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

Abstract

Provided herein are PRAME immunogenic peptides, binding proteins that recognize PRAME immunogenic peptides, and uses thereof. The present invention is based, at least in part, on the discovery of PRAME immunogenic peptides and binding proteins that recognize such PRAME immunogenic peptides based on unbiased functional selection methods used to discover antigens of TCR clonotypes identified from subjects with disorders associated with PRAME expression (e.g., subjects suffering from melanoma, head and neck cancer, lung cancer, leukemia (e.g., leukemia subtypes), ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 413,552, filed October 5, 2022, and U.S. Provisional Application No. 63 / 423,294, filed November 7, 2022, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] The cancer / testis antigen PRAME exemplifies an ideal TCR-T cell therapy target due to its high expression in multiple malignancies and its absence in normal tissues. Initially identified in metastatic cutaneous melanoma (Ikeda et al. (1997) Immunity 6:199-208), PRAME is highly expressed in a variety of additional solid tumors, including lung, head and neck, and ovarian cancers. PRAME plays a pivotal role in multiple cellular processes and has been demonstrated to exhibit pro-tumorigenic functions, primarily through the inhibition of retinoic acid receptor signaling (Epping et al. (2005) Cell 122:835-847). Targeting PRAME in solid tumors represents a promising therapeutic approach for the treatment of many cancer indications, especially when implemented as part of a TCR-T multiplexing strategy. There is a need to develop PRAME-specific TCR immunotherapies, including for treating disorders characterized by PRAME expression. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ikeda et al. (1997) Immunity 6:199-208 [Non-patent document 2] Epping et al. (2005)Cell 122:835-847) Summary of the Invention [Means for solving the problem]

[0004] The present invention is based, at least in part, on the discovery of PRAME immunogenic peptides and binding proteins that recognize such PRAME immunogenic peptides based on an unbiased functional selection method used to discover antigens of TCR clonotypes identified from subjects with disorders associated with PRAME expression (e.g., subjects suffering from melanoma, head and neck cancer, lung cancer, leukemia (e.g., leukemia subtypes), ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma). The identified TCRs recognized PRAME immunogenic peptides, such as those listed in Table 1, in the context of various HLA alleles (e.g., HLA-A*02:01). It has been demonstrated herein that PRAME is selectively expressed in cancer and testicular tissues, but not in normal somatic tissues, making it an ideal target for ACT. The ability of PRAME-binding proteins (e.g., the TCRs described herein) to bind to PRAME immunogenic peptides and induce immune responses that kill cells expressing PRAME (e.g., cancer cells) demonstrates the utility of such binding proteins in a wide variety of applications, including appropriate diagnostic, prognostic, therapeutic, and drug screening methods for disorders characterized by PRAME expression. In one aspect, an immunogenic peptide comprising a peptide epitope selected from the peptide sequences listed in Table 1 is provided.

[0005] In another embodiment, immunogenic peptides consisting of peptide epitopes selected from the peptide sequences listed in Table 1 are provided.

[0006] Numerous further embodiments are provided that may be applied to any aspect encompassed by the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the immunogenic peptide is derived from the PRAME protein, and optionally the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In another embodiment, the immunogenic peptide is capable of eliciting an immune response in a subject against PRAME and / or PRAME-expressing cells, and optionally the immune response is i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing.

[0007] In yet another aspect, there is provided an immunogenic composition comprising at least one immunogenic peptide described herein.

[0008] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the immunogenic composition further comprises an adjuvant. In another embodiment, the immunogenic composition is capable of eliciting an immune response in a subject against PRAME and / or PRAME-expressing cells, optionally the immune response being i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing.

[0009] In yet another aspect, a composition is provided comprising a peptide epitope selected from the peptide sequences listed in Table 1 and an MHC molecule.

[0010] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule is selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18. and optionally the HLA alleles are selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:08, HLA-A*02:09, HLA-A*02:18, HLA-A*02:19, HLA-A*02:20, HLA-A*02:21, HLA-A*02:22, HLA-A*02:23, HLA-A*02:24, HLA-A*02:25, HLA-A*02:26, ​​HLA-A*02:27, HLA-A*02:28, HLA-A*02:29, HLA-A*02:30, HLA-A*02:31, HLA-A*02:32, HLA-A*02:33, HLA-A*02:34, HLA-A*02:35, HLA-A*02:36, HLA-A*02:37, HLA-A*02:38, HLA-A*02:39, HLA-A*02:40, HLA-A*02:41, HLA-A*02:42, HLA-A*02:43, HLA-A*02:44, HLA-A*02:45, HLA-A*02:46, H A-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 Allele, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 allele Child, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 allele, HLA-A*24:02, HLA-A*24:03, HL A-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25,HLA-A*24:26, HLA-A*24:58 allele, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*0 7:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*0 In yet another embodiment, the HLA serotype is selected from the group consisting of HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05, and HLA-C*14:03 alleles. In yet another embodiment, the HLA serotype is HLA-A*02, e.g., HLA-A*02:01.

[0011] In another aspect, there is provided a stable MHC-peptide complex comprising an immunogenic peptide as described herein in the context of an MHC molecule.

[0012] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecules are HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HL A-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20 , HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 allele, HLA-A*03:01, HLA -A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 allele, HLA-A*11:01, HLA-A*11 :02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 allele, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:5 8 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HL A-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03,and HLA-C*14:03 alleles. In yet another embodiment, the peptide epitope and the MHC molecule are covalently linked and / or the alpha and beta chains of the MHC molecule are covalently linked. In another embodiment, the stable MHC-peptide complex comprises a detectable label, and optionally, the detectable label is a fluorophore.

[0013] In yet another aspect, there is provided an immunogenic composition comprising a stable MHC-peptide complex as described herein and an adjuvant.

[0014] In yet another aspect, there is provided an isolated nucleic acid encoding an immunogenic peptide described herein, or a complement thereof.

[0015] In another aspect, a vector is provided that comprises the isolated nucleic acid described herein.

[0016] In yet another aspect, there is provided a cell that a) comprises an isolated nucleic acid as described herein, b) comprises a vector as described herein, and / or c) produces one or more immunogenic peptides as described herein and / or displays one or more stable MHC-peptide complexes as described herein on its cell surface, optionally wherein the cell is genetically engineered.

[0017] In yet another aspect, there is provided a device or kit comprising a) one or more immunogenic peptides as described herein, and / or b) one or more stable MHC-peptide complexes as described herein, the device or kit optionally comprising a reagent for detecting binding of a) and / or b) to a binding protein, optionally wherein the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0018] In another aspect, there is provided a method of detecting T cells that bind to stable MHC-peptide complexes, the method comprising: a) contacting a sample comprising T cells with a stable MHC-peptide complex described herein; and b) detecting binding of the T cells to the stable MHC-peptide complex, and optionally further determining the proportion of stable MHC-peptide-specific T cells that bind the stable MHC-peptide complex, optionally wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

[0019] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, the T cells are CD8+ T cells. In another embodiment, the detecting and / or determining is performed using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, Western blot, or intracellular flow assay. In yet another embodiment, the sample contains T cells that have been in contact with, or are suspected of being in contact with, one or more PRAME proteins or fragments thereof.

[0020] In yet another aspect, a method is provided for determining whether T cells have been exposed to PRAME, comprising: a) incubating a cell population comprising T cells with an immunogenic peptide described herein or a stable MHC-peptide complex described herein; and b) detecting the presence or level of reactivity, wherein the presence or higher level of reactivity compared to a control level indicates that the T cells have been exposed to PRAME, and optionally, wherein the cell population comprising T cells is obtained from a subject.

[0021] In yet another aspect, a method is provided for predicting clinical outcome in a subject suffering from a disorder characterized by PRAME expression, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein; and b) comparing the presence or level of reactivity with that from a control, wherein the control is obtained from a subject with a good clinical outcome, and wherein a presence or higher level of reactivity in the subject sample compared to the control indicates that the subject will have a good clinical outcome.

[0022] In another aspect, a method is provided for assessing the effectiveness of a therapy for a disorder characterized by PRAME expression, comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein in a first sample obtained from the subject before providing at least a portion of the therapy to the subject; and b) determining the presence or level of reactivity between 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 second sample obtained from the subject after providing the therapy to the subject, wherein the presence or higher level of reactivity in the second sample compared to the first sample is an indication that the therapy is effective in treating a disorder characterized by PRAME expression in the subject.

[0023] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the level of responsiveness is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, where optionally, the T cell activation or effector function is T cell proliferation, killing, or cytokine release. In another embodiment, the method further comprises repeating steps a) and b) at a subsequent time point, where, optionally, the subject has received a treatment to ameliorate the disorder characterized by PRAME expression between the first and subsequent time points. In yet another embodiment, the T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, Western blot, or intracellular flow assay. In yet another embodiment, the control level is a reference number. In another embodiment, the control level is the level in a subject without a disorder characterized by PRAME expression.

[0024] In yet another aspect, a method for preventing and / or treating a disorder characterized by PRAME expression in a subject comprises administering to the subject a therapeutically effective amount of a composition described herein.

[0025] In yet another aspect, methods are provided for identifying a peptide-binding molecule, or antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, the method comprising: a) providing a cell that displays a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule on its surface; b) determining the binding of a plurality of candidate peptide-binding molecules, or antigen-binding fragments thereof, to the peptide epitope in the context of the MHC molecule on the cell; and c) identifying one or more peptide-binding molecules, or antigen-binding fragments thereof, that bind the peptide epitope in the context of the MHC molecule.

[0026] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, step a) comprises contacting an MHC molecule on the surface of a cell with a peptide epitope selected from the peptide sequences listed in Table 1. In another embodiment, step a) comprises causing the cell to express a peptide epitope selected from the peptide sequences listed in Table 1 using a vector comprising a heterologous sequence encoding the peptide epitope.

[0027] In another aspect, provided is a method for identifying a peptide-binding molecule, or antigen-binding fragment thereof, that binds to a peptide epitope selected from the peptide sequences listed in Table 1, the method comprising: a) providing a peptide epitope, either alone or in the context of an MHC molecule, selected from the peptide sequences listed in Table 1; b) determining binding of a plurality of candidate peptide-binding molecules, or antigen-binding fragments thereof, to the peptide or stable MHC-peptide complex; and c) identifying one or more peptide-binding molecules, or antigen-binding fragments thereof, that bind to the peptide epitope or stable MHC-peptide complex, wherein optionally the MHC or MHC-peptide complex is as described herein.

[0028] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the plurality of candidate peptide-binding molecules comprises an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain. In another embodiment, the plurality of candidate peptide-binding molecules comprises at least 2, 5, 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7, 10 8 , 10 9 or more different candidate peptide-binding molecules. In yet another embodiment, the plurality of candidate peptide-binding molecules comprises one or more candidate peptide-binding molecules obtained from a sample from a subject or a population of subjects, or the plurality of candidate peptide-binding molecules comprises one or more candidate peptide-binding molecules comprising mutations in a parent scaffold peptide-binding molecule obtained from a sample from a subject. In yet another embodiment, the subject or population of subjects a) is not afflicted with a disorder characterized by PRAME expression and / or has recovered from a disorder characterized by PRAME expression, or b) is afflicted with a disorder characterized by PRAME expression. In another embodiment, the subject or population of subjects has been administered a composition described herein. In yet another embodiment, the subject is an animal model of a disorder characterized by PRAME expression and / or a mammal, optionally, the mammal is a human, primate, or rodent. In yet another embodiment, the subject is an animal model of a disorder characterized by PRAME 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.

[0029] In yet another aspect, there is provided a peptide-binding molecule or antigen-binding fragment thereof identified according to the methods described herein, optionally wherein the peptide-binding molecule or antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0030] In yet another aspect, provided is a method of treating a disorder characterized by PRAME expression in a subject, comprising administering to the subject a therapeutically effective amount of engineered T cells that express a peptide-binding molecule or antigen-binding fragment thereof that i) binds to a peptide epitope selected from the sequences listed in Table 1, ii) is identified according to the methods described herein, and / or iii) binds to a stable MHC-peptide complex comprising a peptide epitope selected from the sequences listed in Table 1 in the context of an MHC molecule; optionally, the peptide-binding molecule or antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain; and optionally, the MHC or MHC-peptide complex is as described herein.

[0031] Numerous embodiments are further provided that may be applied to any aspect encompassed by the invention and / or combined with any other embodiment described herein. For example, in one embodiment, T cells are isolated from a) a subject, b) a donor not afflicted with a disorder characterized by PRAME expression, or c) a donor who has recovered from a disorder characterized by PRAME expression.

[0032] In another aspect, provided is a method of treating a disorder characterized by PRAME expression in a subject, comprising infusing antigen-specific T cells into the subject, wherein the antigen-specific T cells are generated by a) stimulating immune cells from the subject with a composition described herein, and b) expanding the antigen-specific T cells in vitro or ex vivo, optionally wherein i) the immune cells are isolated from the subject before stimulating the immune cells, and / or ii) the immune cells comprise PBMCs, T cells, CD8+ T cells, naive T cells, central memory T cells, and / or effector memory T cells.

[0033] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the agent is contacted under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, immunogenic peptide, stable MHC-peptide complex, T cell receptor, and / or immune cell. In another embodiment, the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor are expressed by cells, and the cells are expanded and / or isolated during one or more steps. In yet another embodiment, the disorder characterized by PRAME expression is cancer or its recurrence, optionally, the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, leukemia (e.g., leukemia subtypes), ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma. In yet another embodiment, the subject is an animal model of a disorder characterized by PRAME expression, and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent.

[0034] In yet another aspect, there is provided a binding protein that binds to a polypeptide comprising an immunogenic peptide sequence described herein, an immunogenic peptide described herein, and / or a stable MHC-peptide complex described herein, optionally wherein the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0035] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, a binding protein comprises a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2, and / or b) a TCR beta chain CDR sequence having at least about 80% identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally, the binding affinity is at least about 5x10 -4 K below M d In another embodiment, the binding protein has a) a TCR alpha chain variable (V) α ) TCR V domain sequences selected from the group consisting of α TCR V with at least about 80% identity to the domain sequence α a) a TCR beta chain variable (V) domain sequence listed in Table 2; β ) TCR V domain sequences selected from the group consisting of β TCR V with at least about 80% identity to the domain sequence β domain sequence, and the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5x10 -4 K below M d In yet another embodiment, the binding protein comprises a) a TCR alpha chain sequence having at least about 80% identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2, and / or b) a TCR beta chain sequence having at least about 80% identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally, the binding affinity is about 5x10 -4 K below Md In yet another embodiment, the binding protein comprises a) a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2, and / or b) a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally, the binding affinity is about 5x10 -4 K below M d In yet another embodiment, the binding protein comprises: a) a TCR alpha chain variable (V) as listed in Table 2; α ) TCR V domain sequences selected from the group consisting of α a) a TCR beta chain variable (V) domain sequence listed in Table 2; β ) TCR V domain sequences selected from the group consisting of β domain sequence, and the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5x10 -4 K below M d In another embodiment, a binding protein is provided comprising a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2, and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity of about 5x10 -4 K below M d In another embodiment, a binding protein is provided having: 1) a TCR alpha chain CDR, a TCR V α and / or 2) the TCR beta chain CDRs, TCR V domains, and / or TCR alpha chain CDRs are encoded by TRAV, TRAJ, and / or TRAC genes or fragments thereof selected from the group consisting of TRAV, TRAJ, and TRAC genes listed in Table 2. β2) the domain, and / or the TCR beta chain are encoded by a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group consisting of the TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof compared to the cognate reference CDR sequence listed in Table 2. In yet another embodiment, the binding protein is chimeric, humanized, or human. In yet another embodiment, the binding protein comprises a binding domain having a transmembrane domain and an intracellular effector domain. In another embodiment, the TCR alpha chain and the TCR beta chain are covalently linked, optionally, the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide. In yet another embodiment, the TCR alpha chain and / or the TCR beta chain are covalently linked to a moiety, optionally, the covalently linked moiety comprises an affinity tag or label. In yet another embodiment, the affinity tag is selected from the group consisting of a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, Halo tag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or the label is a fluorescent protein. In another embodiment, the covalently linked moiety is selected from the group consisting of an inflammatory substance, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof. In yet another embodiment, the binding protein binds to a pMHC complex on the cell surface. In yet another embodiment, the MHC or MHC-peptide complex is described herein. In another embodiment, binding of the binding protein to the PRAME peptide-MHC (pMHC) complex elicits an immune response, optionally the immune response is selected from the group consisting of i) a T cell response and / or a CD8+ T cell response, and / or ii) T cell expansion, cytokine release, and / or cytotoxic killing.In yet another embodiment, the binding protein binds to a PRAME immunogenic peptide-MHC (pMHC) complex at a concentration of about 1x10 -4 M or less, about 5x10 -5 M or less, about 1x10 -5 M or less, about 5x10 -6 M or less, about 1x10 -6 M or less, about 5x10 -7 M or less, about 1x10 -7 M or less, about 5x10 -8 M or less, about 1x10 -8 M or less, about 5x10 -9 M or less, about 1x10 -9 M or less, about 5x10 -10 M or less, about 1x10 -10 M or less, about 5x10 -11 M or less, about 1x10 -11 M or less, about 5x10 -12 M or less, or about 1x10 -12 K below M dIn yet another embodiment, the binding protein has a higher binding affinity for peptide-MHC (pMHC) compared to known T cell receptors, optionally the higher binding affinity is at least 1.05-fold higher. In another embodiment, the binding protein, when contacted with target cells having heterozygous expression of PRAME, induces higher T cell expansion, cytokine release, and / or cytotoxic killing compared to known T cell receptors, optionally the induction is at least 1.05-fold higher. As used herein, in some embodiments, reference to fold change can be compared to any reference mode of interest, such as a comparison with a different binding protein, expression of the same binding protein at different levels in different immune cells, or the same binding protein in a different context, such as in combination with other agents described herein. In yet another embodiment, the cytotoxic killing is of the target cancer cells. In yet another embodiment, the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, leukemia (e.g., leukemia subtypes), ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma. In another embodiment, the binding protein does not bind to peptide-MHC (pMHC) complexes containing PLA2G4E, EFNA1, and / or SLC26A1 peptide epitopes. These genes are well known and art-recognized to be annotated according to the following NCBI gene ID numbers, each available on the World Wide Web at ncbi.nlm.nih.gov / gene: PLA2G4E: Gene ID 123745, EFNA1: Gene ID 1942, and SLC26A1: Gene ID 10861.

[0036] In yet another aspect, there is provided a TCR alpha and / or beta chain selected from the group consisting of the TCR alpha and beta chain sequences listed in Table 2.

[0037] In another aspect, an isolated nucleic acid molecule is provided that i) hybridizes under stringent conditions to the complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, ii) a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, and / or iii) a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide listed in Table 2, optionally comprising 1) a TRAV, TRAJ, and / or TRAC gene or fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) a TRBV, TRBJ, and / or TRBC gene or fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2.

[0038] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the nucleic acid is codon-optimized for expression in a host cell.

[0039] In yet another aspect, provided is a vector comprising an isolated nucleic acid described herein, optionally wherein i) the vector is a cloning vector, expression vector, or viral vector, and / or ii) the vector comprises a vector sequence listed in Table 3.

[0040] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the vector further comprises a nucleic acid sequence encoding CD8α CD8β, a 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 operably linked to a nucleic acid sequence encoding a tag. In yet another embodiment, the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selective protein such that the tag is fused to the N-terminus of the 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 acids described herein, alone or in combination with nucleic acid sequences encoding CD8α, CD8β, DN-TGFβRII, and / or selective protein markers, are interconnected with an internal ribosome entry site or with a nucleic acid sequence encoding a self-cleaving peptide. In yet another embodiment, the self-cleaving peptide is P2A, E2A, F2A, or T2A.

[0041] In yet another aspect, there is provided a host cell comprising the isolated nucleic acid described herein, wherein the host cell comprises a vector described herein and / or expresses a binding protein described herein, optionally wherein the cell is genetically engineered.

[0042] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the host cell comprises a chromosomal gene knockout of a TCR gene, an HLA gene, or both. In another embodiment, the host cell comprises a knockout of an HLA gene selected from the group consisting of the α1 macroglobulin gene, the α2 macroglobulin gene, the α3 macroglobulin gene, the β1 microglobulin gene, the β2 microglobulin gene, and combinations thereof. In yet another embodiment, the host cell comprises a knockout of a TCR gene selected from the group consisting of the TCR α variable region gene, the TCR β variable region gene, the 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 the CD8α, CD8β, DN-TGFβRII, and / or the selective protein marker is fused to a CD34 enrichment tag. In another embodiment, the host cells are enriched using a CD34 enrichment tag. In yet another embodiment, the host cells are hematopoietic progenitor cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells, or immune cells. In yet another embodiment, the immune cells are T cells, cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + T cells, CD8 +The host cells may be T cells, CD4 / CD8 double-negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or a combination thereof. In yet another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or a combination thereof. In another embodiment, the T cells are primary T cells or cells of a T cell line. In yet another embodiment, the T cells do not express an endogenous TCR or have reduced surface expression thereof. In yet another embodiment, the host cells are capable of producing cytokines or cytotoxic molecules when contacted with target cells containing a peptide-MHC (pMHC) complex containing the PRAME peptide epitope in the context of an MHC molecule. In another embodiment, the host cells are contacted with the target cells in vitro, ex vivo, or in vivo. In yet another embodiment, the cytokine is TNF-α, IL-2, and / or IFN-γ. In yet another embodiment, the cytotoxic molecule is perforin and / or a granzyme, and optionally, the cytotoxic molecule is granzyme B. In another embodiment, the host cell is capable of producing higher levels of cytokines or cytotoxic molecules when contacted with target cells having heterozygous expression of PRAME. In yet another embodiment, the host cell is capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules. In yet another embodiment, the host cell is capable of killing target cells containing a peptide-MHC (pMHC) complex that includes a PRAME peptide epitope in the context of an MHC molecule. In another embodiment, killing is determined by a killing assay. In yet another embodiment, the ratio of host cells to target cells in the killing assay is 20:1 to 1:4. In yet another embodiment, the target cells are target cells pulsed with 1 μg / mL to 50 pg / mL of PRAME peptide, and optionally, the target cells are monoallelic with respect to the MHC matched to the PRAME peptide. In another embodiment, the host cell, when contacted with target cells having heterozygous expression of PRAME, is capable of killing a greater number of target cells, optionally, cell killing is at least 1.05-fold greater.In yet another embodiment, the target cells are cell lines (e.g., Hs695T, A375, or NCI-H1563) or primary cells; optionally, the target cells are selected from the group consisting of HEK293-derived cell lines, cancer cell lines, primary cancer cells, transformed cell lines, and immortalized cell lines. In yet another embodiment, the PRAME immunogenic peptide is as described herein, and / or the MHC or MHC-peptide complex is as described herein. In another embodiment, the host cell does not induce T cell expansion, cytokine release, or cytotoxic killing when contacted with a target cell comprising a peptide-MHC (pMHC) complex comprising a PLA2G4E, EFNA1, and / or SLC26A1 peptide epitope. In yet another embodiment, the host cell does not express the PRAME antigen, is not recognized by a binding protein described herein, is not of the serotype HLA-A*02, and / or does not express an HLA-A*02 allele.

[0043] In another aspect, there is provided a population of host cells described herein.

[0044] In yet another aspect, a composition is provided that includes a) a binding protein described herein, b) an isolated nucleic acid described herein, c) a vector described herein, d) a host cell described herein, and / or e) a population of host cells described herein, and a carrier.

[0045] In yet another aspect, there is provided a device or kit comprising a) a binding protein as described herein, b) an isolated nucleic acid as described herein, c) a vector as described herein, d) a host cell as described herein, and / or e) a population of host cells as described herein, the device or kit optionally comprising reagents for detecting binding of a), d) and / or e) to a pMHC complex.

[0046] In another aspect, there is provided a method of producing a binding protein described herein, comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of the binding protein; and (ii) recovering the expressed binding protein.

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

[0048] In yet another aspect, a method is provided for detecting the presence or absence of a PRAME antigen and / or a cell expressing PRAME, optionally wherein the cell is a hyperproliferative cell, and comprising detecting the presence or absence of the PRAME antigen in a sample by using at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, wherein detection of the PRAME antigen indicates the presence of the PRAME antigen and / or a cell expressing PRAME.

[0049] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, at least one binding protein or at least one host cell forms a complex with a PRAME peptide in the context of an MHC molecule, and the complex is detected by fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, Western blot, or intracellular flow assay. In another embodiment, the method further comprises obtaining a sample from a subject.

[0050] In another aspect, a method is provided for detecting the level of a disorder characterized by PRAME expression in a subject, the method comprising: a) contacting a sample obtained from the subject with at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) detecting the level of reactivity, wherein the presence or higher level of reactivity compared to a control level indicates the level of a disorder characterized by PRAME expression in the subject.

[0051] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the control level is a reference number. In another embodiment, the control level is a level from a subject who does not have a disorder characterized by PRAME expression.

[0052] In yet another aspect, a method is provided for monitoring the progression of a disorder characterized by PRAME expression in a subject, comprising: a) detecting in a subject sample the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; b) repeating step a) at a subsequent time point; and c) comparing the levels of PRAME or target cells expressing PRAME detected in steps a) and b) to monitor the progression of the disorder characterized by PRAME expression in the subject, wherein the absence or decrease of the PRAME level or target cells expressing PRAME detected in step b) compared to step a) indicates a suppression of the progression of the disorder characterized by PRAME expression in the subject, and the presence or increase of the PRAME level or target cells expressing PRAME detected in step b) compared to step a) indicates the progression of the disorder characterized by PRAME expression in the subject.

[0053] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the subject has undergone therapy to treat a disorder characterized by PRAME expression between the first time point and the subsequent time point.

[0054] In yet another aspect, a method is provided for predicting clinical outcome in a subject suffering from a disorder characterized by PRAME expression, comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) comparing the presence or level of reactivity with that from a control, wherein the control is obtained from a subject with a good clinical outcome, and wherein an absence or reduced level of reactivity in the subject sample compared to the control indicates that the subject will have a good clinical outcome.

[0055] In another aspect, a method is provided for evaluating the effectiveness of a therapy for a disorder characterized by PRAME expression, comprising: a) determining the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein in a first sample obtained from the subject before providing at least a portion of the therapy for the disorder characterized by PRAME expression to the subject; and b) determining the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein in a second sample obtained from the subject after providing the therapy for the disorder characterized by PRAME expression, wherein an absence or reduced level of reactivity in the second sample compared to the first sample is an indication that the therapy is effective in treating the disorder characterized by PRAME expression in the subject, and a presence or increased level of reactivity in the second sample compared to the first sample is an indication that the therapy is not effective in treating the disorder characterized by PRAME expression in the subject.

[0056] Numerous embodiments are further provided that can be applied to any aspect encompassed by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the level of reactivity is indicated by a) the presence of binding, and / or b) T cell activation and / or effector function, optionally T cell activation or effector function being T cell proliferation, killing, or cytokine release. In another embodiment, T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemically, Western blot, or intracellular flow assay.

[0057] In yet another aspect, a method is provided for preventing and / or treating a disorder characterized by PRAME expression, comprising contacting target cells expressing PRAME with a therapeutically effective amount of a composition comprising cells expressing at least one binding protein described herein, and optionally, the composition is administered to the subject.

[0058] Numerous embodiments are further provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the cells are allogeneic, syngeneic, or autologous cells. In another embodiment, the cells are host cells described herein, or a population of host cells described herein. In yet another embodiment, the target cells are cancer cells that express PRAME. 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 that express PRAME. In yet another embodiment, the cell composition induces an antigen-specific T cell immune response in a subject against target cells that express PRAME. In yet another embodiment, the antigen-specific T cell immune response is a CD4 + and at least one of a helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the method further comprises administering at least one additional treatment for a disorder characterized by PRAME expression, optionally wherein the at least one additional treatment for a disorder characterized by PRAME expression is administered simultaneously or sequentially with the composition. In yet another embodiment, the disorder characterized by PRAME 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, leukemia (e.g., leukemia subtypes), ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma. In yet another embodiment, the subject is an animal model of a disorder characterized by PRAME expression and / or a mammal, optionally wherein the mammal is a human, primate, or rodent. [Brief explanation of the drawings]

[0059] [Figure 1] The PRAME425-433 peptide sequence is shown. [Figure 2A] Figure 1 shows that 392 PRAME425-433-specific TCRs were discovered using the ReceptorScan platform. Figure 2 shows the expansion of target-specific CD8+ T cells. Briefly, CD14+ monocytes were isolated from PBMCs of HLA-A*02:01 healthy donors on day -4 and differentiated into mature DCs. Naive CD8+ T cells were isolated from autologous PBMCs on day -1 and rested overnight. Coculture of naive CD8+ T cells and DCs was performed after a 3-hour pulse of DCs with 1 μg / mL PRAME425-433 (SLLQHLIGL) as part of multiplexed ReceptorScan screening, followed by a 10-day cell expansion phase. [Figure 2B] Figure 1 shows that 392 PRAME425-433-specific TCRs were discovered using the ReceptorScan platform. Figure 2 shows CD8+ cell isolation and single-cell sequencing. Dextramar staining was performed with HLA-A*02:01-specific PRAME425-433 (SLLQHLIGL) dextramers to identify clones. DNA-barcoded dextramers were used to isolate PRAME425-433-specific cells. Sequencing of isolated T cells and pairing of TCR alpha and beta chains were performed using the 10X Genomics platform. [Figure 3-1]This shows that screening of PRAME425-433 TCRs identified seven TCRs with preferred cytotoxic activity relative to the control TCRs. Pan-T cells were transduced to individually express 392 PRAME425-433-specific TCRs, and the engineered T cells were then co-cultured with NucLight™ Red-labeled T2 target cells pulsed with 1 ng / mL of PRAME425-433 peptide. Target cell survival was quantified by time-dependent imaging as a readout of T cell cytotoxicity. Non-transduced cells (NTD) served as a control. Seven (7) of the 392 TCRs were selected for further evaluation of surface expression and cytotoxic potential against PRAME-expressing cell lines. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 4A-1] This figure shows that TCRs 366 and 358 exhibited favorable cytotoxicity against endogenously expressing cell lines relative to the comparison TCR. Pan T cells from an HLA-A*02:01-positive healthy donor were transduced to express the seven PRAME425-433 TCRs selected from the initial cytotoxicity screen using pulsed T2 cells as targets. Comparison TCRs were also expressed. Three (3) TCRs were shown to bind PRAME425-433 (SLLQHLIGL) dextramer and were further evaluated in in vitro cytotoxicity assays compared to the comparison TCR (comparative AE: comparative affinity-enhanced). Surface expression of the seven TCRs and comparison TCRs, assessed by A*02:01-restricted PRAME425-433 (SLLQHLIGL) dextramer staining, gated on live cells, is shown. [Figure 4A-2] Same as above. [Figure 4B-1]This figure shows that TCRs 366 and 358 exhibited favorable cytotoxicity against endogenously expressing cell lines relative to the comparison TCRs. Pan T cells from an HLA-A*02:01-positive healthy donor were transduced to express seven PRAME425-433 TCRs selected from an initial cytotoxicity screen using pulsed T2 cells as targets. Comparison TCRs were also expressed. Three (3) TCRs were shown to bind PRAME425-433 (SLLQHLIGL) dextramers and were further evaluated in in vitro cytotoxicity assays compared to the comparison TCRs (comparative AE: comparative affinity-enhanced). The cytotoxic responses of these TCRs against the target cell lines Hs695T, A375, and pulsed T2 cells are shown. Engineered T cells were co-cultured with NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® instrument as a readout of T cell cytotoxicity. TC366 and 358 showed favorable activity against comparable TCRs, particularly in control A375 cell proliferation, which has lower PRAME expression. [Figure 4B-2] Same as above. [Figure 4B-3] Same as above. [Figure 5A] Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a comparison TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as a PRAME-negative control strain. Expression of the PRAME425-433-specific TCR was assessed by A*02:01-restricted PRAME425-433 (SLLQHLIGL) dextramer staining, gated on live cells (comparative AE: comparative affinity-enhanced). Figure 5A shows the expression of PRAME425-433 TCRs 366 and 358 on the surface of engineered T cells in three healthy donors. [Figure 5B]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5B shows T cell cytotoxicity of Hs695T (HLA-A*02:01+PRAME+) targets at an E:T of 5:1. [Figure 5C]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5C shows T cell cytokine production in response to Hs695T (HLA-A*02:01+PRAME+) targets. [Figure 5D]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5D shows T cell cytotoxicity of A375 (HLA-A*02:01+PRAME+) targets at an E:T of 5:1. [Figure 5E]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5E shows T cell cytokine production in response to A375 (HLA-A*02:01+PRAME+) targets. [Figure 5F]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5F shows T cell cytotoxicity of NCI-H1563 (HLA-A*02:01+PRAME+) targets at an E:T ratio of 5:1. [Figure 5G]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5G shows T cell cytokine production in response to NCI-H1563 (HLA-A*02:01+PRAME+) targets. [Figure 5H]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5H shows T cell cytotoxicity of 647V (HLA-A*02:01+PRAME-) targets at an E:T of 5:1. [Figure 5I]Figure 5 shows the results of functional evaluation of PRAME425-433 TCR. Pan T cells isolated from three HLA-A*02:01-positive healthy donor PBMCs were transduced to express PRAME425-433-specific TCRs 366 and 358, as well as a control TCR. The T cells were evaluated for functional responses against target cells expressing HLA-A*02:01 and various levels of PRAME, as well as PRAME-negative control lines. The results of the PRAME425-433-specific TCR functional responses against the HLA-A*02:01+PRAME+ target cell lines Hs695T (Figures 5B and 5C), A375 (Figures 5D and 5E), NCI-H1563 (Figures 5F and 5G), and the HLA-A*02:01-PRAME-negative control cell line 647V (Figures 5H and 5I) are shown. Engineered T cells were cocultured with NucLight Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified on an IncuCyte® as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production were measured in coculture supernatants at 24 hours (E:T 1:1) (Comparative AE: Comparative Affinity-Enhanced). Figure 5I shows T cell cytokine production in response to 647V (HLA-A*02:01+PRAME-) targets. [Figure 6] The EC50 of TCR 366 is shown to be favorable relative to the control TCR. EC50 values ​​were measured after pulsing Nuclight red-labeled T2 cells with 10-fold serial dilutions of the PRAME425-433 peptide from 1 μM to 10 fM. Pulsed T2 cells were then co-cultured with T cells at a 5:1 ratio of T cell to target, and target cell survival was measured using an Incucyte® instrument as a readout of cytotoxicity. EC50 calculations were performed by fitting the area under the curve (AUC) data using Prism software. [Figure 7]This shows that TCR366 did not exhibit alloreactivity to 103 / 110 MHC molecules tested. TCR366-expressing pan T cells or untransduced control T cells were co-cultured for 48 hours with MHC-null HEK293T cells expressing one of the 110 most frequently occurring class I MHC molecules in the US population. A positive control consisting of HEK293T cells expressing both a fragment of PRAME containing the 425-433 epitope (SLLQHLIGL) and HLA-A*02:01 was included in the screening. After 48 hours of co-culture, inhibition of target cell growth by TCR366-expressing pan T cells compared with untransduced control T cells was measured as a readout of TCR366 reactivity to allogeneic MHC molecules. Positive control and alloreactive alleles (target cell inhibition >20%) were shown. [Figure 8A] Figure 1 shows a genome-wide screen that identified putative off-targets of TCR 366. Figure 2 shows an overview of a unique genome-wide screen. [Figure 8B] Figure 9 shows a genome-wide screen that identified putative off-targets of TCR 366. Screening data for TCR 366 identified seven potential off-targets in a screen of over 600,000 protein fragments spanning all wild-type (wt) human proteins. The screen was designed to over-predict off-targets by overexpressing a 90-aa protein fragment that was processed more efficiently than the full-length protein and was not physiologically recognized in healthy primary human cells (Figure 9 below). Putative off-targets are identified by gene name. [Figure 9A]This shows that TCR 366 showed no reactivity against healthy human primary cells. TCR 366-expressing pan T cells or NTD cells were tested for their reactivity against primary cells derived from healthy HLA-A*02:01+ human donors naturally expressing off-targets identified in genome-wide safety screening. Target cells were pulsed or not with the PRAME425-433 (SLLQHLIGL) peptide and co-cultured with TCR 366 or NTD cells. IFN-γ secretion in the culture supernatant was used as a readout of TCR 366 reactivity against target cells. HLA-A*02:01+PRAME+OVCAR-3 cells were used as a positive control, and HLA-A*02:01+PRAME-CaSki or Loucy cells were used as negative controls. [Figure 9B] This shows that TCR 366 showed no reactivity against healthy human primary cells. TCR 366-expressing pan T cells or NTD cells were tested for their reactivity against primary cells derived from healthy HLA-A*02:01+ human donors naturally expressing off-targets identified in genome-wide safety screening. Target cells were pulsed or not with the PRAME425-433 (SLLQHLIGL) peptide and co-cultured with TCR 366 or NTD cells. IFN-γ secretion in the culture supernatant was used as a readout of TCR 366 reactivity against target cells. HLA-A*02:01+PRAME+OVCAR-3 cells were used as a positive control, and HLA-A*02:01+PRAME-CaSki or Loucy cells were used as negative controls. [Figure 9C]This shows that TCR 366 showed no reactivity against healthy human primary cells. TCR 366-expressing pan T cells or NTD cells were tested for their reactivity against primary cells derived from healthy HLA-A*02:01+ human donors naturally expressing off-targets identified in genome-wide safety screening. Target cells were pulsed or not with the PRAME425-433 (SLLQHLIGL) peptide and co-cultured with TCR 366 or NTD cells. IFN-γ secretion in the culture supernatant was used as a readout of TCR 366 reactivity against target cells. HLA-A*02:01+PRAME+OVCAR-3 cells were used as a positive control, and HLA-A*02:01+PRAME-CaSki or Loucy cells were used as negative controls. [Figure 9D] This shows that TCR 366 showed no reactivity against healthy human primary cells. TCR 366-expressing pan T cells or NTD cells were tested for their reactivity against primary cells derived from healthy HLA-A*02:01+ human donors naturally expressing off-targets identified in genome-wide safety screening. Target cells were pulsed or not with the PRAME425-433 (SLLQHLIGL) peptide and co-cultured with TCR 366 or NTD cells. IFN-γ secretion in the culture supernatant was used as a readout of TCR 366 reactivity against target cells. HLA-A*02:01+PRAME+OVCAR-3 cells were used as a positive control, and HLA-A*02:01+PRAME-CaSki or Loucy cells were used as negative controls. [Figure 10-1] Provides summary data. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above. [Figure 10-7] Same as above. [Figure 11]This figure shows the pMHC dose-dependent function of process-representative TSC-203-A0201 TCR-T cells. T2 cells were pulsed with various concentrations of PRAME peptide and co-cultured with three batches of TSC-203-A0201 process-representative TCR-T cells. The figure shows the relative growth of T2 cells co-cultured with TSC-203-A0201 TCR-T cells at a 2:1 E:T ratio for 72 hours, normalized to t=0. For each donor, co-cultures were performed in triplicate (n=3). Error bars for each data point represent the standard error of the mean (SEM). The area under the curve (AUC) for the resulting growth of T2 cells over 72 hours as a function of peptide concentration was plotted to compare TSC-203-A0201 batches. [Figure 12-1](Figure 12A) TSC-203-A0201 TCR-T cells secrete granzyme B and the proinflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*A02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 hours of coculture, and levels of the inflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated 4-plex ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (thus, values ​​are less accurate), and # indicates samples whose values ​​were below the detection limit. (Figure 12B) This shows that TSC-203-A0201 TCR-T cells secrete granzyme B and the inflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 h of coculture, and levels of the proinflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated quadruple ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (and therefore values ​​are less accurate), and # indicates samples where values ​​were below the limit of detection.(Figure 12C) TSC-203-A0201 TCR-T cells secrete granzyme B and the proinflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*A02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 hours of coculture, and levels of the inflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated 4-plex ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (thus, values ​​are less accurate), and # indicates samples whose values ​​were below the detection limit. (Figure 12D) This shows that TSC-203-A0201 TCR-T cells secrete granzyme B and the inflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 h of coculture, and levels of the proinflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated quadruple ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (and therefore values ​​are less accurate), and # indicates samples where values ​​were below the limit of detection. [Figure 12-2](Figure 12E) TSC-203-A0201 TCR-T cells secrete granzyme B and the proinflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*A02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 hours of coculture, and levels of the inflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated 4-plex ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (thus, values ​​are less accurate), and # indicates samples where values ​​were below the detection limit. (Figure 12F) This shows that TSC-203-A0201 TCR-T cells secrete granzyme B and the inflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 h of coculture, and levels of the proinflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated quadruple ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (and therefore values ​​are less accurate), and # indicates samples where values ​​were below the limit of detection.(Figure 12G) TSC-203-A0201 TCR-T cells secrete granzyme B and the proinflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*A02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 hours of coculture, and levels of the inflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated 4-plex ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (thus, values ​​are less accurate), and # indicates samples where values ​​were below the detection limit. (Figure 12H) This shows that TSC-203-A0201 TCR-T cells secrete granzyme B and the inflammatory cytokines IFN-γ, IL-2, and TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells (Figures 12A-D) or donor-matched UTF control T cells (Figures 12E-H) from three donors (PD314, PD315, and PD317) were cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). Supernatants were collected after 24 h of coculture, and levels of the proinflammatory cytokines IFN-γ, IL-2, and TNF-α, as well as granzyme B, were assessed using an automated quadruple ELISA assay (ELLA, Proteinsimple). * indicates samples that were outside the dynamic range of the assay (and therefore values ​​are less accurate), and # indicates samples where values ​​were below the limit of detection. [Figure 13] TSC-203-A0201 TCR-T cells proliferate in a target-dependent manner. TSC-203-A0201 TCR-T cells (A) or donor-matched transduced control T cells (B) from three T cell batches (PD314, PD315, and PD317) were labeled with CTV dye and cultured in the absence of target cells or cocultured 1:1 with either the HLA-A*A02:01-positive, PRAME-negative target cell line 647v (gray bars) or three different HLA-A*02:01-positive, PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; and SKMEL5, dark blue bars). After 3.5 days of coculture, cells were stained and flow cytometric quantification of T cell proliferation was performed. The graph shows the number of dividing cells (identified as the CTV dim population) normalized to the number of beads counted. The number of dividing cells is shown for the following T cell subsets: total T cells (left panel), helper T cells (middle panel), and cytotoxic T cells (right panel). [Figure 14A]TSC-203-A0201 TCR-T cells exhibit potent and selective cytotoxicity. Three batches of representative TSC-203-A0201 TCR-T cells (blue growth curve) and untransfected (UTF) control T cells (gray growth curve) derived from matched donors were analyzed in an Incucyte®-based cytotoxicity assay for their cytotoxic potential against the indicated target cell lines (A375, Hs695T, and SKMEL5) against an HLA-A*02:01-positive, PRAME-negative control cell line (647v), or three different HLA-A*02:01-positive, PRAME-positive T cells. Effector TCR-T cells and target cells were cocultured over various effector to target ratios (E:T ranging from 5:1 to 0.6:1), and target cell growth was measured over 72 hours. The data presented were obtained using TSC-203-A0201 TCR-T cells and UTF control T cells from batch PD315 and represent data obtained using all three batches of material representative of the process tested. Target cells cultured alone are shown as a negative control (red growth curve). [Figure 14B] We demonstrate that TSC-203-A0201 TCR-T cells exhibit potent and selective cytotoxicity. Cytotoxic activity of three representative batches of TSC-203-A0201 TCR-T cells over a 72-hour period is shown, summarized as the area under the curve (AUC) of the growth curve of target cells co-cultured with TSC-203-A0201 at an E:T ratio of 2.5:1, normalized to the growth curve of target cells co-cultured with the corresponding UTF control cells. [Figure 15]These results demonstrate that TSC-203-A0201 TCR-T cells are resistant to TGFβ-mediated inhibition of cytokine secretion and proliferation. Three batches of process-representative TCR-T cells (PD314, PD315, and PD317) were cocultured with target cells in the presence of 0 or 5 ng / mL of TGFβ1. As controls for adequate TGFβ-mediated T cell inhibition, two batches of process-like TSC-203-A0201 cells lacking DN-TGFβRII (RG2959 164 and 6466 164) and, in Figure 15B, donor-matched process-like TSC-203-A0201 TCR-T cells expressing DN-TGFβRII (RG2959 134 and 6466 134) were included in the assay. (A) TCR-T cells were preincubated with 0 or 5 ng / mL TGFβ1 for 24 hours and then cocultured with peptide-pulsed T2 cells (10 ng / mL PRAME peptide SLLQHLIGL) at a 1:1 E:T ratio for 24 hours. IFN-γ secretion by TCR-T cells was assessed after 24 hours of coculture using an automated ELISA platform (ELLA, ProteinSimple). (B) Flow cytometry assessment of TCR-T cell proliferation after 3.5 days of coculture with the HLA-A*02:01-positive and PRAME-positive cancer cell line SKMEL5 (E:T 1:1). The heatmap shows the percentage of proliferating transduced TCR-T cells observed in cocultures containing 5 ng / mL TGFβ, normalized to the percentage of proliferating TCR-T cells observed in the 0 ng / mL TGFβ condition. Proliferation data are shown for total transduced T cells (TCRαβ+CD34+), transduced helper T cells (TCRαβ+CD34+CD4+CD8+), and transduced cytotoxic T cells (TCRαβ+CD34+CD4-CD8+). Asterisks indicate process-like control TCR- T cells lacking DN-TGFβRII. [Figure 16] The inoculation, administration, and analysis schedule for animals in groups 1 to 7 is shown. [Figure 17]Figures A-D show the in vivo efficacy of TSC-203-A0201. NCG mice were subcutaneously (sc) inoculated with Hs 695T. Upon successful tumor engraftment (tumors reaching an average of 100 mm3 6 days post-inoculation), animals were randomized into different treatment groups. The mean tumor volume for each treatment group in mice (n=12) over time is shown in Figure 17A. The tumor volume over time for individual mice is shown for each individual batch tested (Figures 17B-D). Animals received two iv injections (arrowheads) of process-representative TSC-203-A0201 TCR-T cells, or untransfected (UTF) control T cells from matched donors, or vehicle (PBS) on days 1 and 8 of the study. [Figure 18] Percentage body weight change over time across different groups is shown. NCG mice were inoculated SC with Hs695T. Upon successful tumor engraftment (tumors reaching an average of 100 mm3 6 days post-inoculation), animals were randomized into different treatment groups. Animals received two iv doses of process-representative TSC-203-A0201 TCR-T cells (three batches tested: PD314, 315, and PD317), or control T cells from matched donors, or vehicle (PBS) on day 1. Eight test mean percentages (arrowheads) of body weight per treatment group (n=12) are shown. [Figure 19] FIG. 1 shows a schematic diagram illustrating the principle of target scanning screening. [Figure 20] Figure 1 shows a graphical representation of the results of a target scan screen of mechanistically representative TSC-203-A0201 TCR-T cells. Plotted are the enrichment scores for each of the approximately 600,000 tiles / peptides in the screen, calculated from eight technical replicates, measured against the input. Proteins with overlapping tiles enriched above background are highlighted in matching colors and shown on the graph. [Figure 21] FIG. 1 shows a flowchart illustrating the steps and timeline of a cytokine assay to test the extratumoral reactivity of TSC-203-A0201 TCR-T cells. [Figure 22]Expression of putative off-targets of the therapeutic TCR used in TSC-203-A0201 TCR-T cells in cancer cell lines is shown. RNA was extracted from the cancer cell lines and sequenced. The heatmap shows the TPM (transcripts per million) calculated from the counts. The color scale used in the RNAseq heatmap sets a TPM value of zero to white, and values ​​above zero follow a continuous color scale up to 100 TPM. [Figure 23] Co-culture of TSC-203-A0201 TCR-T cells and UTF T cells with HLA-A*02:01+ cancer cell lines expressing off-targets of the TCR. TSC-203-A0201 TCR-T cells and donor-matched UTF cells were co-cultured with a panel of cancer cell lines, and supernatants were assessed for IFN-γ levels as a measure of T cell reactivity. [Figure 24] Expression of putative off-targets of the therapeutic TCR used in TSC-203-A0201 TCR-T cells in primary and iPSC-derived cells is shown. RNA was extracted from primary and iPSC-derived cells and sequenced. The heatmap shows the TPM (transcripts per million) calculated from the counts. The color scale used in the RNAseq heatmap sets a TPM value of zero to white, and values ​​above zero follow a continuous color scale up to 100 TPM. [Figure 25] TSC-203-A0201 TCR-T cells show no reactivity to HLA-A*02:01+ primary cells. TSC-203-A0201 TCR-T cells and donor-matched UTF cells were co-cultured with a panel of primary cells, and supernatants were assessed for IFN-γ levels as a measure of T cell reactivity. [Figure 26] The steps and timeline of the carcinogenicity assay to evaluate the cytokine dependency of proliferating T cells are shown. T cells are thawed and rested. Cells are labeled with CTV. The different media cultures are listed in Table 14. [Figure 27]Figure 1 shows T cell viability. Data show normalized (using CountBright beads) numbers of viable (eFlour 660-negative) UTF and TSC-203-A0201 TCR-T cells from batches PD314, PD315, and PD317 after 5 days of in vitro culture in the absence (-) or presence (+) of cytokines and ImmunoCult™. Assays were performed in triplicate, and bars indicate the mean and standard error of the mean (SEM). The dotted line represents the initial number of cells (100,000) used in the assay. ****p≦0.0001; ***p≦0.001; **p≦0.01; *p≦0.05; "ns" means not significant, p>0.05. [Figure 28] Figure 1 shows T cell proliferation. Data show normalized (using CountBright beads) numbers of proliferating UTF and TSC-203-A0201 TCR-T cells from batches PD314, PD315, and PD317 after 5 days of in vitro culture in the absence (-) or presence (+) of cytokines or ImmunoCult™. Assays were performed in triplicate, and bars represent the mean and standard error of the mean (SEM). ****p≦0.0001; ***p≦0.001; **p≦0.01; *p≦0.05; "ns" means not significant, p>0.05. [Figure 29] The percentage of proliferating cells is shown. Data show the percentage of proliferating UTF and TSC-203-A0201 TCR-T cells gated on live cells from batches PD314, PD315, and PD317 after 5 days of culture in the absence (-) or presence (+) of cytokines or ImmunoCult™. Assays were performed in triplicate, and bars represent the mean and standard error of the mean (SEM). ****p≦0.0001; ***p≦0.001; **p≦0.01; *p≦0.05; "ns" means not significant, p>0.05. [Figure 30] 1 shows PRAME expression in 48 normal human organs. [Figure 31]A map of the pNVVD134_TSC-203-A02_TCR-366_MSCV-TCR-366-CD8-EF1α-dnTGFbRII-DHFR vector is shown. Legend: CD: Classification of Endogenous Cells; RNA-OUT: antisense RNA against bacterial levansucrase encoded by sacB; SV: simian virus; TCR: T cell receptor; ITR: inverted terminal repeat; QBend: mouse anti-human CD34 antibody; dnTGFbRII: dominant-negative TGF beta receptor II; DHFR: dihydrofolate reductase selection marker. [Figure 32] Figure 1 shows alloreactivity profiling of mechanistically representative TSC-203-A0201 TCR-T cells. Mechanistically representative TSC-203-A0201 TCR-T cells were cocultured with MHC-null HEK293T cells expressing one of the 110 most frequent class I HLAs in the US population for the indicated times. A positive control consisting of HEK293T cells expressing both an HLA-A*02:01-restricted epitope and a fragment of PRAME containing HLA-A*02:01 (red), and a negative control consisting of MHC- / - HEK293T cells (blue) were included in the screen. Inhibition of target cell growth by TCR-T cells compared with that by UTD control T cells was measured over 48 hours of coculture as a readout of the reactivity of mechanistically representative versions of the therapeutic TCR to allogeneic HLA proteins. [Figure 33]Coculture of TSC-203-A0201 TCR-T cells with cancer cell lines expressing putative allogeneic alleles is shown. Mechanistically representative TSC-203-A0201 TCR-T cells and non-transduced (NTD) control T cells were cocultured with cancer cell lines expressing the putative allogeneic alleles HLA-C*16:02, HLA-C*14:02, HLA-C*16:01, HLA-C*01:02, and HLA-C*08:01 for 24 hours, followed by measurement of IFN-γ production in the coculture supernatant. Each cell line was pretreated with 25 ng / mL IFN-γ, washed, and then cocultured with TSC-203-A0201 TCR-T cells or NTD control T cells to examine their reactivity when HLA was upregulated. PRAME-expressing HLA-A*02:01-positive Hs695 T cells were included as a positive control, and PRAME-negative HLA-A*02:01-positive 647V cells were included as a negative control. Experiments were performed with TSC-203-A0201 from two independent donors, and representative data are shown. [Figure 34] Coculture of TSC-203-A0201 TCR-T cells with HEK293T cells overexpressing C*14:03 is shown. Mechanistically representative TSC-203-A0201 TCR-T cells and non-transduced (NTD) control T cells were cocultured with HLA-C*14:03-overexpressing monoallelic HEK293T cells for 24 hours, followed by measurement of IFN-γ production in the coculture supernatant. PRAME ORF-expressing monoallelic A*02:01-overexpressing HEK293T cells were included as a positive control, and monoallelic A*02:01-overexpressing HEK293TT cells in which PRAME was knocked out using CRISPR / Cas9 targeting (PRAME KO HEK) were included as a negative control. Experiments were performed using TSC-203-A0201 from two independent donors, and representative data are shown.

[0060] Unless otherwise noted, for any figures showing bar histograms, curves, or other data associated with a legend, the bars, curves, or other data displayed from left to right in each display correspond directly to the boxes from top to bottom in the legend. DETAILED DESCRIPTION OF THE INVENTION

[0061] The present invention is based, at least in part, on the discovery of PRAME immunogenic peptides (e.g., those comprising or consisting of the sequences listed in Table 1), binding proteins that recognize the PRAME antigen (e.g., those having the sequences listed in Table 2), and uses thereof. A systematic comprehensive search was performed to map the precise T cell targets recognized by the initial pool of T cells of interest.

[0062] Thus, the present invention relates in part to identified epitopes (immunodominant peptides) of the PRAME protein of therapeutic importance, and related compositions (e.g., immunodominant peptides, vaccines, and the like), compositions comprising the immunogenic peptides alone or in combination with MHC molecules, stable MHC-peptide complexes, methods for diagnosing, prognosing, and monitoring immune responses to disorders characterized by PRAME expression, and methods for preventing and / or treating disorders characterized by PRAME expression. The present invention also relates in part to identified binding proteins (e.g., TCRs), host cells expressing the binding proteins (e.g., TCRs), compositions comprising the binding proteins (e.g., TCRs), and host cells expressing the binding proteins (e.g., TCRs), methods for diagnosing, prognosing, and monitoring T-cell responses to cells expressing PRAME, and methods for preventing and / or treating disorders characterized by PRAME expression.

[0063] I. Definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0064] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Additionally, references to tables provided herein include all sub-tables of the table unless otherwise indicated.

[0065] The term "administering" means providing a pharmaceutical agent or pharmaceutical composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration. This includes physically introducing a composition containing a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. In some embodiments, routes of administration for the binding proteins described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration," as used herein, refers to methods of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, intrasternal, and in vivo electroporation. Alternatively, the binding proteins described herein may be administered by parenteral routes, such as topical, epidermal, or mucosal routes, e.g., intranasally, orally, intravaginally, rectally, sublingually, or topically. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0066] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, e.g., a protein, peptide, or hapten. An antigen may be a PRAME antigen or a fragment thereof against which a protective or therapeutic immune response is desired. An "epitope" is the portion of an antigen that is bound by a natural or synthetic substance.

[0067] As used herein, the term "adjuvant" refers to a substance that, when administered before, together with, or after the administration of an antigen, accelerates, prolongs, and / or enhances the quality and / or strength of the immune response to the antigen compared to the administration of the antigen alone. Adjuvants can increase the magnitude and duration of the immune response induced by vaccination.

[0068] As used herein, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion"), or single chains thereof. In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or antigen-binding sites thereof. Each heavy chain comprises a heavy chain variable region (referred to herein as V H In certain naturally occurring antibodies, the heavy chain constant region is composed of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain comprises a light chain variable region (abbreviated herein as V L The light chain constant region is composed of one domain, CL. H and V L The region may be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) separated from each other by more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0069] 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).

[0070] As used herein, the term "antigen-binding portion" of a binding protein, e.g., a TCR, refers to one or more portions of a TCR that retain the ability to bind (e.g., specifically and / or selectively) to an antigen (e.g., a PRAME antigen) and cognate MHC / HLA. Such portions are, for example, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, preferably about 8 to about 300, e.g., 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 a TCR can be performed by fragments of a full-length TCR. Examples of binding portions encompassed by the term "antigen-binding portion" of a TCR include: (i) the V α and V β (ii) an Fv fragment consisting of an isolated complementarity determining region (CDR), or (iii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. α and V β are encoded by separate genes, but recombinant methods can be used to synthesize V α and V βThe regions may be linked by a synthetic linker that allows them to be made as a single protein chain, with pairing to form a monovalent molecule (known as a single-chain TCR (scTCR)). Such single-chain TCRs are also intended to be encompassed by the term "antigen-binding portion" of a TCR. These TCR fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same manner as the intact binding protein. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0071] "Comparative T cell receptor" refers to at least one benchmark T cell receptor (e.g., clone R11P3D3 or R11P3D3 KE) reported in the state of the art, such as U.S. Patent Publication No. 2018 / 0273602. In some embodiments, "comparative" refers to sequence R11P3D3 in U.S. Patent Publication No. 2018 / 0273602. In some embodiments, "comparative affinity enhancement" or "comparative AE" refers to R11P3D3_KE in U.S. Patent Publication No. 2018 / 0273602. Engineered versions of these parent sequences are used in the working examples, and the sequences of such engineered versions are listed in Table 4. In some embodiments, the comparative T cell receptor has the sequence shown in Table 4.

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

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

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

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

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

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

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

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

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

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

[0082] The term "determining a suitable therapeutic regimen for a subject" is understood to mean determining a therapeutic regimen for a subject (e.g., a single therapy or a combination of various therapies used to prevent and / or treat cancer in a subject) that is initiated, modified, and / or terminated based on, or essentially based on, or at least in part based on, the results of an analysis according to the present invention. One example is initiating adjuvant therapy after surgery aimed at reducing the risk of recurrence, and another example is modifying the dose of a particular anti-cancer drug. In addition to the results of an analysis according to the present invention, the decision can be based on the personal characteristics of the subject being treated. In most cases, the actual decision on a suitable therapeutic regimen for a subject will be made by the attending physician or doctor.

[0083] The term "dominant negative TGFβ receptor" or "DN-TGFβR" refers to a variant or mutant of the transforming growth factor (TGF) beta receptor that confers resistance to TGFβ signaling.

[0084] There are five type II receptors (activating receptors) and seven type I receptors (receptors that propagate signaling). Activating TGFβ receptors are heterotetramers consisting of two TGFβ receptor I (TGFβRI) and two TGFβ receptor II (TGFβRII). In some embodiments, the DN-TGFβR is DN-TGFβRII (i.e., a variant or mutant of TGF beta receptor II). In some embodiments, the resistance is to the suppressive effects of TGFβ signaling on immune cells, e.g., T cells, where TGFβ can be produced by cancer cells or by other immune cells in the cellular environment, e.g., stromal cells, macrophages, myeloid cells, epithelial cells, natural killer cells, and the like. TGFβ signaling inhibitors are well known in the art and include, but are not limited to, antibodies that bind to mutant TGFβ, TGFβ, and / or TGFβ receptors (e.g., lerdelimumab, metlimumab, fresolimumab, and the like) that sequester the receptors and thereby inhibit signaling, soluble TGFβ binding proteins, such as portions of the TGFβ receptor that sequester TGFβ (e.g., TGFβRII-Fc fusion proteins), or other binding agents, such as betaglycan. Any and all known TGFβ signaling inhibitors can be used in place of or in addition to the DN-TGFβR (e.g., DN-TGFβRII) described herein. In some embodiments, the DN-TGFβR lacks an intracellular portion required for TGFβ-mediated signaling, such as the entire intracellular domain, the kinase signaling domain, etc.DN-TGFβR constructs are well known in the art (Brand et al. (1993) J. Biol. Chem. 268:11500-11503, Weiser et al. (1993) Mol. Cell Biol. 13:7239-7247, Bollard et al. (2002) Blood 99::3179-3187, PCT Publication No. WO2009 / 152610, PCT Publication No. WO2017 / 156484, Kloss et al. (See representative non-limiting embodiments in PCT Publication Nos. WO2019 / 089884, WO2020 / 042647, and WO2020 / 042648).

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

[0086] As used herein, "homology" refers to the similarity in nucleotide sequence between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. A nucleotide residue position in both regions is homologous at that position if the same nucleotide residue is occupied at that position. A first region is homologous to a second region if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the percentage of nucleotide residue positions in the two regions that are occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, provided that at least about 50%, and in other embodiments at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range therebetween, inclusive, e.g., at least about 80%-100%, of the nucleic acid residue positions of each portion are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions in each portion are occupied by the same nucleotide residue.

[0087] The term "hyperproliferative disorder characterized by expression of the PRAME antigen" can be any hyperproliferative disorder in which the PRAME antigen is present in an MHC (e.g., HLA) complex that is expressed by at least some hyperproliferative cells in a subject. Examples of hyperproliferative disorders characterized by the PRAME:HLA complex include solid malignancies, such as those described in detail below.

[0088] The term "immune response" includes T cell and / or B cell immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses that are indirectly mediated by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0089] An increased ability to stimulate an immune response or the immune system may result from increased agonist activity of T cell costimulatory receptors and / or increased antagonist activity of inhibitory receptors. An increased ability to stimulate an immune response or the immune system may result from increased EC in assays that measure immune responses, such as assays that measure cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly via detecting CD107a or granzymes), and changes in proliferation. 50 The ability to stimulate an immune response or activity of the immune system may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500%, or more.

[0090] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate a host's immune system to generate an immune response against cancer cells in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0091] The term "immune cell" refers to any cell of the immune system that arises from hematopoietic stem cells in the bone marrow, which give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells, e.g., monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes), and lymphoid progenitor cells (which give rise to lymphoid cells, e.g., T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + These include T cells, CD4CD8 double negative T cells, gd T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells are sometimes referred to as "antigen-presenting cells" or "APCs," which are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of the APC, complexed with peptide, interact with TCRs on the surface of the T cell.

[0092] "Isolated protein" refers to a protein that, when isolated from a cell or produced by recombinant DNA techniques, is substantially free of other proteins, cellular material, isolation and culture medium, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. An "isolated" or "purified" protein, or a biologically active portion thereof, is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the binding protein, antibody, polypeptide, peptide, or fusion protein is derived, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of a biomarker polypeptide or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of biomarker proteins or fragments thereof having less than about 30% (by dry weight) non-biomarker protein (also referred to herein as "contaminating proteins"), or in some embodiments about 25%, 20%, 15%, 10%, 5%, 1%, or less, or any ranges therebetween, inclusive, e.g., about less than 1% to 5% non-biomarker protein. When a binding protein, antibody, polypeptide, peptide, or fusion protein, or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it may be substantially free of culture medium, i.e., culture medium may comprise about 20%, 15%, 10%, 5%, 1%, or less, or any ranges therebetween, inclusive, e.g., about less than 1% to 5% of the volume of the protein preparation.

[0093] As used herein, the term "isotype" refers to the class of antibody (e.g., IgM, IgG1, IgG2C, etc.) encoded by heavy chain constant region genes.

[0094] As used herein, "K DThe term "" is intended to refer to the dissociation equilibrium constant of a particular binding protein-antigen interaction. The binding affinity of binding proteins encompassed by the present invention can be measured or determined by standard binding protein-target binding assays, such as competitive assays, saturation assays, or standard immunoassays, such as ELISA or RIA. A relatively lower Kd value indicates a relatively higher binding affinity (e.g., about 5x10 -4 Kd values ​​below M (500uM) are 1x10 -4 M (100uM), where a Kd of 100uM indicates a relatively higher binding affinity compared to a Kd of 500uM).

[0095] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent (e.g., a probe or small molecule for specifically detecting and / or affecting the expression of a marker encompassed by the invention). Kits may be promoted, distributed, or sold as a unit for performing methods encompassed by the invention. Kits may include one or more reagents necessary to express a composition useful in a method encompassed by the invention. In some embodiments, kits may further include a reference standard, e.g., a nucleic acid encoding a protein that does not affect or regulate a signaling pathway controlling cell growth, division, migration, survival, or apoptosis. One of skill in the art can envision many such control proteins, including, but not limited to, common molecular tags (e.g., gre35 TPMen fluorescent protein and beta-galactosidase), proteins that do not fall into any of the pathways encompassing cell growth, division, migration, survival, or apoptosis according to Gene Ontology standards, or ubiquitous housekeeping proteins. Reagents in a kit may be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, instructional materials describing the use of the compositions within the kit may be included.

[0096] As used herein, the term "linked" refers to the association of two or more molecules. The association can be covalent or non-covalent. The association can also be genetic (i.e., recombinantly fused). Such association can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0097] "Linker" can refer, in some embodiments, to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs, and can provide a spacer function that accommodates the interaction of two sub-binding domains so that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., an scTCR) or signaling activity (e.g., a TCR complex). In some embodiments, the linker consists of, for example, about 2 to about 35 amino acids, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids.

[0098] "Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a transmembrane a chain (with three a domains) and a non-covalently associated b2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and b, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides derived from the cytosol to the cell surface, where the peptide antigen-MHC (pMHC) complex is expressed by CD8 + MHC class II molecules deliver peptides from the vesicle system to the cell surface, where they are recognized by CD4 T cells. + Recognized by T cells. Human MHC is called human leukocyte antigen (HLA).

[0099] The term "PRAME" refers to the PRAME nuclear receptor transcription factor, an antigen preferentially expressed in human melanoma and recognized by cytolytic T lymphocytes. It is not expressed in normal tissues except in the testis. The encoded protein likely acts as a repressor of retinoic acid receptors, conferring a growth advantage to cancer cells through this function. Diseases associated with PRAME include, for example, melanoma, choroidal carcinoma, non-small cell lung cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, colon cancer, sarcoma, neuroblastoma, head and neck cancer, ovarian cancer, and several types of leukemia. Human PRAME has multiple transcript variants resulting from alternative splicing, which are known and can be obtained from the NCBI database. Representative human PRAME transcripts include, for example, transcript variant 1 (NM_006115.5) encoding isoform a (NP_006106.1), transcript variant 2 (NM_206953.3) encoding isoform a (NP_996836.1), transcript variant 3 (NM_206954.3) encoding isoform a (NP_996837.1), transcript variant 4 (NM_206955.3) encoding isoform a (NP_996838.1), transcript variant 5 (NM_206956.3) encoding isoform a (NP_996839.1), and transcript variant 6 (NM_206957.3) encoding isoform a (NP_001278644.1). These include variant 6 (NM_001291715.2), transcript variant 7 (NM_001291716.2) encoding isoform a (NP_001278645.1), transcript variant 8 (NM_001291717.2) encoding isoform b (NP_001278646.1), transcript variant 9 (NM_001291719.2) encoding isoform b (NP_001278648.1), transcript variant 10 (NM_001318126.2) encoding isoform b (NP_001305055.1), and transcript variant 11 (NM_001318127.2) encoding isoform b (NP_001305056.1). Representative sequences of PRAME sequences are also shown in Table 3 below.

[0100] As used herein, "PRAME" 425-433 antigen" or "PRAME 425-433 Peptide antigen" or "PRAME" 425-433 containing peptide antigen" or "PRAME 425-433 epitope" or "PRAME 425-433 Peptide epitope" or "PRAME" 425-433 The term "b peptide" refers to a naturally occurring or synthetically produced peptide portion of the PRAME oncoprotein that comprises, consists of, or consists essentially of the sequence SLLQHLIGL.

[0101] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk of or susceptible to developing the disease, disorder, or condition.

[0102] The term "prognosis" includes a prediction of the likely course and outcome of cancer, or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis for cancer for an individual. For example, the prognosis can be surgery, development of a clinical subtype of cancer, occurrence of one or more clinical factors, or recovery from the disease.

[0103] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology," which may be determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This described algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the polynucleotides described herein. BLAST protein searches are performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0104] The phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating substance, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another organ or part of the body.

[0105] The term "ratio" refers to a relationship between two numbers (e.g., a score, a sum, etc.). While ratios may be expressed in a particular order (e.g., a to b, or a:b), one skilled in the art will recognize that the underlying relationship between the numbers may be expressed in any order without losing the significance of the underlying relationship, although the appearance of trends and correlations based on the ratio may be reversed.

[0106] The term "recombinant host cell" (or simply "host cell") refers to a cell containing a nucleic acid that does not naturally occur in the cell, e.g., a cell into which a recombinant expression vector has been introduced. It should be understood that the cells of the invention are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the term cell of the invention.

[0107] The terms "cancer response," "response to immunotherapy," or "response to T-cell-mediated cytotoxicity modulator / immunotherapy combination therapy" refer to any response of a hyperproliferative disorder (e.g., cancer) to a cancer drug, e.g., a T-cell-mediated cytotoxicity modulator, and immunotherapy, preferably to a change in tumor weight and / or volume after initiating neoadjuvant or adjuvant therapy. The term "neoadjuvant therapy" refers to a treatment given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormonal therapy. The response of a hyperproliferative disorder may be evaluated, for example, for efficacy or in the neoadjuvant or adjuvant setting, where the size of the tumor after systemic intervention may be measured by CT, PET, mammogram, ultrasound, or palpation to compare the size and dimensions of the tumor after systemic intervention with the initial size and dimensions. Response may also be evaluated by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative manner, such as the percentage change in tumor volume, or in a qualitative manner, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Assessment of hyperproliferative disorder response can be performed early after the initiation of neoadjuvant or adjuvant therapy, for example, hours, days, weeks, or preferably months. A typical endpoint for response assessment is the end of neoadjuvant chemotherapy or surgical resection of residual tumor cells and / or tumor bed. This is typically 3 months after the initiation of neoadjuvant therapy. In some embodiments, 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 determining the sum of the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the number of patients with stable disease (SD) at least 6 months after the end of treatment. The formula can be simply stated as CBR = CR + PR + SD over a 6-month period.In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating response to cancer therapy relate to "survival," including all of the following: survival until death, also known as overall survival (where death can be either cause-unrelated or tumor-related); "recurrence-free survival" (where the term "recurrence" includes both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term "disease" includes cancer and related diseases). The length of survival can be calculated based on a defined starting point (e.g., time of diagnosis or initiation of treatment) and an end point (e.g., death, recurrence, or metastasis). In addition, criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a specific cancer treatment regimen can be administered to a population of subjects, and the outcome can be correlated with biomarker measurements determined before any cancer treatment is administered. The outcome measurement can be a pathological response to treatment given in a neoadjuvant setting. Alternatively, outcome indicators such as overall survival and disease-free survival can be monitored over a period of time for subjects after cancer treatment for which biomarker measurements are readily available. In certain embodiments, the administered dose is a standard dose known in the art for cancer therapeutic agents. The duration for which subjects are monitored can vary. For example, subjects can 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. Biomarker measurement thresholds that correlate with cancer therapy outcomes can be determined using methods well known in the art, such as those described in the Examples section.

[0108] As indicated, the term can also refer to improved prognosis, for example, as reflected by an increased time to recurrence, which is the time to censoring the first recurrence of a second primary cancer as the first event or death without evidence of recurrence or an increase in overall survival, which is the time from treatment to death from any cause.Responding or having a response means that there is a beneficial endpoint that is achieved when exposed to a stimulus.Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated when exposed to a stimulus.It will be understood that assessing the likelihood that a tumor or subject will respond favorably is equivalent to assessing the likelihood that a tumor or subject will not respond favorably (i.e., showing a lack of response or being non-responsive).

[0109] The term "resistance" refers to the acquired or natural resistance of a cancer sample or mammal to a cancer therapy (i.e., non-responsiveness to therapeutic treatment or a reduced or limited response to therapeutic treatment), for example, a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater decrease in response to therapeutic treatment, such as a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or greater decrease, or any range therebetween, inclusive of the endpoints. The reduced response can be measured by comparing with the same cancer sample or mammal before resistance is acquired, or by comparing with a different cancer sample or mammal known not to be resistant to therapeutic treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance may be mediated by P-glycoprotein or other mechanisms, or may occur when a mammal is infected with a multidrug-resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of a skilled clinician, and may be measured, for example, by cell proliferation and cell death assays, as described herein as "sensitizing." In some embodiments, the term "reversing resistance" refers to the use of a second agent in conjunction with a primary cancer therapy (e.g., chemotherapy or radiation therapy) to significantly reduce tumor volume compared to that of an untreated tumor at a statistically significant level (e.g., p<0.05) in situations where the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone fails to reduce tumor volume statistically significantly compared to that of an untreated tumor. This generally applies to tumor volume measurements performed when untreated tumors are growing exponentially.

[0110] The term "sample" as used to detect or determine the absence or presence, or level, of at least one biomarker typically refers to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., those described above under the definition of "body fluid"), or a tissue sample (e.g., biopsy material), such as skin, a colon sample, or surgically resected tissue. In some embodiments, methods encompassed by the present invention further comprise obtaining a sample from an individual prior to detecting or determining the absence or presence, or level, of at least one marker in the sample.

[0111] The term "sensitize" refers to altering cancer or tumor cells in a way that allows the associated cancer to be more effectively treated with a cancer therapy (e.g., anti-immune checkpoint therapy, chemotherapy, and / or radiation therapy). In some embodiments, normal cells are not affected to the extent that they are unduly harmed by the therapy. Increased or decreased susceptibility to therapeutic treatment is measured according to methods known in the art for the particular treatments and methods described below in this application, including, but not limited to, cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164) and cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., In: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Press). Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90. Sensitivity or resistance can also be measured in animals by measuring the reduction in tumor size over a period of time, e.g., 6 months for humans and 4-6 weeks for mice. A composition or method sensitizes a response to a therapeutic treatment if the increase in therapeutic sensitivity or decrease in resistance compared to the therapeutic sensitivity or resistance in the absence of such composition or method is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any ranges therebetween, inclusive of the endpoints.Determining sensitivity or resistance to therapeutic treatment is routine in the art and within the skill of an ordinary skilled clinician. It should be understood that any method described herein for enhancing the effectiveness of cancer therapy can be equally applied to methods for sensitizing hyperproliferative cells or other cancerous cells (e.g., resistant cells) to cancer therapy.

[0112] The term "small molecule" is a term of the art and includes molecules with a molecular weight of less than about 1000, or molecules with a molecular weight of less than about 500. In one embodiment, a small molecule does not contain exclusively peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63-68), and natural product extract libraries. In another embodiment, the compound is a small organic non-peptide compound. In a further embodiment, the small molecule is not a biosynthetic product.

[0113] The term "specific binding" refers to the binding of a binding protein to a predetermined antigen. Typically, a binding protein binds to a given antigen with a specific binding affinity of approximately 5x10 as determined by a binding assay, for example, surface plasmon resonance (SPR) technology on a BIAcore™ assay instrument, using the antigen of interest as the analyte and the binding protein as the ligand. -4 M or less, about 1x10 -4 M or less, about 5x10 -5 M or less, about 1x10 -5 M or less, about 5x10 -6 M or less, about 1x10 -6 M or less, about 5x10 -7 M or less, about 1x10 -7 M or less, about 5x10 -8 M or less, about 1x10 -8 M or less, about 5x10 -9 M or less, about 1x10 -9 M or less, about 5x10 -10 M or less, about 1x10-10 M or less, about 5x10 -11 M or less, about 1x10 -11 M or less, about 5x10 -12 M or less, about 1x10 -12 The affinity (K) is less than or equal to M, or even less, or any range therebetween, including the endpoints, e.g., about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, etc. D ), In some embodiments, the binding protein binds to a given antigen with an affinity that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 times greater than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). The phrases "binding protein that recognizes an antigen" and "binding protein specific for an antigen" are used interchangeably herein with the term "binding protein that specifically binds to an antigen." Selective binding is a relative term that refers to the binding protein's ability to distinguish binding of one antigen from binding of another antigen, e.g., distinguish binding of a particular family member or antigen target from binding of a related family member or antigen target. For example, the analytical data provided in the Examples section demonstrate that the binding proteins described herein specifically bind to the PRAME immunogenic epitope and / or selectively bind to many related epitopes (e.g., the PRAME immunogenic epitope and closely related sequences), thereby distinguishing such targets from the vast majority of other possible epitopes available in the human genome.

[0114] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a disorder characterized by PRAME expression, such as a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by PRAME expression. The term "subject" is interchangeable with "patient."

[0115] The term "survival" includes all of the following: survival until death, also known as overall survival (where the death can be either cause-unrelated or tumor-related); "recurrence-free survival" (where the term recurrence includes both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease includes cancer and related diseases). The length of survival can be calculated based on a defined starting point (e.g., time of diagnosis or initiation of treatment) and end point (e.g., death, recurrence, or metastasis). In addition, measures of treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0116] The term "synergistic effect" refers to a combined effect of two or more agents (e.g., a PRAME-related agent described herein and a separate therapy, such as an additional PRAME-targeted TCR, anti-cancer therapy, immunotherapy, etc., for treating a disorder characterized by PRAME expression) that is greater than the sum of the individual effects of the cancer agents / therapies alone.

[0117] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, e.g., effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0118] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analog of such RNA or cDNA) that is complementary to or homologous to all or a portion of the mature mRNA produced by transcription of a biomarker nucleic acid and by normal post-transcriptional processing, if any, of the RNA transcript (e.g., splicing), and by reverse transcription of the RNA transcript.

[0119] "T cells" are immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells are naive (not exposed to antigen; T CM increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO compared to normal T cells), memory T cells (T M ) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). M are central memory T cells (T CM , which have increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA compared to naive T cells), and effector memory T cells (T EM , naive T cells or T CM These cells can be further divided into effector T cells (T) and effector T cells (T) (which have decreased expression of CD62L, CCR7, CD28, and CD45RA, and increased expression of CD127 compared to T cells). E ) is T CM These T cells refer to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, and CD28 compared to CD4+ T cells, and are positive for granzymes and perforin. Other exemplary T cells include regulatory T cells, e.g., CD4+ T cells. + CD25 + (Foxp3 + ) Regulatory T cells and Treg17 cells, as well as Tr1, Th3, and CD8 + CD28 and Qa-1 restricted T cells.

[0120] Conventional T cells, also known as Tconv or Teff, have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) and augment immune responses by expressing one or more T cell receptors. Tcon or Teff are generally defined as any T cell population that is not a Treg, including, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated toward, for example, a Th1 lineage or a Th2 lineage. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is a CD4+ Teff or a CD8+ Teff, such as a CD4+ helper T lymphocyte (e.g., Th0, Th1, Tfh, or Th17) and a CD8+ cytotoxic T lymphocyte. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tcon" are CD4+ T cells that have differentiated in the bone marrow and successfully undergone positive and negative central selection processes in the thymus, but have not yet been activated by exposure to antigen. Naive Tcon are generally characterized by the surface expression of L-selectin (CD62L), the absence of activation markers such as CD25, CD44, or CD69, and the absence of memory markers such as CD45RO. Thus, naive Tcon are considered quiescent and non-dividing, and require interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see, at least, WO2010 / 101870). The presence and activity of such cells are undesirable in the context of suppressing immune responses. Unlike Tregs, Tcons are not anergic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637).

[0121] "T Effector" ("T eff " or "T E") cells refer to T cells (e.g., CD4+ and CD8+ T cells) and T helper (Th) cells with cytolytic activity that secrete cytokines to activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0122] "T cell receptor" or "TCR" refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al. (1997) Curr. Biol. Publ. 4:33) that can bind (e.g., specifically and / or selectively) to antigenic peptides bound to an MHC receptor. TCRs can be found on the surface of cells or in soluble form and are generally composed of heterodimers having an alpha chain and a beta chain (also known as TCRα and TCRβ, respectively) or a gamma chain and a delta chain (also known as TCRγ and TCRδ, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portions of TCR chains (e.g., α and β chains) contain two immunoglobulin domains: a variable domain (e.g., an α chain variable domain or V chain) at the N-terminus; α , and a β chain variable domain or V β typically amino acids 1 to 116 based on Kabat numbering (Kabat et al. (1991) "Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 5th ed.) and one constant domain (e.g., an alpha chain constant domain or C chain constant domain) at the C-terminus and proximal to the cell membrane. α , typically amino acids 117-259 based on Kabat, the β chain constant domain or C β, typically amino acids 117-295 based on Kabat). Also similar to immunoglobulins, variable domains contain complementarity-determining regions ("CDRs," also called hypervariable regions or "HVRs") separated by framework regions ("FRs") (see, e.g., Fores et al. (1990) Proc. Natl. Acad Sci. US.A. 87:9138; Chothia et al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol. 27:55). In some embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and associate with the CD3 complex. Sources of TCRs encompassed by the present invention can be derived from various animal species, e.g., humans, mice, rats, rabbits, or other mammals.

[0123] The term "T cell receptor" or "TCR" should be understood to encompass complete TCRs and antigen-binding portions or fragments thereof. In some embodiments, the TCR is a complete or full-length TCR, e.g., an αβ-type TCR or a γδ-type TCR. In some embodiments, the TCR is an antigen-binding portion that is shorter than the full-length TCR but that binds to a specific peptide bound in an MHC molecule, e.g., an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR contains only a portion of the structural domain of a full-length or complete TCR but is still capable of binding to a peptide epitope (e.g., an MHC-peptide complex) that a full-length TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, e.g., the variable α chain and variable β chain of the TCR, to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity-determining regions (CDRs) involved in recognizing peptides, MHC, and / or MHC-peptide complexes.

[0124] Nomenclature has been established by the International Immunogenetics Information System (IMGT) (Scaviner and Lefranc (2000) Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; see also "T Cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). IMGT provides unique sequences used to describe TCRs, and the sequences described herein can be identified by reference to such unique sequences provided herein. TCR sequences are publicly available in the IMGT database at imgt.org.

[0125] As described above, natural alpha / beta heterodimeric TCRs have an alpha chain and a beta chain. Generally, each chain contains a variable region, a joining region, and a constant region. The beta chain usually also contains a short diversity region between the variable and joining regions, although this diversity region is often considered part of the joining region. Each variable region contains three hypervariable CDRs (complementarity-determining regions) embedded in a framework sequence. CDR3 is well known to be the primary mediator of antigen recognition. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions, which are distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. In the IMGT nomenclature, Vα types are designated by unique TRAV numbers. For example, "TRAV4" defines a TCR Vα region with a unique framework, CDR1 and CDR2 sequences, and a CDR3 that is partially defined by amino acid sequences conserved across various TCRs but also contains amino acid sequences that vary from TCR to TCR. Similarly, "TRBV2" defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence. There are known to be 54 alpha variable genes (44 of which are functional) and 67 beta variable genes (42 of which are functional) within the alpha and beta loci, respectively.

[0126] Similarly, the joining regions of the TCR are defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions are defined by the IMGT TRAC and TRBC nomenclature. In the IMGT nomenclature, the diversity region of the beta chain is referred to by the abbreviation TRBD. As noted, the linked TRBD / TRBJ regions are often collectively referred to as the joining region.

[0127] The gene pools encoding the TCR alpha and beta chains are located on different chromosomes and contain distinct V, D, J, and C gene segments, which are brought together by rearrangement during T cell development. This results in a very high degree of diversity for T cell alpha and beta chains due to the numerous potential recombination events that occur between the 54 TCR alpha variable genes and the 61 alpha J genes, or between the 67 beta variable genes, the 2 beta D genes, and the 13 beta J genes. The recombination process is not strict, introducing additional diversity within the CDR3 region. Each alpha and beta variable gene may also contain allelic variants, designated TRAVxx*01 and *02, or TRBVx-x*01 and *02, respectively, in the IMGT nomenclature, thus further increasing the amount of diversity. Similarly, some TRBJ sequences have two known variants. (Note that the absence of the "*" qualifier means that only one allele is known for the relevant sequence.) The natural repertoire of human TCRs resulting from recombination and thymic selection is approximately 10 , determined by CDR3 diversity. 6 It is estimated that the total number of beta chains in a genome contains 100 unique beta chain sequences (Arstila et al. (1999) Science 286:958-961), and may even be higher (Robins et al. (2009) Blood 114:4099-4107). Each beta chain is estimated to pair with at least 25 different alpha chains, thus generating further diversity (Arstila et al. (1999) Science 286:958-961).

[0128] Thus, the term "TCR alpha variable domain" refers to the concatenated TRAV and TRAJ regions, the TRAV region alone, or TRAV and a partial TRAJ region; the term TCR alpha constant domain refers to the extracellular TRAC region, or a C-terminally truncated or full-length TRAC sequence. Similarly, the term "TCR beta variable domain" refers to the concatenated TRBV and TRBD / TRBJ regions, the TRBV and TRBD regions alone, the TRBV and TRBJ regions alone, or TRBV and a partial TRBD and / or TRBJ region; and the term TCR beta constant domain refers to the extracellular TRBC region, or a C-terminally truncated or full-length TRBC sequence. These nomenclatures for TCR alpha variable domain and TCR beta variable domain apply equally to the variable domains of the TCR gamma and TCR delta chains of gamma / delta TCRs. Those skilled in the art can obtain the gene sequences for TRAV, TRAJ, TRAC, TRBV, TRBJ, and TRBC, for example, through the publicly available IMGT database.

[0129] The term "TCR complex" refers to a complex formed by the association of CD3 with a TCR. For example, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0130] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or in promoting desirable physical or mental development and conditions in animals or humans.

[0131] The terms "therapeutically effective amount" and "effective amount" refer to that amount of a substance that produces some desired effect (e.g., a desired local or systemic therapeutic effect) in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any treatment. In some embodiments, the therapeutically effective amount of a substance depends on the substance's therapeutic index, solubility, pharmacokinetics, half-life, etc. Toxicity and therapeutic efficacy of a subject compound can be determined, for example, by the LD 50 and ED 50 The LD can be determined by standard pharmaceutical methods in cell cultures or experimental animals to determine the therapeutic index. In some embodiments, compositions that exhibit large therapeutic indices are used. In some embodiments, the LD 50 (lethal dose) can be measured, for example, for a drug, and can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to not administering the drug. Similarly, the ED 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured, and for example, for a drug, can be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to when the drug is not administered. Similarly, IC 50can be measured, for example, for an agent, the T cell immune response can be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to when the agent is not administered. In some embodiments, the T cell immune response in the assay can be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, a reduction in viral load of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% may be achieved.

[0132] The term "treating" refers to the therapeutic management or amelioration of a symptom of interest (e.g., a disease or disorder). Treatment can include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically performed to alter the course of a disease (this term is used to refer to any disease, disorder, syndrome, or undesirable condition that warrants or potentially warrants therapy) in a manner beneficial to the subject. The effect of treatment can include reversing, alleviating, reducing the severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of the occurrence or recurrence of a disease or one or more symptoms or signs of a disease. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or mitigating the disease state, and achieving remission or improving prognosis. A therapeutic agent can be administered to a subject who has a disease or who is at increased risk of developing a disease compared to members of the general population. In some embodiments, a therapeutic agent can be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent can be administered, for example, to reduce the likelihood of overt disease recurrence. The therapeutic agent can be administered prophylactically, i.e., before the onset of any symptoms or manifestation of the disease. "Prophylactic treatment" refers to providing medical and / or surgical management to a subject who has not developed a disease or who does not subsequently show evidence of the disease, for example, to reduce the likelihood of the disease occurring or to reduce the severity of the disease if it does occur. The subject may have been identified as being at risk for developing a disease (e.g., at increased risk compared to the general population) or as having risk factors that increase the likelihood of developing a disease.

[0133] The term "unresponsiveness" includes the refractoriness of cancer cells to treatment or the unresponsiveness of therapeutic cells (e.g., immune cells) to stimulation (e.g., stimulation via an activating receptor or cytokine). Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigen. As used herein, the terms "anergy" or "tolerance" include unresponsiveness to activating receptor-mediated stimulation. Such unresponsiveness is generally antigen-specific and persists even after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by a lack of cytokine (e.g., IL-2) production. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, if the cells are re-exposed to the same antigen (even if the re-exposure occurs in the presence of a costimulatory polypeptide), they will be unable to produce cytokines and therefore unable to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, a reporter gene construct can be used. For example, anergic T cells cannot initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by multimers of AP1 sequences that can be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0134] The term "vaccine" refers to a pharmaceutical composition that induces an immune response against an antigen of interest. A vaccine can also confer protective immunity in a subject.

[0135] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR) that is involved in binding the immunoglobulin superfamily binding protein to an antigen (e.g., a TCR α or β chain (or the γ and δ chains of a γδ TCR)). The variable domains of the α and β chains of a native TCR (V α and V β ) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. α The domains are encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ), and the V β The domains are encoded by three separate DNA segments: the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). α or V β The V domain may be sufficient to confer antigen-binding specificity. Furthermore, a TCR that binds a particular antigen may have a V domain derived from the TCR that binds the antigen. α or V β The V domains were isolated using complementary V α or V β A library of domains can be screened.

[0136] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, a vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, a vector is capable of autonomous replication and / or expression of nucleic acids to which it is linked. Vector, as used herein, is referred to as an "expression vector," which is capable of directing the expression of genes to which it is operably linked. In general, expression vectors useful in recombinant DNA techniques are often in the form of "plasmids," which generally refer to circular double-stranded DNA loops that, in their vector form, are not bound to the chromosome. As used herein, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. However, as will be understood by those of skill in the art, the present invention is intended to include other forms of expression vectors subsequently known in the art which serve equivalent functions.

[0137] There is a known and definite correspondence, as defined by the genetic code (shown below), between the amino acid sequence of a particular protein and the nucleotide sequence capable of encoding that protein. Similarly, there is a known and definite correspondence, as defined by the genetic code, between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid. [Table 18]

[0138] An important and well-known feature of the genetic code is its redundancy, whereby more than one coding nucleotide triplet can be used for most of the amino acids used to make proteins (as exemplified above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than others). Furthermore, methylated variants of purines or pyrimidines can occasionally be found within a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and its corresponding amino acid.

[0139] In view of the above, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to obtain a polypeptide amino acid sequence using the genetic code to translate DNA or RNA into an amino acid sequence. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence that can encode the polypeptide can be inferred from the genetic code (which, due to its redundancy, will represent multiple nucleic acid sequences for any given amino acid sequence). Thus, any description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, any description and / or disclosure herein of a polypeptide amino acid sequence should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0140] II. Peptides In certain aspects, provided herein are methods and compositions for the treatment and / or prevention of disorders associated with PRAME expression by inducing an immune response against PRAME or cells expressing PRAME, involving the administration of a PRAME immunogenic peptide described herein, a nucleic acid encoding it, and / or a cell expressing it.

[0141] In certain embodiments, a PRAME immunogenic peptide comprises (e.g., consists of) a peptide epitope selected from the peptide sequences listed in Table 1, e.g., Table 1A. The peptide epitopes described herein can be combined with MHC molecules, such as particular HLA molecules having particular HLA alpha chain alleles. For example, the peptides of Table 1A were identified in association with MHC whose alpha chain has the HLA-A*02 serotype, as encoded by the HLA-A*02:01 allele, as further described in the Examples section. In some embodiments, the PRAME immunogenic peptide may be combined with an MHC molecule, the MHC molecule being selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, and an MHC alpha chain that is an HLA serotype selected from the group consisting of HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, and optionally the HLA alleles are HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:08, HLA-A*02:09, HLA-A*03:010, HLA-A*03:020, HLA-A*03:031, HLA-A*03:04, HLA-A*03:05, HLA-A*03:06, HLA-A*03:07, HLA-A*03:08, HLA-A*03:09, HLA-A*03:011, HLA-A*03:012, HLA-A*03:013, HLA-A*03:014, HLA-A*03:015, HLA-A*03:016, HLA-A*03:017, HLA-A*03:018, HLA-A*03:019, HLA-A*03:020, HLA-A*03:021, HLA-A*03:022, HLA-A*03:023, HLA-A*03:024, HLA-A*03:025, HLA-A*03:026, HLA-A*03:027, HLA-A*03:028, HLA-A*03:029, HLA-A*03:03 *02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, H LA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:7 4 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles,HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:1 0, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24: 26, HLA-A*24:58 allele, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HL A-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, H LA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03 , HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:0 2, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05, and HLA-C*14:03 alleles. In some embodiments, the PRAME immunogenic peptide is derived from a human PRAME protein and / or a PRAME protein set forth in Table 3. In some embodiments, one or more PRAME immunogenic peptides are administered alone or in combination with an adjuvant.

[0142] In certain aspects, provided herein are compositions comprising one or more PRAME immunogenic peptides described herein and an adjuvant. [Table 1]

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

[0144] In some embodiments, the PRAME polypeptide comprises at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, In some embodiments, the PRAME polypeptide comprises, consists essentially of, or consists of 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 373, or more contiguous amino acids, or any range therebetween (e.g., 7-25, 8-22, 9-22, etc.), inclusive of the endpoints. In some embodiments, the contiguous amino acids are identical to the amino acid sequence of PRAME shown in Table 3. In some embodiments, the PRAME polypeptide comprises, consists essentially of, or consists of one or more peptide epitopes selected from the group consisting of the PRAME peptide epitopes listed in Table 1, such as Table 1A.

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

[0146] In some embodiments, variations or derivatives of PRAME immunogenic polypeptides are provided herein. A modified polypeptide may have an amino acid sequence altered, for example, by conservative substitution, but still elicit an immune response that reacts with the unmodified protein antigen and is considered a functional equivalent. As used herein, the term "conservative substitution" refers to the replacement of an amino acid residue with another, biologically similar residue. It is well known in the art that amino acids within the same conservative group can typically be substituted for each other without substantially affecting protein function. According to certain embodiments, a derivative, equivalent, variant, or mutant of the ligand-binding domain of a PRAME immunogenic peptide is a polypeptide that is at least 85% homologous to the sequence of a PRAME immunogenic peptide or fragment thereof described herein. In some embodiments, the homology is at least 90%, at least 95%, at least 98%, or more.

[0147] Immunogenic peptides encompassed by the present invention may include peptide epitopes derived from the PRAME protein, such as those listed in Table 1, e.g., 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 conservative or non-conservative mutations. The peptide may include up to 1, 2, 3, 4, or more mutations. In some embodiments, the peptide may include at least 1, 2, 3, 4, or more mutations.

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

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

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

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

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

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

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

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

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

[0157] Binding proteins encompassed by the present invention may 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.

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

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

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

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

[0162] In some embodiments, provided herein are nucleic acids encoding a PRAME immunogenic peptide or fragment thereof described herein, e.g., DNA molecules encoding a PRAME immunogenic peptide. In some embodiments, the compositions include an expression vector containing an open reading frame encoding a PRAME immunogenic peptide or fragment thereof described herein. In some embodiments, the nucleic acid includes regulatory elements necessary for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, an enhancer may be included. These elements may be operably linked to a sequence encoding a PRAME immunogenic polypeptide or fragment thereof. Representative vectors, promoters, regulatory elements, etc. useful for expressing proteins, e.g., peptides, are further described below.

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

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

[0165] The MHC proteins provided and used in the compositions and methods encompassed by the present disclosure can be any suitable MHC molecule known in the art. Generally, they have the formula (α-β-P) n where n is at least 2, e.g., 2 to 10, e.g., 4. α is the α chain of a class I or class II MHC protein. β is the β chain defined herein as a class II MHC protein or β2-microglobulin of an MHC class I protein. P is a peptide antigen.

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

[0167] MHC proteins can be from any mammalian or avian species, such as primates, particularly humans; rodents, including mice, rats, and hamsters; rabbits; horses; cows; dogs; cats; etc. For example, MHC proteins can be derived from human HLA proteins or mouse H-2 proteins. HLA proteins include the class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, as well as the class I proteins HLA-A, HLA-B, HLA-C, and β2-microglobulin. H-2 proteins include the class I subunits H-2K, H-2D, and H-2L, as well as the class II subunits I-Aα, I-Aβ, I-Eα, and I-Eβ, as well as β2-microglobulin. Sequences of several representative MHC proteins can be found in Kabat et al., Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pp. 724-815. MHC protein subunits suitable for use in the present invention are soluble forms of normally membrane-bound proteins, prepared as known in the art, for example, by deletion of transmembrane and cytoplasmic domains.

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

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

[0170] In certain embodiments, the MHC-peptide complex comprises a peptide epitope and an MHC selected from Table 1. In some embodiments, the MHC molecule comprises an MHC alpha chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, and optionally Selectively, the HLA alleles are HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*0 2:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A *02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 allele, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01: 01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 allele, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 allele Gene, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*2 4:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 allele, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15,HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:0 1, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17: In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1A and an MHC having an alpha chain with an HLA-A*02 serotype, as encoded by the HLA-A*02:01 allele.

[0171] To prepare MHC-peptide complexes, the subunits can be combined with antigenic peptides and folded in vitro to form stable heterodimeric complexes with intrachain disulfide-bonded domains. The peptide can be included in the initial folding reaction or added to the empty heterodimer in a later step. In the compositions and methods encompassed by the present invention, this is a PRAME immunogenic peptide or a fragment thereof. Conditions that allow folding and association of the subunits and peptides are known in the art. As an example, approximately equimolar amounts of solubilized α and β subunits can be mixed in a solution of urea. Refolding is initiated by dilution or dialysis into a buffer solution that does not contain urea. The peptide can be introduced into the empty class II heterodimer at a pH of about 5-5.5 for about 1-3 days, followed by neutralization, concentration, and buffer exchange. However, specific folding conditions are not essential for the practice of the present invention.

[0172] The monomeric complex (α-β-P) (herein referred to as "monomer") can be multimerized, for example, to form an MHC tetramer. The resulting multimer is stable over a long period of time. Preferably, the multimer can be formed by binding the monomer to a multivalent entity via specific binding sites on the α or β subunit, as known in the art (e.g., as described in U.S. Pat. No. 5,635,363). MHC proteins in monomeric or multimeric form can also be conjugated to beads or any other support.

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

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

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

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

[0177] In some embodiments, the cells are artificial antigen-presenting cells (aAPCs). Typically, aAPCs include characteristics of natural APCs, such as expression of MHC molecules, stimulatory and costimulatory molecule(s), Fc receptors, adhesion molecule(s), and / or the ability to produce or secrete cytokines (e.g., IL-2). Typically, aAPCs are cell lines that lack expression of one or more of the above, including but not limited to, MHC molecules, low affinity Fc receptors (CD32), high affinity Fc receptors (CD64), costimulatory signals (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, 3 / TR6, or B7-H3 ligand; or CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor, or CD aAPCs are generated by introducing (e.g., by transfection or transduction) one or more of the following missing components: an antibody that specifically binds to a ligand of MHC 1 or 83; a cell adhesion molecule (e.g., ICAM-1, or LFA-3); and / or a cytokine (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon alpha (IFN.alpha), interferon beta (IFN.beta), interferon gamma (IFN.gamma), tumor necrosis factor alpha (TNF.alpha), tumor necrosis factor beta (TNF.beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (GCSF)). In some cases, aAPCs do not normally express MHC molecules but can be engineered to express MHC molecules, or in some cases, can be induced or induced to express MHC molecules, such as by stimulation with cytokines. Optionally, the aAPCs can be loaded with a stimulatory ligand, including, for example, an anti-CD3 antibody, an anti-CD28 antibody, or an anti-CD2 antibody.The exemplary cell line that can be used as a scaffold for generating aAPC is K562 cell line, or fibroblast cell line.Various aAPCs are known in the art, for example, see U.S. Patent No. 8,722,400, published application US2014 / 0212446, Butler and Hirano (2014) Immunol Rev.257:10.1111 / imr.12129, Suhoshki et al. (2007) Mol.Ther.15:981-988).

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

[0179] In some embodiments, cells can be selected to express the desired MHC-restricted MHC alleles. In some embodiments, MHC typing of cells, e.g., cell lines, is well known in the art. In some embodiments, MHC typing of cells, e.g., primary cells obtained from a subject, can be determined by performing tissue typing using procedures well known in the art, for example, molecular haplotype assays (BioTest ABC SSPtray, BioTest Diagnostics Corp., Denville, NJ; SeCore Kits, Life Technologies, Grand Island, NY). In some cases, it is well within the skill of one of ordinary skill in the art to perform standard typing of cells to determine the HLA genotype, for example, by using sequence-based typing (SBT) (Adams et al. (2004) J. Transl. Med., 2:30; Smith (2012) Methods Mol Biol., 882:67-86). In some cases, HLA typing of cells, e.g., fibroblasts, is known. For example, the human fetal lung fibroblast cell line MRC-5 expresses HLA-A*02:01, A29, B13, B44 Cw7 (C*0702), the human foreskin fibroblast cell line Hs68 expresses HLA-A1, A29, B8, B44, Cw7, Cw16, and the WI-38 cell line expresses A*68:01, B*08:01 (Solache et al. (1999) J Immunol, 163:5512-5518; Ameres et al. (2013) PloS Pathog. 9:e1003383). The human transfectant fibroblast cell line M1DR1 / Ii / DM expresses HLA-DR and HLA-DM (Karakikes et al. (2012) FASEB J., 26:4886-96).

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

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

[0182] IV. Immunogenic composition In some aspects, provided herein are pharmaceutical compositions (e.g., vaccine compositions) comprising a PRAME immunogenic peptide and / or a nucleic acid encoding a PRAME immunogenic peptide, and an adjuvant. In some aspects, provided herein are pharmaceutical compositions (e.g., vaccine compositions) comprising a stable MHC-peptide complex comprising a PRAME immunogenic peptide in the context of an MHC molecule, and an adjuvant. In some embodiments, the composition comprises a combination of multiple (e.g., two or more) PRAME immunogenic peptides or nucleic acids, and an adjuvant. In some embodiments, the composition comprises a combination of multiple (e.g., two or more) stable MHC-peptide complexes comprising a PRAME immunogenic peptide in the context of an MHC molecule, and an adjuvant. In some embodiments, the above compositions further comprise a pharmaceutically acceptable carrier.

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

[0184] Methods of preparing these formulations or compositions include the step of bringing into association a PRAME immunogenic peptide and / or nucleic acid described herein with an adjuvant, carrier, and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

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

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

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

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

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

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

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

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

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

[0194] Upon uptake by a cell (e.g., a host cell, an antigen-presenting cell (APC), e.g., a dendritic cell, a macrophage, etc.), the DNA molecule may exist in the cell as an extrachromosomal molecule and / or may be integrated into a chromosome. DNA may be introduced into the cell in the form of a plasmid, which may remain separate genetic material. Alternatively, linear DNA may be introduced into the cell, which may be integrated into a chromosome. Optionally, once the DNA is introduced into the cell, reagents may be added that promote integration of the DNA into a chromosome.

[0195] V. Binding Proteins In some aspects, binding moieties that bind to the peptides described herein and / or stable MHC-peptide complexes described herein are provided, e.g., binding moieties that specifically and / or selectively bind to the peptides and / or stable MHC-peptide complexes, e.g., at least about 10 -4 M or less (e.g., about 10 -4 , 10 -5 , 10 -6 , 10 -7 , about 10 -8 , about 10 -9 , about 10 10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 Binding proteins, such as T cell receptors (TCRs), antibodies and the like, that bind with a Kd of 100 kJ / 200 kcal, 100 kJ / 200 kcal, 100 kcal ...

[0196] In aspects encompassed by the invention, provided herein are binding proteins that bind (e.g., specifically and / or selectively) to peptide-MHC (pMHC) complexes that include a PRAME immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the binding protein binds to about 5x10 -4 M or less, about 1x10 -4 M or less, about 5x10 -5 M or less, about 1x10 -5 M or less, about 5x10 -6 M or less, about 1x10 -6 M or less, about 5x10 -7 M or less, about 1x10 -7 M or less, about 5x10 -8 M or less, about 1x10 -8 M or less, about 5x10 -9 M or less, about 1x10 -9 M or less, about 5x10 -10 M or less, about 1x10 -10 M or less, about 5x10 -11 M or less, about 1x10 -11 M or less, about 5x10 -12 M or less, about 1x10 -12 K less than or equal to M, or any range therebetween, including the endpoints, e.g., about 1-50 micromolar, 1-100 micromolar, 0.1-500 micromolar, etc. dIn some embodiments, the MHC molecule comprises an MHC alpha chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, and optionally Selectively, the HLA alleles are HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*0 2:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A *02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 allele, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01: 01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 allele, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 allele Gene, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*2 4:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 allele, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15,HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:0 1, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17: In some embodiments, the HLA serotype is selected from the group consisting of HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05, and HLA-C*14:03 alleles. In some embodiments, the HLA serotype is HLA-A*02 and / or the HLA allele is an HLA-A*02:01 allele. In some embodiments, the binding proteins provided herein are genetically engineered, isolated, and / or purified.

[0197] In some embodiments, the binding protein has a higher binding affinity for a PRAME peptide-MHC (pMHC) than a known T cell receptor (e.g., a comparative TCR described herein). For example, the binding protein has at least 1.2-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 1000 fold, 5000 fold, 10000 fold, 50000 fold, 100000 fold, 500000 fold, 1000000 fold, or more, or any range therebetween inclusive of the endpoints, e.g., 1.2-2 fold higher, binding affinity for PRAME peptide-MHC (pMHC).

[0198] In some embodiments, the binding protein induces greater T cell expansion, cytokine release, and / or cytotoxic killing than known T cell receptors (e.g., comparative TCRs described herein) when contacted with target cells having expression of PRAME below a certain level. For example, in some embodiments of any aspect described herein, PRAME levels can be expressed as transcripts per million, e.g., 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 10 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or less, or any range therebetween inclusive of the endpoints, e.g., from about 1,000 TPM or less to about 35 TPM or less). In some embodiments, low PRAME expression levels are referred to as "heterozygous expression," meaning about 1 TPM to about 35 TPM, or any range therebetween, inclusive of the endpoints, e.g., 32 TPM or 1 to 32 TPM. Higher expression is 36 TPM or greater.As further described herein, TPM is measured according to well-known techniques, e.g., RNA-Seq, and gene expression TPM data is well known in the art for various cell lines, tissue types, etc. (See, e.g., the Broad Institute Cancer Cell Line Encyclopedia (CCLE) at portals.broadinstitute.org on the world wide web.) In some embodiments, the binding protein, when contacted with a target cell that expresses the PRAME peptide epitope, e.g., expresses a heterozygous form of the PRAME peptide epitope, increases T cell expansion, cytokine release, and / or cytotoxic killing by at least 1.2-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, or more than a known T cell receptor (e.g., a comparative TCR described herein). , 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold or more, or any range therebetween inclusive of the endpoints, e.g., a 1.2- to 2-fold increase.

[0199] In some embodiments, PRAME expression is detected using RNA sequencing (RNA-seq). RNA-seq generally involves the following steps: obtaining a sample containing genetic material, isolating total RNA from the obtained sample, preparing an amplified cDNA library from the total RNA, sequencing the amplified cDNA library, and analyzing and profiling the amplified cDNA to assess the expression levels of various transcripts. The sample may be a cell population, a tissue sample, a biopsy sample, a cell culture, or a single cell. Total RNA can be isolated from a biological sample using any method known in the art. In certain embodiments, total RNA is extracted from plasma. Extraction of plasma RNA is described in Enders et al., "The Concentration of Circulating Corticotropin-Releasing Homer mRNA in Material Plasma Is Inclined in Preclampsia," Clin. 1999, 14:131–132. As described therein, plasma collected after a centrifugation step is mixed with Trizol LS reagent (Invitrogen) and chloroform. The mixture is centrifuged, and the aqueous layer is transferred to a new tube. Ethanol is added to the aqueous layer, and the mixture is then loaded into an RNeasy mini-column (Qiagen) and processed according to the manufacturer's recommendations.

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

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

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

[0203] In certain embodiments, the binding proteins recognize PRAME immunogenic peptides in complex with MHC molecules, such as particular HLA molecules having particular HLA alpha chain alleles. For example, the binding proteins listed in Table 2A have been identified as binders of PRAME immunogenic peptides associated with MHC whose alpha chains have the HLA-A*02 serotype, e.g., encoded by the HLA-A*02:01 allele, as further described in the Examples section. In some embodiments, the binding protein recognizes a complex of a PRAME immunogenic peptide and an MHC molecule, wherein the MHC molecule is selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA and optionally, the HLA allele is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:08, HLA-A*02:11, HLA-A*02:09, HLA-A*03:12, HLA-A*04:13, HLA-A*04:14, HLA-A*04:15, HLA-A*04:16, HLA-A*04:17, HLA-A*04:18, HLA-A*04:19, HLA-A*04:20, HLA-A*04:21, HLA-A*04:22, HLA-A*04:23, HLA-A*04:24, HLA-A*04:25, HLA-A*04:26, HLA-A*04:27, HLA-A*04:28, HLA-A*04:29, HLA-A*04:30, HLA-A*04:31, HLA-A*04:32, HLA-A*04:33, HLA-A*04:34, HLA-A*04:35, HLA-A*04:36, HLA-A*04:37, HLA-A*04:38, HLA-A*04:39, HLA-A*04:40, HLA-A*04:41, HLA-A*04:42, HLA-A*04:43, HLA-A*04:44, HLA-A*04:45, H 2:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02: 22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 allele, HLA-A*03:01, HLA-A* 03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 allele, HLA-A*11:01, HLA -A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 allele, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05,HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A* 24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 allele, HLA-B*0 7:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, H LA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*0 3:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, H LA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*1 2:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05, and HLA-C*14:03 alleles. In some embodiments, the PRAME immunogenic peptides are derived from the human PRAME protein and / or PRAME proteins shown in Table 3. In some embodiments, one or more PRAME immunogenic peptides are administered alone or in combination with an adjuvant.

[0204] In some embodiments, the binding protein does not bind to a peptide-MHC (pMHC) complex, and optionally the peptide is derived from an "off-target" described herein, e.g., PLA2G4E, EFNA1, and / or SLC26A1.

[0205] In some embodiments, the binding protein does not bind to "off-target" peptide-MHC (pMHC) complexes, such as PLA2G4E, EFNA1, and / or SLC26A1-pMHC (pMHC) complexes.

[0206] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151, 152, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 17 %, 99% or more identity to a TCR alpha chain sequence, and / or b) a TCR beta chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chains listed in Table 2.

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

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

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

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

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

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

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

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

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

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

[0217] In some embodiments, the binding proteins provided herein also contain a TCR beta chain constant region (C) listed in Table 2. β ) TCR C sequences selected from the group consisting of β It may comprise (e.g., may comprise, consist essentially of, or consist of) the sequence. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 19-1

Table 19-2

Table 20-1

Table 20-2

Table 21-1

Table 21-2

Table 21-3

Table 21-4

Table 21-5

Table 21-6

[0218] In some embodiments, the binding proteins provided herein comprise constant regions that are chimeric, humanized, human, primate, or rodent (e.g., rat or mouse). For example, a human variable region can be chimerized with a mouse constant region, or a mouse variable region can be humanized with a human constant region and / or human framework regions. In some embodiments, the constant region can be mutated to modify functionality (e.g., introducing non-naturally occurring cysteine ​​substitutions at opposing residue positions in the TCR alpha and beta chains to create disulfide bonds useful for improving affinity between the TCR alpha and beta chains). Similarly, mutations can be made in the transmembrane domains of the constant region to modify functionality (e.g., increasing hydrophobicity by introducing non-naturally occurring substitutions of residues with hydrophobic amino acids). In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof compared to a reference CDR sequence. In some embodiments, mutations can be made in the constant region to increase cell surface expression.

[0219] In some embodiments, the binding proteins disclosed herein may be engineered protein scaffolds, antibodies or antigen-binding fragments thereof, TCR mimetic antibodies, and the like.Such binding moieties can be designed and / or generated against the peptides and / or MHC-peptide complexes described herein using conventional immunological methods, such as immunizing a host, obtaining antibody-producing cells and / or antibodies thereto, and generating hybridomas useful for producing monoclonal antibodies (see, e.g., Watt et al. (2006) Nat. Biotechnol. 24:177-183; Gebauer and Skerra (2009) Curr. Opin. Chem Biol. 13:245-255; Skerra et al. (2008) FEBSJ. 275:2677-2683; Nygren et al. (2008) FEBSJ. 275:2668-2676; Dana et al. (2012) Exp. Rev. Mol. Med. 14:e6; Sergeva et al. (2011) Blood 117:4262-4272, PCT Publication Nos. WO2007 / 143104, PCT / US86 / 02269 and WO86 / 01533, U.S. Patent No. 4,816,567, Better et al. (1988) Science 240:1041-1043, Liu et al. al.(1987) Proc.Natl.Acad.Sci.USA84:3439-3443, Liu et al.(1987) J.Immunol.139:3521-3526, Sun et al.(1987) Proc.Natl.Acad.Sci.84:214-218, Nishimura et al.(1987) Cancer Res.47:999-1005, Wood et al. al. (1985) Nature 314:446-449, Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559), Morrison, SL (1985) Science 229:1202-1207, Oi et al. (1986) Biotechniques 4:214, U.S. Patent No. 5,225,539, Jones et al. (1986) Nature 321:552-525, Verhoeyan et al. (1988) Science 239:1534, and Beidler et al. (1988) J. Immunol. 141:4053-4060.If desired, the binding moiety can be isolated or purified using conventional procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, and high performance liquid chromatography (HPLC) (e.g., Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY).

[0220] The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE), as well as recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, and fragments and derivatives of all of the above, which fragments and derivatives retain at least an antigen-binding site. Antibody derivatives can include proteins or chemical moieties conjugated to antibodies.

[0221] Additionally, intrabodies are well-known antigen-binding molecules that have the characteristics of antibodies but can be expressed intracellularly to bind to and / or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods for adapting antibodies to target (e.g., inhibit) intracellular portions are well known in the art, such as the use of single-chain antibodies (scFv), modifying immunoglobulin VL domains for hyperstability, modifying antibodies to tolerate the reducing intracellular environment, and generating fusion proteins that improve intracellular stability and / or modulate intracellular localization. Intracellular antibodies can also be introduced into and expressed in one or more cells, tissues, or organs of a multicellular organism, e.g., for prophylactic and / or therapeutic purposes (e.g., as gene therapy) (see, at least, PCT Publication Nos. WO 08 / 020079, WO 94 / 02610, WO 95 / 22618, and WO 03 / 014960; U.S. Patent No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.); Kontermann (2004) Methods 34:163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412; Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).

[0222] As used herein, the term "antibody" also includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). The term "antigen-binding portion," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically and / or selectively bind to an antigen (e.g., a peptide and / or MHC-peptide complex described herein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be recombinantly joined by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426, Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a particular scFv can be ligated to human immunoglobulin constant region cDNA or genomic sequence to generate an expression vector encoding an entire IgG polypeptide or other isotype.VH and VL can also be used to create Fab, Fv, or other immunoglobulin fragments using either protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0223] Furthermore, an antibody, or antigen-binding portion thereof, may be part of a larger immunoadhesive polypeptide formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesive polypeptides include the use of a streptavidin core region to generate tetrameric scFv polypeptides (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, protein subunit peptides, and a C-terminal polyhistidine tag to generate bivalent biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions, and immunoadhesin polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

[0224] Antibodies may be polyclonal or monoclonal, and may be xenogeneic, allogeneic, or allogeneic, or may be modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the present invention specifically and / or selectively, or substantially specifically and / or selectively, bind to the peptides and / or MHC-peptide complexes described herein. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one antigen-binding site capable of immunoreacting with a particular epitope of an antigen, whereas the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically exhibits a single binding affinity for the particular antigen to which it immunoreacts.

[0225] As with other binding moieties described herein, antibodies may be "humanized," which is intended to include antibodies made by non-human cells having variable and constant regions that have been modified to more closely resemble antibodies made by human cells (e.g., by modifying the amino acid sequence of a non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences). Humanized antibodies of the invention may contain amino acid residues, for example, in the CDRs, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro / ex vivo random or site-specific mutagenesis or by in vivo somatic mutation). The term "humanized antibody," as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences.

[0226] In some embodiments, the binding proteins disclosed herein may comprise a T cell receptor (TCR), an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In some embodiments, the binding proteins disclosed herein may comprise two polypeptide chains, each of which comprises a variable region comprising the CDR3 of the TCR alpha chain and the CDR3 of the TCR beta chain, or the CDR1, CDR2, and CDR3 of both the TCR alpha chain and the TCR beta chain. In some embodiments, the binding protein comprises a single-chain TCR (scTCR), which comprises a TCR V α and TCR V β domains but only one TCR constant domain (C α or C β The term "chimeric antigen receptor" (CAR) refers to a fusion protein engineered to contain two or more naturally occurring amino acid sequences linked to each other in a manner that does not occur naturally or in a host cell, and that can function as a receptor when present on the surface of a cell. CARs encompassed by the present invention can include an extracellular portion comprising an antigen-binding domain (i.e., an antigen-binding domain derived from or derived from an immunoglobulin or immunoglobulin-like molecule, e.g., an antibody or TCR, or an antigen-binding domain derived from or derived from a killer immune receptor from an NK cell), linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing costimulatory domain(s)) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; and Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0227] In some embodiments, 1) the TCR alpha chain CDRs, TCR V αand / or 2) the TCR beta chain CDRs, TCR V domains and / or TCR alpha chain CDRs are encoded by TRAV, TRAJ, and / or TRAC genes selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, or fragments thereof. β The domain and / or TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof compared to the cognate reference CDR sequence listed in Table 2.

[0228] In some embodiments, the binding proteins disclosed in the present application (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs)) are chimeric (e.g., contain amino acid residues or motifs from more than one donor or species), humanized (e.g., contain residues from a non-human organism that have been modified or substituted to reduce the risk of immunogenicity in humans), or humanized.

[0229] Methods for producing engineered binding proteins, such as TCRs, CARs, and antigen-binding fragments thereof, are well known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 5:3000, U.S. Patent No. 6,410,319, U.S. Patent No. 7,446,191, U.S. Patent Publication No. 2010 / 065818, U.S. Patent No. 8,822,647, PCT Publication No. WO2014 / 031687, U.S. Patent No. 7,514,537, and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0230] In some embodiments, the binding proteins described herein are TCRs or antigen-binding fragments thereof expressed on the cell surface, and the TCRs expressed on the cell surface can associate with CD3 protein more efficiently than endogenous TCRs. When expressed on the surface of a cell, such as a T cell, the binding proteins encompassed by the present invention, e.g., TCRs, may have stronger surface expression on the cell than endogenous binding proteins, e.g., endogenous TCRs. In some embodiments, provided herein are CARs in which the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0231] Further provided are compounds that can be used in combination with the V disclosed herein as starting materials for engineering modified binding proteins that may have altered properties from the starting binding protein. α and / or V β The modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) can be prepared according to well-known methods using binding proteins having one or more of the sequences. The binding proteins can have one or more of the sequences within one or both variable regions (i.e., V α and / or V β ), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, binding proteins can be engineered by modifying residues in the constant region(s).

[0232] Another type of variable region modification is the V α and / or V βAmino acid residues within the CDR1, CDR2, and / or CDR3 regions of a CDR1 protein are mutated to thereby improve one or more binding characteristics (e.g., affinity) of the binding protein of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on protein binding or other functional properties of interest can be assessed in in vitro, ex vivo, or in vivo assays such as those described herein and provided in the Examples. In some embodiments, conservative modifications (as described above) can be introduced. The mutations can be amino acid substitutions, additions, or deletions. In some embodiments, the mutations are substitutions. Furthermore, typically, no more than 1, 2, 3, 4, or 5 residues within the CDR regions are modified.

[0233] In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) described herein may have one or more amino acid substitutions, deletions, or additions compared to naturally occurring TCRs. In some embodiments, each CDR of a binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof compared to the cognate reference CDR sequence listed in Table 2. Conservative amino acid substitutions are well known and may occur naturally or may be introduced when the binding protein is recombinantly produced. Amino acid substitutions, deletions, and additions can be introduced into proteins using mutagenesis methods readily known in the art (e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide-directed site-specific (or segment-specific) mutagenesis procedures can be used to generate modified polynucleotides with specific codons altered according to the desired substitution, deletion, or insertion. Alternatively, immunogenic polypeptide variants can be prepared using random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis (see, e.g., Sambrook et al., supra).

[0234] Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids can be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, although considered more difficult to classify, shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, substitution of glutamine for glutamic acid or asparagine for aspartic acid can be considered similar substitutions in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of a polypeptide and its conserved amino acid substitutes to the sequence of a second polypeptide (e.g., using the GENEWORKS™, Align, BLAST algorithms, or other algorithms described herein and practiced in the art).

[0235] In some embodiments, the encoded binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) may include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). A signal peptide directs newly synthesized polypeptides to their appropriate location inside or outside of a cell. A signal peptide may be removed from a polypeptide during or after completion of localization or secretion. A polypeptide having a signal peptide is referred to herein as a "preprotein," and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In some embodiments, the binding proteins (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) described herein may contain a mature V α Domain, mature V β In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) described herein comprise a mature TCR beta chain, a mature TCR alpha chain, or both.

[0236] In some embodiments, the binding protein is a fusion protein comprising: (a) an extracellular component comprising a TCR or antigen-binding fragment thereof; (b) an intracellular component comprising an effector domain or functional portion thereof; and (c) a transmembrane domain linking the extracellular and intracellular components. In some embodiments, the fusion protein is capable of binding (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex comprising a PRAME immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the MHC molecule comprises an MHC alpha chain that is of the HLA serotype HLA-A*02. In some embodiments, the HLA allele is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, and HLA-A*02:07 alleles. In a specific embodiment, the HLA allele is HLA-A*0201.

[0237] As used herein, an "effector domain" or "immune effector domain" is an intracellular portion or domain of a fusion protein or receptor that can directly or indirectly promote an immune response in a cell upon receiving an appropriate signal. In some embodiments, the effector domain is derived from an immune cell protein or portion thereof or immune cell protein complex that receives a signal upon binding (e.g., CD3ζ) or when the immune cell protein or portion thereof or immune cell protein complex directly binds to a target molecule to trigger signaling from the effector domain in the immune cell.

[0238] An effector domain can directly promote a cellular response if it contains one or more signaling domains or motifs, such as intracellular tyrosine-dependent activation motifs (ITAMs), such as those found in costimulatory molecules. Without wishing to be bound by theory, ITAMs are thought to serve for T cell activation following ligand engagement by the T cell receptor or by a fusion protein containing a T cell effector domain. In some embodiments, the intracellular component or functional portion thereof comprises an ITAM. Exemplary immune effector domains include, but are not limited to, immune effector domains derived from CD3ε, CD3δ, CD3ζ, CD25, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the effector domain comprises a lymphocyte receptor signaling domain (e.g., CD3ζ, or a functional portion or variant thereof).

[0239] In further embodiments, the intracellular component of the fusion protein comprises a costimulatory domain or functional portion thereof selected from a ligand that binds (e.g., specifically and / or selectively) to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-1 (CD54), LFA-1 (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, or a functional variant thereof, or any combination thereof. In some embodiments, the intracellular component comprises a CD28 costimulatory domain or a functional portion or variant thereof (which may optionally contain an LL-GG mutation at positions 186-187 of the native CD28 protein (e.g., Nguyen et al. (2003) Blood 702:4320), a 4-1BB costimulatory domain or a functional portion or variant thereof, or both.

[0240] In some embodiments, the effector domain comprises a CD3ε endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD27 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD28 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a 4-1BB endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an OX40 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD2 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD5 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICAM-1 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an LFA-1 endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICOS endodomain or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof.

[0241] The extracellular and intracellular components encompassed by the present invention are linked by a transmembrane domain. As used herein, a "transmembrane domain" is a portion of a transmembrane protein that can insert into or span a cell membrane. A transmembrane domain is thermodynamically stable in the cell membrane and generally has a three-dimensional structure ranging in length from about 15 amino acids to about 30 amino acids. The structure of a transmembrane domain may comprise an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof. In some embodiments, the transmembrane domain comprises or is derived from a known transmembrane protein (e.g., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, or any combination thereof).

[0242] In some embodiments, the extracellular component of the fusion protein further comprises a linker positioned between the binding domain and the transmembrane domain. As used herein, a "linker," when referring to the component of the fusion protein linking the binding domain and the transmembrane domain, can be an amino acid sequence having from about 2 amino acids to about 500 amino acids that can provide flexibility and room for conformational movement between the two regions, domains, motifs, fragments, or modules connected by the linker. For example, linkers encompassed by the present invention can position the binding domain away from the surface of the host cell expressing the fusion protein, allowing for proper contact, antigen binding, and activation between the host cell and the target cell (Patel et al. (1999) Gene Therapy 6:412-419). The length of the linker can be varied to maximize antigen recognition based on the selected target molecule, the selected binding epitope, or the seize and affinity of the antigen-binding domain (see, e.g., Guest et al. (2005) Immunother. 28:203-11, and PCT Publication No. WO2014 / 031687). Exemplary linkers include Gly x Ser ywherein x and y are each independently an integer of 0 to 10, provided that neither x nor y is 0 (e.g., (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or a combination thereof, such as ((Gly3Ser)2Gly2Ser)).

[0243] The binding proteins may be conjugated to an agent, such as a detection moiety, radiosensitizer, photosensitizer, etc., and / or may be chemically modified as described above for peptides.

[0244] In some embodiments, the binding proteins included in the present invention may be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or label. The affinity tag may be selected from the group consisting of glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, Halo tag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of an inflammatory agent, an anti-inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody, e.g., a single-chain Fv.

[0245] The binding proteins can be conjugated to agents used in imaging, research, therapy, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiation therapy. In some embodiments, the binding proteins can be conjugated or fused to detectable agents, such as fluorophores, near-infrared dyes, contrast agents, nanoparticles, metal-containing nanoparticles, metal chelates, X-ray contrast agents, PET agents, metals, radioisotopes, dyes, radionuclide chelators, or other suitable materials that can be used for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties can be linked to the binding protein. Non-limiting examples of radioisotopes include alpha-emitters, beta-emitters, positron-emitters, gamma-emitters, and the like. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not readily quenched by biological tissues and fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation at wavelengths between 650 nm and 4000 nm, and such radiation is used to detect such agents. Non-limiting examples of fluorescent dyes that can be used as conjugating molecules include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, near-infrared dyes often include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Further non-limiting examples of fluorescent dyes for use as conjugating molecules in accordance with the present invention include:Acradine orange or yellow, Alexa Fluors® (e.g., Alexa Fluor® 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO® dyes and any derivatives thereof, auramine-rhodamine stains and any derivatives thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl) nylanthracene and any derivatives thereof, DAPI, DiOC6, DyLight® Fluors® and any derivatives thereof, epicocconone, ethidium bromide, FlAsH®-EDT2®, Fluo dyes and any derivatives thereof, FluoProbe® and any derivatives thereof, fluorescein and any derivatives thereof, Fura® and any derivatives thereof, GelGreen® and any derivatives thereof, GelRed® and any derivatives thereof, fluorescent proteins and any derivatives thereof, m-isoform proteins and any derivatives thereof, such as mCherry, hetamethine dyes dye) and any derivatives thereof, Hoechst stain, iminocoumarin, Indian yellow, indo-1 and any derivatives thereof, laurdan, lucifer yellow and any derivatives thereof, luciferin and any derivatives thereof, luciferase and any derivatives thereof, mercocyanin and any derivatives thereof, Nile dye and any derivatives thereof, perylene, phloxine, phycodye and any derivatives thereof, propium iodide, pyranine, rhodamine and any derivatives thereof, ribogreen, RoGFP, rubrene, stilbene and any derivatives thereof, sulforhodamine and any derivatives thereof,Other suitable fluorescent dyes include, but are not limited to, SYBR and any derivatives thereof, synaptofluorin, tetraphenylbutadiene, tetrasodium tris, Texas Red, titanium yellow, TSQ, umbelliferone, violanthrone, yellow fluorescent protein, and YOYO-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine, etc.). 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, 500, 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 Suitable detectable agents include FLUOR® dyes (e.g., ALEXA FLUOR® 350, 488, 532, 546, 568, 594, 633, 660, 680, etc.), BODIPY® dyes (e.g., BODIPY® FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), and IRD dyes (e.g., IRD40™, IRD700™, IRD800™, etc.). Additional suitable detectable agents are known in the art (e.g., PCT Publication No. PCT / US14 / 56177). Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters,and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.

[0246] The binding protein can be conjugated to a radiosensitizer or photosensitizer. Radiosensitizers include, but are not limited to, ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleobase derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to, fluorescent molecules or beads that generate heat when irradiated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, and the like. dyes), chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, Nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rose bengals), dimeric and oligomeric porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this approach allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent (e.g., a drug) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the binding protein is fused to or covalently or non-covalently linked to the agent, e.g., directly or via a linker.

[0247] In some embodiments, binding proteins can be chemically modified. For example, binding proteins can be mutated to modify peptide properties, such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is one example of methylation that can occur on binding proteins encompassed by the present invention. In some embodiments, binding proteins can be modified by methylation of free amines, for example, reductive methylation with formaldehyde and sodium cyanoborohydride.

[0248] Chemical modifications can include polymers, polyethers, polyethylene glycol, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains such as palmitate or myristoleate, or albumin. Chemical modifications of binding proteins with Fc regions can be fusion Fc-peptides. Polyamino acids can include, for example, polyamino acid sequences with repeating single amino acids (e.g., polyglycine) and polyamino acid sequences with mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the above.

[0249] In some embodiments, binding proteins encompassed by the invention may be modified. In some embodiments, the modifications produce functional variants that have substantial or significant sequence identity with the parent binding protein and that retain one or more biophysical and / or biological activities of the parent binding protein (e.g., retain pMHC-binding specificity). In some embodiments, the mutations are conservative amino acid substitutions.

[0250] In some embodiments, binding proteins encompassed by the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, oc-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and oc-tert-butylglycine.

[0251] Binding proteins encompassed by the present invention may 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.

[0252] In some embodiments, the half-life of peptides encompassed by the present invention may be extended using hydrophobic moieties, for example, at the N-terminus, C-terminus, or internal amino acid. In other embodiments, the binding protein may contain post-translational modifications (e.g., methylation and / or amidation), which may affect, for example, serum half-life. In some embodiments, a simple carbon chain (e.g., by myristoylation and / or palmitylation) may be conjugated to the binding protein. In some embodiments, a simple carbon chain may allow the binding protein to be easily separated from unconjugated materials. For example, methods that may be used to separate the binding protein from unconjugated materials include, but are not limited to, solvent extraction and reverse-phase chromatography. A lipophilic moiety may extend half-life by reversible binding to serum albumin. The conjugated moiety may be a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety may be cholesterol or a cholesterol derivative, including cholestenone, cholestane, cholestadiene, and oxysterols. In some embodiments, the binding protein may be conjugated to myristic acid (tetradecanoic acid) or its derivatives. In other embodiments, the binding protein may be coupled (e.g., conjugated) to a half-life modifier. Examples of half-life modifiers include, but are not limited to, polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers containing glycine, glutamic acid, and serine, molecules that bind to Fc regions, fatty acids, palmitic acid, or albumin. In some embodiments, a spacer or linker may be attached to the binding protein, such as one, two, three, four, or more amino acid residues that function as a spacer or linker to facilitate conjugation or fusion with other molecules and to facilitate cleavage of the peptide from such conjugated or fused molecules.In some embodiments, the binding proteins may be conjugated with other moieties that can, for example, modify or effect changes in the properties of the binding protein.

[0253] Binding proteins can be produced recombinantly or synthetically, such as by solid-phase or solution-phase peptide synthesis. Polypeptide synthesis can be carried out by known synthetic methods, for example, using fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. Polypeptide fragments can be linked enzymatically or synthetically.

[0254] Within aspects encompassed by the invention, provided herein are methods of producing a binding protein described herein, comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of the binding protein; and (ii) recovering the expressed binding protein.

[0255] A useful method for isolating and purifying recombinantly produced binding proteins can include, for example, obtaining a supernatant from a suitable host cell / vector system that secretes the binding protein into the culture medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as affinity matrices or ion exchange resins. One or more reverse-phase HPLC steps can be utilized to further purify the recombinant polypeptide. These purification methods can also be used when isolating immunogens from their natural environment. Methods for large-scale production of one or more of the binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the binding proteins can be performed according to methods described herein and known in the art.

[0256] In any of the embodiments disclosed herein, the encoded binding protein is capable of binding to a peptide-MHC (pMHC) complex that includes the PRAME immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the MHC molecule includes an MHC alpha chain that is of the HLA serotype HLA-A*02. In some embodiments, the HLA allele is selected from the group consisting of HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0205, HLA-A*0206, and HLA-A*0207 alleles.

[0257] A variety of assays are known for assessing binding affinity and / or for determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand (e.g., a peptide antigen-MHC complex). It is within the level of ordinary skill in the art to determine the binding affinity of a binding protein for a target, e.g., a T-cell peptide epitope of a target polypeptide, for example, by using any of several binding assays known in the art. For example, in some embodiments, a Biacore™ device may be used to determine the binding constant of a complex between two proteins. The dissociation constant (K D) can be determined by monitoring the change in refractive index with time as a buffer flows over the chip. Other assays suitable for measuring the binding of one protein to another include, for example, immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs), or determining binding by monitoring changes in the spectroscopic or optical properties of the proteins by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore™) analysis (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660, Wilson (2002) Science 295:2103, Wolff et al. (1993) Cancer Res. 53:2560, and U.S. Pat. Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, apparent affinity for a target is measured by assessing binding to various concentrations of tetramer, e.g., by flow cytometry using labeled tetramer. In one representative example, the apparent K of a binding protein is determined. D is measured using a range of two-fold dilutions of the labeled tetramer, followed by determination of the binding curve by nonlinear regression, whereas the apparent K D is determined as the concentration of ligand that results in half-maximal binding.

[0258] III. Nucleic Acids and Vectors In aspects encompassed by the present invention, provided herein are nucleic acid molecules encoding the proteins described herein, such as PRAME immunogenic peptides and fragments thereof, MHC molecules, binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, CARs, etc.).

[0259] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to the complement of a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, e.g., over the full length, to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-4.

[0260] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to the complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-4.

[0261] In some embodiments, the nucleic acid molecule comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence that encodes a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-4.

[0262] In some embodiments, the nucleic acid sequence encodes a PRAME immunogenic peptide described herein.

[0263] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α chain CDR shown in Table 2. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α chain CDR shown in Table 2. α TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. αIn some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR alpha chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR alpha chain sequence shown in Table 2.

[0264] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β chain CDR shown in Table 2. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β chain CDR shown in Table 2. β TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. β In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR β chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β chain sequence shown in Table 2.

[0265] The term "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, which may be single- or double-stranded, synthetic or obtained from natural sources (e.g., isolated and / or purified), and which may contain natural, non-natural, or modified nucleotides, and which may include natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides. In one embodiment, nucleic acid includes complementary DNA (cDNA).

[0266] In some embodiments, the nucleic acids encompassed by the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule that can be replicated in a living cell, or (ii) a molecule resulting from replication of the molecule described in (i) above. For purposes herein, replication can be in vitro / ex vivo replication or in vivo replication.

[0267] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green and Sambrook et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to improve the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids encompassed by the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA).

[0268] In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound by any particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence improves the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence may involve replacing a natural codon with another codon that encodes the same amino acid but that can be translated by a more readily available tRNA in the cell, thereby increasing translation efficiency. Optimizing a nucleotide sequence may also reduce secondary structures in the mRNA that would otherwise interfere with translation, thereby increasing translation efficiency. In some embodiments, the nucleotide sequences described herein are codon-optimized for expression in a host cell (e.g., an immune cell such as a T cell).

[0269] The present invention also provides nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0270] Nucleotide sequences that hybridize under stringent conditions can also hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any of the nucleotide sequences of the nucleic acids described herein) with a detectable degree of specificity greater than that observed in nonspecific hybridization. High stringency conditions include conditions that will distinguish polynucleotides with exact complementary sequences or polynucleotides containing only scattered minor mismatches from random sequences that happen to have only small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small complementary regions melt more easily than full-length complements of 14-17 bases or more, making them readily distinguishable by high stringency hybridization. Relatively high stringency conditions may include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions are particularly suitable for detecting expression of any of the TCRs of the present invention, as they tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand. It is widely recognized that conditions can be made more stringent by adding increasing amounts of formamide.

[0271] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids described herein.

[0272] Typically, the nucleic acid is a DNA or RNA molecule that can be contained in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0273] The terms "vector," "cloning vector," and "expression vector" refer to delivery vehicles by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell, thereby transforming the host and promoting expression (e.g., transcription and translation) of the introduced sequences. Accordingly, a further object encompassed by the present invention relates to vectors containing the nucleic acids encompassed by the present invention.

[0274] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to bring about or direct the expression of the polypeptide when administered to a subject. Examples of promoters and enhancers used in expression vectors for animal cells include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and the immunoglobulin heavy chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983).

[0275] Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1 beta d2-4- (Miyaji H et al. 1990). Other representative examples of plasmids include replicative plasmids containing an origin of replication or integrative plasmids, such as pUC, pcDNA, and pBR. Representative examples of viral vectors include adenoviral, retroviral, lentiviral, herpesviral, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses are well known in the art and can be found, for example, in PCT Publication No. WO 95 / 14785, PCT Publication No. WO 96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and PCT Publication No. WO 94 / 19478.

[0276] In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a binding protein or polypeptide, or fragment thereof, described herein. In some embodiments, the nucleic acid comprises regulatory elements required for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, an enhancer may be included. These elements may be operably linked to the sequence encoding the binding protein, polypeptide, or fragment thereof.

[0277] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, a dominant-negative TGFβ receptor (e.g., DN-TGFβRII), a selective protein marker, optionally wherein the selective protein marker is dihydrofolate reductase (DHFR). In certain embodiments, the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβR, and / or the selective protein marker is operably linked to a nucleic acid encoding a tag (e.g., a CD34 enrichment tag). In certain embodiments, the nucleic acid sequences described herein, e.g., the nucleic acid sequences encoding TCRα, TCRβ, CD8α, CD8β, DN-TGFβR, and / or the selective protein marker, are interconnected with a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide such as P2A, E2A, F2A, or T2A.

[0278] In some embodiments, the expression vectors provided herein comprise a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids set forth in Tables 1-3.

[0279] Examples of promoters include, but are not limited to, promoters from simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV), e.g., the HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV), e.g., the CMV immediate early promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Examples of suitable polyadenylation signals include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal.

[0280] In addition to the regulatory elements required for expression, other elements may be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include promoters as described herein. In some embodiments, enhancers / promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers, such as those from CMV, RSV, and EBV.

[0281] In some embodiments, the nucleic acid may be operably incorporated into a carrier or delivery vector, as described further below. Useful delivery vectors include, but are not limited to, biodegradable microcapsules, immunostimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live carriers, such as viruses or bacteria.

[0282] In some embodiments, the vector is a viral vector, such as a lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, fowlpox, AVpox, modified vaccinia Ankara (MVA), and other recombinant viruses. For example, a lentiviral vector can be used to infect T cells.

[0283] In some embodiments, the recombinant expression vector is capable of delivering the polynucleotide to a suitable host cell, e.g., a T cell or an antigen-presenting cell, i.e., a cell that presents peptide / MHC complexes on its cell surface (e.g., a dendritic cell) and lacks CD8. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. For example, the immune system cell is a CD4 + T cells, CD8 +The T cells may be T cells, CD4 / CD8 double-negative T cells, gd T cells, natural killer cells, dendritic cells, or any combination thereof. In some embodiments, the T cells are host, and the T cells may be naive, central memory T cells, effector memory T cells, or any combination thereof. Thus, the recombinant expression vector may also include a lymphoid tissue-specific transcriptional regulatory element (TRE), such as a TRE specific for B lymphocytes, T lymphocytes, or dendritic cells. Lymphoid tissue-specific TREs are known in the art (see, e.g., Thompson et al. (1992) Mol. Cell. Biol. 72:1043; Todd et al. (1993) J. Exp. Med. 777:1663; and Penix et al. (1993) J. Exp. Med. 775:1483).

[0284] In some embodiments, the recombinant expression vector comprises nucleotide sequences encoding a TCR alpha chain, a TCR beta chain, and / or a linker peptide. For example, in some embodiments, the recombinant expression vector comprises nucleotide sequences encoding the full-length TCR alpha and TCR beta chains of a binding protein with a linker therebetween, wherein the nucleotide sequence encoding the beta chain is located 5' to the nucleotide sequence encoding the alpha chain. In some embodiments, the nucleotide sequences encode the full-length TCR alpha and TCR beta chains with a linker therebetween, wherein the nucleotide sequence encoding the TCR beta chain is located 3' to the nucleotide sequence encoding the TCR alpha chain. In some embodiments, the full-length TCR alpha and / or TCR beta chains are replaced with fragments thereof.

[0285] As described further below, another aspect encompassed by the present invention relates to cells transfected, infected, or transformed with a nucleic acid and / or vector according to the present invention. A host cell can include any individual cell or cell culture that can receive vector or nucleic acid and / or protein incorporation, as well as any progeny. The term also includes the descendants of the host cell, whether genetically or phenotypically the same or different. Suitable host cells will depend on the vector and can include mammalian, animal, human, simian, insect, yeast, and bacterial cells. These cells can be induced to take up vectors or other materials by transformation via viral vectors, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2d ed. (Cold Spring Harbor Laboratory)). The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed."

[0286] Nucleic acids encompassed by the invention can be used to produce recombinant polypeptides, including recombinant polypeptides of the invention, in a suitable expression system. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0287] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al. (1980)), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, YB2 / 0 cells are used because the ADCC activity of chimeric or humanized binding proteins is enhanced when expressed in these cells.

[0288] The present invention also encompasses methods for producing recombinant host cells expressing binding proteins, peptides, and fragments thereof, comprising the steps of (i) introducing a recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo, (ii) culturing the resulting recombinant host cell in vitro or ex vivo, and (iii) optionally selecting cells expressing the binding protein, peptide, and fragments thereof. Such recombinant host cells may be used in the diagnostic, prognostic, and / or therapeutic methods encompassed by the present invention.

[0289] In another aspect, the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides included in the present invention can be used to identify, isolate, or amplify partial or full-length clones within a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences complementary to cDNAs isolated from or derived from a human or mammalian nucleic acid library. In some embodiments, the cDNA library contains at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more full-length sequences, or any range therebetween, inclusive of the endpoints, e.g., at least about 80%-100%. The cDNA library can be normalized to enhance the representation of rare sequences. Low or medium stringency hybridization conditions are typically, but not exclusively, used for sequences with low sequence identity relative to the complementary sequence. Medi...

Claims

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

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

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

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

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

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

7. The immunogenic composition of claim 5 or 6, wherein the immunogenic composition is capable of inducing an immune response in a subject against PRAME and / or PRAME-expressing cells, and optionally 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. 9. The composition of claim 8, wherein the MHC molecule is an MHC multimer, and optionally, the MHC multimer is a tetramer.

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

11. The MHC alpha chain, wherein the MHC molecule is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, and optionally, the HLA allele is HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 allele, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 allele, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 allele, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 allele, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02,HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, H LA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HL The composition of any one of claims 9 to 11, wherein the allele is selected from the group consisting of HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05, and HLA-C*14:

03.

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

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

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

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

01.

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

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

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

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

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

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

22. 18. A device or kit comprising a) one or more immunogenic peptides of any one of claims 1 to 4, and / or b) one or more stable MHC-peptide complexes of any one of claims 12 to 17, optionally comprising a reagent for detecting binding of a) and / or b) to a binding protein, optionally wherein the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

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

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

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

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

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

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

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

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

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

32. 32. The method of any one of claims 27 to 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), immunochemically, Western blot, or intracellular flow assay.

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

34. The method of any one of claims 27 to 33, wherein the control level is the level in a subject who does not have the disorder characterized by PRAME expression.

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

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

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

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

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

40. 40. The method of claim 39, wherein the plurality of candidate peptide-binding molecules comprises antibodies, antigen-binding fragments of antibodies, TCRs, antigen-binding fragments of TCRs, single-chain TCRs (scTCRs), chimeric antigen receptors (CARs), or fusion proteins comprising a TCR and an effector domain.

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

42. 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 a sample from a subject or a population of subjects, or wherein the plurality of candidate peptide-binding molecules comprises one or more candidate peptide-binding molecules that comprise mutations in a parent scaffold peptide-binding molecule obtained from a sample from a subject.

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

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

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

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

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

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

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

50. 50. The method of claim 49, wherein the T cells are isolated from a) the subject, b) a donor not afflicted with the disorder characterized by PRAME expression, or c) a donor who has recovered from a disorder characterized by PRAME expression.

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

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

53. 53. The method of claim 51 or 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. 54. The method of any one of claims 23-53, wherein the disorder characterized by PRAME expression is cancer or its recurrence, and optionally the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, leukemia, ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma.

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

56. 18. A binding protein that binds to a polypeptide comprising the immunogenic peptide sequence of any one of claims 1 to 4, the immunogenic peptide of any one of claims 1 to 4, and / or the stable MHC-peptide complex of any one of claims 12 to 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. a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity with a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2, and / or b) a TCR beta chain CDR sequence having at least about 80% identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity of at least about 5x10 －４ K below M ｄ 57. The binding protein of claim 56, having the following structure:

58. a) TCR alpha chain variable (V) α ) TCR V domain sequences selected from the group consisting of α TCR V domain sequence having at least about 80% identity with α domain sequences, and / or b) TCR beta chain variable (V) sequences listed in Table 2 β ) TCR V domain sequences selected from the group consisting of β TCR V domain sequence having at least about 80% identity with β domain sequence, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity of about 5x10 －４ K below M ｄ 57. The binding protein of claim 56, having the following structure:

59. a) a TCR alpha chain sequence having at least about 80% identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2, and / or b) a TCR beta chain sequence having at least about 80% identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity of at least about 5x10 －４ K below M ｄ 57. The binding protein of claim 56, having the following structure:

60. a) a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 2, and / or b) a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity of about 5x10 －４ K below M ｄ 57. The binding protein of claim 56, having the following structure:

61. a) TCR alpha chain variable (V) α ) TCR V domain sequences selected from the group consisting of α domain sequences, and / or b) TCR beta chain variable (V) sequences listed in Table 2 β ) TCR V domain sequences selected from the group consisting of β domain sequence, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity of about 5x10 －４ K below M ｄ 57. The binding protein of claim 56, having the following structure:

62. a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 2, and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity of about 5x10 －４ K below M ｄ 57. The binding protein of claim 56, having the following structure:

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

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

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

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

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

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

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

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

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

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

73. The binding protein binds to the PRAME immunogenic peptide-MHC (pMHC) complex at a concentration of about 1 x 10 －４ M or less, approximately 5x10 －５ M or less, approximately 1x10 －５ M or less, approximately 5x10 －６ M or less, approximately 1x10 －６ M or less, approximately 5x10 －７ MM or less, approximately 1x10 －７ M or less, approximately 5x10 －８ M or less, approximately 1x10 －８ M or less, approximately 5x10 －９ M or less, approximately 1x10 －９ M or less, approximately 5x10 －１０ M or less, approximately 1x10 －１０ M or less, approximately 5x10 －１１ M or less, approximately 1x10 －１１ MM or less, approximately 5x10 －１２ MM or less, or about 1 x 10 －１２ K below M ｄ 73. The binding protein of any one of claims 56 to 72, which is capable of specifically and / or selectively binding to

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

75. 75. The binding protein of any one of claims 56-74, wherein the binding protein, when contacted with a target cell having heterozygous expression of PRAME, induces greater 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 greater.

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

77. 77. The binding protein of claim 76, wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, leukemia, ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma.

78. 78. The binding protein of any one of claims 56 to 77, wherein the binding protein does not bind to a pMHC complex comprising a PLA2G4E, EFNA1, and / or SLC26A1 peptide epitope.

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

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

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

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

83. 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), a selective protein marker, optionally wherein the selective protein marker is dihydrofolate reductase (DHFR).

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

85. 85. The vector of claim 83 or 84, wherein the nucleic acid encoding the tag is 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. 86. The vector of claim 84 or 85, wherein the tag is a CD34 enrichment tag.

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

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

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

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

91. 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 the alpha 1 macroglobulin gene, the alpha 2 macroglobulin gene, the alpha 3 macroglobulin gene, the beta 1 microglobulin gene, the beta 2 microglobulin gene, and combinations thereof.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

4.

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

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

111. 111. The host cell of any one of claims 89-110, wherein the target cell is a cell line or a primary cell, optionally wherein the target cell is selected from the group consisting of a HEK293-derived cell line, a cancer cell line, a primary cancer cell, a transformed cell line, and an immortalized cell line, further optionally wherein the cell line is Hs695T, A375, or NCI-H1563.

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

113. 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 when contacted with a target cell containing a peptide-MHC (pMHC) complex that includes a PLA2G4E, EFNA1, and / or SLC26A1 peptide epitope.

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

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

116. 1. A composition comprising: a) a binding protein according to any one of claims 56 to 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 to 114; and / or e) a population of host cells according to claim 115, and a carrier.

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

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

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

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

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 PRAME peptide in association with an MHC molecule, and the complex is detected by fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay.

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

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

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

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

126. 1. A method of monitoring the progression of a disorder characterized by PRAME expression in a subject, comprising: a) detecting in a subject sample the presence or level of reactivity between a sample obtained from said subject and at least one binding protein of any one of claims 56 to 77, at least one host cell of any one of claims 89 to 114, or a population of host cells of claim 115; b) repeating step a) at a subsequent time point; and c) comparing the levels of PRAME or the target cells expressing PRAME detected in steps a) and b) to monitor the progression of the disorder characterized by PRAME expression in the subject, wherein the absence or decrease of the PRAME level or the target cells expressing PRAME detected in step b) compared to step a) indicates an inhibition of the progression of the disorder characterized by PRAME expression in the subject, and the presence or increase of the PRAME level or the target cells expressing PRAME detected in step b) compared to step a) indicates the progression of the disorder characterized by PRAME expression in the subject.

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

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

129. 1. A method for assessing the efficacy of a therapy for a disorder characterized by PRAME expression, comprising: a) determining in a first sample obtained from a subject prior to providing to the subject at least part of said therapy for said disorder characterized by PRAME expression, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein of any one of claims 56-77, at least one host cell of any one of claims 89-114, or a population of host cells of claim 115; b) determining in a second sample obtained from the subject after providing said therapy for said disorder characterized by PRAME expression the presence or level of said reactivity between the sample obtained from the subject and at least one binding protein of any one of claims 56-77, at least one host cell of any one of claims 89-114, or a population of host cells of claim 115; The method, wherein the absence or reduced level of reactivity in the second sample compared to the first sample is an indication that the therapy is effective in treating the disorder characterized by PRAME expression in the subject, and the presence or increased level of reactivity in the second sample compared to the first sample is an indication that the therapy is not effective in treating the disorder characterized by PRAME expression in the subject.

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

131. 131. The method of any one of claims 120 to 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), immunochemically, Western blot, or intracellular flow assay.

132. 100. A method for preventing and / or treating a disorder characterized by PRAME expression, comprising contacting target cells expressing PRAME with a therapeutically effective amount of a composition comprising cells expressing at least one binding protein of any one of claims 56 to 77, and optionally, administering the composition to a subject.

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

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

135. The method of any one of claims 49 to 55 and 132 to 134, wherein the target cell is a cancer cell that expresses PRAME.

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

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

138. 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 PRAME in the subject.

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

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

141. 141. The method of any one of claims 132-140, wherein the disorder characterized by PRAME expression is cancer or its recurrence, and optionally the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, leukemia, ovarian cancer, renal cell carcinoma (RCC), breast cancer, cervical cancer, or colon cancer, sarcoma, and neuroblastoma.

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

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