Prame immunogenic peptides, binding proteins recogniting the same, and uses thereof

Abstract

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

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Application Serial No. 63 / 413,552, filed on October 5, 2022, and U.S. Provisional Application Serial No. 63 / 423,294, filed on November 7, 2022; the entire content of each of said applications is incorporated herein by reference in its entirety. Background of the Invention

[0004] The cancer / testis antigen PRAME is exemplified as an ideal target for TCR - T cell therapy because it is highly expressed in a variety of malignancies and absent 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 other solid tumors including lung cancer, head and neck cancer, and ovarian cancer. PRAME plays a key role in multiple cellular processes and has been shown to exhibit pro - oncogenic functions mainly by inhibiting retinoic acid receptor signaling (Epping et al. (2005) Cell 122:835 - 847). Targeting PRAME in solid tumors, especially when part of a TCR - T multiplex strategy, represents a promising therapeutic approach for treating multiple cancer indications. There is a need to develop PRAME - specific TCR immunotherapies for treating conditions characterized by PRAME expression. Summary of the Invention

[0005] The present invention is at least in part based on the discovery of PRAME immunogenic peptides and binding proteins that recognize such PRAME immunogenic peptides, based on an unbiased functional screen for antigens of TCR clonotypes identified from subjects suffering from a condition 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). In the case of a variety of HLA alleles (e.g., HLA - A*02:01), the identified TCRs recognize PRAME immunogenic peptides such as those listed in Table 1. It is 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., TCRs as described herein) to bind PRAME immunogenic peptides and initiate an immune response that kills cells expressing PRAME (e.g., cancer cells) demonstrates the utility of such binding proteins in a variety of uses, including methods of diagnosis, prognosis, treating conditions characterized by PRAME expression, and screening for agents associated with said conditions.

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

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

[0008] Also provided are multiple embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the immunogenic peptide is derived from the PRAME protein, optionally wherein the length of the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. In another embodiment, the immunogenic peptide is capable of eliciting an immune response in a subject against PRAME and / or cells expressing PRAME, optionally wherein the immune response is i) a T cell response and / or a CD8+ T cell response and / or ii) selected from the group consisting of T cell expansion (e.g., proliferation), cytokine release and / or cytotoxic killing.

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

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

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

[0012] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally wherein the MHC multimer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule. In another embodiment, the MHC molecule comprises an MHC α-chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 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-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 another embodiment, the HLA serotype is HLA-A*02, such as HLA-A*02:01.,

[0013] In another aspect, there is provided a stabilized MHC-peptide complex comprising an immunogenic peptide as described herein in the context of an MHC molecule.

[0014] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the MHC molecule is an MHC multimer, optionally wherein the MHC multimer is a tetramer. In another embodiment, the MHC molecule is a class I MHC molecule. In another embodiment, the MHC molecule comprises an MHC α-chain that is an HLA serotype selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02,The HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In yet another embodiment, the peptide epitope and the MHC molecule are covalently linked and / or the α-chain and β-chain of the MHC molecule are covalently linked. In another embodiment, the stable MHC-peptide complex comprises a detectable label, optionally wherein the detectable label is a fluorophore.

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

[0016] In yet another aspect, there is provided an isolated nucleic acid encoding an immunogenic peptide as described herein, or its complement.

[0017] In another aspect, there is provided a vector comprising the isolated nucleic acid as described herein.

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

[0019] 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 reagents for detecting the binding of a) and / or b) to a binding protein, optionally wherein the binding protein is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

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

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

[0022] In another aspect, a method for determining whether a T cell has been exposed to PRAME is provided, the method 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 of reactivity or a higher level of reactivity compared to a control level indicates that the T cell has been exposed to PRAME, optionally wherein the cell population comprising T cells is obtained from a subject.

[0023] In yet another aspect, a method for predicting the clinical outcome of a subject suffering from a disorder characterized by PRAME expression is provided, the method comprising: a) determining the presence or level of reactivity between T cells obtained from the subject and one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein; and b) comparing the presence or level of reactivity with the reactivity from a control, wherein the control is obtained from a subject with a favorable clinical outcome, wherein the presence of reactivity or a higher level of reactivity in the subject sample compared to the control indicates that the subject has a favorable clinical outcome.

[0024] In another aspect, a method for assessing the efficacy of a therapy for a disorder characterized by PRAME expression is provided, the method comprising: a) determining the presence or level of reactivity between T cells obtained from a 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 prior to providing at least a portion of the therapy to the subject, and b) determining the presence or level of reactivity between one or more immunogenic peptides described herein or one or more stable MHC-peptide complexes described herein and T cells obtained from the subject, wherein the T cells are present in a second sample obtained from the subject after providing the therapy to the subject, wherein the presence of reactivity or a higher level of reactivity in the second sample compared to the first sample indicates that the therapy is effective in treating the subject's disorder characterized by PRAME expression.

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

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

[0027] In yet another aspect, a method of identifying a peptide-binding molecule or an antigen-binding fragment thereof that binds to a peptide epitope selected from the peptide sequences listed in Table 1 is provided, the method comprising: a) providing a cell that presents on its cell surface a peptide epitope selected from the peptide sequences listed in Table 1 in the context of an MHC molecule; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to the peptide epitope in the context of the MHC molecule on the cell; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to the peptide epitope in the context of the MHC molecule.

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

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

[0030] Also provided are a plurality of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the plurality of candidate peptide-binding molecules comprise antibodies, antigen-binding fragments of antibodies, TCRs, antigen-binding fragments of TCRs, single-chain TCRs (scTCRs), chimeric antigen receptors (CARs), or fusion proteins comprising a TCR and an effector domain. In another embodiment, the plurality of candidate peptide-binding molecules comprise at least 2, 5, 10, 100, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species, 10 9 species or more different candidate peptide-binding molecules. In another embodiment, the plurality of candidate peptide-binding molecules comprise one or more candidate peptide-binding molecules obtained from a sample from a subject or a group of subjects; or the plurality of candidate peptide-binding molecules comprise one or more candidate peptide-binding molecules comprising mutations in a parental scaffold peptide-binding molecule obtained from a sample from a subject. In yet another embodiment, the subject or group of subjects: a) does not have a disorder characterized by PRAME expression and / or has recovered from a disorder characterized by PRAME expression, or b) has a disorder characterized by PRAME expression. In another embodiment, the composition described herein has been administered to the subject or group of subjects. In another embodiment, the subject is an animal model and / or a mammal of a disorder characterized by PRAME expression, optionally wherein the mammal is a human, a primate, or a rodent. In yet another embodiment, the subject is an animal model of a disorder characterized by 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.

[0031] In another aspect, there is provided a peptide-binding molecule or an antigen-binding fragment thereof identified according to the methods described herein, optionally wherein the peptide-binding molecule or an antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0032] In yet another aspect, there is provided a method of treating a subject having a disorder characterized by PRAME expression, the method comprising administering to the subject a therapeutically effective amount of genetically engineered T cells that express a peptide-binding molecule or an antigen-binding fragment thereof, wherein the peptide-binding molecule or an antigen-binding fragment thereof i) binds to a peptide epitope selected from the sequences listed in Table 1, ii) is identified according to the methods described herein, and / or iii) binds to a stable MHC-peptide complex comprising a peptide epitope selected from the sequences listed in Table 1 in the context of an MHC molecule, optionally wherein the peptide-binding molecule or an antigen-binding fragment thereof is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain, optionally wherein the MHC or MHC-peptide complex is as described herein.

[0033] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. By way of example, in one embodiment, the T cells are isolated from: a) a subject, b) a donor not having a disorder characterized by PRAME expression, or c) a donor recovered from a disorder characterized by PRAME expression.

[0034] In another aspect, there is provided a method of treating a subject having a disorder characterized by PRAME expression, the method comprising infusing into the subject antigen-specific T cells, wherein the antigen-specific T cells are generated by: a) stimulating immune cells from the subject with the compositions described herein; and b) expanding the antigen-specific T cells in vitro or ex vivo, optionally i) isolating the immune cells from the subject prior to stimulating the immune cells and / or ii) wherein the immune cells comprise PBMCs, T cells, CD8+ T cells, naive T cells, central memory T cells, and / or effector memory T cells.

[0035] A number of embodiments are also provided, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the agent is contacted and placed under conditions and for a time suitable for forming at least one immune complex between a peptide epitope, an immunogenic peptide, a stable MHC-peptide complex, a T cell receptor, and / or an immune cell. In another embodiment, the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor are expressed by a cell and the cell is amplified and / or isolated during one or more steps. In another embodiment, the disorder characterized by PRAME expression is cancer or its recurrence, 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 and / or a mammal of a disorder characterized by PRAME expression, optionally wherein the mammal is a human, a primate, or a rodent.

[0036] In another aspect, a binding protein is provided 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.

[0037] A number of embodiments are also provided, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the binding protein comprises: a) a T cell receptor (TCR) α-chain CDR sequence that has at least about 80% identity with a TCR α-chain CDR sequence selected from the group consisting of the TCR α-chain CDR sequences listed in Table 2; and / or b) a TCR β-chain CDR sequence that has at least about 80% identity with a TCR β-chain CDR sequence selected from the group consisting of the TCR β-chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d . In another embodiment, the binding protein comprises: a) a T cell receptor (TCR) α-chain variable (V α ) domain sequence that has at least about 80% identity with a TCR V α domain sequence selected from the group consisting of the TCR V α domain sequences listed in Table 2; and / or b) a TCR β-chain variable (Vβ ) A domain sequence that has at least about 80% identity with a TCR V domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 β domain sequences, and the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 β domain sequences, and the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d . In another embodiment, the binding protein comprises: a) a TCR α-chain sequence that has at least about 80% identity with a TCR α-chain sequence selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) a TCR β-chain sequence that has at least about 80% identity with a TCR β-chain sequence selected from the group consisting of the TCR β-chain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d . In yet another embodiment, the binding protein comprises: a) a TCR α-chain CDR sequence selected from the group consisting of the TCR α-chain CDR sequences listed in Table 2; and / or b) a TCR β-chain CDR sequence selected from the group consisting of the TCR β-chain CDR sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d . In yet another embodiment, there is provided a binding protein comprising: a) a TCR α-chain variable (V α ) domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2; and / or b) a TCR β-chain variable (V α ) domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 β ) domain sequences, and the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 β domain sequences, and the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4 M d . In another embodiment, there is provided a binding protein comprising: a) a TCR α-chain sequence selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) a TCR β-chain sequence selected from the group consisting of the TCR β-chain sequences listed in Table 2, wherein the binding protein is capable of binding to a PRAME immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K of less than or equal to about 5×10 -4K of M d 。In another embodiment, 1) the TCRα-chain CDR, TCRV α domain and / or the TCRα-chain is encoded by a TRAV, TRAJ, and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) the TCRβ-chain CDR, TCR V β domain and / or the TCRβ-chain is encoded by a TRBV, TRBJ, and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) compared to the homologous reference CDR sequences listed in Table 2, each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions, or combinations thereof. In another embodiment, the binding protein is chimeric, humanized, or human. In yet another embodiment, the binding protein comprises a binding domain having a transmembrane domain and an intracellular effector domain. In another embodiment, the TCRα-chain and the TCRβ-chain are covalently linked, optionally wherein the TCRα-chain and the TCRβ-chain are covalently linked via a linker peptide. In another embodiment, the TCRα-chain and / or the TCRβ-chain is covalently linked to a moiety, optionally wherein the covalently linked moiety comprises an affinity tag or a label. In yet another embodiment, the affinity tag is selected from the group consisting of: CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or wherein the label is a fluorescent protein. In another embodiment, the covalently linked moiety is selected from the group consisting of: pro-inflammatory factors, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies or antigen-binding fragments thereof. In another embodiment, the binding protein binds to a pMHC complex on the cell surface. In yet another embodiment, the MHC or MHC-peptide complex is as described herein. In another embodiment, the binding of the binding protein to a PRAME peptide-MHC (pMHC) complex elicits an immune response, optionally wherein the immune response is i) a T cell response and / or a CD8+ T cell response and / or ii) selected from the group consisting of T cell expansion, cytokine release, and / or cytotoxic killing. In another embodiment, the binding protein is capable of binding at a concentration less than or equal to about 1×10 -4 M, less than or equal to about 5×10 -5 M, less than or equal to about 1×10 - 5 M, less than or equal to about 5×10 -6 M, less than or equal to about 1×10 -6 M, less than or equal to about 5×10 -7 M, less than or equal to about 1×10 -7M, less than or equal to about 5×10 -8 M, less than or equal to about 1×10 -8 M, less than or equal to about 5×10 -9 M, less than or equal to about 1×10 -9 M, less than or equal to about 5×10 -10 M, less than or equal to about 1×10 -10 M, less than or equal to about 5×10 -11 M, less than or equal to about 1×10 -11 M, less than or equal to about 5×10 -12 M or less than or equal to about 1×10 -12 K of M d Specifically and / or selectively binds to a PRAME immunogenic peptide-MHC (pMHC) complex. In another embodiment, the binding protein has a higher binding affinity for the peptide-MHC (pMHC) compared to a known T cell receptor, optionally wherein the higher binding affinity is at least 1.05-fold higher. In another embodiment, when contacting target cells with heterologous expression of PRAME, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing compared to a known T cell receptor, optionally wherein the induction is at least 1.05-fold higher. As used herein, in some embodiments, references to fold changes can be compared to any reference pattern of interest, such as comparing to different binding proteins; comparing the same binding protein in different contexts, such as in combination with other agents as described herein, where the same binding protein is expressed at different levels in different immune cells; and the like. In another embodiment, the cytotoxic killing is directed against target cancer cells. In 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 a peptide-MHC (pMHC) complex comprising a PLA2G4E, EFNA1, and / or SLC26A1 peptide epitope. These genes are well known and are recognized in the art to be annotated according to the following NCBI gene ID numbers, each of which can be obtained at ncbi.nlm.nih.gov / gene on the World Wide Web: PLA2G4E: gene ID 123745; EFNA1: gene ID 1942; and SLC26A1: gene ID 10861.

[0038] In yet another aspect, provided is a TCR α chain and / or β chain, wherein the TCR α chain and / or β chain is selected from the group consisting of the TCR α chain and β chain sequences listed in Table 2.

[0039] In another aspect, there is provided 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) has a sequence that is at least about 80% homologous to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 2, and / or iii) has a sequence that is at least about 80% homologous to the nucleic acid listed in Table 2, optionally wherein the isolated nucleic acid molecule comprises 1) a TRAV, TRAJ, and / or TRAC gene selected from the group consisting of the TRAV, TRAJ, and TRAC genes listed in Table 2 or a fragment thereof and / or 2) a TRBV, TRBJ, and / or TRBC gene selected from the group consisting of the TRBV, TRBJ, and TRBC genes listed in Table 2 or a fragment thereof.

[0040] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the nucleic acid is codon-optimized for expression in a host cell.

[0041] In another aspect, there is provided a vector that comprises the isolated nucleic acid described herein, optionally wherein i) the vector is a cloning vector, an expression vector, or a viral vector and / or ii) the vector comprises a vector sequence listed in Table 3.

[0042] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, dominant negative TGFβ receptor II (DN-TGFβRII), a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In another embodiment, the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag. In another embodiment, the nucleic acid encoding the tag is at the 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein such that the tag is fused to the N-terminus of CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker. In yet another embodiment, the tag is a CD34 enrichment tag. In another embodiment, the isolated nucleic acid described herein alone or in combination with the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker is ligated to an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide. In another embodiment, the self-cleaving peptide is P2A, E2A, F2A, or T2A.

[0043] In yet another aspect, a host cell is provided, the host cell comprising the isolated nucleic acid described herein, comprising the vector described herein, and / or expressing the binding protein described herein, optionally wherein the cell is genetically engineered.

[0044] A number of embodiments are also provided, which can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the host cell comprises a chromosomal gene knockout of a TCR gene, an HLA gene, or both. In another embodiment, the host cell comprises a knockout of an HLA gene selected from the following: alpha-1 macroglobulin gene, alpha-2 macroglobulin gene, alpha-3 macroglobulin gene, beta-1 microglobulin gene, beta-2 microglobulin gene, and combinations thereof. In another embodiment, the host cell comprises a knockout of a TCR gene selected from the following: TCR alpha variable region gene, TCR beta variable region gene, TCR constant region gene, and combinations thereof. In yet another embodiment, the host cell expresses CD8 alpha, CD8 beta, DN-TGF beta RII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR, and further optionally wherein CD8 alpha, CD8 beta, DN-TGF beta RII, and / or the selectable protein marker are fused to a CD34 enrichment tag. In another embodiment, the host cell is enriched using a CD34 enrichment tag. In another embodiment, the host cell is a hematopoietic progenitor cell, a peripheral blood mononuclear cell (PBMC), a cord blood cell, or an immune cell. In yet another embodiment, the immune cell is a T cell, a cytotoxic lymphocyte, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte progenitor cell, a cytotoxic lymphocyte stem cell, a CD4 + T cell, a CD8 +T cells, CD4 / CD8 double-negative T cells, γδ (gammadelta) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or combinations thereof. In another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or combinations thereof. In another embodiment, the T cells are primary T cells or cells of a T cell line. In another embodiment, the T cells do not express endogenous TCR or have a lower surface expression of endogenous TCR. In another embodiment, the host cell is capable of producing cytokines or cytotoxic molecules when contacted with a target cell comprising a peptide-MHC (pMHC) complex, the peptide-MHC (pMHC) complex comprising a PRAME peptide epitope in the context of an MHC molecule. In another embodiment, the host cell is contacted with the target cell in vitro, ex vivo, or in vivo. In another embodiment, the cytokine is TNF-α, IL-2, and / or IFN-γ. In another embodiment, the cytotoxic molecule is perforin and / or granzyme, optionally wherein the cytotoxic molecule is granzyme B. In another embodiment, the host cell is capable of producing a higher level of cytokines or cytotoxic molecules when contacted with a target cell having a heterozygous expression of PRAME. In another embodiment, the host cell is capable of producing a cytokine or cytotoxic molecule at a level at least 1.05-fold higher. In another embodiment, the host cell is capable of killing a target cell comprising a peptide-MHC (pMHC) complex, the peptide-MHC (pMHC) complex comprising a PRAME peptide epitope in the context of an MHC molecule. In another embodiment, the killing is measured by a killing assay. In another embodiment, the ratio of host cell to target cell in the killing assay is from 20:1 to 1:4. In another embodiment, the target cell is a target cell pulsed with 1 μg / mL to 50 pg / mL PRAME peptide, optionally wherein the target cell is a single-allele cell of an MHC matching the PRAME peptide. In another embodiment, the host cell is capable of killing a higher number of target cells when contacted with a target cell having a heterozygous expression of PRAME, optionally wherein the cell killing is at least 1.05-fold higher. In another embodiment, the target cell is a cell line (such as Hs695T, A375, or NCI-H1563) or a primary cell, optionally wherein the target cell is selected from the group consisting of HEK293-derived cell lines, cancer cell lines, primary cancer cells, transformed cell lines, and immortalized cell lines. In another embodiment, the PRAME immunogenic peptide is as described herein and / or wherein the MHC or MHC-peptide complex is as described herein. In another embodiment, the host cell does not induce T cell expansion, cytokine release, or cytotoxic killing when contacted with a target cell comprising a peptide-MHC (pMHC) complex, the peptide-MHC (pMHC) complex comprising a PLA2G4E, EFNA1, and / or SLC26A1 peptide epitope.In another embodiment, the host cell does not express the PRAME antigen, is not recognized by the binding proteins described herein, does not belong to the HLA-A*02 serotype, and / or does not express the HLA-A*02 allele.

[0045] In another aspect, a population of the host cells described herein is provided.

[0046] In another aspect, a composition is provided, the composition comprising a) a binding protein described herein, b) an isolated nucleic acid described herein, c) a vector described herein, d) a host cell described herein, and / or e) a population of the host cells described herein, and a carrier.

[0047] In yet another aspect, a device or kit is provided, the device or kit comprising a) a binding protein described herein, b) an isolated nucleic acid described herein, c) a vector described herein, d) a host cell described herein, and / or e) a population of the host cells described herein, the device or kit optionally comprising a reagent for detecting the binding of a), d), and / or e) to the pMHC complex.

[0048] In another aspect, a method for producing the binding protein described herein is provided, wherein the method comprises the steps of: (i) culturing a transformed host cell under conditions suitable to permit the expression of the binding protein, the host cell having been transformed with a nucleic acid comprising a sequence encoding the binding protein described herein; and (ii) recovering the expressed binding protein.

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

[0050] In yet another aspect, a method for detecting the presence or absence of the PRAME antigen and / or cells expressing PRAME is provided, optionally wherein the cells are hyperproliferative cells, the method comprising detecting the presence or absence of the PRAME antigen in a sample by using at least one of the binding proteins described herein, at least one of the host cells described herein, or a population of the host cells described herein, wherein detection of the PRAME antigen indicates the presence of the PRAME antigen and / or cells expressing PRAME.

[0051] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. 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 in the form of fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, western blotting, or intracellular flow cytometry. In another embodiment, the method further comprises obtaining a sample from a subject.

[0052] In another aspect, a method for detecting the extent of a disorder characterized by PRAME expression in a subject is provided, 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 a higher level of reactivity compared to a control level indicates the extent of the disorder characterized by PRAME expression in the subject.

[0053] Also provided are a number of embodiments that can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the control level is a reference number. In another embodiment, the control level is the level from a subject not suffering from a disorder characterized by PRAME expression.

[0054] In another aspect, a method for monitoring the progression of a disorder characterized by PRAME expression in a subject is provided, the method comprising: a) detecting the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein in a sample from the subject; b) repeating step a) at a subsequent time point; and c) comparing the PRAME levels or the cells of interest 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 levels or the cells of interest expressing PRAME detected in step b) compared to step a) indicates that the progression of the disorder characterized by PRAME expression in the subject is inhibited, and the presence or increase of the PRAME levels or the cells of interest expressing PRAME detected in step b) compared to step a) indicates that the disorder characterized by PRAME expression in the subject has progressed.

[0055] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, a subject has been treated between a first time point and a subsequent time point to treat a condition characterized by PRAME expression.

[0056] In yet another aspect, provided is a method for predicting the clinical outcome of a subject having a condition characterized by PRAME expression, the method comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) comparing the presence or level of reactivity with the reactivity from a control, wherein the control is obtained from a subject having a favorable clinical outcome; wherein the absence of reactivity or a decrease in the level of reactivity in the subject sample as compared to the control indicates that the subject has a favorable clinical outcome.

[0057] In another aspect, provided is a method for assessing the efficacy of a therapy for a condition characterized by PRAME expression, the method comprising: a) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein in a first sample obtained from the subject prior to providing at least a portion of the therapy for a condition characterized by PRAME expression, and b) determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein in a second sample obtained from the subject after providing the therapy for a condition characterized by PRAME expression, wherein the absence of reactivity or a decrease in the level of reactivity in the second sample as compared to the first sample indicates that the therapy effectively treats the subject's condition characterized by PRAME expression, and wherein the presence of reactivity or an increase in the level of reactivity in the second sample as compared to the first sample indicates that the therapy does not effectively treat the subject's condition characterized by PRAME expression.

[0058] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the level of reactivity is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, optionally wherein the T cell activation or effector function is T cell proliferation, killing, or cytokine release. In another embodiment, T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blotting, or intracellular flow cytometry.

[0059] In another aspect, a method of preventing and / or treating a disorder characterized by PRAME expression is provided, the method comprising contacting a target cell expressing PRAME with a therapeutically effective amount of a composition comprising cells expressing at least one of the binding proteins described herein, optionally wherein the composition is administered to a subject.

[0060] Also provided are a number of embodiments, which can be applied to any aspect covered by the present invention and / or combined with any other embodiment described herein. For example, in one embodiment, the cells are allogeneic cells, syngeneic cells or autologous cells. In another embodiment, the cells are the host cells described herein or a population of the host cells described herein. In another embodiment, the target cell is a cancer cell expressing PRAME. In yet another embodiment, the cell composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the cell composition induces an immune response against the target cells expressing PRAME in a subject. In another embodiment, the cell composition induces an antigen-specific T cell immune response against the target cells expressing PRAME in a subject. In yet another embodiment, the antigen-specific T cell immune response comprises CD4 + helper T lymphocyte (Th) response and at least one of CD8+ cytotoxic T lymphocyte (CTL) response. In another embodiment, the method further comprises administering at least one additional treatment for a disorder characterized by PRAME expression, optionally wherein the at least one additional treatment for a disorder characterized by PRAME expression is administered concurrently or sequentially with the composition. In another embodiment, the disorder characterized by PRAME expression is cancer or its recurrence, 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 and / or a mammal of a disorder characterized by PRAME expression, optionally wherein the mammal is a human, a primate or a rodent. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 shows the PRAME 425-433 peptide sequence.

[0062] Figure 2A and Figure 2B shows that 392 PRAME 425-433 specific TCRs were discovered using the ReceptorScan platform. Figure 2A shows the target-specific CD8 +Expansion of T cells. Briefly, on day -4, CD14 monocytes were isolated from PBMCs of HLA-A*02:01 healthy donors and differentiated into mature DCs. On day -1, naive CD8 T cells were isolated from autologous PBMCs and rested overnight. As part of a multiplex ReceptorScan screen, after pulsing DCs with 1 μg / mL PRAME (SLLQHLIGL) for 3 hours, naive CD8 T cells were co-cultured with DCs followed by a 10-day cell expansion period. + Isolation and single-cell sequencing of CD8 cells. CD8 cells were stained with HLA-A*02:01-specific PRAME (SLLQHLIGL) dextramer to identify clones. DNA barcoded dextramer was used to isolate PRAME-specific cells. The isolated T cells were sequenced using the 10XGenomics platform and TCR α and β chains were paired. 425-433 Figure 2B showed the isolation of CD8 + cells and single-cell sequencing. CD8 cells were stained with HLA-A*02:01-specific PRAME 425-433 (SLLQHLIGL) dextramer to identify clones. DNA barcoded dextramer was used to isolate PRAME 425-433 -specific cells. The isolated T cells were sequenced using the 10XGenomics platform and TCR α and β chains were paired.

[0063] Figure 3 showed that screening of PRAME 425-433 TCR identified 7 TCRs with cytotoxic activity superior to comparator TCRs. Whole T cells were transduced to individually express 392 PRAME 425-433 -specific TCRs, and then the engineered T cells were co-cultured with NucLight 425-433 Red-labeled T2 target cells pulsed with 1 ng / mL PRAME TM peptide. Target cell survival was quantified by time-lapse imaging as a readout of T cell cytotoxicity. Untransduced cells (NTD) were used as controls. Seven (7) TCRs were selected from 392 TCRs for further evaluation of surface expression and cytotoxic potential against PRAME-expressing cell lines.

[0064] Figure 4A and Figure 4B showed that TCRs 366 and 358 showed superior cytotoxicity against endogenously expressed cell lines compared to comparator TCRs. Whole T cells from HLA-A*02:01-positive healthy donors were transduced to express 7 PRAME 425-433 TCRs that were selected from an initial cytotoxicity screen using T2 cells pulsed as targets. Comparator TCRs were similarly expressed. Three (3) TCRs were shown to bind PRAME 425-433 ​(SLLQHLIGL) dextramer and further evaluated in an in vitro cytotoxicity assay, where they were compared to a comparator TCR (comparator AE: affinity enhanced comparator). Figure 4A Shows surface expression of 7 TCRs and the comparator TCR, gated on live cells, assessed by A*02:01-restricted PRAME 425-433 (SLLQHLIGL) dextramer staining. Figure 4B Shows the cytotoxic responses of these TCRs against the target cell lines Hs695T, A375, and T2 cells pulsed with the indicated E:T ratios. Engineered T cells were co-cultured with NucLight TM Red-labeled target cell lines at the indicated E:T ratios and their viability was quantified on an instrument as a readout of T cell cytotoxicity. TCR366 and 358 showed superior activity to the comparator TCR, especially in controlling the growth of A375 cells, which express lower levels of PRAME.

[0065] Figures 5A - 5I Shows the functional evaluation results of PRAME 425-433 -specific TCRs. Total T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express PRAME 425-433 -specific TCR366 and 358, as well as the comparator TCR, and the functional responses of the T cells against target cells expressing HLA-A*02:01 and varying levels of PRAME, and a PRAME-negative control line were assessed. Figure 5A Shows the expression of PRAME 425-433 -specific TCRs, gated on live cells, assessed by A*02:01-restricted PRAME 425-433 (SLLQHLIGL) dextramer staining (comparator AE: affinity enhanced comparator). Figures 5B - 5I Shows the PRAME 425-433 -specific TCRs against HLA-A*02:01 + PRAME + target cell lines Hs695T ( Figure 5B , Figure 5C ), A375 ( Figure 5D , Figure 5E ), NCI-H1563 ( Figure 5F , Figure 5G ) and the HLA-A*02:01 - PRAME-negative control cell line 647V ( Figure 5H , Figure 5I) Results of the functional response. 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 as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B was measured in the co-culture supernatants (E:T 1:1) at 24 hours (comparing AE: affinity-enhanced comparator). Figure 5A Shows the expression of PRAME 425-433 TCR 366 and 358 on the surface of engineered T cells from three healthy donors. Figure 5B Shows T cell cytotoxicity against the Hs695T (HLA-A*02:01 + PRAME + ) target at an E:T of 5:1. Figure 5C Shows cytokine production by T cells in response to the Hs695T (HLA-A*02:01 + PRAME + ) target. Figure 5D Shows T cell cytotoxicity against the A375 (HLA-A*02:01 + PRAME + ) target at an E:T of 5:1. Figure 5E Shows cytokine production by T cells in response to the A375 (HLA-A*02:01 + PRAME + ) target. Figure 5F Shows T cell cytotoxicity against the NCI-H1563 (HLA-A*02:01 + PRAME + ) target at an E:T of 5:1. Figure 5G Shows cytokine production by T cells in response to the NCI-H1563 (HLA-A*02:01 + PRAME + ) target. Figure 5H Shows T cell cytotoxicity against the 647V (HLA-A*02:01 + PRAME - ) target at an E:T of 5:1. Figure 5I Shows cytokine production by T cells in response to the 647V (HLA-A*02:01 + PRAME - ) target.

[0066] Figure 6 Shows that the EC50 of TCR 366 is superior to the comparator TCR. With 1 μM to 10 fM of PRAME 425-433The EC50 value was determined after pulse delivery of 10-fold serial dilutions of the peptide to Nuclight Red-labeled T2 cells. The pulse-delivered T2 cells were then co-cultured with T cells at a T cell to target ratio of 5:1, and target cell survival was measured in the instrument as a readout of cytotoxicity. The EC50 was calculated by fitting the area under the curve (AUC) data using Prism software.

[0067] Figure 7 It was shown that TCR366 did not show alloreactivity against 103 out of 110 tested MHCs. Whole T cells expressing TCR 366 or untransduced control T cells were co-cultured with MHC-free HEK293T cells that re-expressed one of the 110 most common class I MHCs in the US population for 48 hours. A positive control was included in the screen, which consisted of HEK293T cells expressing both a fragment of PRAME containing the 425-433 epitope (SLLQHLIGL) and HLA-A*02:01. After 48 hours of co-culture, the inhibition of target cell growth by whole T cells expressing TCR366 relative to untransduced control T cells was measured as a readout of the reactivity of TCR 366 against allogeneic MHC molecules. Positive controls and alloreactive alleles were indicated (target cell inhibition > 20%).

[0068] Figure 8A and Figure 8B It was shown the putative off-targets of TCR366 identified by whole genome screening. Figure 8A It was shown an overview of the proprietary whole genome screening. Figure 8B It was shown that the screening data of TCR 366 identified seven potential off-targets in a screen of >600,000 protein fragments spanning each wild-type (w.t.) human protein. The screen was designed to over-predict off-targets by overexpressing 90-aa protein fragments that are more efficiently processed than full-length proteins and are not physiologically recognized in healthy human primary cells (Figure 9 below). The putative off-targets were identified by gene name.

[0069] Figures 9A - 9D It was shown that TCR 366 did not show reactivity with healthy human primary cells. The reactivity of whole T cells or NTD cells expressing TCR 366 against primary cells from healthy HLA-A*02:01 + human donors that naturally express the off-targets identified in the whole genome safety screen was tested. With PRAME 425-433(SLLQHLIGL) peptide was used to pulse deliver or not pulse deliver to target cells, and co-cultured with TCR366 or NTD cells. The IFN-γ secretion in the culture supernatant was used as a readout for the reactivity of TCR366 to target cells. HLA-A*02:01 + PRAME + OVCAR-3 cells were used as positive controls, and HLA-A*02:01 + PRAME - CaSki or Loucy cells were used as negative controls.

[0070] Figure 10 Summary data were provided.

[0071] Figure 11 The pMHC dose-dependent function of the process representative TSC-203-A0201 TCR-T cells was shown. T2 cells were pulsed with various concentrations of PRAME peptide and co-cultured with three batches of process representative TSC-203-A0201 TCR-T cells. The figure shows the relative growth of T2 cells during 72-hour co-culture with TSC-203-A0201 TCR-T cells at an E:T ratio of 2:1, normalized to t = 0 h. For each donor, the co-culture was performed in triplicate (n = 3). The error bars for each data point show the standard error of the mean (SEM). The area under the curve (AUC) of the T2 cell growth induced during 72 hours was plotted as a function of peptide concentration to compare the batches of TSC-203-A0201.

[0072] Figures 12A - 12H The TSC-203-A0201 TCR-T cells were shown to secrete granzyme B and the inflammatory cytokines IFN-γ, IL-2, TNF-α in a target-dependent manner. TSC-203-A0201 TCR-T cells from three donors (PD314, PD315, and PD317) ( Figures 12A - 12D ) or donor-matched UTF control T cells ( Figures 12E - 12H) Cultured in the absence of target cells (black bars), or co-cultured at a 1:1 E:T ratio with the HLA-A*A02:01-positive PRAME-negative target cell line 647v (grey bars) or three different HLA-A*02:01-positive PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; SKMEL5, dark blue bars). Supernatants were collected after 24 h of co-culture and assayed for the levels of the inflammatory cytokines IFN-γ, IL-2 and TNF-α, and granzyme B using an automated 4-plex ELISA (ELLA from Proteinsimple). * Indicates samples outside the assay dynamic range (and thus values are less accurate), and # indicates samples with values below the limit of detection.

[0073] Figure 13A and Figure 13B Showed that TSC-203-A0201 TCR-T cells proliferated 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 (black bars), or co-cultured at a 1:1 E:T ratio with the HLA-A*A02:01-positive PRAME-negative target cell line 647v (grey bars) or three different HLA-A*02:01-positive PRAME-positive cell lines (A375, light blue bars; Hs695T, medium blue bars; SKMEL5, dark blue bars). After 3.5 days of co-culture, cells were stained to quantify T cell proliferation by flow cytometry. Plots depict the number of divided cells normalized to counting beads (identified as the CTV dim population). The number of divided 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).

[0074] Figure 14A and Figure 14B Showed that TSC-203-A0201 TCR-T cells demonstrated potent and selective cytotoxicity. Figure 14A Showed that based on In the cytotoxicity assay, three batches of process representative TSC-203-A0201 TCR-T cells (blue growth curves) and untransfected (UTF) control T cells from a matched donor (grey growth curves) were analyzed for their cytotoxic potential against an HLA-A*02:01 positive PRAME negative control cell line (647v) or three different HLA-A*02:01 positive PRAME positive indicator target cell lines (A375, Hs695T, and SKMEL5). Effector TCR-T cells and target cells were co-cultured over a range of effector cell to target ratios (E:T ranging from 5:1 to 0.6:1), and the growth of the target cells was measured over a 72-hour period. The data presented were obtained from TSC-203-A0201 TCR-T cells and UTF control T cells from batch PD315 and are representative of data obtained from all 3 batches of process representative material tested. Target cells cultured alone are shown as a negative control (red growth curve). Figure 14B The cytotoxic activity of three batches of process representative TSC-203-A0201 TCR-T cells over a 72-hour period is shown and is outlined as the area under the curve (AUC) of the growth curve of target cells co-cultured with TSC-203-A0201 at an E:T of 2.5:1, normalized to the growth curve of target cells co-cultured with the corresponding UTF control cells.

[0075] Figure 15A and Figure 15B Shows that TSC-203-A0201 TCR-T cells resist TGFβ-mediated inhibition of cytokine secretion and proliferation. Three batches of process representative TCR-T cells (PD314, PD315, and PD317) were co-cultured with target cells in the presence or absence of 5 ng / mL TGFβ1. Two batches of process-similar TSC-203-A0201 lacking DN-TGFβRII (RG2959164 and 6466164) were included in the assay, as well as Figure 15B donor-matched process-similar TSC-203-A0201 TCR-T cells expressing DN-TGFβRII (RG2959134 and 6466134) in Figure 15A as controls for appropriate TGFβ-mediated T cell inhibition. Shows pre-incubating TCR-T cells with 0 or 5 ng / mL TGFβ1 for 24 hours, then co-culturing with T2 cells pulsed with peptide (10 ng / mL PRAME peptide SLLQHLIGL) at an E:T of 1:1 for 24 hours. IFN-γ secretion by TCR-T cells was evaluated using an automated ELISA platform (ELLA from ProteinSimple) after 24 hours of co-culture. Figure 15BShows the assessment of TCR-T cell proliferation by flow cytometry after co-culture (E:T 1:1) with HLA-A*02:01-positive and PRAME-positive cancer cell line SKMEL5 for 3.5 days. The heatmap depicts the percentage of transduced TCR-T cells that proliferated as observed in co-cultures containing 5 ng / mL TGFβ, normalized relative to the percentage of proliferating TCR-T cells observed in the 0 ng / mL TGFβ condition. Shows the total transduced T cells (TCRαβ + CD34 + ), transduced helper T cells (TCRαβ + CD34 + CD4 + CD8 + ), and transduced cytotoxic T cells (TCRαβ + CD34 + CD4 - CD 8 + ) proliferation data. Asterisks indicate control TCR-T cells with a similar process lacking DN-TGFβRII.

[0076] Figure 16 Shows the schedule of inoculation, administration, and analysis of animals in groups 1 - 7.

[0077] Figures 17A - 17D Shows the in vivo efficacy of TSC-203-A0201. NCG mice were inoculated subcutaneously (s.c.) with Hs 695T. Once tumor engraftment was successful (tumors reached an average of 100 mm 3 , 6 days after inoculation), the animals were randomly assigned to different treatment groups. Figure 17A Shows the average tumor volume over time for mice (n = 12) in each treatment group. Shows the tumor volume over time for individual mice in each individual test batch ( Figures 17B - 17D ). Animals received two intravenous injections of representative TSC-203-A0201 TCR-T cells or untransfected (UTF) control T cells from a matched donor or vehicle (PBS) on day 1 and day 8 of the study (arrows).

[0078] Figure 18 Shows the percentage of body weight development over time across different groups. NCG mice were inoculated subcutaneously with Hs 695T. Once tumor engraftment was successful (tumors reached an average of 100 mm 3, 6 days after inoculation), the animals were randomly assigned to different treatment groups. Animals received two intravenous injections of process representative TSC-203-A0201 TCR-T cells (3 batches tested, PD314, 315 and PD317) or control T cells from matched donors or vehicle (PBS) on study day 1 and day 8 (arrows). The average body weight percentage of each treatment group (n=12) is shown.

[0079] Figure 19 A schematic diagram illustrating the principle of Target Scan screening is shown.

[0080] Figure 20 A graphical representation of the results of a Target Scan screen of mechanistically representative TSC-203-A0201 TCR-T cells is shown. Plotted is the fractional enrichment for each of the ~600,000 tiles / peptides in the screen, calculated from 8 technical replicates, measured relative to the input. Proteins with overlapping tiles enriched above background are highlighted in matching colors, indicated on the graph.

[0081] Figure 21 A flow chart is presented that describes the steps and timeline of a cytokine assay to test the extratumoral reactivity of TSC-203-A0201 TCR-T cells.

[0082] Figure 22 The 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 cancer cell lines and sequenced. The heat map shows the TPM (transcripts per million) calculated from the counts. The color scale used in the RNAseq heat map sets zero TPM values to white, and values above zero follow a continuous color scale until 100 TPM.

[0083] Figure 23 TSC-203-A0201 TCR-T cells and UTF T cells expressing TCR off-target HLA-A*02:01 are shown + Cancer cell line co-culture. 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 responsiveness.

[0084] Figure 24Shows the expression of putative off-targets of the therapeutic TCR used in TSC-203-A0201 TCR-T cells in primary cells and iPSC-derived cells. RNA was extracted from primary cells and iPSC-derived cells and sequenced. The heatmap shows TPM (transcripts per million) calculated from the counts. The color scale used in the RNAseq heatmap sets zero TPM values to white, and values above zero follow a continuous color scale up to 100 TPM.

[0085] Figure 25 Shows that TSC-203-A0201 TCR-T cells did not show reactivity to HLA-A*02:01 + in primary cells. TSC-203-A0201 TCR-T cells and donor-matched UTF cells were co-cultured with a panel of primary cells, and the IFN-γ levels in the supernatant were evaluated as a measure of T cell reactivity.

[0086] Figure 26 Shows the steps and timeline of the oncogenicity assay evaluating cytokine dependence of proliferating T cells. T cells were thawed and rested. Cells were labeled with CTV. Different culture media are described in Table 14.

[0087] Figure 27 Shows T cell viability. Data show the normalized (using CountBright beads) numbers of live (eFlour660 negative) UTF and TSC-203-A0201 TCR-T cells from batch PD314, batch PD315, and batch PD317 after 5 days of in vitro culture in the absence (-) or presence (+) of cytokines and ImmunoCult TM . The assay was performed in triplicate and bars show the mean and standard error of the mean (SEM). The dotted line represents the initial cell number (100,000) used for the assay. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ‘ns’ means not significant, p > 0.05.

[0088] Figure 28 Shows T cell proliferation. Data show the proliferation of T cells in the absence (-) or presence (+) of cytokines and ImmunoCult TMNormalized (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. Assays were performed in triplicate and bars show 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.

[0089] Figure 29 Shows the percentage of proliferating cells. Data show in the absence (-) or presence (+) of cytokines and ImmunoCult TM Percentage (%) of proliferating UTF and TSC-203-A0201 TCR-T gated live cells from batches PD314, PD315, and PD317 after 5 days of culture in the presence or absence of cytokines. Assays were performed in triplicate and bars show 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.

[0090] Figure 30 Shows PRAME expression in 48 normal human organs.

[0091] Figure 31 Shows a diagram of the pNVVD134_TSC-203-A02_TCR-366_MSCV-TCR-366-CD8-EF1α-dnTGFbRII-DHFR vector. Legend: CD: Cluster of Differentiation. RNA-OUT: Antisense RNA against bacterial levansucrase encoded by sacB. SV: Simian virus. TCR: T cell receptor, ITR: Terminal inverted repeat sequence, QBend: Mouse anti-human CD34 antibody, dnTGFbRII: Dominant negative TGFβ receptor II, DHFR: Dihydrofolate reductase selection marker.

[0092] Figure 32Demonstrates the alloreactivity profiling of the engineered representative TSC-203-A0201 TCR-T cells. The engineered representative TSC-203-A0201 TCR-T cells were co-cultured with MHC-free HEK293T cells for the indicated periods of time, which were re-expressing one of 110 class I HLAs most commonly encountered in the US population. The screen included: a positive control (red), consisting of HEK293T cells expressing a PRAME fragment containing an HLA-A*02:01-restricted epitope and HLA-A*02:01; and a negative control (blue), consisting of MHC- / - HEK293T cells. The inhibition of target cell growth by the TCR-T cells during 48 hours of co-culture was measured relative to UTD control T cells as a readout of the reactivity of the engineered representative form of the therapeutic TCR to allogeneic HLA proteins.

[0093] Figure 33 Demonstrates co-culture of TSC-203-A0201 TCR-T cells with cancer cell lines expressing putative allogeneic alleles. Engineered representative TSC-203-A0201 TCR-T cells and untransduced (NTD) control T cells were co-cultured with cancer cell lines expressing 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 co-culture supernatants. Each cell line was also pre-treated with 25 ng / mL IFN-γ, washed, and similarly co-cultured with TSC-203-A0201 TCR-T cells or NTD control T cells to examine reactivity upon HLA upregulation. HLA-A*02:01-positive Hs695T cells expressing PRAME 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; representative data are shown.

[0094] Figure 34Shows co - culture of TSC - 203 - A0201 TCR - T cells with HEK293T cells overexpressing C*14:03. Mechanically representative TSC - 203 - A0201 TCR - T cells or untransduced (NTD) control T cells were co - cultured with mono - allelic HEK293T cells overexpressing HLA - C*14:03 for 24 hours, followed by measuring IFN - γ production in the co - culture supernatant. Mono - allelic HEK293T cells overexpressing A*02:01 and expressing the PRAME ORF were included as a positive control, and mono - allelic HEK293T cells overexpressing A*02:01 in which PRAME had been targeted for knockout using CRISPR / Cas9 (PRAME KO HEK) were included as a negative control. Experiments were performed with TSC - 203 - A0201 from two independent donors; representative data are shown.

[0095] Unless otherwise indicated, for any figure showing a bar graph, curve, or other data related to a legend, each bar, curve, or other data presented from left to right directly and in order corresponds to the boxes in the legend from top to bottom or from left to right. Detailed Description

[0096] The present invention is at least in part based on the discovery of PRAME immunogenic peptides (e.g., peptides comprising or consisting of the sequences listed in Table 1), binding proteins that recognize the PRAME antigen (e.g., binding proteins having the sequences listed in Table 2), and their uses. A systematic and comprehensive investigation was conducted to determine the precise T - cell targets recognized by the initial pool of T cells of interest.

[0097] Accordingly, the present invention is in part directed to the identified epitopes (immunodominant peptides) of the PRAME protein relevant to therapy and related compositions (e.g., immunodominant peptides, vaccines, etc.); compositions comprising immunogenic peptides alone or together with MHC molecules; stabilized MHC - peptide complexes; methods for diagnosing, prognosticating, and monitoring the immune response to disorders characterized by PRAME expression; and methods for preventing and / or treating disorders characterized by PRAME expression. The present invention is also in part directed to the identified binding proteins (e.g., TCRs); host cells expressing the binding proteins (e.g., TCRs); compositions comprising the binding proteins (e.g., TCRs) and host cells expressing the binding proteins (e.g., TCRs); methods for diagnosing, prognosticating, and monitoring the response of T cells to cells expressing PRAME; and methods for preventing and / or treating disorders characterized by PRAME expression.

[0098] I. Definitions

[0099] For convenience, certain terms used in this specification, the examples and the appended claims are collected here.

[0100] The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, “an element” means one element or more than one element. In addition, unless otherwise stated, references to tables provided herein cover all sub-tables of the table.

[0101] The term “administer” means to provide an agent or composition to a subject and includes, but is not limited to, administration by a medical professional and self-administration. This involves physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. In some embodiments, the route of administration of the binding protein described herein includes intravenous, intraperitoneal, intramuscular, subcutaneous, intraspinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase “parenteral administration” means a mode of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the binding protein described herein may be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical. Administration may also be carried out, for example, once, multiple times and / or over one or more extended time periods.

[0102] As used herein, the term “antigen” refers to any natural or synthetic immunogenic substance, such as a protein, peptide or hapten. The antigen may be a PRAME antigen or a fragment thereof, for which a protective or therapeutic immune response is desired. An “epitope” is the part of an antigen to which a natural or synthetic substance binds.

[0103] As used herein, the term “adjuvant” refers to a substance that, when administered before, simultaneously with, or after an antigen, promotes, prolongs, and / or enhances the quality and / or intensity of the immune response to the antigen as compared to administration of the antigen alone. Adjuvants can increase the magnitude and duration of the immune response induced by vaccination.

[0104] The term “antibody” as used herein includes whole antibodies and any antigen-binding fragment (i.e., “antigen-binding portion”) or single chain thereof. In one embodiment, an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H) and a heavy chain constant region. In some naturally occurring antibodies, the heavy chain constant region consists of three domains, CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as V L ) and a light chain constant region. The light chain constant region consists of one domain, CL. V H and V L regions can also be further divided into hypervariable regions, called complementarity-determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). V H and V L Each consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (Clq).

[0105] The term "antigen-presenting cell" or "APC" includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), and other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0106] As used herein, the term "antigen-binding portion" of a binding protein such as a TCR refers to one or more portions of a TCR that retain the ability to bind (e.g., specifically and / or selectively) an antigen (e.g., a PRAME antigen) and a cognate MHC / HLA. Such portions are, for example, from about 8 to about 1500 amino acids in length, suitably from about 8 to about 745 amino acids in length, suitably from about 8 to about 300, such as from about 8 to about 200 amino acids, or from about 10 to about 50 or 100 amino acids. It has been shown that the antigen-binding function of a TCR can be carried out by a fragment of the full-length TCR. Examples of binding portions encompassed by the term "antigen-binding portion" of a TCR include: (i) an Fv fragment consisting of the V α and V β domains of a TCR; (ii) an isolated complementarity-determining region (CDR); or (iii) a combination of two or more isolated CDRs, which can optionally be joined by a synthetic linker. In addition, although V α and V β are encoded by separate genes, they can be joined by recombinant methods using a synthetic linker such that they are able to form a single protein chain, wherein V α and V βThe regions pair to form a monovalent molecule (referred to as a single-chain TCR (scTCR)). Such single-chain TCRs are also intended to be encompassed within the "antigen-binding portion" of the term TCR. These TCR fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as full-length binding proteins. The antigen-binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0107] "Comparative T cell receptor" refers to the current state of the art, such as at least one benchmark T cell receptor (e.g., clone R11P3D3 or R11P3D3 KE) reported in U.S. Patent Publication 2018 / 0273602. In some embodiments, "comparator" refers to sequence R11P3D3 in U.S. Patent Publication 2018 / 0273602. In some embodiments, "affinity-enhanced comparator" or "comparative AE" refers to R11P3D3_KE in U.S. Patent Publication 2018 / 0273602. Engineered forms of these parental sequences are used in the working examples and the sequences of such engineered forms are shown in Table 4. In some embodiments, the comparative T cell receptor has the sequence shown in Table 4.

[0108] The terms "complementary determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to certain non-contiguous amino acid sequences within the variable region of a binding protein, such as a TCR, which confer antigen specificity and / or binding affinity. For a TCR, generally, there are three CDRs (αCDR1, αCDR2, and αCDR3) in each α-chain variable region and three CDRs (βCDR1, βCDR2, and βCDR3) in each β-chain variable region. CDR3 is considered to be the primary CDR responsible for recognizing processed antigen. CDR1 and CDR2 mainly interact with MHC.

[0109] The term "body fluid" refers to fluids excreted or secreted from the body, as well as fluids that are not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's fluid or pre-ejaculatory semen, chyle, chyme, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubricant, vitreous humor, vomit). In some embodiments, the body fluid contains immune cells, optionally wherein the immune cells are cytotoxic lymphocytes, such as cytotoxic T cells and / or NK cells, CD4+ T cells, etc.

[0110] The term "coding region" refers to the region of a nucleotide sequence that contains codons that are translated into amino acid residues, while the term "non-coding region" refers to the region of a nucleotide sequence that is not translated into amino acids (e.g., 5' and 3' untranslated regions).

[0111] 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 that is antiparallel to the first region when the residue in the second nucleic acid region is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can base pair with a residue in a second nucleic acid strand that is antiparallel to the first strand when the residue in the second nucleic acid strand is guanine. Two regions are complementary if at least one nucleotide residue in the first region can base pair with a residue in the second region when the first region of the nucleic acid is arranged antiparallel to the second region of the same or a different nucleic acid. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, wherein when the first portion and the second portion are arranged antiparallel, at least about 50% and in other embodiments at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or any range therebetween (including the endpoints), 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 of the nucleotide residues in the first portion can base pair with the nucleotide residues in the second portion.

[0112] As used herein, the term "co-stimulation" with respect to activated immune cells includes the ability of co-stimulatory molecules to provide a second signal ("co-stimulatory signal") that is non-activating receptor-mediated and can induce proliferation or effector function. For example, a co-stimulatory signal may, for example, cause cytokine secretion in a T cell that has received a T cell receptor-mediated signal. An immune cell that has received a signal mediated by a cell receptor, such as via an activating receptor, is referred to herein as an "activated immune cell".

[0113] "CD3" is known in the art as a multi - protein complex having six chains (see Abbas and Lichtman, Cellular and Molecular Immunology (9th Edition) (2018); Janeway et al. (Immunobiology) (9th Edition) (2016)). In mammals, the complex comprises one CD3γ chain, one CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. The CD3γ chain, CD3δ chain, and CD3ε chain are related cell - surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ chain, CD3δ chain, and CD3ε chain are negatively charged, a feature thought to allow these chains to associate with the positively charged regions or residues of the T - cell receptor chains. The intracellular tails of the CD3γ chain, CD3δ chain, and CD3ε chain each contain a single conserved motif called an immunoreceptor tyrosine - based activation motif or ITAM, while each CD3ζ chain has three ITAMs. Without wishing to be bound by theory, it is believed that the ITAMs are important for the signaling capacity of the TCR complex. The CD3 used according to the present invention can be from various animal species, including humans, mice, rats, or other mammals.

[0114] As used herein, "components of the TCR complex" refers to TCR chains (i.e., TCRα, TCRβ, TCRγ, or TCRδ), CD3 chains (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or 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).

[0115] "Chimeric antigen receptor" or "CAR" refers to a fusion protein engineered to contain two or more amino acid sequences linked in a manner that does not occur naturally or does not occur naturally in a host cell, and the fusion protein can be used as a receptor when present on the cell surface. The CARs covered by the present invention include an extracellular portion that contains an antigen-binding domain (i.e., obtained from or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR specific for the PRAME antigen, a binding protein derived from a single-chain TCR, an scFv derived from an antibody, an antigen-binding domain derived from or obtained from a killer immunoglobulin receptor from NK cells, etc.), and the antigen-binding domain is linked to a transmembrane domain and one or more intracellular signaling domains (such as effector domains, optionally containing co-stimulatory domains) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; see also Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0116] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. The CTL response is mainly mediated by CD8 + T cells.

[0117] The term "consisting essentially of" is not equivalent to "comprising" and refers to the specific materials or steps of a claim, or those materials or steps that do not materially affect the basic characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, or modules) "consists essentially of a specific amino acid sequence" when the amino acid sequences of the domain, region, module, or protein included in the combination account for at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and do not significantly affect (i.e., do not reduce the activity by more than 50%, e.g., no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain, region, module, or protein (e.g., the target-binding affinity of a binding protein), such as an extension, deletion, mutation, or combination thereof (e.g., amino acids or domains between amino acids at the amino terminus or carboxyl terminus).

[0118] The term "determining a treatment regimen suitable for a subject" means determining a treatment regimen for a subject (e.g., a single therapy or a combination of different therapies for preventing and / or treating cancer in a subject), the treatment regimen being initiated, modified, and / or ended based on, substantially based on, or at least partially based on the results of the analysis according to the present invention. One example is starting adjuvant therapy after surgery, the aim of which is to reduce the risk of recurrence, and another example would be modifying the dose of a particular chemotherapy. In addition to the results of the analysis according to the present invention, the determination may also be based on the individual characteristics of the subject to be treated. In most cases, the actual treatment regimen suitable for the subject will be determined by the attending physician or doctor.

[0119] The term "dominant negative TGFβ receptor" or "DN-TGFβR" refers to a transforming growth factor (TGF) β receptor variant or mutant that resists TGFβ signaling.

[0120] There are five type II receptors (activating receptors) and seven type I receptors (signal-propagating receptors). The active TGFβ receptor is a heterotetramer composed of two TGFβ receptor I (TGFβRI) and two TGFβ receptor II (TGFβRII). In some embodiments, the DN-TGFβR is DN-TGFβRII (i.e., a TGFβ receptor II variant or mutant). In some embodiments, resistance to the inhibitory effect of TGFβ signaling on immune cells such as, for example, T cells, where the TGFβ can be produced by cancer cells or by other immune cells within the cellular environment, such as stromal cells, macrophages, myeloid cells, epithelial cells, natural killer cells, etc. TGFβ signaling inhibitors are well known in the art and include, without limitation, mutant TGFβ that chelates the receptor and thus inhibits signaling; antibodies that bind to TGFβ and / or TGFβ receptors (such as lerdelimumab, metlimumab, fressolimumab, etc.); soluble TGFβ-binding proteins, such as portions of TGFβ receptors that chelate TGFβ (such as TGFβRII-Fc fusion proteins); or other binding agents, such as betaglycan. Instead of or in addition to the DN-TGFβR (such as DN-TGFβRII) described herein, any and all known TGFβ signaling inhibitors can be used. In some embodiments, the DN-TGFβR lacks the intracellular portion required for TGFβ-mediated signaling, such as the entire intracellular domain, the kinase signaling domain, etc. DN-TGFβR constructs are well known in the art. (See, for representative non-limiting embodiments, Brand et al. (1993) J. Biol. Chem. 268:11500-11503; Weiser et al. (1993) Mol. Cell Biol. 13:7239-7247; Bollard et al. (2002) Blood 99::3179-3187; PCT Publication WO 2009 / 152610; PCT Publication WO2017 / 156484; Kloss et al. (2018) Mol. Ther. 26:1855-1866; PCT Publication WO.2019 / 089884; PCT Publication WO 2020 / 042647; and PCT Publication WO 2020 / 042648.)

[0121] As used herein, a "hematopoietic progenitor cell" is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiating into mature cell types (such as cells of the immune system). Exemplary hematopoietic progenitor cells include hematopoietic progenitor cells with a CD24 Lo Lin-CD117 + phenotype or hematopoietic progenitor cells found in the thymus (referred to as thymic progenitor cells).

[0122] As used herein, "homologous" refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When the nucleotide residue positions in two regions are occupied by the same nucleotide residue, the regions are homologous at that position. If at least one nucleotide residue position in each region is occupied by the same residue, the first region is homologous to the second region. The homology between two regions is expressed as the proportion of nucleotide residue positions in the two regions that are occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, wherein at least about 50% and in other embodiments at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or any range therebetween (including the endpoints), such as at least about 80%-100% of the nucleotide residue positions in each portion are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions in each portion are occupied by the same nucleotide residue.

[0123] The term "hyperproliferative disorder characterized by PRAME antigen expression" can be any hyperproliferative disorder in which the PRAME antigen is present in an MHC (e.g., HLA) complex expressed by at least some hyperproliferating cells in a subject. Examples of hyperproliferative disorders characterized by the PRAME:HLA complex include solid malignancies, such as those described in detail below.

[0124] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term immune response includes immune responses indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0125] Enhanced agonistic activity of a T cell co-stimulatory receptor and / or enhanced antagonistic activity of an inhibitory receptor may result in enhanced ability to stimulate an immune response or the immune system. Enhanced ability to stimulate an immune response or the immune system can be determined by measuring the EC in an assay of the immune response 50or by the fold increase in the maximum activity level, and the assays measure, for example, changes in cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly via detection of CD107a or granzyme), and proliferation. The ability to stimulate an immune response or immune system activity can be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500% or more.

[0126] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate the host immune system in a subject to produce an immune response against cancer cells. A variety of immunotherapeutic agents can be used in the compositions and methods described herein.

[0127] The term "immune cell" refers to any immune system cell that is derived from hematopoietic stem cells in the bone marrow and that gives rise to two major lineages: myeloid progenitors (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes); and lymphoid progenitors (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 CD8 double-negative T cells, gdT cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells can be referred to as "antigen-presenting cells" or "APCs", which are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor complexed with a peptide on the surface of the APC interacts with the TCR on the surface of the T cell.

[0128] "Isolated protein" means a protein that is isolated from cells or produced by recombinant DNA techniques and is substantially free of other proteins, cellular material, isolation media, and culture media, or a protein that is substantially free of chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or a bioactive portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the binding protein, antibody, polypeptide, peptide, or fusion protein is produced, or is substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a biomarker polypeptide or a fragment thereof, wherein the protein is isolated from the cellular components of the cell from which the protein is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of a biomarker protein or a fragment thereof that have less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as "contaminating protein"), or in some embodiments, less than about 25%, 20%, 15%, 10%, 5%, 1% or less, or any range therebetween (including the endpoints), such as less than about 1% to 5% of non-biomarker protein. When a binding protein, antibody, polypeptide, peptide, or fusion protein or a fragment thereof (e.g., a bioactive fragment thereof) is recombinantly produced, it can be substantially free of culture media, i.e., the culture media accounts for less than about 20%, 15%, 10%, 5%, 1% or less, or any range therebetween (including the endpoints), such as less than about 1% to 5% of the volume of the protein preparation.

[0129] As used herein, the term "isotype" refers to the class of antibody encoded by the heavy chain constant region gene (e.g., IgM, IgG1, IgG2C, etc.).

[0130] As used herein, the term "K D " means the dissociation equilibrium constant of a particular binding protein-antigen interaction. The binding affinity of a binding protein encompassed by the present invention can be measured or determined by standard binding protein-target binding assays, such as competitive assays, saturation assays, or standard immunoassays, such as ELISA or RIA. A relatively low Kd value indicates a relatively high binding affinity (e.g., a Kd value of less than or equal to about 5 × 10 -4 M (500 μM) includes a Kd value of 1 × 10 -4 M (100 μM), and a 100 μM Kd indicates a relatively higher binding affinity compared to a 500 μM Kd).

[0131] "Kit" refers to any article (e.g., a package or container) that contains at least one reagent, such as a probe or small molecule, that is used to specifically detect and / or affect the expression of a biomarker covered by the present invention. Kits can be marketed, resold, or sold as a unit for performing the methods covered by the present invention. A kit can contain one or more reagents necessary for expressing a composition useful in the methods covered by the present invention. In some embodiments, the kit can also contain reference standards, such as nucleic acids encoding proteins that do not affect or regulate signaling pathways that control cell growth, division, migration, survival, or apoptosis. Those skilled in the art can envision many such control proteins, including but not limited to common molecular tags (e.g., gre35 TPMen fluorescent protein and β-galactosidase), proteins not classified by GeneOntology reference into any pathway that covers cell growth, division, migration, survival, or apoptosis, or ubiquitous housekeeping proteins. The reagents in the kit can be provided in separate containers or as a mixture of two or more reagents in a single container. In addition, instructional materials describing the use of the compositions in the kit can be included.

[0132] As used herein, the term "linkage" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage can also be genetic (i.e., recombinant fusion). Such linkages can be achieved using a variety of well-known techniques, such as chemical conjugation and recombinant protein production.

[0133] In some embodiments, a "linker" can refer to an amino acid sequence that links two proteins, polypeptides, peptides, domains, regions, or motifs and can provide a spacer function compatible with the interaction of two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., scTCR) or retains signaling activity (e.g., TCR complex). In some embodiments, the linker is composed of, for example, about 2 to about 35 amino acids or about 4 to about 20 amino acids or about 8 to about 15 amino acids or about 15 to about 25 amino acids.

[0134] "Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a transmembrane chain (with three a domains) and 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 recognized by CD8 + T cells. MHC class II molecules deliver peptides derived from the vesicular system to the cell surface, where they are recognized by CD4 +T cell recognition. The human MHC is known as human leukocyte antigen (HLA).

[0135] The term "PRAME" refers to the PRAME nuclear receptor transcriptional regulator, which is an antigen preferentially expressed in human melanoma and recognized by cytolytic T lymphocytes. It is not expressed in normal tissues, except for the testis. The encoded protein functions as an inhibitor of retinoic acid receptors and may confer a growth advantage to cancer cells via this function. Diseases associated with PRAME include, for example, melanoma, choroid 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 generated by alternative splicing, which are publicly known and available 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); transcript variant 6 (NM_001291715.2) encoding isoform a (NP_001278644.1); 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 the PRAME sequence are also presented in Table 3 below.

[0136] As used herein, the term "PRAME" 425-433 antigen" or "PRAME 425-433 peptide antigen" or "PRAME 425-433 peptide antigen" or "PRAME 425-433 epitope" or "PRAME 425-433 peptide epitope" or "PRAME 425-433The term "b peptide" refers to the native or synthetically produced peptide portion of the PRAME oncoprotein, which comprises, consists of, or consists essentially of the sequence SLLQHLIGL.

[0137] The terms "prevent", "preventing", "prevention", "preventive treatment", etc. refer to reducing the probability that a subject who does not have but is at risk of or susceptible to a disease, disorder, or condition will develop the disease, disorder, or condition.

[0138] The term "prognosis" includes a prediction of the likely course and outcome of cancer or the likelihood of recovery from a disease. In some embodiments, a statistical algorithm is used to provide a prognosis for an individual's cancer. For example, the prognosis can be surgery, the development of a cancer clinical subtype, the development of one or more clinical factors, or recovery from the disease.

[0139] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology" and can be determined using the Karlin and Altschul algorithms (Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)) as modified by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993)). The mentioned algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410 (1990)). The BLAST nucleotide search is performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the polynucleotides described herein. The BLAST protein search is performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the reference polypeptide. To obtain gapped alignments for comparison purposes, gapped BLAST is used as described in Altschul et al. (Nuc. Acids Res. 25:3389-3402 (1997)). When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used.

[0140] The phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting a compound of the invention from one organ or part of the body to another organ or part of the body.

[0141] The term "ratio" refers to the relationship between two numbers (e.g., fractions, sums, etc.). Although a ratio can be expressed in a particular order (e.g., a to b or a:b), one of ordinary skill in the art will recognize that the underlying relationship between the numbers can be expressed in any order and that the underlying relationship does not lose meaning, although the observation and correlation of trends based on the ratio may be reversed.

[0142] The term "recombinant host cell" (or simply "host cell") refers to a cell that contains nucleic acid not naturally present in the cell, e.g., a cell into which a recombinant expression vector has been introduced. It should be understood that the cells according to the invention refer not only to the specific subject cells but also to the progeny of such cells. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parental cell but are still included within the scope of the term cells according to the invention.

[0143] The terms "cancer response", "response to immunotherapy", or "response to a combination therapy of a T cell-mediated cytotoxicity modulator / immunotherapy" refer to any response of a hyperproliferative disease (such as cancer) to a cancer agent (such as a T cell-mediated cytotoxicity modulator) and immunotherapy, preferably a change in tumor mass and / or volume after the initiation of neoadjuvant or adjuvant therapy. The term "neoadjuvant therapy" refers to therapy given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiotherapy, and hormone therapy. The response of a hyperproliferative disorder can be evaluated, for example, for efficacy or in a neoadjuvant or adjuvant setting, where the tumor size after a systemic intervention can be compared to the initial size and dimensions by CT, PET, mammography, ultrasound, or palpation. The response can also be evaluated by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. The response can be recorded quantitatively, such as the percentage change in tumor volume, or qualitatively, such as "pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. The evaluation of the response of a hyperproliferative disorder can be performed early after the initiation of neoadjuvant or adjuvant therapy, for example, after a few hours, days, weeks, or preferably months. The typical endpoint for response evaluation is when neoadjuvant chemotherapy is terminated or when residual tumor cells and / or the tumor bed are surgically resected. This is typically three months after the start of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatment described herein can be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the following: the percentage of patients in complete remission (CR) at a time point at least 6 months from the end of therapy, the number of patients with partial remission (PR), and the number of patients with stable disease (SD). The shorthand for this formula is CBR = CR + PR + SD within 6 months. In some embodiments, the CBR for a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or higher. Other criteria for evaluating the response to cancer therapy are related to "survival" and include all of the following: survival to death, also known as overall survival (where the death may be regardless of cause or related to the tumor); "recurrence-free survival" (where the term recurrence should include local recurrence and distant recurrence); metastasis-free survival; disease-free survival (where the term disease should include cancer and related diseases). The length of the survival can be calculated by referring to a defined starting point (such as the time of diagnosis or the start of treatment) and endpoint (such as death, recurrence, or metastasis). In addition, the criteria for treatment efficacy can be extended to include the response to chemotherapy, the probability of survival, the probability of metastasis within a given time period, and the probability of tumor recurrence.For example, to determine an appropriate threshold, a particular cancer treatment regimen can be administered to a group of subjects and the results can be correlated with biomarker measurements determined prior to the administration of any cancer therapy. The outcome measurement can be the pathologic response to the therapy administered in the neoadjuvant setting. Alternatively, outcome metrics such as overall survival and disease-free survival can be monitored in subjects with known biomarker measurements for a period of time after the cancer therapy. In certain embodiments, the dose administered is a standard dose of a cancer therapeutic agent known in the art. The period of time for monitoring the subjects can vary. For example, the subjects can be monitored for at least 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 14 months, 16 months, 18 months, 20 months, 25 months, 30 months, 35 months, 40 months, 45 months, 50 months, 55 months, or 60 months. Well-known methods in the art, such as those described in the Examples section, can be used to determine the biomarker measurement threshold associated with the outcome of the cancer therapy.

[0144] As noted, the term can also refer to an improved prognosis, such as reflected in an increase in the time to recurrence, which is the period of time to the first recurrence review for a second primary cancer as the first event or death with no evidence of recurrence; or an increase in overall survival, which is the period of time from treatment to death from any cause. Respond or response means achieving a beneficial endpoint when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated when exposed to the stimulus. It should be understood that assessing the likelihood that a tumor or subject will exhibit a favorable response is equivalent to assessing the likelihood that the tumor or subject will not exhibit a favorable response (i.e., will exhibit a lack of response or non-response).

[0145] The term "resistance" refers to acquired or natural resistance of a cancer sample or mammal to a cancer therapy (i.e., non-responsiveness or reduced or limited responsiveness to a therapeutic treatment), such as a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more reduction in response to a therapeutic treatment, such as a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more reduction, or any range therebetween (including the endpoints). The reduction in response can be measured by comparison to the same cancer sample or mammal prior to acquisition of resistance, or by comparison to a different cancer sample or mammal known to be non-resistant to the therapeutic treatment. A typical acquired resistance to chemotherapy is termed "multi-drug resistance". Multi-drug resistance may be mediated by P-glycoprotein, or may be mediated by other mechanisms, or may occur when a mammal is infected with multi-drug resistant microorganisms or combinations of microorganisms. Determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinary clinician, e.g., it can be measured by cell proliferation assays and cell death assays described herein as "sensitization". In some embodiments, the term "reversing resistance" means that in a situation where a single primary cancer therapy (e.g., chemotherapy or radiotherapy) alone does not result in a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor, a second agent in combination with the primary cancer therapy (e.g., chemotherapy or radiotherapy) results in a significant reduction in tumor volume (e.g., p<0.05) to a degree of statistical significance compared to the tumor volume of an untreated tumor. This generally applies to tumor volume measurements taken while the untreated tumor is growing logarithmically.

[0146] The term "sample" for detecting or determining the absence, presence or level of at least one biomarker is generally a brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, feces (e.g., stool), tears and any other body fluid (e.g., as described above under the definition of "body fluid"), or a tissue sample (e.g., a biopsy), e.g., a small intestine, colon sample or surgically removed tissue. In some embodiments, the methods encompassed by the present invention further include obtaining a sample from an individual prior to detecting or determining the absence, presence or level of at least one biomarker in the sample.

[0147] The term "sensitization" means altering cancer cells or tumor cells in a manner that permits more effective treatment of the associated cancer with a cancer therapy (e.g., anti-immune checkpoint, chemotherapy, and / or radiotherapy). In some embodiments, normal cells are not affected to the extent that they are overly damaged by the treatment. The increase or decrease in sensitivity to a therapeutic treatment is measured according to methods known in the art for the particular treatment and the methods described below, including but not limited to cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164) and cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P editors, Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90). Sensitivity or resistance in an animal can also be measured by measuring the decrease in tumor size over a period of time, e.g., 6 months in humans and 4-6 weeks in mice. If the treatment sensitivity is increased or the resistance is decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range therebetween (including the endpoints), compared to the treatment sensitivity or resistance in the absence of the composition or method, then such composition or method sensitizes the response to the therapeutic treatment. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinary clinician. It should be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods of sensitizing hyperproliferative or other cancer cells (e.g., resistant cells) to a cancer therapy.

[0148] The term "small molecule" is a term in the art and includes molecules having a molecular weight of less than about 1000 or less than about 500. In one embodiment, the small molecule does not solely comprise peptide bonds. In another embodiment, the small molecule is not an oligomer. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (such as polyketides) (Cane et al. (1998) Science 282:63-68), and libraries of natural product extracts. In another embodiment, the compound is a small organic non-peptide compound. In another embodiment, the small molecule is not biosynthetic.

[0149] The term "specifically binds" refers to the binding of a binding protein to a predetermined antigen. Generally, when assayed by a binding assay, such as surface plasmon resonance (SPR) technology, using the antigen of interest as the analyte and the binding protein as the ligand in a BIAcore TM assay instrument, the binding protein binds with an affinity (K -4 of less than or equal to about 5×10 -4 M, less than or equal to about 1×10 -5 M, less than or equal to about 5×10 -5 M, less than or equal to about 1×10 -6 M, less than or equal to about 5×10 - 6 M, less than or equal to about 5×10 -7 M, less than or equal to about 1×10 -7 M, less than or equal to about 5×10 -8 M, less than or equal to about 1×10 -8 M, less than or equal to about 5×10 -9 M, less than or equal to about 1×10 -9 M, less than or equal to about 5×10 -10 M, less than or equal to about 1×10 -10 M, less than or equal to about 5×10 -11 M, less than or equal to about 1×10 -11 M, less than or equal to about 5×10 -12 M, less than or equal to about 1×10 -12 D) of less than or equal to about 5×10 D) In some embodiments, the binding protein binds to the predetermined antigen with an affinity that is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold or 10.0-fold greater than its affinity for non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, casein). The phrases "binding protein that recognizes an antigen" and "binding protein specific for an antigen" are used interchangeably herein with the term "binding protein that specifically binds to an antigen". Selective binding is a relative term and refers to the ability of a binding protein to distinguish the binding of one antigen from the binding of another antigen, e.g., the ability to distinguish the binding of a particular family member or antigen target from the binding of a related family member or antigen target. For example, the assay 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 a number of related epitopes (e.g., the PRAME immunogenic epitope and closely related sequences), thereby distinguishing such targets from the numerous other possible epitopes available in the human genome.

[0150] 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 (e.g., a non-malignant disorder, a hyperproliferative disorder or a recurrence of a hyperproliferative disorder characterized by PRAME expression). The term "subject" may be used interchangeably with "patient".

[0151] The term "survival" includes all of the following: survival to death, also known as overall survival (wherein the death may be irrespective of cause or related to the tumor); "recurrence-free survival" (wherein the term recurrence should include local recurrence and distant recurrence); metastasis-free survival; disease-free survival (wherein the term disease should include cancer and diseases associated therewith). The length of the survival can be calculated by reference to a defined starting point (e.g., time of diagnosis or time of initiation of treatment) and an end point (e.g., death, recurrence or metastasis). In addition, the criteria for therapeutic efficacy can be extended to include response to chemotherapy, probability of survival, probability of metastasis within a given time period and probability of tumor recurrence.

[0152] The term "synergy" refers to the combined action of two or more agents (e.g., the PRAME-related agents described herein and another therapy for treating a disorder characterized by PRAME expression, such as another TCR targeting PRAME, an anti-cancer therapy, an immunotherapy, etc.) being greater than the sum of the individual actions of the individual cancer agents / therapies.

[0153] As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4 + cell)-mediated responses. T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0154] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of such RNA or cDNA) that is complementary or homologous to all or a portion of a mature mRNA, the mature mRNA being produced by transcription of a biomarker nucleic acid and (if present) normal post-transcriptional processing (e.g., splicing) of the RNA transcript and reverse transcription of the RNA transcript.

[0155] A "T cell" is a cell of the immune system that matures in the thymus and produces a T cell receptor (TCR). T cells can be naive T cells (not exposed to antigen; compared to T CM cells, have increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO), memory T cells (T M cells) (have experienced antigen and have a long lifespan) and effector cells (have experienced antigen and are cytotoxic). T M cells can also be divided into central memory T cells (T CM cells, 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 cells, which have decreased expression of CD62L, CCR7, CD28, and CD45RA and increased expression of CD127 compared to naive T cells or T CM cells). Effector T cells (T E cells) refer to CD8+ cytotoxic T lymphocytes that have experienced antigen and are positive for granzyme and perforin compared to T CM cells, with decreased expression of CD62L, CCR7, and CD28. Other exemplary T cells include regulatory T cells, such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Tregl7 cells, as well as Trl, Th3, CD8 + CD28 and Qa-1 restricted T cells.

[0156] Conventional T cells (also known as Tconv or Teff) have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) to enhance the immune response by virtue of their expression of one or more T cell receptors. Tcon or Teff are generally defined as any population of T cells that are not Tregs and include, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon that have differentiated into, for example, Th1 or Th2 lineages. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is CD4+ Teff or CD8+ Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T lymphocytes. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. A "naive Tcon" is a CD4 + T cell that has differentiated in the bone marrow and successfully undergone positive and negative central selection processes in the thymus but has not been activated by exposure to an antigen. Naive Tcon are typically characterized by surface expression of L-selectin (CD62L), lack of activation markers (e.g., CD25, CD44, or CD69), and lack of memory markers (e.g., CD45RO). Thus, it is believed that naive Tcon are quiescent and non-dividing and require interleukin 7 (IL-7) and interleukin 15 (IL-15) to maintain homeostatic survival (see, at least, WO2010 / 101870). In the context of suppressing the immune response, the presence and activity of such cells are not desired. Unlike Tregs, Tcon are not anergic and can proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:625-637).

[0157] "T effector" ("T eff " or "T E ") cells refer to T cells (e.g., CD4+ and CD8+ T cells) that have cytolytic activity, as well as T helper (Th) cells that secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0158] "T cell receptor" or "TCR" refers to a member of the immunoglobulin superfamily that is capable of binding (e.g., specifically and / or selectively) an antigenic peptide that binds to an MHC receptor (having variable binding domains, constant domains, transmembrane regions, and short cytoplasmic tails; see, e.g., Janeway et al. (1997) Curr. Biol. Publ. 4:33). The TCR can exist on the cell surface or in a soluble form and is typically composed of a heterodimer having α and β chains (also referred to as TCRα and TCRβ, respectively) or γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portions of the TCR chains (e.g., the α and β chains) contain two immunoglobulin domains: a variable domain at the N-terminus (e.g., the α-chain variable domain or V α and the β-chain variable domain or V β ; typically amino acids 1 to 116 based on Kabat numbering (Kabat et al. (1991) "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service, National Institutes of Health, 5th ed.), and a constant domain at the C-terminus adjacent to the cell membrane (e.g., the α-chain constant domain or C α , typically amino acids 117 to 259 based on Kabat; the β-chain constant domain or C β , typically amino acids 117 to 295 based on Kabat). Also like immunoglobulins, the variable domain contains complementarity-determining regions ("CDRs", also referred to as hypervariable regions or "HVRs") separated by framework regions ("FRs") (see, e.g., Fores et al. (1990) Proc. Natl. Acad Sci. US.A. 87:9138; Chothia et al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol. 27:55). In some embodiments, the TCR is present on the surface of a T cell (or T lymphocyte) and is associated with the CD3 complex. The sources of TCRs encompassed by the present invention can be from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0159] The term "T cell receptor" or "TCR" is to be understood to encompass the full TCR as well as its antigen-binding portion or antigen-binding fragment. In some embodiments, the TCR is a full or full-length TCR, including TCRs in the αβ form or the γδ form. In some embodiments, the TCR is less than full-length but binds to a specific peptide bound in an MHC molecule, such as an antigen-binding portion that binds to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of the TCR may contain only partial domains of the full-length or full TCR but is still capable of binding to the peptide epitope bound by the full TCR, such as an MHC-peptide complex. In some cases, the antigen-binding portion contains the variable domains of the TCR, such as the variable α-chain and variable β-chain of the TCR, sufficient to form a binding site that binds to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain complementarity-determining regions (CDRs) involved in the recognition of the peptide, MHC, and / or MHC-peptide complex.

[0160] Nomenclature is established through the International Immunogenetics Information System (IMGT) (see also Scaviner and Lefranc (2000) Exp. Clin. Immunol. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunol., 17:107-114; "T Cell Receptor Factsbook”, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). IMGT provides unique sequences for describing TCRs, and the sequences described herein can be identified by reference to such unique sequences provided herein. TCR sequences are publicly available in the IMGT database at imgt.org.

[0161] As described above, the native α / β heterodimeric TCR has an α chain and a β chain. Broadly speaking, each chain contains a variable region, a joining region, and a constant region, and the β chain usually also contains a short diversity region between the variable region and the joining region, although this diversity region is generally considered part of the joining region. Each variable region contains three hypervariable CDRs (complementary determining regions) embedded within framework sequences. It is well known that CDR3 is the main mediator of antigen recognition. There are several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions, which are distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. In the IMGT nomenclature, Vα types are designated by unique TRAV numbers. For example, "TRAV4" defines a TCR Vα region that has a unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence that is partially defined by amino acid sequences conserved between TCRs but also includes amino acid sequences that vary between TCRs. Similarly, "TRBV2" defines a TCR Vβ region that has a unique framework and CDR1 and CDR2 sequences but only a partially defined CDR3 sequence. There are known to be 54 α variable genes within the α locus, 44 of which are functional, and 67 β variable genes within the β locus, 42 of which are functional.

[0162] Similarly, the joining region of the TCR is defined by the unique IMGT TRAJ and TRBJ nomenclatures, while the constant region is defined by the IMGT TRAC and TRBC nomenclatures. In the IMGT nomenclature, the β-chain diversity region is abbreviated as TRBD, and as previously described, the tandem TRBD / TRBJ regions are generally considered together as the joining region.

[0163] The gene pools encoding the TCR α and β chains are located on different chromosomes and contain separate V, (D), J, and C gene segments that are brought together by rearrangement during T cell development. The large number of possible recombination events between the 54 TCR α variable genes and 61 α J genes, or between the 67 β variable genes, two β D genes, and 13 β J genes, gives rise to an extremely high diversity of T cell α and β chains. The recombination process is not precise and introduces further diversity within the CDR3 region. Each α and β variable gene can also contain allelic variants, designated as TRAVxx*01 and *02, or TRBVx-x*01 and *02, respectively, in the IMGT nomenclature, thus further increasing the amount of variation. Similarly, some TRBJ sequences have two known variants. (Note that the lack of a "*" qualifier means that only one allele of the relevant sequence is known). The natural repertoire of human TCRs generated by recombination and thymic selection is estimated to contain approximately 10 6A unique β-chain sequence, determined by CDR3 diversity (Arstila et al. (1999) Science 286:958-961), and potentially even higher (Robins et al. (2009) Blood 114:4099-4107). It is estimated that each β-chain pairs with at least 25 different α-chains, generating further diversity (Arstila et al. (1999) Science 286:958-961).

[0164] Thus, the term "TCR α variable domain" refers to the tandem of TRAV and TRAJ regions; the TRAV region only; or the TRAV and part of the TRAJ region, and the term TCR α constant domain refers to the extracellular TRAC region, or the C-terminally truncated or full-length TRAC sequence. Similarly, the term "TCR β variable domain" refers to the tandem of TRBV and TRBD / TRBJ regions; the TRBV and TRBD regions only; the TRBV and TRBJ regions only; or the TRBV region and part of the TRBD and / or TRBJ regions, and the term TCR β constant domain refers to the extracellular TRBC region, or the C-terminally truncated or full-length TRBC sequence. These TCR α variable domain and TCR β variable domain nomenclatures similarly apply to the variable domains of the TCR γ and TCR δ chains of γ / δ TCRs, respectively. A person of ordinary skill in the art can obtain TRAV, TRAJ, TRAC, TRBV, TRBJ, and TRBC gene sequences, for example, through the publicly available IMGT database.

[0165] The term "TCR complex" refers to the complex formed by the association of CD3 with TCR. For example, the TCR complex can consist of one CD3γ chain, one CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, one TCRα chain, and one TCRβ chain. Alternatively, the TCR complex can consist of one CD3γ chain, one CD3δ chain, two CD3ε chains, a homodimer of CD3ζ chains, one TCRγ chain, and one TCRδ chain.

[0166] The term "therapeutic effect" refers to the local or systemic effects caused by a pharmacologically active substance in animals, especially mammals, and more particularly in humans. Thus, the term means any substance intended for diagnosing, curing, alleviating, treating, or preventing diseases in animals or humans or for enhancing their desired physical or mental development and condition.

[0167] The terms "therapeutically effective amount" and "effective amount" mean an amount of a substance that produces some desired effect, such as a desired local or systemic therapeutic effect, in at least one cell subset in an animal at a reasonable benefit / risk ratio suitable for any therapy. In some embodiments, the therapeutically effective amount of a substance will depend on the therapeutic index, solubility, pharmacokinetics, half-life, etc. of the substance. It can be determined in cell cultures or experimental animals by standard pharmaceutical procedures such as those used to determine LD 50 and ED 50 to determine the toxicity and therapeutic efficacy of the subject compound. In some embodiments, compositions exhibiting a large therapeutic index are used. In some embodiments, LD 50 (lethal dose) can be measured, and relative to not administering the agent, it can be reduced by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more when the agent is administered. Similarly, ED 50 (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured, and relative to not administering the agent, it can be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more when the agent is administered. Similarly, IC 50 can also be measured, and relative to not administering the agent, it can be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more when the agent is administered. In some embodiments, in one assay, the T cell immune response can be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100%. In another embodiment, a reduction in viral load of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% can be achieved.

[0168] The term "treatment" refers to the therapeutic management or amelioration of a disorder of interest (e.g., a disease or condition). Treatment can include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is generally carried out in an effort to alter, in a manner beneficial to the subject, the course of a disease (the term being used to denote any disease, condition, syndrome, or adverse condition that requires or may require therapy). Treatment effects can include reversing, alleviating one or more symptoms or manifestations of the disease, reducing its severity, delaying its onset, curing it, inhibiting its progression, and / or reducing the likelihood of its occurrence or recurrence. Desirable treatment effects include, but are not limited to: preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. A therapeutic agent can be administered to a subject having a disease or having an increased risk of disease development relative to a member of the general population. In some embodiments, a therapeutic agent can be administered to a subject who has had a disease but no longer shows signs of the disease. An agent can be administered, for example, to reduce the likelihood of overt disease recurrence. A therapeutic agent can be administered prophylactically, i.e., before any symptoms or manifestations of the disease appear. "Prophylactic treatment" refers to providing medical and / or surgical treatment to a subject who has not yet developed a disease or shown signs of a disease, for example, to reduce the likelihood of disease occurrence or the severity of the disease when it occurs. A subject may have been identified as being at risk of developing a disease (e.g., having an increased risk relative to the general population or having a risk factor that increases the likelihood of developing the disease).

[0169] The term "anergy" encompasses the refractoriness of cancer cells to therapy, or of therapeutic cells, such as immune cells, to stimuli, such as stimuli via activating receptors or cytokines. Anergy can occur, for example, due to exposure to immunosuppressants or to high doses of antigen. As used herein, the terms "incompetence" or "tolerance" encompass refractoriness to stimuli mediated by activating receptors. This refractoriness is typically antigen-specific and persists after cessation of exposure to the tolerizing antigen. For example, T cell anergy (as opposed to unresponsiveness) is characterized by a lack of production of cytokines such as IL-2. T cell anergy occurs when T cells are exposed to antigen and receive a first signal (T cell receptor or CD-3 mediated signal) in the absence of a second signal (co-stimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of co-stimulatory polypeptides) results in an inability to produce cytokines and thus an inability to proliferate. However, anergic T cells may proliferate if cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by measuring the lack of production of IL-2 by T lymphocytes using ELISA or a proliferation assay using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells are unable to initiate IL-2 gene transcription controlled by the 5' IL-2 gene enhancer or induced by a multimer of AP1 sequences that may be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0170] The term "vaccine" refers to a pharmaceutical composition that elicits an immune response to an antigen of interest. A vaccine can also confer protective immunity to a subject.

[0171] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., TCR) that is involved in the binding of the immunoglobulin superfamily binding protein to an antigen (e.g., the TCR α chain or β chain (or γ and δ chains for γδ TCR)). The variable domains of the α and β chains of a native TCR (V α and V β ) generally have a similar structure, each containing four conserved framework regions (FRs) and three CDRs. The V α domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J); the V β domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J). A single V α or V β domain may be sufficient to confer antigen-binding specificity. In addition, V α or V βThe domain separates the TCR that binds a specific antigen from the TCR that binds the antigen, to separately screen libraries of complementary V α or V β domains.

[0172] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, the vector is those vectors capable of autonomous replication and / or expression of the nucleic acid to which it is linked. A vector capable of directing the expression of an operably linked gene is herein referred to as an "expression vector". Generally, expression vectors used in recombinant DNA technology are usually in the form of "plasmids", which generally refer to circular double-stranded DNA rings whose vector form is not bound to chromosomes. In this specification, "plasmid" and "vector" may be used interchangeably because plasmids are the most commonly used vector forms. However, as those skilled in the art will appreciate, the present invention is intended to include other forms of such expression vectors that provide equivalent functions and are subsequently known in the art.

[0173] There is a known and defined correspondence between the amino acid sequence of a specific protein and the nucleotide sequence that can encode the protein, as defined by the genetic code (shown below). Similarly, there is a known and defined correspondence between the nucleotide sequence of a specific nucleic acid and the amino acid sequence encoded by the nucleic acid, as defined by the genetic code.

[0174] Genetic Code

[0175] Alanine (Ala, A) GCA, GCC, GCG, GCT

[0176] Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

[0177] Asparagine (Asn, N) AAC, AAT

[0178] Aspartic acid (Asp, D) GAC, GAT

[0179] Cysteine (Cys, C) TGC, TGT

[0180] Glutamic acid (Glu, E) GAA, GAG

[0181] Glutamine (Gln, Q) CAA, CAG

[0182] Glycine (Gly, G) GGA, GGC, GGG, GGT

[0183] Histidine (His, H) CAC, CAT

[0184] Isoleucine (Ile, I) ATA, ATC, ATT

[0185] Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

[0186] Lysine (Lys, K) AAA, AAG

[0187] Methionine (Met, M) ATG

[0188] Phenylalanine (Phe, F) TTC, TTT

[0189] Proline (Pro, P) CCA, CCC, CCG, CCT

[0190] Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

[0191] Threonine (Thr, T) ACA, ACC, ACG, ACT

[0192] Tryptophan (Trp, W) TGG

[0193] Tyrosine (Tyr, Y) TAC, TAT

[0194] Valine (Val, V) GTA, GTC, GTG, GTT

[0195] Stop signal (End) TAA, TAG, TGA

[0196] An important and well-known feature of the genetic code is its redundancy, i.e., for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (as shown above). Thus, many different nucleotide sequences may code for a given amino acid sequence. These nucleotide sequences are considered to be functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although some organisms may translate some sequences more efficiently than others). In addition, occasional methylated variants of purines or pyrimidines may be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0197] Given the above, a nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any part thereof) can be used to derive a polypeptide amino acid sequence by translating the DNA or RNA into an amino acid sequence using the genetic code. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence that can encode the polypeptide can be inferred from the genetic code (due to its redundancy, multiple nucleic acid sequences will result for any given amino acid sequence). Thus, a description and / or disclosure in this article 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 that nucleotide sequence. Similarly, a description and / or disclosure in this article 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 that amino acid sequence.

[0198] II. Peptide

[0199] In certain aspects, the present disclosure provides methods and compositions for treating and / or preventing a condition associated with PRAME expression by inducing an immune response against PRAME or cells expressing PRAME, which involve administering a PRAME immunogenic peptide, a nucleic acid encoding a PRAME immunogenic peptide, and / or a cell expressing a PRAME immunogenic peptide as described herein.

[0200] In certain embodiments, the PRAME immunogenic peptide comprises a peptide epitope (e.g., consists of) selected from the peptide sequences listed in Table 1, such as those listed in Table 1A. The peptide epitopes described herein can be combined with MHC molecules, such as specific HLA molecules having a particular HLA α-chain allele. By way of example, the peptides of Table 1A were identified as associating with MHC having an HLA-A*02 serotype of the α-chain, such as MHC encoded by the HLA-A*02:01 allele, as further described in the Examples section. In some embodiments, the PRAME immunogenic peptide can be combined with an MHC molecule, wherein the MHC molecule comprises an MHC α-chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20,HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In some embodiments, the PRAME immunogenic peptides are derived from a human PRAME protein and / or the PRAME proteins shown in Table 3. In some embodiments, one or more PRAME immunogenic peptides are administered alone or in combination with an adjuvant.

[0201] In certain aspects, provided are compositions comprising one or more of the PRAME immunogenic peptides described herein and an adjuvant.

[0202] Table 1: PRAME Epitopes

[0203] Table 1A

[0204] PRAME Epitopes Presented by HLA Serotype HLA-A*02

[0205] Peptide Epitope SLLQHLIGL

[0206] *Table 1, for example Table 1A includes peptide epitopes, and polypeptide molecules having an amino acid sequence or a portion thereof that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity to any of the sequences listed in Table 1, for example Table 1A, over its full length. Such polypeptides can have the functions of the full-length peptides or polypeptides further described herein.

[0207] In some embodiments, the present disclosure provides PRAME polypeptides and / or nucleic acids encoding PRAME polypeptides. In some embodiments, the PRAME polypeptide is a polypeptide comprising an amino acid sequence of sufficient length to elicit a PRAME-specific immune response. In certain embodiments, the PRAME polypeptide further 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, the PRAME polypeptide comprises only the amino acid sequence corresponding to the PRAME protein or a fragment thereof.

[0208] In some embodiments, the amino acid sequence of the PRAME polypeptide comprises, consists essentially of, or consists of: at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 373 or more, or any range therebetween (e.g., 7-25, 8-22, 9-22, etc.) (including the endpoints) of contiguous amino acids of the amino acid sequence of PRAME shown in Table 3. 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.

[0209] As is well known to those skilled in the art, polypeptides with significant sequence similarity can elicit the same or very similar immune responses in host animals. Thus, in some embodiments, derivatives, equivalents, variants, fragments, or mutants of the PRAME immunogenic peptides or fragments thereof described herein may also be suitable for the methods and compositions provided herein.

[0210] In some embodiments, variants or derivatives of the PRAME immunogenic polypeptides are provided herein. The altered polypeptides can have, for example, an amino acid sequence altered by conservative substitutions, but still elicit an immune response reactive with the unaltered protein antigen and are considered functional equivalents. As used herein, the term "conservative substitution" means that an amino acid residue is replaced by another biologically similar residue. It is well known in the art that amino acids within the same conservative group can generally be substituted for one another with little or no effect on the function of the protein. According to certain embodiments, 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 the PRAME immunogenic peptide or fragment thereof described herein. In some embodiments, the homology is at least 90%, at least 95%, at least 98%, or higher.

[0211] The immunogenic peptides encompassed by the present invention can comprise peptide epitopes derived from the PRAME protein, such as those listed in Table 1, for example, those listed in 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 can include conservative or non-conservative mutations. The peptide can comprise up to 1, 2, 3, 4, or more mutations. In some embodiments, the peptide can comprise at least 1, 2, 3, 4, or more mutations.

[0212] In some embodiments, the peptide can be chemically modified. For example, the peptide 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 an example of methylation that can occur in the peptides of the present disclosure. In some embodiments, the peptide can be modified by methylation of the free amine, for example, by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0213] Chemical modifications can include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains (such as palmitate or myristate), or albumin. Chemical modification of a peptide having an Fc region can be a fusion Fc - peptide. Polyamino acids can include, for example, polyamino acid sequences having repeating single amino acids (such as polyglycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the foregoing. In some embodiments, the peptides encompassed by the present disclosure can be modified such that the modification increases the stability and / or half - life of the peptide. In some embodiments, attachment of a hydrophobic moiety (such as attachment to the N - terminus, C - terminus, or internal amino acids) can be used to extend the half - life of the peptides encompassed by the present disclosure. In other embodiments, the peptide can include post - translational modifications (such as, methylation and / or amidation) that can affect, for example, the serum half - life. In some embodiments, simple carbon chains (such as, by myristoylation and / or palmitoylation) can be conjugated to a fusion protein or peptide. In some embodiments, simple carbon chains can facilitate separation of the fusion protein or peptide from unconjugated material. For example, methods that can be used to separate a fusion protein or peptide from unconjugated material include, but are not limited to, solvent extraction and reverse - phase chromatography. The lipophilic moiety can extend the half - life by reversible binding to serum albumin. The conjugated moiety can be a lipophilic moiety that extends the half - life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative, including cholestene, cholestane, cholestadiene, and oxysterol. In some embodiments, the peptide can be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptide can be coupled (such as, conjugated) to a half - life modifier. Examples of half - life modifiers include, but are not limited to: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water - soluble polymers, zwitterionic water - soluble polymers, water - soluble poly(amino acids), water - soluble polymers of proline, alanine, and serine, water - soluble polymers containing glycine, glutamic acid, and serine, Fc regions, fatty acids, palmitic acid, or molecules that bind to albumin. In some embodiments, a spacer or linker can be coupled to the peptide, such as 1, 2, 3, 4, or more amino acid residues used as a spacer or linker, to facilitate conjugation or fusion with another molecule, and to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, a fusion protein or peptide can be conjugated to other moieties that can modify or effect a change in the properties of the peptide.

[0214] In some embodiments, the peptide can be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or label. The affinity tag can be selected from the group consisting of: glutathione - S - transferase (GST), calmodulin - binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Tags, His tags, biotin tags, and V5 tags. The label can be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of proinflammatory factors, anti-inflammatory agents, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies, such as single-chain Fvs.

[0215] The peptide can be conjugated to agents for imaging, research, therapeutics, theranostics, pharmacy, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, the peptide can be conjugated or fused to a detectable agent, such as a fluorophore, near-infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radionuclide chelator, or another suitable material for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties can be attached to the peptide. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not readily quenched by biological tissues and body fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation with a wavelength between 650 nm and 4000 nm, and such emission is used to detect such agents. Non-limiting examples of fluorescent dyes that can be used as conjugated molecules include -680, -750, -750, -800, -800, -680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, near-infrared dyes generally include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Additional non-limiting examples of fluorescent dyes used as conjugated molecules in the present disclosure include acridine orange or acridine yellow, Alexa (e.g., Alexa 790, 750, 700, 680, 660, and 647) and any of its derivatives, 7-Actinomycin D, 8-Anilino-1-naphthalenesulfonic acid, ATTO dyes and any of its derivatives, Auramine-Rhodamine stain and any of its derivatives, Bensantrhone, Bimane, 9,10-Bis(phenylethynyl)anthracene, 5,12-Bis(phenylethynyl)naphthacene, Bisbenzimide, Brainbow, Calcein, Carboxyfluorescein and any of its derivatives, 1-Chloro-9,10-bis(phenylethynyl)anthracene and any of its derivatives, DAPI, DiOC6, DyLight Fluors and any of its derivatives, Epicocconone, Ethidium bromide, FlAsH-EDT2, Fluo dyes and any of its derivatives, FluoProbe and any of its derivatives, Fluorescein and any of its derivatives, Fura and any of its derivatives, GelGreen and any of its derivatives, GelRed and any of its derivatives, Fluorescent proteins and any of its derivatives, m isotype proteins and any of its derivatives (e.g., mCherry), Hetamethine dyes and any of its derivatives, Hoeschst stain, Imidacridone, Indian Yellow, Indo-1 and any of its derivatives, Laurdan, Lucifer Yellow and any of its derivatives, Fluorescein and any of its derivatives, Luciferase and any of its derivatives, Merocyanine and any of its derivatives, Nile dyes and any of its derivatives, Perylene, Phloxine, Phycobiliprotein dyes and any of its derivatives, Propidium iodide, Pyranine, Rhodamine and any of its derivatives, Ribogreen, RoGFP, Rubrene, Stilbene and any of its derivatives, Sulfonylrhodamine and any of its derivatives, SYBR TMand any of its derivatives, synapto-pHluorin, tetraphenylbutadiene, tris tetrasodium, Texas Red, Titan Yellow, TSQ, umbelliferone, perylene, yellow fluorescent protein, and YOYO-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), cyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, eosin, eosin Y, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon dye (e.g., Oregon 488, Oregon 500, Oregon 514, etc.), Texas Red, Texas Red-X, SPECTRUMRED, SPECTRUMGREEN, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), ALEXA dye (e.g., ALEXA 350, ALEXA 488, ALEXA 532, ALEXA 546, ALEXA 568, ALEXA 594, ALEXA 633, ALEXA 660, ALEXA 680, etc.), dye (e.g., FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRDye (e.g., IRD40, IRD 700, IRD 800, etc.), etc. Additional suitable detectable agents are described in PCT / US14 / 56177. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of: actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.

[0216] The peptide can be conjugated with a radiosensitizer or a photosensitizer. Examples of radiosensitizers include, but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to: fluorescent molecules or beads, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metal porphyrins, metal phthalocyanines, angelicin, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins, and related compounds (e.g., lumazines and riboflavins), fullerenes, pheophorbide, pyro-pheophorbide, cyanines (e.g., merocyanine 540), pheophytin, sapphyrin, texaphyrin, purpurin, porphycene, phenothiazinium, methylene blue derivatives, naphthalimides, nile blue derivatives, quinones, perylene quinones (e.g., hypericin, hypocrellin, and cercosporin), psoralen, quinones, retinoids, rhodamines, thiophenes, verdin, xanthene dyes (e.g., eosin, erythrosine, rose bengal), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, the method allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent (e.g., a drug) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the peptide is fused with the agent, or covalently or non-covalently linked to the agent, e.g., directly or via a linker.

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

[0218] The chemical modification can include polymers, polyethers, polyethylene glycols, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains (such as palmitate or myristate), or albumin. The chemical modification of a binding protein having an Fc region can be a fusion Fc-protein. The polyamino acids can include, for example, polyamino acid sequences having repeating single amino acids (such as polyglycine), and polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the foregoing.

[0219] In some embodiments, the binding proteins encompassed by the present invention can be modified. In some embodiments, the modification has substantial or significant sequence identity to the parental binding protein to produce a functional variant that maintains one or more biophysical and / or biological activities of the parental binding protein (e.g., maintaining pMHC binding specificity). In some embodiments, the mutation is a conservative amino acid substitution.

[0220] In some embodiments, the binding proteins encompassed by the present invention can include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxy phenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, Ν',Ν'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0221] The binding proteins encompassed by the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0222] In some embodiments, the half-life of the peptides encompassed by the present invention can be extended by attachment of a hydrophobic moiety (e.g., attachment to the N-terminus, C-terminus, or an internal amino acid). In other embodiments, the binding proteins can include post-translational modifications (e.g., methylation and / or amidation) that can affect, for example, the serum half-life. In some embodiments, simple carbon chains (e.g., by myristoylation and / or palmitoylation) can be conjugated to the binding proteins. In some embodiments, the simple carbon chains can facilitate the separation of the binding proteins from unconjugated materials. Methods that can be used to separate the binding proteins from unconjugated materials include, but are not limited to, solvent extraction and reverse-phase chromatography. The lipophilic moiety can extend the half-life by reversible binding to serum albumin. The conjugated moiety can be a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative, including cholestene, cholestane, cholestadiene, and oxysterols. In some embodiments, the binding proteins can be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding proteins can be coupled (e.g., conjugated) to a half-life modifier. Examples of half-life modifiers include, but are not limited to: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers containing glycine, glutamate, and serine, Fc regions, fatty acids, palmitic acid, or molecules that bind to albumin. In some embodiments, a spacer or linker can be coupled to the binding protein, such as 1, 2, 3, 4, or more amino acid residues used as a spacer or linker, to facilitate conjugation or fusion with another molecule, and to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, the binding proteins can be conjugated to other moieties that can modify or effect a change in the properties of the binding proteins.

[0223] Proteins, such as peptides, can be produced recombinantly or synthetically, for example, by solid-phase peptide synthesis or solution-phase peptide synthesis. Protein synthesis can be carried out by known synthetic methods, such as using fluorenylmethoxycarbonyl (Fmoc) chemistry or by tert-butyloxycarbonyl (Boc) chemistry. Protein fragments can be joined together enzymatically or synthetically.

[0224] In one aspect covered by the present invention, provided herein are methods for generating the proteins described herein, the methods comprising the steps of: (i) culturing transformed host cells under conditions suitable to permit expression of the binding protein described herein, the host cells having been transformed with a nucleic acid comprising a sequence encoding the binding protein; and (ii) recovering the expressed binding protein.

[0225] For example, methods for isolating and purifying recombinantly produced binding proteins can include obtaining a supernatant from a suitable host cell / vector system that secretes the binding protein into the culture medium, followed by concentrating the culture medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse-phase HPLC steps can be employed to further purify the recombinant polypeptide. These purification methods can also be used when isolating an immunogen from a natural environment. Methods for large-scale manufacturing of one or more binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. The binding proteins can be purified according to methods described herein and known in the art.

[0226] In some embodiments, provided herein is a nucleic acid encoding a PRAME immunogenic polypeptide or a fragment thereof described herein, such as 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 or a fragment thereof described herein. In some embodiments, the nucleic acid includes regulatory elements necessary for expressing the open reading frame. Such elements can include, for example, a promoter, a start codon, a stop codon, and a polyadenylation signal. Additionally, enhancers can be included. These elements are operably linked to the sequence encoding the PRAME immunogenic polypeptide or a fragment thereof. Representative vectors, promoters, regulatory elements, etc. that can be used for expressing proteins such as peptides are further described below.

[0227] III. MHC-Peptide Complexes

[0228] In certain aspects, provided are compositions comprising a PRAME immunogenic peptide and an MHC molecule described herein. In some embodiments, the PRAME immunogenic peptide forms a stable complex with the MHC molecule.

[0229] The MHC protein can be conjugated to an agent such as a detection moiety, a radiosensitizer, a photosensitizer, etc., and / or can be chemically modified as described above for the peptide.

[0230] The MHC proteins provided and used in the compositions and methods covered by the present disclosure can be any suitable MHC molecule known in the art. Generally, it has the formula (α-β-P) n, where n is at least 2, for example between 2 and 10, for example 4. α is the α chain of a class I or class II MHC protein. β is the β chain, defined herein as the β chain of a class II MHC protein or β2-microglobulin of an MHC class I protein. P is a peptide antigen.

[0231] In some embodiments, the MHC protein is an MHC class I complex, such as an HLA class I complex.

[0232] The MHC protein can be from any mammalian or avian species, such as a primate species, particularly human; a rodent, including mouse, rat, and hamster; rabbit; horse, cow, dog, cat, etc. For example, the MHC protein can be derived from a human HLA protein or a murine H-2 protein. HLA proteins include the class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, and 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, H-2L, and the class II subunits I-Aα, I-Aβ, I-Eα, and I-Eβ, and β2-microglobulin. Sequences of some representative MHC proteins can be found in Kabat et al., Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pages 724-815. The MHC protein subunits suitable for use in the present invention are soluble forms of normal membrane-bound proteins, which are prepared as known in the art, for example, by deletion of transmembrane and cytoplasmic domains.

[0233] For class I proteins, the soluble form can include the α1, α2, and α3 domains. Soluble class II subunits can include the α1 and α2 domains of the α subunit, and the β1 and β2 domains of the β subunit.

[0234] The α and β subunits can be produced separately and allowed to associate in vitro to form a stable heterodimeric complex, or the two subunits can be expressed in a single cell. Methods for producing MHC subunits are known in the art.

[0235] In certain embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1 and an MHC. In some embodiments, the MHC molecule comprises an MHC α-chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01,HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05, and HLA-C*14:03 alleles. In some embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1A and an MHC having an α-chain with an HLA-A*02 serotype, such as an MHC encoded by the HLA-A*02:01 allele.

[0236] To prepare the MHC-peptide complex, the subunits can be combined with an antigenic peptide and allowed to fold in vitro to form a stable heterodimeric complex with an intrastrand disulfide-bonded domain. The peptide can be included in the initial folding reaction or can be added to the empty heterodimer in a subsequent step. In the compositions and methods encompassed by the present invention, the peptide is a PRAME immunogenic peptide or a fragment thereof. Conditions for allowing the subunits and peptide to fold and associate are known in the art. As an example, approximately equimolar amounts of dissolved α and β subunits can be mixed in a urea solution. Refolding is initiated by dilution or dialysis into a urea-free buffer solution. The peptide can be loaded into the empty class II heterodimer at about pH 5 to 5.5 for about 1 to 3 days, followed by neutralization, concentration, and buffer exchange. However, the specific folding conditions are not critical to the practice of the present invention.

[0237] The monomeric complex (α-β-P) (referred to herein as the monomer) can be multimerized, such as into an MHC tetramer. The resulting multimer is stable over a long period of time. Preferably, the multimer can be formed by binding the monomer to a multivalent entity through a specific attachment site on the α or β subunit, as is known in the art (e.g., as described in U.S. Patent No. 5,635,363). Whether in monomeric or multimeric form, the MHC protein can also be conjugated to beads or any other support.

[0238] The multimeric complex can be labeled so that it can be directly detected when used for immunostaining or other methods known in the art, or it can be used in conjunction with a secondary labeled immunological reagent that specifically and / or selectively binds to the complex (e.g., binds to an MHC protein subunit), as known in the art. For example, the detectable label can be a fluorophore, such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin (PE), allophycocyanin (APC), Brilliant Violet TM 421, BrilliantUV TM 395, Brilliant Violet TM 480, Brilliant Violet TM 421 (BV421), Brilliant Blue TM 515, APC-R700 or APC-Fire750. In some embodiments, the multimeric complex is labeled with a moiety capable of specifically and / or selectively binding to another moiety. For example, the label can be biotin, streptavidin, an oligonucleotide, or a ligand. Other labels of interest can include fluorescent dyes, dyes, enzymes, chemiluminescent agents, particles, radioisotopes, or other directly or indirectly detectable agents.

[0239] In some embodiments, cells presenting an immunogenic peptide in the context of an MHC molecule on the cell surface are generated by transfecting or transducing cells with a vector (e.g., a viral vector) that contains nucleic acid encoding a recombinant or heterologous antigen introduced into the cells. In some embodiments, the vector is introduced into the cells under conditions where one or more peptide antigens (in some cases, including one or more peptide antigens of the expressed heterologous protein) are expressed, processed, and presented on the cell surface by the cells in the context of major histocompatibility complex (MHC) molecules.

[0240] Generally, the cells contacted by the vector are MHC-expressing cells, i.e., MHC-expressing cells. The cells can be cells that normally express MHC on the cell surface, cells that are induced to express MHC and / or upregulate MHC expression on the cell surface, or cells that are engineered to express MHC molecules on the cell surface. In some embodiments, the MHC contains polymorphic peptide-binding sites or binding grooves that can, in some cases, complex with peptide antigens of polypeptides, including peptide antigens processed by cellular mechanisms. In some cases, MHC molecules can be presented or expressed on the cell surface, including in the form of a complex with a peptide, i.e., an MHC-peptide complex, for presenting antigens in a conformation recognizable by a TCR or other peptide-binding molecule on a T cell.

[0241] In some embodiments, the cell is a nucleated cell. In some embodiments, the cell is an antigen-presenting cell. In some embodiments, the cell is a macrophage, dendritic cell, B cell, endothelial cell, or fibroblast. In some embodiments, the cell is an endothelial cell, such as an endothelial cell line or primary endothelial cell. In some embodiments, the cell is a fibroblast, such as a fibroblast cell line or primary fibroblast.

[0242] In some embodiments, the cell is an artificial antigen-presenting cell (aAPC). Generally, aAPCs incorporate features of natural APCs, including the expression of MHC molecules, stimulatory and co-stimulatory molecules, Fc receptors, adhesion molecules, and / or the ability to produce or secrete cytokines (e.g., IL-2). Typically, aAPCs are cell lines that lack the expression of one or more of the above and are generated by introducing (e.g., by transfection or transduction) one or more of the following: elements missing in MHC molecules, low-affinity Fc receptors (CD32), high-affinity Fc receptors (CD64), one or more co-stimulatory signals (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, ILT3, ILT4, 3 / TR6, or B7-H3 ligand; or antibodies that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor, or CD83 ligand), cell adhesion molecules (e.g., ICAM-1 or LFA-3), and / or cytokines (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon-α (IFNα), interferon-β (IFNβ), interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), tumor necrosis factor-β (TNFβ), granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (GCSF)). In some cases, aAPCs generally do not express MHC molecules but can be engineered to express MHC molecules or, in some cases, induced or inducible to express MHC molecules, e.g., by stimulation with cytokines. In some cases, aAPCs can also be loaded with stimulatory ligands, which can include, for example, anti-CD3 antibodies, anti-CD28 antibodies, or anti-CD2 antibodies. Exemplary cell lines that can be used as a backbone for generating aAPCs are the K562 cell line or fibroblast cell lines. A variety of aAPCs are known in the art; see, for example, U.S. Patent No. 8,722,400; Published Application No. US2014 / 0212446; Butler and Hirano (2014) Immunol Rev. 257:10.1111 / imr.12129; Suhoshki et al. (2007) Mol. Ther. 15:981-988).

[0243] Determining or identifying specific MHCs or alleles expressed by a cell is well within the level of a skilled artisan. In some embodiments, prior to contacting the cell with a vector, expression of a specific MHC molecule can be evaluated or confirmed, for example, by using an antibody specific for the particular MHC molecule. Antibodies against MHC molecules are known in the art, such as any of the antibodies described below.

[0244] In some embodiments, cells can be selected to express desired MHC-restricted MHC alleles. In some embodiments, MHC typing of cells (e.g., cell lines) is known in the art. In some embodiments, well-known procedures in the art can be used, such as performing tissue typing by using molecular haplotyping (BioTest ABC SSPtray, BioTest Diagnostics, Denville, N.J.; SeCore Kits, Life Technologies, Grand Island, N.Y.) to determine the MHC typing of cells (e.g., primary cells obtained from a subject). In some cases, determining HLA genotypes by performing standard cell typing, for example, by using sequence-based typing (SBT), is well within the level of a skilled artisan (Adams et al. (2004) J. Transl. Med., 2:30; Smith (2012) Methods MolBiol., 882:67-86). In some cases, the HLA typing of cells (e.g., fibroblasts) is known. For example, the human fetal lung fibroblast cell line MRC-5 is HLA-A*02:01, A29, B13, B44 Cw7 (C*0702); the human foreskin fibroblast cell line Hs68 is HLA-A1, A29, B8, B44, Cw7, Cw16; and the WI-38 cell line is A*68:01, B*08:01 (Solache et al. (1999) J Immunol, 163:5512-5518; Ameres et al. (2013) PloS Pathog. 9:e1003383). The human transfectant fibroblast cell line M1DR1 / Ii / DM expresses HLA-DR and HLA-DM (Karakikes et al. (2012) FASEB J., 26:4886-96).

[0245] In some embodiments, the cells contacted with or into which the vector is introduced are cells that have been engineered or transfected to express MHC molecules. In some embodiments, cell lines can be prepared by genetically modifying a parental cell line. In some embodiments, the cells typically lack a specific MHC molecule and are engineered to express such a specific MHC molecule. In some embodiments, the cells are genetically engineered using recombinant DNA technology.

[0246] In some embodiments, the stabilized MHC-peptide complexes described herein are used to detect T cells that bind to the stabilized MHC-peptide complexes. In some embodiments, the stabilized MHC-peptide complexes described herein are used to monitor the T cell response in a subject, for example, by detecting the amount and / or percentage of T cells (e.g., CD8+ T cells) that specifically and / or selectively bind to a fluorescently labeled MHC-peptide complex. Methods for generating, labeling, and using MHC-peptide complexes (e.g., MHC-peptide tetramers) to detect MHC-peptide complex-specific T cells are well known in the art. Additional descriptions can be found, for example, in U.S. Patent No. 7,776,562; U.S. Patent No. 8,268,964; and U.S. Patent Publication 2019 / 0085048.

[0247] IV. Immunogenic Compositions

[0248] In some aspects, the present disclosure provides pharmaceutical compositions (e.g., vaccine compositions) that comprise a PRAME immunogenic peptide and / or a nucleic acid encoding a PRAME immunogenic peptide and an adjuvant. In some aspects, the present disclosure provides pharmaceutical compositions (e.g., vaccine compositions) that include a stabilized 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) stabilized 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.

[0249] The pharmaceutical compositions disclosed herein can be specifically formulated for administration in solid or liquid form, including those forms suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses applied to the tongue, powders, granules, pastes; or (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection in the form of, for example, a sterile solution or suspension or a sustained release formulation.

[0250] Methods for preparing these formulations or compositions include the step of combining a PRAME immunogenic peptide and / or nucleic acid as described herein with an adjuvant, a carrier, and optionally one or more accessory ingredients as selected. Generally, the formulations are prepared by uniformly and intimately bringing the agents as described herein into association with a liquid carrier or a finely divided solid carrier or both, and then shaping the product, if necessary.

[0251] A pharmaceutical composition suitable for parenteral administration comprises a PRAME immunogenic peptide and / or nucleic acid as 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 just prior to use into a sterile injectable solution or dispersion, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0252] Examples of suitable aqueous and non-aqueous carriers for use in pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). For example, appropriate fluidity can be maintained by using coating materials such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0253] Regardless of the route of administration selected, the agents provided herein and / or the pharmaceutical compositions disclosed herein, when presented in a suitable hydrated form, can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0254] In some embodiments, when administered to a subject, the pharmaceutical composition can elicit an immune response against cells infected with PRAME. Such pharmaceutical compositions can be used as prophylactic and / or therapeutic vaccines for treating conditions characterized by PRAME expression.

[0255] In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the adjuvant used increases the immunogenicity of the pharmaceutical composition. Such compounds or adjuvants that stimulate further immune responses can: (i) be mixed into the pharmaceutical composition according to the invention after the peptide is reconstituted and optionally emulsified with an oil-based adjuvant as defined above, (ii) can be part of the reconstituted composition of the invention as defined above, (iii) can be physically linked to the peptide to be reconstituted, or (iv) can be administered separately to the subject, mammal or human to be treated. The adjuvant can be an adjuvant that provides slow release of the antigen (e.g., the adjuvant can be a liposome), or it can be an adjuvant that is immunogenic per se and thus acts synergistically with the antigen (i.e., the antigen present in the PRAME immunogenic peptide). For example, the adjuvant can be a known adjuvant, or other substances that promote antigen uptake, recruit immune system cells to the site of administration, or promote the immune activation of responding lymphoid cells. Adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, dextran, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, synthetic polymers (e.g., polyamino acids and amino acid copolymers), saponins, paraffin oil, and muramyl dipeptide. In some embodiments, the adjuvant is Adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalose dimycolate, or zymosan.

[0256] In some embodiments, the adjuvant is an immunomodulatory molecule. For example, the immunomodulatory molecule can be a recombinant protein cytokine, chemokine, or immunostimulant designed to enhance the immune response, or a nucleic acid encoding a cytokine, chemokine, or immunostimulant.

[0257] Examples of immunomodulatory cytokines include interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, and IL-20), tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1α, MIP-1β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and functional fragments of any of the foregoing.

[0258] In some embodiments, immunomodulatory chemokines that bind to chemokine receptors (i.e., CXC, CC, C, or CX3C chemokine receptors) can also be included in the compositions provided herein. Examples of chemokines include, but are not limited to, Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tarc, Elc, I309, IL-8, Gcp-2 Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), and functional fragments of any of the foregoing.

[0259] In some embodiments, the composition comprises a nucleic acid encoding a PRAME immunogenic polypeptide as described herein, such as a DNA molecule encoding a PRAME immunogenic peptide. In some embodiments, the composition comprises an expression vector that comprises an open reading frame encoding a PRAME immunogenic peptide.

[0260] When taken up by a cell (e.g., a host cell, an antigen-presenting cell (APC), such as a dendritic cell, macrophage, etc.), the DNA molecule can exist as an extrachromosomal molecule in the cell and / or can integrate into the chromosome. The DNA can be introduced into the cell in plasmid form, which can remain as an independent genetic material. Alternatively, linear DNA that can integrate into the chromosome can also be introduced into the cell. Optionally, when introducing the DNA into the cell, a reagent that promotes integration of the DNA into the chromosome can be added.

[0261] V. Binding Proteins

[0262] In some aspects, a binding moiety is provided that binds a peptide and / or a stabilized MHC-peptide complex as described herein. For example, binding proteins such as T cell receptors (TCRs), antibodies, etc. are provided that bind, e.g., with a K -4 less than or equal to about 10 -4 M (e.g., about 10 -5 M, 10 -6 M, 10 -7 M, about 10 -8 M, about 10 -9 M, about 10 10 M, about 10 -11 M, about 10 -12 M, about 10 -13 M, about 10 -14 M, etc.) specifically and / or selectively to the peptide and / or the stabilized MHC-peptide complex. d

[0263] In one aspect encompassed by the present invention, binding proteins are provided herein that bind (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex comprising a PRAME immunogenic peptide in the context of an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the binding protein is capable of binding (e.g., specifically and / or selectively) to the PRAME peptide-MHC (pMHC) complex with a K d less than or equal to about 5×10 -4 M, less than or equal to about 1×10 -4 M, less than or equal to about 5×10 - 5 M, less than or equal to about 1×10 -5 M, less than or equal to about 5×10 -6 M, less than or equal to about 1×10 -6 M, less than or equal to about 5×10 -7 M, less than or equal to about 1×10 -7 M, less than or equal to about 5×10 -8 M, less than or equal to about 1×10 -8 M, less than or equal to about 5×10 -9 M, less than or equal to about 1×10 -9 M, less than or equal to about 5×10 -10 M, less than or equal to about 1×10 -10 M, less than or equal to about 5×10 -11 M, less than or equal to about 1×10 -11 M, less than or equal to about 5×10 -12 M, less than or equal to about 1×10 -12M, or any range therebetween (including the endpoints), such as about 1 - 50 micromoles, 1 - 100 micromoles, 0.1 - 500 micromoles, etc. In some embodiments, the MHC molecule comprises an MHC α-chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18, optionally wherein the HLA allele is selected from the group consisting of: HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20, HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21,HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In some embodiments, the HLA serotype is HLA-A*02 and / or the HLA allele is the HLA-A*02:01 allele. In some embodiments, the binding proteins provided herein are genetically engineered, isolated, and / or purified.

[0264] 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., the comparative TCR described herein). By way of example, the binding protein may have a binding affinity for a PRAME peptide-MHC (pMHC) that is at least 1.2-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 5000-fold, 10000-fold, 50000-fold, 100000-fold, 500000-fold, 1000000-fold or more higher than a known T cell receptor (e.g., the comparative TCR described herein), or any range therebetween (including the endpoints), such as 1.2-fold to 2-fold.

[0265] In some embodiments, when contacting target cells that express PRAME at a certain level or lower, the binding protein induces higher T cell expansion, cytokine release, and / or cytotoxic killing compared to a known T cell receptor (e.g., the comparative TCR described herein). For example, in some embodiments of any of the aspects described herein, the PRAME level can be expressed per million transcripts and can be, for example, less than or equal to about 1,000 transcripts per million transcripts (TPM), 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or any range therebetween (including the endpoints), e.g., less than or equal to about 1,000 TPM to less than or equal to about 35 TPM). In some embodiments, a low PRAME expression level is referred to as "heterozygous expression" and means between about 1 TPM and about 35 TPM, or any range therebetween (including the endpoints), e.g., 32 TPM or 1 - 32 TPM. Higher expression is 36 TPM and higher. As further described herein, TPM is measured according to well-known techniques, such as RNA-Seq, and gene expression TPM data for a variety of cell lines, tissue types, etc. are well-known in the art (see, e.g., the Broad Institute Cancer Cell Line Encyclopedia (CCLE) at portals.broadinstitute.org on the world wide web).In some embodiments, when contacted with target cells expressing a PRAME peptide epitope, such as target cells expressing the PRAME peptide epitope in a heterologous fashion, the binding protein induces at least 1.2-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold or more increase in T cell expansion, cytokine release, and / or cytotoxic killing, as compared to a known T cell receptor (e.g., the comparative TCR described herein), or any range therebetween (including the endpoints), such as 1.2-fold to 2-fold.

[0266] In some embodiments, expression of PRAME is detected using RNA sequencing (RNA-seq). RNA-seq generally includes the steps of: obtaining a sample containing genetic material, isolating total RNA from the obtained sample, preparing an amplified cDNA library from the total RNA, sequencing the amplified cDNA library, and analyzing and profiling the amplified cDNA to assess the expression levels of different transcripts. The sample can be a cell population, a tissue sample, a biopsy sample, a cell culture, or a single cell. Any method known in the art can be used to isolate total RNA from a biological sample. In certain embodiments, total RNA is extracted from plasma. Extraction of plasma RNA is described in Enders et al., “The Concentration of Circulating Corticotropin-Releasing HomermRNA in Material Plasma Is Inclined in Preclampsia”, Clinr. As described therein, plasma collected after a centrifugation step is mixed with Trizol LS reagent (Invitrogen) and chloroform. The mixture is centrifuged and the aqueous layer is transferred to a new tube. Ethanol is added to the aqueous layer. The mixture is then placed in an RNeasy MinElute column (Qiagen) and processed according to the manufacturer's recommendations.

[0267] In some embodiments, the RNA-seq described herein includes the step of preparing amplified cDNA from total RNA. For example, cDNA is prepared and the isolated RNA sample is randomly amplified without dilution, or the mixture of genetic material in the isolated RNA is dispersed into individual reaction samples. In certain embodiments, the amplification randomly starts at the 3' end and traverses the entire transcriptome in the sample to amplify mRNA and non-polyadenylated transcripts. In this way, the double-stranded cDNA amplification products are optimized to generate a sequencing library for next-generation sequencing platforms. Kits suitable for amplifying cDNA by the methods encompassed by the present invention include, for example RNA-Seq system.

[0268] In some embodiments, the RNA-seq described herein includes the step of sequencing the amplified cDNA. Any known sequencing method can be used to sequence the amplified cDNA mixture, including single-molecule sequencing methods. In certain embodiments, the amplified cDNA is sequenced by whole transcriptome shotgun sequencing. Whole transcriptome shotgun sequencing can be performed using various next-generation sequencing platforms, such as genome analysis platforms, ABI SOLiD TM sequencing platform or the 454 sequencing platform of LifeScience.

[0269] In some embodiments, the RNA-seq described herein further includes digital counting and analysis of the cDNA. The number of amplified sequences of each transcript in the amplified sample can be quantified by sequence reads (one read per amplified strand). In some embodiments, transcripts per million (TPM) are used to quantify the expression level of a specific transcript. TPM can be calculated as shown in Wagner et al. (2012) Theory in Biosciences 131:281-285, the content of which is incorporated herein by reference in its entirety.

[0270] In certain embodiments, the binding protein recognizes a PRAME immunogenic peptide in complex with an MHC molecule, such as a specific HLA molecule having a particular HLA α-chain allele. For example, the binding proteins listed in Table 2A were identified as binders of PRAME immunogenic peptides associated with MHC having the HLA-A*02 serotype of the α-chain, such as MHC encoded by the HLA-A*02:01 allele, as further described in the Examples section. In some embodiments, the binding protein recognizes a complex of a PRAME immunogenic peptide and an MHC molecule, wherein the MHC molecule comprises an MHC α-chain that is selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18 HLA serotypes, optionally wherein the HLA allele is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:11, HLA-A*02:12, HLA-A*02:13, HLA-A*02:14, HLA-A*02:16, HLA-A*02:17, HLA-A*02:19, HLA-A*02:20, HLA-A*02:22, HLA-A*02:24, HLA-A*02:30, HLA-A*02:42, HLA-A*02:53, HLA-A*02:60, HLA-A*02:74 alleles, HLA-A*03:01, HLA-A*03:02, HLA-A*03:05, HLA-A*03:07, HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, HLA-A*01:16 alleles, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, HLA-A*11:04, HLA-A*11:05, HLA-A*11:19 alleles, HLA-A*24:02, HLA-A*24:03, HLA-A*24:05, HLA-A*24:07, HLA-A*24:08, HLA-A*24:10, HLA-A*24:14, HLA-A*24:17, HLA-A*24:20,HLA-A*24:22, HLA-A*24:25, HLA-A*24:26, HLA-A*24:58 alleles, HLA-B*07:02, HLA-B*07:04, HLA-B*07:05, HLA-B*07:09, HLA-B*07:10, HLA-B*07:15, HLA-B*07:21, HLA-C*07:02, HLA-C*07:01, HLA-C*04:01, HLA-C*06:02, HLA-C*03:04, HLA-C*05:01, HLA-C*16:01, HLA-C*02:02, HLA-C*03:03, HLA-C*12:03, HLA-C*08:02, HLA-C*01:02, HLA-C*17:01, HLA-C*15:02, HLA-C*14:02, HLA-C*12:02, HLA-C*07:04, HLA-C*08:01, HLA-C*03:02, HLA-C*18:01, HLA-C*15:05, HLA-C*16:02, HLA-C*08:04, HLA-C*03:05 and HLA-C*14:03 alleles. In some embodiments, the PRAME immunogenic peptides are derived from a human PRAME protein and / or a PRAME protein as shown in Table 3. In some embodiments, one or more PRAME immunogenic peptides are administered alone or in combination with an adjuvant.

[0271] In some embodiments, the binding protein does not bind to a peptide-MHC (pMHC) complex, optionally wherein the peptide is derived from an "off-target" as described herein, such as PLA2G4E, EFNA1, and / or SLC26A1.

[0272] In some embodiments, the binding protein does not bind to an "off-target"-peptide-MHC (pMHC) complex, such as a PLA2G4E, EFNA1, and / or SLC26A1-peptide-MHC (pMHC) complex.

[0273] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or comprise the following): a) a TCR α-chain sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR α-chain sequence selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) a TCR β-chain sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR β-chain sequence selected from the group consisting of the TCR β-chain sequences listed in Table 2.

[0274] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or comprise the following): a) a TCR α-chain sequence selected from the group consisting of the TCR α-chain sequences listed in Table 2; and / or b) a TCR β-chain sequence selected from the group consisting of the TCR β-chain sequences listed in Table 2.

[0275] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or comprise the following): a) a TCR α-chain variable (V α ) domain sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR α-chain variable (V α ) domain sequence selected from the group consisting of the TCR V α ) domain sequences listed in Table 2; and / or b) a TCR β-chain variable (V β ) domain sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR β-chain variable (V β ) domain sequence selected from the group consisting of the TCR V β ) domain sequences listed in Table 2.

[0276] In some embodiments, the binding proteins provided herein include the following (e.g., consist of the following, consist essentially of the following, or comprise the following): a) a TCR α-chain variable (V α) A domain sequence selected from the group consisting of TCR V domain sequences listed in Table 2; and / or b) A TCR β-chain variable (V) domain sequence selected from the group consisting of TCR V domain sequences listed in Table 2. α domain sequences; and / or b) A TCR β-chain variable (V β ) domain sequence selected from the group consisting of TCR V β domain sequences.

[0277] In some embodiments, the binding proteins provided herein include (e.g., comprise at least one (e.g., one, two, or three, e.g., CDR3 alone or in combination with CDR1 and CDR2), consist essentially of, or consist of) a TCR α-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 group consisting of TCR α-chain CDR sequences listed in Table 2. CDR3 is considered the main CDR responsible for recognizing processed antigens, and CDR1 and CDR2 mainly interact with MHC. Thus, in some embodiments, binding proteins are provided that contain CDR3 from the TCR α-chain listed in Table 2 alone and / or CDR3 from the TCR β-chain listed in Table 2 alone, each CDR3 having the sequence homology described in this paragraph.

[0278] In some embodiments, the binding proteins provided herein may further include (e.g., comprise at least one (e.g., one, two, or three, e.g., CDR3 alone or in combination with CDR1 and CDR2), consist essentially of, or consist of) a TCR β-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 group consisting of TCR β-chain CDR sequences listed in Table 2. As described above, CDR3 is considered the main CDR responsible for recognizing processed antigens, and CDR1 and CDR2 mainly interact with MHC. Thus, in some embodiments, binding proteins are provided that contain CDR3 from the TCR β-chain listed in Table 2 alone and / or CDR3 from the TCR α-chain listed in Table 2 alone, each CDR3 having the sequence homology described in this paragraph.

[0279] In some embodiments, the binding proteins provided herein include (e.g., comprise at least one (e.g., one, two, or three), consist essentially of, or consist of) the TCR α-chain complementarity determining regions (CDRs) listed in Table 2.

[0280] In some embodiments, the binding proteins provided herein can also include (e.g., comprise at least one (e.g., one, two, or three), consist essentially of, or consist of) the TCRβ chain complementarity determining regions (CDRs) listed in Table 2.

[0281] In some embodiments, the binding proteins provided herein include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCRα chain constant region (C α ) sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the TCR Cα sequence listed in Table 2.

[0282] In some embodiments, the binding proteins provided herein can also include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCR C β sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the TCRβ chain constant region (C β ) sequence listed in Table 2.

[0283] In some embodiments, the binding proteins provided herein include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCRα chain constant region (C α sequence selected from the group consisting of the TCR C α ) sequences listed in Table 2.

[0284] In some embodiments, the binding proteins provided herein can also include the following (e.g., comprise the following, consist essentially of the following, or consist of the following): a TCR C β sequence selected from the group consisting of the TCRβ chain constant region (C β ) sequences listed in Table 2.

[0285] Table 2: TCR Sequences Recognizing the PRAME Antigen

[0286] Table 2A

[0287] TCR Sequences Recognizing the PRAME Antigen Presented by HLA Serotype HLA-A*02

[0288] PRAME-425-366WT Sequence

[0289] Alpha chain:

[0290] TRAV38-2DV8 / TRAJ50 / TRAC

[0291] Alpha chain DNA sequence

[0292]

[0293] Alpha chain protein sequence

[0294]

[0295] Beta chain:

[0296] TRBV13 / TRBJ2-1 / TRBC1

[0297] Beta chain DNA sequence

[0298]

[0299] Beta chain protein sequence

[0300]

[0301] PRAME-425-366MGTM codon-optimized sequence (also known as "366", "TCR 366", TCR expressed by "TSC-203-A02", and TCR expressed by "TSC-203-A0201")

[0302] Alpha chain:

[0303] TRAV38-2DV8 / TRAJ50 / MGTM-modified TRAC

[0304] Alpha chain DNA sequence

[0305]

[0306] Alpha chain protein sequence

[0307]

[0308] Beta chain:

[0309] TRBV13 / TRBJ2-1 / MGTM-modified TRBC

[0310] Beta chain DNA sequence

[0311]

[0312] Beta chain protein sequence

[0313]

[0314]

[0315] Full β and α ORF DNA sequences (the underlined italicized region in the “Furin - P2A” site encodes a sequence that allows the expression of two polypeptide chains in a single cassette)

[0316]

[0317]

[0318] Full β and α ORF protein sequences (the underlined italicized region in the “Furin - P2A” site allows the expression of two polypeptide chains in a single cassette)

[0319]

[0320] PRAME - 425 - 358WT sequence

[0321] α - chain:

[0322] TRAV30 / TRAJ20 / TRAC

[0323] α - chain DNA sequence

[0324]

[0325] α - chain protein sequence

[0326]

[0327] β - chain:

[0328] TRBV27 / TRBJ2 - 7 / TRBC1

[0329] β - chain DNA sequence

[0330]

[0331] β - chain protein sequence

[0332]

[0333] PRAME - 425 - 358MGTM codon - optimized sequence (also known as “358” or “TCR 358”)

[0334] α - chain:

[0335] TRAV30 / TRAJ20 / MGTM - modified TRAC

[0336] α - chain DNA sequence

[0337]

[0338] α-chain protein sequence

[0339]

[0340]

[0341] β-chain:

[0342] TRBV27 / TRBJ2-7 / MGTM-modified TRBC

[0343] β-chain DNA sequence

[0344]

[0345] β-chain protein sequence

[0346]

[0347]

[0348] Complete β and α ORF DNA sequences (the underlined italic region in the "furin-P2A" site encodes a sequence that allows the expression of two polypeptide chains in a single cassette)

[0349]

[0350]

[0351] Complete β and α ORF protein sequences (the underlined italic region in the "furin-P2A" site allows the expression of two polypeptide chains in a single cassette)

[0352]

[0353] *Table 2 provides representative TCR sequences, which are grouped according to MHC serotype presentation and different peptide subgroups presented by MHC serotype and bound by subgrouped TCRs. The description and requirements for individual TCRs, such as those in the representative examples in the table, and the types of binding proteins that bind to the peptide epitope sequences described herein, either alone or in complex with MHC, such as those grouped in the table provided herein. In addition, the TRAV, TRAJ, and TRAC genes of each TCR α-chain described herein, and the TRBV, TRBJ, and TRBC genes of each TCR β-chain described herein are provided. The sequences of each TCR described herein are provided as pairs of homologous α-chain and β-chain for each named TCR. The TCR sequences described herein are annotated. The variable domain sequences are in uppercase. The constant domain sequences are in lowercase. The CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined text. CDR1, CDR2, and CDR3 are shown in the standard order of appearance from left (N-terminus) to right (C-terminus). The TRAV, TRAJ, and TRAC genes of each TCR α-chain described herein, and the TRBV, TRBJ, and TRBC genes of each TCR β-chain described herein are annotated according to the well-known IMGT nomenclature described herein. Similarly, CDR1 and CDR2 of TRAV and TRBV are well known in the art because they are based on well-known and annotated TRAV and TRBV sequences (e.g., as annotated in the databases of IMGT available at imt.org and IEDB available at iedb.org).

[0354] Table 3

[0355] Human PRAME transcript variant 1 (NM_006115.5; CDS: 226-1755)

[0356]

[0357] Human PRAME transcript variant 2 (NM_206953.3; CDS: 840-2369)

[0358]

[0359] Human PRAME transcript variant 3 (NM_206954.3; CDS: 205-1734)

[0360]

[0361] Human PRAME transcript variant 4 (NM_206955.3; CDS: 430-1959)

[0362]

[0363] Human PRAME transcript variant 5 (NM_206956.3; 409-1938)

[0364]

[0365]

[0366] Human PRAME transcript variant 6 (NM_001291715.2; CDS: 187-1716)

[0367]

[0368] Human PRAME transcript variant 7 (NM_001291716.2; 166-1695)

[0369]

[0370] Human PRAME transcript variant 8 (NM_001291717.2; CDS: 359..1840)

[0371]

[0372] Human PRAME transcript variant 9 (NM_001291719.2; CDS: 116-1597)

[0373]

[0374] Human PRAME transcript variant 10 (NM_001318126.2; CDS: 92-414)

[0375]

[0376]

[0377] Human PRAME transcript variant 11 (NM_001318127.2; CDS: 381-1862)

[0378]

[0379]

[0380] Human PRAME isoform a (NP_006106.1; NP_996836.1; NP_996837.1; NP_996838.1; NP_996839.1; NP_001278644.1; NP_001278645.1)

[0381]

[0382] Human PRAME Isoform b (NP_001278646.1; NP_001278648.1; NP_001305055.1; NP_001305056.1)

[0383]

[0384] Representative human HLA-A*02:01 DNA sequence

[0385]

[0386] Representative HLA-A*02:01 protein sequence

[0387] MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSYSQAASSDSAQGSDVSLTACKV*

[0388] Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pTSLV102 - MSCV - HA1 - 10 - 30 - MGTM - Q - CD8

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396] Representative vector (the protein encoding the TCR can be interchanged with any TCR sequence of interest): pHAGE - MSCV - HN - P32 - 41 - P2A - dnTGFbRII (dnTGFbRII is highlighted in bold)

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403] Table 4

[0404] R11P3D3 TCR MGTM codon-optimized sequence (used as "comparative TCR" in the working examples)

[0405] Alpha chain:

[0406] TRAV24 / TRAJ43 / MGTM-modified TRAC

[0407] Alpha chain DNA sequence

[0408]

[0409] Alpha chain protein sequence

[0410]

[0411]

[0412] Beta chain:

[0413] TRBV12-3 / TRBJ2-3 / MGTM-modified TRBC

[0414] Beta chain DNA sequence

[0415]

[0416] Beta chain protein sequence

[0417]

[0418]

[0419] Full beta and alpha ORF DNA sequence (the underlined italic region in the "furin-P2A" site encodes the sequence allowing the expression of two polypeptide chains in a single cassette)

[0420]

[0421]

[0422] Full β and α ORF protein sequences (the underlined italic region in the "furin-P2A" site allows the expression of two polypeptide chains in a single cassette)

[0423]

[0424] R11P3D3-KE TCRMGTM codon-optimized sequence (used as "affinity-enhanced comparator" and "comparative AE TCR" in the working examples)

[0425] α chain:

[0426] TRAV24 / TRAJ43 / MGTM-modified TRAC

[0427] α chain DNA sequence

[0428]

[0429] α chain protein sequence

[0430]

[0431] β chain:

[0432] TRBV12-3 / TRBJ2-3 / MGTM-modified TRBC

[0433] β chain DNA sequence

[0434]

[0435] β chain protein sequence

[0436]

[0437]

[0438] Full β and α ORF DNA sequences (the underlined italic region in the "furin-P2A" site encodes a sequence that allows the expression of two polypeptide chains in a single cassette)

[0439]

[0440]

[0441] Full β and α ORF protein sequences (the underlined italic region in the "furin-P2A" site allows the expression of two polypeptide chains in a single cassette)

[0442]

[0443] Parental original comparative TCR sequence (TCRR11P3D3)

[0444]

[0445]

[0446]

[0447] Comparator TCR sequence with enhanced parental naïve avidity (TCRR11P3D3_KE)

[0448]

[0449]

[0450] Representative vector (protein encoding the TCR can be interchanged with any TCR sequence of interest): pNVVD134_TSC-203-A02_TCR-366_MSCV-TCR-366-CD8-EF1α-dnTGFbRII-DHFR

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458] *For some of the depicted vectors, the MSCV promoter is in bold. The β-chain is annotated in bold and italic text. The α-chain is annotated in bold and underlined text. The CD34-enriching tag (Q-tag) is annotated in italic and underlined text. CD8-α is italic. CD8-β is underlined.

[0459] *Peptide epitopes are included in Tables 1-4, as well as polypeptide molecules having an amino acid sequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity to the amino acid sequence of any of the sequences listed in Tables 1-4 over their full length, or a portion thereof. Such polypeptides can have the function of the full-length peptide or polypeptide further described herein.

[0460] Table 1-4 includes RNA nucleic acid molecules (e.g., thymine is replaced with uracil), nucleic acid molecules encoding orthologs of the encoded protein, and DNA or RNA nucleic acid sequences having a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identical to any of the sequences listed in Table 1-4 over its full length, or a portion thereof. Such nucleic acid molecules can have the functions of the full-length nucleic acids further described herein.

[0461] In some embodiments, the binding proteins provided herein include chimeric, humanized, human, primate or rodent (e.g., rat or mouse) constant regions. For example, a human variable region can be chimerized with a murine constant region, or a murine variable region can be humanized with a human constant region and / or human framework region. In some embodiments, the constant region can be mutated to modify functionality (e.g., introducing a non-naturally occurring cysteine substitution in the relative residue positions in the TCR α and β chains to provide a disulfide bond that can be used to increase the affinity between the TCR α and β chains). Similarly, mutations can be made in the transmembrane domain of the constant region to modify functionality (e.g., increasing hydrophobicity by introducing non-naturally occurring residue substitutions with hydrophobic amino acids). In some embodiments, each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions or combinations thereof as compared to a reference CDR sequence. In some embodiments, the constant region can be mutated to increase cell surface expression.

[0462] In some embodiments, the binding proteins disclosed herein can be engineered protein scaffolds, antibodies or antigen-binding fragments thereof, TCR-mimicking antibodies, etc. Such binding moieties can be designed and / or generated using conventional immunological methods against the peptides and / or MHC-peptide complexes described herein, such as immunizing a host, obtaining antibody-producing cells and / or their antibodies, and generating hybridomas that can be used to produce monoclonal antibodies (e.g., Watt et al. (2006) Nat. Biotechnol. 24:177-183; Gebauer and Skerra (2009) Curr. Opin. Chem Biol. 13:245-255; Skerra et al. (2008) FEBS J. 275:2677-2683; Nygren et al. (2008) FEBS J. 275:2668-2676; Dana et al. (2012) Exp. Rev. Mol. Med. 14:e6; Sergeva et al. (2011) Blood 117:4262-4272; PCT Publication No. WO 2007 / 143104, PCT / US86 / 02269, and WO 86 / 01533; U.S. Patent No. 4,816,567; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S.L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; U.S. Patent No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060).When necessary, conventional procedures can be used to isolate or purify the binding moiety, such procedures as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, and high performance liquid chromatography (HPLC) (e.g., Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y.).

[0463] The term “antibody / antibodies” broadly encompasses naturally occurring antibody forms (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, as well as fragments and derivatives of all of the foregoing antibodies that have at least one antigen-binding site. Antibody derivatives can include a protein or chemical moiety conjugated to the antibody.

[0464] In addition, intracellular antibodies are well-known antigen-binding molecules that have antibody characteristics but are capable of being expressed intracellularly to bind and / or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods for adapting antibodies for targeting (e.g., inhibiting) intracellular compartments are well-known in the art, such as using single-chain antibodies (scFv), modifying immunoglobulin VL domains to obtain hyperstability, modifying antibodies to resist the reducing intracellular environment, generating fusion proteins that increase intracellular stability and / or regulate intracellular localization, and the like. Intracellular antibodies can also be introduced into and expressed in one or more cells, tissues, or organs of a multicellular organism, e.g., for prophylactic and / or therapeutic purposes (e.g., as gene therapy) (see, inter alia, 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).

[0465] As used herein, the term "antibody" also includes an "antigen-binding portion" (or simply "antibody portion") of an antibody. As used herein, the term "antigen-binding portion" refers to one or more fragments of an antibody that retain the ability to specifically and / or selectively bind to an antigen (e.g., a peptide and / or MHC-peptide complex as described herein). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are divalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341:544-546), which consist of a VH domain; and (vi) isolated complementarity-determining regions (CDRs). In addition, although the two domains, VL and VH, of an Fv fragment are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be made as a single protein chain, where the VL region pairs with the VH region to form a monovalent polypeptide (called single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16:778). It is also intended that such single-chain antibodies be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequences of a particular scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences in order to generate an expression vector encoding a complete IgG polypeptide or other isotype. VH and VL can also be used to generate Fab, Fv, or other immunoglobulin fragments using protein chemistry or recombinant DNA techniques. Also encompassed are other forms of single-chain antibodies such as, for example, bispecific antibodies. Bispecific antibodies are bivalent bispecific antibodies in which the VH domain and the VL domain are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain and creating two antigen-binding sites (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0466] In addition, an antibody or an antigen-binding portion thereof can be part of a larger immunoadhesion polypeptide formed by the covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include a tetrameric scFv polypeptide made using a streptavidin core region (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and a bivalent and biotinylated scFv polypeptide made using cysteine residues, protein subunit peptides, and a C-terminal polyhistidine tag (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions such as Fab and F(ab')2 fragments can be prepared from intact antibodies using conventional techniques, such as pepsin digestion or papain digestion of intact antibodies, respectively. In addition, as described herein, antibodies, antibody portions, and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques.

[0467] The antibody can be polyclonal or monoclonal; xenogeneic, allogeneic or syngeneic; or a modified form thereof (e.g., humanized, chimeric, etc.). The antibody can also be fully human. Preferably, the antibodies of the invention bind specifically and / or selectively or substantially specifically and / or selectively to the peptides and / or MHC-peptide complexes described herein. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one antigen-binding site capable of immunoreacting with a specific epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity for the specific antigen with which they immunoreact.

[0468] Similar to the other binding portions described herein, an antibody can also be "humanized", which is intended to include antibodies made by non-human cells having variable and constant regions that have been altered to be more closely similar to antibodies that would be made by human cells. For example, by altering the amino acid sequence of a non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention can include, for example, amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro / in vivo random or site-specific mutagenesis or by in vivo somatic mutation). As used herein, the term "humanized antibody" also includes antibodies in which the CDR sequences from the germline of another mammalian species have been transplanted onto a human framework sequence.

[0469] In some embodiments, the binding proteins disclosed herein can comprise a T cell receptor (TCR), an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In some embodiments, the binding proteins disclosed herein can comprise two polypeptide chains, each of which comprises a variable region that comprises CDR3 of the TCRα chain and CDR3 of the TCRβ chain, or CDR1, CDR2, and CDR3 of both the TCRα chain and the TCRβ chain. In some embodiments, the binding protein comprises a single-chain TCR (scTCR) that comprises both TCR V α and TCR V β domains, but comprises only a single TCR constant domain (C α or C β ). The term "chimeric antigen receptor" (CAR) refers to a fusion protein that is engineered to contain two or more naturally occurring amino acid sequences that are linked together in a non-naturally occurring manner or in a manner that is not naturally occurring in a host cell, and that functions as a receptor when present on the cell surface. The CARs encompassed by the present invention can include an extracellular portion that comprises an antigen-binding domain (i.e., obtained from or derived from an immunoglobulin or immunoglobulin-like molecule, such as an antibody or a TCR, or an antigen-binding domain derived from or obtained from a killer immunoglobulin receptor from a NK cell), which antigen-binding domain is linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing a co-stimulatory domain) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; and Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0470] In some embodiments, 1) the TCRα chain CDR, TCR V α domain, and / or the TCRα chain is encoded by a TRAV, TRAJ, and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 2, and / or 2) the TCRβ chain CDR, TCR V β domain, and / or the TCRβ chain is encoded by a TRBV, TRBJ, and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 2, and / or 3) each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions, or combinations thereof as compared to the homologous reference CDR sequences listed in Table 2.

[0471] In some embodiments, the binding proteins disclosed herein (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs)) are chimeric (e.g., comprising amino acid residues or motifs from more than one donor or species), humanized (e.g., comprising residues from a non-human organism that have been altered or substituted to reduce the risk of human immunogenicity), or human.

[0472] Methods for generating engineered binding proteins (e.g., TCRs, CARs, and antigen-binding fragments thereof) are well known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 5:3000; U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0473] In some embodiments, the binding proteins described herein are TCRs or antigen-binding fragments thereof that are expressed on the cell surface, wherein the cell surface-expressed TCR is capable of associating more effectively with CD3 protein compared to the endogenous TCR. When expressed on the surface of a cell such as a T cell, the binding proteins (e.g., TCRs) encompassed by the present invention may also have higher surface expression on the cell compared to an endogenous binding protein (e.g., an endogenous TCR). In some embodiments, a CAR is provided herein, wherein the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0474] Also provided are modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs), which can be engineered according to well-known methods using the binding proteins disclosed herein having one or more V α and / or V β sequences as starting materials, and the modified binding proteins may have properties that are altered compared to the starting binding protein. The binding protein can be engineered by modifying one or more residues within one or two variable regions (i.e., V α and / or V β ), such as within one or more CDR regions and / or within one or more framework regions. Alternatively or additionally, the binding protein can be engineered by modifying residues within the constant region.

[0475] Another type of variable region modification is to mutate the amino acid residues in the V α and / or V β CDR1, CDR2, and / or CDR3 regions, thereby improving one or more binding properties (e.g., affinity) of the binding protein of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutations, and the effects on protein binding or other functional properties of interest can be evaluated in in vitro, ex vivo, or in vivo assays as described herein and provided in the Examples. In some embodiments, conservative modifications (as discussed above) can be introduced. The mutations can be amino acid substitutions, additions, or deletions. In some embodiments, the mutations are substitutions. Additionally, typically no more than one, two, three, four, or five residues in the CDR region are modified.

[0476] In some embodiments, the binding proteins described herein (e.g., TCR, antigen-binding fragment of a TCR, or CAR) can have one or more amino acid substitutions, deletions, or additions relative to a naturally occurring TCR. In some embodiments, each CDR of the binding protein has at most five amino acid substitutions, insertions, deletions, or combinations thereof compared to the homologous reference CDR sequences listed in Table 2. Conservative substitutions of amino acids are well known and can occur naturally or can be introduced during the recombinant production of the binding protein. Amino acid substitutions, deletions, and additions can be introduced into the protein using mutagenesis methods known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide site-specific (or segment-specific) mutagenesis procedures can be employed to provide an altered polynucleotide having specific codons changed according to the desired substitution, deletion, or insertion. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis, can be used to prepare immunogenic polypeptide variants (see, e.g., Sambrook et al., supra).

[0477] A variety of criteria known to one of ordinary skill in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or conservative amino acid substitution is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids can be included in the following categories: amino acids having a basic side chain (e.g., lysine, arginine, histidine); amino acids having an acidic side chain (e.g., aspartic acid, glutamic acid); amino acids having an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids having a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids having a β-branched side chain (e.g., threonine, valine, isoleucine), and amino acids having an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan). Proline is considered more difficult to classify and shares characteristics with amino acids having an aliphatic side chain (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, substituting glutamine for glutamic acid or asparagine for aspartic acid can be considered a similar substitution because glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As would be understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of the polypeptide and its conservative amino acid substitutions with the sequence of a second polypeptide (e.g., using GENEWORKS TM , Align, BLAST algorithms or other algorithms described herein and practiced in the art).

[0478] In some embodiments, the encoded binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) can comprise a "signal peptide" (also referred to as a leader sequence, leader peptide, or transit peptide). The signal peptide targets the newly synthesized polypeptide to its appropriate location inside or outside the cell. The signal peptide can be removed from the polypeptide during or once localization or secretion is complete. A polypeptide having a signal peptide is referred to herein as a "preprotein" and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In some embodiments, the binding proteins described herein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) comprise a mature V α domain, a mature V β domain, or both. In some embodiments, the binding proteins described herein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) comprise a mature TCR β-chain, a mature TCR α-chain, or both.

[0479] In some embodiments, the binding protein is a fusion protein that comprises: (a) an extracellular component comprising a TCR or an antigen-binding fragment thereof; (b) an intracellular component comprising an effector domain or a functional portion thereof; and (c) a transmembrane domain that links the extracellular component and the intracellular component. In some embodiments, the fusion protein is capable of binding (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex that comprises a 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 α chain that is of HLA serotype HLA-A*02. In some embodiments, the HLA allele is selected from the group consisting of HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, and HLA-A*02:07 alleles. In certain embodiments, the HLA allele is HLA-A*0201.

[0480] As used herein, an "effector domain" or "immune effector domain" is the intracellular portion or domain of a fusion protein or receptor that, upon receipt of an appropriate signal, can directly or indirectly promote an immune response in a cell. In some embodiments, the effector domain is from an immune cell protein or a portion thereof or an immune cell protein complex, and the effector domain receives a signal when binding occurs (e.g., CD3ζ), or when the immune cell protein or a portion thereof or the immune cell protein complex directly binds to a target molecule and triggers signal transduction of the effector domain in an immune cell.

[0481] When the effector domain contains one or more signaling domains or motifs, such as intracellular tyrosine activation motifs (ITAMs), such as those found in co-stimulatory molecules, it can directly promote a cellular response. Without wishing to be bound by theory, it is believed that following engagement of a T cell receptor or a fusion protein containing a T cell effector domain with a ligand, the ITAM can be used for T cell activation. In some embodiments, the intracellular component or a functional portion thereof contains an ITAM. Exemplary immune effector domains include, but are not limited to, those from: CD3ε, CD3δ, CD3ζ, CD25, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the effector domain contains a lymphocyte receptor signaling domain (e.g., CD3ζ or a functional portion or variant thereof).

[0482] In other embodiments, the intracellular component of the fusion protein contains a co-stimulatory domain or a functional portion thereof selected from: CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-l (CD54), LFA-l (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that binds to CD83 (e.g., specifically and / or selectively) or a functional variant thereof, or any combination thereof. In some embodiments, the intracellular component contains a CD28 co-stimulatory domain or a functional portion or variant thereof (which may optionally include the LL-GG mutation at positions 186-187 of the native CD28 protein (e.g., Nguyen et al. (2003) Blood 702:4320), a 4-1BB co-stimulatory domain or a functional portion or variant thereof, or both).

[0483] In some embodiments, the effector domain comprises the intracellular domain of CD3ε or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD27 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD28 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of 4-1BB or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of OX40 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD2 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of CD5 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of ICAM-1 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of LFA-1 or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In other embodiments, the effector domain comprises the intracellular domain of ICOS or a functional (e.g., signaling) portion thereof, or a functional variant thereof.

[0484] The extracellular and intracellular components encompassed by the present invention are linked by a transmembrane domain. As used herein, a "transmembrane domain" is the portion of a transmembrane protein that can be inserted into or span the cell membrane. A transmembrane domain has a three-dimensional structure that is thermodynamically stable in the cell membrane and generally ranges in length from about 15 amino acids to about 30 amino acids. The structure of a transmembrane domain can comprise an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof. In some embodiments, the transmembrane domain comprises or is derived from a known transmembrane protein (e.g., the transmembrane domain of CD4, the transmembrane domain of CD8, the transmembrane domain of CD27, the transmembrane domain of CD28, or any combination thereof).

[0485] In some embodiments, the extracellular component of the fusion protein further comprises a linker disposed between the binding domain and the transmembrane domain. As used herein when referring to the component linking the binding domain and the transmembrane domain in a fusion protein, a "linker" can be an amino acid sequence having from about two amino acids to about 500 amino acids, which can provide flexibility and space for conformational movement between two regions, domains, motifs, fragments, or modules linked by the linker. For example, the linker encompassed by the present invention can position the binding domain away from the surface of the host cell expressing the fusion protein such that proper contact, antigen binding, and activation between the host cell and the target cell can be achieved (Patel et al. (1999) Gene Therapy 6:412-419). The linker length can vary based on the selected target molecule, the selected binding epitope, or the capture and affinity of the antigen-binding domain to maximize antigen recognition (see, for example, Guest et al. (2005) Immunother. 28:203-11, and PCT Publication No. WO 2014 / 031687). Exemplary linkers include those having a glycine-serine amino acid chain having one to about ten Gly x Ser y repeat sequences, where x and y are each independently an integer from 0 to 10, with the proviso that x and y are not both 0 (e.g., (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or combinations thereof, such as ((Gly3Ser)2Gly2Ser)).

[0486] The binding protein can be conjugated to an agent such as a detection moiety, radiosensitizer, photosensitizer, etc., and / or can be chemically modified as described above for peptides.

[0487] In some embodiments, the binding proteins encompassed by the present invention can be covalently linked to a moiety. In some embodiments, the covalently linked moiety comprises an affinity tag or label. The affinity tag can be selected from the group consisting of glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label can be a fluorescent protein. In some embodiments, the covalently linked moiety is selected from the group consisting of pro-inflammatory factors, anti-inflammatory agents, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies, such as single-chain Fv.

[0488] The binding protein can be conjugated to agents for imaging, research, therapeutics, theranostics, pharmacy, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, the binding protein can be conjugated or fused to a detectable agent such as a fluorophore, near-infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radionuclide chelator, or another suitable material for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties can be attached to the binding protein. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not readily quenched by biological tissues and body fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation having a wavelength between 650 nm and 4000 nm, such emission being used to detect such agent. Non-limiting examples of fluorescent dyes that can be used as conjugated molecules include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, the near-infrared dyes generally include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Additional non-limiting examples of fluorescent dyes used as conjugated molecules according to the present invention include acridine orange or acridine yellow, Alexa (e.g., Alexa 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilino-1-naphthalenesulfonic acid, dye and any derivatives thereof, auramine-rhodamine stain and any derivatives thereof, bensantrhone, bimane, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene, bisbenzimide, brainbow, calcein, carboxyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, and any derivatives thereof, epicocconone, ethidium bromide, Fluo dye and any derivatives thereof, and any of its derivatives, fluorescein and any of its derivatives, and any of its derivatives, and any of its derivatives, and any of its derivatives, fluorescent proteins and any of their derivatives, m isotype proteins and any of their derivatives (such as mCherry), hetamethine dyes and any of their derivatives, hoeschst stains, iminocoumarins, Indian yellow, indo-1 and any of its derivatives, laurdan, fluorescein yellow and any of its derivatives, fluorescein and any of its derivatives, luciferase and any of its derivatives, merocyanine and any of its derivatives, nile dyes and any of their derivatives, perylene, phloxine, phycobiliprotein dyes and any of their derivatives, propidium iodide, pyranine, rhodamine and any of its derivatives, ribogreen, RoGFP, rubrene, stilbene and any of its derivatives, sulforhodamine and any of its derivatives, SYBR and any of its derivatives, synapto-pHluorin, tetraphenylbutadiene, tris tetrasodium, TexasRed, TitanYellow, TSQ, umbelliferone, violanthrone, yellow fluorescent protein and YOYO-1. Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green TM dyes (e.g., Oregon Green TM 488, Oregon Green TM 500, OregonGreen TM 514, etc.), Texas Texas -X, SPECTRUM SPECTRUM cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), Alexa Dyes (e.g., Alexa 350, Alexa 488, Alexa 532, Alexa 546, Alexa 568, Alexa 594, Alexa 633, Alexa 660, Alexa 680, etc.), Dyes (e.g., FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRD dyes (e.g., IRD40 TM , IRD700 TM , IRD800 TM , etc.). Other suitable detectable agents are well known in the art (e.g., PCT Publication No. PCT / US14 / 56177). Non-limiting examples of radioisotopes include 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.

[0489] The binding protein can be conjugated with a radiosensitizer or a photosensitizer. Examples of radiosensitizers include, but are not limited to: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to: fluorescent molecules or beads that generate heat upon irradiation, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metal porphyrins, metal phthalocyanines, angelicin, chalcogenopyrylium dyes, chlorophylls, coumarins, flavins, and related compounds (e.g., lumazines and riboflavins), fullerenes, pheophorbide a, pyropheophorbide a, cyanines (e.g., merocyanine 540), pheophytin, thiafurane, tesafurane, purpurin, porphycene, phenothiazinium, methylene blue derivatives, naphthalenedicarboximides, nile blue derivatives, quinones, perylenequinones (e.g., hypericin, hypocrellin, and cercosporin), psoralens, quinones, retinoids, rhodamines, thiophenes, wilting, xanthene dyes (e.g., eosin, erythrosin, rose bengal), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, the method allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent (e.g., a drug) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the binding protein is fused with the agent, or covalently or non-covalently linked to the agent, e.g., directly or via a linker.

[0490] In some embodiments, the binding protein can be chemically modified. For example, the binding protein can be mutated to modify peptide properties such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is an example of methylation that can occur in the binding proteins encompassed by the present invention. In some embodiments, the binding protein can be modified by methylating free amines, e.g., by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0491] The chemical modification can comprise a polymer, a polyether, a polyethylene glycol, a biopolymer, an amphiphilic polymer, a polyamino acid, a fatty acid, a dendrimer, an Fc region, a simple saturated carbon chain (e.g., palmitate or myristate), or albumin. The chemical modification of a binding protein having an Fc region can be a fusion Fc-protein. The polyamino acid can include, for example, a polyamino acid sequence having a repeating single amino acid (e.g., polyglycine), and a polyamino acid sequence having a mixed polyamino acid sequence that may or may not follow a pattern, or any combination of the foregoing.

[0492] In some embodiments, the binding proteins encompassed by the present invention may be modified. In some embodiments, the modification has substantial or significant sequence identity with the parental binding protein to produce a functional variant that maintains one or more biophysical and / or biological activities of the parental binding protein (e.g., maintaining pMHC binding specificity). In some embodiments, the mutation is a conservative amino acid substitution.

[0493] In some embodiments, the binding proteins encompassed by the present invention may contain synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxy phenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0494] The binding proteins encompassed by the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via a disulfide bridge), or converted to an acid addition salt, and / or optionally dimerized or polymerized, or conjugated.

[0495] In some embodiments, attachment of a hydrophobic moiety (e.g., attachment to the N-terminus, C-terminus, or an internal amino acid) can be used to extend the half-life of the peptides encompassed by the present invention. In other embodiments, the binding protein can include post-translational modifications (e.g., methylation and / or amidation) that can affect, for example, serum half-life. In some embodiments, a simple carbon chain (e.g., by myristoylation and / or palmitoylation) can be conjugated to the binding protein. In some embodiments, the simple carbon chain can facilitate separation of the binding protein from unconjugated material. Methods that can be used to separate the binding protein from unconjugated material include, but are not limited to, solvent extraction and reverse-phase chromatography. The lipophilic moiety can extend the half-life by reversible binding to serum albumin. The conjugated moiety can be a lipophilic moiety that extends the half-life of the peptide by reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative, including cholestene, cholestane, cholestadiene, and oxysterol. In some embodiments, the binding protein can be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding protein can be coupled (e.g., conjugated) to a half-life modifier. Examples of half-life modifiers include, but are not limited to: polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers containing glycine, glutamate, and serine, the Fc region, fatty acids, palmitic acid, or a molecule that binds to albumin. In some embodiments, a spacer or linker can be coupled to the binding protein, such as 1, 2, 3, 4, or more amino acid residues that serve as a spacer or linker, to facilitate conjugation or fusion with another molecule and to facilitate cleavage of the peptide from such conjugated or fused molecules. In some embodiments, the binding protein can be conjugated to other moieties that can modify or effect a change in the properties of the binding protein.

[0496] The binding protein can be produced recombinantly or synthetically, for example, by solid-phase peptide synthesis or solution-phase peptide synthesis. Polypeptide synthesis can be carried out by known synthetic methods, such as using fluorenylmethyloxycarbonyl (Fmoc) chemistry or by tert-butyloxycarbonyl (Boc) chemistry. The polypeptide fragments can be joined together enzymatically or synthetically.

[0497] In one aspect encompassed by the present invention, there is provided a method for producing the binding protein described herein, the method comprising the steps of: (i) culturing a transformed host cell under conditions suitable to permit expression of the binding protein described herein, the host cell having been transformed with a nucleic acid comprising a sequence encoding the binding protein; and (ii) recovering the expressed binding protein.

[0498] For example, methods for separating and purifying recombinantly produced binding proteins can include obtaining a supernatant from a suitable host cell / vector system that secretes the binding protein into the culture medium, followed by concentrating the culture medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse-phase HPLC steps can be employed to further purify the recombinant polypeptide. These purification methods can also be used when isolating an immunogen from its natural environment. Methods for large-scale manufacture of one or more binding proteins described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. The binding proteins can be purified according to methods described herein and known in the art.

[0499] In any of the embodiments disclosed herein, the encoded binding protein is capable of conjugating 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 α-chain that is 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.

[0500] There are a variety of assays known for assessing binding affinity and / or determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand (e.g., a peptide antigen-MHC complex). For example, determining the binding affinity of a binding protein for a target (e.g., a T cell peptide epitope of a target polypeptide) is within the level of skill of a person skilled in the art by using any of a variety of binding assays well known in the art. For example, in some embodiments, a Biacore TM machine can be used to determine the binding constant of a complex between two proteins. The dissociation constant (K D ) can be determined by monitoring the change in refractive index over time as buffer passes over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or measuring binding by monitoring changes in the spectral or optical properties of the proteins by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blotting, ELISA, analytical ultracentrifugation, spectroscopic analysis, and surface plasmon resonance (Biacore TM)Analysis (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; Wilson (2002) Science 295:2103; Wolff et al. (1993) Cancer Res. 53:2560; and U.S. Patent Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, apparent affinity for a target is measured by assessing binding to various concentrations of a labeled multimer, such as an MHC-antigen tetramer, e.g., by flow cytometry. In a representative example, a two-fold dilution series of a labeled tetramer at a range of concentrations is used to measure the apparent K D , followed by determination of the binding curve by non-linear regression to determine the apparent K D which is the ligand concentration that produces half-maximal binding.

[0501] III. Nucleic Acids and Vectors

[0502] In one aspect encompassed by the present invention, provided herein are nucleic acid molecules that encode 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.), and the like.

[0503] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to a complement of a sequence having at least about at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-4, e.g., over the full length.

[0504] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to a complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-4.

[0505] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Tables 1-4 (e.g., comprising it, consisting essentially of it, or consisting of it).

[0506] In some embodiments, the nucleic acid sequence encodes a PRAME immunogenic peptide as described herein.

[0507] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one (e.g., one, two, or three) TCRα chain CDRs described in Table 2 (e.g., comprising it, consisting essentially of it, or consisting of it). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCRV α domain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCRV α domain has an amino acid sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the TCRV α domain sequence described in Table 2. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCRα chain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCRα chain has an amino acid sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the TCRα chain sequence described in Table 2.

[0508] In some embodiments, the nucleic acid comprises a nucleotide sequence encoding at least one (e.g., one, two, or three) TCRβ chain CDRs described in Table 2 (e.g., comprising it, consisting essentially of it, or consisting of it). In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCRV β domain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCRV β domain has an amino acid sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the TCR V β domain sequence described in Table 2. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the following TCRβ chain (e.g., comprising it, consisting essentially of it, or consisting of it), wherein the TCRβ chain has an amino acid sequence with at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to the TCRβ chain sequence described in Table 2.

[0509] The term "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally refers to a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources (e.g., isolated and / or purified), which can contain natural, unnatural, or modified nucleotides, and can contain natural, unnatural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, rather than the phosphodiester found between nucleotides in unmodified oligonucleotides. In one embodiment, the nucleic acid comprises complementary DNA (cDNA).

[0510] In some embodiments, the nucleic acids encompassed by the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in a living cell, or (ii) a molecule produced by the replication of the molecules described in (i) above. For the purposes herein, replication can be in vitro / in vitro replication or in vivo replication.

[0511] 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. By way of example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides that are designed to increase the biological stability of the molecule or the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, scytalone, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetate, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more nucleic acids encompassed by the present invention can be purchased from companies such as Integrated DNA Technologies (Coralville, IA).

[0512] In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound by a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence can improve the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but can be translated by a more readily available tRNA within the cell, thereby improving translation efficiency. Optimization of the nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, thereby improving translation efficiency. In some embodiments, the nucleotide sequences described herein are codon-optimized for expression in a host cell (e.g., an immune cell, e.g., a T cell).

[0513] The present invention also provides a nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or that hybridizes to the nucleotide sequence of any nucleic acid described herein under stringent conditions.

[0514] Nucleotide sequences that hybridize under stringent conditions can hybridize under highly stringent conditions. "Highly stringent conditions" mean that a nucleotide sequence hybridizes specifically to a target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. Highly stringent conditions include conditions that distinguish a polynucleotide with an exact complementary sequence or a polynucleotide containing only a few scattered mismatches from a random sequence that has only a few small regions (e.g., 3-10 bases) that match the nucleotides exactly. Such small complementary regions are more readily melted compared to a full-length complement of 14-17 or more bases, and highly stringent hybridization makes it easy to distinguish. Relatively high stringent conditions will include, for example, low salt and / or high temperature conditions, such as provided by about 0.02 - 0.1 M NaCl or equivalents at a temperature of about 50 - 70 °C. Such highly stringent conditions allow little or no mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting the expression of any TCR of the present invention. It is generally understood that the conditions can be made more stringent by adding incremental amounts of formamide.

[0515] The present invention also provides a nucleic acid, the nucleic acid comprising a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to any nucleic acid described herein.

[0516] Generally, the nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0517] The terms "vector", "cloning vector" and "expression vector" mean a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell so as to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Thus, another object encompassed by the present invention relates to a vector comprising a nucleic acid encompassed by the present invention.

[0518] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., to cause or direct the expression of the polypeptide after administration to a subject. Examples of promoters and enhancers for use in animal cell expression vectors include the early promoter and enhancer of SV40 (Mizukami T et al. 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al. 1987), the promoter of immunoglobulin H chain (Mason J O et al. 1985) and enhancer (Gillies SD et al. 1983), etc.

[0519] Any animal cell expression vector can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1βd2-4- (Miyaji H et al. 1990), etc. Other representative examples of plasmids include replicating plasmids containing an origin of replication, or integrating plasmids, such as pUC, pcDNA, pBR, etc. Representative examples of viral vectors include adenovirus, retrovirus, lentivirus, herpesvirus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with a helper plasmid or virus. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses are well known in the art and can be found, for example, in PCT publication WO 95 / 14785, PCT publication WO 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and PCT publication WO 94 / 19478.

[0520] In some embodiments, the composition comprises an expression vector that contains an open reading frame encoding a binding protein or polypeptide or a fragment thereof as described herein. In some embodiments, the nucleic acid includes regulatory elements necessary for expressing the open reading frame. Such elements can include, for example, a promoter, a start codon, a stop codon, and a polyadenylation signal. Additionally, enhancers can be included. These elements are operably linked to the sequence encoding the binding protein, polypeptide, or a fragment thereof.

[0521] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, a dominant negative TGFβ receptor (such as DN-TGFβRII), a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In certain embodiments, the nucleic acid sequences encoding CD8α, CD8β, DN-TGFβR, and / or the selectable protein marker are operably linked to a nucleic acid encoding a tag (such as a CD34 enrichment tag). In specific embodiments, the nucleic acid sequences described herein, such as those encoding TCRα, TCRβ, CD8α, CD8β, DN-TGFβR, and / or the selectable protein marker, are linked to an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, E2A, F2A, or T2A, etc.).

[0522] In some embodiments, the expression vectors provided herein comprise nucleotide sequences that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identical to any of the nucleic acids shown in Tables 1-3.

[0523] Examples of promoters include, but are not limited to, the promoter from Simian Virus 40 (SV40); the Mouse Mammary Tumor Virus (MMTV) promoter; promoters from the Human Immunodeficiency Virus (HIV), such as the HIV Long Terminal Repeat (LTR) promoter; the promoter from Moloney virus; the promoter from Cytomegalovirus (CMV), such as the CMV immediate early promoter; the promoter from Epstein Barr Virus (EBV); the promoter from Rous Sarcoma Virus (RSV); and promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Examples of suitable polyadenylation signals include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal.

[0524] In addition to the regulatory elements required for expression, other elements may also be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include the promoters described above. In some embodiments, the enhancer / promoter includes, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as those from CMV, RSV, and EBV.

[0525] In some embodiments, the nucleic acid is operably incorporated into a carrier or delivery vehicle as further described below. Delivery vehicles that can be used include, but are not limited to, biodegradable microcapsules, immunostimulating complexes (ISCOMs), or liposomes, as well as genetically engineered attenuated live carriers such as viruses or bacteria.

[0526] In some embodiments, the vector is a viral vector, such as a lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, Fowlpox virus, AV-pox virus, Modified Vaccinia Ankara (MVA) virus, and other recombinant viruses. For example, lentiviral vectors can be used to infect T cells.

[0527] In some embodiments, the recombinant expression vector is capable of delivering the polynucleotide to a suitable host cell, such as a T cell or an antigen-presenting cell, i.e., a cell that displays peptide / MHC complexes on its cell surface and lacks CD8 (e.g., dendritic cells). In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. For example, the immune system cell can be a CD4 + T cell, a CD8 + T cell, a CD4 / CD8 double-negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof. In some embodiments, where the T cell is the host, the T cell can be a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. Thus, the recombinant expression vector can also include, for example, a lymphoid tissue-specific transcriptional regulatory element (TRE), such as a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue-specific TREs are known in the art (see, for example, Thompson et al. (1992) Mol. Cell. Biol. 72:1043; Todd et al. (1993) J. Exp. Med. 777:1663; and Penix et al. (1993) J. Exp. Med. 775:1483).

[0528] In some embodiments, the recombinant expression vector contains a nucleotide sequence encoding a TCR α-chain, a TCR β-chain, and / or a linker peptide. For example, in some embodiments, the recombinant expression vector contains a nucleotide sequence encoding full-length TCR α- and TCR β-chains of a binding protein (with a linker therebetween), wherein the nucleotide sequence encoding the β-chain is 5' to the nucleotide sequence encoding the α-chain. In some embodiments, the nucleotide sequence encodes full-length TCR α- and TCR β-chains (with a linker therebetween), wherein the nucleotide sequence encoding the TCR β-chain is 3' to the nucleotide sequence encoding the TCR α-chain. In some embodiments, the full-length TCR α- and / or TCR β-chains are replaced by fragments thereof.

[0529] As further described below, another aspect covered by the present invention relates to a cell that has been transfected, infected, or transformed with a nucleic acid and / or vector according to the present invention. A host cell can include any individual cell or cell culture that can accept a vector or incorporate nucleic acids and / or proteins, as well as any progeny cells. The term also encompasses the progeny of the host cell, whether genetically or phenotypically identical or different. Suitable host cells can depend on the vector and can include mammalian cells, animal cells, human cells, ape cells, insect cells, yeast cells, and bacterial cells. These cells can be induced to incorporate the vector or other materials by using viral vectors, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods (see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory)). The term "transformation" means introducing an "exogenous" (i.e., external or extracellular) gene, DNA, or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence, thereby producing the desired substance, usually a protein or enzyme encoded by the introduced gene or sequence. A host cell that accepts and expresses the introduced DNA or RNA has been "transformed."

[0530] The nucleic acids covered by the present invention can be used to produce recombinant polypeptides covered by the present invention in a suitable expression system. The term "expression system" means a host cell and a compatible vector that, under suitable conditions, express a protein encoded by foreign DNA carried by the vector and introduced into the host cell.

[0531] Common expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli; yeast of the genus Kluyveromyces or Saccharomyces; mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.); and primary or established mammalian cell cultures (e.g., generated from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), dihydrofolate reductase gene (hereinafter referred to as "DHFR gene")-deficient CHO cells (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.

[0532] The present invention also encompasses methods for generating recombinant host cells that express the binding proteins, peptides, and fragments thereof encompassed by the present invention, said methods comprising the steps consisting of: (i) introducing the recombinant nucleic acid or vector as described above in vitro or ex vivo into a competent host cell, (ii) culturing the obtained recombinant host cell in vitro or ex vivo, and (iii) optionally, selecting the cells that express the binding proteins, peptides, and fragments thereof. Such recombinant host cells can be used in the diagnostic, prognostic, and / or therapeutic methods encompassed by the present invention.

[0533] In another aspect, the 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 that contain such polynucleotides. For example, the polynucleotides encompassed by the invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotide is a genomic sequence or cDNA sequence isolated from a human or mammalian nucleic acid library or otherwise complementary to cDNA from said library. In some embodiments, the cDNA library contains at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or any range therebetween (including the endpoints), for example, at least about 80%-100% full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low stringency or moderate stringency hybridization conditions are generally but not limited to sequences having reduced sequence identity relative to the complementary sequence. Moderate stringency and high stringency conditions can optionally be used for sequences having higher identity. Low stringency conditions permit selective hybridization of sequences having about 70% sequence identity and can be used to identify orthologous or paralogous sequences. Optionally, the polynucleotides encompassed by the invention will encode at least a portion of a binding protein encoded by the polynucleotides described herein. The polynucleotides encompassed by the invention include nucleic acid sequences that can be used to selectively hybridize to polynucleotides encoding the binding proteins encompassed by the invention (see, e.g., Ausubel, supra and Colligan, supra).

[0534] IV. Host Cells

[0535] In one aspect encompassed by the invention, provided herein are host cells that express a protein described herein, such as a PRAME immunogenic peptide, a PRAME immunogenic peptide-MHC (pMHC) complex, a PRAME binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, a CAR, or a fusion protein comprising a TCR and an effector domain), etc., described herein. In some embodiments, the host cell contains a nucleic acid or vector described herein.

[0536] In some embodiments, polynucleotides encoding binding proteins are used to transform, transfect or transduce host cells (e.g., T cells) for adoptive transfer therapy. Advances in nucleic acid sequencing and specific TCR sequencing have been described (e.g., Robins et al. (2009) Blood 114:4099; Robins et al. (2010) Sci. Translat. Med. 2:47ra64; Robins et al. (2011) J. Imm. Meth.; and Warren et al. (2011) Genome Res. 21:790) and can be employed in the practice of the embodiments encompassed by the present invention. Similarly, methods for transfecting or transducing T cells with the desired nucleic acid (e.g., U.S. Patent Publication No. US2004 / 0087025), as well as adoptive transfer procedures using T cells with the desired antigen specificity (e.g., Schmitt et al. (2009) Hum. Gen. 20:1240; Dossett et al. (2009) Mol. Ther. 77:742; Till et al. (2008) Blood 772:2261; Wang et al. (2007) Hum. Gene Ther. 18:112; Kuball et al. (2007) Blood 709:2331; U.S. Patent Publication 2011 / 0243972; U.S. Patent Publication 2011 / 0189141; and Leen et al. (2007) Ann. Rev. Immunol. 25:243) are well known in the art.

[0537] Any suitable immune cell can be modified to include the heterologous polynucleotides encompassed by the present invention, including, for example, T cells, NK cells or NK-T cells. In some embodiments, the cells can be primary cells or cells of a cell line. In some embodiments, the modified immune cell comprises CD4 + T cells, CD8 + T cells or both. For the purposes herein, a T cell can be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus or other tissues or body fluids. The T cell can also be enriched or purified. In some embodiments, the T cell is a human T cell. In some embodiments, the T cell is a T cell isolated from a human. The T cell can be any type of T cell and can be at any stage of development, including but not limited to cytotoxic lymphocytes, cytotoxic lymphocyte precursors, cytotoxic lymphocyte progenitors, cytotoxic lymphocyte stem cells, CD4 + / CD8+ Double-positive T cells, CD4 + Helper T cells (e.g., Th1 and Th2 cells), CD4 + T cells, CD8 + T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc.

[0538] Any suitable method can be used to transfect or transduce cells, such as T cells, or to administer a nucleotide sequence or composition encompassed by the methods described herein. Methods for delivering polynucleotides to host cells include, for example, using cationic polymers, lipid molecules, and certain commercial products, such as in Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonoporation, liposome-mediated transfection, receptor-mediated transduction, particle bombardment, transposon-mediated transfer, etc. Further methods for transfecting or transducing host cells employ vectors, which are described in further detail herein.

[0539] The modified immune cells as described herein can be functionally characterized using methods for assaying T cell activity, including assaying T cell binding, activation, or induction, and also including assaying antigen-specific T cell responses. Examples include assaying T cell proliferation, cytokine release from T cells, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., by detecting the 51 Cr release) of pre-loaded target cells, changes in the expression of T cell phenotypic markers, and other measures of T cell function.

[0540] Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998), as well as Current Protocols in Immunology, Weir, (1986) Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA; Mishell and Shigii (eds.) (1979) Selected Methods in Cellular Immunology, Freeman Publishing, ...

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

35. A method of 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 according to any one of claims 1 - 22.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

54. The method according to any one of claims 23-53, wherein the condition characterized by PRAME expression is cancer or its recurrence, optionally 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

4.

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

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

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

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

113. A host cell according to any one of claims 89 - 112, wherein the host cell does not induce T - cell expansion, cytokine release, or cytotoxic killing when contacting a target cell comprising a peptide - MHC (pMHC) complex, the peptide - MHC (pMHC) complex comprising a PLA2G4E, EFNA1, and / or SLC26A1 peptide epitope.

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

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

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

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

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

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

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

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

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

123. A method for detecting the extent 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 according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114, or a population of host cells according to claim 115; and b) detecting the level of reactivity, wherein the presence of reactivity or a higher level of reactivity compared to a control level indicates the extent of the disorder characterized by PRAME expression in the subject.

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

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

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

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

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

129. A method for evaluating the efficacy of a therapy on a disease characterized by PRAME expression, the method comprising: a) Determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114, or a population of host cells according to claim 115 in a first sample obtained from the subject before providing at least a portion of the therapy for the disease characterized by PRAME expression, and b) Determining the presence or level of reactivity between a sample obtained from the subject and at least one binding protein according to any one of claims 56 - 77, at least one host cell according to any one of claims 89 - 114, or a population of host cells according to claim 115 in a second sample obtained from the subject after providing the therapy for the disease characterized by PRAME expression, Wherein the absence of reactivity or a reduced level of reactivity in the second sample relative to the first sample indicates that the therapy is effective in treating the subject's condition characterized by PRAME expression, and wherein the presence of reactivity or an increased level of reactivity in the second sample relative to the first sample indicates that the therapy is not effective in treating the subject's condition characterized by PRAME expression.

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

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

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

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

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

135. The method according to any one of claims 49 - 55 and 132 - 134, wherein the target cells are cancer cells expressing PRAME.

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

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

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

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

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

141. The method according to any one of claims 132 - 140, wherein the disorder characterized by PRAME expression is cancer or its recurrence, optionally 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.

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

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