peptide” refer to the naturally occurring or synthetically produced immunogenic portion of MAGEA1. In some embodiments, the length of the MAGEA1 antigen protein can range from about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids, or any range between these, such as 8-15 amino acids. In some embodiments, the MAGEA1 antigen protein can form a complex with an MHC (e.g., HLA) molecule, such that binding proteins of this disclosure that recognize the MAGEA1 peptide:MHC (e.g., HLA) complex can bind to such complexes (e.g., specifically and / or selectively). Table 1 shows representative MAGEA1 peptide antigen sequences.

[0129] The term "major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers, transmembrane-bound (with three α domains) and non-covalently associated β2 microglobulins. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which cross the membrane. Each chain has two domains. MHC class I molecules deliver cytosolic peptides to the cell surface, where the peptide antigen-MHC (pMHC) complex is activated by CD8+. + T cells recognize MHC class II molecules, which deliver peptides derived from the vesicle system to the cell surface, where they are recognized by CD4+. + T-cell recognition. Human MHC is known as human leukocyte antigen (HLA).

[0130] The terms “preventing” or “prevention” refer to reducing the probability of a subject who does not have a disease, condition, or illness but is at risk or susceptible to developing one.

[0131] The term "prognosis" includes a prediction of the likely course and outcome of cancer or the likelihood of recovery from the disease. In some implementations, statistical algorithms are used to provide a prognosis for an individual's cancer. For example, the prognosis can be surgery, the development of a clinical subtype of cancer, the development of one or more clinical factors, or disease recovery.

[0132] As used herein, the “percentage of identity” between amino acid sequences is synonymous with the “percentage of homology,” which can be determined using the Karlin and Altschul algorithm ((1990)Proc. Natl. Acad. Sci. USA 87:2264-2268) modified by Karlin and Altschul ((1993)Proc. Natl. Acad. Sci. USA 90:5873-5877). The mentioned algorithm was incorporated into the NBLAST and XBLAST programs of Altschul et al. ((1990)J. Mol. Biol. 215:403-410). BLAST nucleotide searches were performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the polynucleotides described herein. BLAST protein searches were performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the reference polypeptide. To obtain empty alignments for comparison purposes, empty BLAST is used, as described by Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402. When using the BLAST and empty BLAST procedures, the default parameters of their respective procedures (e.g., XBLAST and NBLAST) are used.

[0133] The phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, that participates in carrying or transporting the compounds of the present invention from one organ or part of the body to another organ or part of the body.

[0134] The term "ratio" refers to the relationship between two numbers (e.g., fractions, sums, etc.). Although ratios can be expressed in a particular order (e.g., a to b or a:b), those skilled in the art will recognize that the fundamental relationship between the numbers can be expressed in any order without losing its meaning, although observations and correlations based on the trend of the ratio may be reversed.

[0135] The term "recombinant host cell" (or simply "host cell") refers to a cell containing nucleic acids that are not naturally present in cells, such as cells to which a recombinant expression vector has been introduced. It should be understood that the term "cell" according to the invention refers not only to the specific subject cell but also to the progeny of such cells. Because subsequent generations may undergo modifications due to mutations or environmental influences, these progeny cells may actually differ from the parent cells but are still included within the scope of the term "cell" according to the invention.

[0136] The terms “cancer response,” “response to immunotherapy,” or “response to a combination of T-cell-mediated cytotoxic modulators / immunotherapy” refer to any response of a hyperproliferative disease (e.g., cancer) to cancer agents (e.g., T-cell-mediated cytotoxic modulators) and immunotherapy, preferably referring to changes in tumor quality and / or volume after the initiation of neoadjuvant or adjuvant therapy. The term “neoadjuvant therapy” refers to treatment administered prior to primary therapy. Examples of neoadjuvant therapies may include chemotherapy, radiation therapy, and hormone therapy. Response to hyperproliferative diseases can be assessed, for example, for efficacy or in neoadjuvant or adjuvant settings, where the tumor size after systemic intervention can be compared to the initial size and dimensions by means of CT, PET, mammography, ultrasound, or palpation. Response can also be assessed by caliper measurements of the tumor after biopsy or surgical resection or by pathological examination. Responses can be recorded quantitatively, such as as a percentage change in tumor volume, or qualitatively, 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 response to hyperproliferative disorders can be performed early after the initiation of neoadjuvant or adjuvant therapy, such as hours, days, weeks, or preferably months later. The typical endpoint for response assessment is the termination of neoadjuvant chemotherapy or surgical resection of residual tumor cells and / or the tumor bed. This is typically three months after the initiation of neoadjuvant therapy. In some implementations, the clinical efficacy of the therapeutic treatments described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by summing the following: the percentage of patients with complete response (CR), the number of patients with partial response (PR), and the number of patients with stable disease (SD) at a time point at least 6 months after the end of therapy. The abbreviated form of this formula is CBR = CR + PR + SD within 6 months. In some implementations, the CBR for a specific cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or higher. Other criteria for assessing response to cancer therapy relate to “survival” and include all of the following: survival to death, also known as overall survival (where death may be regardless of cause or tumor-related); “recurrence-free survival” (where the term recurrence should include both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease should include both cancer and related diseases). The length of said survival can be calculated by referring to a defined starting point (e.g., time of diagnosis or treatment initiation) and an endpoint (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 time period, and probability of tumor recurrence.For example, to determine an appropriate threshold, a specific cancer treatment regimen may be administered to a subject population and the outcome may be correlated with biomarker measurements determined prior to the administration of any cancer therapy. The outcome measurement may be a pathological response to a therapy administered in a neoadjuvant setting. Alternatively, outcome measures such as overall survival and disease-free survival may be monitored in subjects with known biomarker measurements over a period of time following cancer therapy. In some embodiments, the administered dose is a standard dose of a cancer therapeutic agent known in the art. The time period for monitoring subjects may vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Methods well known in the art, such as those described in the Examples section, may be used to determine the biomarker measurement thresholds associated with cancer therapy outcomes.

[0137] As noted, the term may also refer to improved prognosis, such as reflected in an increased time to recurrence, which is the period from the time of first recurrence review for death from a second primary cancer as a first event or without evidence of recurrence; or an increased overall survival, i.e., the period from treatment to death from any cause. A response or making a response means achieving a beneficial endpoint upon exposure to a stimulus. Alternatively, it means that negative or harmful symptoms are minimized, alleviated, or reduced upon exposure to a stimulus. It should be understood that assessing the likelihood that a tumor or subject will exhibit a favorable response is equivalent to assessing the likelihood that a tumor or subject will not exhibit a favorable response (i.e., will exhibit a lack of response or no response).

[0138] The term "resistance" refers to acquired or natural resistance to cancer therapy in a cancer sample or mammal (i.e., no response to therapeutic treatment or a reduced or limited response), for example, a reduced response to therapeutic treatment by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, such as 2, 3, 4, 5, 10, 15, 20, or more, or any range in between (including the endpoints). The reduction in response can be measured by comparison with the same cancer sample or mammal before resistance was acquired, or by comparison with different cancer samples or mammals known to be resistant to therapeutic treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance may be mediated by P-glycoproteins, or by other mechanisms, or may occur when a mammal is infected with a multidrug-resistant microorganism or combination of microorganisms. Assessing resistance to therapeutic treatment is a routine procedure in the art and can be measured within the skill level of a typical clinician, for example, by cell proliferation and cell death assays as described herein as “sensitization.” In some embodiments, the term “reversal of resistance” means that, where a single primary cancer therapy (e.g., chemotherapy or radiotherapy) fails to produce a statistically significant reduction in tumor volume compared to the untreated tumor volume, the combination of a second agent with the primary cancer therapy (e.g., chemotherapy or radiotherapy) produces a statistically significant reduction in tumor volume (e.g., p < 0.05) compared to the untreated tumor volume. This generally applies to tumor volume measurements performed when the untreated tumor is growing logarithmically.

[0139] The term "sample," used to detect or determine the absence, presence, or level of at least one biomarker, is generally used to refer to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluids (e.g., as described above under the definition of "bodily fluids"), or tissue samples (e.g., biopsies), such as skin, colon samples, or surgically removed tissue. In some embodiments, the methods covered by this invention further include obtaining the sample from the individual prior to detecting or determining the absence, presence, or level of at least one biomarker in the sample.

[0140] The term "sensitization" refers to altering cancer cells or tumor cells in a way that allows the relevant cancer to be treated more effectively with cancer therapies (e.g., anti-immune checkpoint therapy, chemotherapy, and / or radiation therapy). In some implementations, normal cells are not affected to the extent that they would suffer excessive damage from the treatment. The increase or decrease in sensitivity to a therapeutic treatment is measured according to methods known in the art for a specific treatment and the methods described below, including but not limited to cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164) and cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP ed., Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90). Sensitivity or resistance in animals can also be measured by measuring the reduction in tumor size over a period of time, such as 6 months in humans and 4–6 weeks in mice. If, compared to the sensitivity or resistance in the absence of the composition or method, the sensitivity increases or the resistance decreases by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, such as 2, 3, 4, 5, 10, 15, 20, or more, or any range in between (including the endpoints), then such compositions or methods sensitize the response to therapeutic treatment. Assessing sensitivity or resistance to therapeutic treatment is a routine procedure in the art and is within the skill level of a typical clinician. It should be understood that any method described herein for enhancing the efficacy of cancer therapies can also be applied to methods for sensitizing overproliferating or other cancer cells (e.g., resistant cells) to cancer therapies.

[0141] The term "small molecule" is a term used in the art and includes molecules with a molecular weight of less than about 1000 or less than about 500. In one embodiment, a small molecule contains more than just peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptide mimics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63-68), and libraries of natural product extracts. In another embodiment, the compound is a small organic non-peptide compound. In another embodiment, the small molecule is not biosynthesized.

[0142] The term "specific binding" refers to the binding of a binding protein to a predetermined antigen. Typically, when using an antigen of interest as the analyte and a binding protein as the ligand in a BIAcore™ assay instrument via binding assays, such as surface plasmon resonance (SPR) technology, the binding protein binds at a rate of approximately less than or equal to approximately 5 × 10⁻⁶. -4 M, less than or equal to approximately 1 × 10 -4 M, less than or equal to approximately 5 × 10 -5 M, less than or equal to approximately 1 × 10 -5 M, less than or equal to approximately 5 × 10 -6 M, less than or equal to approximately 1 × 10 - 6 M, less than or equal to approximately 5 × 10 -7 M, less than or equal to approximately 1 × 10 -7 M, less than or equal to approximately 5 × 10 -8 M, less than or equal to approximately 1 × 10 -8 M, less than or equal to approximately 5 × 10 -9 M, less than or equal to approximately 1 × 10 -9 M, less than or equal to approximately 5 × 10 -10 M, less than or equal to approximately 1 × 10 -10 M, less than or equal to approximately 5 × 10 -11 M, less than or equal to approximately 1 × 10 -11 M, less than or equal to approximately 5 × 10 -12 M, less than or equal to approximately 1 × 10 -12 M or lower, or any range in between (including the endpoints), such as approximately 1-50 micromoles, 1-100 micromoles, 0.1-500 micromoles, etc., with an affinity (K). DBinding. In some embodiments, the binding protein binds to the predetermined antigen with an affinity 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 that it binds to non-specific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. The phrases “antigen-recognizing binding protein” and “antigen-specific binding protein” are used interchangeably herein with the term “antigen-specific binding protein.” Selective binding is a relative term that refers to the ability of a binding protein to distinguish the binding of one antigen from the binding of another antigen, such as the ability to distinguish the binding of a particular family member or antigen target from the 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 the MAGEA1 immunogenic epitope and / or selectively bind to many related epitopes (e.g., the MAGEA1 immunogenic epitope and closely related sequences), thereby distinguishing such targets from the many other possible epitopes available in the human genome.

[0143] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a condition characterized by MAGEA1 expression (e.g., a non-malignant condition, hyperplastic condition, or recurrent hyperplastic condition characterized by MAGEA1 expression). The term "subject" may be used interchangeably with "patient."

[0144] The term "survival" encompasses all of the following: survival to death, also known as overall survival (where death may be due to no cause or related to the tumor); "recurrence-free survival" (where recurrence should include both local and distant recurrence); metastasis-free survival; and disease-free survival (where disease should include both cancer and related diseases). The length of survival can be calculated by referring to a defined starting point (e.g., time of diagnosis or treatment initiation) and an endpoint (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0145] The term "synergistic effect" refers to the combined effect of two or more agents (e.g., the MAGEA1-related agents described herein and another therapy for treating conditions characterized by MAGEA1 expression, such as additional MAGEA1-targeting TCRs, anticancer therapies, immunotherapies, etc.) that is greater than the sum of the individual effects of individual cancer agents / therapies.

[0146] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8+). + T cells and helper T cells (e.g., CD4 cells) ... + T cell-mediated responses. T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0147] “Transcribed polynucleotides” or “nucleotide transcripts” are polynucleotides (e.g., mRNA, hnRNA, cDNA, or analogs of such RNA or cDNA) that are complementary to or homologous to all or part of mature mRNA, which is produced by transcription of biomarker nucleic acids and (if present) normal post-transcriptional processing (e.g., splicing) of RNA transcripts and reverse transcription of RNA transcripts.

[0148] T cells are immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells can be naïve T cells (unexposed to antigens; similar to T cells) or T cells that have not been exposed to antigens. CM Compared to CD62L, CCR7, CD28, CD3, CD127, and CD45RA, the expression of CD45RO was increased, and the expression of memory T cells (T) was decreased. M (Experienced antigens and have a long lifespan) and effector cells (experienced antigens and are cytotoxic). T M It can also be divided into central memory T cells (T cells). CM Compared to naive T cells, the expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95 was increased, while the expression of CD54RA was decreased. (This is in contrast to effector memory T cells.) EM , with naïve T cells or T CM Compared to CD62L, CCR7, CD28, and CD45RA, the expression of CD127 was reduced, while the expression of CD127 was increased. (This refers to a subset of effector T cells.) E () refers to CD8+ cytotoxic T lymphocytes that have undergone antigen exposure, and T CM In comparison, the expression of CD62L, CCR7, and CD28 was reduced, and the cells were positive for granzyme and perforin. Other exemplary T cells include regulatory T cells, such as CD4+. + CD25 + (Foxp3 + Regulatory T cells and Treg17 cells, as well as Tr1, Th3, and CD8 cells. + CD28 and Qa-1 restricted T cells.

[0149] Conventional T cells (also known as Tconv or Teff) possess effector functions (e.g., secretion of cytokines, cytotoxic activity, anti-self-recognition, etc.) to enhance immune responses by expressing one or more T cell receptors. Tcon or Teff is generally defined as any non-Treg T cell population and includes, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated into, for example, Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is CD4+ Teff or CD8+ Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T lymphocytes. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tcon" is a CD4+ T cell that has differentiated in the bone marrow and has successfully undergone positive and negative central selection in the thymus, but has not yet been activated by exposure to antigens. + T cells. Nascent Tcon are typically characterized by surface expression of L-selectin (CD62L), lack of activation markers (e.g., CD25, CD44, or CD69), and lack of memory markers (e.g., CD45RO). Therefore, nascent Tcon are believed to be quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin-15 (IL-15) to maintain homeostatic survival (see at least WO 2010 / 101870). The presence and activity of these cells are not required in the context of suppressing immune responses. Unlike Tregs, Tcon are not unresponsive and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637).

[0150] "T effect" eff "or "T E T cells refer to T cells with cytolytic activity (such as CD4+ and CD8+ T cells), as well as T helper (Th) cells that secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0151] "T-cell receptor" or "TCR" refers to a member of the immunoglobulin superfamily (possessing a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, for example, Janeway et al. (1997) Curr. Biol. Publ. 4:33) capable of binding (e.g., specifically and / or selectively) antigenic peptides that bind to MHC receptors. TCRs can be present on the cell surface or in soluble form and are typically composed of heterodimers with α and β chains (also referred to as TCRα and TCRβ, respectively) or γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portion of the TCR chain (e.g., the α and β chains) contains two immunoglobulin domains: an N-terminal variable domain (e.g., the α-chain variable domain or V-chain variable domain). α and β-chain variable structural domain or V β Typically, it consists of amino acids 1 to 116 based on Kabat numbering (Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," US Dept. Health and Human Services, Public Health Service, National Institutes of Health, 5th edition), and a constant domain at the C-terminus and adjacent to the cell membrane (e.g., the α-chain constant domain or C-chain constant domain). α Typically, it consists of Kabat-based amino acids 117 to 259; the β-chain constant domain or C β Typically, the amino acid composition is based on Kabat (117 to 295). Furthermore, like immunoglobulins, the variable domain contains complementarity-determining regions (“CDRs”, also known as hypervariable regions or “HVRs”) separated by framework regions (“FRs”) (see, for example, Fores et al. (1990) Proc. Natl. Acad Sci. US.A. 87:9138; Chothia et al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol. 27:55). In some embodiments, the TCR is present on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. The TCRs covered by this invention can be derived from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0152] The term "T-cell receptor" or "TCR" should be understood to encompass the complete TCR as well as its antigen-binding moiety or fragment. In some embodiments, the TCR is a complete or full-length TCR, including TCRs in αβ or γδ form. In some embodiments, the TCR is a smaller than full-length TCR that binds to a specific peptide in an MHC molecule, such as an antigen-binding moiety that binds to an MHC-peptide complex. In some cases, the antigen-binding moiety or fragment of the TCR may contain only a portion of the domains of the full-length or complete TCR, but may still be able to bind to the peptide epitope bound by the complete TCR, such as an MHC-peptide complex. In some cases, the antigen-binding moiety contains variable domains of the TCR, such as variable α-chains and variable β-chains of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain complementarity-determining regions (CDRs) involved in the recognition of peptides, MHC, and / or MHC-peptide complexes.

[0153] Nomenclature was established using the International Immunogenetic Information System (IMGT) (see also Scaviner and Lefranc (2000) Exp. Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; T Cell Receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). IMGT provides unique sequences for describing TCRs, and the sequences described herein can be identified by referring to these unique sequences. TCR sequences are publicly available in the IMGT database at imgt.org.

[0154] As described above, the natural α / β heterodimer TCR has an α chain and a β chain. Broadly speaking, each chain contains a variable region, a conjugation region, and a constant region, and the β chain typically contains a short diversification region between the variable and conjugation regions, but this diversification region is usually considered part of the conjugation region. Each variable region contains three hypervariable CDRs (complementarity-determining regions) embedded in the frame sequence. CDR3 is known to be the primary mediator of antigen recognition. Several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions exist, distinguished by their frame, CDR1 and CDR2 sequences, and a partially defined CDR3 sequence. In IMGT nomenclature, Vα types are represented by unique TRAV numbers. For example, “TRAV4” defines a TCR Vα region with a unique frame and CDR1 and CDR2 sequences, and a CDR3 sequence defined by a conserved amino acid sequence between TCRs but also including amino acid sequences that change between TCRs. Similarly, “TRBV2” defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence. It is known that there are 54 α-variable genes, 44 of which are functional, and 67 β-variable genes, 42 of which are functional, within the α and β loci, respectively.

[0155] Similarly, the junction region of the TCR is defined by the unique IMGT TRAJ and TRBJ nomenclature, while the constant region is defined by the IMGTTRAC and TRBC nomenclature. In the IMGT nomenclature, the β-chain diversity region is simply referred to as TRBD, and as previously mentioned, tandem TRBD / TRBJ regions are usually considered together as junction regions.

[0156] The gene pools encoding the TCR α and β chains are located on different chromosomes and contain independent V, (D), J, and C gene segments that cluster together through rearrangement during T cell development. This results in extremely high diversity of the T cell α and β chains due to numerous possible recombination events between the 54 TCR α variants and 61 α J genes, or between the 67 β variants, two β D genes, and 13 β J genes. Recombination processes are not precise and introduce further diversity within the CDR3 region. Each α and β variant can also contain allelic variants, designated in IMGT nomenclature as TRAVxx*01 and *02, or TRBVx-x*01 and *02, respectively, further increasing the amount of variation. Similarly, some TRBJ sequences have two known variants. (Note that the absence of the "*" qualifier indicates that only one allele is known for the relevant sequence). The natural pedigree of the human TCR, resulting from recombination and thymic selection, is estimated to contain approximately 10... 6Each β-chain has a unique sequence, determined by CDR3 diversity (Arstila et al. (1999) Science 286:958-961), and possibly even higher (Robins et al. (2009) Blood 114:4099-4107). It is estimated that each β-chain pairs with at least 25 different α-chains, resulting in further diversity (Arstila et al. (1999)). Science 286:958-961).

[0157] Therefore, the term "TCR α variable domain" refers to the tandem of the TRAV and TRAJ regions; the TRAV region only; or the TRAV and part of the TRAJ region, and the term "TCR α constant domain" refers to the extracellular TRAC region, or a C-terminal truncated or full-length TRAC sequence. Similarly, the term "TCR β variable domain" refers to the tandem of the TRBV and TRBD / TRBJ regions; the TRBV and TRBD regions only; the TRBV and TRBJ regions only; or the TRBV and part of the TRBD and / or TRBJ regions, and the term "TCR β constant domain" refers to the extracellular TRBC region, or a C-terminal truncated or full-length TRBC sequence. These TCR α and TCR β variable domain nomenclatures are similarly applied to the variable domains of the TCR γ and TCR δ chains of γ / δ TCRs, respectively. Gene sequences of TRAV, TRAJ, TRAC, TRBV, TRBJ, and TRBC can be obtained, for example, from the publicly available IMGT database.

[0158] The term "TCR complex" refers to a complex formed by the association of CD3 and TCR. For example, a TCR complex can consist of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of the CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex can consist of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of the CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0159] The term "therapeutic effect" refers to the local or systemic effects caused by a pharmacologically active substance in animals, especially mammals, and even more particularly in humans. Therefore, the term means any substance intended for the diagnosis, cure, relief, treatment, or prevention of disease in animals or humans, or for enhancing their desired physical or intellectual development and condition.

[0160] The terms "therapeutic effective amount" and "effective amount" refer to the amount of a substance that produces some desired effect, such as a desired local or systemic therapeutic effect, in at least one cell subpopulation in an animal with a reasonable benefit / risk ratio suitable for any treatment. In some embodiments, the therapeutic effective amount of a substance will depend on the substance's therapeutic index, solubility, pharmacokinetics, half-life, etc. It can be determined in cell cultures or laboratory animals, for example, by using methods such as determining LD50. 50 and ED 50 Standard pharmaceutical procedures are used to determine the toxicity and therapeutic efficacy of the subject compound. In some embodiments, compositions exhibiting a large therapeutic index are used. In some embodiments, the LD50 can be measured. 50 (Lethal dose), and compared to no administration, it can be reduced by, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more when the drug is administered. Similarly, ED can be measured. 50 (i.e., the concentration at which half-maximal inhibition of symptoms is achieved), and compared to no medication, it can increase by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more when medication is administered. Similarly, IC50 can also be measured. 50 And compared to no medication, the effect of medication application can increase the effect by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. In some embodiments, in one assay, the T-cell immune response can be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, a viral load reduction 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%.

[0161] The term "treatment" refers to the therapeutic management or improvement of a condition of interest (e.g., a disease or symptom). Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically performed in an effort to alter the course of a disease (the term is used to indicate any disease, symptom, syndrome, or adverse condition that requires or may require a therapy) in a manner beneficial to the subject. Therapeutic effects may include reversing, alleviating, or reducing one or more symptoms or manifestations of a disease, reducing its severity, delaying its onset, curing it, inhibiting its progression, and / or reducing its likelihood of occurrence or recurrence. Desired therapeutic effects include, but are not limited to: preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. Therapeutic agents may be administered to subjects who have the disease or who have an increased risk of disease development relative to members of the general population. In some embodiments, therapeutic agents may be administered to subjects who have had the disease but no longer show signs of the disease. Agents may be administered, for example, to reduce the likelihood of a significant recurrence of the disease. Therapeutic agents may be administered preventively, i.e., before any symptoms or manifestations of the disease appear. "Preventive treatment" refers to providing medical and / or surgical treatment to subjects who have not yet developed the disease or show no signs of it, in order to, for example, reduce the likelihood of the disease developing or reduce the severity of the disease when it does develop. Subjects may have been identified as being at risk of developing the disease (e.g., having an increased risk relative to the general population or having risk factors that increase the likelihood of developing the disease).

[0162] The term "unresponsiveness" includes the refractive index of cancer cells to therapy, or the refractive index of therapeutic cells, such as immune cells, to stimulation, such as stimulation via activating receptors or cytokines. Unresponsiveness can occur, for example, due to exposure to immunosuppressants or to high doses of antigens. As used herein, the terms "incompetent" or "tolerant" include the refractive index of activating receptor-mediated stimulation. This refractive index is typically antigen-specific and persists after cessation of exposure to the tolerant antigen. For example, T cell incompetence (as opposed to unresponsiveness) is characterized by the lack of production of cytokines such as IL-2. T cell incompetence occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, re-exposure to the same antigen (even in the presence of co-stimulatory peptides) results in the inability to produce cytokines and therefore cannot proliferate. However, if cultured with cytokines (e.g., IL-2), incompetent T cells may proliferate. For example, T cell anergy can be observed by measuring the lack of IL-2 production in T lymphocytes using ELISA or a proliferation assay using indicator cell lines. Alternatively, a reporter gene construct can be used. For example, anergic T cells are unable to initiate IL-2 gene transcription induced by a heterologous promoter controlled by the 5' IL-2 gene enhancer or by an AP1 sequence multimer that may be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0163] The term "vaccine" refers to a pharmaceutical composition that elicits an immune response to an antigen of interest. Vaccines can also confer protective immunity to a subject.

[0164] The term "variable region" or "variable domain" refers to a domain of an immunoglobulin superfamily binding protein (e.g., the α or β chain of a TCR (or the γ and δ chains for γδTCR)) that is involved in the binding of an immunoglobulin superfamily binding protein (e.g., TCR) to an antigen. The α and β chains of the native TCR (V...) α and V β The variable domains of V typically have similar structures, with each domain containing four conservative frame regions (FRs) and three conserved frame regions (CDRs). α The domain is encoded by two separate DNA segments: the variable gene segment and the conjugation gene segment (VJ); V β The domain is encoded by three separate DNA segments: the variable gene segment, the diversity gene segment, and the conjugation gene segment (VDJ). A single V... α or V β The structural domain is sufficient to confer antigen-binding specificity. Furthermore, V can be used. α or V βThe domain separates TCRs that bind specific antigens from antigen-binding TCRs to screen complementary V-type TCRs. α or V β A library of structural domains.

[0165] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a free organism, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, the vector is one capable of autonomous replication and / or expression of the nucleic acid to which it is linked. A vector capable of guiding the expression of a gene with operatively linked structures is referred to herein as an "expression vector." Generally, expression vectors used in recombinant DNA technology are typically in the form of "plasmids," which generally refer to circular double-stranded DNA loops whose vector form does not bind to chromosomes. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, as those skilled in the art will appreciate, the present invention is intended to include other forms of such expression vectors that provide equivalent functionality and are subsequently known in the art.

[0166] There is a known and established correspondence between the amino acid sequence of a specific protein and the nucleotide sequence that encodes that protein, as defined by the genetic code (as shown below). Similarly, there is a known and established correspondence between the nucleotide sequence of a specific nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0167] A key and well-known feature of the genetic code is its redundancy; that is, for most amino acids used to make proteins, there may be more than one coding nucleotide triplet (as shown above). Therefore, many different nucleotide sequences may encode a given amino acid sequence. These nucleotide sequences are considered functionally equivalent because they result in the same amino acid sequence in all organisms (although some organisms may translate some sequences more efficiently than others). Furthermore, occasionally methylated variants of purines or pyrimidines may be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0168] Given the above, a polypeptide amino acid sequence can be derived using the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any part thereof), and by translating the DNA or RNA into an amino acid sequence using the genetic code. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence encoding that polypeptide can be deduced from the genetic code (due to its redundancy, multiple nucleic acid sequences will be generated for any given amino acid sequence). Therefore, the description and / or disclosure of nucleotide sequences encoding polypeptides herein should be considered to also include the description and / or disclosure of amino acid sequences encoded by those nucleotide sequences. Likewise, the description and / or disclosure of polypeptide amino acid sequences herein should be considered to also include the description and / or disclosure of all possible nucleotide sequences that can encode those amino acid sequences.

[0169] II. Peptides In some respects, this article provides methods and compositions for treating and / or preventing conditions associated with MAGEA1 expression by inducing an immune response against MAGEA1 or cells expressing MAGEA1, which involve administering the MAGEA1 immunogenic peptide described herein, nucleic acids encoding the MAGEA1 immunogenic peptide, and / or cells expressing the MAGEA1 immunogenic peptide.

[0170] In some embodiments, the MAGEA1 immunogenic peptide comprises a peptide epitope (e.g., composed of) peptide sequences selected from those listed in Table 1, such as Table 1A. The peptide epitopes described herein may combine with MHC molecules, such as specific HLA molecules having a specific HLA α-chain allele. For example, the peptides in Table 1A are identified as associating with MHCs whose α-chain has the HLA-A*01 serotype, such as MHCs encoded by the HLA-A*01:01 allele, as further described in the Examples section. In some embodiments, the MAGEA1 immunogenic peptide may combine with an MHC molecule containing an MHC α-chain of the HLA serotype HLA-A*01, optionally wherein the HLA allele is HLA-A*01:01. In some embodiments, the MAGEA1 immunogenic peptide is derived from human MAGEA1 protein and / or the MAGEA1 protein shown in Table 3. In some embodiments, one or more MAGEA1 immunogenic peptides are administered alone or in combination with an adjuvant.

[0171] In some respects, compositions comprising one or more of the MAGEA1 immunogenic peptides described herein and an adjuvant are provided.

[0172] Table 1: MAGEA1 Tabletops Table 1A MAGEA1 epitope presented by HLA serotype HLA-A*01

[0173] *Table 1, such as Table 1A, includes peptide epitopes, and polypeptide molecules containing amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identical in length to any of the sequences listed in Table 1, such as Table 1A. Such polypeptides may have the functions of the full-length peptides or polypeptides further described herein.

[0174] In some embodiments, this document provides a MAGEA1 polypeptide and / or a nucleic acid encoding a MAGEA1 polypeptide. 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 some embodiments, the MAGEA1 polypeptide also includes amino acids that do not correspond to said amino acid sequence (e.g., a fusion protein comprising the 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 an amino acid sequence corresponding to the MAGEA1 protein or a fragment thereof.

[0175] In some embodiments, the amino acid sequence of the MAGEA1 polypeptide comprises, is substantially composed of, or consists of the following: the amino acid sequence of the MAGEA1 protein, for example, 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 amino acids as shown in Table 3. 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 373 or more, or any range in between (e.g., 7-25, 8-22, 9-22, etc.) (including the endpoints) consecutive amino acids. In some embodiments, the consecutive amino acids are identical to the amino acid sequence of MAGEA1 shown in Table 3. In some embodiments, the MAGEA1 polypeptide comprises one or more peptide epitopes selected from the group consisting of MAGEA1 peptide epitopes listed in Table 1, such as Table 1A, substantially consisting of or consisting of them.

[0176] As is well known to those skilled in the art, polypeptides with significant sequence similarity can elicit the same or very similar immune responses in host animals. Therefore, in some embodiments, derivatives, equivalents, variants, fragments, or mutants of the MAGEA1 immunogenic peptide or fragments thereof described herein may also be suitable for the methods and compositions provided herein.

[0177] In some embodiments, this document provides variant forms or derivatives of the MAGEA1 immunogenic peptide. The altered peptide may have an altered amino acid sequence, for example, through conserved substitution, but still elicit an immune response to an unaltered protein antigen and is considered a functional equivalent. As used herein, the term "conserved substitution" means that an amino acid residue is replaced by another biologically similar residue. It is well known in the art that amino acids within the same conserved group can often be substituted for each other without substantially affecting the function of the protein. According to some embodiments, derivatives, equivalents, variants, or mutants of the ligand-binding domain of the MAGEA1 immunogenic peptide are peptides that are at least 85% sequence homologous to the MAGEA1 immunogenic peptide or fragment thereof described herein. In some embodiments, the homology is at least 90%, at least 95%, at least 98%, or higher.

[0178] The immunogenic peptides covered by this invention may comprise peptide epitopes derived from the MAGEA1 protein, such as those listed in Table 1, such as Table 1A. 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 conserved or non-conserved mutations. The peptide may contain up to 1, 2, 3, 4, or more mutations. In some embodiments, the peptide may contain at least 1, 2, 3, 4, or more mutations.

[0179] In some embodiments, the peptide may be chemically modified. For example, the peptide may be mutated to modify peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation sites, pH, and function. N-methylation is an example of methylation that can occur in the peptides disclosed herein. In some embodiments, the peptide may be modified by methylating a free amine, for example by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0180] Chemical modifications may include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendritic polymers, Fc regions, simple saturated carbon chains (e.g., palmitate or myristate), or albumin. Chemical modifications of peptides having Fc regions may be fused Fc-peptides. Polyamino acids may include, for example, polyamino acid sequences having repeating single amino acids (e.g., polyglycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the foregoing. In some embodiments, the peptides covered by this disclosure may be modified such that the modifications increase the stability and / or half-life of the peptide. In some embodiments, the attachment of hydrophobic portions (e.g., to the N-terminus, C-terminus, or internal amino acids) may be used to extend the half-life of the peptides covered by this disclosure. In other embodiments, the peptide may include post-translational modifications (e.g., methylation and / or amidation) that affect, for example, serum half-life. In some embodiments, simple carbon chains (e.g., by myristylation and / or palmitylation) may be conjugated to fusion proteins or peptides. In some embodiments, a simple carbon chain facilitates the separation of the fusion protein or peptide from the unconjugated material. For example, methods for separating the fusion protein or peptide from the unconjugated material include, but are not limited to, solvent extraction and reversed-phase chromatography. The lipophilic moiety can have its half-life extended by reversible binding to serum albumin. The conjugated moiety can be a lipophilic moiety whose half-life is extended by reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or cholesterol derivatives, including cholesterolene, cholesterolane, cholesteroldiene, and oxidized sterols. In some embodiments, the peptide can be conjugated with myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptide can be coupled (e.g., conjugated) with a half-life modifier. Examples of half-life modifiers include, but are not limited to: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers containing proline, alanine, and serine, water-soluble polymers containing glycine, glutamic acid, and serine, Fc regions, fatty acids, palmitic acid, or molecules bound to albumin. In some embodiments, spacers or linkers may be coupled to peptides, for example, one, two, three, four, or more amino acid residues serving as spacers or linkers, to facilitate conjugation or fusion with another molecule and to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, fusion proteins or peptides may be conjugated to other parts, for example, that can modify or achieve changes in the properties of the peptide.

[0181] In some embodiments, the peptide may be covalently linked to a portion. In some embodiments, the covalently linked portion 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, HaloTag, HA tag, Flag® tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently linked portion may be selected from the group consisting of: pro-inflammatory cytokines, anti-inflammatory agents, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies, such as single-chain Fv.

[0182] Peptides can be conjugated to agents used in imaging, research, therapeutics, therapeutic diagnostics, pharmacology, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, the peptide can be conjugated to or fused with a detectable agent, such as a fluorophore, near-infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radionuclide chelator, or another suitable material that can be used for imaging. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more detectable moieties can 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 radioactive isotope 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 radioactive isotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not easily quenched by biological tissues and body fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation with wavelengths between 650 nm and 4000 nm; this type of emission is used for the detection of such agents. Non-limiting examples of fluorescent dyes that can be used as conjugated molecules include DyLight®-680, DyLight®-750, VivoTag®-750, DyLight®-800, IRDye®-800, VivoTag®-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, the near-infrared dye typically includes cyanine dyes (e.g., Cy7, Cy5.5, and Cy5).Further non-limiting examples of fluorescent dyes used as conjugating molecules in this disclosure include acridine orange or acridine yellow, Alexa Fluors® (e.g., Alexa Fluor® 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dyes and any derivatives thereof, auramine-rhodamine staining agents and any derivatives thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthobenzene, bisbenzoimide, brain rainbow, calcein, carboxyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, and DyLight. Fluors and any of their derivatives, epicoconone, ethidium bromide, FlasH-EDT2, Fluo dyes and any of their derivatives, FluoProbe and any of its derivatives, fluorescein and any of its derivatives, Fura and any of its derivatives, GelGreen and any of its derivatives, GelRed and any of its derivatives, fluorescent proteins and any of their derivatives, m-isotype proteins and any of their derivatives (e.g., mCherry), hetamethine dyes and any of their derivatives, hoeschst staining agents, iminocoumarin, Indian yellow, indo-1 and any of its derivatives, laurdan, fluorescein yellow and any of its derivatives, fluorescein and any of its derivatives, luciferase and any of its derivatives, cyanide and any of its derivatives, Nile dyes dyes and any derivatives thereof, perylene, phloxine, algal dyes and any derivatives thereof, propidium iodide, pyranine, rhodamine and any derivatives thereof, ribose green, RoGFP, rubrene, stilbene and any derivatives thereof, sulfonyl rhodamine and any derivatives thereof, SYBR™ and any derivatives thereof, synapto-pH-sensitive green fluorescent protein, tetraphenylbutadiene, tris tetrasodium, Texas Red, Titan Yellow, TSQ, umbelliferone, violet anthrone, yellow fluorescent protein, and YOYO-1.Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbonyl cyanide, styrene dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissaminerhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), and Oregon Green® dyes (e.g., Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, etc.), Texas Red, Texas Red-X, SPECTRUM RED, SPECTRUM GREEN, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA 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® Examples of suitable detectable agents include 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, etc., and IRDye (e.g., IRD40, IRD 700, IRD 800, etc.). Other suitable detectable agents are described in PCT / US14 / 56177. Non-limiting examples of radioactive isotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioactive isotope 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 implementations, the radioactive isotope is actinium-225 or lead-212.

[0183] Peptides can be conjugated with radiosensitizers or photosensitizers. Examples of radiosensitizers include, but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to: fluorescent molecules or beads that generate heat upon irradiation, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorophyll, bacteriochlorin, isochlorophyll, phthalocyanine, and naphthylphthalocyanine), metalloporphyrins, metal phthalocyanines, angelicin, and chalcogenapyrrillium dyes. Dyes, chlorophyll, coumarin, flavins and related compounds (e.g., chlorophyll and riboflavin), fullerene, pheophorbide, pyropheophorbide, anthocyanins (e.g., cyanin 540), pheophorbide, sapphyrin, texaphyrin, purpurin, porphyrin, phenothiazinium, methylene blue derivatives, naphthalene dicarboximide, Nile blue derivatives, quinones, perylene quinones (e.g., hypericin, hypocrellin, and cercosporin), psoralen, quinones, retinoids, rhodamine, thiophene, verdin, xanthene dyes (e.g., eosin, erythrosine, rose bengal), etc. The method utilizes porphyrins in dimer and oligomeric forms, such as bengal, and prodrugs, such as 5-aminolevulinic acid. Advantageously, this method allows for highly specific targeting of cells of interest (e.g., immune cells) using both therapeutic agents (e.g., drugs) and electromagnetic energy (e.g., radiation or light). In some embodiments, the peptide is fused to the agent or covalently or non-covalently linked to the agent, such as directly or via a linker.

[0184] In some embodiments, the binding protein may be chemically modified. For example, the binding protein may be mutated to modify peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation sites, pH, and function. N-methylation is an example of methylation that can occur in the binding proteins covered by this invention. In some embodiments, the binding protein may be modified by methylating a free amine, for example by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0185] Chemical modifications may include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendritic polymers, Fc regions, simple saturated carbon chains (e.g., palmitate or myristate), or albumin. Chemical modifications of binding proteins with Fc regions may be fused Fc-proteins. Polyamino acids may include, for example, polyamino acid sequences having repeating single amino acids (e.g., polyglycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination thereof.

[0186] In some embodiments, the binding protein covered by this invention may be modified. In some embodiments, the modification has substantial or significant sequence identity with the parent binding protein to produce a functional variant that maintains one or more biophysical and / or biological activities of the parent binding protein (e.g., maintaining pMHC binding specificity). In some embodiments, mutations are made to conserved amino acid substitutions.

[0187] In some embodiments, the binding proteins covered by this invention may comprise 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, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indole. Phosphoric acid-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-phenylmethyl-N'-methyl-lysine, N',N'-diphenylmethyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, oc-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norborneane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and oc-tert-butylglycine.

[0188] The binding proteins covered by this invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0189] In some embodiments, the attachment of a hydrophobic portion (e.g., to the N-terminus, C-terminus, or internal amino acid) can be used to extend the half-life of the peptides covered by this invention. In other embodiments, the binding protein may include post-translational modifications (e.g., methylation and / or amidation) that can affect, for example, serum half-life. In some embodiments, a simple carbon chain (e.g., by myristylation and / or palmitoylation) may be conjugated to the binding protein. In some embodiments, a simple carbon chain may facilitate the separation of the binding protein from unconjugated material. For example, methods that can be used to separate the binding protein from unconjugated material include, but are not limited to, solvent extraction and reversed-phase chromatography. The lipophilic portion can extend the half-life by reversible binding to serum albumin. The conjugated portion can be a lipophilic portion that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic portion can be cholesterol or cholesterol derivatives, including cholesterolene, cholesterolane, cholesteroldiene, and oxidized sterols. In some embodiments, the binding protein may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding protein may be coupled (e.g., conjugated) to a half-life modifier. Examples of half-life modifiers include, but are not limited to: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers containing 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, spacers or linkers may be coupled to the binding protein, for example, one, two, three, four, or more amino acid residues serving as spacers or linkers, to facilitate conjugation or fusion with another molecule and to facilitate peptide cleavage from such conjugated or fused molecules. In some embodiments, the binding protein may be conjugated to other parts, for example, that can modify or achieve changes in the properties of the binding protein.

[0190] Proteins, such as peptides, can be produced, for example, through solid-phase peptide synthesis or solution-phase peptide synthesis, either recombinantly or synthetically. Protein synthesis can be performed using known synthetic methods, such as fluorenylmethoxycarbonyl (Fmoc) chemistry or butyloxycarbonyl (Boc) chemistry. Protein fragments can be joined together by enzymatic or synthetic means.

[0191] In one aspect covered by the present invention, a method for producing the protein described herein is provided, the method comprising the steps of: (i) culturing transformed host cells under conditions suitable for allowing expression of the binding protein described herein, the host cells having been transformed with nucleic acids containing a sequence encoding the binding protein; and (ii) recovering the expressed binding protein.

[0192] For example, methods for isolating and purifying recombinant-derived binding proteins may include obtaining a supernatant from a suitable host cell / carrier system that secretes the binding protein into a culture medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reversed-phase HPLC steps may be used to further purify the recombinant peptide. These purification methods can also be used when isolating immunogens from the natural environment. Methods for large-scale production of one or more binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Binding proteins may be purified according to methods described herein and known in the art.

[0193] In some embodiments, this document provides a nucleic acid encoding the MAGEA1 immunogenic peptide or a fragment thereof described herein, such as a DNA molecule encoding the MAGEA1 immunogenic peptide. In some embodiments, the composition comprises an expression vector containing an open reading frame encoding the MAGEA1 immunogenic peptide or a fragment thereof described herein. In some embodiments, the nucleic acid includes regulatory elements necessary for the expression of the open reading frame. Such elements may include, for example, promoters, start codons, stop codons, and polyadenylation signals. Additionally, enhancers may be included. These elements are operatively linked to a sequence encoding the MAGEA1 immunogenic peptide or a fragment thereof. Representative vectors, promoters, regulatory elements, etc., that can be used to express proteins such as peptides are further described below.

[0194] III. MHC-peptide complex In some aspects, compositions comprising the MAGEA1 immunogenic peptide described herein and an MHC molecule are provided. In some embodiments, the MAGEA1 immunogenic peptide forms a stable complex with the MHC molecule.

[0195] MHC proteins can be conjugated to agents such as detection modulonucleotides, radiosensitizers, and photosensitizers, and / or can be chemically modified as described above regarding peptides.

[0196] The MHC protein provided and used in the compositions and methods covered in this disclosure can be any suitable MHC molecule known in the art. Generally, it has the formula (α-β-P). n, where n is at least 2, for example, between 2 and 10, such as 4. α is the α chain of a class I or class II MHC protein. β is the β chain, defined herein as the β chain of a class II MHC protein or the β2 microglobulin of a class I MHC protein. P is a peptide antigen.

[0197] In some implementations, the MHC protein is an MHC class I complex, such as an HLA I complex.

[0198] MHC proteins can originate from any mammalian or avian species, such as primates, especially humans; rodents, including mice, rats, and hamsters; rabbits; horses, cattle, dogs, cats, etc. For example, MHC proteins can be derived from human HLA proteins or mouse H-2 proteins. HLA proteins include class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, and class I proteins HLA-A, HLA-B, HLA-C, and β2-microglobulin. H-2 proteins include class I subunits H-2K, H-2D, and H-2L, and class II subunits I-Aα, I-Aβ, I-Eα, and I-Eβ, and β2-microglobulin. Sequences of some representative MHC proteins can be found in Kabat et al., Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pp. 724-815. The MHC protein subunits suitable for use in this invention are soluble forms of normal membrane-bound proteins, prepared as known in the art, for example by the deletion of transmembrane and cytoplasmic domains.

[0199] For class I proteins, the soluble form may include α1, α2, and α3 domains. Soluble class II subunits may include the α1 and α2 domains of the α subunit and the β1 and β2 domains of the β subunit.

[0200] The α and β subunits can be generated separately and allowed to associate in vitro to form a stable heteroduplex complex, or both subunits can be expressed in a single cell. Methods for generating MHC subunits are known in the art.

[0201] In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1 and an MHC. In some embodiments, the MHC molecule comprises an MHC α chain of serotype HLA-A*01, optionally wherein the HLA allele is HLA-A*01:01. In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1A and an MHC α chain having the HLA-A*01 serotype, such as an MHC encoded by the HLA-A*01:01 allele.

[0202] To prepare MHC-peptide complexes, subunits can be combined with antigenic peptides and allowed to fold 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 an empty heterodimer in a subsequent step. In the compositions and methods covered by this invention, the peptide is a MAGEA1 immunogenic peptide or a fragment thereof. Conditions that allow the subunits and peptide to fold and associate are known in the art. As an example, approximately equimolar amounts of dissolved α and β subunits can be mixed in a urea solution. Refolding is initiated by dilution or dialyzing to a urea-free buffer solution. The peptide can be loaded into an empty class II heterodimer at about pH 5 to 5.5 for about 1 to 3 days, followed by neutralization, concentration, and buffer exchange. However, specific folding conditions are not critical to the practice of this invention.

[0203] Monomer complexes (α-β-P) (monomers herein) can be polymerized, for example, into MHC tetramers. The resulting polymers are stable over long periods. Preferably, polymers can be formed by binding the monomer to a multivalent entity via specific attachment sites on the α or β subunits, as known in the art (e.g., as described in U.S. Patent No. 5,635,363). MHC proteins can also be conjugated to beads or any other support, whether in monomeric or polymeric form.

[0204] The multimeric complex can be labeled for direct detection in immunostaining or other methods known in the art, or, as known in the art, in combination with immunoassay reagents that specifically and / or selectively bind the complex (e.g., to MHC protein subunits). For example, the detectable label can be a fluorophore such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin (PE), allophycocyanin (APC), Brilliant Violet™ 421, Brilliant UV™ 395, Brilliant Violet™ 480, Brilliant Violet™ 421 (BV421), Brilliant Blue™ 515, APC-R700, or APC-Fire750. In some embodiments, the multimeric complex is labeled with a portion capable of specifically and / or selectively binding to another portion. For example, the label can be biotin, streptavidin, oligonucleotides, or ligands. Other markers of interest may include fluorescent dyes, dyes, enzymes, chemiluminescent agents, particles, radioactive isotopes, or other directly or indirectly detectable agents.

[0205] In some embodiments, cells are generated by transfecting or transducing them with a vector (e.g., a viral vector) containing nucleic acids encoding recombinant or heterologous antigens introduced into the cells, thereby producing cells that present immunogenic peptides on the cell surface against a background of MHC molecules. In some embodiments, the vector is introduced into the cells under conditions in which one or more peptide antigens (in some cases, including one or more peptide antigens of expressed heterologous proteins) are expressed, processed, and presented on the cell surface by the cells against a background of major histocompatibility complex (MHC) molecules.

[0206] Generally, the cells contacted by the carrier are MHC-expressing cells, i.e., MHC-expressing cells. These cells can be cells that normally express MHC on their cell surface, cells induced to express MHC on their cell surface and / or with upregulated MHC expression, or cells engineered to express MHC molecules on their cell surface. In some embodiments, MHC contains polymorphic peptide-binding sites or binding grooves, which in some cases can complex with peptide antigens of polypeptides, including peptide antigens processed through cellular mechanisms. In some cases, MHC molecules can be presented or expressed on the cell surface, including in the form of peptide complexes, i.e., MHC-peptide complexes, for presenting antigens in a conformation that can be recognized by the TCR or other peptide-binding molecules on T cells.

[0207] In some embodiments, the cells are nucleated cells. In some embodiments, the cells are antigen-presenting cells. In some embodiments, the cells are macrophages, dendritic cells, B cells, endothelial cells, or fibroblasts. In some embodiments, the cells are endothelial cells, such as endothelial cell lines or primary endothelial cells. In some embodiments, the cells are fibroblasts, such as fibroblast cell lines or primary fibroblasts.

[0208] In some implementations, the cells are artificial antigen-presenting cells (aAPCs). Typically, aAPCs include characteristics of native APCs, including the ability to express MHC molecules, stimulatory and co-stimulatory molecules, Fc receptors, adhesion molecules, and / or produce or secrete cytokines (e.g., IL-2). Typically, aAPCs are cell lines lacking expression of one or more of the above and are generated by introducing (e.g., through transfection or transduction) one or more of the following: missing elements in the MHC molecule, low-affinity Fc receptor (CD32), high-affinity Fc receptor (CD64), and one or more co-stimulatory signals (e.g., CD7, B7-1). (CD80), B7-2(CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, ILT3, ILT4, 3 / TR6 or B7-H3 ligand; or antibodies that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor or CD83 ligand), cell adhesion molecules (e.g., ICAM-1 or LFA-3) and / or cytokines (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon-α) The expression of MHC molecules includes interferon-α (IFNα), interferon-β (IFNβ), interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), tumor necrosis factor-β (TNFβ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (GCSF). In some cases, aAPCs do not typically express MHC molecules but can be engineered to express them, or in some cases, they are induced to express or can be induced to express MHC molecules, for example, by stimulation with cytokines. In some cases, aAPCs may also be loaded with stimulating ligands, which may include, for example, anti-CD3 antibodies, anti-CD28 antibodies, or anti-CD2 antibodies. Exemplary cell lines that can be used as the backbone for generating aAPCs are the K562 cell line or fibroblast cell lines. Various aAPCs are known in the art, see, for example, U.S. Patent No. 8,722,400; Publication No. US2014 / 0212446; Butler and Hirano (2014) Immunol Rev. 257:10. 1111 / imr.12129; Suhoshki et al. (2007) Mol. Ther. 15:981-988.

[0209] Assessing or identifying specific MHC or allele expression in cells is entirely within the scope of a skilled craftsman. In some embodiments, the expression of a specific MHC molecule may be assessed or confirmed, for example, by using an antibody specific to that specific MHC molecule, before contacting the cells with the vector. Antibodies against MHC molecules are known in the art, such as any antibodies described below.

[0210] In some embodiments, cells may be selected to express the desired MHC-restricted MHC alleles. In some embodiments, the MHC typing of the cells (e.g., cell lines) is known in the art. In some embodiments, the MHC typing of cells (e.g., primary cells obtained from a subject) may be determined using procedures well known in the art, such as tissue typing performed by molecular haplotype assays (BioTest ABC SSPtray, BioTestDiagnostics, Denville, NJ; SeCore Kits, Life Technologies, Grand Island, NY). In some cases, determining HLA genotypes, for example, by performing standard cell typing using sequence-based typing (SBT), is entirely within the capabilities of a skilled technician (Adams et al. (2004) J. Transl. Med., 2:30; Smith (2012) MethodsMol Biol., 882:67-86). In some cases, the HLA typing of the cells (e.g., fibroblasts) is known. For example, the human fetal lung fibroblast cell line MRC-5 contains HLA-A*02:01, A29, B13, B44, and Cw7 (C*0702); the human foreskin fibroblast cell line Hs68 contains HLA-A1, A29, B8, B44, Cw7, and Cw16; and the WI-38 cell line contains A*68:01 and B*08:01 (Solache et al. (1999) J Immunol, 163:5512-5518; Amers et al. (2013) PloS Pathog.9:e1003383). The human transfected fibroblast cell line M1DR1 / Ii / DM expresses HLA-DR and HLA-DM (Karakikes et al. (2012) FASEB J., 26:4886-96).

[0211] In some embodiments, the cells to which the vector is contacted or introduced are cells engineered or transfected to express MHC molecules. In some embodiments, cell lines can be prepared by genetically modifying parental cell lines. In some embodiments, cells typically lack a specific MHC molecule and are engineered to express that specific MHC molecule. In some embodiments, recombinant DNA technology is used to genetically engineer the cells.

[0212] In some embodiments, the stable MHC-peptide complex described herein is used to detect T cells that bind to the stable MHC-peptide complex. In some embodiments, the stable MHC-peptide complex described herein is used to monitor 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 the fluorescently labeled MHC-peptide complex. Methods for generating, labeling, and using MHC-peptide complexes (e.g., MHC-peptide tetramers) to detect MHC-peptide complex-specific T cells are well known in the art. Further descriptions can be found, for example, in U.S. Patent No. 7,776,562; U.S. Patent No. 8,268,964; and U.S. Patent Publication 2019 / 0085048.

[0213] IV. Immunogenic Compositions In some aspects, this document provides 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, this document provides pharmaceutical compositions (e.g., vaccine compositions) comprising a stable MHC-peptide complex of a MAGEA1 immunogenic peptide contained in an MHC molecular background 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 of a MAGEA1 immunogenic peptide contained in an MHC molecular background and an adjuvant. In some embodiments, the above-described compositions further comprise a pharmaceutically acceptable carrier.

[0214] The pharmaceutical compositions disclosed herein can be specifically formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, such as oral enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those intended for absorption via the buccal, sublingual, and systemic routes), granules, powders, pellets, or pastes applied to the tongue; or (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection in the form of, for example, sterile solutions or suspensions or sustained-release formulations.

[0215] Methods for preparing these formulations or compositions include the steps of conjugating the MAGEA1 immunogenic peptide and / or nucleic acid described herein with an adjuvant, a carrier, and optionally one or more auxiliary components. Generally, the formulations are prepared by uniformly and tightly conjugating the agent described herein with a liquid carrier or a finely dispersed solid carrier, or both, and then, if necessary, shaping the product.

[0216] Pharmaceutical compositions suitable for parenteral administration comprise the combination of the MAGEA1 immunogenic peptide and / or nucleic acid described herein with an adjuvant, and one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions just before use. These compositions may contain sugars, alcohols, antioxidants, buffers, antibacterial agents, solutes that make the formulation isotonic with the blood of the intended recipient, or suspending agents or thickeners.

[0217] Examples of suitable aqueous and non-aqueous carriers for pharmaceutical compositions include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). For example, in the case of dispersions, the desired particle size can be maintained by using a coating material such as lecithin, and appropriate flowability can be maintained by using surfactants.

[0218] Regardless of the chosen route of administration, the agents provided herein and / or the pharmaceutical compositions disclosed herein, which can be used in a suitable hydrated form, can be formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0219] In some embodiments, when administered to a subject, the pharmaceutical composition can elicit an immune response against cells infected with MAGEA1. Such pharmaceutical compositions can be used as vaccine compositions for the prophylactic and / or therapeutic treatment of conditions characterized by MAGEA1 expression.

[0220] In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the adjuvant used increases the immunogenicity of the pharmaceutical composition. Such compounds or adjuvants that stimulate further immune responses may: (i) be mixed into the pharmaceutical composition according to the invention after peptide reconstitution and optionally emulsification with an oil-based adjuvant as defined above; (ii) be part of the reconstitution composition of the invention as defined above; (iii) be substantially linked to the peptide to be reconstituted; or (iv) be separately administered to a subject, mammal, or human to be treated. The adjuvant may be an adjuvant that provides a slow release of the antigen (e.g., the adjuvant may be a liposome), or it may be an adjuvant that is itself immunogenic, thereby acting synergistically with the antigen (i.e., the antigen present in the MAGEA1 immunogenic peptide). For example, the adjuvant may be a known adjuvant, or other substances that promote antigen uptake, recruit immune system cells to the site of application, or promote immune activation of responding lymphoid cells. Adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, dextran, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, synthetic polymers (e.g., polyamino acids and amino acid copolymers), saponins, paraffin oils, and muramyl dipeptides. In some embodiments, adjuvants are adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramycin-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalose dimethicone, or yeast polysaccharide.

[0221] In some implementations, the adjuvant is an immunomodulatory molecule. For example, the immunomodulatory molecule may be a recombinant protein cytokine, chemokine, or immunostimulant designed to enhance the immune response, or a nucleic acid encoding a cytokine, chemokine, or immunostimulant.

[0222] Examples of immunomodulatory cytokines include interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, and IL-20), tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1α, MIP-1β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as functional fragments of any of the foregoing.

[0223] 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 foregoing.

[0224] In some embodiments, the composition comprises a nucleic acid encoding the MAGEA1 immunogenic peptide described herein, such as a DNA molecule encoding the MAGEA1 immunogenic peptide. In some embodiments, the composition comprises an expression vector containing an open reading frame encoding the MAGEA1 immunogenic peptide.

[0225] When taken up by cells (e.g., host cells, antigen-presenting cells (APCs), such as dendritic cells, macrophages, etc.), DNA molecules can exist as extrachromosomal molecules within the cell and / or integrate into the chromosome. DNA can be introduced into the cell in plasmid form, which can remain as independent genetic material. Alternatively, linear DNA that can integrate into the chromosome can also be introduced into the cell. Optionally, when DNA is introduced into the cell, agents that promote DNA integration into the chromosome may be added.

[0226] V. Binding protein In some aspects, a binding portion is provided that binds the peptides and / or stable MHC-peptide complexes described herein. For example, binding proteins such as T-cell receptors (TCRs) and antibodies are provided, for instance, in a concentration of less than or equal to about 10... -4 M (for example, about 10) -4 10 -5 10 -6 10 -7 Approximately 10 -8 Approximately 10 -9 Approximately 10 10 Approximately 10 -11 Approximately 10 -12 Approximately 10 -13 Approximately 10 -14 K (etc.) d It binds specifically and / or selectively to peptides and / or stable MHC-peptide complexes.

[0227] In one aspect covered by the present invention, a binding protein is provided herein that binds (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex containing the MAGEA1 immunogenic peptide in a background of MHC molecules (e.g., MHC class molecules). In some embodiments, the binding protein is capable of binding (e.g., specifically and / or selectively) to the MAGEA1 peptide-MHC (pMHC) complex, K d Less than or equal to approximately 5 × 10 -4 M, less than or equal to approximately 1 × 10 -4 M, less than or equal to approximately 5 × 10 -5 M, less than or equal to approximately 1 × 10 -5 M, less than or equal to approximately 5 × 10 -6 M, less than or equal to approximately 1 × 10 -6 M, less than or equal to approximately 5 × 10 -7 M, less than or equal to approximately 1 × 10 -7 M, less than or equal to approximately 5 × 10 -8 M, less than or equal to approximately 1 × 10 -8 M, less than or equal to approximately 5 × 10 -9 M, less than or equal to approximately 1 × 10 -9 M, less than or equal to approximately 5 × 10 -10 M, less than or equal to approximately 1 × 10 -10 M, less than or equal to approximately 5 × 10 -11 M, less than or equal to approximately 1 × 10 -11 M, less than or equal to approximately 5 × 10 -12 M, less than or equal to approximately 1 × 10 -12M, or any range in between (including endpoints), such as about 1-50 micromoles, 1-100 micromoles, 0.1-500 micromoles, etc. In some embodiments, the MHC molecule comprises an MHC α chain of HLA-A*01 serotype, optionally wherein the HLA allele is HLA-A*01:01. In some embodiments, the HLA serotype is HLA-A*01 and / or the HLA allele is the HLA-A*01:01 allele. In some embodiments, the binding protein provided herein is genetically engineered, isolated, and / or purified.

[0228] In some embodiments, the binding affinity of the binding protein for MAGEA1 peptide-MHC (pMHC) is higher than that of known T-cell receptors (e.g., TCRs from van Kunert et al. (2016) J. Immunol. 197:2541-2552 or other TCRs described herein). For example, the binding affinity of the binding protein for MAGEA1 peptide-MHC (pMHC) may be at least 1.2, 1.5, 1.8, 2.0, 2.2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, or 16 times higher than that of known T-cell receptors. 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times, 5000 times, 10000 times, 50000 times, 100000 times, 500000 times, 1000000 times or more, or any range in between (including the endpoints), such as 1.2 times to 2 times.

[0229] In some implementations, when contacted with target cells expressing MAGEA1 at a certain level or lower, the binding protein induces greater T cell expansion, cytokine release, and / or cytotoxic killing compared to known T cell receptors (see Examples section for representative cell lines expressing MAGEA1 at different levels). For example, in some embodiments of any aspect described herein, the MAGEA1 level may be expressed in terms of per million transcripts and may be, for example, less than or equal to about 1,000 transcripts per million transcripts (TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or any range in between (including endpoints), such as less than or equal to about 1,000 TPM to less than or equal to about 35 TPM. In some embodiments, low MAGEA1 expression levels are referred to as "heterozygous expression," meaning between about 1 TPM and about 35 TPM, or any range in between (including endpoints), such as 32 TPM or 1–32 TPM. Higher expression is defined as 36 TPM and above. As further described herein, TPM is measured using well-known techniques such as RNA-Seq, and gene expression TPM data for various cell lines, tissue types, etc., are well-known in the field (see, for example, the Broad Institute Cancer Cell Line Encyclopedia (CCLE) at portals.broadinstitute.org on the World Wide Web).In some embodiments, when contacted with target cells expressing the MAGEA1 peptide epitope, such as those heterozygous for MAGEA1 peptide epitope expression, the binding protein induces at least a 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, and 5-fold increase in T cell proliferation, cytokine release, and / or cytotoxic killing compared to known T cell receptors (e.g., the comparative TCRs described herein). 5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 1000 times or more, or any range in between (including the endpoints), such as 1.2 times to 2 times.

[0230] In some embodiments, MAGEA1 expression is detected using RNA sequencing (RNA-seq). RNA-seq generally includes the following steps: obtaining a sample containing genetic material, isolating total RNA from the obtained sample, preparing an amplified cDNA library from the total RNA, sequencing the amplified cDNA library, and analyzing and dissecting the amplified cDNA to assess the expression levels of different transcripts. The sample can be a cell population, tissue sample, biopsy sample, cell culture, or single cell. Total RNA can be isolated from the biological sample using any method known in the art. In some embodiments, total RNA is extracted from plasma. The extraction of plasma RNA is described in Enders et al., “The Concentration of Circulating Corticotropin-Releasing HomermRNA in Material Plasma Is Inclined in Preclampsia”, Clinr. As described therein, the plasma collected after the 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 microcolumn (Qiagen) and processed according to the manufacturer's recommendations.

[0231] In some embodiments, the RNA-seq described herein includes the step of preparing amplified cDNA from total RNA. For example, cDNA is prepared and isolated RNA samples are randomly amplified without dilution, or a mixture of genetic material from isolated RNA is dispersed into individual reaction samples. In some embodiments, amplification begins randomly at the 3' end and continues throughout the entire transcriptome in the sample to amplify both mRNA and unpolyadenylated transcripts. In this way, the double-stranded cDNA amplification products are optimized to produce sequencing libraries for next-generation sequencing platforms. Kits suitable for amplifying cDNA using the methods covered by this invention include, for example, the Ovation® RNA-Seq system.

[0232] In some embodiments, the RNA-seq described herein includes the step of sequencing the amplified cDNA. Any known sequencing method can be used to sequence the amplified cDNA mixture, including single-molecule sequencing methods. In some embodiments, the amplified cDNA is sequenced using whole transcriptome shotgun sequencing. Whole transcriptome shotgun sequencing can be performed using various next-generation sequencing platforms, such as the Illumina® Genome Analysis Platform, the ABI SOLiD™ Sequencing Platform, or the LifeScience 454 Sequencing Platform.

[0233] In some implementations, the RNA-seq described herein also includes digital counting and analysis of cDNA. The number of amplified sequences for each transcript in a sample can be quantified by sequence reads (one read per amplified strand). In some implementations, transcripts per million (TPM) is used to quantify the expression level of a specific transcript. TPM can be calculated as shown in Wagner et al. (2012) Theory in Biosciences 131:281-285, the contents of which are incorporated herein by reference in their entirety.

[0234] In some embodiments, the binding protein recognizes a MAGEA1 immunogenic peptide in the form of a complex with an MHC molecule, such as a specific HLA molecule having a specific HLA α-chain allele. For example, the binding proteins listed in Table 2A are identified as binders to MAGEA1 immunogenic peptides associated with an MHC α-chain having the HLA-A*01 serotype, such as an MHC encoded by the HLA-A*01:01 allele, as further described in the Examples section. In some embodiments, the binding protein recognizes a complex of the MAGEA1 immunogenic peptide with an MHC molecule, wherein the MHC molecule comprises an MHC α-chain of the HLA-A*01 HLA serotype, optionally wherein the HLA allele is HLA-A*01:01. In some embodiments, the MAGEA1 immunogenic peptide is derived from human MAGEA1 protein and / or the MAGEA1 protein shown in Table 3. In some embodiments, one or more MAGEA1 immunogenic peptides are administered alone or in combination with an adjuvant.

[0235] In some implementations, the binding protein does not bind to the peptide-MHC (pMHC) complex, optionally wherein the peptide is derived from “off-target” as described herein.

[0236] In some implementations, the binding protein does not bind to the “off-target” described herein, which is complexed with the MHC peptide-MHC (pMHC) complex.

[0237] In some embodiments, the binding proteins provided herein include (e.g., comprising, substantially consisting of, or consisting of): a) a TCR α chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with a TCR α chain sequence selected from the group of TCR α sequences listed in Table 2; and / or b) a TCR β chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with a TCR β chain sequence selected from the group of TCR β chain sequences listed in Table 2.

[0238] In some embodiments, the binding proteins provided herein include (e.g., comprising, substantially consisting of, or composed of): a) a TCR α chain sequence selected from the group consisting of the TCR α chain sequences listed in Table 2; and / or b) a TCR β chain sequence selected from the group consisting of the TCR β chain sequences listed in Table 2.

[0239] In some embodiments, the binding proteins provided herein include the following (e.g., comprising, substantially composed of, or composed of the following): a) a variable TCR α chain (V α The sequence of structural domains, which are selected from the TCR V listed in Table 2 α The TCR α chain of the group composed of domain sequences is variable (V) α The domain sequences have at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity; and / or b) the TCR β chain is variable (V β The sequence of structural domains, which are selected from the TCR V listed in Table 2 β The TCR β chain of the group composed of domain sequences is variable (V) β The domain sequences have 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.

[0240] In some embodiments, the binding proteins provided herein include the following (e.g., comprising, substantially composed of, or composed of the following): a) a variable TCR α chain (V α The domain sequence is selected from the TCR V listed in Table 2. α Groups consisting of domain sequences; and / or b) TCR β-chain variable (V β The domain sequence is selected from the TCR V listed in Table 2. β A group consisting of a sequence of structural domains.

[0241] In some embodiments, the binding proteins provided herein include (e.g., comprising at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2), substantially composed of, or composed of) a TCR α chain CDR 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 with the TCR α chain CDR sequences selected from those listed in Table 2. CDR3 is considered the primary CDR responsible for recognizing processed antigens, and CDR1 and CDR2 primarily interact with the MHC; therefore, in some embodiments, binding proteins are provided comprising individual CDR3s from the TCR α chains listed in Table 2 and / or individual CDR3s from the TCR β chains listed in Table 2, each CDR3 having sequence homology as described in this paragraph.

[0242] In some embodiments, the binding protein provided herein may also include (e.g., comprising or consisting substantially of at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2) TCR β chain CDR sequences selected from the group consisting of TCR β chain CDR sequences listed in Table 2) TCR β chain 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 with TCR β chain complementarity-determining region (CDR) sequences. As mentioned above, CDR3 is considered to be the major CDR responsible for recognizing processed antigens, and CDR1 and CDR2 primarily interact with MHC. Therefore, in some embodiments, binding proteins comprising individual CDR3s from the TCR β chains listed in Table 2 and / or individual CDR3s from the TCR α chains listed in Table 2 are provided, each CDR3 having sequence homology as described in this paragraph.

[0243] In some embodiments, the binding proteins provided herein include (e.g., comprising at least one (e.g., one, two, or three), substantially composed of, or composed of) the TCR α-chain complementarity-determining region (CDR) listed in Table 2.

[0244] In some embodiments, the binding protein provided herein may also include (e.g., comprising, substantially comprising, or consisting of) the TCR β chain complementarity-determining region (CDR) listed in Table 2.

[0245] In some embodiments, the binding protein provided herein comprises (e.g., includes, is substantially composed of, or is composed of) a TCR α chain constant region (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 TCR Cα sequences listed in Table 2. α )sequence.

[0246] In some embodiments, the binding protein provided herein may also include the following (e.g., comprising, substantially consisting of, or consisting of the following): TCR C listed in Table 2 β The TCR β-chain constant region (C) with at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity. β )sequence.

[0247] In some implementations, the binding proteins provided herein include the following (e.g., comprising, substantially consisting of, or composed of the following): selected from the TCR C listed in Table 2. α The TCR α-chain constant region (C) of the group composed of sequences α )sequence.

[0248] In some implementations, the binding protein provided herein may also include (e.g., comprising, substantially consisting of, or consisting of) TCR C selected from those listed in Table 2. β The TCR β chain constant region (C) of the sequence group β )sequence.

[0249] Table 2: TCR sequences recognizing the MAGEA1 antigen Table 2A TCR sequence that recognizes the MAGEA1 antigen presented by HLA serotype HLA-A*01 MAGEA1-161-458 WT sequence alpha chain: TRAV21 / TRAJ47 / TRAC α-strand DNA sequence ATGGAAACCCTGCTGGGACTGCTGATCCTGTGGCTGCAGCTTCAGTGGGTGTCCTCTAAGCAAGAAGTGACACAGATCCCTGCCGCACTGTCTGTGCCTGAGGGCGAAAACCTGGTGCTGAACTGCTCCTTCACC GATAGCGCT ATTTACAAC CTGCAGTGGTTCAGACAGGACCCCGGCAAGGGACTGACAAGCCTGCTGCTC ATTCAGTCAAGTCAGA GAGAG CAGACCAGCGGCAGACTGAATGCCAGCCTGGATAAGTCCTCCGGCAGAAGCACCCTGTATATCGCCGCTTCTCAGCCAGGCGATAGCGCCACATACCTG TGTGCTGTCCAATATGGAAACAAGCTGGTCTTT GGCGCAGGAACCATTCTGAGAGTCAAGTCCTatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc Alpha chain protein sequence METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CAVQYGNKLVFGAGTILRVKSYiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpspesscdvklveksfetdtnlnfqnlsvigfrilllkvagfnllmtlrlwss Beta chain: TRBV5-4 / TRBJ2-7 / TRBC2 Beta chain DNA sequence ATGGGACCCGGCCTTCTGTGTTGGGCCCTGCTTTGCCTTCTCGGAGCTGGCTCTGTGGAAACCGGCGTGACACAGTCTCCTACACATCTGATCAAAACTCGCGGGCAACAAGTGACTCTGCGGTGTAGCTCTCAG TCTGGGCAC AACACT GTTTCTTGGTATCAGCAAGCTCTCGGCCAGGGGCCACAGTTTATTTTCCAG TATTATAGGGAGGAAGAG AACGGCAGAGGCAACTTTCCACCTCGGTTTTCCGGACTGCAGTTTCCCAACTACTCCTCCGAGCTGAACGTGAACGCCTTGGAGCTGGATGATAGTGCCCTCTACCTCT GTGCCAGCAGCACGGACATAACCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAAgatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggc β-chain protein sequence MGPGLLCWALLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQ SGHNT VSWYQQALGQGPQFIFQ Y YREEE NGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYL CASSTDITYEQY FGPGTRLTVTEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsatfwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsg MAGEA1-161-458 is an MGTM codon-optimized sequence (also known as clones of "MAGE-A1-458", "TCR 458", "458", TCR expressed by "TSC-204-A01", TCR expressed by "TSC-204-A01:01", and TCR expressed by "TSC-204-A0101"). alpha chain: TRAV21 / TRAJ47 / MGTM modified TRAC α-strand DNA sequence ATGGAAACCCTGCTGGGACTGCTGATCCTGTGGCTGCAGCTTCAGTGGGTGTCCTCTAAGCAAGAAGTGACACAGATCCCTGCCGCACTGTCTGTGCCTGAGGGCGAAAACCTGGTGCTGAACTGCTCCTTCACC GACTCCGCC ATCTACAAC CTGCAGTGGTTCAGACAGGACCCCGGCAAGGGACTGACAAGCCTGCTGCTC ATTCAGAGCAGCCAGA GAGAG CAGACCAGCGGCAGACTGAATGCCAGCCTGGATAAGTCCTCCGGCAGAAGCACCCTGTATATCGCCGCTTCTCAGCCAGGCGATAGCGCCACATACCTG TGCGCTGTGCAATATGGGAATAAACTCGTTTTTCGGCGCAGGAACCATTCTGAGAGTCAAGTCCTacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagcagc Alpha chain protein sequence METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CAVQYGNKLVF GAGTILRVKSYiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlwss Beta chain: TRBV5-4 / TRBJ2-7 / MGTM modified TRBC Beta chain DNA sequence ATGGGACCCGGCCTTCTGTGTTGGGCCCTGCTTTGCCTTCTCGGAGCTGGCTCTGTGGAAACCGGCGTGACACAGTCTCCTACACATCTGATCAAAACTCGCGGGCAACAAGTGACTCTGCGGTGTAGCTCTCAG TCCGGCCAC AACACA GTTTCTTGGTATCAGCAAGCTCTCGGCCAGGGGCCACAGTTTATTTTCCAG TACTACCGCGAGGAAGAG AACGGCAGAGGCAACTTTCCACCTCGGTTTTCCGGACTGCAGTTTCCCAACTACTCCTCCGAGCTGAACGTGAACGCCTTGGAGCTGGATGATAGTGCCCTCTACCTC TGCGCTTCCAGCACAGACATCACCTATGAGCAGTACTTC GGGCCGGGCACCAGGCTCACGGTCACAGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggc β-chain protein sequence MGPGLLCWALLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQ SGHNT VSWYQQALGQGPQFIFQ Y YREEE NGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYL CASSTDITYEQYFGPGTRLTVTEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsat fwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsg The complete β and α ORF DNA sequences (the underlined italicized region at the "Furin-P2A" site encodes a sequence that allows expression of two polypeptide chains in a single cassette) ATGGGACCCGGCCTTCTGTGTTGGGCCCTGCTTTGCCTTCTCGGAGCTGGCTCTGTGGAAACCGGCGTGACACAGTCTCCTACACATCTGATCAAAACTCGCGGGCAACAAGTGACTCTGCGGTGTAGCTCTCAG TCCGGCCAC AACACA GTTTTCTTGGTATCAGCAAGCTCTCGGCCAGGGGCCACAGTTTATTTTCCAG TACTACCGCGAGGAAGAG AACGGCAGAGGCAACTTTCCACCTCGGTTTTCCGGACTGCAGTTTCCCAACTCCTCCGAGCTGAACGTGAACGCCTTGGAGCTGGATGATAGTGCCCTCTACCTC TGCGCTTCCAGCACAGACATCACCTATGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggc agagccaaaaggtccgggagcggtGCGACAAACTTTAGCCTGTTGAA ACAAGCCGGCGACGTTGAAGAGAACCCCGGACCT ATGGAAACCCTGCTGGGACTGCTGATCCTGTGGCTGCAGCTTCAGTGGGTGTCCTCTAAGCAAGAAGTGACACAGATCCCTGCCGCACTGTCTGTGCCTGAGGGCGAAAACCTGGTGCTGAACTGCTCCTTCACC GACTCCGCCATCTACAAC CTGCAGTGGTTCAGACAGGACCCCGGCAAGGGACTGACAAGCCTGCTGCTC ATTCAGAGCAGCCAGAGAGAG CAGACCAGCGGCAGACTGAATGCCAGCCTGGATAAGTCCTCCGGCAGAAGCACCCTGTATATCGCCGCTTCTCAGCCAGGCGATAGCGCCACATACCTG TGCGCTGTGCAATATGGGAATA AACTCGTTTTCGGCGCAGGAACCATTCTGAGAGTCAAGTCCTacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagcagc Full β and α ORF protein sequences (the italicized regions underlined in the "furin-P2A" site allow the expression of two polypeptide chains in a single cassette) MGPGLLCWALLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQ SGHNT VSWYQQALGQGPQFIFQ Y YREEE NGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYL CASSTDITYEQYF GPGTRLTVTEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsatfwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsg rakrsgsgATNFSLLKQAGDVEENPGP METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CA VQYGNKLVF GAGTILRVKSYiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlwss MAGEA1-161-747 WT sequence Alpha chain: TRAV21 / TRAJ54 / TRAC Alpha chain DNA sequence ATGGAAACCCTGCTGGGACTGCTGATCCTGTGGCTGCAGCTTCAGTGGGTGTCCTCTAAGCAAGAAGTGACACAGATCCCTGCCGCACTGTCTGTGCCTGAGGGCGAAAACCTGGTGCTGAACTGCTCCTTCACC GACTCCGCC ATCTACAAC CTGCAGTGGTTCAGACAGGACCCCGGCAAGGGACTGACAAGCCTGCTGCTC ATTCAGAGCAGCCAGA GAGAG CAGACCAGCGGCAGACTGAATGCCAGCCTGGATAAGTCCTCCGGCAGAAGCACCCTGTATATCGCCGCTTCTCAGCCAGGCGATAGCGCCACATACCTG TGCGCCGTCCGCCCACAGCAAGGCGCACAGAAGTTGGTGTTCGGCCAAGGAACCAGGCTGACTATCAACCCAAACatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc Alpha chain protein sequence METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CAVRPQQGAQKLVF GQGTRLTINPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpspesscdvklveksfetdtnlnfqnlsvigfrilllkvagfnllmtlrlwss Beta chain: TRBV5-4 / TRBJ2-7 / TRBC2 Beta chain DNA sequence ATGGGACCCGGCCTTCTGTGTTGGGCCCTGCTTTGCCTTCTCGGAGCTGGCTCTGTGGAAACCGGCGTGACACAGTCTCCTACACATCTGATCAAAACTCGCGGGCAACAAGTGACTCTGCGGTGTAGCTCTCAG TCCGGCCACAACACA GTTTCTTGGTATCAGCAAGCTCTCGGCCAGGGGCCACAGTTTATTTTCCAG TACTACCGCGAGGAAGAG AACGGCAGAGGCAACTTTCCACCTCGGTTTTCCGGACTGCAGTTTCCCAACTACTCCTCCGAGCTGAACGTGAACGCCTTGGAGCTGGATGATAGTGCCCTCTACCTC TGCGCAAGCAGCCTGGACCGCACATACGAGCAATACTTC GGGCCGGGCACCAGGCTCACGGTCACAGAAgacctgaacaaggtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttcttccctgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacggacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggctttacctcggtgtcctaccagcaaggggtcctgtctgccaccatcctctatgagatcctgctagggaaggccaccctgtatgctgtgctggtcagcgcccttgtgttgatggccatggtcaagagaaaggatttc β-chain protein sequence MGPGLLCWALLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQ SGHNT VSWYQQALGQGPQFIFQ Y YREEE NGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYL CASSLDRTYEQYFGPGTRLTVTEdlnkvfppevavfepseaeishtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsatfwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgftsvsyqqgvlsatilyeillgkatlyavlvsalvlmamvkrkdf MAGEA1 - 161 - 747 MGTM codon - optimized sequence (also known as clone "MAGE - A1 - 747", "TCR 747" and "747" Alpha chain: TRAV21 / TRAJ54 / MGTM - modified TRAC Alpha - chain DNA sequence ATGGAAACCCTGCTGGGACTGCTGATCCTGTGGCTGCAGCTTCAGTGGGTGTCCTCTAAGCAAGAAGTGACACAGATCCCTGCCGCACTGTCTGTGCCTGAGGGCGAAAACCTGGTGCTGAACTGCTCCTTCACC GACTCCGCC ATCTACAAC CTGCAGTGGTTCAGACAGGACCCCGGCAAGGGACTGACAAGCCTGCTGCTC ATTCAGAGCAGCCAGA GAGAG CAGACCAGCGGCAGACTGAATGCCAGCCTGGATAAGTCCTCCGGCAGAAGCACCCTGTATATCGCCGCTTCTCAGCCAGGCGATAGCGCCACATACCTG TGCGCCGTCCGCCCACAGCAAGGCGCACAGAAGTTGGTGTTCGGCCAAGGAACCAGGCTGACTATCAACCCAAacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagcagc Alpha chain protein sequence METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CAVRPQQGAQKLVF GQGTRLTINPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlwss Beta chain: TRBV5-4 / TRBJ2-7 / MGTM modified TRBC Beta chain DNA sequence ATGGGACCCGGCCTTCTGTGTTGGGCCCTGCTTTGCCTTCTCGGAGCTGGCTCTGTGGAAACCGGCGTGACACAGTCTCCTACACATCTGATCAAAACTCGCGGGCAACAAGTGACTCTGCGGTGTAGCTCTCAGTCCGGCCAC AACACA GTTTCTTGGTATCAGCAAGCTCTCGGCCAGGGGCCACAGTTTATTTTCCAG TACTACCGCGAGGAAGAG AACGGCAGAGGCAACTTTCCACCTCGGTTTTCCGGACTGCAGTTTCCCAACTACTCCTCCGAGCTGAACGTGAACGCCTTGGAGCTGGATGATAGTGCCCTCTACCTC TGCGCAAGCAGCCTGGACCGCACATACGAGCAATACTTC GGGCCGGGCACCAGGCTCACGGTCACAGAAgatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggc β-chain protein sequence MGPGLLCWALLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQ SGHNT VSWYQQALGQGPQFIFQ Y YREEE NGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYL CASSLDRTYEQYFGPGTRLTVTEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsat fwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsg The complete β and α ORF DNA sequences (the underlined italicized region at the "furin protease-P2A" site encodes a sequence that allows expression of two polypeptide chains in a single cassette) ATGGGACCCGGCCTTCTGTGTTGGGCCCTGCTTTGCCTTCTCGGAGCTGGCTCTGTGGAAACCGGCGTGACACAGTCTCCTACACATCTGATCAAAACTCGCGGGCAACAAGTGACTCTGCGGTGTAGCTCTCAG TCCGGCCAC AACACA GTTTTCTTGGTATCAGCAAGCTCTCGGCCAGGGGCCACAGTTTATTTTCCAG TACTACCGCGAGGAAGAG AACGGCAGAGGCAACTTTCCACCTCGGTTTTCCGGACTGCAGTTTCCCAACTCCTCCGAGCTGAACGTGAACGCCTTGGAGCTGGATGATAGTGCCCTCTACCTC TGCGCAAGCAGCCTGGACCGCACATACGAGCAATACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAAgatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggcagagccaaaaggtccgggagcggt GCGACAAACTTTAGCCTGTTGAA ACAAGCCGGCGACGTTGAAGAGAACCCCGGACCT ATGGAAACCCTGCTGGGACTGCTGATCCTGTGGCTGCAGCTTCAGTGGGTGTCCTCTAAGCAAGAAGTGACACAGATCCCTGCCGCACTGTCTGTGCCTGAGGGCGAAAACCTGGTGCTGAACTGCTCCTTCACC GACTCCGCCATCTACAAC CTGCAGTGGTTCAGACAGGACCCCGGCAAGGGACTGACAAGCCTGCTGCTC ATTCAGAGCAGCCAGAGAGAG CAGACCAGCGGCAGACTGAATGCCAGCCTGGATAAGTCCTCCGGCAGAAGCACCCTGTATATCGCCGCTTCTCAGCCAGGCGATAGCGCCACATACCTG TGCGCCGTCCGCCCACAGCAAG GCGCACAGAAGTTGGTGTTCGGCCAAGGAACCAGGCTGACTATCAACCCAAacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagcagc Full β and α ORF protein sequences (the italicized regions underlined in the "furin - P2A" site allow the expression of two polypeptide chains in a single cassette) MGPGLLCWALLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQ SGHNT VSWYQQALGQGPQFIFQ Y YREEE NGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYL CASSLDRTYEQYF GPGTRLTVTEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsatfwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsgrakrsgsg ATNFSLLKQAGDVEENPGP METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CA VRPQQGAQKLVF GQGTRLTINPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlwss *Table 2 provides representative TCR sequences, grouped according to MHC serotype and grouped by different peptides bound to the TCRs in the subgroups. Individual TCRs are described and requested, such as those represented in the table, and the types of binding proteins that bind to the peptide epitopes described herein, either alone or in combination with MHC, such as those grouped in the table provided herein. Additionally, the TRAV, TRAJ, and TRAC genes for each TCR α chain and the TRBV, TRBJ, and TRBC genes for each TCR β chain described herein are provided. The sequence of each TCR described herein is provided as a homologous α and β chain pair for each named TCR. The TCR sequences described herein are annotated. Variable domain sequences are capitalized. Constant domain sequences are lowercase. CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined text. CDR1, CDR2, and CDR3 are shown in standard order of appearance from left (N-terminus) to right (C-terminus). The TRAV, TRAJ, and TRAC genes of each TCR α strand described herein, and the TRBV, TRBJ, and TRBC genes of each TCR β strand described herein, are annotated according to the well-known IMGT nomenclature described herein. Similarly, the CDR1 and CDR2 of TRAV and TRBV are well-known in the art because they are based on well-known and annotated TRAV and TRBV sequences (e.g., annotated in databases such as IMGT, available at imt.org, and IEDB, available at iedb.org).

[0250] Table 3 Representative human MAGEA1 cDNA sequence atgtctcttgagcagaggagtctgcactgcaagcctgaggaagcccttgaggcccaacaagaggccctgggcctggtgtgtgtgcaggctgccacctcctcctcctctcctctggtcctgggcaccctggaggaggtgcccactgctgggtcaacagatcctccccagagtcctcagggagcctccgcctttcccactaccatcaacttcactcgacagaggcaacccagtgagggttccagcagccgtgaagaggaggggccaagcacctcttgtatcctggagtccttgttccgagcagtaatcactaagaaggtggctgatttggttggttttctgctcctcaaatatcgagccagggagccagtcacaaaggcagaaatgctggagagtgtcatcaaaaattacaagcactgttttcctgagatcttcggcaaagcctctgagtccttgcagctggtctttggcattgacgtgaag gaagcagaccccaccggccactcctat gtccttgtcacctgcctaggtctctcctatgatggcctgctgggtgataatcagatcatgcccaagacaggcttcctgataattgtcctggtcatgattgcaatggagggcggccatgctcctgaggaggaaatctgggaggagctgagtgtgatggaggtgtatgatgggagggagcacagtgcctatggggagcccaggaagctgctcacccaagatttggtgcaggaaaagtacctggagtaccggcaggtgccggacagtgatcccgcacgctatgagttcctgtggggtccaagggccctcgctgaaaccagctatgtgaaagtccttgagtatgtgatcaaggtcagtgcaagagttcgctttttcttcccatccctgcgtgaagcagctttgagagaggaggaagagggagtctga Representative human MAGEA1 protein sequence MSLEQRSLHCKPEEALEAQQEALGLVCVQAATSSSSPLVLGTLEEVPTAGSTDPPQSPQGASAFPTTINFTRQRQPSEGSSSREEEGPSTSCILESLFRAVITKKVADLVGFLLLKYRAREPVTKAEMLESVIKNYKHCFPEIFGKASESLQLVFGIDVKEADPTGHSYVLVTCLGLSYDGLLGDNQIMPKTGFLIIVLVMIAMEGGHAPEEEIWEELSVMEVYDGREHSAYGEPRKLLTQDLVQEKYLEYRQVPDSDPARYEFLWGPRALAETSYVKVLEYVIKVSARVRFFFPSLREAALREEEEGV* Representative human HLA-A*01:01 DNA sequence Representative human HLA-A*01:01 protein sequence MAVMAPRTLLLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQEGPEYWDQETRNMKAHSQTDRANLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAVHAAEQRRV YLEGRCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSYTQAASSDSAQGSDVSLTACKV Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pTSLV102-MSCV-HA1-10-30-MGTM-Q-CD8 ATGGAAACCCTcTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAA TGGGTGAGCAGCAAACAGGAGGTGACTCAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCA ACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGCCTCACATCTCT GTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGACGCCTTAATGCCTCTCTGGATAAATCATCAGGACGC AGTACTCTTTACATTGCAGCTTCTCAGCCTGGTGATTCAGCCACCTACCTGTGCGCTGTGAGGGGTGGTACCTCAG GAACCTACAAATACATCTTTGGAACAGGCACCAGGCTGAAGGTTCTTGCAAACATCCAGAACCCCGACCCCGCCGT GTACCAGCTGAGGGACTCCAAGTCCAGCGACAAGAGCGTGTGTCTGTTTACGGACTTCGACAGCCAGACCAACGTG AGTCAAAGCAAGGACAGCGACGTCTACATAACGGATAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCA ACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGA CACCTTCTTCCCCAGCAGCGACGTGCCCTGCGACGTGAAACTGGTGGAGAAGTCCTTCGAGACAGACACCAATCTG AACTTTCAGAACCTGCTGGTGATCGTGCTGCGGATTCTGCTGCTGAAAGTGGCCGGCTTCAATCTGCTGATGACCC TGCGGCTGTGGAGCAGC AGGGCTAAGAGGTCCGGCAGCGGAGCCACCAATTTTTCCCTGCTGAAACAGGCTGGTGACGTGGAAGAAAACCCTGGCCCCATGGCGCTGCCCGTCACCGCGCTGCTGCTGCCCCTGGCGCTGCTGTTACACGCCGCTCGGCCA GAGCTTCCCACCCAGGGCACATTCTCCAACGTGTCCACCAATGTGTCG GGAGGCGGCGGATCGTCCCAGTTCAGAGTGTCCCCTCTGGACCGCACCTGGAACCTGGGCGAGACCGTGGAGCTGAAATGTCAGGTCCTGCTGAGCAACCCGACCTCCGGGTGCAGTTGGCTGTTCCAGCCGCGTGGTGCTGCCGCAAGCCCTACGTTCCTGCTTTACCTGAGCCAGAACAAGCCCAAGGCGGCCGAGGGCCTGGACACCCAGAGATTCTCCGGCAAGCGCCTGGGGGACACATTCGTGCTTACTTTGAGCGATTTCCGCAGAGAGAACGAGGGCTACTATTTCTGTTCGGCGCTGAGCAATTCCATCATGTATTTCAGCCACTTTGTGCCAGTGTTCCTGCCTGCCAAGCCTACCACAACACCAGCTCCCCGTCCCCCGACTCCGGCGCCTACCATCGCGAGTCAACCGTTGAGCCTGAGGCCTGAGGCTTGTCGGCCCGCTGCGGGGGGTGCCGTCCACACCAGGGGCCTCGACTTTGCGTGCGACATCTATATTTGGGCGCCTCTGGCGGGTACCTGCGGGGTGCTGCTGCTGTCATTGGTGATTACCCTGTACTGCAATCACCGCAACCGCCGGCGGGTCTGTAAGTGCCCACGGCCTGTGGTCAAGTCCGGTGACAAACCGTCGCTCTCGGCTCGCTACGTGCGCGCTAAGCGCAGCGGTTCCGGGGCCACCAACTTTTCATTGCTGAAGCAGGCCGGTGATGTGGAGGAGAATCCAGGGCCC ATGCGCCCCAGGCTTTGGCTCCTTCTTGCTGCTCAGCTCAC TGTCTTGCATGGCAACTCCGTTCTGCAGCAGACTCCCGCCTACATCAAGGTGCAGACGAACAAGATGGTGATGCTG TCATGCGAGGCCAAGATCTCTCTTTCAAATATGAGAATTTATTGGCTACGACAGCGCCAGGCCCCCTCCAGCGACA GCCACCACGAGTTCCTGGCGCTTTGGGATTCTGCTAAAGGCACCATCCATGGAGAGGAGGTGGAACAGGAGAAGAT AGCTGTCTTCCGCGACGCATCCCGCTTCATCCTGAACCTGACCAGCGTGAAGCCGGAGGACAGCGGCATCTACTTC TGTATGATCGTTGGCTCCCCCGAGCTGACCTTCGGCAAAGGCACCCAGCTGTCCGTGGTGGACTTCCTGCCCACCA CAGCCCAGCCAACCAAGAAATCCACCCTCAAGAAGCGCGTGTGCCGACTGCCCCGCCCTGAAACCCAGAAGGGCCC TCTGTGCTCCCCCATCACCCTTGGACTGCTGGTGGCGGGAGTCCTGGTGCTGCTCGTATCTCTGGGTGTCGCCATC CACCTGTGCTGCCGCCGCCGCCGCGCCCGCCTGAGGTTTATGAAACAGTTTTACAAGT Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pHAGE-MSCV-HN-P32-41-P2A-dnTGFbRII (dnTGFbRII is highlighted in bold). Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pNVVD136_TSC-204-A02_TCR-1479_MSCV-TCR-1479-CD8-EF1α-dnTGFbRII-DHFR ATGGAAAAAATGCTCGAGTGCGCCTTCATCGTGCTTTGGCTGCAG CTCGGATGGCTGAGCGGAGAGGATCAAGTGACACAGTCTCCCGAGGCTCTGAGGCTGCAAGAGGGCGAAAGCAGCTC CCTGAATTGCAGCTACACCGTGTCTGGCCTGAGGGGCCTGTTTTGGTACAGACAAGACCCTGGCAAGGGACCCGAGT TCCTGTTCACACTGTACTCTGCCGGCGAAGAAAAAGAGAAAGAGCGCCTGAAAGCAACCCTGACCAAGAAAGAGAGC TTCCTGCACATCACAGCCCCTAAGCCAGAGGACAGCGCTACTTACCTGTGTGCCGTTTCATACGGCCAGAATTTCGT TTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGAGGGACT CCAAGTCCAGCGACAAGAGCGTGTGTCTGTTTACGGACTTCGACAGCCAGACCAACGTGAGTCAAAGCAAGGACAGC GACGTCTACATAACGGATAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTC CAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCAGCG ACGTGCCCTGCGACGTGAAACTGGTGGAGAAGTCCTTCGAGACAGACACCAATCTGAACTTTCAGAACCTGCTGGTG ATCGTGCTGCGGATTCTGCTGCTGAAAGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGAGCAGC AGGGCTAAGAGGTCCGGCAGCGGAGCCACCAATTTTTCCCTGCTGAAACAGGCTGGTGACGTGGAAGAAAACCCTGGCCCCATGGCGCTGCCCGTCACCGCGCTGCTGCTGCCCCTGGCGCTGCTGTTACACGCCGCTCGGCCA GAGCTTCCCACCCAG GGCACATTCTCCAACGTGTCCACCAATGTGTCG GGAGGCGGCGGATCGTCCCAGTTCAGAGTGTCCCCTCTGGACCGCACCTGGAACCTGGGCGAGACCGTGGAGCTGAAATGTCAGGTCCTGCTGAGCAACCCGACCTCCGGGTGCAGTTGGCTGTTCCAGCCGCGTGGTGCTGCCGCAAGCCCTACGTTCCTGCTTTACCTGAGCCAGAACAAGCCCAAGGCGGCCGAGGGCCTGGACACCCAGAGATTCTCCGGCAAGCGCCTGGGGGACACATTCGTGCTTACTTTGAGCGATTTCCGCAGAGAGAACGAGGGCTACTATTTCTGTTCGGCGCTGAGCAATTCCATCATGTATTTCAGCCACTTTGTGCCAGTGTTCCTGCCTGCCAAGCCTACCACAACACCAGCTCCCCGTCCCCCGACTCCGGCGCCTACCATCGCGAGTCAACCGTTGAGCCTGAGGCCTGAGGCTTGTCGGCCCGCTGCGGGGGGTGCCGTCCACACCAGGGGCCTCGACTTTGCGTGCGACATCTATATTTGGGCGCCTCTGGCGGGTACCTGCGGGGTGCTGCTGCTGTCATTGGTGATTACCCTGTACTGCAATCACCGCAACCGCCGGCGGGTCTGTAAGTGCCCACGGCCTGTGGTCAAGTCCGGTGACAAACCGTCGCTCTCGGCTCGCTACGTGCGCGCTAAGCGCAGCGGTTCCGGGGCCACCAACTTTTCATTGCTGAAGCAGGCCGGTGATGTGGAGGAGAATCCAGGGCCC ATGCGCCCCAGGCTTTGGCTCCTTCTTGCTGCTCAGCTCACTGTCTTGCATGGCAACTCCGTTCTGCAGCAGACTC CCGCCTACATCAAGGTGCAGACGAACAAGATGGTGATGCTGTCATGCGAGGCCAAGATCTCTCTTTCAAATATGAGA ATTTATTGGCTACGACAGCGCCAGGCCCCCTCCAGCGACAGCCACCACGAGTTCCTGGCGCTTTGGGATTCTGCTAA AGGCACCATCCATGGAGAGGAGGTGGAACAGGAGAAGATAGCTGTCTTCCGCGACGCATCCCGCTTCATCCTGAACC TGACCAGCGTGAAGCCGGAGGACAGCGGCATCTACTTCTGTATGATCGTTGGCTCCCCCGAGCTGACCTTCGGCAAA GGCACCCAGCTGTCCGTGGTGGACTTCCTGCCCACCACAGCCCAGCCAACCAAGAAATCCACCCTCAAGAAGCGCGT GTGCCGACTGCCCCGCCCTGAAACCCAGAAGGGCCCTCTGTGCTCCCCCATCACCCTTGGACTGCTGGTGGCGGGAG TCCTGGTGCTGCTCGTATCTCTGGGTGTCGCCATCCACCTGTGCTGCCGCCGCCGCCGCGCCCGCCTGAGGTTTATG AAACAGTTTTACAAGTGA Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pNVVD236_TSC-204-A01_TCR-458_MSCV-TCR-458-CD8-EF1a-dnTGFbRII-DHFR ATGGAAACCCTGCTGGGACTGCTGATCCTGTGGCTGCAGCTTCAGTGG GTGTCCTCTAAGCAAGAAGTGACACAGATCCCTGCCGCACTGTCTGTGCCTGAGGGCGAAAACCTGGTGCTGAACTG CTCCTTCACCGACTCCGCCATCTACAACCTGCATGGTTCAGACAGGACCCCGGCAAGGGACTGACAAGCCTGCTGC TCATTCAGAGCAGCCAGAGAGGCAGACCAGCGGCAGACTGAATGCCAGCCTGGATAAGTCCTCCGGCAGAAGCACC CTGTATATCGCCGCTTCTCAGCCAGGCGATAGCGCCACATACCTGTGCGCTGTGCAATATGGGAATAAACTCGTTTT CGGCGCAGGAACCATTCTGAGAGTCAAGTCCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGAGGGACTCCA AGTCCAGCGACAAGAGCGTGTGTCTGTTTACGGACTTCGACAGCCAGACCAACGTGAGTCAAAGCAAGGACAGCGAC GTCTACATAACGGATAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAA CAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCAGCGACG TGCCCTGCGACGTGAAACTGGTGGAGAAGTCCTTCGAGACAGACACCAATCTGAACTTTCAGAACCTGCTGGTGATC GTGCTGCGGATTCTGCTGCTGAAAGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGAGCAGC AGGGCTAAGAGGTCCGGCAGCGGAGCCACCAATTTTTCCCTGCTGAAACAGGCTGGTGACGTGGAAGAAAACCCTGGCCCCATGGCGCTGCCCGTCACCGCGCTGCTGCTGCCCCTGGCGCTGCTGTTACACGCCGCTCGGCCA GAGCTTCCACCACCAGGGC ACATTCTCCAACGTGTCCACCAATGTGTCGGGAGGCGGCGGATCGTCCCAGTTCAGAGTGTCCCCTCTGGACCGCACCTGGAACCTGGGCGAGACCGTGGAGCTGAAATGTCAGGTCCTGCTGAGCAACCCGACCTCCGGGTGCAGTTGGCTGTTCCAGCCGCGTGGTGCTGCCGCAAGCCCTACGTTCCTGCTTTACCTGAGCCAGAACAAGCCCAAGGCGGCCGAGGGCCTGGACACCCAGAGATTCTCCGGCAAGCGCCTGGGGGACACATTCGTGCTTACTTTGAGCGATTTCCGCAGAGAGAACGAGGGCTACTATTTCTGTTCGGCGCTGAGCAATTCCATCATGTATTTCAGCCACTTTGTGCCAGTGTTCCTGCCTGCCAAGCCTACCACAACACCAGCTCCCCGTCCCCCGACTCCGGCGCCTACCATCGCGAGTCAACCGTTGAGCCTGAGGCCTGAGGCTTGTCGGCCCGCTGCGGGGGGTGCCGTCCACACCAGGGGCCTCGACTTTGCGTGCGACATCTATATTTGGGCGCCTCTGGCGGGTACCTGCGGGGTGCTGCTGCTGTCATTGGTGATTACCCTGTACTGCAATCACCGCAACCGCCGGCGGGTCTGTAAGTGCCCACGGCCTGTGGTCAAGTCCGGTGACAAACCGTCGCTCTCGGCTCGCTACGTGCGCGCTAAGCGCAGCGGTTCCGGGGCCACCAACTTTTCATTGCTGAAGCAGGCCGGTGATGTGGAGGAGAATCCAGGGCCC AT GCGCCCCAGGCTTTGGCTCCTTCTTGCTGCTCAGCTCACTGTCTTGCATGGCAACTCCGTTCTGCAGCAGACTCCCG CCTACATCAAGGTGCAGACGAACAAGATGGTGATGCTGTCATGCGAGGCCAAGATCTCTCTTTTCAAATATGAGAATT TATTGGCTACGACAGCGCCAGGCCCCCTCCAGCGACAGCCACCACGAGTTCCTGGCGTTGGGATTCTGCTAAAGG CACCATCCATGGAGAGGAGGTGGAACAGGAGAAGATAGCTGTCTTCCGCGACGCATCCCGCTTCATCCTGAACCTGA CCAGCGTGAAGCCGGAGGACAGCGGCATCTACTTCTGTATGATCGTTGGCTCCCCCGAGCTGACCTTCGGCAAAGGC ACCCAGCTGTCCGTGGTGGACTTCCTGCCCACCACAGCCCAGCCAACCAATCCACCCTCAAGAAGCGCGTGTG CCGACTGCCCCGCCCTGAAACCCAGAAGGGCCCTCTGTGCTCCCCCATCACCCTTGGACTGCTGGTGGCGGGAGTCC TGGTGCTGCTCGTATCTCTGGGTGTCGCCATCCACCTGTGCTGCCGCCGCCGCCGCGCCCGCCTGAGGTTTATGAAA CAGTTTTACAAGTGA *For some depicted vectors, the MSCV promoter is in bold. The β chain uses bold and italic text annotations. The α chain uses bold and underlined text annotations. CD34 enrichment tags (Q tags) use italic and underlined text annotations. CD8-α is italicized. CD8-β is underlined.

[0251] Table 4 Immatics-based R37P1C9 TCR MGTM codon optimization sequence alpha chain: TRAV26-2 / TRAJ21 / MGTM modified TRAC α-strand DNA sequence ATGAAGCTGGTGACCAGCATCACCGTGCTGCTGAGCCTGGGCATCATGGGCGACGCCAAGACCACCCAGCCCAACAGCATGGAGAGCAACGAGGAGGAGCCCGTGCACCTGCCCTGCAACCACAGC ACCATCAGCGGCACCGAC TAC ATCCACTGGTACAGGCAGCTGCCCAGCCAGGGCCCCGAGTACGTGATCCAC GGCCTGACCAGCAAC GTGAACAACAGGATGGCCAGCCTGGCCATCGCCGAGGACAGGAAGAGCAGCACCCTGATCCTGCACAGGGCCACCCTGAGGGACGCCGCCGTGTACTAC TGCATCCTGTTCAACTTCAAACAAGTTCTACTTCGGCAGCGGCACCAAGCTGAACGTGAAGCCCAacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagc Alpha chain protein sequence MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHS TISGTDY IHWYRQLPSQGPEYVIH GL TSN VNNRMASLAIAEDRKSSTLILHRATLRDAAVYY CILFNFNKFYF GSGTKLNVKPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlws Beta chain: TRBV15 / TRBJ1-4 / MGTM modified TRBC Beta chain DNA sequence ATGGGCCCCGGCCTGCTGCACTGGATGGCCCTGTGCCTGCTGGGCACCGGCCACGGCGACGCCATGGTGATCCAGAACCCCAGGTACCAGGTGACCCAGTTCGGCAAGCCCGTGACCCTGAGCTGCAGCCAGACC CTGAACCAC AACGTG ATGTACTGGTACCAGCAGAAGAGCAGCCAGGCCCCCAAGCTGCTGTTCCAC TACTACGACAAGGACTTC AACAACGAGGCCGACACCCCCGACAACTTCCAGAGCAGGAGGCCCAACACCAGCTTCTGCTTCCTGGACATCAGGAGCCCCGGCCTGGGCGACGCCGCCATGTACCTG TGCGCCCACCAGCAGCGGCGAGACCAACGAGAAGCTGTTCTTC GGCAGCGGCACCCAGCTGAGCGTGCTGGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggcagagccaaaaggtccgggagcggt β-chain protein sequence MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQT LNHNV MYWYQQKSSQAPKLLFH Y YDKDF NNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYL CATSSGETNEKLFFGSGTQLSVLEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsatfwqn prnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsgrakrsgsg The complete β and α ORF DNA sequences (the underlined italicized region at the "furin protease-P2A" site encodes a sequence that allows expression of two polypeptide chains in a single cassette) ATGGGCCCCGGCCTGCTGCACTGGATGGCCCTGTGCCTGCTGGGCACCGGCCACGGCGACGCCATGGTGATTCCAGAACCCCAGGTACCAGGTGACCCAGTTCGGCAAGCCCGTGACCCTGAGCTGCAGCCAGACC CTGAACCAC AACGTG ATGTACTGGTACCAGCAGAAGAGCAGCCAGGCCCCCAAGCTGCTGTTCCAC TACTACGACAAGGACTTC AACAACGAGGCCGACACCCCCGACAACTTCCAGAGCAGGAGGCCCAACACCAGCTTCTGCTTCCTGGACATCAGGAGCCCCGGCCTGGGCGACGCCGCCATGTACCTG TGCGCCCACCAGCAGCGGCGAGACCAACGAGAAGCTGTTCTTCGGCAGCGGCACCCAGCTGAGCGTGCTGGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggc agagccaaaaggtccggggagcggtGCGACAAACTTTAGCCTGTT GAAACAAGCCGGCGACGTTGAAGAGAACCCCGGACCT ATGAAGCTGGTGACCAGCATCACCGTGCTGCTGAGCCTGGGCATCATGGGCGACGCCAAGACCACCCAGCCCAACAGCATGGAGAGCAACGAGGAGGAGCCCGTGCACCTGCCCTGCAACCACAGC ACCATCAGCGGCACCGACTAC ATCCACTGGTACAGGCAGCTGCCCAGCCAGGGCCCCGAGTACGTGATCCAC GGCCTGACCAGCAAC GTGAACAACAGGATGGCCAGCCTGGCCATCGCCGAGGACAGGAAGAGCAGCACCCTGATCCTGCACAGGGCCACCCTGAGGGACGCCGCCGTGTACTAC TGCATCCTGTTCAACTTCAAACAAGTTCTACT TCGGCAGCGGCACCAAGCTGAACGTGAAGCCCAacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggact tcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctgg tccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagt ccttcgagacagaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagc Complete β and α ORF protein sequences (the underlined italicized region at the "furin protease-P2A" site allows for the expression of two polypeptide chains in a single cassette) MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQT LNHNV MYWYQQKSSQAPKLLFH Y YDKDF NNEADTPDNFQSRRPNTSFFCFLDIRSPGLGDAAMYL CATSSGETNEKLFF GSGTQLSVLEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsat fwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsg rakrsgsgATNFSLLKQAGDVEENPGP MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHS TISGTDY IHWYRQLPSQGPEYVIH GLTSN VNNRMASLAIAEDRKSSTLILHRATLRDAAVYY CILFN FNKFYF GSGTKLNVKPNiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlws T1367 TCR MGTM Codon-Optimized Sequence Based on T-Knife Alpha chain: TRAV5 / TRAJ41 / MGTM-Modified TRAC Alpha chain DNA sequence ATGAAGACCTTCGCCGGCTTCAGCTTCCTGTTCCTGTGGCTGCAGCTGGACTGCATGAGCAGGGGCGAGGACGTGGAACAGAGCCTGTTTCTGAGCGTGCGCGAGGGCGACAGCAGCGTGATCAATTGCACCTACACC GACAGC TCCAGCACCTAC CTGTACTGGTACAAGCAGGAACCTGGCGCCGGACTGCAGCTGCTGACCTAC ATCTTCAGCAACA TGGACATG AAGCAGGACCAGAGACTGACCGTGCTGCTGAACAAGAAGGACAAGCACCTGAGCCTGCGGATCGCCGATACCCAGACAGGCGACAGCGCCATCTACTTT TGCGCCGAGAGCATCGGCAGCAACAGCGGCTACGCCCTGAACTTCGGCAAGGGCACAAGCCTGCTCGTGACCCCTCacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagc Alpha chain protein sequence MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTY IFSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAESIGSNSGYALNF GKGTSLLVTPHiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlws Beta chain: TRBV28 / TRBJ2-7 / MGTM-modified TRBC Beta chain DNA sequence ATGGGAATCAGACTGCTGTGCAGAGTGGCCTTCTGCTTCCTGGCCGTGGGCCTGGTGGACGTGAAAGTGACCCAGAGCAGCAGATACCTCGTGAAGCGGACCGGCGAGAAGGTGTTCCTGGAATGCGTGCAGGAC ATGGACCAC GAGAAT ATGTTCTGGTACAGACAGGACCCCGGCCTGGGCCTGCGGCTGATCTACTTC AGCTACGACGTGAAGATG AAGGAAAAGGGCGACATCCCCGAGGGCTACAGCGTGTCCAGAGAGAAGAAAGAGCGGTTCAGCCTGATCCTGGAAAGCGCCAGCACCAACCAGACCAGCATGTACCTG TGCGCCAGCAGAGGCCTGGCCGGCTACGAGCAGTATTTT GGCCCTGGCACCCGGCTGACCGTGACCGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggcagagccaaaaggtccgggagcggt β-chain protein sequence MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQD MDHEN MFWYRQDPGLGLRLIYF S YDVKM KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYL CASRGLAGYEQYFGPGTRLTVTEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsatfwqn prnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsgrakrsgsg The complete β and α ORF DNA sequences (the underlined italicized region at the "furin protease-P2A" site encodes a sequence that allows expression of two polypeptide chains in a single cassette) ATGGGAATCAGACTGCTGTGCAGAGTGGCCTTCTGCTTCCTGGCCGTGGGCCTGGTGGACGTGAAAGTGACCCAGAGCAGCAGATACCTCGTGAAGCGGACCGGCGAGAAGGTGTTCCTGGAATGCGTGCAGGAC ATGGACCAC GAGAAT ATGTTCTGGTACAGACAGGACCCCGGCCTGGGCCTGCGGCTGATCTACTTC AGCTACGACGTGAAGATG AAGGAAAAGGGCGACATCCCCGAGGGCTACAGCGTGTCCAGAGAGAAGAAAGAGCGGTTCAGCCTGATCCTGGAAAGCGCCAGCACCAACCAGACCAGCATGTACCTG TGCGCCAGCAGAGGCCTGGCCGGCTACGAGCAGTATTTTGGCCCTGGCACCCGGCTGACCGTGACCGaagatctgaacaaggtgttccctccagaggtggccgtgttcgagccttctaaggccgagatcgcccacacacaaaaagccaccctcgtgtgcctggccaccggctttttccccgaccacgtggaactgtcttggtgggtcaacggcaaagaggtgcactccggcgtgtcaacggatccccagcctctgaaagaacagcctgccctgaacgacagccggtactgcctgagctccagactgagagtgtccgccaccttctggcagaacccccggaaccacttcagatgccaggtgcagttttacggcctgagcgagaacgacgagtggacccaggacagagccaagcccgtgacacaaatcgtgtctgccgaagcctggggaagagccgattgcggcatcaccagcgcctcctatcaccagggcgtgctgagcgccacaatcctgtacgaaatcctgctgggcaaggccaccctgtacgccgtgctggtgtctgctctggtgctgatggccatggtcaagcggaaggactttggcagcggc agagccaaaaggtccgggagcggtGCGACAAACTTTAGCCTGTTGAA ACAAGCCGGCGACGTTGAAGAGAACCCCGGACCT ATGAAGACCTTCGCCGGCTTCAGCTTCCTGTTCCTGTGGCTGCAGCTGGACTGCATGAGCAGGGGCGAGGACGTGGAACAGAGCCTGTTTCTGAGCGTGCGCGAGGGCGACAGCAGCGTGATCAATTGCACCTACACC GACAGCTCCAGCACCTAC CTGTACTGGTACAAGCAGGAACCTGGCGCCGGACTGCAGCTGCTGACCTAC ATCTTCAGCAACATGGACATG AAGCAGGACCAGAGACTGACCGTGCTGCTGAACAAGAAGGACAAGCACCTGAGCCTGCGGATCGCCGATACCCAGACAGGCGACAGCGCCATCTACTTT TGCGCCGAGAGCATCGGCA GCAACAGCGGCTACGCCCTGAACTTCGGCAAGGGCACAAGCCTGCTCGTGACCCCTCacatccagaaccccgaccccgccgtgtaccagctgagggactccaagtccagcgacaagagcgtgtgtctgtttacggacttcgacagccagaccaacgtgagtcaaagcaaggacagcgacgtctacataacggataagaccgtgctggacatgcggagcatggacttcaagagcaacagcgccgtggcctggtccaacaagagcgacttcgcctgcgccaacgccttcaacaacagcatcatccccgaggacaccttcttccccagcagcgacgtgccctgcgacgtgaaactggtggagaagtccttcgagacagacaccaatctgaactttcagaacctgctggtgatcgtgctgcggattctgctgctgaaagtggccggcttcaatctgctgatgaccctgcggctgtggagc Full β and α ORF protein sequences (italicized regions underlined in the "furin-P2A" site allow expression of two polypeptide chains in a single cassette) MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQD MDHEN MFWYRQDPGLGLRLIYF S YDVKM KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYL CASRGLAGYEQYF GPGTRLTVTEdlnkvfppevavfepskaeiahtqkatlvclatgffpdhvelswwvngkevhsgvstdpqplkeqpalndsryclssrlrvsatfwqnprnhfrcqvqfyglsendewtqdrakpvtqivsaeawgradcgitsasyhqgvlsatilyeillgkatlyavlvsalvlmamvkrkdfgsg rakrsgsgATNFSLLKQAGDVEENPGP MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTY IFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF C AESIGSNSGYALNF GKGTSLLVTPHiqnpdpavyqlrdskssdksvclftdfdsqtnvsqskdsdvyitdktvldmrsmdfksnsavawsnksdfacanafnnsiipedtffpssdvpcdvklveksfetdtnlnfqnllvivlrilllkvagfnllmtlrlws *For some depicted vectors, the MSCV promoter is in bold. The β chain uses bold and italic text annotations. The α chain uses bold and underlined text annotations. CD34 enrichment tags (Q tags) use italic and underlined text annotations. CD8-α is italicized. CD8-β is underlined.

[0252] *Tables 1-4 include peptide epitopes, and polypeptide molecules, or portions thereof, comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity in length with any of the sequences listed in Table 1. Such polypeptides may have the functions of the full-length peptides or polypeptides further described herein.

[0253] *Tables 1-4 include RNA nucleic acid molecules (e.g., thymine replaced by uracil), nucleic acid molecules encoding orthologs of the encoded proteins, and DNA or RNA nucleic acid sequences, or portions thereof, containing nucleic acid sequences that have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity in full length with any of the sequences listed in Tables 1-4. Such nucleic acid molecules may have the functions of the full-length nucleic acids further described herein.

[0254] In some embodiments, the binding protein provided herein comprises a chimeric, humanized, human, primate, or rodent (e.g., rat or mouse) constant region. For example, a human variable region may be chimeric with a mouse constant region, or a mouse variable region may be humanized using a human constant region and / or a human framework region. In some embodiments, the constant region may be mutated to modify functionality (e.g., introducing non-naturally occurring cysteine ​​substitutions at relative residue positions in the TCR α and β chains to provide disulfide bonds that can be used to increase affinity between the TCR α and β chains). Similarly, the transmembrane domain of the constant region may be mutated to modify functionality (e.g., increasing hydrophobicity by introducing non-naturally occurring residue substitutions with hydrophobic amino acids). In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof, as compared to a reference CDR sequence. In some embodiments, the constant region may be mutated to increase cell surface expression.

[0255] In some implementations, the binding protein disclosed herein may be an engineered protein scaffold, an antibody or its antigen-binding fragment, a TCR mimic antibody, etc. Such binding moieties can be designed and / or generated using conventional immunological methods targeting the peptides and / or MHC-peptide complexes described herein, such as immunizing a host, obtaining antibody-producing cells and / or their antibodies, and generating hybridomas that can be used to generate monoclonal antibodies (e.g., Watt et al. (2006) Nat. Biotechnol. 24:177-183; Gebauer and Skerra (2009) Curr. Opin. Chem Biol. 13:245-255; Skerra et al. (2008) FEBS J. 275:2677-2683; Nygren et al. (2008) FEBS J. 275:2668-2676; Dana et al. (2012) Exp. Rev. Mol. Med. 14:e6; Sergeva et al. (2011) Blood 117:4262-4272; PCT Publication No. WO 2007 / 143104, PCT / US86 / 02269 and WO 86 / 01533; US Patent No. 4,816,567; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, 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 necessary, conventional procedures can be used to separate or purify the bound moiety, such as protein A-agarose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate 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).

[0256] The term "antibody" broadly encompasses naturally occurring antibody forms (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, as well as fragments and derivatives of all the aforementioned antibodies having at least one antigen-binding site. Antibody derivatives may contain a protein or chemical moiety conjugated to an antibody.

[0257] Furthermore, intracellular antibodies are well-known antigen-binding molecules that possess antibody characteristics 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, such as using single-chain antibodies (scFv), modifying the VL domain of immunoglobulins to obtain hyperstability, modifying antibodies to resist the reducing intracellular environment, generating fusion proteins that increase intracellular stability and / or regulate intracellular localization, etc. Intracellular antibodies can also be introduced into and expressed in one or more cells, tissues, or organs of a multicellular organism, for purposes such as prevention and / or treatment (e.g., as gene therapy) (see at least PCT Publications Nos. WO 08 / 020079, WO 94 / 02610, WO 95 / 22618, and WO 03 / 014960; U.S. Patent No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.); Kontermann (2004) Methods 34:163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412; Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303: 19-39).

[0258] As used herein, the term "antibody" also includes the "antigen-binding portion" (or simply "antibody portion") of an antibody. As used herein, the term "antigen-binding portion" refers to one or more segments of an antibody that retain the ability to bind specifically and / or selectively to an antigen (e.g., the peptide and / or MHC-peptide complex described herein). It has been shown that the antigen-binding function of an antibody can be performed by segments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments connected by disulfide bridges of hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of VH domains; and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be conjugated using recombinant methods via synthetic linkers that allow them to be made into a single protein chain, where the VL region pairs with the VH region to form a monovalent polypeptide (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). It is also intended that such single-chain antibodies be encompassed within the term "antigen-binding portion" of antibody. Any VH and VL sequence of a particular scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used to generate Fab, Fv, or other immunoglobulin fragments using protein chemistry or recombinant DNA techniques. It also includes other forms of single-chain antibodies, such as bifunctional antibodies. Bifunctional antibodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to pair between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of the other chain and creating two antigen-binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0259] Additionally, the antibody or its antigen-binding moiety can be part of a larger immunoadhesion polypeptide formed by the covalent or non-covalent association of the antibody or antibody moiety with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include tetrameric scFv polypeptides prepared using the streptavidin core region (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and divalent and biotinylated scFv polypeptides prepared using cysteine ​​residues, protein subunit peptides, and C-terminal multihistidine tags (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody moieties, for example, of the Fab and F(ab')2 fragments can be prepared from whole antibodies using conventional techniques, such as papain digestion or pepsin digestion of the whole antibody, respectively. Furthermore, as described herein, antibodies, antibody moieties, and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques.

[0260] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or homogenotyped; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies can also be fully human. Preferably, the antibodies of the present invention bind specifically and / or selectively, or substantially specifically and / or selectively, to the peptides and / or MHC-peptide complexes described herein. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a group of antibody peptides containing only one antigen-binding site capable of responding to an immune response to a specific epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a group of antibody peptides containing multiple antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity to the specific antigen with which they respond to an immune response.

[0261] Similar to other binding components described herein, antibodies can also be “humanized,” which are intended to comprise antibodies made from non-human cells having variable and constant regions that have been modified to more closely resemble antibodies to be made from human cells. For example, this can be achieved by altering the amino acid sequence of a non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of this invention may, for example, include amino acid residues in the CDR that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through in vitro / in vitro random or site-specific mutagenesis or through in vivo somatic mutations). As used herein, the term “humanized antibody” also includes antibodies in which a CDR sequence derived from the germline of another mammalian species has been grafted onto a human framework sequence.

[0262] In some embodiments, the binding protein 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 protein disclosed herein may comprise two polypeptide chains, each containing a variable region comprising CDR3 of the TCR α chain and CDR3 of the TCR β chain, or CDR1, CDR2, and CDR3 of both the TCR α and TCR β chains. In some embodiments, the binding protein comprises a single-chain TCR (scTCR) containing TCR V. α and TCR V β Both domains exist, but only a single TCR constant domain (C) is contained. α Or C β The term "chimeric antigen receptor" (CAR) refers to a fusion protein engineered to contain two or more naturally occurring amino acid sequences linked together in a non-natural manner or in a non-natural manner within the host cell, which can function as a receptor when present on the cell surface. CARs covered by this invention may include an extracellular portion comprising an antigen-binding domain (i.e., an antigen-binding domain derived from or derived from immunoglobulins or immunoglobulin-like molecules, such as antibodies or TCRs, or derived from or derived from cytotoxic immune receptors from NK cells), the antigen-binding domain being linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing a co-stimulatory domain) (see, for example, 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).

[0263] In some implementations, 1) TCR α chain CDR, TCR V α The domain and / or TCR α chain is encoded by the TRAV, TRAJ, and / or TRAC genes or fragments thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) TCR β chain CDR, TCR V β The domain and / or TCR β chain are encoded by TRBV, TRBJ and / or TRBC genes or fragments 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 homologous reference CDR sequences listed in Table 2.

[0264] In some embodiments, the binding proteins disclosed herein (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs)) are chimeric (e.g., containing amino acid residues or motifs from more than one donor or species), humanized (e.g., containing residues from non-human organisms, said residues being altered or substituted to reduce the risk of immunogenicity in humans), or human.

[0265] Methods for generating engineered binding proteins (e.g., TCRs, CARs, and their antigen-binding fragments) 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. WO 2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0266] In some embodiments, the binding protein described herein is a TCR or its antigen-binding fragment expressed on the cell surface, wherein the cell surface-expressed TCR is able to associate more effectively with CD3 protein compared to the endogenous TCR. When expressed on the surface of cells such as T cells, the binding protein (e.g., TCR) covered by the present invention may also have higher surface expression on the cell compared to the endogenous binding protein (e.g., endogenous TCR). In some embodiments, this document provides a CAR wherein the binding domain of the CAR includes an antigen-specific TCR binding domain (see, for example, Walseng et al. (2017) Scientific Reports 7:10713).

[0267] Modified binding proteins (e.g., TCR, antigen-binding fragments of TCR, or CAR) are also provided, which can be used according to well-known methods, using the one or more V disclosed herein. α and / or V β The modified binding protein is prepared by engineering a sequence-binding protein as a starting material. The modified binding protein may possess properties altered compared to the starting binding protein. This can be achieved by modifying one or two variable regions (i.e., V...). α and / or V β The binding protein can be engineered by modifying residues within a region, such as one or more CDR regions and / or one or more frame regions. Alternatively, the binding protein can be engineered by modifying residues within a constant region.

[0268] Another type of variable region modification is to make V α and / or V β Mutations are made to amino acid residues within the CDR1, CDR2, and / or CDR3 regions, thereby modifying one or more binding properties (e.g., affinity) of the binding protein of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effects on protein binding or other functional properties of interest can be evaluated in in vitro, ex vivo, or in vivo assays as described herein and in the examples. In some embodiments, conserved modifications (as discussed above) may be introduced. Mutations can be amino acid substitutions, additions, or deletions. In some embodiments, the mutation is a substitution. Furthermore, typically no more than one, two, three, four, or five residues within the CDR region are modified.

[0269] In some embodiments, the binding protein described herein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) may have one or more amino acid substitutions, deletions, or additions relative to a naturally occurring TCR. In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof compared to the homologous reference CDR sequences listed in Table 2. Conserved substitutions of amino acids are well known and may be naturally occurring or introduced during recombinant production of the binding protein. Amino acid substitutions, deletions, and additions may be introduced into the protein using mutagenesis methods known in the art (see, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY). Site-specific (or segment-specific) oligonucleotide mutagenesis procedures may be used to provide altered polynucleotides having specific codons that have been altered according to the desired substitution, deletion, or insertion. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide directed mutagenesis, may be used to prepare immunogenic peptide variants (see, for example, Sambrook et al., ibid.).

[0270] Several criteria known to those skilled in the art indicate whether an amino acid substituted at a specific position in a peptide or polypeptide is conserved (or similar). For example, a similar amino acid or conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Similar amino acids can include 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 nonpolar 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 β-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline is considered difficult to classify because it shares characteristics with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, substitution of glutamic acid with glutamine or aspartic acid with asparagine can be considered a similar substitution, since glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As will be understood in the art, “similarity” between two peptides can be determined by comparing the amino acid sequence of a peptide and its conserved amino acid substitutions with the sequence of a second peptide (e.g., using GNEWORKS™, Align, BLAST algorithms, or other algorithms described herein and practiced in the art).

[0271] 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 transport peptide). The signal peptide directs the newly synthesized polypeptide to its appropriate location inside or outside the cell. The signal peptide can be removed from the polypeptide during or after localization or secretion. A polypeptide containing a signal peptide is referred to herein as a "preprotein," and a polypeptide with the signal peptide removed is referred herein as a "mature" protein or polypeptide. In some embodiments, the binding protein described herein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) includes a mature V α Structural domain, mature V β Domains or both. In some embodiments, the binding protein described herein (e.g., TCR, antigen-binding fragment of TCR, or CAR) comprises a mature TCR β-chain, a mature TCR α-chain, or both.

[0272] In some embodiments, the binding protein is a fusion protein comprising: (a) an extracellular component containing a TCR or an antigen-binding fragment thereof; (b) an intracellular component containing an effector domain or a functional portion thereof; and (c) a transmembrane domain connecting the extracellular and intracellular components. In some embodiments, the fusion protein is capable of binding to a peptide-MHC (pMHC) complex (e.g., specifically and / or selectively), the peptide-MHC complex comprising the MAGEA1 immunogenic peptide in a background of MHC molecules (e.g., MHC class I molecules). In some embodiments, the MHC molecule comprises an MHC α chain of HLA serotype HLA-A*01. In some embodiments, the HLA alleles are selected from the group consisting of HLA-A*01:01.

[0273] As used herein, an "effect domain" or "immune effector domain" is an intracellular portion or domain of a fusion protein or receptor that, when received with an appropriate signal, can directly or indirectly promote an immune response in the cell. In some embodiments, the effector domain is derived from an immune cell protein or a portion thereof or an immune cell protein complex, which receives a signal when it binds (e.g., CD3ζ), or when the immune cell protein or a portion thereof or an immune cell protein complex directly binds to a target molecule and triggers signal transduction of the effector domain in the immune cell.

[0274] When an effector domain contains one or more signal transduction domains or motifs, such as intracellular tyrosine activation motifs (ITAMs), as found in co-stimulatory molecules, it can directly promote cellular responses. Without being bound by theory, it is believed that ITAMs can be used for T cell activation after the T cell receptor or a fusion protein containing a T cell effector domain is linked to a ligand. In some embodiments, an intracellular component or its functional portion contains an ITAM. Exemplary immune effector domains include, but are not limited to, those 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).

[0275] In other embodiments, the intracellular component of the fusion protein comprises a co-stimulatory domain selected from the following or a functional portion thereof: 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, a ligand that binds to CD83 (e.g., specifically and / or selectively) 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 include an LL-GG mutation at position 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).

[0276] In some embodiments, the effector domain comprises an intracellular domain of CD3ε or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises an intracellular domain of CD27 or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises an intracellular domain of CD28 or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises an intracellular domain of 4-1BB or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises an intracellular domain of OX40 or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises an intracellular domain of CD2 or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises an intracellular domain of CD5 or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises an intracellular domain of ICAM-1 or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises the LFA-1 intracellular domain or its functional (e.g., signal transduction) portion, or a functional variant thereof. In other embodiments, the effector domain comprises the ICOS intracellular domain or its functional (e.g., signal transduction) portion, or a functional variant thereof.

[0277] The extracellular and intracellular components covered by this invention are connected via transmembrane domains. As used herein, a "transmembrane domain" is a portion of a transmembrane protein that can insert into or cross the cell membrane. Transmembrane domains have a three-dimensional structure that is thermodynamically stable in the cell membrane and generally ranges in length from about 15 to about 30 amino acids. The structure of a transmembrane domain may include α-helices, β-barrels, β-sheets, β-helices, or any combination thereof. In some embodiments, the transmembrane domain comprises or is derived from known transmembrane proteins (e.g., CD4 transmembrane domain, CD8 transmembrane domain, CD27 transmembrane domain, CD28 transmembrane domain, or any combination thereof).

[0278] In some embodiments, the extracellular component of the fusion protein further includes a linker positioned between the binding domain and the transmembrane domain. As used herein when referring to the component of the fusion protein that connects the binding domain and the transmembrane domain, a “linker” can be an amino acid sequence of about two to about 500 amino acids, which can provide flexibility and space for conformational movement between two regions, domains, motifs, segments, or modules connected by the linker. For example, the linker covered by this invention can localize the binding domain away from the surface of the host cell expressing the fusion protein to enable proper contact, antigen binding, and activation between the host cell and the target cell (Patel et al. (1999) Gene Therapy 6:412-419). The linker length can be varied based on the selected target molecule, the selected binding epitope, or the capture and affinity of the antigen-binding domain to maximize antigen recognition (see, for example, Guest et al. (2005) Immunother. 28:203-11, and PCT Publication No. WO 2014 / 031687). Exemplary linkers include those having a glycine-serine amino acid chain having one to about ten Gly. x Ser y A repeating sequence in which x and y are each an independent integer from 0 to 10, with the constraint that x and y are not both 0 (e.g., (Gly4Ser)2, (Gly3Ser)2, Gly2Ser or combinations thereof, such as ((Gly3Ser)2Gly2Ser)).

[0279] The binding protein can be conjugated to agents such as detection modulonucleotides, radiosensitizers, photosensitizers, etc., and / or can be chemically modified as described above regarding peptides.

[0280] In some embodiments, the binding protein covered by the present invention may be covalently linked to a portion. In some embodiments, the covalently linked portion 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, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently linked portion may be selected from the group consisting of: pro-inflammatory factors, anti-inflammatory agents, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies, such as single-chain Fv.

[0281] The binding protein may be conjugated to agents used in imaging, research, therapeutics, therapeutic diagnostics, pharmacology, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, the binding protein may be conjugated to or fused with a detectable agent, such as a fluorophore, near-infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radionuclide chelator, or another suitable material that can be used for imaging. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more detectable moieties may be linked to the binding protein. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioactive isotope 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 radioactive isotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not easily quenched by biological tissues and body fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation with wavelengths between 650 nm and 4000 nm; this type of emission is used for the detection of such agents. Non-limiting examples of fluorescent dyes that can be used as conjugated molecules include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, the near-infrared dye typically includes cyanine dyes (e.g., Cy7, Cy5.5, and Cy5).Further non-limiting examples of fluorescent dyes used as conjugated molecules according to the present invention include acridine orange or acridine 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 staining agent and any derivatives thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthobenzene, bisbenzoimide, brain rainbow, calcein, carboxyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, and DyLight®. Fluors® and any of its derivatives, epicoconone, ethidium bromide, FlasH-EDT2®, Fluo dyes and any of their derivatives, FluoProbe® and any of their derivatives, fluorescein and any of its derivatives, Fura® and any of its derivatives, GelGreen® and any of its derivatives, GelRed® and any of its derivatives, fluorescent proteins and any of their derivatives, m-isotype proteins and any of their derivatives (e.g., mCherry), hetamethine dyes and any of their derivatives, hoeschst staining agents, iminocoumarin, Indian yellow, indo-1 and any of its derivatives, laurdan, fluorescein yellow and any of its derivatives, fluorescein and any of its derivatives, luciferase and any of its derivatives, cyanide and any of its derivatives, Nile dyes dyes and any derivatives thereof, perylene, phloxine, algal dyes and any derivatives thereof, propidium iodide, pyranine, rhodamine and any derivatives thereof, ribose green, RoGFP, rubrene, stilbene and any derivatives thereof, sulfonyl rhodamine and any derivatives thereof, SYBR and any derivatives thereof, synapto-pH-sensitive green fluorescent protein, tetraphenylbutadiene, tris tetrasodium, Texas Red, Titan Yellow, TSQ, umbelliferone, violet anthrone, yellow fluorescent protein, and YOYO-1.Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbonyl cyanide, styrene dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green™ dyes (e.g., Oregon Green™ 488, Oregon Green™ 500, Oregon Green™ 514, etc.), Texas Red®, Texas Red®-X, SPECTRUM RED®, SPECTRUM GREEN®, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), Alexa 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, BODIPY® 558 / 568, BODIPY® Examples of suitable detectable reagents include 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, etc., and IRD dyes (e.g., IRD40™, IRD700™, IRD800™, etc.). Other suitable detectable reagents are well known in the art (e.g., PCT Publication No. PCT / US14 / 56177). Non-limiting examples of radioactive isotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioactive isotope 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 radioactive isotope is actinium-225 or lead-212.

[0282] The binding protein can be conjugated to a radiosensitizer or a photosensitizer. Examples of radiosensitizers include, but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, ethamidazole, misoprostazole, teirazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to: fluorescent molecules or beads that generate heat upon irradiation, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorophyll, mycochlorophyll, isochlorophyll, phthalocyanine, and naphthalenephthalocyanine), metalloporphyrins, metal phthalocyanines, angelicin, sulfhydryl pyranonium dyes, chlorophyll, coumarins, flavins and related compounds (e.g., pyrazine and riboflavin), fullerenes, pheophytin, pyropheophytin, and anthocyanins (e.g., cyanine 540). Phthalate, thiafuroline, tesafuroline, violetin, porphyrin, phenothiazine, methylene blue derivatives, naphthalenedicarboximide, Nile blue derivatives, quinones, perylenequinones (e.g., hyperoside, styracin, and cercariaein), psoralen, quinones, visual pigments, rhodamine, thiophene, veldine, xanthan dyes (e.g., eosin, erythrosine, and Rosemary red), dimers and oligomers of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this method allows for highly specific targeting of cells of interest (e.g., immune cells) using both therapeutic agents (e.g., drugs) and electromagnetic energy (e.g., radiation or light). In some embodiments, the binding protein is fused to the agent or covalently or non-covalently linked to the agent, e.g., directly or via a linker.

[0283] In some embodiments, the binding protein may be chemically modified. For example, the binding protein may be mutated to modify peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation sites, pH, and function. N-methylation is an example of methylation that can occur in the binding proteins covered by this invention. In some embodiments, the binding protein may be modified by methylating a free amine, for example by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0284] Chemical modifications may include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendritic polymers, Fc regions, simple saturated carbon chains (e.g., palmitate or myristate), or albumin. Chemical modifications of binding proteins with Fc regions may be fused Fc-proteins. Polyamino acids may include, for example, polyamino acid sequences having repeating single amino acids (e.g., polyglycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination thereof.

[0285] In some embodiments, the binding protein covered by this invention may be modified. In some embodiments, the modification has substantial or significant sequence identity with the parent binding protein to produce a functional variant that maintains one or more biophysical and / or biological activities of the parent binding protein (e.g., maintaining pMHC binding specificity). In some embodiments, mutations are made to conserved amino acid substitutions.

[0286] In some embodiments, the binding proteins covered by this invention may comprise 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, leucine, α-aminodecanoic acid, homoserine, S-acetaminomethylcysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indole. Phosphoric acid-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-phenylmethyl-N'-methyl-lysine, N',N'-diphenylmethyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, oc-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norborneane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and oc-tert-butylglycine.

[0287] The binding proteins covered by this invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0288] In some embodiments, the attachment of a hydrophobic portion (e.g., to the N-terminus, C-terminus, or internal amino acid) can be used to extend the half-life of the peptides covered by this invention. In other embodiments, the binding protein may include post-translational modifications (e.g., methylation and / or amidation) that can affect, for example, serum half-life. In some embodiments, a simple carbon chain (e.g., by myristylation and / or palmitoylation) may be conjugated to the binding protein. In some embodiments, a simple carbon chain may facilitate the separation of the binding protein from unconjugated material. For example, methods that can be used to separate the binding protein from unconjugated material include, but are not limited to, solvent extraction and reversed-phase chromatography. The lipophilic portion can extend the half-life by reversible binding to serum albumin. The conjugated portion can be a lipophilic portion that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic portion can be cholesterol or cholesterol derivatives, including cholesterolene, cholesterolane, cholesteroldiene, and oxidized sterols. In some embodiments, the binding protein may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding protein may be coupled (e.g., conjugated) to a half-life modifier. Examples of half-life modifiers include, but are not limited to: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers containing 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, spacers or linkers may be coupled to the binding protein, for example, one, two, three, four, or more amino acid residues serving as spacers or linkers, to facilitate conjugation or fusion with another molecule and to facilitate peptide cleavage from such conjugated or fused molecules. In some embodiments, the binding protein may be conjugated to other parts, for example, that can modify or achieve changes in the properties of the binding protein.

[0289] Bound proteins can be produced, for example, through solid-phase peptide synthesis or solution-phase peptide synthesis, either recombinantly or synthetically. Peptides can be synthesized using known synthetic methods, such as fluorenylmethoxycarbonyl (Fmoc) chemistry or butyloxycarbonyl (Boc) chemistry. Peptide fragments can be joined together via enzymatic or synthetic methods.

[0290] In one aspect covered by the present invention, a method for generating the binding protein described herein is provided, the method comprising the steps of: (i) culturing transformed host cells under conditions suitable for allowing expression of the binding protein described herein, said host cells having been transformed with nucleic acids containing a sequence encoding said binding protein; and (ii) recovering the expressed binding protein.

[0291] For example, methods for isolating and purifying recombinant-derived binding proteins may include obtaining a supernatant from a suitable host cell / vector system that secretes the binding protein into a culture medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse-phase HPLC steps may be used to further purify the recombinant peptide. These purification methods may also be used when isolating immunogens from the natural environment. Methods for large-scale production of one or more binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Binding proteins may be purified according to methods described herein and known in the art. In any embodiment disclosed herein, the encoded binding protein is capable of binding to a peptide-MHC (pMHC) complex of the MAGEA1 immunogenic peptide contained in a background of MHC molecules (e.g., MHC class I molecules). In some embodiments, the MHC molecule comprises an MHC α chain for the HLA serotype HLA-A*01. In some embodiments, the HLA allele is HLA-A*01:01.

[0292] Various assays are well known for assessing binding affinity and / or determining whether a binding molecule binds to a specific ligand (e.g., a peptide antigen-MHC complex) (e.g., specifically and / or selectively). For example, the binding affinity of a binding protein to a target (e.g., a T-cell peptide epitope of a target polypeptide) can be determined at the level of a skilled technician by using any of a variety of binding assays well known in the art. For instance, in some embodiments, a Biacore™ instrument can be used to determine the binding constan...

Claims

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

2. An immunogenic peptide, said immunogenic peptide comprising peptide epitopes selected from the peptide sequences listed in Table 1.

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

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

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

6. The immunogenic composition of claim 5, wherein the immunogenic composition further comprises an adjuvant.

7. The immunogenic composition of claim 5 or 6, wherein the immunogenic composition is capable of inducing an immune response against MAGEA1 and / or cells expressing MAGEA1 in a subject, optionally wherein the immune response is i) a T cell response and / or a CD8+ T cell response and / or ii) selected from the group consisting of T cell expansion, cytokine release and / or cytotoxic killing.

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

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

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

11. The composition of any one of claims 9-11, wherein the MHC molecule comprises an MHC α chain, the chain being an HLA serotype of HLA-A*01, and optionally wherein the HLA allele is HLA-A*01:

01.

12. A stable MHC-peptide complex, said stable MHC-peptide complex comprising an immunogenic peptide according to any one of claims 1-4 in an MHC molecular background.

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

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

15. The stable MHC-peptide complex of any one of claims 12-14, wherein the MHC molecule comprises an MHCα chain, the chain being an HLA serotype of HLA-A*01, and optionally wherein the HLA allele is HLA-A*01:

01.

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

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

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

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

20. A vector comprising the isolated nucleic acid as described in claim 19.

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

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

23. A method for detecting T cells bound to a stable MHC-peptide complex, the method comprising: a) Contact a sample containing T cells with a stable MHC-peptide complex according to any one of claims 12-17; as well as b) Detect the binding of T cells to the stable MHC-peptide complex, and optionally further determine the percentage of stable MHC-peptide-specific T cells that bind to the stable MHC-peptide complex, wherein the sample optionally includes peripheral blood mononuclear cells (PBMCs).

24. The method of claim 23, wherein the T cell is a CD8+ T cell.

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

26. The method of any one of claims 22-25, wherein the sample comprises T cells that have been in contact with or suspected of being in contact with one or more MAGEA1 proteins or fragments thereof.

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

28. A method for predicting clinical outcomes in subjects suffering from a condition characterized by MAGEA1 expression, the method comprising: a) Determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides according to any one of claims 1-4 or one or more stable MHC-peptide complexes according to any one of claims 12-17; and b) Compare the presence or level of said responsiveness to responsiveness from a control obtained from a subject with good clinical outcomes. The presence of reactivity or a higher level of reactivity in the subject's sample compared to the control indicates that the subject has good clinical outcomes.

29. A method for evaluating the efficacy of a therapy for a condition characterized by MAGEA1 expression, the method comprising: a) In a first sample obtained from the subject prior to administering at least a portion of the therapy, 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 1-4 or stable MHC-peptide complexes according to any one of claims 12-17 is determined, and b) Determine the presence or level of reactivity between one or more immunogenic peptides according to any one of claims 1-4 or one or more stable MHC-peptide complexes according to any one of claims 12-17 and T cells obtained from the subject, said T cells being present in a second sample obtained from the subject after the therapy has been administered to the subject. The presence of reactivity or a higher level of reactivity in the second sample compared to the first sample indicates that the therapy is effective in treating the subject's condition characterized by MAGEA1 expression.

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

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

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

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

34. The method of any one of claims 27-33, wherein the control level is the level of a subject who does not suffer from the condition characterized by MAGEA1 expression.

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

36. 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, the method comprising: a) Provide cells that present peptide epitopes selected from peptide sequences listed in Table 1 in the background of MHC molecules on the surface of the cells. b) Determine the binding of multiple candidate peptide-binding molecules or their antigen-binding fragments on the cells to the peptide epitopes in the MHC molecular background; and c) Identify one or more peptide-binding molecules or their antigen-binding fragments that bind to the peptide epitope in the MHC molecular background.

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

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

39. A method for identifying a peptide-binding molecule or an antigen-binding fragment thereof that binds to a peptide epitope selected from peptide sequences listed in Table 1, the method comprising: a) Provide peptide epitopes, either alone or in the form of stable MHC-peptide complexes, comprising peptide epitopes, either alone or in the MHC molecular background, selected from peptide sequences listed in Table 1. b) Determine the binding of various candidate peptide-binding molecules or their antigen-binding fragments to the peptide or the stable MHC-peptide complex; and c) Identify one or more peptide-binding molecules or antigen-binding fragments thereof that bind to the peptide epitope or the stable MHC-peptide complex, optionally wherein the MHC or the MHC-peptide complex is as claimed in any one of claims 8-17.

40. The method of claim 39, wherein the plurality of candidate peptide-binding molecules comprises an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

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

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

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

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

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

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

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

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

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

50. The method of claim 49, wherein the T cells are isolated from: a) the subject, b) a donor not suffering from the condition characterized by MAGEA1 expression, or c) a donor who has recovered from the condition characterized by MAGEA1 expression.

51. A method of treating a subject for a condition characterized by MAGEA1 expression, the method comprising infusing the subject with antigen-specific T cells, wherein the antigen-specific T cells are generated by: a) Stimulating immune cells from a subject with the composition according to any one of claims 1-22; and b) Expanding antigen-specific T cells in vitro or ex vivo, optionally i) isolating immune cells from the subject prior to stimulation of the immune cells and / or ii) wherein the immune cells comprise PBMCs, T cells, CD8+ T cells, IT cells, central memory T cells and / or effector memory T cells.

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

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

54. The method of any one of claims 23-53, wherein the condition 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 urothelial carcinoma of the bladder.

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

56. A binding protein that binds a polypeptide comprising an immunogenic peptide sequence according to any one of claims 1 to 4, an immunogenic peptide according to any one of claims 1-4, and / or a stable MHC-peptide complex according to any one of claims 12-17, optionally wherein the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

57. The binding protein of claim 56, wherein the binding protein comprises: a) A T-cell receptor (TCR) α-chain CDR sequence that has at least approximately 80% identity with a TCR α-chain CDR sequence selected from the group of TCR α-chain CDR sequences listed in Table 2; and / or b) A TCR β-chain CDR sequence that has at least about 80% identity with a TCR β-chain CDR sequence selected from the group of TCR β-chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a binding affinity of less than or equal to about 5 × 10⁻⁶. -4 M of K d .

58. The binding protein of claim 56, wherein the binding protein comprises: a) TCR α chain variable (V α The sequence of structural domains, which are selected from the TCR V listed in Table 2 α TCR V of the group composed of domain sequences α The domain sequences have at least approximately 80% identity; and / or b) TCR β chain variable (V β The sequence of structural domains, which are selected from the TCR V listed in Table 2 β TCR V of the group composed of domain sequences β The domain sequences have at least about 80% identity, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has less than or equal to about 5 × 10⁻⁶. -4 M of K d .

59. The binding protein of claim 56, wherein the binding protein comprises: a) A TCR α-chain sequence that has at least approximately 80% identity with the TCR α-chain sequences selected from the group of TCR α-chain sequences listed in Table 2; and / or b) A TCR β-chain sequence having at least about 80% identity with a TCR β-chain sequence selected from the group of TCR β-chain sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a binding affinity of less than or equal to about 5 × 10⁻⁶. -4 M of K d .

60. The binding protein of claim 56, wherein the binding protein comprises: a) TCR α-chain CDR sequences, selected from the group consisting of the TCR α-chain CDR sequences listed in Table 2; and / or b) A TCR β-chain CDR sequence selected from the group consisting of TCR β-chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a binding affinity of less than or equal to about 5 × 10⁻⁶. -4 M of K d .

61. The binding protein of claim 56, wherein the binding protein comprises: a) TCR α chain variable (V α The domain sequence is selected from the TCR V listed in Table 2. α A group consisting of a sequence of structural domains; and / or b) TCR β chain variable (V β The domain sequence is selected from the TCR V listed in Table 2. β A group of domain sequences, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a value of less than or equal to about 5 × 10⁻⁶. -4 M of K d .

62. The binding protein of claim 56, wherein the binding protein comprises: a) TCR α-chain sequences, selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) A TCR β chain sequence selected from the group consisting of TCR β chain sequences listed in Table 2, wherein the binding protein is capable of binding to the MAGEA1 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a binding affinity of less than or equal to about 5 × 10⁻⁶. -4 M of K d .

63. The binding protein according to any one of claims 56-62, wherein 1) the TCR α chain CDR, the TCR V α The domain and / or the TCR α 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 β chain CDR, the TCR V β The domain and / or the TCRβ chain are encoded by TRBV, TRBJ and / or TRBC genes or fragments 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 homologous reference CDR sequences listed in Table 2.

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

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

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

67. The binding protein of any one of claims 56-66, wherein the TCR α chain and / or the TCR β chain are covalently linked to a portion, optionally wherein the covalently linked portion comprises an affinity tag or marker.

68. The binding protein of claim 67, wherein the affinity tag is selected from the group consisting of: CD34 enrichment tag, glutathione S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag and V5 tag, and / or wherein the tag is a fluorescent protein.

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

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

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

72. The binding protein of any one of claims 56-71, wherein the binding of the binding protein to the MAGEA1 peptide-MHC (pMHC) complex triggers an immune response, optionally wherein the immune response is i) a T cell response and / or a CD8+ T cell response and / or ii) selected from the group consisting of T cell expansion, cytokine release and / or cytotoxic killing.

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

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

75. The binding protein of any one of claims 56-74, wherein when contacted with target cells having MAGEA1 heterozygous expression, the binding protein induces higher T cell expansion, cytokine release and / or cytotoxic killing compared to known T cell receptors, optionally wherein the induction is at least 1.05 times higher.

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

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, colorectal cancer, gastrointestinal cancer, invasive breast cancer, and urothelial carcinoma of the bladder.

78. The binding protein of any one of claims 56-77, wherein the binding protein does not bind to a pMHC complex comprising the peptide epitopes MAGEA10, MAGEA11, RPS6KA2, RPS6KA3, RPS6KA6, STIL, TECPR1, WDR45, CCDC168, SIRPB1, TENM1, ADAMTS20, CPO, SPATA22 and / or ZNF202.

79. A TCR α chain and / or β chain, said TCR α chain and / or β chain being selected from the group consisting of TCR α chain and β chain sequences listed in Table 2.

80. An isolated nucleic acid molecule, said isolated nucleic acid molecule: i) hybridizes with complement of a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2 under stringent conditions, ii) has at least about 80% homology with a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2, and / or iii) has at least about 80% homology with a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 2, optionally said isolated nucleic acid molecule comprises: 1) TRAV, TRAJ and / or TRAC genes or fragments thereof selected from the group consisting of TRAV, TRAJ and TRAC genes listed in Table 2 and / or 2) TRBV, TRBJ and / or TRBC genes or fragments thereof selected from the group consisting of TRBV, TRBJ and TRBC genes listed in Table 2.

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

82. A vector comprising the isolated nucleic acid as described in claim 80 or 81, 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.

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

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

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

86. The carrier as claimed in claim 84 or 85, wherein the tag is a CD34 enrichment tag.

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

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

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

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

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

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

93. The host cell of any one of claims 89-92, wherein the host cell expresses CD8α, CD8β, DN-TGFβRII and / or a selective protein marker, optionally wherein the selective protein marker is DHFR, and further optionally wherein the CD8α, CD8β, the DN-TGFβRII and / or the selective protein marker are fused with 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 89-94, 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 host cell of claim 95, wherein 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, or a CD4+ cell. + T cells, CD8 + T cells, CD4 / CD8 double-negative T cells, γδ (gamma delta) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or combinations thereof.

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

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

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

100. The host cell of any one of claims 89-99, wherein the host cell is capable of producing cytokines or cytotoxic molecules upon contact with a target cell containing a peptide-MHC (pMHC) complex, wherein the peptide-MHC (pMHC) complex contains a MAGEA1 peptide epitope in an MHC molecular background.

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

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

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

104. The host cell of any one of claims 89-103, wherein the host cell is capable of producing higher levels of cytokines or cytotoxic molecules when in contact with target cells having MAGEA1 heterozygous expression.

105. The host cell of claim 104, wherein the host cell is capable of producing at least 1.05 times higher levels of cytokines or cytotoxic molecules.

106. The host cell of any one of claims 89-103, wherein the host cell is capable of killing target cells comprising a peptide-MHC (pMHC) complex containing the MAGEA1 peptide epitope in a background of MHC molecules.

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

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

4.

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

110. The host cell of any one of claims 106-109, wherein the host cell is capable of killing a higher number of target cells when in contact with target cells having MAGEA1 heterozygous expression, optionally wherein the cell killing is at least 1.05 times higher.

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: HEK293-derived cell lines, cancer cell lines, primary cancer cells, transformed cell lines and immortalized cell lines.

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

113. The host cell of any one of claims 89-112, wherein the host cell does not induce T cell expansion, cytokine release, or cytotoxic killing upon contact with a target cell containing a peptide-MHC (pMHC) complex, wherein the peptide-MHC (pMHC) complex comprises MAGEA10, MAGEA11, RPS6KA2, RPS6KA3, RPS6KA6, STIL, TECPR1, WDR45, CCDC168, SIRPB1, TENM1, ADAMTS20, CPO, SPATA22, and / or ZNF202 peptide epitopes.

114. The host cell of any one of claims 89-113, wherein the host cell does not express the MAGEA1 antigen, is not recognized by the binding protein of any one of claims 56-78, does not belong to the serotype HLA-A*01, and / or does not express the HLA-A*01 allele.

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

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

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

118. A method for producing a binding protein according to any one of claims 56-77, wherein the method comprises the following steps: (i) culturing transformed host cells under conditions suitable for allowing expression of the binding protein, the host cells having been transformed with nucleic acids containing a sequence encoding the binding protein according to any one of claims 56-77; and (ii) recovering the expressed binding protein.

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

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

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 an MHC molecular background, and the complex is detected by fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting, or intracellular flow cytometry.

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

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

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

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

126. A method for monitoring the progression of a disease characterized by MAGEA1 expression in subjects, the method comprising: a) Detecting the presence or level of reactivity in a subject sample between a sample obtained from the subject and at least one binding protein according to any one of claims 56-77, at least one host cell according to any one of claims 89-114, or a population of host cells according to claim 115; b) Repeat step a) at subsequent time points; and c) Compare the MAGEA1 levels or cells of interest expressing MAGEA1 detected in steps a) and b) to monitor the progression of the disease characterized by MAGEA1 expression in the subject, wherein the absence or reduction of the MAGEA1 levels or the cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates that the progression of the disease characterized by MAGEA1 expression in the subject is suppressed, and the presence or increase of the MAGEA1 levels or the cells of interest expressing MAGEA1 detected in step b) compared to step a) indicates that the disease characterized by MAGEA1 expression in the subject is progressing.

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

128. A method for predicting clinical outcomes in subjects suffering from a condition characterized by MAGEA1 expression, the method comprising: a) Determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 56-77, at least one host cell according to any one of claims 89-114, or a population of host cells according to claim 115; and b) Compare the presence or level of said responsiveness to responsiveness from a control obtained from a subject with good clinical outcomes; The absence of reactivity or a reduced level of reactivity in the subject's sample compared to the control indicates that the subject has good clinical outcomes.

129. A method for evaluating the efficacy of a therapy for a condition characterized by MAGEA1 expression, the method comprising: a) In a first sample obtained from the subject prior to administering at least a portion of the therapy to the subject for the condition 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 56-77, at least one host cell according to any one of claims 89-114, or a population of host cells according to claim 115, and b) In a second sample obtained from the subject after the treatment is administered for the condition 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 56-77, at least one host cell according to any one of claims 89-114, or a population of host cells according to claim 115 is determined. The absence of reactivity or a decrease in reactivity level in the second sample relative to the first sample indicates that the therapy is effective in treating the subject's condition characterized by MAGEA1 expression, and the presence of reactivity or an increase in reactivity level in the second sample relative to the first sample indicates that the therapy is not effective in treating the subject's condition characterized by MAGEA1 expression.

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

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

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

133. The method of any one of claims 49-55 and 132, wherein the cell is an allogeneic cell, a cell of the same genotype, or an autologous cell.

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

135. The method of any one of claims 49-55 and 132-134, 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 pharmaceutically acceptable carrier.

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

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

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

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

141. The method of any one of claims 132-140, wherein the condition 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, invasive breast cancer, and urothelial carcinoma of the bladder.

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

Images