Binding protein that recognizes HA-2 antigen and uses thereof

Abstract

The present application provides binding proteins that recognize the HA-2 antigen, and uses thereof.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Application No. 63 / 174,818, filed April 14, 2021, U.S. Provisional Application No. 63 / 215,760, filed June 28, 2021, U.S. Provisional Application No. 63 / 288,078, filed December 10, 2021, and U.S. Provisional Application No. 63 / 329,560, filed April 11, 2022; the entire contents of each of which are incorporated herein by this reference in their entirety. [Background technology]

[0002] Approximately 50% of AML patients relapse after allogeneic hematopoietic stem cell transplantation, leaving very few treatment options available (Rautenberg et al. (2019) Int. J. Mol. Sci. 20:228). The rare patients who spontaneously develop mild antigen-specific graft-versus-leukemia T-cell responses exhibit substantially lower relapse rates (Marijt et al. (2003) Proc. Natl. Acad. Sci. USA 100:2742-2747; Spierings et al. (2013) Biol. BloodMarrowTransplant. 19:1244-1253). HA-2 (YIGEVLVSV, genotype RS_61739531 C / C or T / C) is a genotype associated with HLA-A *HA-2 is a non-critical histocompatibility antigen (derived from the class I myosin protein, MYO1G; Pierce et al. (2001) J. Immunol. 167:3223-3230) that is hematopoietically restricted, making it an ideal candidate for TCR immunotherapy of tumors. Patients receiving donor lymphocyte infusions from HA-2-mismatched donors who develop HA-2-specific T cells exhibit graft-versus-leukemia responses and experience long-term remissions (Marijt et al. (2003) Proc. Natl. Acad. Sci. USA 100:2742-2747). The development of HA-2-specific TCR immunotherapies is warranted, including for the treatment of disorders characterized by HA-2 antigen expression. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Rautenberg et al. (2019) Int. J.Mol. Sci. 20:228 [Non-patent document 2] Marijt et al. (2003) Proc. Natl. Acad. Sci. USA 100:2742-2747 [Non-patent document 3] Spierings et al. (2013) Biol. BloodMarrowTransplant. 19:1244-1253 [Non-patent document 4] Pierce et al. (2001) J. Immunol. 167:3223-3230 [Non-Patent Document 5] Marijt et al. (2003) Proc. Natl. Acad. Sci. USA 100:2742-2747) [Non-patent document 6] Marijt et al. (2003) Proc. Natl. Acad. Sci. USA 100:2742-2747 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention is based, at least in part, on the discovery of binding proteins comprising a T cell receptor (TCR) that recognize the HA-2 antigen (eg, YIGEVLVSV and / or YIGEVLVSM). [Means for solving the problem]

[0005] In one embodiment, the binding protein comprises: a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity to a TCR alpha chain CDR sequence selected from the group consisting of the T cell receptor (TCR) alpha chain CDR sequences listed in Table 1; and / or b) a TCR beta chain CDR sequence having at least about 80% identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 1, wherein the binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity is about 5×10 -4 K below M d having to provide. In another embodiment, a binding protein comprising: a) TCR alpha chain variable (V) α ) TCR V domain sequences selected from the group consisting of α TCR V with at least about 80% identity to the domain sequence α domain sequences; and / or b) TCR beta chain variable (V) as listed in Table 1 β ) TCR V domain sequences selected from the group consisting of βTCR V with at least about 80% identity to the domain sequence β domain sequence, wherein the binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity is about 5×10 -4 K below M d having to provide.

[0006] In yet another embodiment, a binding protein comprising: a) a TCR alpha chain sequence having at least about 80% identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 1; and / or b) a TCR beta chain sequence having at least about 80% identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 1, wherein the binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity is about 5×10 -4 K below M d having to provide.

[0007] In yet another embodiment, a binding protein comprising: a) a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 1; and / or b) a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 1, wherein the binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity is about 5×10 -4 K below M d having to provide.

[0008] In another embodiment, a binding protein comprising: a) TCR alpha chain variable (V) α) TCR V domain sequences selected from the group consisting of α domain sequences; and / or b) TCR beta chain variable (V) β ) TCR V domain sequences selected from the group consisting of β domain sequence, wherein the binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity is about 5×10 -4 K below M d having to provide.

[0009] In yet another embodiment, a binding protein comprising: a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 1; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 1, wherein the binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity is about 5×10 -4 K below M d having to provide.

[0010] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described in this application. For example, in one embodiment: 1) The TCR alpha chain CDR, TCR V α the domain and / or TCR alpha chain is encoded by a TRAV, TRAJ, and / or TRAC gene, or a fragment thereof, selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 1; and / or 2) The TCR beta chain CDR, TCR V βThe domain and / or TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 1; and / or 3) Each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof compared to a cognate reference CDR sequence, as shown in Table 1. In another embodiment, the HA-2 immunogenic peptide comprises the amino acid sequence YIGEVLVSV or YIGEVLVSM. In yet another embodiment, the binding protein is chimeric, humanized, or human. In yet another embodiment, the binding protein is 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 binding domain comprises a transmembrane domain and an intracellular effector domain. In yet another embodiment, the TCR alpha chain and the TCR beta chain are covalently linked, optionally wherein the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide. In another embodiment, the TCR alpha chain and / or the TCR beta chain are covalently linked to a substructure, optionally wherein the covalently linked substructure comprises an affinity tag or label. In yet another embodiment, the affinity tag is selected from the group consisting of a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, Halo tag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or wherein the label is a fluorescent protein. In another embodiment, the covalently linked substructure is selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof. In another embodiment, a binding protein binds to the pMHC complex on the cell surface. In yet another embodiment, the MHC is an MHC multimer, optionally wherein the MHC multimer is a tetramer. In yet another embodiment, the MHC is an MHC class I molecule. In another embodiment, the MHC is an HLA serotype HLA-A *In yet another embodiment, the HLA allele comprises an MHC alpha chain that is HLA-A. * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0205, HLA-A * 0206, and HLA-A * 207 alleles. In yet another embodiment, binding of a binding protein described herein to an HA-2 peptide-MHC (pMHC) complex elicits an immune response, optionally wherein the immune response is a T cell response. In another embodiment, the T cell response is selected from the group consisting of T cell proliferation, cytokine release, and / or cytotoxic damage. In another embodiment, the binding protein binds to an HA-2 immunogenic peptide-MHC (pMHC) complex at a concentration of about 1 x 10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, or approximately 1 x 10 -12 M and below, K dIn yet another embodiment, the binding protein has a higher binding affinity for peptide-MHC (pMHC) than known T-cell receptors. In another embodiment, the binding protein has a binding affinity for peptide-MHC (pMHC) that is at least 1.05-fold higher than known T-cell receptors. In yet another embodiment, the binding protein induces greater T-cell proliferation, cytokine release, and / or cytotoxicity when contacted with target cells heterozygously expressing HA-2 than known T-cell receptors. In yet another embodiment, the binding protein induces at least 1.05-fold increased T-cell proliferation, cytokine release, and / or cytotoxicity when contacted with target cells heterozygously expressing HA-2 than known T-cell receptors. As used in this application, reference to fold change can, in some embodiments, be a comparison to any reference modality of interest (e.g., a comparison to a different binding protein; a comparison to the same binding protein under different circumstances, such as expression of the same binding protein in different immune cells, at different levels, in combination with other agents described herein; etc.). In another embodiment, the target cell is a DEL, THP-1, or TF-1 cell line. In yet another embodiment, the target cell is a cancer cell. In yet another embodiment, the cancer is a hematological malignancy. In another aspect, the hematological malignancy is a leukemia, lymphoma, myelodysplastic disorder, myeloproliferative neoplasm, or myeloma.

[0011] In yet another aspect, there is provided a TCR alpha chain and / or TCR beta chain selected from the group consisting of the TCR alpha chain sequences and TCR beta chain sequences listed in Table 1.

[0012] In another embodiment, an isolated nucleic acid molecule that 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 1, or to a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1; 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 1, and / or 2) comprising a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 1; to provide.

[0013] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the nucleic acid is codon-optimized for expression in a host cell.

[0014] In yet another aspect, there is provided a vector comprising the isolated nucleic acid described herein.

[0015] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, the vector is a cloning vector, an expression vector, or a viral vector. In another embodiment, the vector further comprises a nucleic acid sequence encoding CD8α and / or CD8β. In yet another embodiment, the nucleic acid sequence encoding CD8α or CD8β is operably linked to a nucleic acid encoding a tag. In yet another embodiment, the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding the CD8α or CD8β, such that the tag is fused to the N-terminus of the CD8α or CD8β. In another embodiment, the tag is a CD34 enrichment tag. In yet another embodiment, the isolated nucleic acid described herein and the nucleic acid sequence encoding the CD8α and / or CD8β are interconnected with an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide. In yet another embodiment, the self-cleaving peptide is P2A, E2A, F2A, or T2A.

[0016] In yet another aspect, a host cell comprising an isolated nucleic acid described herein, comprising a vector described herein, and / or expressing a binding protein described herein, optionally wherein the cell is genetically engineered. to provide.

[0017] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, the host cell comprises a chromosomal gene knockout of a TCR gene, an HLA gene, or both. In yet another embodiment, the host cell comprises a knockout of an HLA gene selected from the group consisting of α1 macroglobulin gene, α2 macroglobulin gene, α3 macroglobulin gene, β1 microglobulin gene, β2 microglobulin gene, and combinations thereof. In yet another embodiment, the host cell comprises a knockout of a TCR gene selected from the group consisting of a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and combinations thereof. In another embodiment, the host cell expresses CD8α and / or CD8β. In another embodiment, CD8α and / or CD8β are fused to a CD34 enrichment tag. In yet another embodiment, the host cell has been enriched using a CD34 enrichment tag. In yet another embodiment, the host cell is a hematopoietic progenitor cell, a peripheral blood mononuclear cell (PBMC), an umbilical cord blood cell, or an immune cell. In another embodiment, the immune cell is a cytotoxic lymphocyte, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte progenitor cell, a cytotoxic lymphocyte stem cell, a CD4 + T cells, CD8 +The host cells may be T cells, CD4 / CD8 double-negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or a combination thereof. In yet another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or a combination thereof. In yet another embodiment, the T cells are primary T cells or cells of a T cell line. In yet another embodiment, the T cells do not express an endogenous TCR or have low cell surface expression of an endogenous TCR. In yet another embodiment, the host cells are capable of producing cytokines or cytotoxic molecules when contacted with target cells containing a peptide-MHC (pMHC) complex containing the HA-2 peptide epitope within the MHC molecule. In yet another embodiment, the host cells are contacted with the target cells 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 perforins and / or granzymes, optionally wherein the cytotoxic molecule is granzyme B. In yet another embodiment, the host cells are capable of producing increased levels of cytokines or cytotoxic molecules when contacted with target cells heterozygously expressing HA-2. In yet another embodiment, the host cells are capable of producing at least 1.05-fold increased levels of cytokines or cytotoxic molecules. In another embodiment, the host cells are capable of killing target cells containing peptide-MHC (pMHC) complexes that include an HA-2 peptide epitope in the MHC molecule. In yet another embodiment, killing is measured by a killing assay. In another embodiment, the ratio of host cells to target cells in the killing assay is between 20:1 and 0.625:1. In yet another embodiment, the target cells are T2 cells pulsed with 1 μg / mL to 50 pg / mL of HA-2 peptide.In yet another embodiment, the host cells are capable of killing a greater number of target cells when contacted with target cells heterozygously expressing HA-2. In another embodiment, the host cells are capable of killing at least 1.05-fold more target cells. In yet another embodiment, the target cells are THP-1 or TF1 cell lines. In yet another embodiment, the HA-2 immunogenic peptide comprises the amino acid sequence YIGEVLVSV or YIGEVLVSM. In yet another embodiment, the MHC molecule is an MHC class I molecule. In another embodiment, the MHC molecule is of the HLA serotype HLA-A. * In yet another embodiment, the HLA allele comprises an MHC alpha chain that is HLA-A. * 02:01, HLA-A * 0202, HLA-A * 0203, HLA-A * 0205, HLA-A * 0206, and HLA-A * 0207 allele. In another embodiment, the target cell is a cell line selected from the group consisting of Del, THP-1, and TF-1 cell lines, is a hematopoietic cancer cell that expresses HA-2, or is not NB4. In yet another embodiment, the host cell is a hematopoietic cancer cell that does not express the HA-2 antigen, which is not recognized by the binding proteins described herein, and is of the serotype HLA-A * Not 02 cells and / or HLA-A * 02 allele (e.g., HLA-A * 02:01 and / or HLA-A * 0206, etc.) is not expressed.

[0018] In another aspect, a population of host cells described herein is provided.

[0019] In yet another aspect, A composition comprising: a) a binding protein as described in the present application; b) an isolated nucleic acid described in the present application; c) a vector as described in the present application; d) a host cell as described in the present application, and / or e) a population of host cells described in the present application; and a carrier, to provide.

[0020] In yet another aspect, A device or kit that includes: a) a binding protein as described in the present application; b) an isolated nucleic acid described in the present application; c) a vector as described in the present application; d) a host cell as described in the present application, and / or e) a population of host cells described in the present application; wherein the device or kit optionally comprises reagents for detecting binding of a), d) and / or e) to a pMHC complex; to provide.

[0021] In another aspect, A method for producing a binding protein as described in the present application, wherein the method comprises the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of the binding protein; and (ii) recovering the expressed binding protein; to provide.

[0022] In yet another aspect, A method for producing a host cell that expresses the binding protein described in the present application, the method comprising the steps of: (i) introducing into said host cell a nucleic acid comprising a sequence encoding a binding protein described herein; (ii) culturing the transformed host cells under conditions suitable to allow expression of the binding protein; to provide.

[0023] In yet another aspect, A method for detecting the presence or absence of HA-2 antigen and / or cells expressing HA-2, optionally wherein the cells are hyperproliferative cells, the method comprising detecting the presence or absence of the HA-2 antigen in a sample by using at least one binding protein described herein or at least one host cell described herein, wherein detecting the HA-2 antigen is an indication that the HA-2 antigen and / or cells expressing HA-2 are present. to provide.

[0024] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, at least one binding protein described herein or at least one host cell described herein forms a complex with an HA-2 peptide in an MHC molecule, and the complex is detected by fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow assay. In another embodiment, the method further comprises obtaining a sample from the subject. In yet another embodiment, the method further comprises identifying cells expressing HA-2 by bone marrow biopsy.

[0025] In another aspect, 1. A method for detecting the level of a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of HA-2 antigen in a subject, comprising: a) contacting a sample obtained from the subject with at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein; and b) detecting a level of responsiveness, wherein a higher level of responsiveness compared to the control level indicates a level of a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen in the subject.

[0026] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, the control level is a reference number. In another embodiment, the control level is the level in a subject free of a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen.

[0027] In yet another aspect, 1. A method for monitoring the progression of a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder, characterized by expression of HA-2 antigen in a subject, the method comprising: a) detecting the level of HA-2 antigen, or a cell of interest that expresses HA-2, as described herein, in a sample from a subject at a first time point; b) repeating step a) at a subsequent time point; and and c) comparing the levels of HA-2 antigen or cells of interest expressing HA-2 detected in step a) and step b) to monitor the progression of a non-malignant disorder, hyperproliferative disorder, or recurrence of a hyperproliferative disorder in the subject characterized by expression of the HA-2 antigen, wherein an absent or reduced level of HA-2 antigen or cells of interest expressing HA-2 detected in step b) compared to step a) indicates that the progression of a non-malignant disorder, hyperproliferative disorder, or recurrence of a hyperproliferative disorder in the subject characterized by expression of the HA-2 antigen has been inhibited.

[0028] Numerous further embodiments are provided that may be applied to any aspect encompassed by the present invention and / or may be combined with any other embodiment described herein. For example, in one embodiment, between the first time point and the subsequent time point, the subject has undergone treatment to treat a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen.

[0029] In yet another aspect, 1. A method for assessing the efficacy of a therapy for a non-malignant, hyperproliferative, or recurring hyperproliferative disorder characterized by expression of the HA-2 antigen, including: a) determining the presence or level of responsiveness 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 the subject with at least part of a therapy for a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen; and b) determining the presence or level of responsiveness between 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 obtained from the subject and a second sample obtained from the subject after providing at least part of a therapy for a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen;

[0030] wherein the absence or decreased level of responsiveness in the second sample compared to the first sample indicates that the therapy is effective for treating a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen in the subject. to provide.

[0031] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, the level of responsiveness is indicated by a) the presence of binding and / or b) T cell activation and / or effector function, optionally T cell activation or effector function being T cell proliferation, killing, or cytokine release. In another embodiment, T cell activation or effector function is T cell proliferation, killing, or cytokine release. In yet another embodiment, T cell binding, activation, and / or effector function are detected using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow assay.

[0032] In another aspect, A method for preventing and / or treating a non-malignant disorder, a hyperproliferative disorder, or the recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising cells expressing at least one binding protein described herein.

[0033] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, the cells are allogeneic, syngeneic, or autologous cells. In another embodiment, the cells are genetically modified. In yet another embodiment, the cells comprise a chromosomal gene knockout of a TCR gene, an HLA gene, or both a TCR gene and an HLA gene. In yet another embodiment, the cells comprise a knockout of an HLA gene selected from the group consisting of the α1 macroglobulin gene, the α2 macroglobulin gene, the α3 macroglobulin gene, the β1 microglobulin gene, the β2 microglobulin gene, and combinations thereof. In yet another embodiment, the cells comprise a knockout of a TCR gene selected from the group consisting of the TCR α variable region gene, the TCR β variable region gene, the TCR constant region gene, and combinations thereof. In another embodiment, the cells express CD8α and / or CD8β, optionally wherein the CD8α and / or CD8β are fused to a CD34 enrichment tag. In yet another embodiment, the cells are enriched using a CD34 enrichment tag. In yet another embodiment, the cells are hematopoietic progenitor cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells, or immune cells. In another embodiment, the immune cells are cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + T cells, CD8 +The T cells are T cells, CD4 / CD8 double-negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or a combination thereof. In another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or a combination thereof. In yet another embodiment, the T cells are primary T cells or cells of a T cell line. In yet another embodiment, the T cells do not express an endogenous TCR or have low cell surface expression of an endogenous TCR. In another embodiment, the cells are capable of producing cytokines or cytotoxic molecules when contacted with target cells containing a peptide-MHC (pMHC) complex that includes HA-2 among the MHC molecules. In yet another embodiment, the cytokine is TNF-α, IL-2, and / or IFN-γ. In yet another embodiment, the cytotoxic molecule is perforin and / or granzyme, optionally, where the cytotoxic molecule is granzyme B. In another embodiment, the cytokine is TNF-α, IL-2, and / or IFN-γ. In yet another embodiment, the cytotoxic molecule is perforin and / or granzyme, optionally, where the cytotoxic molecule is granzyme B. In yet another embodiment, the cells are capable of producing higher levels of cytokines or cytotoxic molecules when contacted with target cells heterozygously expressing HA-2. In another embodiment, the cells are capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules. In yet another embodiment, the host cells are capable of killing target cells containing peptide-MHC (pMHC) complexes that include HA-2 among their MHC molecules. In yet another embodiment, the host cells are capable of killing a greater number of target cells when contacted with target cells heterozygously expressing HA-2. In yet another embodiment, the host cells are capable of killing at least 1.05 times more target cells.In yet another embodiment, the HA-2 immunogenic peptide comprises the amino acid sequence YIGEVLVSV or YIGEVLVSM. In another embodiment, the MHC molecule is an MHC class I molecule. In yet another embodiment, the MHC molecule is of the HLA serotype HLA-A. * In yet another embodiment, the HLA allele comprises an MHC alpha chain that is HLA-A. * 02:01, HLA-A * 0202, HLA-A * 0203, HLA-A * 0205, HLA-A * 0206, and HLA-A * 0207 allele. In another embodiment, the target cell is a non-malignant cell or a hyperproliferative cell that expresses the HA-2 antigen in the subject. In yet another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In yet another embodiment, the composition induces an antigen-specific T cell immune response against a non-malignant cell or a hyperproliferative cell that expresses the HA-2 antigen in the subject. In another embodiment, the composition induces an antigen-specific T cell immune response against a non-malignant cell or a hyperproliferative cell that expresses the HA-2 antigen in the subject. In yet another embodiment, the antigen-specific T cell immune response is selected from the group consisting of CD4 +The hyperproliferative disorder comprises at least one of a helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In yet another embodiment, the hyperproliferative disorder comprises a hematological malignancy. In yet another embodiment, the hematological malignancy comprises a leukemia, lymphoma, myelodysplastic disorder, myeloproliferative neoplasm, or myeloma. In another embodiment, the hematological malignancy comprises a leukemia. In yet another embodiment, the leukemia is selected from acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia, or chronic lymphocytic leukemia (CLL). In yet another embodiment, the hematological disorder comprises lymphoma. In yet another embodiment, the lymphoma is Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma, or leukemia. lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt's lymphoma.In yet another embodiment, the hematological malignancy comprises a myelodysplastic disorder (MDS), optionally wherein the MDS is selected from the group consisting of refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts - thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD), and refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD). In yet another embodiment, the non-malignant disorder is selected from the group consisting of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Omenn syndrome, X-linked lymphoproliferative syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, DiGeorge syndrome, and an indication for hematopoietic stem cell transplantation (HCT).In yet another embodiment, the non-malignant disorder is a non-malignant hematologic disorder, optionally selected from the group consisting of sickle cell anemia, thalassemia, aplastic anemia, hemophagocytic lymphohistiocytosis (HLH), severe aplastic anemia, bone marrow failure syndrome, Fanconi anemia, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. In another embodiment, the non-malignant disorder is an autoimmune disorder, optionally wherein the autoimmune disorder is systemic sclerosis or multiple sclerosis. In yet another embodiment, the subject is undergoing or has previously undergone hematopoietic cell transplantation (HCT), optionally wherein the HCT does not express the HA-2 antigen, a serotype HLA-A that is not recognized by the binding proteins described herein. * Not 02 cells and / or HLA-A *The HCT comprises cells that do not express the 02:01 allele. In yet another embodiment, the HCT comprises donor hematopoietic cells comprising a chromosomal knockout of a gene encoding an HLA component, a chromosomal knockout of a gene encoding a TCR component, or both. In another embodiment, the subject has previously undergone lymphocyte-depleting chemotherapy. In yet another embodiment, the lymphocyte-depleting chemotherapy comprises cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof. In yet another embodiment, the method further comprises prescribing to the subject at least one additional treatment for the non-malignant disorder, the hyperproliferative disorder, or a recurrence of the hyperproliferative disorder. In another embodiment, the at least one additional treatment for the non-malignant disorder, the hyperproliferative disorder, or a recurrence of the hyperproliferative disorder is prescribed simultaneously or sequentially with the composition. In yet another embodiment, the subject is an animal model of a disorder characterized by expression of the HA-2 antigen, and / or the subject is a mammal, optionally wherein the mammal is a human, a primate, or a rodent.

[0034] In yet another aspect, an expression vector is provided comprising a promoter operably linked to a nucleic acid sequence encoding CD8α and / or CD8β.

[0035] Numerous further embodiments are provided that can be applied to any aspect encompassed by the present invention and / or can be combined with any other embodiment described herein. For example, in one embodiment, a nucleic acid sequence encoding CD8α or CD8β is operably linked to a nucleic acid encoding a tag, such that the tag is fused to CD8α or CD8β. In another embodiment, the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding CD8α or CD8β, such that the tag is fused to the N-terminus of CD8α or CD8β. In yet another embodiment, the tag is a CD34 enrichment tag. In yet another embodiment, the vector further comprises a nucleic acid sequence encoding TCRα and / or TCRβ. In another embodiment, the TCRα and / or TCRβ comprise a mutated transmembrane domain and / or a mutated constant domain. In yet another embodiment, the mutated transmembrane domain and / or mutated constant domain enhance cell surface expression of TCRα and / or TCRβ while reducing endogenous expression of TCRα and / or TCRβ. In yet another embodiment, the nucleic acid sequences encoding CD8α and / or CD8β and the nucleic acid sequences encoding TCRα and / or TCRβ are interconnected with an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide. In another embodiment, the self-cleaving peptide is P2A, E2A, F2A, or T2A.In yet another embodiment, the vector further comprises a nucleic acid sequence encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1, or a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1, 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 1, 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 1. In yet another embodiment, the vector has a nucleic acid sequence set forth in Table 2. [Brief explanation of the drawings]

[0036] [Figure 1] A schematic diagram of the TCR discovery platform is shown. The platform identified 1,302 novel HA-2 TCRs. CD14+ monocytes were isolated from PBMCs of six HA-2-negative healthy donors on day -4 and differentiated into mature DCs. Naive CD8+ T cells were isolated from autologous PBMCs on day -1 and allowed to rest overnight. DCs were pulsed with 1 μg / mL of HA-2REF peptide (YIGEVLVSV) for 3 hours, followed by co-culture of CD8+ T cells and DCs for 11 days in the cell expansion phase. Dextramers were used to identify clones using A*02:01-specific HA-2REF (YIGEVLVSV) and HA-2 SNP (YIGEVLVSM) dextramers, and then the clones were subjected to single-cell TCR sequencing.

[0037] [Figure 2]The results of DexScan, which performed functional evaluation of the 380 TCRs identified as shown in Figure 1, using a library of individually cloned HA-2 TCR viruses, are shown. Briefly, Pan-T cells isolated from HLA-A*01:03 / A*03:01-positive healthy donor PBMCs were transduced to express a pool of HA-2-specific TCRs and stained with various dilutions of HA-2 REF(YIGEVLVSV) dextramer. Dextramer-bound cells were sorted and sequenced. TCR enrichment (i.e., the change in TCR percentages in sorted samples and materials) was used to select TCRs for functional evaluation. The figure shows the TCRs selected by DexScan for functional evaluation.

[0038] [Figure 3] HA-2 TCR is expressed on the surface of genetically engineered T cells. Pan-T cells were transduced to express 15 HA-2-specific TCRs selected by DexScan screen. Dot plots show surface expression of A*02:01-specific HA-2 REF (YIGEVLVSV) TCRs as assessed by dextramer staining.

[0039] [Figure 4A] Functional evaluation of HA-2 TCRs is shown. Pan-T cells isolated from PBMCs of HLA-A*01:03 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs and evaluated for functional responses against target cells expressing HA-2 and HLA-A*02:01. Dot plots are shown demonstrating representative expression of three HA-2-specific TCRs as assessed by A*2:12-specific HA-02 REF (YIGEVLVSV) dextramer staining.

[0040] [Figure 4B]Figure 1 shows functional assessment results of HA-2 TCR. Functional responses of HA2-specific TCRs to HLA-A*02:01* target cell lines, DEL (high MHC, high HA-2), THP-1 (moderate MHC, low HA-2), TF-1 (low MHC, low HA-2), and negative control cell lines, and NB4 (irrelevant MHC) are shown. Results are shown for engineered T cells co-cultured with IncuCyte®, NucLight™ Red-labeled target cell lines at the indicated E:T ratios. Cell viability was quantified with an IncuCyte® instrument as a readout of T cell cytotoxicity.

[0041] [Figure 4C-1] Functional evaluation of HA-2 TCR is shown. Functional responses of HA2-specific TCR to HLA-A*02:01* target cell lines DEL (high MHC, high HA-2), THP-1 (moderate MHC, low HA-2), TF-1 (low MHC, low HA-2), and negative control cell lines NB4 (irrelevant MHC) are shown. IFN-γ, IL-2, TNF-α, and granzyme B production in 24-hour coculture supernatants are shown (E:T 1:1). T cell-only conditions were used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. [Figure 4C-2] Same as above.

[0042] [Figure 4D-1]Functional evaluation of the HA-2 TCR is shown. Functional responses of the HA2-specific TCR to the HLA-A*02:01* target cell lines, DEL (high MHC, high HA-2), THP-1 (moderate MHC, low HA-2), TF-1 (low MHC, low HA-2), and negative control cell lines, and NB4 (irrelevant MHC), are shown. Proliferation of HA-2-specific TCR-expressing T cells is shown. Engineered T cells were labeled with a proliferation dye and cocultured with the target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD4+ and CD4+ T cell proliferation. Prior to flow cytometry analysis, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. The dotted line represents the highest level of proliferation with T cells alone. TCRs were compared by ANOVA followed by Tukey's multiple comparison test. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001. These experiments were performed on 2-3 donors, and representative data from one donor are shown. [Figure 4D-2] Same as above.

[0043] [Figure 5]This figure shows the alloreactivity landscape of MJ2-DP8 and MJ7-DP19 TCRs tested in an alloreactivity assay. Endogenous MHC was knocked out in HEK293T cells using CRISPR / Cas9-gRNA ribonucleoprotein delivery. MHC-null HEK293T cells were transduced with 110 independent MHCs representing the most prevalent HLA types using IncuCyte®, NucLight™ Red, and lentiviral transduction. A cell line stably expressing A*02:01 and a 90-residue protein fragment containing the HA-2 REF (YIGEVLVSV) sequence were used as positive controls. Cells were plated in a 96-well format and incubated with human primary CD8+ T cells expressing MJ2-DP9 and MJ7-DP19 TCRs for 48 hours. Target cell numbers were measured over time using an IncuCyte® instrument by counting the number of IncuCyte® NucLight™ Red-positive cells. The 48-hour cell inhibition for each TCR for each MHC in the assay was calculated as 1 - (target cell doublings [incubated with TCR-expressing T cells] / target cell doublings [incubated with T cell media]). The 48-hour cell inhibition for each TCR for each MHC in the assay was calculated as 1 - (target cell doublings [incubated with TCR-expressing T cells] / target cell doublings [incubated with T cell media]). The effects of non-MHC-related target cell growth inhibition, such as T cell nutrient competition and nonspecific cell killing by donor T cells, were subtracted, and the batch effect of each 96-well plate was corrected for. The cell inhibition value for each well (each MHC) of the 96-well plate was subtracted by the median cell inhibition for each well of the 96-well plate.

[0044] [Figure 6]DexScan results were performed on 11,742 TCRs identified as shown in Figure 1 for functional evaluation using the HA-2 TCR viral library pool. Briefly, Pan-T cells isolated from HLA-A*01:01 / A*03:01-positive healthy donor PBMCs were transduced to express the HA-2 TCR library pool and stained with HA-2 REF(YIGEVLVSV) dextramer. Dextramer-bound cells were sorted and sequenced. TCR enrichment (i.e., the change in TCR percentage in sorted samples and materials) was used to select TCRs for functional evaluation. The figure shows the DexScan-selected TCRs for functional evaluation.

[0045] [Figure 7A] Results of HA-2 TCR functional assessment are shown, presented using standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses were assessed with target cells expressing HA-2 and HLA-A*02:01. Dot plots showing representative expression of 15 HA-2-specific TCRs assessed by A*02:01-specific HA-2 REF (YIGEVLVSV) dextramer staining are shown.

[0046] [Figure 7B]Functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines are shown: DEL (high MHC, high HA-2) (B–D), THP-1 (moderate MHC, moderate HA-2) (E–G), TF1 (low MHC, low HA-2) (H–J), and the negative control cell line NB4 (irrelevant MHC) (K–M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B in the supernatants of 24-hour cocultures is shown (E:T 1:1). The T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (E:T 1:1) for 96 hours. Dye dilutions were used to assess CD8+ and CD4+ T cell proliferation. Counting beads were added to samples before analysis by flow cytometry, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates a significant difference. Statistical significance is indicated, and key statistics are reported in each figure as follows: a) The bar graphs in the left panel of B show that the results for MJ2-DP22, MJ9-DP5, and MJ7-DP19 were significantly different from those for MJ14-SP4, MJ13-SP43, MJ14-DP162, and NTD, respectively (P<0.05). b) The bar graphs in the right panel of B show that the results for MJ14-DP317, MJ2-DP8, and MJ7-DP19 were significantly different from those for MJ14-DP33, MJ13-DP32, and NTD, respectively (P<0.05).

[0047] [Figure 7C]Functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines are shown: DEL (high MHC, high HA-2) (B–D), THP-1 (moderate MHC, moderate HA-2) (E–G), TF1 (low MHC, low HA-2) (H–J), and the negative control cell line NB4 (irrelevant MHC) (K–M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B in the supernatants of 24-hour cocultures is shown (E:T 1:1). The T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (E:T 1:1) for 96 hours. Dye dilutions were used to assess CD8+ and CD4+ T cell proliferation. Counting beads were added to samples before analysis by flow cytometry, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates a significant difference. Statistical significance is indicated, and key statistics are reported in each figure as follows: c) The bar graph of interferon-γ in C shows that the results of MJ2-DP22, MJ14-DP317, and MJ2-DP8 were significantly different from those of MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, MJ14-DP33, MJ2-DP24, MJ13-DP148, MJ13-DP32, and NTD, respectively (P<0.05), while the results of MJ9-DP5 and MJ7-DP19 were significantly different from those of all TCRs, except for MJ14-DP137 (P<0.05).d) The bar graphs for IL-2 and TNF-α in C show that the results for MJ2-DP22, MJ9-DP5, MJ14-DP317, MJ2-DP8, and MJ7-DP19 were significantly different from the results for MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, MJ14-DP33, MJ2-DP24, MJ13-DP148, MJ13-DP32, and NTD, respectively (P<0.05). e) The bar graph for granzyme B in C shows the results for MJ2-DP22, MJ14-DP317, MJ2-DP8, and MJ7-DP19. The results for MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, MJ14-DP33, MJ2-DP24, MJ13-DP148, MJ13-DP32, and NTD were significantly different (P<0.05), whereas the results for MJ9-DP5 were significantly different from the results for all TCRs except MJ14-DP137 (P<0.05).

[0048] [Figure 7D]Functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines are shown: DEL (high MHC, high HA-2) (B–D), THP-1 (moderate MHC, moderate HA-2) (E–G), TF1 (low MHC, low HA-2) (H–J), and the negative control cell line NB4 (irrelevant MHC) (K–M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B in the supernatants of 24-hour cocultures is shown (E:T 1:1). The T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (E:T 1:1) for 96 hours. Dye dilutions were used to assess CD8+ and CD4+ T cell proliferation. Counting beads were added to samples before analysis by flow cytometry, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates a significant difference. Statistical significance is indicated, and key statistics are reported in each figure as follows: f) The bar graph of CD8+ T cell proliferation in D shows that the results of MJ14-DP317, MJ9-DP5, MJ2-DP8, and MJ7-DP19 were significantly different from the results of MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, MJ14-DP33, MJ2-DP24, MJ13-DP148, MJ13-DP32, and NTD, respectively, except for MJ14-DP137 (P<0.05).g) The bar graph of CD4 T cell proliferation in D shows that the results for MJ14-DP317 and MJ2-DP8 were significantly different from those for MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, MJ14-DP33, MJ2-DP24, MJ13-DP148, MJ13-DP32, and NTD (P<0.05), whereas the results for MJ2-DP22, MJ9-DP5, and MJ7-DP19 were significantly different from those for all TCRs except for MJ14-DP137 (P<0.05).

[0049] [Figure 7E]Functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines are shown: DEL (high MHC, high HA-2) (B–D), THP-1 (moderate MHC, moderate HA-2) (E–G), TF1 (low MHC, low HA-2) (H–J), and the negative control cell line NB4 (irrelevant MHC) (K–M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B in the supernatants of 24-hour cocultures is shown (E:T 1:1). The T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (E:T 1:1) for 96 hours. Dye dilutions were used to assess CD8+ and CD4+ T cell proliferation. Counting beads were added to samples before analysis by flow cytometry, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates a significant difference. Statistical significance is indicated, and key statistics are reported in each figure as follows: h) The bar graphs in the left panel of E show that the results for MJ2-DP22, MJ9-DP5, and MJ7-DP19 are significantly different from those for MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, and NTD, respectively (P<0.05). i) The bar graphs in the right panel of E show that the results for MJ14-DP317, MJ2-DP8, and MJ7-DP19 are significantly different from those for MJ14-DP33, MJ12-DP24, MJ13-DP148, MJ13-DP32, and NTD, respectively (P<0.05).

[0050] [Figure 7F]Functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines are shown: DEL (high MHC, high HA-2) (B–D), THP-1 (moderate MHC, moderate HA-2) (E–G), TF1 (low MHC, low HA-2) (H–J), and the negative control cell line NB4 (irrelevant MHC) (K–M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B in the supernatants of 24-hour cocultures is shown (E:T 1:1). The T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (E:T 1:1) for 96 hours. Dye dilutions were used to assess CD8+ and CD4+ T cell proliferation. Counting beads were added to samples before analysis by flow cytometry, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates a significant difference. Statistical significance is indicated, and key statistics are reported in each figure as follows: j) The bar graphs for interferon-γ, TNF-α, and granzyme B in F show that the results for MJ2-DP22, MJ9-DP5, MJ14-DP317, MJ2-DP8, and MJ7-DP19 were different from those for MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, MJ14-DP33, MJ2-DP24, MJ13-DP148, MJ13-DP32, and NTD, respectively (P<0.05), and only MJ9-DP5, MJ14-DP317, and MJ2-DP8 produced IL-2 above background levels.

[0051] [Figure 7G] Functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines are shown: DEL (high MHC, high HA-2) (B–D), THP-1 (moderate MHC, moderate HA-2) (E–G), TF1 (low MHC, low HA-2) (H–J), and the negative control cell line NB4 (irrelevant MHC) (K–M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B in the supernatants of 24-hour cocultures is shown (E:T 1:1). The T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (E:T 1:1) for 96 hours. Dye dilutions were used to assess CD8+ and CD4+ T cell proliferation. Counting beads were added to samples before analysis by flow cytometry, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates a significant difference. Statistical significance is indicated, and key statistics are reported in each figure as follows: k) The CD8+ T cell proliferation graph and CD4+ T cell proliferation graph in G show that the results of MJ2-DP22, MJ14-DP317, MJ9-DP5, MJ2-DP8, and MJ7-DP19 were significantly different from those of MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, MJ14-DP33, MJ2-DP24, MJ13-DP148, MJ13-DP32, and NTD, respectively (P<0.05).

[0052] [Figure 7H]Functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines are shown: DEL (high MHC, high HA-2) (B–D), THP-1 (moderate MHC, moderate HA-2) (E–G), TF1 (low MHC, low HA-2) (H–J), and the negative control cell line NB4 (irrelevant MHC) (K–M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratio, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. Production of IFN-γ, IL-2, TNF-α, and granzyme B in the supernatants of 24-hour cocultures is shown (E:T 1:1). The T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine levels in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (E:T 1:1) for 96 hours. Dye dilutions were used to assess CD8+ and CD4+ T cell proliferation. Counting beads were added to samples before analysis by flow cytometry, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates a significant difference. Statistical significance is indicated, and key statistics are reported in each figure as follows: l) The bar graphs in the left panel of H show that the results for MJ2-DP22, MJ9-DP5, and MJ7-DP19 were significantly different from those for MJ14-SP4, MJ13-SP43, MJ14-DP137, MJ14-DP162, and NTD, respectively (P<0.05). M) The bar graphs in the right panel of H show that the results for MJ14-DP317, MJ2-DP8, and MJ7-DP19 were significantly different from those for MJ14-DP33, MJ12-DP24, MJ13-DP148, MJ13-DP32, and NTD, respectively (P<0.05). [Figure 7I] Same as above. [Figure 7J] Same as above. [Figure 7K] Same as above. [Figure 7L] Same as above. [Figure 7M] Same as above.

[0053] [Figure 8A] Similarly, the results of HA-2 TCR functional assessment for seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses were assessed against target cells expressing HA-2 and HLA-A*02:01. Dot plots showing representative expression of seven HA-2-specific TCRs assessed by A*02:01-specific HA-2 REF (YIGEVLVSV) dextramer staining are shown.

[0054] [Figure 8B]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated, and key statistics are reported in each figure as follows: a) The bar graphs in the left and right panels of B show that the results for MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8, respectively, were significantly different from the results for MJ14-SP4 and NTD, respectively (P<0.05).

[0055] [Figure 8C]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated and key statistics are reported in each figure as follows: b) The bar graph in C shows that the results for MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8, respectively, were significantly different (P<0.05) from the results for all cytokines in both donors for MJ14-DP137, MJ14-SP4, and NTD, respectively, while only the results for MJ14-DP317 did not differ from the results for MJ14-DP137 for TNF-α in donor 3.

[0056] [Figure 8D]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (intermediate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated and key statistics are reported in each figure as follows: c) For donor 2 in D, the results of the CD8+ T cell proliferation panels MJ14-DP317, MJ9-DP5, and MJ2-DP22 were significantly different from those of MJ14-SP4 and NTD (P<0.05), while the results of MJ7-DP19 and MJ2-DP8 were not significantly different from those of MJ14-DP137, MJ14-SP4, and NTD (P<0.05). d) The results of the CD4 T cell proliferation panels MJ14-DP317, MJ2-DP22, MJ7-DP19, and MJ2-DP8 for donor 2 in D were significantly different from the results of MJ14-DP317, MJ14-SP4, and NTD, respectively (P<0.05), while the results of MJ9-DP5 were not significantly different from the results of MJ14-SP4 and NTD, respectively (P<0.05). e) The CD8 T cell proliferation panel and CD4 T cell proliferation panel for donor 3 in D were significantly different from the results of MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8, respectively (P<0.05).

[0057] [Figure 8E]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated, and key statistics are reported in each figure as follows: f) The bar graphs in the left and right panels of E show that the results for MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8 were significantly different from the results for MJ14-DP137, MJ14-SP4, and NTD for each donor (P<0.05).

[0058] [Figure 8F]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated, and key statistics are reported in each figure as follows: g) The bar graph in F shows that the results of MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8, respectively, were significantly different (P<0.05) from the results of MJ14-DP137, MJ14-DP4, and NTD, respectively, for IFN-γ, IL-2, and TNF-α of both donors. The respective results for donor 2 with respect to granzyme B were significantly different from those for MJ14-DP137, MJ14-SP4, and NTD, respectively (P<0.05), and the respective results for MJ9-DP5, MJ7-DP19, and MJ2-DP8 were significantly different from those for donor 3 with respect to granzyme B, respectively (P<0.05).

[0059] [Figure 8G]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated and key statistics are reported in each figure as follows: h) The bar graph in G shows that the results of MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8 were significantly different from the results of MJ14-DP137, MJ14-SP4, and NTD for CD8 T cell proliferation and CD4 T cell proliferation, respectively, for both donors (P<0.05).

[0060] [Figure 8H]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated, and key statistics are reported in each figure as follows: i) The bar graph for donor 2 in H shows that the results for MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8 were significantly different from those for MJ14-DP137, MJ14-SP4, and NTD (P<0.05). j) The bar graph for donor 3 in H shows that the results for MJ9-DP5, MJ7-DP19, and MJ2-DP8 were significantly different from those for MJ14-DP137, MJ14-SP4, and NTD (P<0.05), while the results for MJ2-DP22 were not significantly different from those for MJ14-DP137 and NTD (P<0.05).

[0061] [Figure 8I]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated, and key statistics are reported in each figure as follows: k) The CD8 T cell proliferation bar graph for J donor 2 was significantly different from the results of MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8 (P<0.05), while the CD4 T cell proliferation bar graph for J donor 2 was not significantly different (P<0.05) among TCRs.

[0062] [Figure 8J]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. *P<0.05.Statistical significance is indicated, and key statistics are reported in each figure as follows: k) The CD8+ T cell proliferation bar graph for J's donor 2 was significantly different from the results of MJ14-DP317, MJ9-DP5, MJ2-DP22, MJ7-DP19, and MJ2-DP8 (P<0.05), while the CD4+ T cell proliferation bar graph for J's donor 2 was not significantly different among TCRs (P<0.05). l) The CD8+ T cell proliferation and CD4+ T cell proliferation bar graphs for J's donor 3 were significantly different from the results of MJ2-DP8, MJ14-DP137, MJ14-SP4, and NTD (P<0.05), and the results of MJ2-DP22 were significantly different from the results of MJ14-DP137 and NTD (P<0.05).

[0063] [Figure 8K]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates statistical significance, and key statistics are reported in each figure as follows:

[0064] [Figure 8L]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates statistical significance, and key statistics are reported in each figure as follows:

[0065] [Figure 8M]Similarly, the results of HA-2 TCR functional assessment against seven HA-2 TCRs for two unique donors selected based on the data in Figures 7A-7M are shown. Bar graphs show standard error of the mean (SEM) values. Pan-T cells isolated from PBMCs of HLA-A*01:01 / A*03:01-positive healthy donors were transduced to express HA-2-specific TCRs, and functional responses against target cells expressing HA-2 and HLA-A*02:01 were assessed. BM shows functional responses of HA2-specific TCRs against the following HLA-A*02:01+HA-2+ target cell lines: DEL (high MHC, high HA-2) (B-D), THP-1 (moderate MHC, moderate HA-2) (E-G), TF1 (low MHC, low HA-2) (H-J), and the negative control cell line NB4 (irrelevant MHC) (K-M). Engineered T cells were cocultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their survival was quantified using an IncuCyte® instrument as a readout of T cell cytotoxicity. IFN-γ, IL-2, TNF-α, and granzyme B production in the supernatants of 24-hour cocultures is shown (E:T 1:1). A T cell-only condition was used to determine background levels of cytokine production. The dotted line represents the highest cytokine level in the T cell-only condition. To measure proliferation of HA-2-specific TCR-expressing T cells, engineered T cells were labeled with a proliferation dye and cocultured with target cell lines for 96 hours (E:T 1:1). Dye dilution was used to assess CD8+ and CD4+ T cell proliferation. Before analysis by flow cytometry, counting beads were added to the samples, and absolute numbers of dividing CD8+ and CD4+ T cells were determined. The T cell-only condition was used to determine background levels of proliferation. As above, the dotted line represents the highest level of proliferation in the T cell-only condition. TCRs were compared using ANOVA followed by Tukey's multiple comparison test. P<0.05 indicates statistical significance, and key statistics are reported in each figure as follows:

[0066] [Figure 9]The alloreactivity landscapes of MJ9-DP5, MJ2-DP22, and MJ14-DP317 tested in an alloreactivity assay are shown. Endogenous MHC was knocked out in HEK293T cells using CRISPR / Cas9-gRNA ribonucleoprotein delivery. MHC-null HEK293T cells were transduced with 110 independent MHCs representing the most prevalent HLA types using IncuCyte®, NucLight™ Red, and lentiviral transduction. A cell line stably expressing A*02:01 and a 90-residue protein fragment containing the HA-2 REF (YIGEVLVSV) sequence were used as positive controls. Cells were plated in a 384-well format and incubated with human primary CD8+ T cells expressing MJ9-DP5, MJ2-DP22, and MJ14-DP317 for 48 hours. Target cell numbers were measured over time using an IncuCyte® instrument by counting the number of IncuCyte® NucLight™ Red-positive cells. The 48-hour cell inhibition of TCRs for each MHC in the assay was calculated as 1 - (target cell doublings [incubated with TCR-expressing T cells] / target cell doublings [incubated with T cell medium]). After subtracting the effects of non-MHC-related target cell growth inhibition, such as T cell nutrient competition and nonspecific cell killing by donor T cells, and correcting for batch effects in each 384-well plate, the cell inhibition value for each well (each MHC) in the 394-well plate was subtracted by the median cell inhibition value for each well in the 384-well plate.

[0067] [Figure 10](Figure 10A) The strategy for the implementation and outcome of the off-target screen assay is shown. A schematic overview of the genome-wide safety screening assay used is shown. In this genome-wide safety screen, T cells expressing HA2-MJ14-DP317 are cocultured with a library of HLA-A*02:01+ target cells, each expressing a different 90-amino acid (90-aa) protein fragment. In essence, this library contains fragments that are tiles that can map to any protein in 22-amino acid steps. The fragments are naturally processed by the target cells, and the resulting peptides appear on the cell surface MHC. When T cells recognize their targets, they attempt to kill the target cells, thereby activating a fluorescent reporter. By isolating the fluorescent cells and sequencing their expression cassettes, the natural target(s) of the TCR are revealed. Because this screen was designed to overpredict off-targets by overexpressing a 90-aa protein fragment that is processed more efficiently than the full-length protein, the identified hits would not be expected to be physiologically relevant off-targets. Kula et al. (2019) Cell 178:1016-1028 provide additional details.

[0068] (Figure 10B) Strategy for the implementation and performance of the off-target screen assay. Multiple tiles containing the HA-2 epitope were significantly enriched in the screen with HA2-MJ14-DP317 (TCR-101), and multiple genes were identified as potential off-targets. The x-axis represents protein fragments within the peptidome library, and the y-axis represents the fold enrichment of individual tiles in the input library.

[0069] [Figure 11](Figure 11A) TSC-101 has at least 100- to 3,000-fold less affinity for putative off-target peptides compared to the target HA-2 peptide. The cytotoxic activity of TSC-101 TCR-T cells was tested against T2 cells pulsed with different on-target and off-target peptides. (A) The affinity of TCR-101 for on-target and off-target peptides was measured by calculating the area under the curve (AUC) for the proliferation of T2 target cells cocultured with TSC-101 T cells, measured as a function of peptide dose over a 72-hour period.

[0070] (Figure 11B) shows that TSC-101 has at least 100-3,000-fold less affinity for putative off-target peptides compared to the target HA-2 peptide. The cytotoxic activity of TSC-101 TCR-T cells was tested against T2 cells pulsed with different on-target and off-target peptides. HA-2 + Peptide, HA-2 - Individual EC values ​​for eight-point titrations of peptides, putative off-target MYO3A / B peptides, and putative off-target MALTI peptides 50 The EC50 determinations for the remaining peptides (MTA1, KLHL30, and HUWE1) showed that their 3-point titrations yielded EC 50 Please note that the calculation of is not possible and is therefore not shown.

[0071] [Figure 12A]We demonstrate that TSC-101 has potent cytotoxicity against primary hematologic tumor samples, including acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) tumor samples. TSC-101 and non-genetically engineered control (NC) T cells were co-cultured with HLA-A*01:12-positive PBMCs or BMMCs from AML (Figure 12A) or ALL (Figure 12B) patients at an E:T ratio of 10:1 for 1 hour. Target viability in co-culture with TSC-101 T cells was then assessed by flow cytometry and normalized to target viability in co-culture with NC T cells. Data are representative of three donors.

[0072] [Figure 12B] We demonstrate that TSC-101 has potent cytotoxicity against primary hematologic tumor samples, including acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) tumor samples. TSC-101 and non-genetically engineered control (NC) T cells were co-cultured with HLA-A*01:12-positive PBMCs or BMMCs from AML (Figure 12A) or ALL (Figure 12B) patients at an E:T ratio of 10:1 for 1 hour. Target viability in co-culture with TSC-101 T cells was then assessed by flow cytometry and normalized to target viability in co-culture with NC T cells. Data are representative of three donors.

[0073] [Figure 13-1] Summary data is shown. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above. [Figure 13-5] Same as above.

[0074] [Figure 14A]Panels A and B show that TSC-101 lacks reactivity to non-hematopoietic primary human cells. Data show IFN-γ concentrations measured in supernatants obtained from 48-hour cocultures of TSC-101 with different target cells corrected for background (background IFN-γ was measured with effector cells alone (Figure 14A)).

[0075] [Figure 14B] Panels A and B show that TSC-101 lacks reactivity with non-hematopoietic primary human cells. Target cells included NB-4 (negative control) and THP-1 (positive control), as well as HLA-A*02:01-positive primary human cells, such as PBMCs (green bars), endothelial cells (HUVECs), skeletal muscle cells (SKMCs), keratinocytes (NHEKs), bronchial epithelial cells (HBECs), melanocytes (NHEMs), pigmented retinal epithelial cells (RPECs), small intestinal epithelial cells (SIECs), GABA neurons, hepatocytes, gastric epithelial cells (GECs), astrocytes, cardiomyocytes (HCMs), cardiac fibroblasts (HCFs), dermal fibroblasts (HDFs), and lung fibroblasts (HPFs). Each co-culture performed is indicated by a dashed line. Non-hematopoietic cells were pulsed with 100 ng / mL of HA-2+ peptide before co-culture (Figure 14B). DETAILED DESCRIPTION OF THE INVENTION

[0076] [Detailed Description of the Invention] The present invention is based, at least in part, on the discovery of binding proteins, such as T cell receptors (TCRs), that recognize the HA-2 antigen (eg, an immunogenic peptide comprising the amino acid sequence YIGEVLVSV or YIGEVLVSM).

[0077] The present invention relates, in part, to isolated binding proteins (e.g., TCRs), host cells expressing the binding proteins (e.g., TCRs), compositions comprising the binding proteins (e.g., TCRs) and host cells expressing the binding proteins (e.g., TCRs), methods for diagnosing, prognosing, and monitoring T cell responses to cells expressing the HA-2 antigen, and methods for preventing and / or treating non-malignant, hyperproliferative, or recurrence of hyperproliferative disorders characterized by expression of the HA-2 antigen by administering host cells expressing the binding proteins (e.g., TCRs).

[0078] I. Definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0079] The articles "a" and "an" are used in this application to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or two or more elements.

[0080] The term "administering" means providing a pharmaceutical agent or pharmaceutical composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration. This includes physically introducing a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. In some embodiments, routes of administration for the binding proteins described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, such as by injection or infusion. The term "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the binding proteins described herein may be administered by a non-parenteral route, such as a topical, epidermal, or mucosal route, such as intranasal, oral, vaginal, rectal, sublingual, or topical administration. Administration may also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0081] As used in this application, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. The antigen may be the HA-2 antigen, or a fragment thereof, against which a protective or therapeutic immune response is desired.

[0082] As used herein, the term "adjuvant" refers to a substance that, when administered before, together with, or after the administration of an antigen, accelerates, prolongs, and / or enhances the quality and / or strength of the immune response to that antigen compared to the administration of the antigen alone. Adjuvants can increase the strength and duration of the immune response induced by vaccination.

[0083] As used in this application, the term "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion"), or single chains thereof. In one embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding sites thereof, inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H In certain naturally occurring antibodies, the heavy chain constant region is composed of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain is composed of a light chain variable region (abbreviated as V in this application). L The V is composed of a V domain (abbreviated as V) and a light chain constant region. The light chain constant region is composed of one domain, CL. H and V L The regions may be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0084] The term "antigen-presenting cell" or "APC" includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0085] As used herein, the term "antigen-binding portion" with respect to a binding protein (e.g., a TCR, etc.) refers to one or more portions of a TCR that retain the ability to bind (e.g., specifically and / or selectively) to an antigen (e.g., an HA-2 antigen). Such portions are, for example, between about 8 and about 1500 amino acids in length, preferably between about 8 and about 745 amino acids in length, preferably between about 8 and about 300 amino acids in length, such as from about 8 to about 200 amino acids in length, or from about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of a TCR can be performed by fragments of a full-length TCR. Examples of binding portions encompassed within the term "antigen-binding portion" of a TCR include: (i) the V α and V β (ii) an isolated complementarity-determining region (CDR); or (iii) a combination of two or more isolated CDRs, optionally linked by a synthetic linker. α and V β are encoded by separate genes, but using recombinant methods, V α and V β , a single protein chain (in this single protein chain, V α and V β The V domains are linked by a synthetic linker that allows them to pair together to form a monovalent molecule (known as a single-chain TCR (scTCR)). α and V βSuch single-chain TCRs are also intended to be encompassed within the term "antigen-binding portion" in reference to a TCR. These TCR fragments can be obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as the entire binding protein. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0086] The terms "complementarity determining region" and "CDR" are known in the art to be synonymous with "hypervariable region" or "HVR" and refer to non-contiguous sequences of amino acids within a particular binding protein (such as a TCR variable region) that confer antigen specificity and / or binding affinity. For TCRs, there are generally three CDRs in each α-chain variable region (αCDR1, αCDR2, and αCDR3), and three CDRs in each β-chain variable region (βCDR1, βCDR2, and βCDR3). CDR3 is considered to be the primary CDR involved in recognizing processed antigen. CDR1 and CDR2 primarily interact with MHC.

[0087] 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 or pre-ejaculatory fluid, chyle, chyme, feces, female ejaculate (squirt), interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In some embodiments, the body fluid comprises immune cells, optionally wherein the immune cells are cytotoxic lymphocytes, e.g., cytotoxic T cells, and / or NK cells, CD4+ T cells, etc.

[0088] 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., the 5' and 3' untranslated regions).

[0089] The term "complementary" refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue in a first nucleic acid region can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region antiparallel to the first region if that residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid strand can form specific hydrogen bonds ("base pairing") with a residue in a second nucleic acid region antiparallel to the first region if that residue is guanine. A first region of nucleic acid is complementary to a second region of the same or a different nucleic acid if the two regions are in an antiparallel orientation and at least one nucleotide residue in the first region can base pair with a residue in the second region. In some embodiments, the first region comprises a first portion, and the second region comprises a second portion, such that when the first portion and the second portion are in an antiparallel orientation, at least about 50%, and in other embodiments at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range therebetween, inclusive, e.g., at least about 80%-100%, etc., of the nucleotide residues of the first portion can base pair with the nucleotide residues in the second portion. In some embodiments, the nucleotide residues of the first portion can base pair with the nucleotide residues in the second portion.

[0090] As used herein, the term "co-stimulate," with respect to activated immune cells, includes that a costimulatory molecule can provide a second, non-activating receptor-mediated signal (a "co-stimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal. An immune cell that has received a cell-receptor-mediated signal, e.g., via an activating receptor, is referred to herein as an "activated immune cell."

[0091] "CD3" is known in the art as a six-chain multi-protein complex (Abbas and Lichtman, Cellular and Molecular Immunology (9) th Edition)(2018);Janeway et al.(Immunobiology)(9 th (See, e.g., 2016 Edition). In mammals, the complex comprises a homodimer of the CD3γ chain, the CD3δ chain, two CD3ε chains, and the CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that is thought to enable these chains to associate with positively charged regions or residues of T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), while each CD3ζ chain possesses three ITAMs. Without wishing to be bound by theory, it is believed that such ITAMs are important for the signaling capacity of the TCR complex. The CD3 used in accordance with the present invention may be derived from a variety of animal species, such as, for example, human, mouse, rat, or other mammals.

[0092] As used in this application, "a component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0093] The term "chimeric antigen receptor" or "CAR" refers to a fusion protein engineered to contain two or more amino acid sequences linked together in a manner that does not occur naturally or naturally in a host cell, such that the fusion protein can function as a receptor when present on the surface of a cell. CARs encompassed by the present invention comprise an extracellular portion comprising an antigen-binding domain (i.e., derived from or derived from an immunoglobulin or immunoglobulin-like molecule, e.g., a TCR specific for the HA-2 antigen, a single-chain TCR-derived binding protein, an scFv derived from an antibody, an antigen-binding domain derived from a killer immune receptor of an NK cell, etc.), linked to a transmembrane domain and one or more intracellular signaling domains (e.g., an effector domain, optionally including costimulatory domain(s)) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; Harris and Kranz (2016) Trends Pharmacol. Sci. 37: 220; see also, Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0094] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are primarily CD8 + Mediated by T cells.

[0095] The term "consisting essentially of" is not equivalent to "comprising" and refers to specified materials or steps of a claim or to materials or steps that do not substantially 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 particular amino acid sequence if the amino acid sequence of the domain, region, module, or protein includes an extension, deletion, mutation, or combination thereof (e.g., of amino acids at the amino terminus, or carboxy terminus, or between domains), and the extension, deletion, mutation, or combination thereof is not affected. These combinations, in combination, contribute up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain, region, module or protein, and do not substantially affect the activity (e.g., target binding affinity of a binding protein) of the domain(s), region(s), module(s) or protein (i.e., do not reduce said activity by more than 50%, e.g., by 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% or less).

[0096] The term "determining a suitable treatment regimen for the subject" is understood to mean determining a treatment regimen for the subject (i.e., a single therapy or a combination of various therapies used to prevent and / or treat a viral infection in the subject) to be initiated, modified, and / or terminated based on, or based essentially on, or at least in part on, the results of an analysis according to the present invention. For example, initiating adjuvant therapy after surgery to reduce the risk of recurrence, or another example, modifying the dose of a particular chemotherapy, may be included. Such a decision may be based on the results of an analysis according to the present invention as well as the personal characteristics of the subject to be treated. In most cases, the attending physician or doctor will actually determine the treatment regimen appropriate for the subject.

[0097] As used herein, a "hematopoietic progenitor cell" is a cell that may be derived from hematopoietic stem cells or fetal tissue and that can further differentiate into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include those expressing CD24 Lo Lin-CD117 + These include those with a phenotype that is specific to the thymus, or those found in the thymus (called primitive thymocytes).

[0098] As used herein, "homology" refers to the similarity in nucleotide sequence between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. A nucleotide residue position in both regions is homologous at that position if the same nucleotide residue is occupied by that position. A first region is homologous to a second region if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the percentage of nucleotide residue positions in the two regions that are occupied by the same nucleotide residue. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. In some embodiments, the first region comprises a first portion, and the second region comprises a second portion, whereby 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, such as at least about 80%-100%, inclusive, of the nucleic acid residue positions of each of the portions are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0099] As used herein, the terms "HA-2 antigen" or "HA-2 peptide antigen" or "HA-2-containing peptide antigen" or "HA-2 epitope" or "HA-2 peptide epitope" or "HA-2 peptide" refer to the naturally occurring or synthetically produced peptide portion of a member of the class I myosin family known as MYO1G (Pierce et al. (2001) J. Immunol. 167:3223-3230). This gene is located on the short arm of human chromosome 7, and its expression is restricted to cells of hematopoietic origin. MY01G is a diallelic gene encoding two genetic variants, designated MY01G(V) and MY01G(M). MYO1G(V) encodes the YIGEVLVSV HA-2 epitope, and MYO1G(M) encodes the YIGEVLVSM HA-2 epitope. A single amino acid change between two peptides matches the class I MHC restriction element HLA-A (e.g., HLA-A * 0201) and does not significantly affect T cell recognition when exogenously added to target cells. However, peptides encoded by MYO1G(M) are not expected to be displayed on the surface of cells that endogenously express the MYO1G(M) allele. HA-2 antigen proteins can range in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, or up to about 20 amino acids, and form complexes with MHC (e.g., HLA) molecules such that binding proteins of the present disclosure specific for HA-2 peptide:MHC (e.g., HLA) complexes can bind (specifically and / or selectively) to such complexes. Exemplary HA-2 peptide antigens include peptides having the amino acids YIGEVLVSV or YIGEVLVSM. In some embodiments, the YIGEVLVSV peptide sequence is referred to as "HA-2 REF" and the YIGEVLVSM sequence is referred to as "HA-2 SNP."

[0100] The term "hyperproliferative disorder characterized by expression of the HA-2 antigen" can refer to any hyperproliferative disorder in which the HA-2 antigen is present in an MHC (e.g., HLA) complex expressed by at least some hyperproliferative cells in a subject. Examples of hyperproliferative disorders characterized by an HA-2:HLA complex include hematological malignancies. In certain embodiments, the hematological malignancies include leukemias (e.g., acute leukemia or chronic leukemia). In certain embodiments, the leukemias include acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia, or chronic lymphocytic leukemia (CLL). In certain embodiments, the hematological malignancy comprises lymphoma. In certain embodiments, the lymphoma comprises Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma, or leukemia. lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, extraskeletal plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, B-cell hyperplasia of unspecified grade, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.In certain embodiments, the hematological malignancy is a myelodysplastic disorder (MDS), such as refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts - thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (refractory anemia with excess blasts). blasts (RAEB)), unclassifiable myelodysplastic syndrome, and refractory cytopenia of childhood, myelodysplastic syndrome with isolated deletion of the long arm of chromosome 5 (MDS with isolated del(5q)), and other known MDS subtypes (see, e.g., the 2016 World Health Organization MDS classification, available on the World Wide Web at nbci.nlm.nih.gov / pmc / articles / PMC655460 / ). In certain embodiments, the hematological malignancy includes myeloproliferative neoplasms (MPN) (e.g., myelofibrosis, polycythemia vera, essential thrombocythemia, chronic myelomonocytic leukemia, chronic myeloproliferative disorder-unclassifiable, chronic neutrophilic leukemia, chronic eosinophilic leukemia, and MPN-not otherwise specified, etc.). In a further embodiment, the hematological malignancy comprises myeloma.

[0101] 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 that are indirectly effected by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0102] An increased ability to stimulate an immune response or the immune system may result from increased agonist activity of T cell costimulatory receptors and / or increased antagonist activity of inhibitory receptors. An increased ability to stimulate an immune response or the immune system may result from increased EC in assays that measure immune responses, such as assays that measure changes in cytokine or chemokine release, cytolytic activity (measured directly on target cells or indirectly via detecting CD107a or granzymes), and proliferation. 50 The ability to stimulate an immune response or the immune system may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500%, or more.

[0103] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent that can stimulate a host's immune system to generate an immune response to a viral infection in a subject. A variety of immunotherapeutic agents are useful in the compositions and methods described herein.

[0104] The term "immune cell" refers to any cell of the immune system that arises from hematopoietic stem cells in the bone marrow, which give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells, such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes); and lymphoid progenitor cells (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 + These include T cells, CD4+CD8 double-negative T cells, gdT cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells are sometimes called "antigen-presenting cells" or "APCs," and are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of APCs complexed with peptides interact with TCRs on the surface of T cells.

[0105] "Isolated protein" refers to a protein that, when isolated from a cell or produced by recombinant DNA technology, is substantially free of other proteins, cellular material, isolation and culture medium, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. By "isolated" or "purified," a protein, or biologically active portion thereof, is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the binding protein, antibody, polypeptide, peptide, or fusion protein is derived, or, when chemically synthesized, is substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of biomarker polypeptides, or fragments thereof, in which the protein is separated from cellular components of the cells from which it is isolated or from cells from which it is recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of biomarker proteins, or fragments thereof, having less than about 30% (by dry weight) non-biomarker proteins (also referred to herein as "contaminating proteins"), or in some embodiments, about 25%, 20%, 15%, 10%, 5%, 1%, or less, or any range therebetween, inclusive, non-biomarker proteins. When a binding protein, antibody, polypeptide, peptide, or fusion protein, or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it may be substantially free of culture medium, i.e., culture medium represents about 20%, 15%, 10%, 5%, 1%, or less, or any range therebetween, inclusive, of the volume of the protein preparation.

[0106] As used in this application, the term "isotype" refers to the class of antibody (e.g., IgM, IgG1, IgG2C, etc.) that is encoded by heavy chain constant region genes.

[0107] As used in this application, the term "K D " is intended to refer to the dissociation equilibrium constant of a particular binding protein-antigen interaction. The binding affinity of binding proteins encompassed by the present invention may be measured or determined by standard binding protein-target binding assays, such as competitive assays, saturation assays, or standard immunoassays, such as ELISA or RIA. A relatively lower Kd value indicates a relatively higher binding affinity (e.g., about 5 x 10 -4 The Kd value for M (500uM) or less is 1×10 -4 M (100uM), where a Kd of 100uM indicates a relatively higher binding affinity compared to a Kd of 500uM).

[0108] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent (e.g., a probe or small molecule for specifically detecting and / or affecting the expression of a marker encompassed by the invention). The kit may be advertised, distributed, or sold as a unit for performing a method encompassed by the invention. The kit may include one or more reagents necessary to represent a composition useful in a method encompassed by the invention. In some embodiments, the kit may further include a reference standard (e.g., a nucleic acid encoding a protein that does not affect or modulate signaling pathways controlling cell growth, division, migration, survival, or apoptosis). Those skilled in the art can envision many such control proteins, including, but not limited to, general molecular tags (e.g., green fluorescent protein and beta-galactosidase), proteins unclassified by GeneOntology in any pathway involving cell growth, division, migration, survival, or apoptosis, or ubiquitous housekeeping proteins. Reagents in the kit may be provided in individual containers, or mixtures of two or more reagents may be provided in a single container. Additionally, kits may include instructional materials describing the use of the compositions.

[0109] As used in this application, the term "linked" refers to the association of two or more molecules. The association may be covalent or non-covalent. The association may also be genetic (i.e., recombinantly fused). Such association can be achieved using a variety of art-recognized techniques, including, for example, chemical conjugation and recombinant protein production.

[0110] "Linker" may, in some embodiments, refer to an amino acid sequence that links two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function that accommodates the interaction of two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., an scTCR) or retains signaling activity (e.g., a TCR complex). In some embodiments, the linker is composed of about 2 to about 35 amino acids, e.g., about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids.

[0111] "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 domains) and a non-covalently associated b2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and b, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides from the cytosol to the cell surface, where the peptide antigen-MHC (pMHC) complex is expressed by CD8 + MHC class II molecules deliver peptides from the vesicle system to the cell surface, where they are recognized by CD4 T cells. + Human MHC is recognized by T cells and is called human leukocyte antigen (HLA).

[0112] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject (who does not have the disease, disorder, or condition, but who is at risk of or susceptible to developing the disease, disorder, or condition).

[0113] The term "prognosis" includes a prediction of the likely course and outcome of a viral infection or the likelihood of recovery from the disease. In some embodiments, a statistical algorithm is used to predict a prognosis of a viral infection in an individual. For example, the prognosis can be surgery, development of a clinical subtype of the viral infection, the occurrence of one or more clinical factors, or recovery from the disease.

[0114] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology" and is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This described algorithm has been incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the polynucleotides described in this application. BLAST protein searches are performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0115] The phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another organ or part of the body, that encapsulates a substance, etc.

[0116] The term "recombinant host cell" (or simply "host cell") refers to a cell containing a nucleic acid that does not naturally occur in the cell, such as a cell into which a recombinant expression vector has been introduced. It should be understood that the cells of the invention include not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the term cell of the invention.

[0117] The terms "cancer response," "response to immunotherapy," or "response to modulators of T-cell-mediated cytotoxicity / immunotherapy combination therapy" refer to any response of a hyperproliferative disorder (e.g., cancer) to a cancer agent, e.g., a modulator of T-cell-mediated cytotoxicity and immunotherapy, preferably a change in tumor weight and / or volume after initiation of neoadjuvant or adjuvant therapy. The term "neoadjuvant therapy" refers to a treatment given before primary treatment. Examples of neoadjuvant therapy include chemotherapy, radiation therapy, and hormonal therapy. The responsiveness of a hyperproliferative disorder may be evaluated, for example, for efficacy or in the neoadjuvant or adjuvant setting, where tumor size after systemic intervention may be compared to the initial size and dimensions, as measured by CT, PET, mammogram, ultrasound, or palpation. Response may also be assessed by caliper measurement of tumors after biopsy or surgical resection or by pathological examination. Response may be recorded in a quantitative manner, such as percentage change in tumor volume, or in a qualitative manner, 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. Assessment of responsiveness to a hyperproliferative disorder may be performed early, e.g., hours, days, weeks, or preferably months, after initiation of neoadjuvant or adjuvant therapy.A typical endpoint for assessing response is the end of neoadjuvant chemotherapy or the time of surgical resection of residual tumor cells and / or the tumor bed. This is typically three months after the start of neoadjuvant therapy. In some embodiments, the clinical effectiveness of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients in complete remission (CR), the number of patients in partial remission (PR), and the number of patients with stable disease (SD) at least six months after the end of treatment. Expressed simply, this formula is CBR = CR + PR + SD over six months. In some embodiments, the CBR of a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for assessing responsiveness to cancer therapy relate to "survival," which includes all of the following: survival until death, also known as overall survival (where the death is either causal or tumor-related); "recurrence-free survival" (where the term recurrence includes both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease includes cancer and related diseases). The length of survival may be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy may be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a particular cancer treatment regimen may be prescribed to a population of subjects, and the outcome may be correlated with biomarker measurements determined prior to prescribing any cancer treatment.The outcome measure may be pathological responsiveness to prescribed treatment in a neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival, may be monitored over a period of time for a subject following cancer therapy for which biomarker measurements are known. In certain embodiments, the administered dose is a standard dose known in the art for the cancer treatment drug. The period of time for monitoring a subject may vary. For example, a subject may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. The threshold value of biomarker measurement that correlates with the outcome of cancer therapy may be determined using methods known in the art, such as those described in the Examples section.

[0118] As indicated, these terms can also refer to improved prognosis, for example, as reflected by an increased time to recurrence (which is the time to first recurrence, censored by a second primary cancer as the first event or death without evidence of recurrence) or an increased overall survival (which is the time from treatment to death from any cause). To respond or be responsive means that upon exposure to a stimulus, there is a beneficial endpoint that is achieved. Alternatively, upon exposure to a stimulus, negative or adverse symptoms are minimized, alleviated, or attenuated. It is understood that assessing the likelihood that a tumor or subject will exhibit a favorable response is equivalent to assessing the likelihood that a tumor or subject will not exhibit a favorable response (i.e., exhibit a lack of response or exhibit non-responsiveness).

[0119] The term "resistance" refers to an acquired or natural resistance (i.e., failure to respond to therapeutic treatment or a reduced or limited response to therapeutic treatment) of a cancer sample or mammal to a cancer therapy, e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, e.g., a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more, or any range therebetween, inclusive, decrease in responsiveness to therapeutic treatment. The decrease in responsiveness may be measured by comparing with the same cancer sample or mammal before resistance was acquired, or by comparing with a different cancer sample or mammal known not to have resistance to the therapeutic treatment. Typical acquired resistance to chemotherapy is called "multidrug resistance." Multidrug resistance may be mediated by P-glycoprotein or other mechanisms, or may occur when a mammal is infected with a multidrug-resistant microorganism or combination of microorganisms. Determining resistance to therapeutic treatment is routine in the art and within the skill of a skilled clinician, and may be measured, for example, by cell proliferation and cell death assays, as described herein as "sensitizing." In some embodiments, the term "reverses resistance" means that the use of a second agent in conjunction with a primary cancer therapy (e.g., chemotherapy or radiation therapy) can significantly reduce tumor volume compared to the tumor volume of an untreated tumor at a statistically significant level (e.g., p<0.05) in situations where the primary cancer therapy (e.g., chemotherapy or radiation therapy) alone fails to achieve a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor. This generally applies to measurements of tumor volume taken when untreated tumors are growing exponentially.

[0120] The term "sample" as used to detect or determine the absence or presence, or level, of at least one biomarker, typically refers to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., as described above under the definition of "body fluid"), or a tissue sample (e.g., biopsy), such as a small intestine, large intestine sample, or surgically resected tissue. In some embodiments, methods encompassed by the invention further comprise obtaining the sample from the individual prior to detecting or determining the absence or presence, or level, of at least one marker in the sample.

[0121] The term "sensitize" refers to altering cancer or tumor cells in a way that allows them to be more effectively treated for the associated cancer with a cancer therapy (e.g., anti-immune checkpoint therapy, chemotherapy, and / or radiation therapy), in some embodiments, such that normal cells are not affected to the extent that they are unduly harmed by the therapy. Increased or decreased sensitivity to therapeutic treatment can be assessed by, for example, but not limited to, cell proliferation assays (Tanigawa et al. (1982) Cancer Res. 42:2159-2164) and cell death assays (Weisenthal et al. (1984) Cancer Res. 94:161-173; Weisenthal et al. (1985) Cancer Treat Rep. 69:615-632; Weisenthal et al., In: Kaspers GJL, Pieters R, Twentyman PR, Weisenthal LM, Veerman AJP, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, PA: Harwood Academic Publishers, 1993:415-432; Weisenthal (1994) Contrib. Gynecol. Obstet. 19:82-90). etc., as determined according to methods known in the art for the particular treatment and method, as described below in this application. Sensitivity or resistance may also be determined in animals by measuring the reduction in tumor size over a period of time (e.g., 6 months for humans and 4-6 weeks for mice).A composition or method sensitizes a subject to a therapeutic treatment if there is a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more increase in sensitivity to the treatment, or a decrease in resistance, compared to the sensitivity or resistance to the treatment in the absence of such composition or method, or any range therebetween, inclusive. Determining sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It should be understood that any of the methods described herein for increasing the effectiveness of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative cells or other cancerous cells (e.g., resistant cells) to that cancer therapy.

[0122] The term "small molecule" is a term of the art and includes molecules with a molecular weight of less than about 1000, or molecules with a molecular weight of less than about 500. In one embodiment, a small molecule is not composed solely of peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63-68), and natural product extract libraries. In another embodiment, the compound is a small organic non-peptide compound. In a further embodiment, the small molecule is not biosynthetic.

[0123] The term "specific binding" refers to the binding of a binding protein to a predetermined antigen. Typically, the binding protein binds to approximately 5×10 mAb or less when measured in a binding assay, such as surface plasmon resonance (SPR) technology on a BIAcore™ assay instrument, using the antigen of interest as the analyte and the binding protein as the ligand. -4 M or less, approximately 1×10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, approximately 1×10 -12 Affinity (K) of about 1-50 μM (micromolar), 1-100 μM (micromolar), 0.1-500 μM (micromolar), etc. DIn some embodiments, the binding protein binds to a predetermined antigen with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The phrases "binding protein that recognizes an antigen" and "binding protein specific for an antigen" are used interchangeably in this application with the term "binding protein that specifically binds to an antigen." Selective binding is a related term that refers to the ability of a binding protein to distinguish binding of one antigen from binding of another antigen (e.g., binding of a particular family member or antigen target from binding of related family members or antigen targets). For example, the analytical data provided in the Examples section demonstrate that the binding proteins described herein specifically bind to the HA-2 immunogenic epitope and / or selectively bind to many related epitopes (e.g., the HA-2 immunogenic epitope and closely related sequences), distinguishing such targets from the majority of other possible epitopes available in the human genome.

[0124] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen. The term "subject" is interchangeable with "patient."

[0125] The term "survive" includes all of the following: survival until death, also known as overall survival (where the death may be unrelated to the cause or tumor-related); "recurrence-free survival" (where the term recurrence includes both local and distant recurrence); metastasis-free survival; and disease-free survival (where the term disease includes cancer and related diseases). The length of survival can be calculated by reference to a defined starting point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy can be expanded to include responsiveness to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0126] The term "synergistic effect" refers to a combined effect of two or more agents (e.g., an HA-2-related agent described in this application and another therapy for treating a disorder characterized by HA-2 expression) that is greater than the sum of the separate effects of the cancer agents / therapies alone.

[0127] As used in this application, the term "T cell-mediated response" refers to a response mediated by T cells, e.g., effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0128] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or analog of such RNA or cDNA) that is complementary to or homologous to all or a portion of the mature mRNA produced by transcription of a biomarker nucleic acid and normal post-transcriptional processing, if any, of the RNA transcript (e.g., splicing) and reverse transcription of the RNA transcript.

[0129] "T cells" are immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells are naive (not exposed to antigen; T CM increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO, compared with normal T cells), memory T cells (T M ) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). M Central memory T cells (T CM , which have increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA, compared with naive T cells, as well as effector memory T cells (T EM , naive T cells or T CM They can be further divided into effector T cells (T) and effector T cells (T) (which have decreased expression of CD62L, CCR7, CD28, and CD45RA, and increased expression of CD127, compared to T cells). E ) is T CM CD4+ T cells are antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, and CD28, and are positive for granzymes and perforin, compared to CD4+ T cells. Other exemplary T cells include regulatory T cells, e.g., CD4+ T cells. + CD25 + (Foxp3 +) Regulatory T cells and Tregl7 cells, as well as Trl, Th3, and CD8 + CD28 and Qa-1 restricted T cells.

[0130] Conventional T cells (also known as Tconv or Teff) have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self-recognition, etc.) and augment immune responses by expressing one or more T cell receptors. Tcon or Teff is generally defined as any T cell population that is not a Treg, including, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated toward, for example, a Th1 lineage or a Th2 lineage. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is a CD4+ Teff or CD8+ Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17), and CD8+ cytotoxic T lymphocytes. As further described herein, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tcon" refers to CD4 T cells differentiated in the bone marrow. +Naive T cells are T cells that have successfully undergone the positive and negative processes of central selection in the thymus but have not yet been activated by exposure to antigen. Naive T cells are generally characterized by the surface expression of L-selectin (CD62L), the absence of activation markers such as CD25, CD44, or CD69, and the absence of memory markers such as CD45RO. Naive T cells are therefore quiescent and non-dividing and are thought to require interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see, at least, WO 2010 / 101870). The presence and activity of such cells is undesirable in situations where an immune response is suppressed. 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. Biol. Sci. 356:625-637).

[0131] "T Effector" ("T eff " or "T E ") cells refer to T cells with cytolytic activity (e.g., CD4+ and CD8+ T cells) and T helper (Th) cells, which secrete cytokines and activate and direct other immune cells, but do not include regulatory T cells (Treg cells).

[0132] A "T cell receptor" or "TCR" is a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al. (1997) Curr. Biol. Publ. 4:33) that can bind (e.g., specifically and / or selectively) to antigenic peptides bound to an MHC receptor. TCRs can be found on the surface of cells or in soluble form and generally consist of a heterodimer having an alpha chain and a beta chain (also known as TCRα and TCRβ, respectively) or a gamma chain and a delta chain (also known as TCRγ and TCRδ, respectively). Like immunoglobulins (e.g., antibodies), the extracellular portion of a TCR chain (e.g., α chain and β chain) contains two immunoglobulin domains: an N-terminus, a variable domain (e.g., an α-chain variable domain or V-chain), and a β-chain. α、 and the β-chain variable domain or V β typically amino acids 1 to 116 based on Kabat numbering (Kabat et al. (1991) "Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 5th ed.)), and at the C-terminus and proximal to the cell membrane, one constant domain (e.g., an 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, TCRs comprise amino acids 117 to 295 based on Kabat. Also similar to immunoglobulins, the variable domains comprise complementarity-determining regions ("CDRs," also called hypervariable regions or "HVRs") separated by framework regions ("FRs") (see, e.g., Fores et al. (1990) Proc. Natl. Acad Sci. USA 87:9138; Chothia et al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol. 27:55). In some embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and are associated with the CD3 complex. Sources of TCRs encompassed by the present invention may be derived from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0133] The term "T cell receptor" or "TCR" should be understood to encompass complete TCRs as well as antigen-binding portions or fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, such as an αβ TCR or a γδ TCR. In some embodiments, the TCR is an antigen-binding portion that is shorter than the full-length TCR but binds to a specific peptide bound in an MHC molecule, e.g., an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR contains only a portion of the structural domain of a full-length or intact TCR, but is still capable of binding to a peptide epitope (e.g., an MHC-peptide complex) that a full-length TCR binds. In some cases, the antigen-binding portion includes sufficient variable domains of the TCR (e.g., the variable α chain and variable β chain of the TCR) to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chain of a TCR comprises complementarity determining regions (CDRs), which are involved in the recognition of peptides, MHC and / or MHC-peptide complexes.

[0134] The nomenclature has been established by the International Immunogenetics Information System (IMGT) (Scaviner and Lefranc (2000) Exp. Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; see also "T Cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). The IMGT provides unique sequences used to describe TCRs, and sequences described in this application may be identified by reference to such unique sequences provided in this application. TCR sequences are publicly available in the IMGT database at imgt.org.

[0135] As described above, natural alpha / beta heterodimeric TCRs have an alpha chain and a beta chain. Generally, each chain contains a variable region, a joining region, and a constant region. The beta chain also usually contains a short diversity region between the variable and joining regions, although this diversity region is often considered part of the joining region. Each variable region contains three hypervariable CDRs (complementarity-determining regions) embedded in a framework sequence. CDR3 is well known to be the primary mediator of antigen recognition. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions, which are distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. The Vα types are referred to by unique TRAV numbers in the IMGT nomenclature. For example, "TRAV4" defines a TCR Vα region with a unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence that is partially defined by amino acid sequences that are conserved from TCR to TCR, but also includes amino acid sequences that vary from TCR to TCR. Similarly, "TRBV2" defines a TCR Vβ region with a unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence. There are known to be 54 alpha variable genes (44 of which are functional) and 67 beta variable genes (42 of which are functional) within the alpha and beta loci, respectively.

[0136] Similarly, the joining regions of the TCR are defined by the specific IMGTTRAJ and TRBJ nomenclature, and the constant regions are defined by the IMGTTRAC and TRBC nomenclature. The diversity region of the beta chain is referred to by the abbreviation TRBD in the IMGT nomenclature, and as noted, the joined TRBD / TRBJ regions are often considered together as the joining region.

[0137] The gene pool encoding the TCR alpha and beta chains is located on various chromosomes and contains distinct V, D, J, and C gene segments that are brought together by rearrangement during T cell development. This results in a very high degree of diversity for T cell alpha and beta chains due to the numerous recombination events that can occur between the 54 TCR alpha variable genes and the 61 alpha J genes, or between the 67 beta variable genes, the 2 beta D genes, and the 13 beta J genes. The recombination process is not precise, introducing additional diversity within the CDR3 region. Each alpha and beta variable gene may also contain allelic variants, designated in the IMGT nomenclature as TRAVxx*01 and *02, or TRBVx-x*01 and *02, respectively, thus further increasing the amount of diversity. Similarly, some TRBJ sequences have two known variants. (Note that the absence of the "*" qualifier means that only one allele is known for the relevant sequence.) The natural repertoire of human TCRs resulting from recombination and thymic selection consists of approximately 10 6 It is estimated that each beta chain contains a unique beta chain sequence for each species (Arstila et al. (1999) Science 286:958-961), and may even be higher (Robins et al. (2009) Blood 114:4099-4107). Each beta chain is estimated to pair with at least 25 different alpha chains, thus generating a wide variety of DNA (Arstila et al. (1999) Science 286:958-961).

[0138] Thus, the term "TCR alpha variable domain" refers to the TRAV and TRAJ regions linked together; the TRAV region alone; or the TRAV and a partial TRAJ region, and the term TCR alpha constant domain refers to the extracellular TRAC region, or a C-terminally truncated or full-length TRAC sequence. Similarly, the term "TCR beta variable domain" refers to the TRBV and TRBD / TRBJ regions linked together; the TRBV and TRBD regions alone; the TRBV and TRBJ regions alone; or the TRBV and a partial TRBD and / or TRBJ region. And the term TCR beta constant domain refers to the extracellular TRBC region, or a C-terminally truncated or full-length TRBC sequence. These TCR alpha variable domain and TCR beta variable domain nomenclature similarly apply to the variable domains of the TCR gamma chain and TCR delta chain, respectively, for gamma / delta TCRs. One of skill in the art can obtain the gene sequences for TRAV, TRAJ, TRAC, TRBV, TRBJ, and TRBC, for example, through the publicly available IMGT database.

[0139] The term "TCR complex" refers to a complex formed by the association of CD3 with a TCR. For example, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0140] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmaceutically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or in enhancing desirable physical or mental development and conditions in animals or humans.

[0141] The terms "therapeutically effective amount" and "effective amount" refer to that amount of a substance that produces some desired effect (e.g., a desired local or systemic therapeutic effect) in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any treatment. In some embodiments, the therapeutically effective amount of a substance depends on the substance's therapeutic index, solubility, pharmacokinetics, half-life, etc. Toxicity and therapeutic efficacy of subject compounds can be assessed by standard pharmaceutical methods (e.g., LD) in cell cultures or experimental animals. 50 and ED 50 In some embodiments, compounds that exhibit large therapeutic indices are used. In some embodiments, the LD 50 The lethal dose (ED) may be measured, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, for the drug compared to not administering the drug. 50 The IC (i.e., the concentration capable of inhibiting symptoms half-maximally) may be measured and may, for example, be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more for the drug compared to not administering the drug. Similarly, the IC 50may be measured, for example, by at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increase for the agent compared to not administering the agent. In some embodiments, the T cell immune response in the assay may be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, the viral load may be reduced 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%.

[0142] The term "treating" refers to the therapeutic management or amelioration of a symptom of interest (e.g., a disease or disorder). Treatment includes, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically performed to alter the course of a disease (this term is used to refer to any disease, disorder, syndrome, or undesirable condition that warrants or potentially warrants therapy) in a manner beneficial to the subject. The effect of treatment includes reversing, alleviating, reducing the severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of a disease, or one or more symptoms or manifestations of a disease. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. Therapeutic agents may be administered to subjects who have a disease or who are at increased risk of developing a disease compared to members of the general population. In some embodiments, therapeutic agents may be administered to subjects who previously had a disease but no longer show evidence of the disease. The agent may be administered, for example, to reduce the likelihood of a definite recurrence of the disease. Therapeutic agents may be administered prophylactically, i.e., before the onset of any symptoms or manifestations of the disease. "Prophylactic treatment" refers to providing medical and / or surgical treatment to a subject who does not have a disease or shows no evidence of the disease, for example, to reduce the likelihood of the disease occurring or to reduce the severity of the disease if it does occur. The subject may have been identified as being at risk for developing the disease (e.g., as being at increased risk compared to the general population or having risk factors that increase the likelihood of developing the disease).

[0143] The term "unresponsiveness" includes the refractoriness of cancer cells to therapy or the refractoriness of therapeutic cells (e.g., immune cells) to stimulation (e.g., stimulation via an activating receptor or cytokine). Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigen. As used herein, the terms "anergy" or "tolerance" include refractoriness to activating receptor-mediated stimulation. Such refractoriness is broadly antigen-specific and persists even after exposure to the tolerizing antigen has ceased. For example, anergy (as opposed to unresponsiveness) in T cells is characterized by a lack of cytokine (e.g., IL-2) production. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (a T cell receptor- or CD3-mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, when the cells are re-exposed to the same antigen (even if re-exposure occurs in the presence of a costimulatory polypeptide), they are unable to produce cytokines and therefore unable to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes, as measured by ELISA or proliferation assays using indicator cell lines. Alternatively, a reporter gene construct can be used. For example, anergic T cells are unable to initiate transcription of the IL-2 gene induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by multimers of AP1 sequences that can be found within the enhancer (Kang et al. (1992) Science 257:1134).

[0144] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR α-chain or β-chain [or the γ-chain and δ-chain of a γδ TCR]) that is involved in binding the immunoglobulin superfamily binding protein (e.g., a TCR) to an antigen. The variable domains of the α-chain and β-chain of a native TCR (V, V ... α and V β ) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs. α The domains are encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ); β The domains are encoded by three separate DNA segments: the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). α or V β The V domain may be sufficient to confer antigen-binding specificity. Furthermore, a TCR that binds to a particular antigen may be identified by a V domain derived from the TCR that binds that antigen. α or V β Using the V domains, isolate and differentiate the complementary V α or V β A library of domains may be screened.

[0145] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, a vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, a vector is a vector capable of autonomously replicating and / or expressing a nucleic acid to which it is linked. A vector capable of directing the expression of a gene (the gene to which it is operably linked) is referred to herein as an "expression vector." Generally, expression vectors useful in recombinant DNA techniques are often in the form of "plasmids," which generally refer to circular double-stranded DNA loops that, in their vector form, are not bound to the chromosome. As used herein, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. However, as will be understood by those of skill in the art, the present invention is intended to include other forms of expression vectors that serve equivalent functions and that subsequently become known in the art.

[0146] There is a known, definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequence capable of encoding that protein, as defined by the genetic code (see below).Similarly, there is a known, definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0147] [Table 4]

[0148] An important and well-known feature of the genetic code is its redundancy, whereby more than one coding nucleotide triplet can be used for most of the amino acids used to make proteins (see above). Thus, several different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although some organisms can translate certain sequences more efficiently than others). Furthermore, methylated variants of purines or pyrimidines may occasionally be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and its corresponding amino acid.

[0149] In view of the above, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) may be used to derive a polypeptide / amino acid sequence using the genetic code for translating DNA or RNA into an amino acid sequence. Similarly, for a polypeptide / amino acid sequence, the corresponding nucleotide sequence that may encode the polypeptide may be deduced from the genetic code (which, due to its redundancy, generates multiple nucleic acid sequences for any given amino acid sequence). Thus, any description and / or disclosure herein of a nucleotide sequence encoding a polypeptide should be considered to also include a description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, any description and / or disclosure herein of a polypeptide / amino acid sequence should be considered to also include a description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

[0150] II. Binding Proteins In aspects encompassed by the present invention, the present application provides binding proteins that bind to peptide-MHC (pMHC) complexes (e.g., MHC complexes that stably present peptides) that include an HA-2 immunogenic peptide in an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the binding proteins are present in a concentration of about 5×10 -4 M or less, approximately 1×10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, approximately 1×10 -12 In some embodiments, the MHC molecule is capable of binding (e.g., specifically and / or selectively) to an HA-2 peptide-MHC (pMHC) complex with a Kd of less than or equal to 1 μM, or any range therebetween, inclusive, e.g., between about 1-50 μM, 1-100 μM, 0.1-500 μM, etc. In some embodiments, the MHC molecule is of the HLA serotype HLA-A. * In some embodiments, the HLA allele is HLA-A. * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0205, HLA-A * 0206, and HLA-A * 207 alleles. In certain embodiments, the HLA allele is selected from the group consisting of HLA-A *0201. In some embodiments, the binding proteins provided herein are engineered, isolated, and / or purified.

[0151] In some embodiments, the binding protein has a higher binding affinity for HA-2 peptide-MHC (pMHC) than does a known T-cell receptor. For example, the binding protein may bind to HA-2 peptide-MHC (pMHC) in a manner similar to that of a known T-cell receptor (e.g., a TCR from van Loenen et al. (2010) Proc. Natl. Acad. Sci. USA 107:10972-10977). or other described herein) has at least 1.2, 1.5, 1.8, 2.0, 2.2, 2.5, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 9x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 5000x, 10000x, 50000x, 100000x, 500000x, 1000000x, or more, or any range therebetween, inclusive, such as from 1.2x to 2x, etc., higher binding affinity.

[0152] In some embodiments, the binding protein induces greater T cell proliferation, cytokine release, and / or cytotoxic killing than known T-cell receptors when contacted with target cells that express HA-2 at or below a certain level (see, e.g., Table 4 for representative cell lines expressing HA-2 at various levels). For example, in some embodiments of any aspect described herein, HA-2 levels may be expressed as transcripts per million, and may be, for example, about 1000 transcripts per million. perMillionTranscripts(TPM)) (approximately 1000TPM) or less, 950TPM, 900TPM, 850TPM, 800TPM, 750TPM, 700TPM, 650TPM, 600TPM, 550TPM, 500TPM, 450TPM, 400TPM, 350TPM, 300TPM, 250TPM, 200TPM, 150TPM, 100TPM, 95TPM, 90TPM, 85TPM, 80TPM, 75TPM, 70TPM, 65TPM, 60TPM, 55TPM, 50TPM, 45TPM, 40TPM, 35TPM, 34TPM , 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, inclusive, for example, from about 1000 TPM or less to about 35 TPM or less. In some embodiments, low HA-2 expression levels are referred to as "heterozygous expression," meaning between about 1 TPM and about 35 TPM, or any range therebetween, inclusive, e.g., 32 TPM or 1-32 TPM, etc. Higher expression is 36 TPM or greater.As further described herein, TPM is measured according to well-known techniques (e.g., RNA-Seq, etc.), and gene expression TPM data is well-known in the art for various cell lines, tissue types, etc. (See, e.g., the Broad Institute Cancer Cell Line Encyclopedia (CCLE), available on the World Wide Web at portals.broadinstitute.org). In some embodiments, the binding protein, when contacted with target cells heterozygously expressing HA-2, increases T cell proliferation, cytokine release, and / or cytotoxicity by at least 1.2-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x or more, or any range therebetween, inclusive, for example, from 1.2x to 2x increase.

[0153] In some embodiments, HA-2 expression is detected using RNA sequencing (RNA-seq). RNA-seq generally involves obtaining a sample containing genetic material, isolating total RNA from the obtained sample, preparing an amplified cDNA library from the total RNA, sequencing the amplified cDNA library, and analyzing and profiling the amplified cDNA to assess the expression levels of various transcripts. The sample can be a population of cells, a tissue sample, a biopsy sample, a cell culture, or a single cell. Total RNA can be isolated from biological samples using any method known in the art. In certain embodiments, total RNA is extracted from plasma. Extraction of plasma RNA is described in Enders et al., "The Concentration of Circulating Corticotropin-Releasing Homer mRNA in Material Plasma Is Inclined in Preclampsia," Clin. As described therein, plasma collected after the centrifugation step is mixed with Trizol LS reagent (Invitrogen) and chloroform. The mixture is centrifuged, and the aqueous layer is transferred to a new tube. Ethanol is added to this aqueous layer. The mixture is then placed in an RNeasy mini-column (Qiagen) and processed according to the manufacturer's recommendations.

[0154] In some embodiments, the RNA-seq method described herein involves preparing amplified cDNA from total RNA. For example, cDNA is prepared and the isolated RNA sample is randomly amplified undiluted, or the mixture of genetic material in the isolated RNA is dispersed into individual reaction samples. In certain embodiments, amplification is randomly initiated at the 3' end of the transcripts and throughout the transcripts in the sample to amplify both mRNA and non-polyadenylated transcripts. In this manner, double-stranded cDNA amplification products are optimized to generate sequencing libraries for next-generation sequencing platforms. Kits suitable for amplifying cDNA using methods encompassed by the present invention include, for example, the Ovation® RNA-Seq System.

[0155] In some embodiments, the RNA-seq described herein includes sequencing the amplified cDNA. The amplified cDNA mixture may be sequenced using any known sequencing method, including, for example, single molecule sequencing. In certain embodiments, the amplified cDNA is sequenced by whole transcriptome shotgun sequencing. Whole transcriptome shotgun sequencing may be performed using various next-generation sequencing platforms (e.g., the Illumina® Genome Analyzer platform, the ABI SOLiD™ sequencing platform, or the Life Sciences 454 sequencing platform, etc.).

[0156] In some embodiments, the RNA-seq method described herein further includes digitally counting and analyzing the cDNA. The number of amplified sequences for each transcript in the amplified sample may be quantified by sequence reading (one reading per amplified strand). In some embodiments, transcripts per million (TPM) are used to quantify the expression level of a particular transcript. TPM can be calculated as shown in Wagner et al. (2012) Theory in Biosciences 131:281-285, the contents of which are incorporated herein by reference in their entirety.

[0157] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha sequences listed in Table 1; and / or b) A TCR beta chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chains listed in Table 1.

[0158] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 1; and / or b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 1.

[0159] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR V listed in Table 1 α TCR alpha chain variable (V) domain sequences selected from the group consisting of α a TCR alpha chain variable (V) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the TCR alpha chain variable (V) domain sequence; α ) domain sequence; and / or b) a TCR V listed in Table 1 β TCR beta chain variable (V) domain sequences selected from the group consisting of β a TCR beta chain variable (V) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the V β ) domain array.

[0160] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of): a) a TCR V listed in Table 1 α TCR alpha chain variable (V) domain sequences selected from the group consisting of α a) a domain sequence; and / or b) a TCR V listed in Table 1 β TCR beta chain variable (V) domain sequences selected from the group consisting of β ) domain array.

[0161] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of) at least one (e.g., one, two, or three, e.g., CDR3 alone or in combination with CDR1 and CDR2) TCR alpha chain complementarity determining region (CDR) sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 1. CDR3 is considered the primary CDR responsible for recognizing processed antigen, and CDR1 and CDR2 primarily interact with MHC. Thus, in some embodiments, binding proteins are provided that comprise a CDR3 from the TCR alpha chain alone and / or a CDR3 from the TCR beta chain alone, as listed in Table 1, each CDR3 having a sequence homology as listed in this paragraph.

[0162] In some embodiments, the binding proteins provided herein may also comprise (e.g., may comprise, consist essentially of, or consist of) at least one (e.g., one, two, or three, e.g., CDR3 alone or in combination with CDR1 and CDR2) TCR beta chain complementarity determining region (CDR) sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 1. As noted above, CDR3 is considered the primary CDR responsible for recognizing processed antigen, and CDR1 and CDR2 primarily interact with MHC. Thus, in some embodiments, binding proteins are provided that comprise a CDR3 from the TCR beta chain alone and / or a CDR3 from the TCR alpha chain alone, as listed in Table 1, each CDR3 having a sequence homology as listed in this paragraph.

[0163] In some embodiments, the binding proteins provided herein comprise (e.g., comprise, consist essentially of, or consist of) at least one (e.g., one, two, or three) TCR alpha chain complementarity determining region (CDR) listed in Table 1.

[0164] In some embodiments, the binding proteins provided herein may also comprise (e.g., comprise, consist essentially of, or consist of) at least one (e.g., one, two, or three) complementarity determining region (CDR) of a TCR beta chain listed in Table 1.

[0165] In some embodiments, the present application provides a binding protein comprising a TCR C αa TCR alpha chain constant region (C) having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence α ) sequence, comprising (e.g., comprising, consisting essentially of, or consisting of).

[0166] In some embodiments, the present application provides a binding protein comprising a TCR C β a TCR beta chain constant region (C) having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the sequence β ) sequences, such as (e.g., may comprise, consist essentially of, or consist of).

[0167] In some embodiments, the present application provides a binding protein comprising a TCR C α a TCR alpha chain constant region (C) selected from the group consisting of α ) sequence, comprising (e.g., comprising, consisting essentially of, or consisting of).

[0168] In some embodiments, the present application provides a binding protein comprising a TCR C β a TCR beta chain constant region (C) selected from the group consisting of β ) sequences, such as (e.g., may comprise, consist essentially of, or consist of).

[0169] In some embodiments, the binding proteins provided herein comprise a constant region that is chimeric, humanized, human, primate, or rodent (e.g., rat or mouse). For example, a human variable region may be chimerized with a mouse constant region, or a mouse variable region may be humanized with a human constant region and / or human framework region. In some embodiments, the constant region may be mutated to alter function (e.g., introducing non-naturally occurring cysteine ​​substitutions at opposing residue positions in the TCR alpha and beta chains to provide disulfide bonds useful for increasing affinity between the TCR alpha and beta chains). Similarly, mutations may be made in the transmembrane domain of the constant region to alter function (e.g., increasing hydrophobicity by introducing non-naturally occurring substitutions at certain residues with hydrophobic amino acids). In some embodiments, mutations may be made in the constant region to increase cell surface expression.

[0170] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

[0171] Schematic diagram of the TSC-101 npDNA transposon vector cassette: [ka]

[0172] Map of the TSC-100 npDNA transposon vector cassette: [ka]

[0173] The TSC-101 npDNA transposon is a 5,721-bp supercoiled Nanoplasmid™ DNA (npDNA) containing a minimal R6K bacterial origin of replication and an antisense RNA-based antibiotic-free selectable marker (RNA-OUT). The terminal inverted repeats (ITRs) of the armyworm Spodoptera frugiperda transposon (required for transposase-mediated integration) are ligated to the two ends of the exogenous DNA sequence. The TSC-101 HA-2-specific TCR α- and β-chain genes and codon-diversified CD8 α- and β-chain genes are placed under the control of the murine stem cell virus (MSCV) promoter. The constant regions of the TCR α- and β-chains contain amino acid substitutions that stabilize their expression on the surface of engineered T cells and help prevent mispairing of the endogenous TCR α- and β-chains. These four open reading frames (TCRβ, TCRα, CD8α, and CD8β) are encoded on a single mRNA molecule along with the open reading frame of the woodchuck post-transcriptional regulatory element (WPRE), which is known to enhance gene expression (Loeb, 1999). Ribosomal skipping, which occurs on the self-cleaving peptide P2A, results in the production of four separate polypeptides. A 16-amino acid CD34 epitope recognized by the mouse monoclonal antibody QBend / 10, was fused to the N-terminus of the exogenous CD8 α-chain, allowing for the purification of transgene-expressing cells and thereby increasing the safety and efficacy of the drug substance. The Nanoplasmid vector backbone (<500 base pairs) is substantially smaller than conventional plasmids (>1500 base pairs), it does not contain antibiotic resistance genes, and the replication origin (R6K) required for plasmid preparation can support propagation in a narrower range of bacterial species than the commonly used pUC origin.

[0174] *Table 1 includes peptide epitopes and polypeptide molecules comprising amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity over their entire length to the amino acid sequence of any SEQ ID NO: listed in Table 1, or to a portion thereof. Such polypeptides may have the function of the full-length peptide or polypeptide, as further described herein.

[0175] * Table 2 includes RNA nucleic acid molecules (e.g., in which thymine is substituted with uredine), nucleic acid molecules encoding orthologs of the encoded proteins, and DNA or RNA nucleic acid sequences, including nucleic acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over their entire length to any of the sequences, or a portion thereof, listed in Table 2. Such nucleic acid molecules may have the function of the full-length nucleic acid, as further described in this application.

[0176] In some embodiments, the binding proteins disclosed herein may comprise a T cell receptor (TCR), an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In some embodiments, the binding proteins disclosed herein may comprise two polypeptide chains, each of which comprises a variable region comprising the CDR3 of the TCR alpha chain and the CDR3 of the TCR beta chain, or the CDR1, CDR2, and CDR3 of both the TCR alpha chain and the TCR beta chain. In some embodiments, the binding protein comprises a single-chain TCR (scTCR), which comprises a TCR V α and TCR V β domains, but only one TCR constant domain (C αor C β The term "chimeric antigen receptor" (CAR) refers to a fusion protein engineered to contain two or more naturally occurring amino acid sequences linked together in a manner that does not occur naturally or in a host cell, where the fusion protein can function as a receptor when present on the surface of a cell. CARs encompassed by the present invention may comprise an extracellular portion comprising an antigen-binding domain (i.e., an antigen-binding domain derived from or obtained from an immunoglobulin or immunoglobulin-like molecule (e.g., an antibody or TCR, or a killer immune receptor from an NK cell)) linked to a transmembrane domain and one or more intracellular signaling domains (optionally including costimulatory domain(s)) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; and Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0177] In some embodiments, 1) the TCR alpha chain CDRs, TCR V α 1) the TCR alpha chain CDRs are encoded by a TRAV, TRAJ, and / or TRAC gene, or a fragment thereof, selected from the group consisting of the TRAV, TRAJ, and TRAC genes listed in Table 1; and / or 2) the TCR beta chain CDRs, TCR V β 2) the domain and / or TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group consisting of the TRBV, TRBJ, and TRBC genes listed in Table 1; and / or 3) each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof, compared to the cognate reference CDR sequence listed in Table 1.

[0178] In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs)) disclosed in the present application are chimeric (e.g., containing amino acid residues or motifs from more than one donor or species), humanized (e.g., containing residues from a non-human organism that have been modified or substituted to reduce the risk of immunogenicity in humans), or human.

[0179] Methods for producing engineered binding proteins (e.g., TCRs, CARs, and antigen-binding fragments thereof, etc.) are well known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 5:3000; US Pat. No. 6,410,319; US Pat. No. 7,446,191; US ​​Pat. Publ. No. 2010 / 065818; US Pat. No. 8,822,647; PCT Publ. No. WO 2014 / 031687; US Pat. No. 7,514,537; and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0180] In some embodiments, the binding proteins described herein are TCRs, or antigen-binding fragments thereof, expressed on the cell surface, where the TCR expressed on the cell surface can associate with CD3 protein more efficiently than endogenous TCRs. When expressed on the surface of a cell, such as a T cell, the binding proteins (e.g., TCRs) encompassed by the present invention have higher surface expression on the cell than endogenous binding proteins (e.g., endogenous TCRs). In some embodiments, the present application provides CARs, where the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0181] The V disclosed herein may also be used as starting material for engineering modified binding proteins, which may have altered properties from the starting binding protein. α and / or V β Also provided are modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) that may be prepared according to well-known methods using binding proteins having one or more of the sequences. α and / or V β ), e.g., in one or more CDR regions and / or in one or more framework regions. Additionally or alternatively, binding proteins may be engineered by modifying residues in the constant region(s).

[0182] Another type of variable region modification is V α and / or V βMutating amino acid residues within the CDR1, CDR2, and / or CDR3 regions of a CDR1 protein to thereby improve one or more binding characteristics (e.g., affinity) of a binding protein of interest. Site-directed mutagenesis(s) or PCR-mediated mutagenesis may be performed to introduce the mutations, and the effect on protein binding or other functional properties of interest may be assessed in in vitro, ex vivo, or in vivo assays, as described herein and provided in the Examples. In some embodiments, conservative modifications (as described above) may be introduced. The mutations may be amino acid substitutions, additions, or deletions. In some embodiments, the mutations are substitutions. Furthermore, typically, no more than 1, 2, 3, 4, or 5 residues within the CDR regions are modified.

[0183] In some embodiments, the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) described herein may have one or more amino acid substitutions, deletions, or additions compared to naturally occurring TCRs. In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof compared to the cognate reference CDR sequence listed in Table 1. Conservative amino acid substitutions are well known and may occur naturally or may be introduced when the binding protein is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into proteins using mutagenesis methods known in the art (e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide-directed site-specific (or segment-specific) mutagenesis may be used to provide modified polynucleotides with specific codons altered according to the desired substitution, deletion, or insertion. Alternatively, immunogenic polypeptide variants may be prepared using random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis (see, e.g., Sambrook et al., supra).

[0184] Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid substitution or conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids may be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered more difficult to classify, shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, the substitution of glutamine with glutamic acid or asparagine with aspartic acid can be considered a similar substitution, in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions to the sequence of a second polypeptide (e.g., using the GENEWORKS™, Align, BLAST algorithms, or other algorithms described in this application and practiced in the art).

[0185] In some embodiments, the encoded binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) may include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). The signal peptide targets a newly synthesized polypeptide to an appropriate location inside or outside of a cell. The signal peptide may be removed from the polypeptide during localization or secretion, or once localization or secretion is complete. A polypeptide having a signal peptide is referred to herein as a "pre-protein," and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In some embodiments, the binding proteins (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) described herein contain a mature V protein or polypeptide. α Domain, mature V β In some embodiments, a binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) described herein comprises a mature TCR β-chain, a mature TCR α-chain, or both.

[0186] In some embodiments, the binding protein is a fusion protein comprising: (a) an extracellular component comprising a TCR or an antigen-binding fragment thereof; (b) an intracellular component comprising an effector domain or a functional portion thereof; and (c) a transmembrane domain connecting the extracellular and intracellular components. In some embodiments, the fusion protein is capable of binding (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex comprising the HA-2 immunogenic peptide in an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the MHC molecule is of the HLA serotype HLA-A. * In some embodiments, the HLA allele is HLA-A. * 0201, HLA-A * 0202, HLA-A *0203, HLA-A * 0205, HLA-A * 0206, and HLA-A * 207 alleles. In certain embodiments, the HLA allele is selected from the group consisting of HLA-A * The time is 0201.

[0187] As used herein, an "effector domain" or "immune effector domain" is an intracellular portion or domain of a fusion protein or receptor that, upon receiving an appropriate signal, can directly or indirectly stimulate an immune response in a cell. In some embodiments, the effector domain is derived from an immune cell protein or portion thereof or immune cell protein complex that receives a signal upon binding (e.g., CD3ζ) or when the immune cell protein or portion thereof or immune cell protein complex directly binds to a target molecule, triggering signaling from the effector domain in the immune cell.

[0188] An effector domain may directly promote a cellular response if it contains one or more signaling domains or motifs, such as intracellular tyrosine-based activation motifs (ITAMs), such as those found in costimulatory molecules. Without wishing to be bound by theory, ITAMs are believed to be beneficial to T cell activation following ligand engagement by the T cell receptor or by fusion proteins containing T cell effector domains. In some embodiments, the intracellular component or functional portion thereof comprises an ITAM. Exemplary immune effector domains include, but are not limited to, immune effector domains derived from CD3ε, CD3δ, CD3ζ, CD25, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the effector domain comprises a lymphocyte receptor signaling domain (e.g., CD3ζ, or a functional portion or variant thereof).

[0189] In further embodiments, the intracellular component of the fusion protein comprises a costimulatory domain or functional portion thereof selected from a ligand that binds (e.g., specifically and / or selectively) to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-1 (CD54), LFA-1 (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, or a functional variant thereof, or any combination thereof. In some embodiments, the intracellular component comprises a CD28 costimulatory domain, or a functional portion or variant thereof (which may optionally contain an LL-GG mutation at positions 186-187 of the native CD28 protein [e.g., Nguyen et al. (2003) Blood 702:4320]), a 4-1BB costimulatory domain, or a functional portion or variant thereof, or both.

[0190] In some embodiments, the effector domain comprises a CD3ε endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD27 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD28 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In still further embodiments, the effector domain comprises a 4-1BB endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an OX40 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD2 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises a CD5 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICAM-1 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an LFA-1 endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICOS endodomain or a functional (e.g., signaling) portion thereof, or a functional variant thereof.

[0191] The extracellular and intracellular components encompassed by the present invention are connected by transmembrane domains. A "transmembrane domain," as used herein, is a portion of a transmembrane protein that can be inserted into or span a cell membrane. A transmembrane domain has a three-dimensional structure that is thermodynamically stable in the cell membrane and generally ranges in length from about 15 to about 30 amino acids. The structure of a transmembrane domain may include an alpha helix, a beta barrel, a beta sheet, a beta helix, or any combination thereof. In some embodiments, the transmembrane domain comprises or is derived from a known transmembrane protein (e.g., a CD4 transmembrane domain, a CD8 transmembrane domain, a CD27 transmembrane domain, a CD28 transmembrane domain, or any combination thereof).

[0192] 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 to refer to the fusion protein component connecting the binding domain and the transmembrane domain, a "linker" can be an amino acid sequence having from about 2 amino acids to about 500 amino acids, which can provide flexibility and room for conformational movement between the two regions, domains, motifs, fragments, or modules connected by the linker. For example, linkers encompassed by the present invention can position the binding domain away from the surface of a host cell expressing the fusion protein, allowing for proper contact, antigen binding, and activation between the host cell and target cell (Patel et al. (1999) GeneTherapy 6:412-419). Linker length can be varied to maximize antigen recognition based on the selected target molecule, the selected binding epitope, or the capture and affinity of the antigen-binding domain (e.g., Guest et al. (2005) Immunother. 28:203-11 and PCT Publ. No. WO 2014 / 031687). Exemplary linkers include Gly x Ser y where x and y are each independently an integer from 0 to 10, provided that both x and y are not 0 (e.g., (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or a combination thereof [e.g., ((Gly3Ser)2Gly2Ser)]).

[0193] Binding proteins encompassed by the present invention may, in some embodiments, be covalently attached to a moiety. In some embodiments, the covalently attached moiety comprises an affinity tag or label. The affinity tag may be selected from the group consisting of glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently attached moiety is selected from the group consisting of an inflammatory agent, an anti-inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody, such as a single-chain Fv.

[0194] The binding proteins may be conjugated to agents used in imaging, research, therapy, theranostics, medicine, chemotherapy, chelation therapy, targeted drug delivery, and radiation therapy. In some embodiments, the binding proteins may be conjugated or fused to a detectable agent, such as a fluorophore, a near-infrared dye, a contrast agent, a nanoparticle, a metal-containing nanoparticle, a metal chelate, an X-ray contrast agent, a PET agent, a metal, a radioisotope, a dye, a radionuclide chelator, or another suitable substance that may be used for imaging. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more detectable moieties may be linked to the binding protein. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not readily quenched by biological tissues and fluids. In some embodiments, the fluorophore has a wavelength range between 650 nm and 4000 nm.Near-infrared dyes are fluorescent agents that emit electromagnetic radiation at wavelengths between 1000 and 1500 nm, and such radiation is used to detect such agents. Non-limiting examples of fluorescent dyes that may be used as conjugate molecules include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG). In some embodiments, near-infrared dyes often include cyanine dyes (e.g., Cy7, Cy5.5, and Cy5). Additional non-limiting examples of fluorescent dyes for use as conjugate molecules in accordance with the present invention include acrazine orange or yellow, Alexa Fluors® (e.g., Alexa Fluor® 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO® dye and any derivatives thereof, auramine-rhodamine stain and any derivatives thereof, bensantrhone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naththacene, bisbenzimide, brainbow, calcein, carbodyfluorescein,) and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, DyLight® Fluors® and any derivatives thereof, epicocconone, ethidium bromide, FlAsH-EDT2®, Fluo dyes and any derivatives thereof, FluoProbe® and any derivatives thereof, fluorescein and any derivatives thereof, Fura® and any derivatives thereof, GelGreen® and any derivatives thereof, GelRed® and any derivatives thereof, fluorescent proteins and any derivatives thereof, for example, m isoform proteins such as mCherry and any derivatives thereof, hetamethine dyes and any derivatives thereof, Hoeschst stain, iminocoumarin, Indian Yellow yellow), indo-1 and any derivatives thereof, laurdan, lucifer yellow and any derivatives thereof, luciferin and any derivatives thereof, luciferase and any derivatives thereof, mercocyanin and any derivatives thereof, Nile dye and any derivatives thereof, perylene, phloxine, phyco dye and any derivatives thereof, propium iodide, pyranine, rhodamine and any derivatives thereof, ribogreen, RoGFP, rubrene, stilbene and any derivatives thereof, sulforhodamine and any derivatives thereof, SYBR and any derivatives thereof, synapto-pHluorin, tetraphenyl butadiene, tetrasodium tris(tris), Texas red, titanium yellow, TSQ, umbelliferone, violanthrone, yellow fluorescent proteinOther suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, etc.). Red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green™ dyes (e.g., Oregon Green™ 488, 500, 514, etc.), Texas Red™, Texas Red™-X, SPECTRUM RED™, SPECTRUM GREEN™, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), Alexa Fluor™ dyes (e.g., Alexa Fluor™Suitable detectable agents include radioisotopes (e.g., 350, 488, 532, 546, 568, 594, 633, 660, 680, etc.), BODIPY® dyes (e.g., BODIPY® FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRD dyes (e.g., IRD40™, IRD700™, IRD800™, etc.), and the like. Additional suitable detectable agents are known in the art (e.g., PCT Publication No. PCT / US14 / 56177). Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.

[0195] The binding protein may be conjugated to a radiosensitizer or photosensitizer. Radiosensitizers include, but are not limited to, ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleobase derivatives (e.g., halogenated purines or pyrimidines such as 5-fluorodeoxyuridine). Examples of photosensitizers include, but are not limited to, fluorescent molecules or beads that generate heat when irradiated, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, and the like. dyes), chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, Nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rose bengals), dimeric and oligomeric porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this approach allows for highly specific targeting of cells of interest (e.g., immune cells) using both a therapeutic agent (e.g., a drug) and electromagnetic energy (e.g., irradiation or light) simultaneously. In some embodiments, the binding protein is fused to, or covalently or non-covalently linked to, the agent, e.g., directly or via a linker.

[0196] In some embodiments, the binding proteins may be chemically modified. For example, the binding proteins may be mutated to alter peptide properties, such as detectability, stability, biodistribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc. N-methylation is one example of methylation that may occur in binding proteins encompassed by the present invention. In some embodiments, the binding proteins may be modified by methylation on free amines, such as by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0197] Chemical modifications may include polymers, polyethers, polyethylene glycol, biopolymers, amphoteric polymers, polyamino acids, fatty acids, dendrimers, Fc regions, monosaturated carbon chains such as palmitic acid or myristoleic acid, or albumin. Chemical modifications of binding protein Fc regions may be fusion Fc-proteins. Polyamino acids may include, for example, polyamino acid sequences with repeating single amino acids (e.g., polyglycine), and polyamino acid sequences with interleaved polyamino acid sequences that may or may not follow a pattern, or any combination of the above.

[0198] In some embodiments, binding proteins encompassed by the present invention may be modified. In some embodiments, the modifications produce functional variants that have substantial or significant sequence identity with the parent binding protein and that retain one or more biophysical and / or biological activities of the parent binding protein (e.g., retain pMHC binding specificity). In some embodiments, the mutations are conservative amino acid substitutions.

[0199] In some embodiments, binding proteins encompassed by the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, oc-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and oc-tert-butylglycine.

[0200] Binding proteins encompassed by the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted to acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0201] In some embodiments, attachment of a hydrophobic moiety, e.g., to the N-terminus, C-terminus, or internal amino acid, may be used to extend the half-life of peptides encompassed by the present invention. In other embodiments, the binding protein may contain post-translational modifications (e.g., methylation and / or amidation) that may affect, for example, serum half-life. In some embodiments, a simple carbon chain may be conjugated to the binding protein (e.g., by myristoylation and / or palmitylation). In some embodiments, the simple carbon chain may allow the binding protein to be easily separated from unconjugated substances. For example, methods that may be used to separate the binding protein from unconjugated substances include, but are not limited to, solvent extraction and reverse-phase chromatography. Lipid-soluble moieties may extend half-life via reversible binding to serum albumin. The conjugated moiety can be a lipid-soluble moiety that extends the half-life of the peptide through reversible binding to serum albumin. In some embodiments, the lipid-soluble moiety can be cholesterol or a cholesterol derivative, such as cholestenes, cholestanes, cholestadiene, and oxysterols. 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-modifying agent. Half-life-modifying agents include, but are not limited to, polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers containing glycine, glutamic acid, and serine, Fc regions, fatty acids, palmitic acid, or molecules that bind to albumin.In some embodiments, a spacer or linker may be coupled to the binding protein, such as one, two, three, four, or more amino acid residues that act as a spacer or linker to facilitate conjugation or fusion to another molecule and to facilitate cleavage of the peptide from such a conjugated or fused molecule. In some embodiments, the binding protein may be conjugated to other moieties that can, for example, modify or alter the properties of the binding protein.

[0202] Binding proteins may be produced recombinantly or synthetically, for example, by solid-phase peptide synthesis or solution-phase peptide synthesis. Polypeptide synthesis may be performed by known synthetic methods, for example, using fluorenylmethyloxycarbonyl (Fmoc) chemistry or by butyloxycarbonyl (Boc) chemistry. Polypeptide fragments may be linked together enzymatically or synthetically.

[0203] In aspects encompassed by the present invention, the present application provides a method for producing a binding protein described herein, the method comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of the binding protein; and (ii) recovering the expressed binding protein.

[0204] Useful methods for isolating and purifying recombinantly produced binding proteins may include, for example, obtaining a supernatant from a host cell / vector system suitable for secreting the binding protein into culture medium and then concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a suitable purification matrix or a series of suitable matrices, such as affinity matrices or ion exchange resins. One or more reverse-phase HPLC steps may be used to further purify the recombinant polypeptide. These purification methods may also be used when isolating immunogens from their natural environment. Large-scale production of one or more binding proteins described herein may involve batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the binding protein may be performed according to methods described herein and known in the art.

[0205] In any of the embodiments disclosed herein, the encoded binding protein is capable of binding to a peptide-MHC (pMHC) complex that includes the HA-2 immunogenic peptide in an MHC molecule (e.g., an MHC class I molecule). In some embodiments, the MHC molecule is of the HLA serotype HLA-A. * In some embodiments, the HLA allele is HLA-A. * 0201, HLA-A * 0202, HLA-A * 0203, HLA-A * 0205, HLA-A * 0206, and HLA-A * Selected from the group consisting of 207 alleles.

[0206] A variety of assays are known for assessing binding affinity and / or for determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand (e.g., a peptide antigen-MHC complex). It is within the level of ordinary skill in the art to determine the binding affinity of a binding protein for a target, e.g., a target polypeptide, e.g., a T cell peptide epitope, by, for example, using any of a number of binding assays known in the art. For example, in some embodiments, Biacore TM The device may be used to determine the binding constant of a complex between two proteins. The dissociation constant (K D ) may 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 immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs), or measuring binding by monitoring changes in the spectroscopic or optical properties of the proteins, for example, by fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blots, ELISAs, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore). TM ) analysis (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660, Wilson (2002) Science 295:2103, Wolff et al. (1993) Cancer Res. 53:2560, and U.S. 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 tetramers, e.g., by flow cytometry using labeled multimers, such as MHC-antigen tetramers. In one representative example, the apparent K of a binding protein is measured. Dis measured using two-fold dilutions of the labeled tetramer over a range of concentrations, followed by determination of the binding curve by non-linear regression. D is determined as the concentration of ligand that results in half-maximal binding.

[0207] III. Nucleic Acids and Vectors In aspects encompassed by the present invention, provided herein are nucleic acid molecules encoding the binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, CARs, etc.), peptides, and fragments thereof, described herein.

[0208] In some embodiments, the nucleic acid molecule hybridizes complementary to a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, e.g., over the entire length, to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1 under stringent conditions.

[0209] In some embodiments, the nucleic acid molecule hybridizes, under stringent conditions, to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1.

[0210] In some embodiments, the nucleic acid molecule comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1.

[0211] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α-chain CDR listed in Table 1. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR α-chain CDR listed in Table 1. α TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. α In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR α-chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR α-chain sequence set forth in Table 1.

[0212] In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β-chain CDR listed in Table 1. In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding at least one (e.g., one, two, or three) TCR β-chain CDR listed in Table 1. β TCR V having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the domain sequence. βIn some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR β-chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β-chain sequence set forth in Table 1.

[0213] The term "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA. It can be single-stranded or double-stranded, can be synthetic, or can be obtained from natural sources (e.g., isolated and / or purified), can contain natural, non-natural, or modified nucleotides, and can contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages in place of phosphodiester linkages found between nucleotides in unmodified oligonucleotides. In one embodiment, the nucleic acid comprises complementary DNA (cDNA).

[0214] In some embodiments, nucleic acids encompassed by the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside of a living cell by joining natural or synthetic nucleic acid segments to a nucleic acid molecule capable of replicating in a living cell, or (ii) a molecule resulting from replication of a molecule described in (i) above. For purposes of this application, such replication may be in vitro / ex vivo replication or in vivo replication.

[0215] The nucleic acids can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. See, e.g., Green and Sambrook et al., supra. For example, nucleic acids may be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that may be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids encompassed by the present invention can be purchased from a company such as, for example, Integrated DNA Technologies (Coralville, IA).

[0216] In one embodiment, the nucleic acid comprises a codon-optimized nucleotide sequence. Without being bound by any particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence increases the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence involves replacing a native codon with another codon that encodes the same amino acid but that may be translated by a more readily available tRNA in the cell, thus increasing translation efficiency. Optimizing a nucleotide sequence may also reduce secondary structures in the mRNA that may interfere with translation, thus increasing translation efficiency. In some embodiments, the nucleotide sequences described herein are codon-optimized for expression in a host cell (e.g., an immune cell such as a T cell).

[0217] The present invention also provides nucleic acids comprising a nucleotide sequence complementary to, or that hybridizes under stringent conditions to, the nucleotide sequence of any of the nucleic acids described herein.

[0218] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes to a target sequence (a nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions under which polynucleotides with exact complementary sequences or containing only scattered minor mismatches can be distinguished from random sequences that happen to match only a small region (e.g., 3-10 bases) to the nucleotide sequence. Such minor regions of complementarity melt more easily than full-length complements of 14-17 bases or more, and can be easily distinguished by high stringency hybridization. Relatively high stringency conditions may include, for example, low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70° C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the TCRs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0219] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids described in this application.

[0220] Typically, the nucleic acid is a DNA or RNA molecule, which may be contained in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0221] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which DNA or RNA sequences (e.g., foreign genes) can be introduced into a host cell, thereby transforming the host and promoting expression (e.g., transcription and translation) of the introduced sequences. Accordingly, a further object encompassed by the present invention relates to vectors containing nucleic acids encompassed by the present invention. In some embodiments, the vector is selected from the group of vectors set forth in Table 2.

[0222] Such vectors may contain regulatory elements, such as promoters, enhancers, terminators, etc., which may induce or direct expression of the polypeptide upon administration to a subject. Examples of promoters and enhancers used in animal cell expression vectors include the SV40 early promoter and enhancer (Mizukami T. et al. 1987), the Moloney murine leukemia virus LTR promoter and enhancer (Kuwana Y et al. 1987), and the immunoglobulin heavy chain promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983).

[0223] Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1 beta d2-4- (Miyaji H et al. 1990). Other representative examples of plasmids include replicative plasmids containing an origin of replication, or integrative plasmids such as pUC, pcDNA, and pBR. Representative examples of viral vectors include adenoviral, retroviral, lentiviral, herpesvirus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses are 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.

[0224] In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a binding protein or polypeptide, or fragment thereof, described herein. In some embodiments, the nucleic acid comprises regulatory elements necessary for expression of the open reading frame. Such elements include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. Enhancers may also be included. These elements may be operably linked to the sequence encoding the binding protein, polypeptide, or fragment thereof.

[0225] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8α and / or CD8β. In certain embodiments, the nucleic acid sequence encoding CD8α or CD8β is operably linked to a nucleic acid encoding a tag (e.g., a CD34 enrichment tag). In particular implementations, the nucleic acid sequence encoding CD8α and / or CD8β is interconnected with an internal ribosome entry site or with a nucleic acid sequence encoding a self-cleaving peptide, such as P2A, E2A, F2A, or T2A.

[0226] In some embodiments, the expression vectors provided in the present application comprise a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids set forth in Table 2.

[0227] Examples of promoters include, but are not limited to, promoters derived from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney Virus, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein-Barr Virus (EBV), and Rous Sarcoma Virus (RSV), as well as promoters derived 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, SV40 polyadenylation signals and LTR polyadenylation signals.

[0228] In addition to the regulatory elements required for expression, other elements may be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include promoters described herein. In some embodiments, enhancers / promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers, such as those from CMV, RSV, and EBV.

[0229] In some embodiments, the nucleic acid may be operably incorporated into a carrier or delivery vector, as further described below. Useful delivery vectors include, but are not limited to, biodegradable microcapsules, immuno-stimulating complexes (ISCOMs) or liposomes, and genetically engineered, attenuated biological carriers such as viruses or bacteria.

[0230] In some embodiments, the vector is a viral vector, such as a lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, fowlpox, AV-pox, modified vaccinia Ankara (MVA), and other recombinant viruses. For example, a lentiviral vector may be used to infect T cells.

[0231] In some embodiments, the recombinant expression vector can deliver the polynucleotide to a suitable host cell, e.g., a T cell or an antigen-presenting cell, i.e., a cell that presents peptide / MHC complexes on its cell surface (e.g., a dendritic cell) and lacks CD8. In some embodiments, the host cell is a hematopoietic progenitor cell or a cell of the human immune system. For example, the immune system cell can be a CD4 + T cells, CD8 +The T cells may be T cells, CD4 / CD8 double-negative T cells, gdT cells, natural killer cells, dendritic cells, or any combination thereof. In some embodiments, where the T cells are host, the T cells may be naive, central memory T cells, effector memory T cells, or any combination thereof. Accordingly, the recombinant expression vector may also include lymphoid tissue-specific transcriptional regulatory elements (TREs), such as TREs specific for B lymphocytes, T lymphocytes, or dendritic cells. Lymphoid tissue-specific TREs are known in the art (see, e.g., Thompson et al. (1992) Mol. Cell. Biol. 72:1043, Todd et al. (1993) J. Exp. Med. 777:1663, and Penix et al. (1993) J. Exp. Med. 775:1483).

[0232] In some embodiments, the recombinant expression vector comprises nucleotide sequences encoding a TCR alpha chain, a TCR beta chain, and / or a linker peptide. For example, in some embodiments, the recombinant expression vector comprises nucleotide sequences encoding the full-length TCR alpha and TCR beta chains of the binding protein and a linker positioned therebetween, wherein the nucleotide sequence encoding the beta chain is located 5' to the nucleotide sequence encoding the alpha chain. In some embodiments, the nucleotide sequences encode the full-length TCR alpha and TCR beta chains and a linker positioned therebetween, wherein the nucleotide sequence encoding the TCR beta chain is located 3' to the nucleotide sequence encoding the TCR alpha chain. In some embodiments, the full-length TCR alpha and / or TCR beta chains are replaced with fragments thereof.

[0233] As described further below, another aspect encompassed by the present invention relates to cells transfected, infected, or transformed with a nucleic acid and / or vector of the present invention. A host cell includes any individual cell or cell culture capable of receiving a vector or incorporating a nucleic acid and / or protein, as well as any progeny. The term also encompasses progeny of a host cell, whether genetically or phenotypically identical or different. Suitable host cells may depend on the vector, and include mammalian, animal, human, simian, insect, yeast, and bacterial cells. These cells can be induced to incorporate the vector or other material by transformation via viral vectors, calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory)). The term "transformation" refers to the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme, encoded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed."

[0234] Nucleic acids encompassed by the invention may be used to produce recombinant polypeptides encompassed by the invention in a suitable expression system. The term "expression system" refers, for example, to a host cell and a compatible vector under suitable conditions for expressing proteins encoded by foreign DNA carried by the vector and introduced into the host cell.

[0235] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") (Urlaub G et al. (1980)), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"), etc. In some embodiments, the YB2 / 0 cells are used because the ADCC activity of chimeric or humanized binding proteins is enhanced when expressed in these cells.

[0236] The present invention also encompasses methods for producing recombinant host cells expressing the binding proteins, peptides, and fragments thereof, comprising the steps of (i) introducing, in vitro or ex vivo, a recombinant nucleic acid or vector as described above into a competent host cell, (ii) culturing, in vitro or ex vivo, the resulting recombinant host cell, and (iii) optionally selecting cells expressing the binding protein, peptide, and fragments thereof. Such recombinant host cells can be used in diagnostic, prognostic, and / or therapeutic methods encompassed by the present invention.

[0237] In another aspect, the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment may be used to isolate, detect, and / or quantify nucleic acids that contain such polynucleotides. For example, polynucleotides encompassed by the present invention may be used to identify, isolate, or amplify partial or full-length clones in a stored library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated from, or otherwise complementary to, cDNAs derived from a human or mammalian nucleic acid library. In some embodiments, the cDNA library contains at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range therebetween, inclusive, e.g., at least about 80%-100%, of full-length sequences. The cDNA library is normalized to increase the representation of rare sequences. Low or medium stringency hybridization conditions are typically, but not exclusively, used with sequences that have low sequence identity relative to complementary sequences. Medium and high stringency conditions can optionally be used for sequences with higher identities. Low stringency conditions allow selective hybridization for sequences with about 70% sequence identity and may be used to identify orthologous or paralogous sequences. Optionally, polynucleotides encompassed by the invention encode at least a portion of a binding protein encoded by a polynucleotide described in this application.Polynucleotides encompassed by the present invention include nucleic acid sequences that can be used for selective hybridization to polynucleotides encoding binding proteins encompassed by the present invention (see, e.g., Ausubel, supra, and Colligan, supra).

[0238] IV. Host cells In aspects encompassed by the present invention, provided herein are host cells that express a 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) described herein. In some embodiments, the host cell comprises a nucleic acid or vector described herein.

[0239] In some embodiments, polynucleotides encoding the binding proteins are used to transform, transfect, or transduce host cells (e.g., T cells) for use in adoptive transfer therapy. Advances in nucleic acid sequencing and specific TCR sequencing have been described (e.g., Robins et al. (2009) Blood 114:4099; Robins et al. (2010) Sci. Translat. Med. 2:47ra64; Robins et al. (2011) J. Imm. Meth.; and Warren et al. (2011) Genome Res. 21:790) and may be used in the course of practicing embodiments encompassed by the present invention. Similarly, methods for transfecting or transducing T cells with a desired nucleic acid are known in the art (e.g., U.S. Patent Application Publication No. 2004 / 0087025), allowing for adoptive transfer methods using T cells of a desired antigen specificity (e.g., Schmitt et al. (2009) Hum. Gen. 20:1240, Dossett et al. (2009) Mol. Ther. 77:742, Till et al. (2008) Blood 772:2261, Wang et al. (2007) Hum. Gene Ther. 18:112, Kuball et al. (2007) Blood 709:2331, US Pat. Publ. 2011 / 0243972, US Pat. Publ. 2011 / 0189141, and Leen et al. (2007) Ann. Rev. Immunol. 25:243).

[0240] Any suitable immune cell may be modified to contain a heterologous polynucleotide encompassed by the present invention, such as a T cell, an NK cell, or an NK-T cell. In some embodiments, the cell may be a primary cell or a cell line. In some embodiments, the modified immune cell is a CD4 + T cells, CD8 +T cells, or both. For purposes of this application, the T cells can be any T cells, e.g., cultured T cells, e.g., primary T cells, or cells from a cultured T cell line, e.g., Jurkat, SupT1, etc., or T cells obtained from a mammal. If obtained from a mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are T cells isolated from a human. The T cells can be any type of T cell and at any developmental stage, including, but not limited to, cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (tumor infiltrating lymphocytes (TILs)), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc.

[0241] Any suitable method may be used to transfect or transduce the cells (e.g., T cells) or administer the nucleotide sequences or compositions encompassed by the methods described herein. Methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercially available products, such as in vivo-jetPEI®. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, etc. Still further methods of transfecting or transducing host cells use vectors, which are described in more detail herein.

[0242] The modified immune cells described herein may be functionally characterized using methodologies to assay T cell activity (e.g., measuring T cell binding, activation, or induction, and also measuring antigen-specific T cell responses, etc.). Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., 51 These include measuring T-cell function (by detecting Cr release from pre-loaded target cells), changes in T-cell phenotypic marker expression, and other measures of T-cell function.

[0243] Procedures for performing these and similar assays may 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, San Francisco, CA; Green and Reed (1998) Science 281:1309, and references cited therein.

[0244] In some embodiments, apparent affinity for a binding protein (e.g., a TCR or antigen-binding portion thereof) can be measured by assessing binding to various concentrations of MHC multimers. "MHC-peptide multimer staining" refers to an assay used to detect antigen-specific T cells, which in some embodiments features a tetramer of MHC molecules, each containing an identical peptide having an amino acid sequence that is homologous (e.g., identical or related) to at least one antigen (e.g., an HA-2 immunogenic peptide), where the complex can bind to a binding protein (e.g., a TCR or antigen-binding portion thereof) that recognizes the cognate antigen. Each of the MHC molecules may be tagged with a biotin molecule. The biotinylated MHC / peptide may be multimerized (e.g., tetramerized) by adding streptavidin (which may be fluorescently labeled).

[0245] The multimers can be detected by flow cytometry via fluorescent labeling. In some embodiments, pMHC multimer assays are used to detect or select binding proteins (e.g., TCRs or antigen-binding portions thereof) with enhanced affinity encompassed by the invention. In some instances, the apparent K D is measured using two-fold dilutions of labeled multimer at a range of concentrations, and then the apparent K is calculated by determining the binding curve by non-linear regression. D is determined as the concentration of ligand that results in half-maximal binding.

[0246] Cytokine levels may be determined using methods described herein, such as ELISA, ELISPOT, intracellular cytokine staining, and flow cytometry, and combinations thereof (e.g., intracellular cytokine staining and flow cytometry).

[0247] Immune cell proliferation and clonal expansion resulting from induction or stimulation of an antigen-specific immune response may be determined by isolating lymphocytes (e.g., circulating lymphocytes in a sample of peripheral blood cells or cells from lymph nodes, etc.), stimulating the cells with an antigen, and measuring cytokine production, cell proliferation, and / or cell viability (e.g., by tritiated thymidine incorporation or a non-radioactive assay such as an MTT assay, etc.). The effect of the immunogens described herein on the balance between Th1 and Th2 immune responses may be examined, for example, by determining the levels of Th1 cytokines (e.g., IFN-γ, IL-12, IL-2, and TNF-β, etc.) and type 2 cytokines (e.g., IL-4, IL-5, IL-9, IL-10, and IL-13, etc.).

[0248] Host cells encompassed by the present invention may contain a single polynucleotide encoding a binding protein described in this application, or the binding protein may be encoded by more than one polynucleotide. In other words, components or portions of a binding protein may be encoded by more than one polynucleotide, which may be contained on a single nucleic acid molecule or may be contained on more than one nucleic acid molecule.

[0249] In some embodiments, polynucleotides encoding two or more components or portions of a binding protein encompassed by the invention comprise two or more coding sequences operably linked in a single open reading frame. Advantageously, such an arrangement allows for coordinated expression of desired gene products (e.g., simultaneous expression of the alpha- and beta-chains of a TCR, resulting in production in an approximately 1:1 ratio). In some embodiments, two or more replaceable gene products of a binding protein encompassed by the invention (e.g., a TCR [e.g., an alpha- and beta-chain] or a CAR) are expressed as separate molecules and post-translationally associate. In further embodiments, two or more replaceable gene products of a binding protein encompassed by the invention are expressed as a single peptide with portions separated by a cleavable or removable segment. For example, self-cleaving peptides useful for expressing separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, porcine Teschovirus-1 2A (P2A) peptide, thoseaasigna virus 2A (T2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, and foot-and-mouth disease virus 2A (F2A) peptide.

[0250] In some embodiments, binding proteins encompassed by the invention comprise one or more linking amino acids. A "linking amino acid" or "linking amino acid residue" refers to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide (e.g., between a binding domain and an adjacent constant domain, or between a TCR chain and an adjacent self-cleaving peptide, etc.). Linking amino acids can result from the design of a construct encoding a fusion protein (e.g., amino acid residues resulting from the use of restriction enzyme sites during construction of a nucleic acid molecule encoding a fusion protein), or from cleavage of a self-cleaving peptide adjacent to one or more domains of an encoded binding protein encompassed by the invention (e.g., a P2A peptide located between the TCR a-chain and the TCR β-chain, whose self-cleavage can leave one or more linking amino acids in the a-chain, the TCR β-chain, or both).

[0251] Engineered immune cells encompassed by the present invention may be administered as a therapeutic agent for, for example, non-malignant disorders, hyperproliferative disorders, or recurrent hyperproliferative disorders characterized by expression of the HA-2 antigen. In some situations, it may be desirable to reduce or eliminate activity associated with cellular immunotherapy. Thus, in some embodiments, engineered immune cells encompassed by the present invention comprise heterologous polynucleotides encoding binding proteins and accessory proteins (e.g., safety switch proteins, etc.) that can be targeted with the same drugs or other compounds to selectively modulate (e.g., reduce or eliminate) the activity of such cells, if desired. Safety switch proteins that may be used in this regard include, for example, truncated EGF receptor polypeptides (huEGFRt) that lack the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retain the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux), caspase polypeptides (e.g., iCasp9; Straathof et al. (2005) Blood 105:4247-4254, Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Zhou and Brenner (2016) Hematol. pii:S0301-472X:30513-30516), RQR8 (Philip et al. (2014) Blood 124:1277-1287), and human c-myc protein tag (Kieback et al. (2008) Proc. Natl. Acad. Sci. USA 105:623-628).

[0252] Other accessory components useful for therapeutic cells include tags or selectable markers (e.g., CD34 enrichment tags) that allow the cells to be identified, sorted, isolated, enriched, or tracked. For example, marked immune cells with desired properties (e.g., antigen-specific TCR and safety switch proteins) can be sorted from unmarked cells in a sample, more efficiently activated, and expanded for inclusion in a therapeutic product of desired purity.

[0253] As used herein, the term "selection marker" includes a nucleic acid construct that confers an identifiable change to immune cells transduced with a polynucleotide containing the selection marker, allowing for detection and positive selection of the cells. For example, RQR is a selection marker that contains the major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, the polynucleotide encoding RQR contains a polynucleotide encoding a 16-amino acid CD34 minimal epitope. In some embodiments (e.g., certain embodiments provided in the Examples), the CD34 minimal epitope is incorporated into the amino-terminal position of the CD8 stalk domain (Q8). In further embodiments, the CD34 minimal binding site sequence can be combined with a target epitope of CD20 to form a compact marker / suicide gene for T cells (RQR8) (Philip et al. 2014). This construct allows for the selection of immune cells expressing the construct using, for example, a CD34-specific antibody (Miltenyi) coupled to magnetic beads, and the selective elimination of engineered T cells expressing the transgene using the clinically approved pharmaceutical antibody, rituximab (e.g., Philip et al. (2014) Blood 124:1277-1287; U.S. Patent Application Publication No. 2015-0093401; and U.S. Patent Application Publication No. 2018-0051089).

[0254] Further exemplary selection markers include several truncated type I transmembrane proteins not normally expressed on T cells: truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (e.g., Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Mavilio et al. (1994) Blood 83:1988-1997, and Fehse et al. (2000) Mol. Ther. 7:448-456). A particularly attractive feature of CD19 and CD34 is the availability of the Miltenyi CliniMACs™ selection system, which can target these markers for clinical-grade selection. However, CD19 and CD34 are relatively large surface proteins, which may impair vector packaging capacity and transcription efficiency of integrated vectors. Extracellular, non-signaling domain, or surface markers, including various proteins (e.g., CD19, CD34, LNGFR, etc.), may also be used. Any selectable marker may be used and should be acceptable to good manufacturing practices. In some embodiments, a selectable marker is co-expressed with a polynucleotide encoding a gene product of interest (e.g., a binding protein encompassed by the invention, such as a TCR or CAR, or an antigen-binding fragment thereof). Further examples of selectable markers include reporters such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In some embodiments, for example, cells express a selectable marker such as CD34, and the CD34 may be used to select, enrich, or isolate transduced cells of interest (e.g., by immunomagnetic selection) for use in the methods described herein.As used in this application, the CD34 marker is distinguished from an anti-CD34 antibody, or, for example, an scFv, TCR, or other antigen recognition moiety that binds to CD34.

[0255] In some embodiments, the selectable marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.

[0256] As background, CD4 + Inclusion of T cells in immunotherapy cell products allows for antigen-induced secretion of IL-2 and inhibits the expression of transferred cytotoxic CD8 + T cell persistence and function can be enhanced (e.g., Kennedy et al. (2008) Immunol. Rev. 222:129 and Nakanishi et al. Nature (2009) 52:510). In some embodiments, CD4 + Class I-restricted TCRs in T cells may require the introduction of a CD8 co-receptor. The CD4 co-receptor is structurally distinct from CD8 and cannot effectively replace the CD8 co-receptor (e.g., Stone & Kranz (2013) Front. Immunol. 4:244 and Cole et al. (2012) Immunology 737:139). Therefore, another accessory protein for use in the compositions and methods encompassed by the present invention includes the CD8 co-receptor or a component thereof. Engineered immune cells comprising a heterologous polynucleotide encoding a binding protein encompassed by the present invention may, in some embodiments, further comprise a heterologous polynucleotide encoding the CD8 co-receptor protein or a component of its beta-chain or alpha-chain.

[0257] The host cell may be efficiently transduced to contain and efficiently express a single polynucleotide encoding the binding protein, the safety switch protein, the selectable marker, and the CD8 co-receptor protein.

[0258] In one embodiment, a host cell encompassed by the invention further comprises a nucleic acid encoding a costimulatory molecule such that the engineered T cells express the costimulatory molecule. In some embodiments, the costimulatory domain is selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1, and PD1L.

[0259] In any of the foregoing embodiments, the host cell expressing the binding protein described herein can be a universal immune cell. A "universal immune cell" includes immune cells modified to reduce or eliminate expression of one or more endogenous genes encoding polypeptide products selected from PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA molecule, a TCR molecule, or any combination thereof. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins can downregulate the immune activity of the modified immune cell (e.g., PD-1, LAG-3, CTLA4, TIGIT) or interfere with the binding activity of heterologously expressed binding proteins encompassed by the invention (e.g., an endogenous TCR that binds to a non-HA-2 antigen and prevents the modified immune cell from binding to target cells expressing an HA-2 antigen (e.g., an HA-2 immunogenic peptide comprising the amino acid sequence YIGEVLVSV or YIGEVLVSM in its MHC molecule). Furthermore, endogenous proteins (e.g., immune cell proteins, e.g., HLA alleles, etc.) expressed on the donor immune cells may be recognized as foreign by the allogeneic host, which may result in the modified donor immune cells being eliminated or suppressed by the allogeneic host.

[0260] Thus, by reducing or eliminating the expression or activity of such endogenous genes or proteins, the activity, tolerance, or persistence of engineered immune cells can be improved in an autologous or allogeneic host environment, and the cells can be universally administered (e.g., to any recipient, regardless of HLA type). In some embodiments, the cells of the present invention are syngeneic, i.e., they are genetically identical or sufficiently identical and immunologically compatible, thereby enabling transplantation. In some embodiments, the universal immune cells are donor cells (e.g., allogeneic) or autologous cells. In some embodiments, modified immune cells (e.g., universal immune cells) encompassed by the invention comprise a chromosomal gene knockout for one or more genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g., a gene encoding alpha 1 macroglobulin, alpha 2 macroglobulin, alpha 3 macroglobulin, beta 1 microglobulin, or beta 2 microglobulin), or a TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region). (See, e.g., Torikai et al. (2016) Nature Sci. Rep. 6:21757; Torikai et al. (2012) Blood 179:5697; and Torikai et al. (2013) Blood 722:1341, which also provide representative and exemplary gene editing techniques, compositions, and adoptive cell therapies useful in accordance with the invention.)

[0261] As used herein, the term "chromosomal gene knockout" refers to a genetic modification or introduced inhibitory agent in a host cell that prevents (e.g., reduces, delays, suppresses, or eliminates) the production of a functionally active endogenous polypeptide product by the host cell. Modifications that result in a chromosomal gene knockout can include, for example, the introduction of nonsense mutations (e.g., introduction of a stop codon), missense mutations, gene deletions, and strand breaks, as well as the heterologous expression of an inhibitory nucleic acid molecule that inhibits endogenous gene expression in the host cell.

[0262] In some embodiments, chromosomal gene knockouts or gene knock-ins may be generated by chromosomal editing of a host cell. Chromosomal editing can be performed, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of phosphodiester bonds in a polynucleotide chain. In some embodiments, the endonuclease can cleave a target gene, thereby inactivating or "knocking out" the target gene. The endonuclease can be a natural, recombinant, genetically modified, or fusion endonuclease. Nucleic acid strand breaks caused by the endonuclease are generally repaired via different mechanisms: homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, donor nucleic acid molecules may be used to "knock in" a donor gene, to "knock out" a target gene, and, optionally, to inactivate a target gene via a donor gene knock-in event or a target gene knock-out event. NHEJ is an error-prone repair process that often results in a change in the DNA sequence at the break site (e.g., a substitution, deletion, or addition of at least one nucleotide). NHEJ may be used to "knock out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, megaTALs, etc.

[0263] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein containing a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain (e.g., Fok1 endonuclease). Each zinc finger motif of approximately 30 amino acids binds to approximately three base pairs of DNA, with amino acids at specific residues being varied to alter triplet sequence specificity (e.g., Desjarlais et al. (1993) Proc. Natl. Acad. Sci. 90:2256-2260 and Wolfe et al. (1999) J. Mol. Biol. 255:1917-1934). Multiple zinc finger motifs can be linked in tandem to create binding specificity for a desired DNA sequence (e.g., a region having a length ranging from about 9 to about 18 base pairs). By way of background, ZFNs mediate genome editing by catalyzing the creation of site-specific DNA double strand breaks (DSBs) in the genome, and targeted integration of transgenes containing flanking sequences homologous to the genome at the site of the DSB is facilitated by homology-directed repair. Alternatively, ZFN-generated DSBs can result in knockout of the target gene via repair by non-homologous end joining (NHEJ), an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the break site. In some embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, created using a ZFN molecule.

[0264] As used herein, "transcription activator-like effector nuclease" (TALEN) refers to a fusion protein containing a TALE DNA-binding domain and a DNA cleavage domain (e.g., Fok1 endonuclease). A "TALE DNA-binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each generally having a highly conserved 33-35 amino acid sequence with variable 12th and 13th amino acids. The TALE repeat domain is responsible for TALE binding to target DNA sequences. The variable amino acid residues (called repeat variable diresidues (RVDs)) correlate with specific nucleotide recognition. The natural (canonical) codes for DNA recognition of these TALEs have been determined, with the HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE causing the TALE to bind to cytosine (C), NG (asparagine-glycine) causing the TALE to bind to a T nucleotide, NI (asparagine-isoleucine) causing the TALE to bind to an A, NN (asparagine-asparagine) causing the TALE to bind to a G or A nucleotide, and NG (asparagine-glycine) causing the TALE to bind to a T nucleotide. Non-canonical (atypical) RVDs are also known in the art (e.g., U.S. Patent Application Publication No. 2011 / 0301073, the entire disclosure of which is incorporated herein by reference). TALENs may be used to direct site-specific double-strand breaks (DSBs) in the genome of T cells. Non-homologous end joining (NHEJ) ligates DNA on either side of a double-strand break where there is little or no sequence overlap to anneal, thereby introducing an error that knocks out gene expression.Alternatively, a transgene may be introduced at the site of the DSB (provided homologous flanking sequences present in the transgene) by homology directed repair. In some embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, and is generated using a TALEN molecule.

[0265] As used herein, the term "clustered regularly interspaced short palindromic repeats / Cas" (CRISPR / Cas) nuclease system refers to a system that uses a CRISPR RNA (crRNA)-guided Cas nuclease to recognize a target site (known as a protospacer) within the genome through base-pairing complementarity and then cleaves DNA if a short, conserved protospacer-associated motif (PAM) follows immediately 3' of the complementary target sequence. CRISPR / Cas systems are classified into three types (i.e., Type I, Type II, and Type III) based on the sequence and structure of the Cas nuclease. Type I and Type III crRNA-guided surveillance complexes require multiple Cas subunits. Type II systems (the most studied) contain at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA contains a duplex-forming region. The crRNA and tracrRNA form a duplex that can interact with the Cas9 nuclease and guide the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between a spacer on the crRNA and a protospacer on the target DNA upstream from the PAM. The Cas9 nuclease cleaves a double-strand break within the region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions, disrupting expression of the target locus. Alternatively, a transgene with homologous flanking sequences can be introduced at the site of the DSB via homology-directed repair.The crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (e.g., Jinek et al. (2012) Science 337:816-821). Furthermore, the region of the guide RNA complementary to the target site can be modified or programmed to target a desired sequence (Xie et al. (2014) PLOS One 9:el00448, U.S. Patent Application Publication No. US 2014 / 0068797, U.S. Patent Application Publication No. US 2014 / 0186843, U.S. Patent No. 8,697,359, and PCT Application Publication No. WO 2015 / 071474). In some embodiments, gene knockouts, including insertions, deletions, mutations, or combinations thereof, are generated using the CRISPR / Cas nuclease system.

[0266] Exemplary gRNA sequences and exemplary methods of using gRNA sequences to knock out endogenous genes encoding immune cell proteins include those described in Ren et al. (2017) Clin. Cancer Res. 23:2255-2266, which provides representative, exemplary gRNA, CAS9 DNA, vector, and gene knockout techniques.

[0267] As used herein, "meganuclease" (also referred to as "homing endonuclease") refers to an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, RI-Sce, I-ScelV, I-Csml, I-Panl, I-Scell, I-Ppol, I-SceIII, I-Crel, I-Tevl, I-TevII, and I-TevIII, and their recognition sequences are known (e.g., U.S. Patents 5,420,032 and 6,833,252; Belfort et al. (1997) Nucl. Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 52:115-118; Perler et al. (1994) Nucl. Acids Res. 22:1125-1127; Jasin (1996) Trends Genet. 72:224-228, Gimble et al. (1996) J. Mol. Biol. 263:163-180, and Argast et al. (1998) J. Mol. Biol. 280: 345-353).

[0268] In some embodiments, naturally occurring meganucleases may be used to facilitate site-specific genomic modification of a target of interest (e.g., an immune checkpoint, an HLA-encoding gene, or a TCR component-encoding gene, etc.).

[0269] In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genome modification (see, e.g., Porteus et al. (2005) Nat. Biotechnol. 23:967-73, Sussman et al. (2004) J. Mol. Biol. 342:31-41, Epinat et al. (2003) Nucl. Acids Res. 37:2952-2962, Chevalier et al. (2002) Mol. Cell 70:895-905, Ashworth et al. (2006) Nature 441:656-659, Paques et al. (2007) Curr. GeneTher. 7:49-66, as well as U.S. Patent Application Publications US 2007 / 0117128, US 2006 / 0206949, US (See US 2006 / 0153826, US 2006 / 0078552, and US 2004 / 0002092.) In a further embodiment, homing endonucleases in which the modular DNA-binding domain of TALENs has been engineered to generate fusion proteins known as megaTALs are used to generate chromosomal gene knockouts. megaTALs may be used not only to knock out one or more target genes, but also to introduce (knock in) heterologous or exogenous polynucleotides when used in conjunction with an exogenous donor template encoding a polypeptide of interest.

[0270] In some embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that binds (e.g., specifically and / or selectively) to the HA-2 antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor, and wherein the encoded target-specific inhibitor inhibits expression of an endogenous gene (i.e., an immune checkpoint, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.

[0271] Chromosomal gene knockout may be confirmed directly by DNA sequencing of the host immune cells following the knockout procedure or use of the agent.

[0272] A chromosomal gene knockout may also be inferred from the absence of gene expression following the knockout (eg, the absence of mRNA or polypeptide product encoded by the gene).

[0273] In some embodiments, host cell targeting encompassed by the present invention may involve specifically and / or selectively killing 50% or more of target cells containing peptide-MHC (pMHC) complexes that contain the HA-2 immunogenic peptide in the MHC molecule.

[0274] In some embodiments, the modified immune cells are capable of producing cytokines when contacted with target cells containing peptide-MHC (pMHC) complexes that include the HA-2 immunogenic peptide in the MHC molecule.

[0275] In some embodiments, the cytokine comprises IFN-γ or IL2, hi some embodiments, the cytokine comprises TNF-α.

[0276] In some embodiments, the host cell has a transcript per Million Transcripts (TPM) of about 1000 TPM or less, such as 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 10 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 less, inclusive of any range therebetween, for example, from about 1000 TPM or less to about 35 TPM or less, can produce higher levels of cytokines or cytotoxic molecules when contacted with target cells expressing HA-2. In some embodiments, low HA-2 expression levels are referred to as "heterozygous expression" and refer to between about 1 TPM and about 35 TPM, or any range therebetween, inclusive, such as 1-32 TPM. For example, the host cell may produce at least 1.2x, 1.5x, 1.8x, 2.0x, 2.2x, 2.5x, 2.8x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or more, or any range therebetween, inclusive, such as from 1.2x to 2x higher levels of a cytokine or cytotoxic molecule.

[0277] In some embodiments, the host cell is capable of specifically and / or selectively killing target cells that express HA-2 (e.g., hyperproliferative cells that express HA-2). In certain embodiments, the target cells express: (i) a polypeptide comprising or consisting of the amino acid sequence YIGEVLVSV or YIGEVLVSM; and (ii) a matched MHC molecule.

[0278] In some embodiments, the host cell does not express the HA-2 antigen, a serotype HLA-A that is not recognized by the binding protein of any one of claims 1 to 30. * Not 02 and / or HLA-A * 02 allele, e.g., HLA-A * 02:01, HLA-A * 0202, HLA-A * 0203, HLA-A * 0205, HLA-A * 0206 or HLA-A * For example, the patient may be HA-2 REF-negative (e.g., does not express the HA-2 SNP / SNP genotype and / or the HA2-REF), or may be HLA-A *Host cells may be received from a healthy donor who is 02:01-negative. Stem cells (e.g., hematopoietic stem cells) isolated from the donor (or engineered autologous cells) may be used as the source of transplant material. In parallel, T cells isolated from the same donor may be genetically modified to recognize HA-2, for example, by expressing an HA-2-binding protein described herein. Donor stem cells may be used to engraft a cell population (e.g., a reconstituted immune system) in the patient, and host cells may be injected into the patient to induce a highly specific anti-tumor effect. The engineered donor T cells may be designed to recognize and eliminate HA-2-expressing cells, such as all of the patient's natural blood cells, including cancer cells such as residual leukemia cells (which are HA-2-positive), thereby preventing recurrence and promoting a complete cure. The patient's new healthy blood cells are derived from the donor and are therefore HA-2-negative, HLA-A * 02 serotype negative and / or HLA-A * 02:01-negative, thus minimizing the toxic side effects of the engineered cells described herein. Such patient-matched host cells and therapeutic methods may be used in accordance with the therapeutic methods described further below.

[0279] In some embodiments, injury is determined by an injury assay, which is performed by co-culturing the host cells and target cells at a ratio of 20:1 to 0.625:1 (e.g., 15:1 to 1.25:1, 10:1 to 1.5:1, 8:1 to 3:1, 6:1 to 5:1, 20:1 to 5:1, 10:1 to 2.5:1, etc.). In some embodiments, the target cells are pulsed with 1 μg / mL to 50 pg / mL (e.g., 1 μg / mL to 10 ng / mL, 500 ng / mL to 0.5 ng / mL, 10 ng / mL to 10 pg / mL, 250 ng / mL to 1 ng / mL, 50 ng / mL to 5 ng / mL, 20 ng / mL to 10 ng / mL, etc.) of HA-2 peptide.

[0280] In some embodiments, the host cell has a transcript per Million Transcripts (TPM) of about 1000 TPM or less, such as 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 1000 TPM or less, or any range therebetween, inclusive, such as from about 1000 TPM or less to about 16 TPM or less, can injure more target cells when contacted with target cells having HA-2 levels of 1000 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 less, inclusive, such as from about 1000 TPM or less to about 16 TPM or less. In some embodiments, low HA-2 expression levels are referred to as "heterozygous expression" and refer to between about 1 TPM and about 35 TPM, or any range therebetween, inclusive, such as 1-32 TPM. For example, the host cells can kill at least 1.2x, 1.5x, 1.8x, 2.0x, 2.2x, 2.5x, 2.8x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, or more, or any range therebetween, inclusive, such as from 1.2x to 2x more target cells.

[0281] The present invention further provides a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population, comprising host cells comprising any of the described recombinant expression vectors, in addition to at least one other cell (e.g., a host cell (e.g., a T cell) that does not comprise any of the recombinant expression vectors, or a cell other than a T cell (e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc.)). Alternatively, the population of cells may be a substantially homogeneous population (in which the population primarily comprises (e.g., consists essentially of) host cells comprising the recombinant expression vector). The population may also be a clonal population of cells (in which all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise the recombinant expression vector). In one embodiment encompassed by the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0282] In embodiments encompassed by the present invention, the number of cells in the population may be rapidly expanded. Expansion of T cells may be achieved by a number of methods known in the art (e.g., U.S. Patents 8,034,334 and 8,383,099, U.S. Patent Application Publication No. 2012 / 0244133, Dudley et al. (2003) J. Immunother. 26:332-242, and Riddell et al. (1990) J. Immunol. Methods 128:189-201). For example, expansion of T cells may be performed by culturing T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0283] V. Pharmaceutical Compositions In another aspect encompassed by the present invention, the present application provides a pharmaceutical composition comprising a composition described herein (e.g., a binding protein, a nucleic acid, a cell, etc.) and a pharmaceutically acceptable carrier, diluent, or excipient.

[0284] The term "pharmaceutically acceptable" refers to agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0285] Medicaments and other compositions encompassed by the present invention may be specially formulated for administration in solid or liquid form, e.g., suitable for various routes of administration, such as (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, or pastes; (2) parenteral administration, e.g., as sterile solutions or suspensions, e.g., subcutaneous, intramuscular, or intravenous injection; (3) topical administration, e.g., as applied to the skin, creams, ointments, or sprays; (4) vaginal or rectal administration, e.g., as pessaries, creams, or foams; or (5) aerosols, e.g., as aqueous aerosols, liposomal formulations, or solid particles containing the compounds. Any suitable form factor for the medicaments or compositions described herein is contemplated, such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, or enemas.

[0286] Pharmaceutical compositions encompassed by the present invention may be provided as discrete dosage forms (e.g., capsules, sachets, or tablets, or liquids or aerosol sprays, each containing a predetermined amount of the active ingredient, as powders, or granules, solutions, aqueous or non-aqueous suspensions, oil-in-water emulsions, water-in-oil liquid emulsions, powders for reconstitution, powders for oral ingestion, bottles (e.g., powders or liquids in bottles), melt-in-the-mouth films, troches, pastes, tubes, gums, and packs). Such dosage forms may be prepared by any of the methods of pharmacy.

[0287] Suitable excipients include water, saline, dextrose, glycerol, etc., and combinations thereof. In some embodiments, the compositions comprising the host cells, binding proteins, or fusion proteins disclosed herein further comprise a suitable infusion medium. The suitable infusion medium may be any isotonic medium formulation, typically normal saline, Normosol™-R (Abbott), or Plasma-Lyte™ A (Baxter), 5% dextrose in water, or lactated Ringer's solution. The infusion medium may be supplemented with human serum albumin or other human serum components. Unit doses comprising an effective amount of host cells are also contemplated.

[0288] The present application also provides a unit dose comprising an effective amount of host cells or an effective amount of a composition comprising host cells. As described in the present application, host cells include immune cells, T cells (CD4 + T cells and / or CD8+ T cells), cytotoxic lymphocytes (e.g., cytotoxic T cells and / or natural killer (NK) cells), etc. For example, in some embodiments, a unit dose comprises a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% engineered cells, alone or in combination with other cells (e.g., comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% other cells). In some embodiments, undesirable cells are present in reduced amounts or are substantially absent (e.g., less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the population of cells in the composition, etc.).

[0289] The amount of cells in a composition or in a unit dose may be at least one cell (e.g., at least one modified CD8 + T cells, modified CD4+ T cells, and / or NK cells), or more typically, 10 2 More than 10 cells, e.g., up to 10 6 , up to 10 7 , up to 10 8 cells, up to 10 9 cells, or 10 10 In some embodiments, the cells are greater than about 10 to about 10 10 cells / m 2 range, e.g., about 10 5 from about 10 9 cells / m 2 The number of cells will depend on the end use for which the formulation is intended, as well as the type of cells contained therein. For example, cells modified to contain a binding protein specific for a particular antigen include cell populations containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of such cells. For uses provided herein, the cells will generally be in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In embodiments, the desired cell density is typically 10 4 cells / ml, and generally, 10 7 cells / ml, generally above 10 8 The cells may be administered as a single infusion or as multiple infusions over a range of time periods. Clinically relevant numbers of immune cells are administered cumulatively at doses of 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 In some embodiments, a unit dose of engineered immune cells may be co-administered with hematopoietic stem cells from an allogeneic donor (e.g., simultaneously or contemporaneously).

[0290] Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented), as determined by one skilled in the medical arts. The appropriate dose, as well as the suitable duration and frequency of administration of the composition, will be determined by factors such as the patient's health, the patient's size (i.e., weight, size, or body surface), the type and severity of the patient's condition, the particular form of the active ingredient, and the method of administration. Generally, an appropriate dose and treatment regimen will provide a sufficient amount of the composition(s) to provide a therapeutic and / or prophylactic benefit (e.g., improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or reduced symptom severity, as described herein).

[0291] An effective amount of a pharmaceutical composition, as used herein, refers to an amount sufficient, at dosages and for time periods required, to achieve a desired clinical result or beneficial treatment. An effective amount may be delivered in one or more administrations. When the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutically effective amount" may be used in reference to treatment, while a "prophylactically effective amount" may be used to describe administering an effective amount to a subject susceptible to or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative measure.

[0292] The pharmaceutical compositions described herein may be provided in unit-dose or multi-dose containers (e.g., sealed ampoules or vials). Such containers may be frozen to maintain the stability of the formulation until infusion into the patient. In some embodiments, a unit dose contains about 10 host cells described herein. 7 cells / m 2 from about 10 11 cells / m 2Suitable dosing and treatment regimens will be developed for use with the particular compositions described in this application in a variety of treatment regimens (e.g., parenteral or intravenous administration or formulation, etc.).

[0293] When the subject compositions are administered parenterally, they may also comprise sterile aqueous or oily solutions or suspensions. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol in a mixture with water. Aqueous solutions or suspensions may further contain one or more buffering agents (e.g., sodium acetate, sodium citrate, sodium borate, or sodium tartrate, etc.). Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts used. Additionally, active compounds may be incorporated into sustained-release preparations and formulations. Dosage unit form, as used in this application, refers to physically discrete units suited as unitary dosages for subjects to be treated; each unit may contain a predetermined quantity of engineered immune cells or active compound calculated to produce the desired effect, in association with a suitable pharmaceutical carrier.

[0294] In some embodiments, the described pharmaceutical compositions, when administered to a subject, are capable of eliciting an immune response against cells of interest that express HA-2. Such pharmaceutical compositions may be useful as vaccines for the prophylactic and / or therapeutic treatment of non-malignant disorders, hyperproliferative disorders, or recurrence of hyperproliferative disorders characterized by expression of the HA-2 antigen.

[0295] In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the use of the adjuvant increases the immunogenicity of the pharmaceutical composition. Such additional immune response-stimulating compounds or adjuvants can (i) be mixed with the pharmaceutical composition according to the invention after reconstitution of the peptide, optionally emulsified with an oil-based adjuvant as defined above, (ii) be part of the reconstituted composition encompassed by the invention as defined above, (iii) be physically linked to the peptide(s) to be reconstituted, or (iv) be administered separately to the subject, mammal or human, to be treated. The adjuvant can provide sustained release of the antigen (e.g., the adjuvant can be a liposome) or can be an adjuvant that is itself immunogenic and thereby functions synergistically with the antigen. For example, the adjuvant can be a known adjuvant or other substance that promotes antigen uptake, recruits immune system cells to the site of administration, or promotes immune activation of responding lymphoid cells. Adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, glucans, dextran sulfate, iron oxide, sodium alginate, bacto-adjuvants, synthetic polymers such as polyamino acids and amino acid co-polymers, saponins, paraffin oil, and muramyl dipeptides. In some embodiments, the adjuvant is Adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptides, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide TM ), N-acetyl-muramyl-L-alanyl-D-isoglutamine, pam3CSK4, quil A, trehalose dimycolate, or zymosan.

[0296] In some embodiments, the adjuvant is an immunomodulatory molecule, for example, the immunomodulatory molecule can be a recombinant protein cytokine, chemokine, or immunostimulatory agent, or a nucleic acid encoding a cytokine, chemokine, or immunostimulatory agent, designed to enhance the immune response.

[0297] Examples of immunomodulatory cytokines include interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, and IL-20), tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1 alpha, MIP-1β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as functional fragments of any of these.

[0298] In some embodiments, immunomodulatory chemokines that bind to chemokine receptors (ie, CXC, CC, C, or CX3C chemokine receptors) may also be included in the compositions provided herein. Examples of chemokines include, but are not limited to, Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, McP-1, McP-2, McP-3, McP-4, McP-5, Eotaxin, Tarc, Elc, I309, IL-8, Gcp-2, Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), as well as functional fragments of any of these.

[0299] In some embodiments, the composition comprises a 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), a TCRα and / or TCRβ polypeptide, described herein. In some embodiments, the composition comprises a nucleic acid encoding a binding protein, a TCRα and / or TCRβ polypeptide, described herein, e.g., a DNA molecule encoding a binding protein, a TCRα and / or TCRβ polypeptide. In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a binding protein, a TCRα and / or TCRβ polypeptide.

[0300] When taken up by a cell (e.g., a T cell, a NK cell, etc.), the DNA molecule may be present in the cell as an extrachromosomal molecule and / or may be integrated into a chromosome. DNA may be introduced into the cell in the form of a plasmid, which remains separate genetic material. Alternatively, linear DNA may be introduced into the cell, which can be integrated into a chromosome. Optionally, when introducing DNA into a cell, a reagent may be added that promotes integration of the DNA into a chromosome.

[0301] VI. Use and Methods The compositions described herein can be used in a variety of diagnostic, prognostic, and therapeutic applications. In any of the methods described herein (e.g., diagnostic, prognostic, or therapeutic methods, or a combination thereof, etc.), all steps of the method may be performed by a single actor or by two or more actors. For example, diagnosis may be performed directly by the actor providing the therapeutic treatment. Alternatively, the provider of the therapeutic agent may request that a diagnostic assay be performed. A diagnostician and / or interventionist may interpret the results of the diagnostic assay to determine a treatment strategy. Similarly, such alternative processes may be applied to other assays (e.g., prognostic assays, etc.).

[0302] Some uses and methods encompassed by the present invention utilize a subject or a subject sample. In some embodiments, the subject is an animal. The animal may be of either sex and at any stage of development. In some embodiments, the animal is a vertebrate (e.g., a mammal, etc.). In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a domesticated animal (e.g., a dog, cat, cow, pig, horse, sheep, or goat, etc.). In some embodiments, the subject is a companion animal (e.g., a dog or cat, etc.). In some embodiments, the subject is a livestock animal (e.g., a cow, pig, horse, sheep, or goat, etc.). In some embodiments, the subject is a zoo animal. In some embodiments, the subject is a research animal (e.g., a rodent (e.g., a mouse or rat), a dog, a pig, or a non-human primate, etc.). In some embodiments, the animal is a genetically engineered animal. In some embodiments, the animal is a transgenic animal (e.g., a transgenic mouse or a transgenic pig). In some embodiments, the subject is a fish or a reptile.

[0303] In some embodiments, the subject is a rodent (e.g., a mouse). In some such embodiments, the mouse is a transgenic mouse (e.g., a mouse expressing human MHC [i.e., HLA] molecules) (e.g., Nicholson et al. (2012) Adv. Hematol. 2012:404081). In some embodiments, the subject is a transgenic mouse expressing a human TCR or is an antigen-negative mouse (e.g., Li et al. (2010) Nat. Med. 16:1029-1034 and Obenaus et al. (2015) Nat. Biotechnol. 33:402-407). In some embodiments, the subject is a transgenic mouse expressing a human HLA molecule and a human TCR. In some embodiments, for example, when the subject is a genetically modified HLA mouse, the identified TCR is modified (e.g., chimeric or humanized). In some embodiments, the TCR scaffold is modified (eg, similar to known binding protein humanization methods).

[0304] In some embodiments, the subject is a human. In some embodiments, the subject is an animal model of a disease characterized by expression of HA-2 (e.g., a non-malignant disorder, a hyperproliferative disorder, or a recurrent hyperproliferative disorder characterized by expression of the HA-2 antigen). For example, the animal model can be an orthotopic xenograft animal model of a human-derived cancer.

[0305] In some embodiments, the subject is a human (eg, a human having a disorder characterized by expression of HA-2).

[0306] The methods described herein may be used to treat a subject in need thereof. As used herein, a "subject in need thereof" includes a subject having a disorder characterized by HA-2 expression, a subject having a recurrence of a disorder characterized by HA-2 expression, and...

Claims

1. Binding proteins, including: １） a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity to a TCR alpha chain CDR sequence selected from SEQ ID NO: 93 or selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 1; and / or b) a TCR beta chain CDR sequence having at least about 80% identity to a TCR beta chain CDR sequence selected from SEQ ID NO:92 or selected from the group consisting of the TCR beta chain CDR sequences listed in Table 1, wherein said binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity is about 5×10 －４ K below M ｄ having ２） a) a TCR alpha chain variable (V α ) domain sequence having at least about 80% identity to a TCR V α domain sequence selected from SEQ ID NO: 93 or selected from the group consisting of the TCR alpha chain variable (V α ) domain sequences listed in Table 1; and / or b) a TCR beta chain variable (V β ) domain sequence having at least about 80% identity to a TCR V β domain sequence selected from SEQ ID NO:92 or selected from the group consisting of the TCR V β ) domain sequences listed in Table 1, wherein said binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K d of about 5×10 −4 M or less; ３） a) a TCR alpha chain sequence having at least about 80% identity to a TCR alpha chain sequence selected from SEQ ID NO: 93 or selected from the group consisting of the TCR alpha chain sequences listed in Table 1; and / or b) a TCR beta chain sequence having at least about 80% identity to a TCR beta chain sequence selected from SEQ ID NO:92 or selected from the group consisting of the TCR beta chain sequences listed in Table 1, wherein said binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K d of about 5×10 −4 M or less; ４） a) a TCR alpha chain CDR sequence selected from SEQ ID NO: 93 or selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 1; and / or b) a TCR beta chain CDR sequence selected from SEQ ID NO:92 or selected from the group consisting of the TCR beta chain CDR sequences listed in Table 1, wherein said binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K d of about 5×10 −4 M or less; ５） a) a TCR alpha chain variable (V α ) domain sequence selected from SEQ ID NO: 93 or selected from the group consisting of the TCR alpha chain variable (V α ) domain sequences listed in Table 1; and / or b) a TCR beta chain variable (V β ) domain sequence selected from SEQ ID NO: 92 or selected from the group consisting of TCR V β ) domain sequences listed in Table 1, wherein said binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K d of about 5×10 −4 M or less; or ６） a) a TCR alpha chain sequence selected from SEQ ID NO: 93 or selected from the group consisting of the TCR alpha chain sequences listed in Table 1; and / or b) a TCR beta chain sequence selected from SEQ ID NO:92 or selected from the group consisting of the TCR beta chain sequences listed in Table 1, wherein said binding protein is capable of binding to an HA-2 immunogenic peptide-MHC (pMHC) complex, optionally wherein the binding affinity has a K d of about 5×10 −4 M or less.

2. 2. The binding protein of claim 1, wherein 1) The TCR alpha chain CDR, TCR V α The domain and / or the TCR alpha chain is encoded by a TRAV, TRAJ, and / or TRAC gene, or a fragment thereof, selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 1; 2) the TCR beta chain CDR, TCR V β domain, and / or TCR beta chain are 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 1; 3) each CDR of said binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof, compared to the cognate reference CDR sequence listed in Table 1; 4) the HA-2 immunogenic peptide comprises the amino acid sequence YIGEVLVSV or YIGEVLVSM; 5) the binding protein is chimeric, humanized, or human; 6) the binding protein is 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 where the binding domain comprises a transmembrane domain and an intracellular effector domain; 7) the TCR alpha chain and the TCR beta chain are covalently linked, optionally wherein the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide; 8) The TCR alpha chain and / or the TCR beta chain are covalently linked to a substructure, optionally wherein the covalently linked substructure is a) an affinity tag or label, optionally 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, Halo tag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or wherein the label is a fluorescent protein; and / or b) selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof; 9) the binding protein binds to the pMHC complex on the cell surface; 10) the MHC is an MHC multimer, optionally wherein the MHC multimer is a tetramer; 11) The MHC is an MHC class I molecule; 12) the MHC comprises an MHC alpha chain that is of HLA serotype HLA-A*02, optionally wherein the MHC alpha chain is encoded by an HLA allele selected from the group consisting of HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0205, HLA-A*0206, and HLA-A*207 alleles; 13) binding of the binding protein to the HA-2 peptide-MHC (pMHC) complex elicits an immune response, optionally wherein the immune response is a T cell response, optionally wherein the T cell response is selected from the group consisting of T cell proliferation, cytokine release, and / or cytotoxicity, and / or the binding protein binds to the HA-2 immunogenic peptide-MHC (pMHC) complex at about 1x10-4 M or less, about 5x10-5 M or less, about 1x10-5 M or less, about 5x10-6 M or less, about 1x10-6 M or less, about 5x10-7 M or less, about 1x10-7 M or less, about 5x10-8 M or less, about 1x10-8 M or less, about 5x10-9 M or less, about 1 ... specifically and / or selectively bind with a K d of about 1×10 −10 M or less, about 5×10 −10 M or less, about 1×10 −10 M or less, about 5×10 −11 M or less, about 1×10 −11 M or less, about 5×10 −12 M or less, or about 1×10 −12 M or less; 14) the binding protein has a higher binding affinity for the peptide-MHC (pMHC) than known T-cell receptors, optionally the binding protein has at least a 1.05-fold higher binding affinity for the peptide-MHC (pMHC) than known T-cell receptors; and / or 15) the binding protein, when contacted with a target cell heterozygously expressing HA-2, induces higher T cell proliferation, cytokine release, and / or cytotoxicity than known T cell receptors; optionally, the binding protein, when contacted with a target cell heterozygously expressing HA-2, induces at least 1.05-fold increased T cell proliferation, cytokine release, and / or cytotoxicity than known T cell receptors; further optionally, the target cell is a) a DEL, THP-1 or TF1 cell line; or b) a cancer cell; optionally, the cancer is a hematological malignancy; further, the hematological malignancy is a leukemia, lymphoma, myelodysplastic disorder, myeloproliferative disorder, or myeloma.

3. A TCR alpha chain and / or a TCR beta chain selected from the group consisting of the TCR alpha chain sequences and the TCR beta chain sequences listed in Table 1.

4. An isolated nucleic acid molecule which 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 1, or to a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1. 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 1, and / or 2) A nucleic acid comprising a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 1, optionally wherein the nucleic acid is codon-optimized for expression in a host cell.

5. A vector comprising the isolated nucleic acid of claim 4, optionally comprising: a) the vector is a cloning vector, an expression vector, or a viral vector; b) the vector further comprises a nucleic acid sequence encoding CD8α and / or CD8β, optionally the nucleic acid sequence encoding CD8α or CD8β is operably linked to a nucleic acid encoding a tag, optionally the nucleic acid encoding the tag is 5′ upstream of the nucleic acid sequence encoding CD8α or CD8β, such that the tag is fused at the N-terminus of CD8α or CD8β, further optionally, the tag is a CD34 enrichment tag; and / or c) the isolated nucleic acid and the nucleic acid sequence encoding CD8α and / or CD8β are interconnected with an internal ribosome entry site or with a nucleic acid sequence encoding a self-cleaving peptide, optionally the self-cleaving peptide is P2A, E2A, F2A, or T2A.

6. A host cell comprising the isolated nucleic acid of claim 4, comprising the vector of claim 5, and / or expressing the binding protein of claim 1 or 2, optionally wherein the cell is genetically engineered.

7. 7. The host cell of claim 6, wherein the host cell comprises: 1) Includes chromosomal gene knockouts of TCR genes, HLA genes, or both; 2) knockout of an HLA gene selected from the group consisting of the α1 macroglobulin gene, the α2 macroglobulin gene, the α3 macroglobulin gene, the β1 microglobulin gene, the β2 microglobulin gene, and combinations thereof; 3) knockout of a TCR gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and a combination thereof; 4) expressing CD8α and / or CD8β, optionally wherein the CD8α and / or CD8β are fused to a CD34 enrichment tag, and further optionally, the host cells have been enriched using the CD34 enrichment tag; 5) hematopoietic progenitor cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells, or immune cells, optionally the immune cells are cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + T cells, CD8 + T cells, CD4 / CD8 double negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or combinations thereof, optionally the T cells are a) naive T cells, central memory T cells, effector memory T cells, or a combination thereof; b) a primary T cell, or a cell of a T cell line; and / or c) does not express an endogenous TCR or has lower cell surface expression of an endogenous TCR; and / or 6) capable of producing cytokines or cytotoxic molecules when contacted with a target cell that contains a peptide-MHC (pMHC) complex that contains the HA-2 peptide epitope in the MHC molecule; and optionally a) the host cell is contacted with the target cell in vitro, ex vivo, or in vivo; b) the cytokine is TNF-α, IL-2, and / or IFN-γ; c) the cytotoxic molecules are perforins and / or granzymes, optionally wherein the cytotoxic molecule is granzyme B; d) the host cell is capable of producing higher levels of a cytokine or cytotoxic molecule when contacted with a target cell that heterozygously expresses HA-2, optionally the host cell is capable of producing at least 1.05-fold higher levels of a cytokine or cytotoxic molecule; e) the host cell is capable of killing a target cell that contains a peptide-MHC (pMHC) complex that contains an HA-2 peptide epitope in an MHC molecule; and optionally i) the injury is measured by an injury assay; ii) the ratio of host cells to target cells in the killing assay is between 20:1 and 0.625:1; iii) the target cells are T2 cells pulsed with 1 μg / mL to 50 pg / mL of HA-2 peptide; iv) the host cell, when contacted with target cells that heterozygously express HA-2, is capable of killing a greater number of target cells, optionally the host cell is capable of killing at least 1.05-fold greater number of target cells; and / or v) the target cell is a DEL, THP-1 or TF-1 cell line; f) the HA-2 immunogenic peptide comprises the amino acid sequence YIGEVLVSV or YIGEVLVSM; g) the MHC molecule is an MHC class I molecule; h) said MHC molecule comprises an MHC alpha chain that is of the HLA serotype HLA-A*02; i) said MHC alpha chain is encoded by an HLA allele 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; j) the target cell is a cell line selected from the group consisting of Del, THP-1, and TF-1 cell lines, is a hematopoietic cancer cell expressing HA-2, or is not NB4, optionally the cancer cell is a cell of a hematological malignancy selected from the group consisting of leukemia, lymphoma, myelodysplastic disorder, myeloproliferative neoplasm, or myeloma; and / or k) the host cell does not express the HA-2 antigen, which is not recognized by the binding protein of claim 1 or 2, and / or does not express the serotype HLA-A*02 allele.

8. A population of host cells according to claim 6 or 7.

9. A composition comprising: a) a binding protein according to claim 1 or 2, b) an isolated nucleic acid according to claim 4; c) the vector according to claim 5 ; d) a host cell according to claim 6 or 7, and / or e) a population of host cells according to claim 8; As well as carriers.

10. A device or kit that includes: a) a binding protein according to claim 1 or 2, b) an isolated nucleic acid according to claim 4; c) the vector according to claim 5 ; d) a host cell according to claim 6 or 7, and / or e) a population of host cells according to claim 8; wherein the device or kit optionally comprises reagents for detecting binding of a), d) and / or e) to a pMHC complex.

11. 3. A method for producing a binding protein according to claim 1 or 2, wherein the method comprises the steps of: (i) culturing a transformed host cell, transformed with a nucleic acid comprising a sequence encoding a binding protein according to claim 1 or 2, under suitable conditions allowing the binding protein to be expressed; and (ii) recovering the expressed binding protein.

12. 3. A method for producing a host cell expressing a binding protein according to claim 1 or 2, wherein the method comprises the steps of: (i) introducing into said host cell a nucleic acid comprising a sequence encoding a binding protein according to claim 1 or 2; (ii) culturing the transformed host cell under suitable conditions to allow the binding protein to be expressed.

13. 1. A method for detecting the presence or absence of HA-2 antigen and / or cells expressing HA-2, optionally wherein the cells are hyperproliferative cells, the method comprising detecting the presence or absence of the HA-2 antigen in a sample by using at least one binding protein according to claim 1 or 2, or at least one host cell according to claim 6 or 7, wherein detecting the HA-2 antigen is an indication of the presence of HA-2 antigen and / or cells expressing HA-2, optionally wherein the at least one binding protein or the at least one host cell forms a complex with the HA-2 peptide in an MHC molecule, and the complex is sorted by fluorescence activated cell sorting. The detection may be in the form of a fluorescence in situ hybridization (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay.

14. 14. The method of claim 13, wherein the method comprises: 1) obtaining said sample from a subject, optionally wherein said subject is an animal model of a disorder characterized by expression of HA-2, and / or a mammal, optionally wherein said mammal is a human, a primate, or a rodent; and / or 2) Identifying cells expressing HA-2 by bone marrow biopsy Further includes:

15. 1. A method for detecting the level of a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder, characterized by expression of HA-2 antigen in a subject, comprising: a) contacting a sample obtained from the subject with at least one binding protein of claim 1 or 2, at least one host cell of claim 6 or 7, or a population of host cells of claim 8; and b) detecting a level of responsiveness, wherein a higher level of responsiveness compared to a control level indicates a level of a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen in the subject. Optionally, i) the control level is A) It is a reference figure, or B) in subjects free of said non-malignant disorder, hyperproliferative disorder, or recurrence of said hyperproliferative disorder, characterized by expression of the HA-2 antigen; ii) the level of responsiveness is A) the presence of a bond, and / or B) T cell activation and / or effector function, optionally as indicated by: 1) the T cell activation or effector function results in T cell proliferation, killing, or cytokine release; and / or 2) detecting the T cell binding, activation, and / or effector function using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay; iii) the hyperproliferative disorder comprises a hematological malignancy, optionally wherein the hematological malignancy comprises a leukemia, lymphoma, myelodysplastic disorder, myeloproliferative neoplasm, or myeloma, optionally wherein the hematological malignancy comprises A) leukemia, optionally said leukemia including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), mixed phenotypic acute leukemia (mixed phenotype acute leukemia (MPAL)), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia (prolymphocytic leukemia), hairy cell leukemia (hairy cell leukemia) cell leukemia), or chronic lymphocytic leukemia (chronic lymphocytic leukemia (CLL)), B) lymphoma, optionally including Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma, lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt's lymphoma, or C) myelodysplastic disorder (MDS), optionally including refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts-Thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (refractory cytopenia with ring sideroblasts-Thrombocytosis (RARS-t) ... selected from: multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplastic syndrome, refractory cytopenia of childhood, myelodysplastic syndrome with isolated del(5q) of the long arm of chromosome 5; iv) the non-malignant disorder is A) an immunodeficiency disorder, optionally the immunodeficiency disorder is selected from the group consisting of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Omenn syndrome, X-linked lymphoproliferative syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, DiGeorge syndrome, and an indication for hematopoietic stem cell Transplantation (HCT); B) a non-malignant hematological disorder, optionally wherein said non-malignant hematological disorder is selected from the group consisting of sickle cell anemia, thalassemia, aplastic anemia, hemophagocytic lymphohistiocytosis (HLH), severe aplastic anemia, bone marrow failure syndromes, Fanconi anemia, Diamond-Blackfan anemia, and Schwachman Diamond syndrome; or v) is an autoimmune disorder, optionally wherein the autoimmune disorder is systemic sclerosis or multiple sclerosis; and / or vi) said subject is an animal model of a disorder characterized by expression of HA-2, and / or said mammal, optionally said mammal is a human, a primate, or a rodent.

16. 1. A method for monitoring the progression of a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder, characterized by expression of HA-2 antigen in a subject, said method comprising: a) detecting the level of an HA-2 antigen according to any one of claims 13 to 15, or the level of a cell of interest expressing HA-2, in a sample from a subject at a first time point; b) repeating step a) at a subsequent time point; and c) comparing the levels of HA-2 antigen or the levels of cells of interest expressing HA-2 detected in step a) and step b) to monitor the progression of a non-malignant disorder, hyperproliferative disorder, or recurrence of a hyperproliferative disorder in said subject, characterized by expression of the HA-2 antigen, wherein an absent or reduced level of HA-2 antigen or of cells of interest expressing HA-2 detected in step b) compared to step a) indicates that the progression of a non-malignant disorder, hyperproliferative disorder, or recurrence of a hyperproliferative disorder in said subject, characterized by expression of the HA-2 antigen, has been inhibited; and optionally i) between said first time point and said subsequent time point, said subject has undergone a treatment to treat said non-malignant disorder, said hyperproliferative disorder, or a recurrence of said hyperproliferative disorder, characterized by expression of HA-2 antigen; ii) the level of the HA-2 antigen or the level of the cells of interest expressing HA-2 A) the presence of a bond, and / or B) T cell activation and / or effector function, optionally as indicated by 1) the T cell activation or effector function results in T cell proliferation, killing, or cytokine release; and / or 2) detecting the T cell binding, activation, and / or effector function using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay; iii) the hyperproliferative disorder comprises a hematological malignancy, optionally wherein the hematological malignancy comprises a leukemia, lymphoma, myelodysplastic disorder, myeloproliferative neoplasm, or myeloma, optionally wherein the hematological malignancy comprises A) leukemia, optionally said leukemia comprising acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), mixed phenotypic acute leukemia (mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia (hairy cell leukemia) cell leukemia), or chronic lymphocytic leukemia (chronic lymphocytic leukemia (CLL)), B) lymphoma, optionally including Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma, lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt's lymphoma, or C) myelodysplastic disorder (MDS), optionally including refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts-Thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (refractory cytopenia with ring sideroblasts-Thrombocytosis (RARS-t) ... selected from: multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplastic syndrome, refractory cytopenia of childhood, myelodysplastic syndrome with isolated del(5q) of the long arm of chromosome 5; iv) the non-malignant disorder is A) an immunodeficiency disorder, optionally the immunodeficiency disorder is selected from the group consisting of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Omenn syndrome, X-linked lymphoproliferative syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, DiGeorge syndrome, and an indication for hematopoietic stem cell Transplantation (HCT); B) a non-malignant hematological disorder, optionally wherein said non-malignant hematological disorder is selected from the group consisting of sickle cell anemia, thalassemia, aplastic anemia, hemophagocytic lymphohistiocytosis (HLH), severe aplastic anemia, bone marrow failure syndromes, Fanconi anemia, Diamond-Blackfan anemia, and Schwachman Diamond syndrome; or C) an autoimmune disorder, optionally wherein the autoimmune disorder is systemic sclerosis or multiple sclerosis; and / or v) said subject is an animal model of a disorder characterized by expression of HA-2, and / or said mammal, optionally said mammal is a human, a primate, or a rodent.

17. A method for assessing the efficacy of a therapy for a non-malignant, hyperproliferative, or recurrent hyperproliferative disorder characterized by expression of the HA-2 antigen, including: a) determining in a first sample obtained from a subject the presence or level of responsiveness between a sample obtained from the subject and at least one binding protein of claim 1 or 2, at least one host cell of claim 6 or 7, or a population of host cells of claim 8, prior to providing to the subject at least part of a therapy for a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of an HA-2 antigen; and b) determining the presence or level of responsiveness between the sample obtained from the subject and at least one binding protein of claim 1 or 2, at least one host cell of claim 6 or 7, or a population of host cells of claim 8, in a second sample obtained from the subject after providing at least part of a therapy for a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder characterized by expression of the HA-2 antigen; wherein the absence or decreased level of responsiveness in the second sample compared to the first sample indicates that the therapy is effective for treating a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder, characterized by expression of HA-2 antigen in the subject. i) the level of responsiveness is A) the presence of a bond; and / or B) T cell activation and / or effector function; Optionally, 1) the T cell activation or effector function results in T cell proliferation, killing, or cytokine release; and / or 2) detecting the T cell binding, activation, and / or effector function using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay; ii) the hyperproliferative disorder comprises a hematological malignancy, optionally wherein the hematological malignancy comprises a leukemia, lymphoma, myelodysplastic disorder, myeloproliferative neoplasm, or myeloma, optionally wherein the hematological malignancy comprises A) leukemia, optionally said leukemia comprising acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), mixed phenotypic acute leukemia (mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia (hairy cell leukemia) cell leukemia), or chronic lymphocytic leukemia (chronic lymphocytic leukemia (CLL)), B) lymphoma, optionally including Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma, lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt's lymphoma, or C) myelodysplastic disorder (MDS), optionally including refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts-Thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (refractory cytopenia with ring sideroblasts-Thrombocytosis (RARS-t) ... selected from: multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplastic syndrome, refractory cytopenia of childhood, myelodysplastic syndrome with isolated del(5q) of the long arm of chromosome 5; iii) the non-malignant disorder is A) an immunodeficiency disorder, optionally the immunodeficiency disorder is selected from the group consisting of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Omenn syndrome, X-linked lymphoproliferative syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, DiGeorge syndrome, and an indication for hematopoietic stem cell Transplantation (HCT); B) a non-malignant hematological disorder, optionally wherein said non-malignant hematological disorder is selected from the group consisting of sickle cell anemia, thalassemia, aplastic anemia, hemophagocytic lymphohistiocytosis (HLH), severe aplastic anemia, bone marrow failure syndromes, Fanconi anemia, Diamond-Blackfan anemia, and Schwachman Diamond syndrome; or C) an autoimmune disorder, optionally wherein the autoimmune disorder is systemic sclerosis or multiple sclerosis; and / or iv) the subject is an animal model of a disorder characterized by expression of HA-2, and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent.

18. 1. A composition for use in a method of preventing and / or treating a non-malignant disorder, a hyperproliferative disorder, or the recurrence of a hyperproliferative disorder, characterized by expression of the HA-2 antigen in a subject, wherein the composition comprises a cell expressing at least one binding protein according to claim 1 or 2, and optionally 1) the cells are allogeneic, syngeneic, or autologous; 2) the cell is genetically engineered; 3) the cell comprises a chromosomal knockout of a TCR gene, of an HLA gene, or of both a TCR gene and an HLA gene; 4) the 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; 5) The cell comprises a knockout of a TCR gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and a combination thereof. 6) the cells express CD8α and / or CD8β, optionally, where the CD8α and / or CD8β are fused to a CD34 enrichment tag, and further optionally, the cells have been enriched using a CD34 enrichment tag; 7) The cells are hematopoietic progenitor cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells, or immune cells; Optionally, the immune cells are cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + T cells, CD8 + T cells, CD4 / CD8 double negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells, or combinations thereof; Optionally, the T cells are a) naive T cells, central memory T cells, effector memory T cells, or a combination thereof; b) primary T cells, or cells of a T cell line; and / or c) does not express an endogenous TCR or has lower cell surface expression of an endogenous TCR; 8) the cells are capable of producing cytokines or cytotoxic molecules when contacted with a target cell that contains a peptide-MHC (pMHC) complex that contains HA-2 among its MHC molecules; and optionally, a) the cytokine is TNF-α, IL-2, and / or IFN-γ; and / or b) the cytotoxic molecules are perforins and / or granzymes, optionally wherein the cytotoxic molecule is granzyme B; 9) the cells are capable of producing higher levels of cytokines or cytotoxic molecules when contacted with a target cell that heterozygously expresses HA-2, optionally the cells are capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules; 10) the host cell is capable of killing a target cell that contains a peptide-MHC (pMHC) complex that contains HA-2 among its MHC molecules; 11) the host cell, when contacted with a target cell that heterozygously expresses HA-2, is capable of killing a greater number of target cells, optionally the host cell is capable of killing at least 1.05-fold more target cells; 12) The HA-2 immunogenic peptide comprises the amino acid sequence YIGEVLVSV or YIGEVLVSM; 13) The MHC molecule is an MHC class I molecule; 14) The MHC molecule comprises an MHC alpha chain that is of the HLA serotype HLA-A*02; 15) 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; 16) The target cell is a non-malignant or hyperproliferative cell that expresses the HA-2 antigen in the subject; 17) the composition further comprises a pharma- ceutically acceptable carrier; 18) the composition induces an immune response against the non-malignant or hyperproliferative cells expressing the HA-2 antigen in the subject, optionally wherein the composition induces an antigen-specific T cell immune response against the non-malignant or hyperproliferative cells expressing the HA-2 antigen in the subject, optionally 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; 19) The hyperproliferative disorder comprises a hematological malignancy, optionally comprising a leukemia, lymphoma, myelodysplastic disorder, myeloproliferative neoplasm, or myeloma; Optionally, the hematological malignancy is: a) leukemia, optionally said leukemia is acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), mixed phenotypic acute leukemia (mixed phenotype acute leukemia (MPAL)), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia (prolymphocytic leukemia), hairy cell leukemia (hairy cell leukemia) cell leukemia), or chronic lymphocytic leukemia (chronic lymphocytic leukemia (CLL)), b) lymphoma, optionally including Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma, lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt's lymphoma, or c) myelodysplastic disorder (MDS), optionally including refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts -Thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (refractory cytopenia with ring sideroblasts -Thrombocytosis (RARS-t) ... selected from: multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplastic syndrome, refractory cytopenia of childhood, myelodysplastic syndrome with isolated del(5q); 20) The non-malignant disorder is a) an immunodeficiency disorder, optionally the immunodeficiency disorder is selected from the group consisting of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Omenn syndrome, X-linked lymphoproliferative syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, DiGeorge syndrome, and an indication for hematopoietic stem cell Transplantation (HCT); b) a non-malignant hematological disorder, optionally wherein said non-malignant hematological disorder is selected from the group consisting of sickle cell anemia, thalassemia, aplastic anemia, hemophagocytic lymphohistiocytosis (HLH), severe aplastic anemia, bone marrow failure syndromes, Fanconi anemia, Diamond-Blackfan anemia, and Schwachman Diamond syndrome, or c) an autoimmune disorder, optionally wherein the autoimmune disorder is systemic sclerosis or multiple sclerosis; 21) The subject is undergoing or has previously undergone hematopoietic cell transplantation (HCT), optionally where the HCT comprises cells that do not express the HA-2 antigen, are not recognized by the binding protein of claim 1 or 2, are not cells of serotype HLA-A*02, and / or do not express the HLA-A*02:01 allele, optionally where the HCT comprises donor hematopoietic cells that comprise a chromosomal knockout of a gene encoding an HLA component, a chromosomal knockout of a gene encoding a TCR component, or both; 22) the subject has previously undergone lymphodepleting chemotherapy, optionally, the lymphodepleting chemotherapy comprising cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof; 23) the method further comprises the step of prescribing to the subject at least one additional treatment for a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder, optionally the at least one additional treatment for a non-malignant disorder, a hyperproliferative disorder, or a recurrence of a hyperproliferative disorder is prescribed simultaneously or sequentially with the composition; and / or 24) The subject is an animal model of a disorder characterized by expression of HA-2, and / or the subject is a mammal, optionally wherein the mammal is a human, a primate, or a rodent.

19. an expression vector comprising a promoter operably linked to a nucleic acid sequence encoding CD8α and / or CD8β, optionally 1) the nucleic acid sequence encoding said CD8α or CD8β is operably linked to a nucleic acid encoding a tag, such that said tag is fused to said CD8α or CD8β; 2) the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding the CD8α or CD8β, such that the tag is fused at the N-terminus of CD8α or CD8β; 3) the tag is a CD34 enrichment tag; 4) the vector further comprises a nucleic acid sequence encoding TCRα and / or TCRβ; 5) the TCR alpha and / or the TCR beta comprises a transmembrane domain having a mutation and / or a constant domain having a mutation; 6) the mutated transmembrane domain and / or the mutated constant domain enhance cell surface expression of TCRα and / or TCRβ while reducing endogenous expression of TCRα and / or TCRβ; 7) the nucleic acid sequence encoding CD8α and / or CD8β, and the nucleic acid sequence encoding TCRα and / or TCRβ, are interconnected with an internal ribosome entry site or with a nucleic acid sequence encoding a self-cleaving peptide; 8) The self-cleaving peptide is P2A, E2A, F2A, or T2A; 9) the vector further comprises a nucleic acid sequence encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1, or a sequence having at least about 80% homology to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1, optionally wherein the isolated nucleic acid molecule comprises: a) a TRAV, TRAJ, and / or TRAC gene, or a fragment thereof, selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 1; and / or b) a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 1; and / or 10) The vector has a nucleic acid sequence as set forth in Table 2.

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