Binding protein that recognizes HPV16 E7 antigen and uses thereof

Abstract

Provided herein are binding proteins that recognize the HPV16 E7 antigen, and uses thereof. In one aspect, a binding protein is provided that comprises: a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity with a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha α chain CDR sequences listed in Table 1, and / or b) a TCR beta chain CDR sequence having at least about 80% identity with a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 1, wherein the binding protein is a binding protein that recognizes the HPV16 E7 11-19 capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity K d is about 5×10 -4 It is below M.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 342,479, filed May 16, 2022, U.S. Provisional Application No. 63 / 317,326, filed March 7, 2022, and U.S. Provisional Application No. 63 / 277,901, filed November 10, 2021, the entire contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Human papillomavirus (HPV) is an oncogenic virus present in many solid tumors (e.g., 20,000–30,000 cases of HPV-associated cancers are diagnosed annually in the United States), and is responsible for a variety of malignancies, including over 25% of head and neck cancers, over 70% of oropharyngeal cancers, over 90% of cervical and anal cancers, and over 60% of vaginal, vulvar, and penile cancers. HPV antigens, such as HPV E6 and E7 proteins, are attractive targets for several reasons: 1) HPV proteins promote tumorigenesis and are essential for cancer cell survival; 2) HPV proteins are expressed in a wide range of tumor cells, allowing for uniform target expression; and 3) HPV proteins are not expressed in critical healthy tissues, thereby avoiding toxicity to healthy tissues when targeting HPV. Early clinical data from TCR-T cell therapy targeting HPV antigens demonstrated tumor shrinkage and an objective response rate in 50% (6 of 12) of patients treated in a phase 1 trial (study NCT02858310). Complete regression of one or more tumors was observed in 25% (3 of 12) of patients. The development of HPV-specific TCR immunotherapies is warranted, for example, to treat diseases characterized by HPV antigen expression. Summary of the Invention

[0003] The present invention is based, at least in part, on HPV16 E7 11-19 It is based on the discovery of binding proteins containing T cell receptors (TCRs) that recognize antigens.

[0004] In one aspect, a binding protein is provided comprising: a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha α 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 HPV16 E7 11-19 capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity of K d is about 5 × 10 -4 It is below M.

[0005] In another embodiment, a binding protein is provided that comprises: a) a TCR alpha chain variable (V) antibody listed in Table 1 α ) TCR V domain sequences selected from the group consisting of α TCR V with at least about 80% identity to the domain sequence α a) a TCR beta chain variable (V) domain sequence listed in Table 1; β ) TCR V domain sequences selected from the group consisting of β TCR V with at least about 80% identity to the domain sequence β domain sequence, where the binding protein is HPV16 E7 11-19 capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity of K d is about 5 × 10 -4 It is below M.

[0006] In yet another aspect, a binding protein is provided comprising: a) a TCR alpha chain sequence that is at least about 80% identical 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 selected from the group consisting of the TCR beta chain sequences listed in Table 1, wherein the binding protein is selected from the group consisting of HPV16 E7 11-19capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity of K d Paper approximately 5 x 10 -4 It is below M.

[0007] In yet another aspect, a binding protein is provided 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 a binding protein of HPV16 E7 11-19 capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity of K d is approximately 5 x 10 -4 It is below M.

[0008] In another embodiment, a binding protein is provided that comprises: a) a TCR alpha chain variable (V) antibody listed in Table 1 α ) TCR V domain sequences selected from the group consisting of α domain sequence; and / or b) a TCR beta chain variable (V β ) TCR V domain sequences selected from the group consisting of β domain sequence, where the binding protein is HPV16 E7 11-19 capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity of K d is approximately 5 x 10 -4 It is below M.

[0009] In yet another aspect, a binding protein is provided comprising: a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 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 selected from the group consisting of HPV16 E7 11-19 capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity of K d is approximately 5 x 10 -4 It is below M.

[0010] Numerous further embodiments are provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, 1) 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 of TRAV, TRAJ, and TRAC genes listed in Table 1; and / or 2) the TCR beta chain CDRs, TCR V β 3) the domain and / or TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene, or a fragment thereof, selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 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 listed in Table 1. In another embodiment, the HPV16 E7 11-19The immunogenic peptide comprises the amino acid sequence YMLDLQPET. 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, wherein the binding domain comprises a transmembrane domain and an intracellular effector domain. In another embodiment, the TCR alpha chain and the TCR beta chain are covalently linked, and optionally, the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide. In yet another embodiment, the TCR alpha chain and / or the TCR beta chain are covalently linked to a moiety, and optionally, the covalently linked moiety comprises an affinity tag or label. In yet another embodiment, the affinity tag is selected from the group consisting of a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or the label is a fluorescent protein. In another embodiment, the covalently attached moiety is selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof. In yet another embodiment, the binding protein binds to a pMHC complex on the cell surface. In yet another embodiment, the MHC is an MHC multimer, and optionally, the MHC multimer is a tetramer. In another embodiment, the MHC is an MHC class I molecule. In yet another embodiment, the MHC is of the 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 * In another embodiment, the binding proteins described herein are selected from the group consisting of HPV16 E7 and 0207 alleles. 11-19Binding to the peptide-MHC (pMHC) complex elicits an immune response, and optionally the immune response is a T cell response. In yet another embodiment, the T cell response is selected from the group consisting of T cell expansion (e.g., proliferation), cytokine release, and / or cytotoxic killing. In yet another embodiment, the binding protein is HPV16 E7. 11-19 capable of specifically and / or selectively binding to immunogenic peptide-MHC (pMHC) complexes, d is approximately 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, about 5 x 10 -12 M or less, or approximately 1 x 10 -12M or less. In 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 at least 1.05-fold higher binding affinity for peptide-MHC (pMHC) than known T cell receptors. In yet another embodiment, the binding protein induces greater T cell proliferation, cytokine release, and / or cytotoxic killing than known T cell receptors. In yet another embodiment, the binding protein induces at least a 1.05-fold increase in T cell proliferation, cytokine release, and / or cytotoxic killing than known T cell receptors. As used herein, in some embodiments, reference to fold change may be in comparison to any reference modality of interest, such as a comparison with a different binding protein, expression of the same binding protein at different levels in different immune cells, or the same binding protein under different circumstances, such as in combination with other agents described herein. In another embodiment, the target cells are CaSki, SCC152, or SCC090 cell lines. In yet another embodiment, the target cell is a cancer cell, and optionally the cancer cell is a head and neck cancer cell, anal cancer cell, vaginal cancer cell, vulvar cancer cell, or penile cancer cell. In yet another embodiment, the binding protein described herein does not bind to a peptide-MHC (pMHC) complex, and the peptide comprises the amino acid sequence YMLDLQPET. In yet another embodiment, the binding protein described herein does not bind to a peptide-MHC (pMHC) complex, and optionally the peptide is derived from SPTA1, MPL, HERC1, CPAMD8, INTS4, NUTM1, or XM_00172256. These genes are well known and recognized in the art to be annotated according to the following NCBI gene ID numbers, which are available on the World Wide Web at ncbi.nlm.nih.gov / gene. SPTA1: Gene ID 6708, MPL: Gene ID 4352, HERC1: Gene ID 8925, CPAMD8: Gene ID 27151, INTS4: Gene ID 92105, NUTM1: Gene ID 256646.XM_00172256: Mapped to the heterochromatic centromeric region of chromosome 20 and removed from the RefSeq annotation, indicating a lack of evidence for its expression.

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

[0012] In another aspect, an isolated nucleic acid molecule is provided 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, and optionally 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.

[0013] Numerous embodiments are further provided that can be applied to any aspect of the invention and / or can 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 of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the vector is a cloning vector, expression vector, or viral vector. In another embodiment, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, dominant-negative TGFβ receptor II (DN-TGFβRII), and a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR). In yet another embodiment, the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag. In yet another embodiment, the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker, and the tag is fused to the N-terminus of CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker. In another embodiment, the tag is a CD34 enrichment tag. In yet another embodiment, the isolated nucleic acids described herein are interconnected with a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide, either alone (e.g., encoding TCRα and / or TCRβ) or in combination with a nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker. In yet another embodiment, the self-cleaving peptide is P2A, E2A, F2A, or T2A.

[0016] In yet another aspect, there is provided a host cell that comprises an isolated nucleic acid described herein, that comprises a vector described herein, and / or that expresses a binding protein described herein, and optionally is a genetically engineered cell.

[0017] Numerous further embodiments are provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the host cell comprises a chromosomal gene knockout of a TCR gene, an HLA gene, or both. In another embodiment, the host cell comprises a knockout of an HLA gene selected from the group consisting of the α1 macroglobulin gene, the α2 macroglobulin gene, the α3 macroglobulin gene, the β1 microglobulin gene, the β2 microglobulin gene, and combinations thereof. In yet another embodiment, the host cell comprises a knockout of a TCR gene selected from the group consisting of a TCR α variable region gene, a TCR β variable region gene, a TCR constant region gene, and combinations thereof. In yet another embodiment, the host cell expresses CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally, the selectable protein marker is DHFR. In another embodiment, the CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker are fused to a CD34 enrichment tag. In yet another embodiment, the host cell is enriched using a CD34 enrichment tag. In yet another embodiment, the host cell is an immune cell. In another embodiment, the immune cell is a cytotoxic lymphocyte, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte stem cell, a 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 endogenous TCR or have low surface expression. In another embodiment, the host cells contain HPV16 E7 in the MHC molecule. 11-19Upon contact with target cells containing peptide-MHC (pMHC) complexes containing the peptide epitope, cytokines or cytotoxic molecules can be produced. In yet another embodiment, the host cells are contacted with the target cells in vitro, ex vivo, or in vivo. In yet another embodiment, the cytokine is TNF-α, IL-2, and / or IFN-γ. In another embodiment, the cytotoxic molecule is perforin and / or a granzyme, and optionally, the cytotoxic molecule is granzyme B. In yet another embodiment, the host cells are contacted with target cells containing HPV16 E7 11-19 Upon contact with target cells expressing the peptide epitope, the host cell may produce higher levels of cytokines or cytotoxic molecules. In yet another embodiment, the host cell may produce at least 1.05-fold higher levels of cytokines or cytotoxic molecules. In another embodiment, the host cell may express HPV16 E7 in its MHC molecule. 11-19 In yet another embodiment, the killing is determined by a killing assay. In yet another embodiment, the ratio of host cells to target cells in the killing assay is between 20:1 and 0.625:1. In another embodiment, the target cells are infected with 1 μg / mL to 50 pg / mL of HPV16 E7. 11-19 In yet another embodiment, the host cells are HPV16 E7 peptide-pulsed T2 cells. 11-19 Upon contact with target cells expressing the peptide epitope, the host cell is able to kill a greater number of target cells. In yet another embodiment, the host cell is able to kill at least 1.05 times more target cells. In another embodiment, the target cells are CaSki, SCC152, or SCC090 cell lines. In yet another embodiment, the HPV16 E7 11-19 The immunogenic peptide comprises the amino acid sequence YMLDLQPET. 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 * 02:02, HLA-A * 02:03, HLA-A * 02:05, HLA-A * 02:06, and HLA-A * In yet another embodiment, the target cell is a cell line selected from the group consisting of CaSki, SCC152, and SCC090 cell lines, and is selected from the group consisting of HPV16 E7 11-19 In another embodiment, the cancer cells are selected from the group consisting of head and neck cancer cells, oropharyngeal cancer cells, cervical cancer cells, anal cancer cells, vaginal cancer cells, vulvar cancer cells, and penile cancer cells. In yet another embodiment, a) the host cells do not induce T cell proliferation, cytokine release, or cytotoxic killing when contacted with target cells containing a peptide-MHC (pMHC) complex that includes the SPTA1, MPL, HERC1, CPAMD8, INTS4, NUTM1, and / or XM_00172256 peptide epitope in an MHC molecule, and / or b) the host cells are not HPV16 E7 11-19 does not express the antigen and is not recognized by the binding proteins described herein, and is of the serotype HLA-A * Not 02 and / or HLA-A * 02:01 and / or HLA-A * HLA-A such as 02:06 * Does not express the 02 allele.

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

[0019] In yet another aspect, there is provided a composition comprising: a) a binding protein described herein; b) an isolated nucleic acid described herein; c) a vector described herein; d) a host cell described herein; and / or e) a population of host cells described herein; and a carrier.

[0020] In yet another aspect, there is provided a device or kit comprising a) a binding protein described herein, b) an isolated nucleic acid described herein, c) a vector described herein, d) a host cell described herein, and / or e) a population of host cells described herein, and optionally, reagents for detecting binding of a), d) and / or e) to a pMHC complex.

[0021] In another aspect, there is provided a method of producing a binding protein described herein, comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding a binding protein described herein under conditions suitable to allow expression of said binding protein; and (ii) recovering the expressed binding protein.

[0022] In yet another aspect, there is provided a method of producing a host cell that expresses a binding protein described herein, the method comprising the steps of: (i) introducing into a host cell a nucleic acid comprising a sequence encoding a binding protein described herein; and (ii) culturing the transformed host cell under conditions suitable to allow expression of the binding protein.

[0023] In yet another embodiment, HPV16 E7 11-19 Antigen and / or HPV16 E7 11-19 1. A method for detecting the presence or absence of a cell expressing HPV16 E7 in a sample, optionally wherein the cell is a hyperproliferative cell, by using at least one binding protein described herein, or at least one host cell described herein. 11-19 detecting the presence or absence of the HPV16 E7 antigen 11-19 Antigen detection is HPV16 E7 11-19 antigen and / or HPV16 E7 11-19 Methods are provided for indicating the presence of cells expressing

[0024] Numerous further embodiments are provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, at least one binding protein or at least one host cell contains, in an MHC molecule, HPV16 E7 11-19 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 comprises detecting HPV16 E7 by bone marrow biopsy. 11-19 The method further comprises identifying cells that express the

[0025] In another embodiment, HPV16 E7 in a subject 11-19 1. A method for detecting a level of a non-malignant disease, hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of an HPV16 E7 antigen, comprising: a) contacting a sample obtained from a 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 reactivity, wherein a higher level of reactivity compared to a control level indicates HPV16 E7 in the subject. 11-19 Methods are provided that indicate the level of a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of an antigen.

[0026] Numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the control level is a reference number. In another embodiment, the control level is a reference number. 11-19 The level is in subjects with a non-malignant disease, a hyperproliferative disease, or no recurrence of a hyperproliferative disease characterized by expression of the antigen.

[0027] In yet another embodiment, HPV16 E7 in a subject 11-19In a method for monitoring the progression of a non-malignant disease, hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of an antigen, the method includes: a) detecting HPV16 E7 in a sample from a subject at a first time point, as described herein; 11-19 Antigen or HPV16 E7 11-19 b) repeating step a) at a later time point; and c) detecting the target cells expressing HPV16 E7 detected in steps a) and b). 11-19 antigen, or HPV16 E7 11-19 and comparing target cells expressing HPV16 E7 in the subject. 11-19 and monitoring the progression of a non-malignant disease, hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of HPV16 E7 detected in step b) compared to step a). 11-19 Antigen or HPV16 E7 11-19 The absence or reduced levels of target cells expressing HPV16 E7 11-19 It shows that the progression of a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of the antigen is inhibited.

[0028] Numerous embodiments are further provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the subject receives HPV16 E7 HIV between the first and subsequent time points. 11-19 The patient is undergoing treatment to treat a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of the antigen.

[0029] In yet another embodiment, HPV16 E7 11-19 1. A method for assessing the efficacy of a treatment for a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of an antigen, the method comprising: a) detecting HPV16 E7 11-19and b) determining in a first sample obtained from the subject the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, prior to providing the subject with at least part of a treatment for a non-malignant disease, hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of an HPV16 E7 antigen. 11-19 and determining, in a second sample obtained from the subject after providing a portion of a treatment for a non-malignant disease, hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of an antigen, the presence or level of reactivity between the sample obtained from the subject and at least one binding protein described herein, at least one host cell described herein, or a population of host cells described herein, wherein a lack of reactivity or a reduced level of reactivity in the second sample compared to the first sample indicates HPV16 E7 in the subject. 11-19 The therapeutic method is effective for treating a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of the antigen.

[0030] Numerous further embodiments are provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the level of reactivity is indicated by a) the presence of binding and / or b) T cell activation and / or effector function. 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), immunochemical methods, Western blotting, or intracellular flow assays.

[0031] In another embodiment, HPV16 E7 in a subject 11-19

[0013] A method for preventing and / or treating a non-malignant disease, hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of at least one binding protein described herein comprises administering to a subject a therapeutically effective amount of a composition comprising cells expressing at least one binding protein described herein.

[0032] Numerous further embodiments are provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, the cells are allogeneic, syngeneic, or autologous. 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 aspect, the cells comprise 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. In another embodiment, the cells comprise a knockout of a TCR gene selected from the TCR α variable region gene, the TCR β variable region gene, the TCR constant region gene, and combinations thereof. In yet another embodiment, the cells express CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR, and further optionally wherein the CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker is fused to a CD34 enrichment tag. In yet another embodiment, the cells are enriched using a CD34 enrichment tag. In another embodiment, the immune cells are selected from the group consisting of 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 yet another embodiment, the T cells are naive T cells, central memory T cells, effector memory T cells, or a combination thereof. In another embodiment, the T cells are primary T cells or cells of a T cell line. In yet another embodiment, the T cells do not express or have reduced surface expression of an endogenous TCR. In another embodiment, the cells express HPV16 E7 in the MHC molecule. 11-19 Upon contact with target cells containing a peptide-MHC (pMHC) complex containing the peptide epitope, cytokines or cytotoxic molecules can be produced. 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, and optionally, the cytotoxic molecule is granzyme B. In yet another embodiment, the cells are HPV16 E7 11-19 Upon contact with target cells expressing the peptide epitope, the cells are capable of producing increased levels of cytokines or cytotoxic molecules. In another embodiment, the cells are capable of producing at least 1.05-fold increased levels of cytokines or cytotoxic molecules. In yet another embodiment, the host cells express HPV16 E7 in MHC molecules. 11-19 In yet another embodiment, the host cell is a target cell that contains a peptide-MHC (pMHC) complex that contains the peptide epitope. 11-19 Upon contact with target cells expressing the peptide epitope, the host cell is capable of killing more target cells. In another embodiment, the host cell is capable of killing at least 1.05 times as many target cells. In yet another embodiment, the host cell is capable of killing at least 1.05 times as many target cells as the HPV16 E7 11-19 The immunogenic peptide comprises the amino acid sequence YMLDLQPET. 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 * 02:02, HLA-A * 02:03, HLA-A * 02:05, HLA-A * 02:06, and HLA-A * In yet another embodiment, the target cell is selected from the group consisting of the HPV16 E7 allele of the subject. 11-19 In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In yet another embodiment, the composition is administered to a subject to detect HPV16 E7 in a non-malignant or hyperproliferative cell that expresses the antigen. 11-19 In yet another embodiment, the composition induces an immune response against non-malignant or hyperproliferative cells that express the antigen. 11-19 In another embodiment, the antigen-specific T cell immune response is induced against non-malignant or hyperproliferative cells expressing the antigen. + Helper T lymphocyte (Th) response and CD8 + In yet another embodiment, the disorder is associated with HPV infection, such as HPV16 infection. In another embodiment, the cancer is head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), oropharyngeal cancer, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, and / or penile cancer. In yet another embodiment, the subject is undergoing or has previously undergone hematopoietic cell transplantation (HCT), optionally wherein the HCT is with or without HPV16 E7. 11-19 including cells that do not express an antigen, not recognized by the binding proteins described herein, and serotype HLA-A * Not 02 and / or HLA-A *The HCT does not express the 02:01 allele. In another embodiment, the HCT includes 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 yet another embodiment, the subject has previously undergone lymphodepleting chemotherapy. In another embodiment, the lymphodepleting chemotherapy included cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof. In yet another embodiment, the method further includes administering to the subject at least one additional treatment for a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease. In yet another embodiment, the at least one additional treatment for a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease is administered simultaneously or sequentially with the composition. In another embodiment, the subject is vaccinated against HPV16 E7 11-19 The subject is an animal model of the disease characterized by the expression, and / or the subject is a mammal, optionally, the mammal is a human, a primate, or a rodent.

[0033] In yet another aspect, an expression vector is provided comprising a promoter operably linked to a nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR.

[0034] Numerous further embodiments are provided that can be applied to any aspect of the invention and / or combined with any other embodiment described herein. For example, in one embodiment, a nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or a selectable protein marker is operably linked to a nucleic acid encoding a tag, such that the tag is fused to the CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker. In another embodiment, the nucleic acid encoding the tag is 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker, and the tag is fused to the N-terminus of the CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker. 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, TCRβ, and / or DN-TGFβRII comprises 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, TCRβ, and / or DN-TGFβRII, while decreasing expression of endogenous TCRα, TCRβ, and / or TGFβRII. In yet another embodiment, the nucleic acid sequences encoding CD8α, CD8β, DN-TGFβRII, a selectable protein marker, TCRα, and / or TCRβ are interconnected with a nucleic acid sequence encoding an internal ribosome entry site or an autocleaving peptide. In another embodiment, the autocleaving 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, and optionally, 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 or comprises a nucleic acid sequence, or a fragment thereof, set forth in Table 3, and optionally, the fragment encodes DN-TGFβRII.

[0035] Unless otherwise noted below, an MGTM modified version of the TCR (e.g., E7-11-28 MGTM) was used to generate the data shown in the figures and illustrated in the Examples. [Brief explanation of the drawings]

[0036] [Figure 1] The HPV16 E711-19 peptide sequence is shown. [Figure 2]We demonstrate the selection of various TCRs that recognize HPV16 E711-19. Using a dedicated ReceptorScan platform, we identified 459 HPV16 E711-19 (YMLDLQPET)-specific TCRs. Briefly, CD14+ monocytes were isolated from PBMCs of HLA-A*02:01 healthy donors on day -4 and differentiated into mature DCs. On day -1, naive CD8+ T cells were isolated from autologous PBMCs and allowed to rest overnight. As part of multiplexed ReceptorScan screening, DCs were pulsed with 1 μg / mL HPV16 E711-19 peptide for 3 hours, followed by co-culture with naive CD8+ T cells and DCs for an 11-day cell expansion phase. To identify clones, we performed dextramer staining using HLA-A*02:01-specific HPV16 E711-19 (YMLDLQPET) dextramer. HPV16 E711-19-specific cells were isolated using DNA barcoded dextramers. TCR alpha-beta pairs were identified using the 10x genomics platform. Pan-T cells were individually transfected to express 293 HPV-specific TCRs. TCR surface expression was assessed by HPV16 E711-19 dextramers staining. The engineered T cells were co-cultured with IncuCyte® NucLight Red-labeled target cells, such as T2 cells or CaSki cells, loaded with 1 ng / mL HPV16 E711-19 peptide. Target cell survival was quantified by time-dependent imaging as a readout of T cell cytotoxicity. Non-transduced cells (NTD) were used as a control. Fifty-nine of the 293 TCRs listed in Figure 2 were selected for further evaluation of their surface expression and cytotoxic potential against HPV16- and HLA-A*02:01-positive and -negative cell lines. [Figure 3A-1]The results of selecting HPV16 E711-19 TCRs based on expression and cytotoxic function are shown. Pan-T cells from HLA-A*02:01-positive healthy donors were transduced to express 59 HPV16 E711-19 TCRs selected from the VAYG screening described in Figure 2 above. Of the 59 TCRs, 24 were selected based on strong surface binding of HPV16 E711-19 (YMLDLQPET) dextramers and further evaluated in in vitro cytotoxicity assays and compared with a "comparator TCR." Dot plots of the surface expression of the 24 TCRs assessed by A*02:01-specific HPV16 E711-19 (YMLDLQPET) dextramers staining are shown. The cytotoxic responses of these TCRs against the HLA-A*02:01+HPV16+ target cell lines, CaSki (Figure 3B), SCC152 (Figure 3C), and SCC090 (Figure 3D), are shown. Engineered T cells were co-cultured with Incucyte® NucLight Red-labeled target cell lines at the indicated effector-to-target (E:T) ratios, and their viability was quantified by IncuCyte® as a readout of T cell cytotoxicity. [Figure 3A-2]The results of selecting HPV16 E711-19 TCRs based on expression and cytotoxic function are shown. Pan-T cells from HLA-A*02:01-positive healthy donors were transduced to express 59 HPV16 E711-19 TCRs selected from the VAYG screening described in Figure 2 above. Of the 59 TCRs, 24 were selected based on strong surface binding of HPV16 E711-19 (YMLDLQPET) dextramers and further evaluated in in vitro cytotoxicity assays and compared with a "comparator TCR." Dot plots of the surface expression of the 24 TCRs assessed by A*02:01-specific HPV16 E711-19 (YMLDLQPET) dextramers staining are shown. The cytotoxic responses of these TCRs against the HLA-A*02:01+HPV16+ target cell lines, CaSki (Figure 3B), SCC152 (Figure 3C), and SCC090 (Figure 3D), are shown. Engineered T cells were co-cultured with Incucyte® NucLight Red-labeled target cell lines at the indicated effector-to-target (E:T) ratios, and their viability was quantified by IncuCyte® as a readout of T cell cytotoxicity. [Figure 3B] We show the results of selecting HPV16 E711-19 TCRs based on expression and cytotoxic function. Pan-T cells from HLA-A*02:01-positive healthy donors were transduced to express 59 HPV16 E711-19 TCRs selected from the VAYG screening described in Figure 2 above. Of the 59 TCRs, 24 were selected based on strong surface binding of the HPV16 E711-19 (YMLDLQPET) dextramers and further evaluated in in vitro cytotoxicity assays and compared with "comparator TCRs." The cytotoxic responses of these TCRs against the HLA-A*02:01+ HPV16+ target cell lines, CaSki (Figure 3B), SCC152 (Figure 3C), and SCC090 (Figure 3D), are shown. Engineered T cells were co-cultured with Incucyte® NucLight Red-labeled target cell lines at the indicated effector cell to target cell (E:T) ratios, and their viability was quantified by IncuCyte® as a readout of T cell cytotoxicity. [Figure 3C] We show the results of selecting HPV16 E711-19 TCRs based on expression and cytotoxic function. Pan-T cells from HLA-A*02:01-positive healthy donors were transduced to express 59 HPV16 E711-19 TCRs selected from the VAYG screening described in Figure 2 above. Of the 59 TCRs, 24 were selected based on strong surface binding of the HPV16 E711-19 (YMLDLQPET) dextramers and further evaluated in in vitro cytotoxicity assays and compared with "comparator TCRs." The cytotoxic responses of these TCRs against the HLA-A*02:01+ HPV16+ target cell lines, CaSki (Figure 3B), SCC152 (Figure 3C), and SCC090 (Figure 3D), are shown. Engineered T cells were co-cultured with Incucyte® NucLight Red-labeled target cell lines at the indicated effector cell to target cell (E:T) ratios, and their viability was quantified by IncuCyte® as a readout of T cell cytotoxicity. [Figure 3D] We show the results of selecting HPV16 E711-19 TCRs based on expression and cytotoxic function. Pan-T cells from HLA-A*02:01-positive healthy donors were transduced to express 59 HPV16 E711-19 TCRs selected from the VAYG screening described in Figure 2 above. Of the 59 TCRs, 24 were selected based on strong surface binding of the HPV16 E711-19 (YMLDLQPET) dextramers and further evaluated in in vitro cytotoxicity assays and compared with "comparator TCRs." The cytotoxic responses of these TCRs against the HLA-A*02:01+ HPV16+ target cell lines, CaSki (Figure 3B), SCC152 (Figure 3C), and SCC090 (Figure 3D), are shown. Engineered T cells were co-cultured with Incucyte® NucLight Red-labeled target cell lines at the indicated effector cell to target cell (E:T) ratios, and their viability was quantified by IncuCyte® as a readout of T cell cytotoxicity. [Figure 4A]Functional evaluation of HPV16 E711-19 TCRs is shown. Pan-T cells isolated from the PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR." Functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. (A) Dot plots displaying the expression of E711-19-specific TCRs assessed by A*02:01-specific HPV16 E711-19 (YMLDLQPET) dextramer staining are shown. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4B]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4C-1]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4C-2]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4D]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4E-1]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4E-2]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4F]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4G-1]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4G-2]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4H]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4I-1]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4I-2]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4J]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4K-1]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 4K-2]Functional evaluation of HPV16 E711-19 TCRs. Pan-T cells isolated from PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express HPV16 E711-19-specific TCRs, E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR," and functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were evaluated. Functional responses of E711-19-specific TCRs to the HLA-A*02:01+HPV16+ target cell lines CaSki (B and C), SCC152 (D and E), and SCC090 (F and G), the HLA-A*02:01-HPV16+ negative control cell line SiHa (H and I), and the HLA-A*02:01+HPV16- cell line NCI-H1792 (J and K) are shown. Engineered T cells were cocultured with Incuyte® NucLight Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified using IncuCyte® as a readout of T cell cytotoxicity. The production of IFN-γ, IL-2, TNF-α, and granzyme B in the coculture supernatants (E:T 1:1) after 24 hours was analyzed and shown. Background levels of cytokine production were determined using the T cell-only condition. The dotted line represents the highest level of cytokines in the T cell-only condition (i.e., background level of cytokine production or proliferation by T cells). For cytotoxicity studies, means were compared using one-way ANOVA, followed by Dunnett's multiple comparison test to compare TCRs to the "comparator TCR." For CaSki, SCC152, and SCC090 cell lines, only non-significant differences are displayed. The remaining differences are significant at P < 0.05. For SiHa and NCI-H1792 cell lines, none of the differences were significant. [Figure 5]This shows that TCR E7-11-28 is not alloreactive to 108 of 110 HLA types. Pan-T cells expressing TCR E7-11-28 or untransduced control T cells were cocultured for 48 hours with MHC-null HEK293T cells expressing one of the 110 most frequently occurring class I MHC alleles in the U.S. population. A positive control consisting of HEK293T cells expressing both a fragment of HPV16-E7 containing the E711-19 epitope (YMLDLQPET) and HLA-A*02:01 was included in the screen. Target cell proliferation inhibition by pan-T cells expressing TCR E7-11-28 compared with untransduced control T cells was measured after 48 hours of coculture as a readout of TCR E7-11-28's reactivity to cognate MHC molecules. Positive control and alloreactive alleles (target cell inhibition >20%) are shown. [Figure 6A] Further selection of various TCRs recognizing HPV16 E711-19 is shown. Pan T cells isolated from PBMCs of HLA-A*02:01-positive healthy donors were transduced to express each of 161 HPV16 E711-19-specific TCRs and a "comparator TCR." (A) Representative data are shown for TCR surface expression assessed by HPV16 E711-19 dextramers staining and the cytotoxic response against T2 cells pulsed with 200 pg / ml of HPV16 E711-19 peptide and the HLA-A*02:01+HPV16+ target cell line SCC152. (B) 15 of the 161 TCRs were selected for further evaluation of surface expression and cytotoxic potential against HPV16 and HLA-A*02:01-positive and -negative cell lines. [Figure 6B]Further selection of various TCRs recognizing HPV16 E7 11-19 is shown. Pan T cells isolated from PBMCs of HLA-A*02:01-positive healthy donors were transduced to express each of 161 HPV16 E7 11-19-specific TCRs and a "comparator TCR." (A) Representative data are shown for TCR surface expression assessed by HPV16 E7 11-19 dextramer staining and cytotoxic responses against T2 cells pulsed with 200 pg / ml of HPV16 E7 11-19 peptide and the HLA-A*02:01 + HPV16 + target cell line SCC152. (B) 15 of the 161 TCRs were selected for further evaluation of surface expression and cytotoxic potential against HPV16 and HLA-A*02:01-positive and -negative cell lines. [Figure 7-1] We show the results of selecting HPV16 E711-19 TCRs based on expression and cytotoxic function. Pan T cells isolated from HLA-A*02:01-positive healthy donor PBMCs were transduced to express the HPV16 E711-19-specific TCRs E7-11-28 and E7-11-455, as well as a "comparator TCR," and the cytotoxic response against HPV16 and HLA-A*02:01-positive and -negative target cells was assessed. Functional responses of the E711-19-specific TCR against the HLA-A*02:01+HPV16+ target cell lines CaSki, SCC152, and SCC090, the HLA-A*02:01-HPV16+ negative control cell line SiHa, and the HLA-A*02:01+HPV16- cell line, NCI-H1792, are shown. Engineered T cells were co-cultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified with IncuCyte® as a readout of T cell cytotoxicity. For cytotoxicity studies, TCRs were compared to "comparator TCRs" using one-way ANOVA followed by Dunnett's multiple comparison test. * indicates p<0.05. [Figure 7-2]We show the results of selecting HPV16 E711-19 TCRs based on expression and cytotoxic function. Pan T cells isolated from HLA-A*02:01-positive healthy donor PBMCs were transduced to express the HPV16 E711-19-specific TCRs E7-11-28 and E7-11-455, as well as a "comparator TCR," and the cytotoxic response against HPV16 and HLA-A*02:01-positive and -negative target cells was assessed. Functional responses of the E711-19-specific TCR against the HLA-A*02:01+HPV16+ target cell lines CaSki, SCC152, and SCC090, the HLA-A*02:01-HPV16+ negative control cell line SiHa, and the HLA-A*02:01+HPV16- cell line, NCI-H1792, are shown. Engineered T cells were co-cultured with IncuCyte® NucLight™ Red-labeled target cell lines at the indicated E:T ratios, and their viability was quantified with IncuCyte® as a readout of T cell cytotoxicity. For cytotoxicity studies, TCRs were compared to "comparator TCRs" using one-way ANOVA followed by Dunnett's multiple comparison test. * indicates p<0.05. [Figure 8-1] Summary results are shown showing that TCR-28 exhibits comparable cytotoxicity and superior effector function compared to comparator TCRs. [Figure 8-2] Summary results are shown showing that TCR-28 exhibits comparable cytotoxicity and superior effector function compared to comparator TCRs. [Figure 9A]Figure 1 shows T cell proliferative responses generated by the HPV16 E711-19 TCR. Pan T cells isolated from the PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express the HPV16 E711-19-specific TCRs E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR." Functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were assessed. To determine proliferation of HPV16 E711-19-specific TCR-expressing T cells, the engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (CaSki, A; SCC152, B; SCC090, C; SiHa, D; and NCI-H1792, E) for 96 hours (E:T 1:1). These cell lines have the following characteristics: CaSki, SCC152, and SCC090 are HLA-A*02:01+HPV16+; SiHa is HLA-A*02:01-HPV16+; and NCI-H1792 is HLA-A*02:01+HPV16-. CD8+ and CD4+ T cell proliferation was assessed using the dye dilution method. Counting beads were added to samples before flow cytometry analysis, and the absolute numbers of dividing CD8+ and CD4+ T cells were counted. The T cell-only condition was used to determine background levels of proliferation. The dotted line represents the highest proliferation level in the T cell-only condition. Mean values ​​were compared using one-way ANOVA, followed by Dunnett's multiple comparison test, to compare TCRs with the "comparator TCR." For CaSki (A), SCC152 (B), and SCC090 (C) cell lines, only non-significant differences (ns) are shown. The remaining differences were significant at P<0.05. [Figure 9B]Figure 1 shows T cell proliferative responses generated by the HPV16 E711-19 TCR. Pan T cells isolated from the PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express the HPV16 E711-19-specific TCRs E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR." Functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were assessed. To determine proliferation of HPV16 E711-19-specific TCR-expressing T cells, the engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (CaSki, A; SCC152, B; SCC090, C; SiHa, D; and NCI-H1792, E) for 96 hours (E:T 1:1). These cell lines have the following characteristics: CaSki, SCC152, and SCC090 are HLA-A*02:01+HPV16+; SiHa is HLA-A*02:01-HPV16+; and NCI-H1792 is HLA-A*02:01+HPV16-. CD8+ and CD4+ T cell proliferation was assessed using the dye dilution method. Counting beads were added to samples before flow cytometry analysis, and the absolute numbers of dividing CD8+ and CD4+ T cells were counted. The T cell-only condition was used to determine background levels of proliferation. The dotted line represents the highest proliferation level in the T cell-only condition. Mean values ​​were compared using one-way ANOVA, followed by Dunnett's multiple comparison test, to compare TCRs with the "comparator TCR." For CaSki (A), SCC152 (B), and SCC090 (C) cell lines, only non-significant differences (ns) are shown. The remaining differences were significant at P<0.05. [Figure 9C]Figure 1 shows T cell proliferative responses generated by the HPV16 E711-19 TCR. Pan T cells isolated from the PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express the HPV16 E711-19-specific TCRs E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR." Functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were assessed. To determine proliferation of HPV16 E711-19-specific TCR-expressing T cells, the engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (CaSki, A; SCC152, B; SCC090, C; SiHa, D; and NCI-H1792, E) for 96 hours (E:T 1:1). These cell lines have the following characteristics: CaSki, SCC152, and SCC090 are HLA-A*02:01+HPV16+; SiHa is HLA-A*02:01-HPV16+; and NCI-H1792 is HLA-A*02:01+HPV16-. CD8+ and CD4+ T cell proliferation was assessed using the dye dilution method. Counting beads were added to samples before flow cytometry analysis, and the absolute numbers of dividing CD8+ and CD4+ T cells were counted. The T cell-only condition was used to determine background levels of proliferation. The dotted line represents the highest proliferation level in the T cell-only condition. Mean values ​​were compared using one-way ANOVA, followed by Dunnett's multiple comparison test, to compare TCRs with the "comparator TCR." For CaSki (A), SCC152 (B), and SCC090 (C) cell lines, only non-significant differences (ns) are shown. The remaining differences were significant at P<0.05. [Figure 9D]Figure 1 shows T cell proliferative responses generated by the HPV16 E711-19 TCR. Pan T cells isolated from the PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express the HPV16 E711-19-specific TCRs E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR." Functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were assessed. To determine proliferation of HPV16 E711-19-specific TCR-expressing T cells, the engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (CaSki, A; SCC152, B; SCC090, C; SiHa, D; and NCI-H1792, E) for 96 hours (E:T 1:1). These cell lines have the following characteristics: CaSki, SCC152, and SCC090 are HLA-A*02:01+HPV16+; SiHa is HLA-A*02:01-HPV16+; and NCI-H1792 is HLA-A*02:01+HPV16-. CD8+ and CD4+ T cell proliferation was assessed using the dye dilution method. Counting beads were added to samples before flow cytometry analysis, and the absolute numbers of dividing CD8+ and CD4+ T cells were counted. The T cell-only condition was used to determine background levels of proliferation. The dotted line represents the highest proliferation level in the T cell-only condition. Mean values ​​were compared using one-way ANOVA, followed by Dunnett's multiple comparison test, to compare TCRs with the "comparator TCR." For CaSki (A), SCC152 (B), and SCC090 (C) cell lines, only non-significant differences (ns) are shown. The remaining differences were significant at P<0.05. [Figure 9E]Figure 1 shows T cell proliferative responses generated by the HPV16 E711-19 TCR. Pan T cells isolated from the PBMCs of three HLA-A*02:01-positive healthy donors were transduced to express the HPV16 E711-19-specific TCRs E7-11-194, E7-11-176, and E7-11-28, as well as a "comparator TCR." Functional responses against HPV16 and HLA-A*02:01-positive and -negative target cells were assessed. To determine proliferation of HPV16 E711-19-specific TCR-expressing T cells, the engineered T cells were labeled with a proliferation dye and cocultured with target cell lines (CaSki, A; SCC152, B; SCC090, C; SiHa, D; and NCI-H1792, E) for 96 hours (E:T 1:1). These cell lines have the following characteristics: CaSki, SCC152, and SCC090 are HLA-A*02:01+HPV16+; SiHa is HLA-A*02:01-HPV16+; and NCI-H1792 is HLA-A*02:01+HPV16-. CD8+ and CD4+ T cell proliferation was assessed using the dye dilution method. Counting beads were added to samples before flow cytometry analysis, and the absolute numbers of dividing CD8+ and CD4+ T cells were counted. The T cell-only condition was used to determine background levels of proliferation. The dotted line represents the highest proliferation level in the T cell-only condition. Mean values ​​were compared using one-way ANOVA, followed by Dunnett's multiple comparison test, to compare TCRs with the "comparator TCR." For CaSki (A), SCC152 (B), and SCC090 (C) cell lines, only non-significant differences (ns) are shown. The remaining differences were significant at P<0.05. [Figure 10A]

[0023] Figure 1 shows the results of a genome-wide SafetyScan screen to identify putative off-targets of TCR E7-11-28.

[0024] Figure 2 shows a representative, non-limiting overview of a genome-wide SafetyScan screen. [Figure 10B]We present the results of a genome-wide SafetyScan screening to identify putative off-targets of TCR E7-11-28. SafetyScan screening data for TCR E7-11-28 identifies seven potential off-targets in a screen of over 600,000 protein fragments covering all wild-type (wt) human proteins. The screen was designed to overpredict off-targets by overexpressing a 90-amino acid protein fragment, which is processed more efficiently than the full-length protein. Putative off-targets are identified by gene name. XM_0017722256 maps to the heterochromatic centromeric region of chromosome 20 and has been removed from the RefSeq annotation, indicating a lack of evidence for its expression. RNA-seq analysis of 51 samples, including normal tissue samples, cancer cell lines, and tumor samples, did not detect expression of this gene. [Figure 11A] This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11B]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11C]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11D]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11E]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11F]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11G]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11H]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11I]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 11J]This figure shows that TCR E7-11-28 does not exhibit reactivity against cancer cell lines expressing putative off-targets. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against HLA-A*02:01+ cancer cell lines that naturally express off-targets identified in genome-wide safety screening. Panels A, C, E, G, and I show the results of co-culture of target cells pulsed or not with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. Peptide-pulsed T2 cells were used as a positive control. Where indicated, HLA-A*02:01+HPV16+SCC152 cells were used as an additional positive control. For cell lines expressing multiple off-targets, co-culture was performed only once and is shown in multiple figures. B, D, F, H, and J show the expression of HERC1, INTS4, CPAMD8, MPL, and SPTA1, respectively, measured in target cells relative to the control gene TBP. [Figure 12A] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12B]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12C] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12D]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12E] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12F]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12G] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12H]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12I] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12J]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12K] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12L]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12M] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12N]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12O] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPSC-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. Panels A-O show the results of co-culture of target cells pulsed or unpulsed with E711-19 peptide with TCR E7-11-28 or NTD cells. IFNγ secretion in the culture supernatant was used as a readout of TCR E7-11-28 reactivity against target cells. HLA-A*02:01+HPV16+SCC152 cells were used as a positive control, and HLA-A*02:01+HPV16-NCI-H1792 cells or unpulsed T2 or OVCAR-3 cells were used as negative controls. [Figure 12P]This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPS-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. P and Q indicate HERC1 and INTS4 expression, respectively, measured in target cells relative to the control gene TBP. RT = reverse transcriptase. [Figure 12Q] This shows that TCR E7-11-28 is not reactive against healthy human primary cells. Pan T cells or NTD cells expressing TCR E7-11-28 were tested for reactivity against a panel of primary or iPS-derived cells from healthy HLA-A*02:01+ human donors, including cells naturally expressing putative off-targets identified in genome-wide safety screening. P and Q indicate HERC1 and INTS4 expression, respectively, measured in target cells relative to the control gene TBP. RT = reverse transcriptase. [Figure 13A] These results demonstrate that TCR E7-11-28 effectively controls tumor growth in vivo. NCG mice were subcutaneously injected with 1 x 10 Caski cells or 1 x 10 SCC152 cells per mouse (8 mice per group). On day 10, when tumors reached 95 ± 15 mm, mice were randomized and treated with 20 x 10 TCR E7-11-28, NTD, or vehicle on day 11. Panels A and C show the results of treatment with 20 x 10 TCR E7-11-28 cells, demonstrating potent inhibition of tumor growth in vivo. *p<0.05, one-way ANOVA, Holm-Sidak correction for in vivo multiple comparisons. Data line labels are indicated in panels A and C. [Figure 13B]These results demonstrate that TCR E7-11-28 effectively controls tumor growth in vivo. NCG mice were subcutaneously injected with 1 x 10 Caski cells or 1 x 10 SCC152 cells per mouse (8 mice per group). When tumors reached 95 ± 15 mm on day 10, mice were randomized and treated with 20 x 10 TCR E7-11-28, NTD, or vehicle on day 11. Panels B and D show tumor growth results for individual mice in each group over time. *p<0.05, one-way ANOVA, Holm-Sidak correction for in vivo multiple comparisons. Data line labels are shown in A and C. [Figure 13C] These results demonstrate that TCR E7-11-28 effectively controls tumor growth in vivo. NCG mice were subcutaneously injected with 1 x 10 Caski cells or 1 x 10 SCC152 cells per mouse (8 mice per group). On day 10, when tumors reached 95 ± 15 mm, mice were randomized and treated with 20 x 10 TCR E7-11-28, NTD, or vehicle on day 11. Panels A and C show the results of treatment with 20 x 10 TCR E7-11-28 cells, demonstrating potent inhibition of tumor growth in vivo. *p<0.05, one-way ANOVA, Holm-Sidak correction for in vivo multiple comparisons. Data line labels are indicated in panels A and C. [Figure 13D] These results demonstrate that TCR E7-11-28 effectively controls tumor growth in vivo. NCG mice were subcutaneously injected with 1 x 10 Caski cells or 1 x 10 SCC152 cells per mouse (8 mice per group). When tumors reached 95 ± 15 mm on day 10, mice were randomized and treated with 20 x 10 TCR E7-11-28, NTD, or vehicle on day 11. Panels B and D show tumor growth results for individual mice in each group over time. *p<0.05, one-way ANOVA, Holm-Sidak correction for in vivo multiple comparisons. Data line labels are shown in A and C. [Figure 14] We demonstrate that dominant-negative TGFβ receptor II (DN-TGFβRII) confers resistance to the inhibitory effects of TGFβ signaling in DN-TGFβRII-expressing cells (e.g., DN-TGFβRII confers resistance to TGFβ-mediated suppression in TCR E7-11-28). T cells were co-transduced with lentiviruses encoding TCR E7-11-28 and DN-TGFβRII, respectively, and sorted by FACS into DN-TGFβRII-positive and DN-TGFβRII-negative fractions. Intracellular IFNγ levels in TCR-expressing T cells were quantified after 24 hours of co-culture with peptide-pulsed T2 cells + / - 5 ng / mL TGFβ. [Figure 15A] Representative results of expression and functional evaluation of TCR E7-11-28 and DN-TGFβRII in the pNVVD154 and pNVVD160 vectors are shown. PBMCs from an HLA-A*02:01-positive healthy donor were transfected with the pNVVD154 and pNVVD160 vectors to express HPV16 TCR E7-11-28. Untransfected (UTF) PBMCs from the same donor were used as a control. (A) Dot plots of surface expression of TGFβRII, CD34, and TCR E7-11-28 assessed by A*02:01-specific HPV16 E711-19 (YMLDLQPET) dextramer staining. The top panel of A shows the results of modified T cells co-cultured with IncuCyte® NucLight™ Red-labeled target cell lines CaSki and NCI-H1792 (E:T ratio 10:1), and SCC-152 (E:T ratio 5:1), and their viability was quantified by IncuCyte® as a readout of T cell cytotoxicity. [Figure 15B]Representative results of expression and functional evaluation of TCR E7-11-28 and DN-TGFβRII in the pNVVD154 and pNVVD160 vectors are shown. PBMCs from an HLA-A*02:01-positive healthy donor were transfected with the pNVVD154 and pNVVD160 vectors to express HPV16 TCR E7-11-28. Untransfected (UTF) PBMCs from the same donor were used as a control. (B) shows the cytotoxic response of TCR E7-11-28-modified T cells against the HLA-A*02:01+HPV16+ target cell lines CaSki and SCC152, and the HLA-A*02:01+HPV16- target cell line NCI-H1792. The bottom panel in (B) shows the target cell viability after 96 hours for CaSki, SCC-152, and NCI-H1792 cell lines. [Figure 15C] Representative results of expression and functional evaluation of TCR E7-11-28 and DN-TGFβRII in the pNVVD154 and pNVVD160 vectors are shown. PBMCs from an HLA-A*02:01-positive healthy donor were transfected with the pNVVD154 and pNVVD160 vectors to express HPV16 TCR E7-11-28. Untransfected (UTF) PBMCs from the same donor were used as a control. Figure C shows the results of an assay testing T cell affinity. For example, HPV16 TCR E7-11-28-expressing T cells were co-cultured with E711-19 peptide-pulsed (0-1,000 pg / ml E7 peptide) IncuCyte® NucLight™-expressing T2 cells at an E:T ratio of 5:1. The graph shows the area under the curve (AUC) of T2 cell proliferation from 0 to 96 hours of co-culture. Experiments were performed in duplicate. A Mann-Whitney t-test was performed. [Figure 16]Figure 1 shows a map of the pNVVD154_TSC-200-A02_TCR-28_MSCV—TCR28-CD8-EF1a-TGFR-DHFR vector. Abbreviations: CD: cluster of differentiation, RNA-OUT: antisense RNA against bacterial levansucrase encoded by sacB, SV: simian virus, TCR: T cell receptor, TIR: inverted terminal repeat, QBend: mouse anti-human CD34 antibody, dnTGFbRII: dominant-negative TGF beta receptor II, DHFR: dihydrofolate reductase selectable marker. [Figure 17] Figure 1 shows a map of the pNVVD160_TSC-200-A02_TCR-28_MSCV-TCR28-CD8-EF1a-TGFR-DHFR vector. Abbreviations: CD: cluster of differentiation, RNA-OUT: antisense RNA against bacterial levansucrase encoded by sacB, SV: simian virus, TCR: T cell receptor, TIR: terminal inverted repeat, QBend: mouse anti-human CD34 antibody, dnTGFbRII: dominant-negative TGF beta receptor II, DHFR: dihydrofolate reductase selection marker.

[0037] Unless otherwise noted, for any figure showing a bar histogram, curve, or other data associated with a legend, the bars, curves, or other data displayed from left to right in each display directly correspond to the order of the boxes in the legend, from top to bottom or left to right. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention relates, at least in part, to HPV16 E7 11-19 It is based on the discovery of a binding protein containing a T cell receptor (TCR) that recognizes an antigen (e.g., an immunogenic peptide containing the amino acid sequence YMLDLQPET).

[0039] Accordingly, the present invention provides, in part, identified binding proteins (e.g., TCRs), host cells expressing the binding proteins (e.g., TCRs), compositions comprising the binding proteins (e.g., TCRs) and host cells expressing the binding proteins (e.g., TCRs), HPV16 E711-19 Methods for diagnosing, prognosing, and monitoring T cell responses to cells expressing an antigen, and methods for detecting HPV16 E7 by administering host cells expressing a binding protein (e.g., TCR) 11-19 The present invention relates to methods for preventing and / or treating non-malignant diseases, hyperproliferative diseases, or recurrence of hyperproliferative diseases characterized by expression of an antigen.

[0040] I. Definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0041] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0042] As used herein, 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 involves physically introducing a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. In some embodiments, routes of administration for the binding proteins described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration," as used herein, refers to methods of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, intrasternal, and in vivo electroporation. Alternatively, the binding proteins described herein can be administered by parenteral routes, such as topical, epidermal, or mucosal routes, e.g., intranasally, orally, intravaginally, rectally, sublingually, or topically. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0043] As used herein, the term "antigen" refers to a natural or synthetic immunogenic substance, such as a protein, peptide, hapten, etc. An antigen is an HPV16 E7 antigen against which a protective or therapeutic immune response is desired. 11-19 It may be an antigen or a fragment thereof.

[0044] As used herein, the term "adjuvant" refers to a substance that, when administered before, concurrently with, or after administration of an antigen, accelerates, prolongs, and / or enhances the quality and / or strength of the immune response to the antigen compared to administration of the antigen alone. Adjuvants can increase the magnitude and duration of the immune response elicited by vaccination.

[0045] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" in some embodiments comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain comprises a heavy chain variable region (herein referred to as V H In certain naturally occurring antibodies, the heavy chain constant region consists of three domains, CH1, CH2, and CH3. In certain naturally occurring antibodies, each light chain consists of a light chain variable region (abbreviated herein as V L The light chain constant region consists of one domain, CL. H Area and V L The regions can 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 binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0046] The term "antigen-presenting cells" or "APCs" includes not only professional antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), but also other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

[0047] As used herein, the term "antigen-binding portion" of a binding protein, such as a TCR, refers to a portion of a protein that binds to an antigen (e.g., HPV16 E7 11-19 "antigen-binding portion" refers to one or more portions of a TCR that retain the ability to bind (e.g., specifically and / or selectively) to an antigen and cognate MHC / HLA. Such portions are, for example, about 8 to about 1500 amino acids in length, suitably about 8 to about 745 amino acids in length, suitably about 8 to about 300, e.g., about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of a TCR can be performed by fragments of a full-length TCR. Examples of binding portions encompassed by the term "antigen-binding portion" of a TCR include (i) the V of the TCR α and V β (ii) an Fv fragment consisting of an isolated complementarity determining region (CDR), or (iii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. α and V β Although the V are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods. α and V β The regions pair to form a monovalent molecule (known as a single-chain TCR (scTCR)). Such single-chain TCRs are also intended to be encompassed by the term "antigen-binding portion" of a TCR. These TCR fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as the intact binding protein. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact protein.

[0048] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids within certain binding proteins, such as TCR variable regions, that confer antigen specificity and / or binding affinity. In the case of TCRs, there are generally three CDRs (αCDR1, αCDR2, αCDR3) in each α chain variable region and three CDRs (βCDR1, βCDR2, βCDR3) in each β chain variable region. CDR3 is considered to be the primary CDR that recognizes processed antigens. CDR1 and CDR2 primarily interact with MHC.

[0049] 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-ejaculate fluid, chyle, chyme, stool, female semen, interstitial fluid, intracellular fluid, lymph, menstruation, breast milk, mucus, pleural effusion, pus, saliva, semen, serum, sweat, synovial fluid, tears, urine, vaginal fluid, vitreous humor, vomit). In some embodiments, the body fluid comprises immune cells, and optionally, the immune cells are cytotoxic T cells and / or cytotoxic lymphocytes, such as NK cells, CD4+ T cells, etc.

[0050] 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).

[0051] 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 anti-parallel 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 anti-parallel 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 anti-parallel 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.

[0052] The term "costimulation" used in reference to activated immune cells includes the ability of a costimulatory polypeptide to provide a second, non-activating receptor-mediated signal ("costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion in, for example, a T cell that has received a T cell receptor-mediated signal. Immune cells that have received a cell receptor-mediated signal, for example, via an activating receptor, are referred to herein as "activated immune cells."

[0053] "CD3" is known in the art as a multiprotein complex consisting of six chains (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 property thought to enable these chains to bind to positively charged regions or residues on T cell receptor chains. The intracellular ends 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 has three ITAMs. Without wishing to be bound by theory, ITAMs are thought to be important for the signaling capabilities of the TCR complex. The CD3 used in the present invention can be derived from various animal species, including humans, mice, rats, or other mammals.

[0054] As used herein, "a component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0055] "Chimeric antigen receptor" or "CAR" refers to a fusion protein engineered to contain two or more amino acid sequences linked in a manner that does not occur naturally or naturally within a host cell, such that the fusion protein can function as a receptor when presented on the surface of a cell. CARs encompassed by the present invention include an antigen-binding domain (i.e., HPV16 E7 11-19 These antibodies include an extracellular portion in which an antigen-specific TCR, a single-chain TCR-derived binding protein, an scFv derived from an antibody, an antigen-binding domain derived from or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR specific for an antigen, a single-chain TCR-derived binding protein, an scFv derived from an antibody, or an antigen-binding domain derived from or derived from a killer immune receptor from an NK cell, is linked to a transmembrane domain, and one or more intracellular signaling domains (including an effector domain and, optionally, a costimulatory domain) (see, e.g., Sadelain et al. (2013) Cancer Discov. 3:388; see also, Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220; Stone et al. (2014) Cancer Immunol. Immunother 63:1163).

[0056] 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.

[0057] The term "consisting essentially of" is not the same as "comprising" and refers to particular materials or steps in a claim or that do not materially affect the essential characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or protein (which may have one or more domains, regions, or modules) may be defined as "a domain, region, module, or protein whose amino acid sequence includes extensions, deletions, mutations, or combinations thereof (e.g., amino- or carboxy-terminal amino acids, or inter-domain amino acids) that 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 (i.e., do not reduce activity by more than 50%, e.g., by 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% or less) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

[0058] The term "determining a suitable therapeutic regimen for a subject" is understood to mean determining a subject's therapeutic regimen (i.e., a single therapy or a combination of different therapies used to prevent and / or treat viral infection in a subject) that is initiated, modified, and / or terminated based on, or essentially based on, or at least in part based on, the results of the analysis of the present invention. One example is initiating adjuvant therapy after surgery aimed at reducing the risk of recurrence, and another example is changing the dose of a specific anticancer drug. In addition to the results of the analysis of the present invention, the decision can be based on the personal characteristics of the subject being treated. In most cases, the actual decision on a suitable therapeutic regimen for a subject will be made by the attending physician or doctor.

[0059] The term "dominant negative TGFβ receptor" or "DN-TGFβR" refers to a variant or mutant of the transforming growth factor (TGF) beta receptor that confers resistance to TGFβ signaling.

[0060] There are five type-II receptors (activating receptors) and seven type-I receptors (signal-transmitting receptors). Activated TGFβ receptors are heterotetramers consisting of two TGFβ receptor I (TGFβRI) and two TGFβ receptor II (TGFβRII). In some embodiments, the DN-TGFβR is DN-TGFβRII (i.e., a variant or mutant of TGF beta receptor II). In some embodiments, the tolerance is to the inhibitory effects of TGFβ signaling on immune cells, such as T cells, where TGFβ may be produced by cancer cells or by other immune cells in the cellular environment, such as stromal cells, macrophages, myeloid cells, epithelial cells, natural killer cells, etc. TGFβ signaling inhibitors are well known in the art and include, but are not limited to, mutant TGFβ, antibodies that bind to TGFβ and / or TGFβ receptors (e.g., lerdelimumab, metlimumab, fressolimumab, etc.) that sequester the receptor and inhibit signaling, soluble TGFβ binding proteins (e.g., TGFβRII-Fc fusion proteins) that sequester TGFβ, such as portions of the TGFβ receptor, or other binding agents such as betaglycan. Any known TGFβ signaling inhibitor can be used in place of or in addition to the DN-TGFβR (e.g., DN-TGFβRII) described herein. In some embodiments, the DN-TGFβR lacks the intracellular portion required for TGFβ-mediated signaling, such as the entire intracellular domain or the kinase signaling domain.DN-TGFβR constructs are well known in the art (Brand et al. (1993) J. Biol. Chem. 268:11500-11503; Weiser et al. (1993) Mol. Cell Biol. 13:7239-7247; Bollard et al. (2002) Blood 99::3179-3187; PCT Publication No. WO2009 / 152610; PCT Publication No. WO2017 / 156484; Kloss et al. (See representative non-limiting embodiments of PCT Publication No. WO.2019 / 089884; PCT Publication No. WO2020 / 042647; and PCT Publication No. WO2020 / 042648).

[0061] In some embodiments, immune cell products (e.g., engineered T cells) comprising one or more binding proteins (e.g., TCRs) described herein are resistant to β-mediated immunosuppression. As described above and further herein, TGFβ is an immunosuppressive cytokine produced by tumor cells and cells in the tumor microenvironment. TGFβ inhibits the function and proliferation of cytotoxic T cells and Th1 helper T cells, suppressing tumor-specific T cell responses (Dahmani and Delisle (2018) Cancers 10:194). TGFβ signaling in T cells is inhibited by expression of the dominant-negative TGFβ type II receptor (DN-TGFβRII) (Wieser et al. (1993) Mol. Cell Biol. 13:7239-7247; Bollard et al. (2002) Blood 99:3179-3187). Upon binding to TGFβ, wild-type TGFβRII is phosphorylated, thereby activating TGFβRI and initiating intracellular signaling. This signaling cascade is disrupted in cells expressing a truncated form of TGFβRII (DN-TGFβRII) that lacks the intracellular kinase domain, rendering the cells resistant to inhibition by TGFβ. DN-TGFβRII inhibits TGFβ signaling in engineered T cells (both CAR-T cells and TCR-T cells) (Bollard et al. (2002) Blood 99:3179-3187; Foster et al. (2008) J. Immunother. 31:500-505; Kloss et al. (2018) Mol. Ther. 26:1855-1866; Alabanza et al. (2022) Front Immunol. 13:832645; Silk et al. (2022) J. Immunol. 208:169-180; Li et al. (2020) Fromt. Oncol. 10:1117). A representative study evaluated EBV-specific T cells loaded with DN-TGFβRII for the treatment of Hodgkin lymphoma and showed that DN-TGFβRII-modified T cells were safe and effective (Bollard et al. (2018) J. Clin. Oncol. 36:1128-1139).

[0062] "Kite T cell receptor" or "comparator T cell receptor" refers to at least one benchmark T cell receptor (e.g., "Kite-439") reported in U.S. Patent No. 10,174,098 and U.S. Patent Application Nos. 62 / 004,335; 61 / 846,167; and 61 / 846,161. In some embodiments, the "Kite" or "comparator" T cell receptor has a sequence set forth in Table 2.

[0063] As used herein, "homology" refers to the similarity of nucleotide sequences between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. If a nucleotide residue position in both regions is occupied by the same nucleotide residue, the regions are homologous at that position. If at least one nucleotide residue position in each region is occupied by the same residue, a first region is homologous to a second region. The homology between two regions is expressed by the percentage of nucleotide residue positions occupied by the same nucleotide residue in the two regions. As an 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, such that at least about 50%, and in some 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 inclusive therein, e.g., at least 80%-100%, of the nucleotide residue positions in each portion are occupied by the same nucleotide residue. In some embodiments, all nucleotide residue positions in each portion are occupied by the same nucleotide residue.

[0064] The term "human papillomavirus" or "HPV" refers to a family of viruses in which infection with some subtypes, such as HPV16 (NCBI Ref.Seq.NC_001526.4), is associated with several disorders, including cancer. In some cases, such disorders are associated with expression of the HPV oncoprotein E7, which is thought to target, for example, tumor-suppressor signaling pathways that regulate cell growth control.

[0065] As used herein, the term "HPV16 E7 11-19 antigen" or "HPV16 E7 11-19 peptide antigen" or "HPV16 E7 11-19 containing peptide antigen" or "HPV16 E7 11-19 epitope" or "HPV16 E7 11-19 peptide epitope" or "HPV16 E7 11-19 "b peptide" refers to a naturally or synthetically produced peptide portion of the HPV16 E7 oncoprotein that comprises, consists of, or consists essentially of the sequence YMLDLQPET.

[0066] "HPV16 E7 11-19 The term "hyperproliferative disease characterized by expression of an antigen" refers to a disorder characterized by the expression of an HPV16 E7 antigen in an MHC (e.g., HLA) complex expressed by at least some hyperproliferative cells of a subject. 11-19 It can be any hyperproliferative disease for which the antigen is present. HPV16 E7 11-19 Examples of hyperproliferative disorders characterized by HLA complexes include solid malignancies, as detailed below.

[0067] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cytotoxicity. Additionally, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral response) and activation of cytokine-responsive cells, e.g., macrophages.

[0068] The increased ability to stimulate an immune response or the immune system may result from enhanced agonist activity of T cell costimulatory receptors and / or enhanced antagonist activity of inhibitory receptors. The increased ability to stimulate an immune response or the immune system may be observed by measuring the release of ECs in assays that measure immune responses (e.g., assays that measure cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly by detecting CD107a or granzymes), and changes in proliferation). 50 or may be reflected by a fold increase in maximum activity level. The ability to stimulate an immune response or immune system activity may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 500% or more.

[0069] The term "immunotherapeutic agent" can include any molecule, peptide, antibody, or other agent capable of stimulating the host 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.

[0070] The term "immune cells" refers to cells of the immune system that are derived from hematopoietic stem cells in the bone marrow and 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). Examples of 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 the major histocompatibility complex (MHC) receptors on the surface of the APC interact with the TCRs on the surface of the T cell in complex with peptides.

[0071] "Isolated protein" refers to a protein that is isolated from a cell or produced by recombinant DNA techniques and is substantially free of other proteins, cellular material, separation medium, and culture medium, or, if chemically synthesized, chemical precursors or other chemicals. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the binding protein, antibody, polypeptide, peptide, or fusion protein is derived, or, if chemically synthesized, is substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" includes preparations of a biomarker polypeptide or fragment thereof in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of biomarker proteins or fragments thereof having less than about 30% (by dry weight) non-biomarker proteins (also referred to herein as "contaminating proteins"), or in some embodiments, about 25%, 20%, 15%, 10%, 5%, 1% or less, or any range thereof inclusive, e.g., less than about 1% to 5% 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 is preferably substantially free of culture medium, i.e., culture medium represents about 20%, 15%, 10%, 5%, 1% or less, or any range thereof inclusive, e.g., less than about 1% to 5% of the volume of the protein preparation.

[0072] As used herein, the term "isotype" refers to the antibody class (e.g., IgM, IgG1, IgG2C, etc.) that is encoded by heavy chain constant region genes.

[0073] As used herein, "K DThe term "" is intended to refer to the dissociation equilibrium constant of a particular binding protein-antigen interaction. The binding affinity of the binding proteins encompassed by the present invention can be measured or determined by standard binding protein target binding assays, e.g., 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 1 × 10 for Kd values ​​below M (500uM) -4 Kd values ​​of M (100uM) are included, where a Kd of 100uM indicates a relatively higher binding affinity compared to a Kd of 500uM).

[0074] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent, such as a probe or small molecule for specifically detecting and / or affecting the expression of a marker encompassed by the present invention. A kit may be promoted, distributed, or sold as a unit for performing a method encompassed by the present invention. A kit may also contain one or more reagents necessary to deliver a composition useful in a method encompassed by the present invention. In some embodiments, a kit may further include a reference standard, e.g., a nucleic acid encoding a protein that does not affect or regulate a signaling pathway controlling cell growth, division, migration, survival, or apoptosis. One skilled in the art can envision many such regulatory proteins, including, but not limited to, common molecular tags (e.g., green fluorescent protein and beta-galactosidase), proteins that do not fall into any of the pathways involving cell growth, division, migration, survival, or apoptosis by reference to Gene Ontology, or ubiquitous housekeeping proteins. Reagents in a kit may be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, instructional materials describing the use of the compositions in the kit may be included.

[0075] As used herein, the term "linked" refers to the attachment of two or more molecules. The attachment can be covalent or non-covalent. The attachment can also be genetic (i.e., recombinant fusion). Such attachment can be achieved using a variety of techniques recognized in the art, including chemical conjugation and recombinant protein production.

[0076] In some embodiments, a "linker" refers to an amino acid sequence connecting two proteins, polypeptides, peptides, domains, regions, or motifs, and can provide a spacer function compatible with the interaction of two sub-binding domains so that the resulting polypeptide retains a particular binding affinity for a target molecule (e.g., an scTCR) or signaling activity (e.g., a TCR complex). In some embodiments, the linker is composed of, for example, about 2 to about 35 amino acids, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids.

[0077] "Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a membrane-bound, non-covalently associated b2 microglobulin spanning chains (containing three a domains). MHC class II molecules are composed of two transmembrane glycoproteins, a and b, each of which has two domains. MHC class I molecules deliver peptides derived from the cytoplasm to the cell surface, where the peptide antigen-MHC (pMHC) complex is expressed by CD8 + MHC class II molecules deliver peptides generated in the vesicle system to the cell surface, where they are recognized by CD4 T cells. + Recognized by T cells. Human MHC is called human leukocyte antigen (HLA).

[0078] The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk of or susceptible to developing the disease, disorder, or condition.

[0079] The term "prognosis" includes a prediction of the expected course and outcome of a viral infection, or the likelihood of recovery from the disease. In some embodiments, the use of a statistical algorithm provides a prognosis for a viral infection in an individual. For example, the prognosis can be surgery, development of a clinical subtype of the viral infection, development of one or more clinical factors, or recovery from the disease.

[0080] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology," which can be determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 (modified by Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877). The described algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the polynucleotides described herein. BLAST protein searches are performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic 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.

[0081] 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.

[0082] The term "recombinant host cell" (or simply "host cell") refers to a cell that contains a nucleic acid that is not naturally occurring within the cell, such as a cell into which a recombinant expression vector has been introduced. It should be understood that the cells of the present invention are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the cells herein.

[0083] The terms "cancer response," "response to immunotherapy," or "response to a modulator of T-cell-mediated cytotoxicity / immunotherapy combination therapy" refer to any response of a hyperproliferative disorder (e.g., cancer) to a cancer mediator, such as a modulator of T-cell-mediated cytotoxicity, and a change in tumor mass and / or volume after the initiation of neoadjuvant or adjuvant therapy, such as immunotherapy. The term "neoadjuvant therapy" refers to treatment given before primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormonal therapy. Hyperproliferative disorder response can be assessed, for example, for efficacy or in the neoadjuvant or adjuvant setting, and tumor size after systemic intervention can be compared to the initial size and dimensions measured by CT, PET, mammogram, ultrasound, or palpation. Response can also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Responses may be recorded in a quantitative manner, such as the percent change in tumor volume, or in a qualitative manner, such as "pathological complete response" (pCR), "clinical complete response" (cCR), "clinical partial response" (cPR), "clinical stable state" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. Assessment of hyperproliferative disorder response may be performed early, for example, hours, days, weeks, or preferably months, after the initiation of neoadjuvant or adjuvant therapy. A typical endpoint for response assessment is the end of neoadjuvant chemotherapy or surgical removal of residual tumor cells and / or tumor bed, which is typically 3 months after the initiation of neoadjuvant therapy. In some embodiments, the clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the percentage (%) of patients in complete remission (CR), the number of patients in partial remission (PR), and the number of patients with stable disease (SD) at least 6 months after the end of treatment. A shorthand notation for this formula is CBR = CR + PR + SD over 6 months.In some embodiments, the CBR for a particular cancer treatment regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating response to cancer treatment relate to "survival," which includes all of the following: survival until death, also known as overall survival (death can be cause-unrelated or tumor-related); "recurrence-free survival" (the term recurrence is intended to include both local and distant recurrence); metastasis-free survival; and disease-free survival (the term disease is intended to include cancer and its associated diseases). The length of survival can be calculated by reference to a defined starting point (e.g., diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, criteria for treatment efficacy can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence. For example, to determine an appropriate threshold, a specific cancer treatment regimen can be administered to a population of subjects, and the outcome can be correlated with biomarker measurements determined before any cancer treatment was administered. The outcome measurement can be a pathological response to treatment given in a neoadjuvant setting. Alternatively, outcome indicators such as overall survival and disease-free survival can be monitored over a period of time for subjects after cancer treatment for which biomarker measurements are readily available. In certain embodiments, the administered dose is a standard dose known in the art for cancer therapeutic agents. The duration for which subjects are monitored can vary. For example, subjects can be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement thresholds that correlate with cancer treatment outcomes can be determined using methods well known in the art, such as those described in the Examples section.

[0084] As indicated, the term can also refer to improved prognosis, for example, as reflected by an increased time to recurrence, which is the time to censoring the first recurrence of a second primary cancer as a first event or death without evidence of recurrence or an increase in overall survival, which is the time from treatment to death from any cause.Responding or having a response means that there is a beneficial endpoint achieved when exposed to a stimulus. Alternatively, negative or adverse symptoms are minimized, alleviated, or attenuated when exposed to a stimulus.It will be understood that assessing the likelihood that a tumor or subject will respond favorably is equivalent to assessing the likelihood that a tumor or subject will not respond favorably (i.e., showing a lack of response or not responding).

[0085] The term "resistance" (i.e., no response or reduced or limited response to therapeutic treatment) refers to acquired or natural resistance of a cancer sample or mammal to cancer treatment, such as a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more reduction in response to cancer treatment, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, or more, or any range therebetween (including borderlines). The reduced response can be measured by comparing the same cancer sample or mammal with that before resistance was acquired, or by comparing with a different cancer sample or mammal known not to be resistant to therapeutic treatment. Typical acquired resistance to chemotherapy is referred to as "multidrug resistance." Multidrug resistance can be mediated by P-glycoprotein or other mechanisms, or can 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 artisan, and can be measured, for example, by cell proliferation and cell death assays, described herein as "sensitizing." In some embodiments, the term "reverse resistance" refers to the use of a second agent in combination with a primary cancer treatment (e.g., chemotherapy or radiation therapy) that can produce a significant reduction in tumor volume at a statistically significant level (e.g., p<0.05) compared to the tumor volume of an untreated tumor in situations where the primary cancer treatment (e.g., chemotherapy or radiation therapy) alone fails to produce a statistically significant reduction in tumor volume compared to the tumor volume of an untreated tumor. This generally applies to tumor volume measurements taken when an untreated tumor is growing exponentially.

[0086] The term "sample" as used to detect or determine the absence, presence or level of at least one biomarker typically refers to brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., those described in the definition of "bodily fluid" above), or a tissue sample (e.g., biopsy) such as a small intestine, colon sample, or surgically resected tissue. In some embodiments, methods encompassed by the invention further comprise obtaining a sample from an individual prior to detecting or determining the absence, presence, or level of at least one marker in the sample.

[0087] The term "sensitization" refers to modifying cancer or tumor cells in a manner that allows for more effective treatment of the associated cancer with cancer therapies (e.g., anti-immune checkpoint agents, chemotherapy, and / or radiation therapy). In some embodiments, normal cells are not affected to the extent that they are unduly damaged by the treatment. Increased or decreased sensitivity to therapeutic treatment can be measured using 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. Langhome PA; Harwood Academic Sensitivity or resistance can be measured according to methods known in the art for the particular treatment and methods described below, including, but not limited to, steroid therapy (e.g., steroid therapy, anti-inflammatory drug ... A composition or method sensitizes a response to a therapeutic treatment if the increase in therapeutic sensitivity or decrease in resistance is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, or any range therebetween (inclusive), compared to the therapeutic sensitivity or resistance in the absence of such composition or method. 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 enhancing the effectiveness of cancer treatment are equally applicable to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to cancer treatment.

[0088] The term "small molecule" is a term of the art and includes molecules with a molecular weight of less than about 1000 or less than about 500. In one embodiment, a small molecule is not composed exclusively of peptide bonds. In another embodiment, a small molecule is not an oligomer. Exemplary small molecule compounds that can be screened for activity include, but are not limited to, peptides, 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 biosynthesized.

[0089] The term "specific binding" refers to the binding of a binding protein to a predetermined antigen. Typically, when measured in a binding assay, such as by 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, the binding protein binds to a specific antigen at 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 -12M or less, approximately 1×10 -12 M or lower, or even lower affinity (K D ), or any range of affinity (K ) therebetween (including boundaries), such as about 1-50 micromolar, 1-100 micromolar, or 0.1-500 micromolar. D ) binding affinity. In 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 times greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "binding protein that specifically binds to an antigen." Selective binding is a relative term that refers to the ability of a binding protein to distinguish between the binding of one antigen and other antigens (e.g., a particular family member or antigen target versus a related family member or antigen target). For example, analytical data provided in the Examples section demonstrate that the binding proteins described herein bind to HPV16 E7. 11-19 Specific binding to an immunogenic epitope and / or multiple related epitopes (e.g., HPV16 E7 11-19 It has been demonstrated that these targets selectively bind to specific epitopes (immunogenic epitopes and closely related sequences) and distinguish such targets from the majority of other potential epitopes available in the human genome.

[0090] The term "subject" refers to any healthy animal, mammal, or human, or to a subject infected with HPV16 E7 11-19 "Subject" refers to any animal, mammal, or human suffering from a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of an antigen. The term "subject" is interchangeable with "patient."

[0091] The term "survival" includes all of the following: survival until death, also known as overall survival (death may not be related to either the associated cause or the tumor), "recurrence-free survival" (the term recurrence includes both local and distant recurrence), metastasis-free survival, and disease-free survival (the term disease includes cancer and related diseases). The length of survival may be calculated by reference to a defined starting point (e.g., diagnosis or start of treatment) and end point (e.g., death, recurrence, or metastasis). Furthermore, the criteria for treatment efficacy may be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given period, and probability of tumor recurrence.

[0092] The term "synergistic effect" refers to a combination of two or more drugs (e.g., HPV16 E7 as described herein) that is greater than the sum of the individual effects of the cancer drugs / therapies alone. 11-19 Related drugs and HPV16 E7 11-19 The term "combined therapy" refers to the combined effect of a therapeutic agent and another therapeutic agent for treating a disorder characterized by the development of a

[0093] As used herein, the term "T cell-mediated response" refers to a response mediated by effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + The term "T cell-mediated response" refers to responses mediated by T cells, including T cells. T cell-mediated responses include, for example, T cell cytotoxicity and proliferation.

[0094] A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of such RNA or cDNA) that is complementary 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.

[0095] "T cells" are immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells are classified as naive T cells (those that have not been exposed to antigens, i.e., T CM expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA is increased and expression of CD45RO is decreased compared to normal T cells), memory T cells (T M ) (antigen-experienced and long-lived), and effector cells (antigen-experienced and cytotoxic). M Furthermore, central memory T cells (T CM , which have increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95 and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (T EM , naive T cells or T CM They are divided into effector T cells (T E ) refers to antigen-experienced CD8+ cytotoxic T lymphocytes, and T CM Other exemplary T cells include CD4 + CD25 + (Foxp3 + ) Regulatory T cells, such as regulatory T cells and Treg17 cells, as well as Tr1, Th3, and CD8 + CD28 and Qa-1 restricted T cells.

[0096] Conventional T cells, also known as Tconv or Teff, have effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) and augment immune responses by expressing one or more T cell receptors. Tcon or Teff are generally defined as any T cell population that is not a Treg, including, for example, naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated, e.g., into the Th1 or 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 positive and negative central selection processes in the thymus but have not yet been activated by exposure to antigen. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), the absence of activation markers such as CD25, CD44, and CD69, and the absence of memory markers such as CD45RO. Thus, naive T cells are considered quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see at least WO 2010 / 101870). The presence and activity of such cells are undesirable in situations where immune responses are to be suppressed. Unlike Tregs, T cells 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).

[0097] "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 induce other immune cells, but do not include regulatory T cells (Treg cells).

[0098] "T cell receptor" or "TCR" refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al. (1997) Curr. Biol. Publ. 4:33) that can bind (e.g., specifically and / or selectively) to antigenic peptides bound to MHC receptors. TCRs exist on the cell surface or in soluble form and are generally heterodimers with an alpha and a beta chain (also called TCRα and TCRβ, respectively), or gamma and delta chains (also called TCRγ and TCRδ, respectively). Similar to immunoglobulins (e.g., antibodies), the extracellular portions of TCR chains (e.g., α and β chains) contain variable domains (e.g., α chain variable domains or V chains) at the N-terminus and a V-terminal end. α and β chain variable domain or V β : Usually, Kabat numbering (Kabat et al. (1991) "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 5 th ed.)) and one constant domain (e.g., an α chain constant domain or C α , usually amino acids 117-259 according to Kabat numbering, the β chain constant domain or C β, typically amino acids 117-295 according to Kabat numbering. Similarly to immunoglobulins, variable domains also contain complementarity-determining regions ("CDRs," also called hypervariable regions or "HVRs") separated by framework regions ("FRs") (see, e.g., Fores et al. (1990) Proc. Natl. Acad Sci. USA 87:9138; Chothia et al. (1988) EMBO J. 7:3745; Lefranc et al. (2003) Dev. Comp. Immunol. 27:55). In some embodiments, TCRs are present on the surface of T cells (or T lymphocytes) and associate with the CD3 complex. Sources of TCRs encompassed by the present invention may be derived from a variety of animal species, including humans, mice, rats, rabbits, and other mammals.

[0099] The term "T cell receptor" or "TCR" should be understood to include not only complete TCRs, but also antigen-binding portions or fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including αβ or γδ TCRs. In some embodiments, the TCR is an antigen-binding portion that is shorter than the entire full-length TCR but binds to a specific peptide bound to an MHC molecule, e.g., binds to an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR may include only a portion of the structural domains of a full-length or intact TCR, yet is still capable of binding to a peptide epitope, such as an MHC-peptide complex, bound by a full-length TCR. In some cases, the antigen-binding portion includes a variable domain of the TCR, such as the variable α or β chain of the TCR, which is large enough to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity-determining regions (CDRs) involved in recognizing peptides, MHC, and / or MHC-peptide complexes.

[0100] Nomenclature established by the International Immunogenetics Information System (IMGT) (Scaviner and Lefranc (2000) Exp. Clin. Immunogenet. 17:83-96 and 97-106; Folch and Lefranc (2000) Exp. Clin. Immunogenet, 17:107-114; see also "T Cell Receptor Factsbook", (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8). IMGT provides unique sequences used to describe TCRs, and the sequences described herein can be identified by reference to such unique sequences provided herein. TCR sequences are publicly available in the IMGT database at imgt.org.

[0101] As mentioned above, naturally occurring alpha / beta heterodimeric TCRs consist of an alpha chain and a beta chain. Generally, each chain is composed of a variable region, a binding region, and a constant region. Beta chains typically contain a short diversity region between the variable and binding regions, although this diversity region is often considered part of the binding region. Each variable region is composed of three hypervariable CDRs (complementarity-determining regions) embedded in a framework sequence. CDR3s are well-known as the primary mediators of antigen recognition. There are several types of alpha chain variable regions (Vα) and several types of beta chain variable regions (Vβ), distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences. Vα types are designated by unique TRAV numbers in the IMGT nomenclature. For example, "TRAV4" defines a TCR Vα region with unique framework and CDR1 and CDR2 sequences, and a CDR3 sequence that is partially defined by amino acid sequences conserved among TCRs but also contains amino acid sequences that vary among TCRs. Similarly, "TRBV2" defines a TCR Vβ region with unique framework and CDR1 and CDR2 sequences, but only a partially defined CDR3 sequence. The α and β loci are known to contain 54 α variable genes (44 of which are functional) and 67 β variable genes (42 of which are functional), respectively.

[0102] The binding regions of TCRs are similarly defined by the unique IMGT TRAJ and TRBJ nomenclature, and the constant regions are defined by the IMGT TRAC and TRBC nomenclature. The beta chain diversity region is referred to by the abbreviation TRBD in the IMGT nomenclature, and as mentioned above, the concatenated TRBD / TRBJ regions are often considered together as the binding region.

[0103] The gene pools encoding the TCR alpha and beta chains are located on different chromosomes and contain separate V, D, J, and C gene segments that are brought together by rearrangement during T cell development. This results in a great deal of diversity in T cell alpha and beta chains, with numerous potential recombination events occurring 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, resulting in additional diversity within the CDR3 region. Each alpha and beta variable gene is designated TRAVxx in the IMGT nomenclature, respectively. * 01 and * 02, or TRBVx-x * 01 and * They may also contain an allelic variant designated 02, which further increases the amount of variation. Similarly, some TRBJ sequences have two known variations (see * (The absence of a 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 alleles determined by CDR3 diversity. 6 It is estimated that each beta chain is composed of a unique beta chain sequence for each species (Arstila et al. (1999) Science 286:958-961), and possibly many more (Robins et al. (2009) Blood 114:4099-4107). Each beta chain is estimated to pair with at least 25 different alpha chains, thereby generating further diversity (Arstila et al. (1999) Science 286:958-961).

[0104] Thus, the term "TCR alpha variable domain" refers to the concatenation of the TRAV and TRAJ regions, the TRAV region alone, or the TRAV region and a partial TRAJ region; the term TCR alpha constant domain refers to the extracellular TRAC region, or a C-terminal truncated or full-length TRAC sequence. Similarly, the term "TCR beta variable domain" refers to the concatenation of the TRBV and TRBD / TRBJ regions, the TRBV and TRBD regions alone, the TRBV and TRBJ regions alone, or TRBV and a partial TRBD and / or TRBJ region; and the term TCR beta constant domain refers to the extracellular TRBC region, or a C-terminal truncated or full-length TRBC sequence. These nomenclatures for TCR alpha variable domain and TCR beta variable domain apply equally to the variable domains of the TCR gamma and TCR delta chains of gamma / delta TCRs. Those skilled in the art can obtain TRAV, TRAJ, TRAC, TRBV, TRBJ, and TRBC gene sequences, for example, through the publicly available IMGT database.

[0105] The term "TCR complex" refers to a complex formed by binding of CD3 and 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 TCRδ.

[0106] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. Thus, the term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desired physical or mental development and conditions in animals or humans.

[0107] The terms "therapeutically effective amount" and "effective amount" refer to an amount of a substance effective to produce 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 its therapeutic index, solubility, pharmacokinetics, half-life, etc. Toxicity and therapeutic efficacy of a subject compound can be determined, for example, by the LD 50 and ED 50 The LD can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the therapeutic index. In some embodiments, compositions that exhibit large therapeutic indices are used. In some embodiments, the LD 50 The lethal dose (ED) can 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 when the drug is not administered. 50 The IC (i.e., the concentration that achieves half-maximal inhibition of symptoms) can be measured and can be, for example, 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 when the drug is not administered. Similarly, the IC 50can be measured and can be increased by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more for the agent compared to not administering the agent. In some embodiments, the T cell immune response in the assay can be increased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, a reduction in viral load of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% can be achieved.

[0108] The term "treating" refers to the therapeutic management or amelioration of a desired condition (e.g., a disease or disorder). Treatment may include, but is not limited to, the administration of an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically performed in an attempt to alter the course of a disease in a manner beneficial to the subject (the term is used to refer to a disease, disorder, syndrome, or undesirable condition that warrants, or potentially warrants, treatment). The effect of treatment may include reversing, alleviating, reducing the severity of, delaying the onset of, curing, inhibiting progression of, and / or reducing the likelihood of occurrence or recurrence of a disease or one or more symptoms or signs of a disease. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or temporarily alleviating the condition, and achieving remission or improving prognosis. A therapeutic agent may be administered to a subject who has a disease or who is at increased risk of developing a disease compared to members of the general population. In some embodiments, a therapeutic agent can be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent can be administered, for example, to reduce the likelihood of overt disease recurrence. A therapeutic agent can be administered prophylactically, i.e., before the onset of any symptoms or the manifestation of the disease. "Prophylactic treatment" refers to providing medical and / or surgical management to a subject who has not developed a disease or does not subsequently show evidence of the disease, for example, to reduce the likelihood of the disease occurring or to reduce the severity of the disease if it does occur. A subject can be identified as being at risk of developing a disease (e.g., at an increased risk compared to the general population or having risk factors that increase the likelihood of developing a disease).

[0109] The term "unresponsiveness" includes the unresponsiveness of cancer cells to treatment or the unresponsiveness of therapeutic cells, such as immune cells, to stimulation, e.g., stimulation via an activating receptor or cytokine. Unresponsiveness can occur, for example, due to exposure to immunosuppressants or high doses of antigen. As used herein, the terms "anergy" or "tolerance" include unresponsiveness to activating receptor-mediated stimulation. Such unresponsiveness is generally antigen-specific and persists even after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by a lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of a costimulatory polypeptide) results in failure to produce cytokines and therefore failure to proliferate. However, anergic T cells can proliferate when cultured with cytokines (e.g., IL-2). For example, T cell anergy can be observed by the lack of IL-2 production by T lymphocytes measured by ELISA or by proliferation assays using indicator cell lines. Alternatively, reporter gene constructs can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by multimers of AP1 sequences found within heterologous promoters or enhancers under the control of the 5' IL-2 gene enhancer (Kang et al. (1992) Science 257:1134).

[0110] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR) that is involved in binding the protein to an antigen (e.g., a TCR α or β chain (or γ and δ chains in the case of a γδ TCR)). The variable domains of the α and β chains of a native TCR (V, V ... α and V β) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. α The domains are encoded by two separate DNA segments: the variable gene segment and the joining gene segment (VJ): β The domains are encoded by three separate DNA segments: the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). To confer antigen-binding specificity, a single V α or V β Furthermore, a TCR that binds to a specific antigen can be isolated by using a V domain derived from the TCR that binds to that antigen. α or V β The domains were isolated using the complementary V α or V β A library of domains may be screened.

[0111] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, a vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. In some embodiments, a vector is capable of autonomous replication and / or expression of nucleic acids to which it is linked. As used herein, vectors capable of directing the expression of genes to which they are operably linked are referred to as "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of "plasmids," which generally refer to circular double-stranded DNA loops that, in their vector form, are not bound to the chromosome. As used herein, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. However, as will be understood by those of skill in the art, the invention is intended to include other forms of expression vectors that serve equivalent functions and which subsequently become known in the art.

[0112] There is a known and 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 and 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.

[0113] genetic code Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp, D) GAC, GAT Cysteine ​​(Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signals (stop) TAA, TAG, TGA

[0114] An important and well-known feature of the genetic code is its redundancy, which allows more than one coding nucleotide triplet to be used for most amino acids used to make proteins (as shown above). Thus, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent because they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than others). Furthermore, occasionally, methylated variants of purines or pyrimidines can be found within a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

[0115] In view of the above, the nucleotide sequence of DNA or RNA encoding a biomarker nucleic acid (or any portion thereof) can be used to derive a polypeptide amino acid sequence by translating the DNA or RNA into an amino acid sequence using the genetic code. Similarly, for a polypeptide amino acid sequence, the corresponding nucleotide sequence capable of encoding the polypeptide can be deduced from the genetic code (due to its redundancy, multiple nucleic acid sequences are generated 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.

[0116] II. Binding Proteins In one embodiment of the present invention, HPV16 E7 is included in an MHC molecule (e.g., an MHC class I molecule) as described herein. 11-19Binding proteins are provided that bind (e.g., specifically and / or selectively) to peptide-MHC (pMHC) complexes that include immunogenic peptides. In some embodiments, the binding proteins are HPV16 E7 11-19 Approximately 5 × 10 -4 M or less, approximately 1×10 -4 M or less, about 5 x 10 -5 M or less, approximately 1×10 -5 M or less, about 5 x 10 -6 M or less, approximately 1×10 -6 M or less, about 5 x 10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, approximately 1×10 -9 M or less, about 5 x 10 -10 M or less, approximately 1×10 -10 M or less, about 5 x 10 -11 M or less, approximately 1×10 -11 M or less, about 5 x 10 -12 M or less, approximately 1×10 -12 K below M d or any range therebetween (including limits), such as about 1 to 50 micromolar, 1 to 100 micromolar, or 0.1 to 500 micromolar. d In some embodiments, the MHC molecule is of the HLA serotype HLA-A * In some embodiments, 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 * In certain embodiments, the HLA allele is selected from the group consisting of HLA-A 0207 alleles. * 0201. In some embodiments, the binding proteins provided herein are engineered, isolated, and / or purified.

[0117] In some embodiments, the binding protein binds to HPV16 E7 T cell receptors rather than known T cell receptors (e.g., Kite TCRs described herein). 11-19 have high binding affinity to peptide-MHC (pMHC). For example, the binding protein has 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, 5000x, 10000x, 50000x, 100000x, 500000x, 1000000x, or more, or any range therebetween (including boundaries), such as 1.2x to 2x, inclusive.

[0118] In some embodiments, the binding protein is at or below a particular level of HPV16 E7 11-19 When contacted with a target cell having expression of HPV16 E7, the T cell receptor induces greater T cell proliferation, cytokine release, and / or cytotoxic killing than known T cell receptors (e.g., Kite TCRs described herein). For example, in some embodiments of any aspect described herein, the T cell receptor induces greater T cell proliferation, cytokine release, and / or cytotoxic killing when contacted with a target cell having expression of HPV16 E7. 11-19Levels can be expressed as transcripts per million, for example, about 1,000 transcripts per million (TPM) or less, 950 TPM, 900 TPM, 850 TPM, 800 TPM, 750 TPM, 700 TPM, 650 TPM, 600 TPM, 550 TPM, 500 TPM, 450 TPM, 400 TPM, 350 TPM, 300 TPM, 250 TPM, 200 TPM, 150 TPM, 100 TPM, 95 TPM, 90 TPM, 85 TPM, 80 TPM, 75 TPM, 70 TPM, 65 TPM, 60 TPM, 55 TPM, 50 TPM, 45 TPM, 40 TPM, The HPV concentration can be 35 TPM, 34 TPM, 33 TPM, 32 TPM, 31 TPM, 30 TPM, 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or any range therebetween (inclusive), such as about 1,000 TPM or less to about 35 TPM or less. 11-19 The expression level is referred to as "heterozygous expression" and means about 1 TPM to about 35 TPM, or 32 TPM, or any range therebetween (inclusive), such as 1-32 TPM. Higher expression is 36 TPM or greater. As further described herein, TPM is measured according to well-known techniques, such as RNA-Seq, and gene expression TPM data is well known in the art for various cell lines, tissue types, and the like (see, e.g., the Broad Institute Cancer Cell Line Encyclopedia (CCLE) at portals.broadinstitute.org on the World Wide Web). In some embodiments, the binding protein is HPV16 E7. 11-19When contacted with a target cell expressing the peptide epitope, the peptide is transduced into a known T cell receptor (e.g., a Kite as described herein). TCR), or by at least a 1.2-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold or more, or any range therebetween, such as 1.2-fold to 2-fold (inclusive).

[0119] In some embodiments, HPV16 E7 11-19Expression of 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 cell population, a tissue sample, a biopsy sample, a cell culture, or a single cell. Total RNA can be isolated from a biological sample using any method known in the art. In certain embodiments, total RNA is extracted from plasma. Plasma RNA extraction is described in Enders et al., "The Concentration of Circulating Corticotropin-Releasing Homer mRNA in Material Plasma Is Inclined in Preclampsia," Clinr. As described therein, plasma collected after a centrifugation step is mixed with Trizol LS reagent (Invitrogen) and chloroform. The mixture is centrifuged, and the aqueous layer is transferred to a new tube. Ethanol is added to the aqueous layer. The mixture is then loaded into an RNeasy mini-column (Qiagen) and processed according to the manufacturer's recommendations.

[0120] In some embodiments, RNA-seq as described herein includes preparing amplified cDNA from total RNA. For example, cDNA is prepared and the isolated RNA sample is randomly amplified without dilution, or the mixture of genetic material within the isolated RNA is dispersed into individual reaction samples. In certain embodiments, amplification is initiated randomly at the 3' end of the sample and across the entire transcriptome, amplifying both mRNA and non-polyadenylated transcripts. In this way, the double-stranded cDNA amplification product is optimized for generating sequencing libraries for next-generation sequencing platforms. Kits suitable for amplifying cDNA using the methods encompassed by the present invention include, for example, the Ovation® RNA-Seq System.

[0121] In some embodiments, the RNA-seq described herein comprises sequencing the amplified cDNA. To determine the sequence of the amplified cDNA mixture, any known sequencing method, including single molecule sequencing, can be used. In certain embodiments, the amplified cDNA is sequenced by whole transcriptome shotgun sequencing. Whole transcriptome shotgun sequencing can be performed using various next-generation sequencing platforms, such as the Illumina® Genome Analyzer platform, the ABI SOLiD™ sequencing platform, or Life Science's 454 sequencing platform.

[0122] In some embodiments, the RNA-seq method described herein further comprises performing digital counting and analysis on the cDNA. The number of amplified sequences for each transcript in an amplified sample can be quantified by sequence reads (one read per amplified strand). In some embodiments, transcripts per million (TPM) are used to quantify the expression level of a particular transcript. TPM can be calculated as shown in Wagner et al. (2012) Theory in Biosciences 131:281-285, the contents of which are incorporated herein by reference in their entirety.

[0123] In some embodiments, the binding protein does not bind to a peptide-MHC (pMHC) complex, and optionally the peptide is derived from SPTA1, MPL, HERC1, CPAMD8, INTS4, NUTM1, or XM_00172256.

[0124] In some embodiments, the binding protein does not bind to SPTA1-, MPL-, HERC1-, CPAMD8-, INTS4-, NUTM1-, and / or XM_00172256-peptide-MHC (pMHC) complexes.

[0125] 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 sequences listed in Table 1 and at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, a) a TCR alpha chain sequence having 98%, 99%, or greater identity; 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 greater identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 1.

[0126] In some embodiments, the binding proteins provided herein include (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.

[0127] In some embodiments, the binding proteins provided herein include (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 ) 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 a TCR alpha chain variable (V 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 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 variable (V β ) domain array.

[0128] In some embodiments, the binding proteins provided herein include (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.

[0129] 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. Because CDR3 is believed to be the primary CDR that recognizes processed antigens, while CDR1 and CDR2 primarily interact with MHC, in some embodiments, binding proteins are provided that include only CDR3s from the TCR alpha chain and / or only CDR3s from the TCR beta chain listed in Table 1, each having the sequence homology listed in this paragraph.

[0130] 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 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, e.g., the TCR beta chain CDR sequences listed in Table 1. As noted above, CDR3 is believed to be the primary CDR that recognizes processed antigens, while CDR1 and CDR2 primarily interact with MHC; therefore, in some embodiments, binding proteins are provided that comprise only CDR3s from the TCR beta chain and / or only CDR3s from the TCR alpha chain listed in Table 1, each having the sequence homology listed in this paragraph.

[0131] 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) of) the TCR alpha chain complementarity determining regions (CDRs) listed in Table 1.

[0132] 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) of) the TCR beta chain complementarity determining regions (CDRs) listed in Table 1.

[0133] In some embodiments, the binding proteins provided herein comprise a TCR alpha chain constant region (Cα) 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 Cα sequence listed in Table 1.α ) sequence.

[0134] In some embodiments, the binding proteins provided herein bind to 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 a sequence β ) sequences.

[0135] In some embodiments, the binding proteins provided herein include those containing a TCR C α A TCR alpha chain constant region (C) selected from the group consisting of α ) sequence.

[0136] In some embodiments, the binding proteins provided herein include those containing a TCR C β A TCR beta chain constant region (C) selected from the group consisting of β ) sequences.

[0137] In some embodiments, the binding proteins provided herein comprise chimeric, humanized, human, primate, or rodent (e.g., rat or mouse) constant regions. For example, a human variable region is chimerized with a mouse constant region, or a mouse variable region is humanized with a human constant region and / or human framework regions. In some embodiments, the constant region can be mutated to alter functionality (e.g., introducing non-natural 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, the transmembrane domain of the constant region can be mutated to alter functionality (e.g., increasing hydrophobicity by introducing non-natural substitutions at residues with hydrophobic amino acids). In some embodiments, the constant region can be mutated to increase cell surface expression. [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 2-1 Table 2-2 Table 2-3 *Representative TCR sequences provided in Table 1 are grouped according to MHC serotype presentation and subgrouped according to the different peptides presented by the MHC serotypes and bound by the subgrouped TCRs. Individual TCRs, such as the representative ones illustrated in the table, are described and claimed, as are types of binding proteins that bind to the peptide epitope sequences described herein, either alone or in complexes with MHC as grouped in the tables described herein. Additionally, the TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein are provided. The sequences of each TCR described herein are provided as the cognate alpha and beta chain pair for each TCR listed therein. The TCR sequences described herein are annotated. Variable domain sequences are written in uppercase. Constant domain sequences are in lowercase. CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined text. CDR1, CDR2, and CDR3 are listed in standard order of appearance from left (N-terminus) to right (C-terminus). The TRAV, TRAJ, and TRAC genes for each TCR alpha chain and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein are annotated according to the well-known IMGT nomenclature described herein. Similarly, CDR1 and CDR2 of TRAV and TRBV are well known in the art, as they are based on well-known annotated TRAV and TRBV sequences (e.g., as annotated in databases such as IMGT, available at imt.org, and IEDB, available at iedb.org). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] * Annotation of the vectors in Table 3 is as follows: MSCV promoter is shown in bold. Beta chains are annotated using bold and italic letters. Alpha chains are annotated using bold and underlined letters. Q tags are annotated using italic and underlined letters. CD8-alpha is shown in italics. CD8-beta is underlined. *Tables 1-3 herein include peptide epitopes and polypeptide molecules comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity over its entire length to the amino acid sequence of any of the SEQ ID NOs: listed in Tables 1-4, or a portion thereof. Such polypeptides can have the function of the full-length peptide or polypeptide, as further described herein. * Tables 1-3 include RNA nucleic acid molecules (e.g., in which thymidines are substituted with uridines), nucleic acid molecules encoding orthologs of the encoded proteins, and DNA or RNA nucleic acid sequences that contain a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over its entire length to the nucleic acid sequence of any of the sequences listed in Tables 1-4, or a portion thereof. Such nucleic acid molecules can have the function of the full-length nucleic acid, as further described herein.

[0138] 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 comprise two polypeptide chains, each comprising a variable region comprising the CDR3 of the TCR alpha chain and the CDR3 of the TCR beta chain, or the CDR1, CDR2, and CDR3 of both the TCR alpha chain and the TCR beta chain. In some embodiments, the binding proteins comprise a TCR V α and TCR V β domains, but a single TCR constant domain (C α or C β"Chimeric antigen receptor" (CAR) refers to a fusion protein that has been engineered to contain two or more naturally occurring amino acid sequences linked in a manner that does not occur naturally or within a host cell, and which can function as a receptor when presented 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 obtained or derived 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 a 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).

[0139] In some embodiments, 1) the TCR alpha chain CDRs, TCR V α and / or 2) the TCR beta chain CDRs, TCR V, are encoded by a TRAV, TRAJ, and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ, and TRAC genes listed in Table 1. β The domain and / or TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 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 sequences listed in Table 1.

[0140] In some embodiments, the binding proteins disclosed herein (e.g., TCRs, antigen-binding fragments of TCRs, or chimeric antigen receptors (CARs)) are chimeric (e.g., containing amino acid residues or motifs from multiple donors 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.

[0141] Methods for generating engineered binding proteins such as TCRs, CARs, and antigen-binding fragments thereof are well known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 5:3000; U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO2014 / 031687, U.S. Patent No. 7,514,537, and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0142] In some embodiments, the binding protein described herein is a TCR or antigen-binding fragment thereof expressed on the cell surface, and the cell surface-expressed TCR can associate with CD3 protein more efficiently than an endogenous TCR. When expressed on the surface of a cell, such as a T cell, the binding protein, e.g., a TCR, included in the present invention may also have higher surface expression on the cell than an endogenous binding protein, e.g., an endogenous TCR. In some embodiments, the present invention provides a CAR in which the binding domain of the CAR comprises an antigen-specific TCR binding domain (see, e.g., Walseng et al. (2017) Scientific Reports 7:10713).

[0143] Also disclosed herein are V α and / or V βAlso provided are modified binding proteins (e.g., TCRs, antigen-binding fragments of TCRs, or CARs) that can be prepared according to well-known methods using as a starting material a binding protein having one or more of the sequences to engineer a modified binding protein that may have altered properties from the starting binding protein. The binding protein may have one or more variable regions (i.e., V α and / or V β ), for example, one or more residues in one or more CDR regions and / or one or more framework regions. Additionally, or alternatively, binding proteins can be engineered by modifying residues in the constant region(s).

[0144] Another type of variable region modification is V α and / or V β Amino acid residues within the CDR1, CDR2, and / or CDR3 regions of a CDR1 protein are mutated to thereby improve one or more binding characteristics (e.g., affinity) of the binding protein of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on protein binding or other functional properties of interest can be assessed in in vitro, ex vivo, or in vivo assays, as described herein and illustrated in the Examples. In some embodiments, conservative modifications (as explained above) may be introduced. The mutations can be amino acid substitutions, additions, or deletions. In some embodiments, the mutations are substitutions. Furthermore, typically, no more than 1, 2, 3, 4, or 5 residues within the CDR regions are modified.

[0145] 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 sequences 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 can be introduced into proteins using mutagenesis methods known in the art (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY). Oligonucleotide-directed site-specific (or segment-specific) mutagenesis can be used to provide modified polynucleotides in which specific codons are altered according to the desired substitution, deletion, or insertion. Alternatively, immunogenic polypeptide variants can be prepared using random or saturation mutagenesis techniques such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis (see, e.g., Sambrook et al., supra).

[0146] Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered more difficult to classify, shares properties with amino acids with aliphatic side chains (e.g., leucine, valine, isoleucine, alanine, etc.). In some embodiments, substituting glutamine for glutamic acid or asparagine for aspartic acid may 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 a 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 herein and practiced in the art).

[0147] In some embodiments, the encoded binding protein (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) can include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). The signal peptide directs a newly synthesized polypeptide to the appropriate location inside or outside the cell. The signal peptide may be removed from the polypeptide during localization or secretion, or once localization or secretion is complete. A polypeptide having a signal peptide is referred to herein as a "preprotein," and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In some embodiments, the binding proteins (e.g., a TCR, an antigen-binding fragment of a TCR, or a CAR) described herein 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 beta chain, a mature TCR alpha chain, or both.

[0148] In some embodiments, the binding protein is a fusion protein that includes (a) an extracellular component comprising a TCR or antigen-binding fragment thereof, (b) an intracellular component comprising an effector domain or functional portion thereof, and (c) a transmembrane domain linking the extracellular and intracellular components. In some embodiments, the fusion protein binds HPV16 E7 in an MHC molecule (e.g., an MHC class I molecule). 11-19 In some embodiments, the MHC molecule is capable of binding (e.g., specifically and / or selectively) to a peptide-MHC (pMHC) complex comprising an immunogenic peptide. In some embodiments, the MHC molecule is of the HLA serotype HLA-A. * In some embodiments, 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* In certain embodiments, the HLA allele is selected from the group consisting of HLA-A 0207 alleles. * The time is 0201.

[0149] As used herein, an "effector domain" or "immune effector domain" is an intracellular portion or domain of a fusion protein or receptor that can directly or indirectly stimulate an immune response within a cell upon receiving an appropriate signal. In some embodiments, the effector domain is derived from an immune cell protein or portion thereof or immune cell protein complex that receives a signal upon binding (e.g., CD3ζ) or when the immune cell protein or portion thereof or immune cell protein complex directly binds to a target molecule, triggering signaling in the immune cell from the effector domain.

[0150] Effector domains may directly promote cellular responses if they contain one or more signaling domains or motifs, such as intracellular tyrosine-based activation motifs (ITAMs) found in costimulatory molecules. Without wishing to be bound by theory, ITAMs are believed to be useful in activating T cells following ligand binding by a T cell receptor or fusion protein containing a T cell effector domain. In some embodiments, the intracellular component or functional portion thereof comprises an ITAM. Examples of 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).

[0151] 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 include 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.

[0152] 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., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an LFA-1 endodomain, or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof. In further embodiments, the effector domain comprises an ICOS endodomain, or a functional (e.g., signal transduction) portion thereof, or a functional variant thereof.

[0153] The extracellular and intracellular components included in the present invention are connected by a transmembrane domain. As used herein, a "transmembrane domain" refers to a portion of a transmembrane protein that can insert into or span a cell membrane. A transmembrane domain has a thermodynamically stable three-dimensional structure within the cell membrane and generally ranges in length from about 15 to about 30 amino acids. The structure of a transmembrane domain may be 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).

[0154] In some embodiments, the extracellular component of the fusion protein further comprises a linker positioned between the binding domain and the transmembrane domain. As used herein, when referring to the component of the fusion protein connecting the binding domain and the transmembrane domain, a "linker" is an amino acid sequence of from about 2 to about 500 amino acids that can provide flexibility and room for conformational movement between the two regions, domains, motifs, fragments, or modules connected by the linker. For example, a linker included in the present invention can position the binding domain away from the surface of the host cell expressing the fusion protein, allowing for proper contact, antigen binding, and activation between the host cell and the target cell (Patel et al. (1999) Gene Therapy 6:412-419). The length of the linker can be varied to maximize antigen recognition based on the capture and affinity of the selected target molecule, selected binding epitope, or antigen-binding domain (see, e.g., Guest et al. (2005) Immunother. 28:203-11 and PCT Publication No. WO2014 / 031687). Exemplary linkers include Gly x Ser ywherein x and y are each independently an integer between 0 and 10, but x and y are not both 0 (e.g., (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or a combination thereof such as ((Gly3Ser)2Gly2Ser)).

[0155] In some embodiments, the binding proteins included in the present invention may be covalently attached to a moiety. In some embodiments, the covalently attached moiety comprises an affinity tag or label. The affinity tag can be selected from the group consisting of glutathione S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label can be a fluorescent protein. In some embodiments, the covalently 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.

[0156] The binding proteins may be conjugated to agents used in imaging, research, therapy, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiation therapy. In some embodiments, the binding proteins may be conjugated or fused to a detectable agent such as a fluorophore, near-infrared dye, contrast agent, nanoparticle, metal-containing nanoparticle, metal chelate, X-ray contrast agent, PET agent, metal, radioisotope, dye, radionuclide chelator, or another suitable material that can be used for imaging. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more detectable moieties may be linked to the binding protein. Non-limiting examples of radioisotopes include alpha-emitters, beta-emitters, positron-emitters, gamma-emitters, and the like. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye is not readily quenched by biological tissues and fluids. In some embodiments, the fluorophore is a fluorescent agent that emits electromagnetic radiation at wavelengths between 650 nm and 4000 nm, and such radiation is used to detect such agents. Non-limiting examples of fluorescent dyes that can be used as binding 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 that can be used as binding molecules in accordance with the present invention include acradin orange or yellow, Alexa Fluors® (e.g., Alexa Fluor® 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO® dyes and any derivatives thereof, auramine-rhodamine stains and any derivatives thereof, bensanthrone, bimane, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene, bisbenzimide, Brainbow, calcein, carbodifluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, DyLight® (Typical) 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, and m isoform proteins and any derivatives thereof, such as mCherry, hetamethine dyes and any derivatives thereof, Hoechst dyes, iminocoumarin, Indian Yellow, Indo-1 and derivatives thereof, laurdan, lucifer yellow and any derivatives thereof, luciferin and any derivatives thereof, luciferase and any derivatives thereof, mercocyanin and derivatives thereof, Nile dyes and derivatives thereof, perylene, phloxine, phycodyes 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, tetraphenylbutadiene, tris tetrasodium, Texas Red, titanium yellow, TSQ, umbelliferone, violanthrone, yellow fluorescent protein, and YOYO-1.Other suitable fluorescent dyes include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine, or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green™ dyes (Oregon Green™ dyes), and the like. Green™ 488, 500, 514, etc.), Texas Red®, Texas Red®-X, SPECTRUM RED®, SPECTRUM GREEN®, cyanine dyes (CY-3, Cy-5, CY-3.5, CY-5.5, etc.), Alexa Fluor® dyes (Alexa Fluor® 350, 488, 532, 546, 568, 594, 633, 660, 680, etc.), BODIPY® dyes (BODIPY® FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, etc.), 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRD dyes (e.g., IRD40™, IRD700™, IRD800™, etc.). Additional suitable detectable agents are well known in the art (e.g., PCT Publication No. PCT / US14 / 56177). Non-limiting examples of radioisotopes include alpha-emitters, beta-emitters, positron-emitters, gamma-emitters, etc.In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.

[0157] The binding protein can be conjugated to a radiosensitizer or photosensitizer. Examples of radiosensitizers include, but are not limited to, ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and 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, naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicin, chalcogenapyrylium dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazines and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., Examples include merocyanine 540), pheophytin, sapphyrin, texaphyrin, purpurin, porphycene, phenothiazinium, methylene blue derivatives, naphthalimides, Nile blue derivatives, quinones, perylenequinones (e.g., hypericin, hypocrellin, cercosporin), psoralens, quinones, retinoids, rhodamines, thiophenes, verdine, xanthine dyes (e.g., eosin, erythrosine, rose bengal), dimeric and oligomeric forms of 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 therapeutic agents (e.g., drugs) and electromagnetic energy (e.g., radiation or light) simultaneously. In some embodiments, the binding protein is fused to, or covalently or non-covalently linked to, the agent, e.g., directly or via a linker.

[0158] In some embodiments, binding proteins may be chemically modified. For example, binding proteins may be mutated to alter peptide properties such as detectability, stability, in vivo distribution, pharmacokinetics, half-life, surface charge, hydrophobicity, binding 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, binding proteins may be modified by methylation of free amines, such as reductive methylation with formaldehyde and sodium cyanoborohydride.

[0159] Chemical modifications may include polymers, polyethers, polyethylene glycol, biopolymers, zwitterionic polymers, polyamino acids, fatty acids, dendrimers, Fc regions, simple saturated carbon chains such as palmitic acid or myristoleic acid, or albumin. Chemical modifications of binding proteins having an Fc region may be fusion Fc proteins. Polyamino acids may include, for example, polyamino acid sequences in which a single amino acid is repeated (e.g., polyglycine), polyamino acid sequences containing mixed polyamino acid sequences with or without a pattern, or any combination of the foregoing.

[0160] In some embodiments, binding proteins encompassed by the invention can be modified, in some embodiments, with substantial or significant sequence identity to the parent binding protein to generate functional variants 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.

[0161] In some embodiments, binding proteins encompassed by the invention can 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-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indolylalanine, α-isopropyl ... cyclohexane-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 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.

[0162] The binding proteins encompassed by the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0163] In some embodiments, attachment of a hydrophobic moiety, such as to the N-terminus, C-terminus, or internal amino acid, can be used to extend the half-life of a peptide 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 (e.g., by myristoylation and / or palmitylation) can be attached to the binding protein. In some embodiments, a simple carbon chain allows for easy separation of the binding protein from unbound substances. For example, methods that can be used to separate the binding protein from unbound substances include, but are not limited to, solvent extraction and reverse-phase chromatography. The lipophilic moiety can extend half-life through reversible binding to serum albumin. The binding moiety can be a lipophilic moiety that extends the half-life of the peptide through reversible binding to serum albumin. In some embodiments, the lipophilic moiety can be cholesterol or a cholesterol derivative, including cholestene, cholestane, cholestadiene, and oxysterols. In some embodiments, the binding protein may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the binding protein may be conjugated (e.g., linked) to a half-life modifying agent. Examples of half-life modifying agents include, but are not limited to, polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, zwitterionic water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers comprising glycine, glutamic acid, and serine, molecules that bind to Fc regions, fatty acids, palmitic acid, or albumin. In some embodiments, a spacer or linker may be attached to the binding protein, such as one, two, three, four, or more amino acid residues that function as a spacer or linker to facilitate conjugation or fusion to other molecules, as well as to facilitate cleavage of the peptide from such conjugated or fused molecules.In some embodiments, the binding proteins may be conjugated with other moieties that can, for example, modify or change the properties of the binding protein.

[0164] Binding proteins can be produced recombinantly or synthetically, such as by solid-phase or solution-phase peptide synthesis. Polypeptides can be synthesized by known synthetic methods, such as fluorenylmethyloxycarbonyl (Fmoc) or butyloxycarbonyl (Boc) chemistry. Polypeptide fragments may be linked together enzymatically or synthetically.

[0165] Aspects included herein provide methods of producing a binding protein described herein, the methods comprising: (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.

[0166] A useful method for isolating and purifying recombinantly produced binding proteins includes, for example, obtaining supernatant from a suitable host cell / vector system that secretes the binding protein into the culture medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as affinity matrices or ion exchange resins. One or more reverse-phase HPLC steps may be employed 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 of the binding proteins described herein involves batch cell cultures that are monitored and controlled to maintain appropriate culture conditions. Purification of the binding proteins can be performed according to methods described herein and known in the art.

[0167] In any of the embodiments disclosed herein, the encoded binding protein is incorporated into an MHC molecule (e.g., an MHC class I molecule) as HPV16 E7. 11-19 In some embodiments, the MHC molecule is capable of binding to a peptide-MHC (pMHC) complex containing an immunogenic peptide. * In some embodiments, 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 * 0207 alleles.

[0168] A variety of assays are well known for assessing binding affinity and / or determining whether a binding molecule binds (e.g., specifically and / or selectively) to a particular ligand (e.g., a peptide antigen-MHC complex). It is within the level of ordinary skill in the art to determine the binding affinity of a binding protein for a target, such as a T cell peptide epitope of a target polypeptide, by using any of a number of binding assays well known in the art. For example, in some embodiments, a Biacore™ instrument can be used to determine the binding constant of a complex between two proteins. The dissociation constant (K D) can be determined by monitoring the change in refractive index over time as buffer solution passes over the chip. Other suitable assays for measuring binding of one protein to another include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or radioimmunoassays (RIAs), or measuring binding by monitoring changes in the spectroscopic or optical properties of the proteins by fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore™) analysis (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660, Wilson (2002) Science 295:2103, Wolff et al. (1993) Cancer Res. 53:2560, and U.S. Pat. Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, apparent affinity for a target is measured by flow cytometry using labeled multimers, such as MHC antigen tetramers, by assessing various concentrations of tetramers. In one representative example, the apparent K of a binding protein is determined by flow cytometry using labeled multimers, such as MHC antigen tetramers, by assessing various concentrations of tetramers. D was measured using two-fold dilutions of various concentrations of labeled tetramer, and the binding curve was subsequently determined by nonlinear regression to give the apparent K D is determined as the concentration of ligand that resulted in half-maximal binding.

[0169] III. Nucleic Acid Vectors In one aspect of 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.

[0170] In some embodiments, a nucleic acid molecule hybridizes under stringent conditions to the complement of a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over its entire length to a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1.

[0171] In some embodiments, the nucleic acid molecule hybridizes under stringent conditions to the complement of a nucleic acid encoding a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1.

[0172] In some embodiments, the nucleic acid molecule comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence that encodes a polypeptide selected from the group consisting of the polypeptide sequences listed in Table 1.

[0173] 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. αIn some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR alpha chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR alpha chain sequence set forth in Table 1.

[0174] 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. β In some embodiments, the nucleic acid comprises (e.g., comprises, consists essentially of, or consists of) a nucleotide sequence encoding a TCR β chain having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β chain sequence set forth in Table 1.

[0175] The term "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA that is single- or double-stranded, synthetic or obtained (e.g., isolated and / or purified) from natural sources, and may contain natural, non-natural, or modified nucleotides, including natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides. In one embodiment, nucleic acid includes complementary DNA (cDNA).

[0176] In some embodiments, the nucleic acids encompassed by the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining natural or synthetic nucleic acid segments to a nucleic acid molecule capable of replicating within the living cell, or (ii) a molecule resulting from replication of the molecule described in (i) above. For purposes herein, replication can be in vitro / ex vivo replication or in vivo replication.

[0177] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green and Sambrook et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylketone, inosine, N-acetyl-3-methyl-4-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-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queusine, 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 included in the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA).

[0178] 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 can increase translation efficiency by replacing a native codon with another codon that encodes the same amino acid but that can be translated by a more readily available tRNA in the cell. Optimizing a nucleotide sequence may also increase translation efficiency by reducing secondary structures in the mRNA that interfere with translation. 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).

[0179] The present invention also provides nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0180] Nucleotide sequences that hybridize under stringent conditions may also hybridize under high stringency conditions. "High stringency conditions" refers to a nucleotide sequence that specifically hybridizes to a target sequence (a nucleotide sequence of any nucleic acid described herein) in an amount detectably stronger than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences, or polynucleotides containing only scattered mismatches, from random sequences with a few small regions (e.g., 3-10 bases) that coincidentally match the nucleotide sequence. Such small regions of complementarity are more easily resolved than full-length complementary regions of 14-17 bases or more, and can be easily distinguished by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by a concentration of about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions tolerate little 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 recognized that conditions can be made more stringent by adding increasing amounts of formamide.

[0181] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the nucleic acids described herein.

[0182] Typically, the nucleic acid is a DNA or RNA molecule that may be contained in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0183] The terms "vector," "cloning vector," and "expression vector" refer to vehicles for introducing DNA or RNA sequences (e.g., foreign genes) into a host cell, transforming the host, and promoting expression (e.g., transcription and translation) of the introduced sequences. Accordingly, a further object of the present invention relates to vectors containing the nucleic acids encompassed by the present invention.

[0184] Such vectors may contain regulatory elements such as promoters, enhancers, and terminators to cause or induce expression of the polypeptide when administered 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).

[0185] 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 adenovirus, retrovirus, lentivirus, herpesvirus, and AAV vectors. Such recombinant viruses can be produced by techniques known in the art, such as transfection of packaging cells or transient transfection with helper plasmids or viruses. Representative examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, and 293 cells. Detailed protocols for producing such replication-defective recombinant viruses are well known in the art and can be found, for example, in PCT Publication No. WO95 / 14785, PCT Publication No. WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056, and PCT Publication No. WO94 / 19478.

[0186] In some embodiments, the expression vector is a nanoplasmid. As used herein, the term "nanoplasmid" refers to a circular DNA sequence with a reduced bacterial sequence, providing a smaller plasmid with a desired cargo insert. The reduced vector size may limit DNA-induced toxicity during transfection, potentially extend intracellular persistence, improve post-transfection survival, and increase translocation efficiency. In some embodiments, the nanoplasmid is a nanoplasmid that does not contain an antibiotic resistance marker. In some embodiments, the nanoplasmid contains a selectable marker and / or a nonsense suppressor marker. Due to their small backbone size (e.g., <500 bp backbone), such nanoplasmids maximize the size of the desired cargo insert. The desired cargo insert (e.g., a eukaryotic transgene) can be any size that can be delivered to a target cell, for example, up to 50 kb, 45 kb, 40 kb, 35 kb, 30 kb, 25 kb, 20 kb, 18 kb, 15 kb, 12 kb, 10 kb, 5.0 kb, 4.5 kb, 4.0 kb, 3.5 kb, 3.0 kb, 2.8 kb, 2.5 kb, 2.2 kb, 2 kb, 1.8 kb, 1.5 kb, 1.2 kb, 1 kb, or any range in between, such as 8-12 kb. They also provide high gene expression and reduced gene silencing due to tunable integration efficiency of the cargo associated with random integration of the cargo into the genome of the genetically modified cell.

[0187] In some embodiments, a Nanoplasmid can contain elements shown in the vectors provided in Table 3 (e.g., R6K and RNA-OUT). For example, in some embodiments, a Nanoplasmid contains a minimized bacterial ColE1 or R6K origin of replication (which allows such Nanoplasmids to replicate in bacterial host strains), a selectable marker (e.g., a bacterial RNA selectable marker), and a eukaryotic gene region. An RNA selectable marker is a non-translated RNA expressed by the vector that controls a target gene expressed on the chromosome, allowing for selection of the vector. It can also be a plasmid-derived nonsense-suppressing tRNA that controls a nonsense-suppressing selectable chromosomal target, as described in U.S. Patent No. 6,977,174, incorporated herein by reference. This can be a plasmid-borne antisense repressor RNA, an RNA-OUT gene that represses an RNA-IN regulatory target, an RNAI encoded pMB1 plasmid origin that represses an RNAII regulatory target, an RNAI encoded IncB plasmid pMU720 origin that represses an RNAII regulatory target, the ParB locus Sok of plasmid RI that represses a Hok regulatory target, the Flm locus FlmB of the F plasmid that represses a flmA regulatory target, a natural antisense repressor RNA as described, for example, in Wagner et al. (2002) Adv. Genet. 46:361, and Franch and Gerdes (2000) Current Opin. Microbiol. 3:159, or an engineered repressor RNA such as a small synthetic small RNA such as the SgrS, MicC, or MicF scaffold described in Park et al. (2013) Nature Biotechnology 31:170-174.

[0188] For exemplary nanoplasmids produced by the antibiotic-free RNA-OUT selection system and methods for producing such nanoplasmids, see, e.g., PCT Publication No. WO2008153733, U.S. Patent No. 9,737,620, U.S. Patent Publication No. 2010 / 0303859, and U.S. Patent No. 9,109,012, each of which is incorporated by reference in its entirety. Additional exemplary nanoplasmids are described, e.g., in PCT Application Nos. PCT / US2013 / 000259, PCT / US2013 / 00067, and PCT / US2013 / 00068, and U.S. Patent Application Publication No. 2015 / 0275221, each of which is incorporated by reference in its entirety.

[0189] Nanoplasmids are commercially available; for example, Nature Technology Corporation, a subsidiary of Aldevron, offers Nanoplasmid vectors that combine an RNA selectable marker with an R6K, ColE2, or ColE2-related origin of replication. These nanoplasmid vectors include, for example, NTC9385C, NTC9685C, NTC9385R, NTC9685R vectors, and variants thereof, such as those described in PCT Application No. PCT / US13 / 00068; NTC9385R-BE, NTC9385Ra-O1, and NTC9385Ra-O2 vectors, such as those described in U.S. Pat. No. 10,144,935; and NTC9385C2, NTC9385C2a, NTC9385R2, NTC9385R2a, NTC9385R2b, NTC9385Ra, NTC9385RaF, and NTC9385RbF replicating minicircular vectors, such as those described in U.S. Patent Publication No. 2021 / 0189407, each of which is incorporated herein by reference in its entirety.

[0190] In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a binding protein or polypeptide described herein, or a fragment thereof. In some embodiments, the nucleic acid comprises regulatory elements necessary for expression of the open reading frame. Such elements may include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers may be included. These elements may be operably linked to the sequence encoding the binding protein, polypeptide, or fragment thereof.

[0191] In some embodiments, the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, a dominant-negative TGFβ receptor (e.g., DN-TGFβRII), a selectable protein marker, and optionally, the selectable protein marker is dihydrofolate reductase (DHFR). In certain embodiments, the nucleic acid sequence encoding CD8α, CD8β, DN-TGFβR, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag (e.g., a CD34 enrichment tag). In certain embodiments, the nucleic acid sequences described herein, e.g., the nucleic acid sequences encoding TCRα, TCRβ, CD8α, CD8β, DN-TGFβR, and / or the selectable protein marker, are interconnected with a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide, such as P2A, E2A, F2A, or T2A.

[0192] In some embodiments, the expression vectors provided herein comprise a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to any of the nucleic acids set forth in SEQ ID NO:27.

[0193] Examples of promoters include, but are not limited to, Simian Virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV) promoters, such as the HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV) promoters, such as the CMV immediate early promoter, Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoters, and promoters of human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Examples of suitable polyadenylation signals include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal.

[0194] In addition to the regulatory elements required for expression, other elements may also be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include promoters as described herein. In some embodiments, enhancers / promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as those derived from CMV, RSV, and EBV.

[0195] 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, immunostimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live carriers such as viruses or bacteria.

[0196] 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.

[0197] 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 a cell that lacks CD8. In some embodiments, the host cell is an immune cell, such as a human immune system cell. 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 in which T cells are the host, the T cells may be naive T cells, central memory T cells, effector memory T cells, or any combination thereof. Thus, the recombinant expression vector may also include a lymphoid tissue-specific transcriptional regulatory element (TRE), such as, for example, a B lymphocyte-, T lymphocyte-, or dendritic cell-specific TRE. 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).

[0198] 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 chain and TCR beta chain of the binding protein and a linker disposed therebetween, wherein the nucleotide sequence encoding the beta chain is disposed 5' to the nucleotide sequence encoding the alpha chain. In some embodiments, the nucleotide sequences encode the full-length TCR alpha chain and TCR beta chain and a linker disposed therebetween, wherein the nucleotide sequence encoding the TCR beta chain is disposed 3' to the nucleotide sequence encoding the TCR alpha chain. In some embodiments, the full-length TCR alpha chain and / or TCR beta chain is replaced with a fragment thereof.

[0199] As discussed further below, another aspect encompassed by the present invention relates to cells transfected, infected, or transformed with the nucleic acids and / or vectors of the present invention. A host cell includes an individual cell or cell culture that may receive the vector, nucleic acid, and / or protein, as well as progeny. The term also includes the descendants of the host cell, whether genetically or phenotypically the same or different. Suitable host cells vary depending on the vector and include mammalian, animal, human, simian, insect, yeast, bacterial, and the like. These cells can be induced to take up vectors or other materials by transformation using 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" means the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA, or RNA sequence into a host cell, such that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA has been "transformed."

[0200] For example, in some embodiments, engineered immune cells (e.g., T cells) are pan T cells (CD4 T cells) engineered by transposon / transposase-mediated gene delivery to express gene cargo encoding elements such as the alpha and beta chains of a recombinant T cell receptor (TCR) specific for a given target antigen presented on a particular MHC (e.g., class I HLA). + T cells and CD8 + The vector may contain a) CD4 T cells. +Additional elements can be expressed, such as one or more elements selected from the group consisting of: a) CD8α and CD8β co-receptors to enable T cell engagement; b) a QBEND / 10 epitope tag derived from CD34 fused to the amino terminus of CD8α to allow for in vitro and in vivo tracking of engineered cells; c) a dominant-negative type II TGFβ receptor (DN-TGFβRII) to overcome tumor-mediated immunosuppression; and d) a selectable marker, such as a mutant form of dihydrofolate reductase (DHFRdm), to facilitate enrichment of engineered cells during the manufacturing process. The exogenous TCR α and β chains and CD8 α and β chains can be encoded by a single mRNA molecule under the control of a single promoter, such as the murine stem cell virus (MSCV) promoter. Post-translational processing at self-cleaving peptide elements, such as P2A sites, can result in independent polypeptides, e.g., four separate polypeptides corresponding to each element in the vector can be generated. Similarly, DN-TGFβRII and DHFRdm could be encoded by a single mRNA molecule driven by a single promoter, such as the human elongation factor 1α (EF1α) promoter. Post-translational processing at self-cleaving peptide elements, such as P2A sites, could result in independent polypeptides corresponding to each individual element in the vector.

[0201] Nucleic acids encompassed by the invention can be used in a suitable expression system to produce recombinant polypeptides encompassed by the invention. The term "expression system" refers to a host cell and a compatible vector under suitable conditions for the expression of proteins encoded by foreign DNA, for example, carried by the vector and introduced into the host cell.

[0202] 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 (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neuronal cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al (1980)), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"). In some embodiments, YB2 / 0 cells are used because expression in YB2 / 0 cells enhances the ADCC activity of chimeric or humanized binding proteins.

[0203] The present invention also encompasses methods for producing recombinant host cells expressing the binding proteins, peptides, and fragments thereof encompassed by the present invention, comprising the steps of (i) introducing the above-described recombinant nucleic acid or vector into competent host cells in vitro or ex vivo, (ii) culturing the resulting recombinant host cells in vitro or ex vivo, and (iii) optionally selecting cells expressing the binding proteins, peptides, and fragments thereof. Such recombinant host cells can be used in diagnostic, prognostic, and / or therapeutic methods encompassed by the present invention.

[0204] In another aspect, the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to the polynucleotides disclosed herein. Thus, the polynucleotides of this embodiment can be used to isolate, detect, and / or quantify nucleic acids that contain such polynucleotides. For example, the polynucleotides included in the present invention can be used to identify, isolate, or amplify partial or full-length clones within a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated from, or otherwise complementary to, a cDNA in, a human or mammalian nucleic acid library. In some embodiments, the cDNA library contains at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more full-length sequences, or any range therebetween, e.g., at least about 80%-100%. The cDNA library may be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, used for sequences with low sequence identity relative to the complementary sequence. Moderate and high stringency conditions can optionally be employed for sequences with higher identity. Low stringency conditions allow for selective hybridization of sequences with about 70% sequence identity and can be used to identify orthologous or paralogous sequences. Optionally, polynucleotides encompassed by the present invention will encode at least a portion of a binding protein encoded by a polynucleotide described herein. Polynucleotides encompassed by the present invention include nucleic acid sequences that can be used for selective hybridization to polynucleotides encoding binding proteins encompassed by the present invention (see, e.g., Ausubel, supra, and Colligan, supra).

[0205] IV host cell In one aspect encompassed by the present invention, provided herein is a host cell that expresses a binding protein described herein (e.g., a TCR, an antigen-binding fragment of a TCR, a CAR, or a fusion protein comprising a TCR and an effector domain). In some embodiments, the host cell comprises a nucleic acid or vector described herein.

[0206] 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 sequence analysis, particularly TCR sequence analysis, are described (e.g., Robins et al. (2009) Blood 114:4099; Robins et al. (2010) Sci. Translat. Med. 2:47ra64; Robins et al. (2011) J. Imm. Meth.; and Warren et al. (2011) Genome Res. 21:790) and can be utilized in the course of practicing embodiments encompassed by the present invention. Similarly, methods for transfecting or transducing T cells with a desired nucleic acid are well known in the art (e.g., U.S. Patent Application Publication No. 2004 / 0087025), as are adoptive transfer procedures using T cells with a desired antigen specificity (e.g., Schmitt et al. (2009) Hum. Gen. 20:1240; Dossett et al. (2009) Mol. Ther. 77:742; Till et al. (2008) Blood 772:2261; Wang et al. (2007) Hum. Gene Ther. 18:112; Kuball et al. (2007) Blood 709:2331; U.S. Patent Application Publication No. 2011 / 0243972; U.S. Patent Application Publication No. 2011 / 0189141; and Leen et al. al.(2007)Ann.Rev.Immunol.25:243).

[0207] Any suitable immune cell can be modified to contain a heterologous polynucleotide encompassed by the present invention, including, for example, 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 + For purposes herein, T cells may be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. T cells obtained from a mammal can be obtained from a variety of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, and other tissues and fluids. T cells may be enriched or purified. In some embodiments, the T cells are human T cells. In some embodiments, the T cells are T cells isolated from a human. T cells may be any type of T cell, at any developmental stage, including cytotoxic lymphocytes, cytotoxic lymphocyte precursor cells, cytotoxic lymphocyte progenitor cells, cytotoxic lymphocyte stem cells, CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, such as Th1 cells and Th2 cells, CD4 + T cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, and the like.

[0208] Any suitable method can be used to transfect or transduce cells, e.g., T cells, or administer a nucleotide sequence or composition 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, liposome-mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated delivery, and the like. Yet another method for transfecting or transducing host cells uses vectors, which are described in more detail herein.

[0209] The engineered immune cells described herein can be functionally characterized using methodologies to assay T cell activity, including measuring T cell binding, activation or induction, and measuring antigen-specific T cell responses. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., from pre-loaded target cells), and the like. 51 These include measuring T cell function through the detection of T cell proliferation (by detecting Cr release), changes in T cell phenotypic marker expression, and other indicators of T cell function.

[0210] Procedures for performing these and similar assays are described, for example, in Lefkovits (Immunology Methods Manual: A Comprehensive Sourcebook of Techniques, 1998), 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.

[0211] In some embodiments, apparent affinity for a binding protein, such as 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, each of which in some embodiments binds at least one antigen (e.g., HPV16 E7 11-19 The complex is characterized by a tetramer of MHC molecules containing identical peptides with cognate (e.g., identical or related) amino acid sequences (immunogenic peptides), and the complex is capable of binding to a binding protein, such as a TCR or antigen-binding portion thereof, that recognizes the cognate antigen. Each MHC molecule may be tagged with a biotin molecule. The biotinylated MHC / peptide may be multimerized (e.g., tetramerized) by the addition of streptavidin, which may be fluorescently labeled.

[0212] Multimers can be detected by flow cytometry via fluorescent labeling. In some embodiments, pMHC multimer assays are used to detect or select binding proteins with enhanced affinity, such as TCRs or antigen-binding portions thereof encompassed by the present invention. In some instances, the apparent K of a binding protein, e.g., a TCR or its antigen-binding protein, is detected. D was measured using two-fold dilutions of various concentrations of labeled multimer, followed by nonlinear regression to determine the binding curve and the apparent K D is determined as the concentration of ligand that resulted in half-maximal binding.

[0213] Cytokine levels can be determined using methods described herein, such as ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and combinations thereof (eg, intracellular cytokine staining and flow cytometry).

[0214] Immune cell proliferation and clonal expansion resulting from elicitation or stimulation of an antigen-specific immune response can be determined by isolating lymphocytes, such as circulating lymphocytes in a sample of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation, and / or cell viability, such as by non-radioactive assays such as tritiated thymidine incorporation or MTT assays. The effect of the immunogens described herein on the balance between Th1 and Th2 immune responses can be examined by measuring, for example, the levels of Th1 cytokines, such as IFN-γ, IL-12, IL-2, and TNF-β, and type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

[0215] Host cells encompassed by the present invention may contain a single polynucleotide encoding a binding protein described herein, or the binding protein may be encoded by multiple polynucleotides, 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 on more than one nucleic acid molecule.

[0216] Furthermore, as further described below and in the Examples, host cells encompassed by the invention can encode and / or express useful accessory proteins, in addition to the binding proteins described herein, on the same or different polynucleotides as the binding proteins or components thereof. For example, the host cells can encode and / or express CD8α, CD8β, DN-TGFβR (e.g., DN-TGFβRII), and / or a selectable protein marker, optionally DHFR.

[0217] 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 within a single open reading frame. Such an arrangement advantageously allows for coordinated expression of desired gene products, such as coexpression of the alpha and beta chains of a TCR, so that they are produced in a ratio of about 1:1. In some embodiments, two or more replacement gene products of a binding protein encompassed by the invention, such as a TCR (e.g., alpha and beta chains) or a CAR, are expressed as separate molecules and linked post-translationally. In further embodiments, two or more replacement 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 expression of separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, the porcine teschovirus-1 2A (P2A) peptide, the thoseaasigna virus 2A (T2A) peptide, the equine rhinitis A virus (ERAV) 2A (E2A) peptide, and the foot-and-mouth disease virus 2A (F2A) peptide.

[0218] In some embodiments, binding proteins encompassed by the invention comprise one or more junction amino acids. A "junction amino acid" or "junction amino acid residue" refers to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, such as between a binding domain and an adjacent constant domain, or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids may 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 may result from cleavage of a self-cleaving peptide adjacent to one or more domains of an encoded binding protein encompassed by the invention (e.g., a P2A peptide positioned between the TCRβ chain and the TCRβ chain, whose self-cleavage may leave one or more junction amino acids in the a chain, the TCRβ chain, or both).

[0219] The engineered immune cells encompassed by the present invention include, for example, HPV16 E7 11-19They can be administered as a treatment for non-malignant diseases, hyperproliferative diseases, or recurrence of hyperproliferative diseases characterized by antigen expression. In some situations, it may be desirable to reduce or stop activity associated with cellular immunotherapy. Thus, in some embodiments, engineered immune cells encompassed by the present invention contain heterologous polynucleotides encoding accessory proteins, such as binding proteins and safety switch proteins, which can be targeted using cognate drugs or other compounds to selectively modulate (e.g., reduce or eliminate) the activity of such cells as needed. Safety switch proteins used in this regard include, for example, truncated EGF receptor polypeptide (huEGFRt), which lacks the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a structurally intact binding epitope for the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux), tEGF receptor (tEGFr; Wang et al. (2011) Blood 118:1255-1263), caspase polypeptides (e.g., iCasp9; Straathof et al. (2005) Blood 105:4247-4254, Di Stasi et al. (2011) N. Engl. J. Med. 365:1673-1683, Zhou and Brenner (2016) Hematol. pii:S0301-472X:30513-30516), RQR8 (Philip et al. (2014) Blood 124:1277-1287), and the human c-myc protein tag (Kieback et al. (2008) Proc. Natl. Acad. Sci. USA 105:623-628).

[0220] Other useful adjuncts to therapeutic cells include tags or selectable markers (e.g., CD34 enrichment tags) that allow for cell identification, sorting, separation, enrichment, and tracking. For example, labeled immune cells with desired properties (e.g., antigen-specific TCRs or safety switch proteins) can be sorted from unlabeled cells in a sample, more efficiently activated and expanded, and included in a therapeutic product of desired purity.

[0221] As used herein, the term "selection marker" includes nucleic acid constructs that confer a distinguishable change to cells, allowing for the detection and positive selection of immune cells transduced with a polynucleotide containing the selection marker. For example, RQR is a selection marker composed of the major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, the polynucleotide encoding RQR includes a polynucleotide encoding a 16-amino acid CD34 minimal epitope. In some embodiments, such as the specific embodiment provided in the Examples herein, 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 T cell marker / suicide gene (RQR8) (Philip et al. 2014). This construct allows for selection of immune cells expressing the construct using, for example, CD34-specific antibodies coupled to magnetic beads (Miltenyi) and selective deletion 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).

[0222] Further exemplary selection markers include several truncated type I transmembrane proteins not normally expressed in T cells, namely, 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 pre-made Miltenyi CliniMACs™ selection system, which allows these markers to be targeted 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. Surface markers containing extracellular non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR, etc.) may also be used. Any selectable marker may be used, provided it complies with good manufacturing practice. In some embodiments, the selectable marker is expressed in a polynucleotide encoding a gene product of interest (e.g., a binding protein encompassed by the present invention, e.g., a TCR or CAR, or an antigen-binding fragment thereof). Further examples of selectable markers include reporters such as GFP, EGFP, β-gal, chloramphenicol acetyltransferase (CAT), etc. In some embodiments, a selectable marker, such as CD34, is expressed by the cells, and CD34 can be used to select, enrich, or isolate (e.g., by immunomagnetic selection) transduced cells of interest for use in the methods described herein. As used herein, the CD34 marker is distinguished from anti-CD34 antibodies, or, for example, scFvs, TCRs, or other antigen-recognition moieties that bind to CD34.

[0223] 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.

[0224] As background, immunotherapy cell products contain CD4 + The inclusion of T cells provides antigen-induced IL-2 secretion and transfers cytotoxic CD8 + T cell persistence and function are enhanced (e.g., Kennedy et al. (2008) Immunol. Rev. 222:129 and Nakanishi et al. Nature (2009) 52:510). In some embodiments, CD4 + Class I-restricted TCRs in T cells may require the transfer of a CD8 coreceptor to enhance TCR sensitivity to class I HLA-peptide complexes. The CD4 coreceptor is structurally distinct from CD8 and cannot effectively replace the CD8 coreceptor (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 coreceptor or a component thereof. Engineered immune cells comprising a heterologous polynucleotide encoding a binding protein encompassed by the present invention may, in some embodiments, further comprise a heterologous polynucleotide encoding a CD8 coreceptor protein or a beta or alpha chain component thereof.

[0225] Host cells can be efficiently transduced to contain and efficiently express a single polynucleotide encoding the binding protein, the safety switch protein, the selectable marker, and the CD8 co-receptor protein.

[0226] In one embodiment, the host cells encompassed by the invention further comprise a nucleic acid encoding a costimulatory molecule, and 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.

[0227] In any of the foregoing embodiments, the host cells expressing the binding proteins described herein may be universal immune cells. "Universal immune cells" include immune cells that have been modified to reduce or eliminate expression of one or more endogenous genes encoding polypeptide products selected from PD-1, LAG-3, CTLA4, TIM3, TIGIT, HLA molecules, TCR molecules, or any combination thereof. Without wishing to be bound by theory, it is possible that certain endogenously expressed immune cell proteins may downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, TIGIT), or heterologously expressed binding proteins encompassed by the invention (e.g., non-HPV16 E7). 11-19 HPV16 E7, which binds to antigen and contains the amino acid sequence YMLDLQPET in the MHC molecule 11-19 HPV16 E7, such as immunogenic peptides 11-19 These proteins may interfere with the binding activity of endogenous TCRs, preventing the modified immune cells from binding to antigen-expressing target cells. Furthermore, endogenous proteins (e.g., immune cell proteins such as HLA alleles) expressed on donor immune cells may be recognized as foreign by the allogeneic host, resulting in the elimination or suppression of the modified donor immune cells by the allogeneic host.

[0228] Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerance, or persistence of the engineered immune cells in an autologous or allogeneic host environment, allowing for universal administration of the cells (e.g., administration to any recipient regardless of HLA type). In some embodiments, the cells according to the invention are syngeneic cells, meaning that they are genetically identical or sufficiently identical and immunologically compatible to allow transplantation. In some embodiments, the universal immune cells are donor cells (e.g., allogeneic) or autologous cells. In some embodiments, modified immune cells (e.g., universal immune cells) encompassed by the invention comprise one or more chromosomal gene knockouts of genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g., a gene encoding alpha 1 macroglobulin, alpha 2 macroglobulin, alpha 3 macroglobulin, beta 1 microglobulin, or beta 2 microglobulin), or a TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai et al (2016) Nature Sci. Rep. 6:21757, Torikai et al (2012) Blood 179:5697, and Torikai et al (2013) Blood 722:1341, which also provide representative and exemplary gene editing techniques, compositions, and adoptive cell therapies useful in accordance with the invention).

[0229] As used herein, the term "chromosomal gene knockout" refers to a genetic modification or introduced inhibitor in a host cell that prevents (e.g., reduces, delays, suppresses, or abolishes) the production of a functionally active endogenous polypeptide product by the host cell. Changes that result in a chromosomal gene knockout include, for example, nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, strand breaks, and heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0230] In some embodiments, chromosomal gene knockout or gene knockin can be achieved by chromosomal editing of a host cell. Chromosomal editing can be performed, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of phosphodiester bonds within a polynucleotide chain. In some embodiments, the endonuclease can cleave a target gene, thereby inactivating or "knocking out" the target gene. The endonuclease can be a naturally occurring endonuclease, an recombinant endonuclease, a recombinant endonuclease, or a fusion endonuclease. Nucleic acid strand breaks caused by endonucleases are usually repaired by different mechanisms: homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, a donor nucleic acid molecule is used to "knock in" a donor gene, "knock out" a target gene, or inactivate a target gene through a donor gene knock-in or target gene knock-out event. NHEJ is an error-prone repair process that often results in changes to the DNA sequence at the cut site (substitution, deletion, or addition of at least one nucleotide). NHEJ can potentially be used to "knock out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0231] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein containing a zinc finger DNA-binding domain fused to a nonspecific DNA cleavage domain, such as a Fokl endonuclease. Each zinc finger motif, consisting of approximately 30 amino acids, binds to approximately three base pairs of DNA, and triplet sequence specificity can be altered by changing the amino acids at specific residues (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 desired DNA sequences, such as regions ranging in length from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and targeted integration of transgenes containing flanking sequences homologous to the genome into the DSB site is promoted by homology-directed repair. Alternatively, ZFN-generated DSBs can cause 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, performed using a ZFN molecule.

[0232] 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., Fokl endonuclease). A "TALE DNA-binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each of which typically has a highly conserved 33-35 amino acid sequence with different 12th and 13th amino acids. The TALE repeat domain is responsible for the binding of the TALE to the target DNA sequence. Different amino acid residues, called repeat variable dimers (RVDs), correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined to be that the HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE directs the TALE to bind to cytosine (C), NG (asparagine-glycine) binds to T nucleotides, NI (asparagine-isoleucine) binds to A, NN (asparagine-asparagine) binds to G or A nucleotides, and NG (asparagine-glycine) binds to T nucleotides. Non-canonical (atypical) RVDs are also well known in the art (e.g., U.S. Patent Application Publication No. 2011 / 0301073, atypical RVDs, incorporated herein by reference in their entirety). TALENs can be used to induce site-specific double-strand breaks (DSBs) in the genome of T cells. Non-homologous end joining (NHEJ) joins DNA on either side of a double-strand break with little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homology-directed repair can be used to introduce a transgene into the DSB site if a homologous flanking sequence is present within the transgene. In some embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, and is generated using a TALEN molecule.

[0233] 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-pair complementarity and then cleaves DNA when a short, conserved protospacer-associated motif (PAM) follows immediately 3' of the complementary target sequence. CRISPR / Cas systems are classified into three types (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. The most studied type II system consists of at least three elements: 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 and interact with the Cas9 nuclease, directing the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA upstream of the PAM. The Cas9 nuclease then cleaves the double strand within the region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions, disrupting expression of the target locus. Alternatively, transgenes with homologous flanking sequences can be introduced into the DSB site via homology-directed repair. The crRNA and tracrRNA can be incorporated into a single guide RNA (sgRNA or gRNA) (e.g., Jinek et al. (2012) Science 337:816-821). Additionally, the region of the guide RNA complementary to the target site can be altered or programmed to target a desired sequence (Xie et al. (2014) PLOS One 9:el00448, U.S. Patent Application Publication No. US2014 / 0068797, U.S. Patent Application Publication No. US2014 / 0186843, U.S. Patent No. 8,697,359, and PCT Publication No. WO 2015 / 071474).In some embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, and is generated using a CRISPR / Cas nuclease system.

[0234] Exemplary gRNA sequences and methods for using them 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 and exemplary gRNAs, CAS9 DNA, vectors, and gene knockout techniques.

[0235] As used herein, "meganuclease," also known as "homing endonuclease," refers to an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of approximately 12 to 40 base pairs). Meganucleases are classified into five families based on their sequences 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, whose recognition sequences are well known (e.g., U.S. Pat. Nos. 5,420,032 and 6,833,252; Belfort et al. (1997) Nucl. Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 52:115-118; Perler et al. (1994) Nucl. Acids Res. 22:1125-1127; Jasin (1996) Trends Genet.72:224-228, Gimble et al. (1996) J.Mol.Biol.263:163-180, and Argast et al. (1998) J.Mol.Biol.280: 345-353).

[0236] In some embodiments, natural meganucleases can be used to facilitate site-specific genomic modification of targets of interest, such as immune checkpoints, HLA-encoding genes, or TCR component-encoding genes.

[0237] In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genome modification (e.g., Porteus et al. (2005) Nat. Biotechnol. 23:967-73; Sussman et al. (2004) J. Mol. Biol. 342:31-41; Epinat et al. (2003) Nucl. Acids Res. 37:2952-2962; Chevalier et al. (2002) Mol. Cell 70:895-905; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Curr. Gene Ther. 7:49-66, and U.S. Patent Application Publication Nos. US2007 / 0117128, US2006 / 0206949, US2006 / 0153826, US2006 / 0078552, and US2004 / 0002092. In a further embodiment, homing endonucleases engineered with modular DNA-binding domains of TALENs are used to generate chromosomal gene knockouts, creating fusion proteins called megaTALs. MegaTALs can 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 combination with an exogenous donor template encoding a polypeptide of interest.

[0238] In some embodiments, the chromosomal gene knockout is HPV16 E7 11-19The present invention includes an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) that comprises a heterologous polynucleotide encoding an antigen-specific receptor that binds (e.g., specifically and / or selectively) to an antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor, and the encoded target-specific inhibitor inhibits endogenous gene expression (i.e., an immune checkpoint, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.

[0239] Chromosomal gene knockout may be directly confirmed by DNA sequencing of host immune cells after use of the knockout procedure or drug.

[0240] A chromosomal gene knockout can also be inferred from the lack of gene expression following the knockout (eg, the lack of mRNA or polypeptide product encoded by the gene).

[0241] In some embodiments, host cells encompassed by the present invention contain HPV16 E7 in their MHC molecules. 11-19 It is possible to specifically and / or selectively detect 50% or more of the target cells containing peptide-MHC (pMHC) complexes that contain an immunogenic peptide.

[0242] In some embodiments, the modified immune cells contain HPV16 E7 in the MHC molecule. 11-19 Upon contact with target cells containing peptide-MHC (pMHC) complexes containing immunogenic peptides, cytokines can be produced.

[0243] In some embodiments, the cytokine comprises IFN-γ or IL2. In some embodiments, the cytokine is TNF-α.

[0244] In some embodiments, the host cell has about 1000 transcripts per million (TPM), 950 TPM, or 、900TPM, 850TPM, 800TPM, 750TPM, 700TPM, 650TPM, 600TPM, 550TPM, 500TPM, 450TPM, 400TPM, 350TPM, 300TPM, 250TPM, 200TPM, 150TPM, 1 00TPM, 95TPM, 90TPM, 85TPM, 80TPM, 75TPM, 70TPM, 65TPM, 60TPM, 55TPM, 50TPM, 45TPM, 40TPM, 35TPM, 34TPM, 33TPM, 32TPM, 31TPM, 30TPM , 29 TPM, 28 TPM, 27 TPM, 26 TPM, 25 TPM, 24 TPM, 23 TPM, 22 TPM, 21 TPM, 20 TPM, 19 TPM, 18 TPM, 17 TPM, 16 TPM, 15 TPM, 14 TPM, 13 TPM, 12 TPM, 11 TPM, 10 TPM, 9 TPM, 8 TPM, 7 TPM, 6 TPM, 5 TPM, 4 TPM, 3 TPM, 2 TPM, and 1 TPM, or any range therebetween (including boundary values), for example, about 1,000 TPM or less to about 35 TPM or less, and HPV16 E7 11-19 In some embodiments, the low HPV E7 gene may produce higher levels of cytokines or cytotoxic molecules when contacted with target cells expressing HPV E7. 11-19 The expression level is referred to as "heterozygous expression" and means about 1 TPM to about 35 TPM, or any range therebetween (including boundaries), for example, 1 to 32 TPM. For example, the host cell may produce levels of a cytokine or cytotoxic molecule that are 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), e.g., 1.2x to 2x.

[0245] In some embodiments, the host cell is HPV16 E7 11-19Target cells expressing HPV16 E7 11-19 In certain embodiments, the target cells express (i) a polypeptide comprising or consisting of the amino acid sequence YMLDLQPET, and (ii) a matching MHC molecule.

[0246] In some embodiments, the host cell is HPV16 E7 11-19 does not express an antigen, is not recognized by the binding protein of any one of claims 1 to 30, and is of the serotype HLA-A * Not 02 and / or HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:05, HLA-A * 02:06, or HLA-A * 02:07HLA-A alleles * does not express the O2 allele. For example, patients with HPV16 E7 11-19 Negative or HLA-A * Host cells may be received from a healthy donor who is O2:01 negative. Stem cells, such as hematopoietic stem cells (or engineered autologous cells), isolated from the donor can be used as a source of transplant material. In parallel, T cells isolated from the same donor can be transfected with HPV16 E7 as described herein. 11-19 HPV16 E7 binding protein 11-19 Donor stem cells can be used to transplant cell populations, such as a reconstituted immune system, into patients, and host cells can be infused into patients to elicit highly specific anti-tumor effects. Engineered donor T cells can recognize HPV16 E7. 11-19 expressing cells (all natural blood cells of the patient, e.g., HPV16 E7 11-19The vaccine is designed to recognize and eliminate cancer cells (including residual leukemia cells) that are positive for HPV16 E7, thereby preventing recurrence and promoting a complete cure. The patient's new healthy blood cells are derived from a donor and therefore contain HPV16 E7. 11-19 Negative, HLA-A * 02 Seronegative and / or HLA-A * Because they are 02:01 negative, the engineered cells described herein may have minimal toxic side effects. Such patient-matched host cells and treatment methods may be used in accordance with the treatment methods described further below.

[0247] In some embodiments, the killing is determined by a killing assay. In some embodiments, the killing assay is performed by co-culturing 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 co-cultured with 1 μg / mL to 50 pg...

Claims

1. A binding protein comprising: 1) a) a T cell receptor (TCR) alpha chain variable domain comprising: a) a CDR1 having residues 45-49 of SEQ ID NO:19, a CDR2 having residues 67-73 of SEQ ID NO:19, and a CDR3 having residues 107-120 of SEQ ID NO:19; and b) a TCR beta chain variable domain comprising a CDR1 having residues 46-50 of SEQ ID NO:21, a CDR2 having residues 68-73 of SEQ ID NO:21, and a CDR3 having residues 111-123 of SEQ ID NO:21; 2) a) a TCR alpha chain variable (V α ) domain comprising an amino acid sequence having at least about 80% identity to residues 1-131 of SEQ ID NO:19; and b) a TCR beta chain variable (Vβ) domain comprising an amino acid sequence having at least about 80% identity to residues 1-133 of SEQ ID NO:21; 3) a) a TCR alpha chain comprising an amino acid sequence having at least about 80% identity to the sequence of SEQ ID NO: 19; and b) a TCR beta chain comprising an amino acid sequence having at least about 80% identity to the sequence of SEQ ID NO: 21; 4) a) a T cell receptor (TCR) alpha chain CDR sequence having at least about 80% identity to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 1; and / or b) a TCR beta chain CDR sequence having at least about 80% identity with a TCR beta chain CDR sequence selected from the group consisting of the TCR beta chain CDR sequences listed in Table 1, wherein the binding protein is HPV16 E7 １１－１９ capable of binding to an immunogenic peptide-MHC (pMHC) complex, and optionally having a binding affinity of K ｄ is about 5 x 10 －４ M or less; 5) a) a TCR alpha chain variable (Vα) domain sequence having at least about 80% identity to a TCR Vα domain sequence selected from the group consisting of the TCR Vα 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 the group consisting of the TCR V β domain sequences listed in Table 1, wherein said binding protein is capable of binding to an HPV16 E7 11-19 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity K d of about 5×10 −4 M or less; 6) 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 said binding protein is capable of binding to an HPV16 E7 11-19 immunogenic peptide-MHC (pMHC) complex, and optionally the binding affinity, K d , is about 5×10 −4 M or less; 7) 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 said binding protein is capable of binding to an HPV16 E7 11-19 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity K d of about 5×10 −4 M or less; 8) a) a TCR alpha chain variable (Vα) domain sequence selected from the group consisting of the TCR Vα domain sequences listed in Table 1; and / or b) a TCR beta chain variable (V β ) domain sequence 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 HPV16 E7 11-19 immunogenic peptide-MHC (pMHC) complex, optionally with a binding affinity K d of about 5×10 −4 M or less; and / or 9) 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 said binding protein is capable of binding to an HPV16 E7 11-19 immunogenic peptide-MHC (pMHC) complex, and optionally has a binding affinity, K d , of about 5×10 −4 M or less.

2. 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 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 The domain, and / or the TCR beta chain is encoded by a TRBV, TRBJ, and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ, and TRBC genes listed in Table 1; 3) each CDR of said binding protein has up to five amino acid substitutions, insertions, deletions, or a combination thereof, compared to the cognate reference CDR sequences listed in Table 1; 4) the HPV16 E7 11-19 immunogenic peptide comprises the amino acid sequence YMLDLQPET; 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, and optionally, 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, and optionally, 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 moiety, and optionally, the covalently linked moiety is a) comprising an affinity tag or label, optionally wherein the affinity tag is selected from the group consisting of a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag, and / or the label is a fluorescent protein; and / or b) selected from the group consisting of an inflammatory substance, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof; 9) the binding protein binds to the pMHC complex on the cell surface; 10) the MHC is an MHC multimer, and optionally, the MHC multimer is a tetramer; 11) The MHC is an MHC class I molecule; 12) the MHC comprises an MHC alpha chain of HLA serotype HLA-A*02, and optionally the HLA allele is selected from the group consisting of HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0205, HLA-A*0206, and HLA-A*0207 alleles; 13) The binding of the binding protein to the HPV16 E7 11-19 peptide-MHC (pMHC) complex elicits an immune response, optionally the immune response is a T cell response, and further optionally the T cell response is selected from the group consisting of T cell proliferation, cytokine release, and / or cytotoxic killing; 14) The binding protein has a concentration of about 1 x 10 -4 M or less, about 5 x 10 -5 M or less, about 1 x 10 -5 M or less, about 5 x 10 -6 M or less, about 1 x 10 -6 M or less, about 5 x 10 -7 with respect to the HPV16 E7 11-19 immunogenic peptide-MHC (pMHC) complex. M or less, about 1 x 10 -7 M or less, about 5 x 10 -8 M or less, about 1 x 10 -8 M or less, about 5 x 10 -9 M or less, about 1 x 10 -9 M or less, about 5 x 10 -10 M or less, about 1 x 10 -10 M or less, about 5 x 10 -11 M or less, about 1 x 10 -11 M or less, about 5×10 −12 M or less, or about 1×10 capable of specifically and / or selectively binding with a K d of −12 M or less; 15) the binding protein has a higher binding affinity for the peptide-MHC (pMHC) than any known T-cell receptor, and optionally, the binding protein has at least a 1.05-fold higher binding affinity for the peptide-MHC (pMHC) than any known T-cell receptor; 16) the binding protein, when contacted with a target cell expressing the HPV16 E7 11-19 peptide epitope, induces greater T cell proliferation, cytokine release, and / or cytotoxic killing than known T cell receptors, optionally, the binding protein, when contacted with a target cell expressing the HPV16 E7 11-19 peptide epitope, induces at least 1.05-fold increased T cell proliferation, cytokine release, and / or cytotoxic killing than known T cell receptors, and further optionally, the target cell is a) a CaSki, SCC152, or SCC090 cell line, or b) a cancer cell, and optionally, the cancer cell is a head and neck cancer cell, anal cancer cell, vaginal cancer cell, vulvar cancer cell, or penile cancer cell; 17) the binding protein does not bind to a peptide-MHC (pMHC) complex, and optionally the peptide is derived from SPTA1, MPL, HERC1, CPAMD8, INTS4, NUTM1, or XM_00172256; and / or 18) The binding protein of claim 1, wherein the binding protein does not bind to SPTA1-, MPL-, HERC1-, CPAMD8-, INTS4-, NUTM1-, and / or XM_00172256-peptide-MHC (pMHC) complexes.

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

4. 1. 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 the complement of 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 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 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. 5. A vector comprising the isolated nucleic acid of claim 4, optionally comprising: 1) the vector is a cloning vector, an expression vector, or a viral vector; 2) the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, dominant-negative TGFβ receptor II (DN-TGFβRII), and / or a selectable protein marker; Optionally, a) the selectable protein marker is dihydrofolate reductase (DHFR), and / or b) the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag; Optionally, i) the nucleic acid encoding a tag is 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII and / or the selectable protein marker, such that the tag is fused to the N-terminus of CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker; and / or ii) the tag is a CD34 enrichment tag, and / or 3) The vector, wherein the isolated nucleic acid, and / or the nucleic acid sequence encoding TCRα, TCRβ, CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker is interconnected with a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide, and optionally the self-cleaving peptide is P2A, E2A, F2A, or T2A.

6. A host cell, (i) the host cell comprises 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 the complement of 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 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 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 the host cell; (ii) the host cell contains a vector comprising the isolated nucleic acid molecule of (i), and optionally 1) the vector is a cloning vector, an expression vector, or a viral vector; 2) the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, dominant-negative TGFβ receptor II (DN-TGFβRII), and / or a selectable protein marker; Optionally, a) the selectable protein marker is dihydrofolate reductase (DHFR), and / or b) the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag, and optionally i) the nucleic acid encoding a tag is 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII and / or the selectable protein marker, such that the tag is fused to the N-terminus of CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker; and / or ii) the tag is a CD34 enrichment tag, and / or 3) the isolated nucleic acid, and / or the nucleic acid sequence encoding TCRα, TCRβ, CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker, are interconnected with a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide, and optionally the self-cleaving peptide is P2A, E2A, F2A, or T2A; and / or 3) The host cell, wherein the host cell expresses the binding protein of claim 1, and optionally the cell is genetically engineered.

7. The host cell 1) Includes chromosomal gene knockout 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, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR, and optionally wherein the CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker are fused to a CD34 enrichment tag, and further optionally wherein the host cells are enriched using the CD34 enrichment tag; 5) 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; Further optionally, the T cells are a) naive T cells, central memory T cells, effector memory T cells, or a combination thereof; b) is a primary T cell or a cell of a T cell line; and / or c) does not express endogenous TCR or has low surface expression of endogenous TCR; 6) capable of producing cytokines or cytotoxic molecules upon contact with target cells containing peptide-MHC (pMHC) complexes containing the HPV16 E7 11-19 peptide epitope in the MHC molecule; 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 molecule is perforin and / or a granzyme, optionally, the cytotoxic molecule is granzyme B; and / or d) said host cells are capable of producing increased levels of cytokines or cytotoxic molecules upon contact with target cells expressing the HPV16 E7 11-19 peptide epitope, optionally said host cells are capable of producing at least 1.05-fold increased levels of cytokines or cytotoxic molecules; e) said host cells are capable of killing target cells that contain a peptide-MHC (pMHC) complex that contains the HPV16 E7 11-19 peptide epitope in an MHC molecule; and optionally i) the killing is determined by a killing assay; ii) the ratio of the host cells to the target cells in the killing assay is 20:1 to 0.625:1, and optionally the target cells are T2 cells pulsed with 1 μg / mL to 50 pg / mL of HPV16 E7 11-19 peptide; iii) when the host cell is contacted with target cells expressing the HPV16 E7 11-19 peptide epitope, it is capable of killing more target cells, optionally, the host cell is capable of killing at least 1.05 times more target cells; and / or iv) the target cell is a CaSki, SCC152, or SCC090 cell line; f) the HPV16 E7 11-19 immunogenic peptide comprises the amino acid sequence YMLDLQPET; g) the MHC molecule is an MHC class I molecule; h) said MHC molecule comprises an MHC alpha chain of HLA serotype HLA-A*02; i) said 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; j) the target cells are cancer cells that are selected from the group consisting of CaSki, SCC152 and SCC090 cell lines and express HPV16 E7 11-19 immunogenic peptide, or are not SiHa cell lines and / or are not NCI-H1792 cell lines, optionally the cancer cells are selected from the group consisting of head and neck cancer cells, oropharyngeal cancer cells, cervical cancer cells, anal cancer cells, vaginal cancer cells, vulvar cancer cells and penile cancer cells; k) the host cell does not induce T cell proliferation, cytokine release, or cytotoxic killing when contacted with a target cell containing a peptide-MHC (pMHC) complex that includes a SPTA1, MPL, HERC1, CPAMD8, INTS4, NUTM1, or XM_00172256 peptide epitope in its MHC molecule; and / or l) The host cell of claim 6, wherein the host cell does not express the HPV16 E7 11-19 antigen, is not recognized by the binding protein of claim 1, is not of serotype HLA-A*02, and / or does not express the HLA-A*02 allele, and optionally the HLA-A*02 allele is HLA-A*02:01 and / or HLA-A*02:

06.

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

9. 10. A composition comprising: a) a binding protein according to claim 1 or 2; b) an isolated nucleic acid according to claim 4; c) a 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; and a carrier.

10. 10. A device or kit comprising a) a binding protein according to claim 1 or 2, b) an isolated nucleic acid according to claim 4, c) a 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, said device or kit optionally comprising 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, comprising the steps of: (i) culturing a transformed host cell transformed with a nucleic acid comprising a sequence encoding the binding protein according to claim 1 or 2 under conditions suitable to allow expression of the binding protein; and (ii) recovering the expressed binding protein.

12. 10. A method for producing a host cell that expresses a binding protein of claim 1 or 2, the method comprising the steps of: (i) introducing into the host cell a nucleic acid comprising a sequence encoding the binding protein of claim 1 or 2; and (ii) culturing the transformed host cell under conditions suitable to allow expression of the binding protein.

13. HPV16 E7 １１－１９ antigen and / or HPV16 E7 １１－１９ 10. A method for detecting the presence or absence of a cell expressing HPV16 E7 in a sample, optionally wherein the cell is a hyperproliferative cell, the method comprising detecting the presence or absence of the HPV16 E7 in a sample using at least one binding protein according to claim 1 or 2, or at least one host cell according to claim 6 or 7. １１－１９ detecting the presence or absence of the HPV16 E7 antigen; １１－１９ Antigen detection is HPV16 E7 １１－１９ antigen, and / or HPV16 E7 １１－１９ and optionally, said at least one binding protein or said at least one host cell forms a complex with the HPV16 E7 11-19 peptide in an MHC molecule, said complex being detected by fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow assay.

14. 1) obtaining the sample from a subject, optionally wherein the subject is an animal model of a disease characterized by HPV16 E7 11-19 expression, and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent; and / or 2) HPV16 E7 by bone marrow biopsy １１－１９ Identifying cells that express 14. The method of claim 13, further comprising:

15. HPV16 E7 in the subject １１－１９ 1. A method for detecting the level of a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of an antigen, 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 the level of reactivity; wherein the high level of reactivity compared to the control level is a result of HPV16 E7 in said subject. １１－１９ indicating the level of non-malignant disease, hyperproliferative disease, or recurrence of hyperproliferative disease characterized by expression of an antigen; Optionally, 1) The control level is: a) the number of references, or b) the level in subjects free of said non-malignant disease, hyperproliferative disease, or recurrence of said hyperproliferative disease characterized by expression of the HPV16 E7 11-19 antigen. is; 2) the level of reactivity is: a) the presence of a bond; and / or b) T cell activation and / or effector function and optionally, i) the T cell activation or effector function is T cell proliferation, killing, or cytokine release; and / or ii) the T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow assay; 3) the disease is associated with HPV infection, and optionally, the HPV infection is HPV16 infection; 4) the hyperproliferative disease is head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), oropharyngeal cancer, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, or penile cancer); and / or 5) The method, wherein the subject is an animal model of a disease characterized by HPV16 E7 11-19 expression and / or a mammal, and optionally the mammal is a human, a primate, or a rodent.

16. HPV16 E7 in the subject １１－１９ 1. A method for monitoring the progression of a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease characterized by expression of an antigen, comprising: a) detecting the HPV16 E7 in a subject sample according to claim 13; １１－１９ antigen or HPV16 E7 １１－１９ detecting a level of cells of interest expressing the gene at a first time point; b) repeating step a) at a later time point; and c) HPV16 E7 detected in steps a) and b) １１－１９ antigen, or HPV16 E7 １１－１９ and comparing the levels of the cells of interest that express HPV16 E7 in the subject. １１－１９ monitoring the progression of a non-malignant disease, hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of an antigen; and the HPV16 E7 detected in step b) is compared with step a). １１－１９ antigen, or HPV16 E7 １１－１９ The absence or reduced level of the target cells expressing HPV16 E7 in the subject １１－１９ inhibiting the progression of said non-malignant disease, said hyperproliferative disease, or said recurrence of said hyperproliferative disease characterized by expression of an antigen; Optionally, 1) between the first time point and the subsequent time point, the subject is undergoing treatment to treat the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease characterized by expression of an HPV16 E7 11-19 antigen; 2) the disease is associated with HPV infection, and optionally, the HPV infection is HPV16 infection; 3) the hyperproliferative disease is head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), oropharyngeal cancer, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, or penile cancer); and / or 4) The method, wherein the subject is an animal model of a disease characterized by HPV16 E7 11-19 expression and / or a mammal, and optionally the mammal is a human, a primate, or a rodent.

17. HPV16 E7 １１－１９ 1. A method for assessing the effect of a treatment on the recurrence of a non-malignant disease, hyperproliferative disease, or hyperproliferative disease characterized by expression of an antigen, comprising: a) HPV16 E7 １１－１９ determining in a first sample obtained from the subject the presence or level of reactivity 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, prior to administering to the subject at least a portion of the treatment for the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease characterized by expression of an antigen; and b) HPV16 E7 １１－１９ determining the presence or level of reactivity 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 in a second sample obtained from the subject after a portion of the treatment for the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease characterized by expression of an antigen; wherein the absence or reduced level of reactivity in the second sample compared to the first sample indicates HPV16 E7 in the subject. １１－１９ demonstrating that the treatment is effective in treating the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease characterized by expression of an antigen; Optionally, 1) the level of reactivity is: a) the presence of a bond; and / or b) T cell activation and / or effector function and optionally i) the T cell activation or effector function is T cell proliferation, killing, or cytokine release; and / or ii) the T cell binding, activation, and / or effector function is detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow assay; 2) the disease is associated with HPV infection, and optionally, the HPV infection is HPV16 infection; 3) the hyperproliferative disease is head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), oropharyngeal cancer, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, or penile cancer); and / or 4) The method, wherein the subject is an animal model of a disease characterized by HPV16 E7 11-19 expression and / or a mammal, and optionally the mammal is a human, a primate, or a rodent.

18. HPV16 E7 in subjects １１－１９ 10. A method for preventing and / or treating a non-malignant disease, a hyperproliferative disease, or the recurrence of a hyperproliferative disease characterized by expression of an antigen, comprising administering to said subject a therapeutically effective amount of a composition comprising cells expressing at least one binding protein of claim 1 or 2; Optionally, 1) the cells are allogeneic, syngeneic, or autologous; 2) the cells are genetically modified; 3) the cells comprise a chromosomal gene knockout of a TCR gene, an HLA gene, or both a TCR gene and an HLA gene; 4) 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; 5) the cells comprise 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, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR, and further optionally wherein the CD8α, CD8β, DN-TGFβRII, and / or the selectable protein marker are fused to a CD34 enrichment tag, and optionally wherein the cells are enriched using the CD34 enrichment tag; 7) The cells are immune cells, and 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, and further optionally the T cells are a) naive T cells, central memory T cells, effector memory T cells, or a combination thereof; b) is a primary T cell or a cell of a T cell line; and / or c) do not express endogenous TCR or have lower surface expression of endogenous TCR; 8) the cells are capable of producing cytokines or cytotoxic molecules upon contact with target cells containing peptide-MHC (pMHC) complexes containing the HPV16 E7 11-19 peptide epitope in the MHC molecule, and optionally a) the cytokine is TNF-α, IL-2, and / or IFN-γ; b) the cytotoxic molecule is perforin and / or granzyme, optionally, the cytotoxic molecule is granzyme B; 9) the cells are capable of producing higher levels of cytokines or cytotoxic molecules upon contact with target cells expressing the HPV16 E7 11-19 peptide epitope, 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 target cells containing peptide-MHC (pMHC) complexes that contain the HPV16 E7 11-19 peptide epitope in the MHC molecule; 11) the host cell, upon contact with target cells expressing the HPV16 E7 11-19 peptide epitope, is capable of killing a greater number of target cells, and optionally, the host cell is capable of killing at least 1.05-fold greater number of target cells; 12) The HPV16 E7 11-19 immunogenic peptide comprises the amino acid sequence YMLDLQPET; 13) The MHC molecule is an MHC class I molecule; 14) The MHC molecule comprises an MHC alpha chain of 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 HPV16 E7 11-19 antigen in the subject; 17) the composition further comprises a pharmaceutically acceptable carrier; 18) The composition induces an immune response in the subject against the non-malignant cells or the hyperproliferative cells expressing the HPV16 E7 11-19 antigen, and optionally, the composition induces an antigen-specific T cell immune response in the subject against the non-malignant cells or the hyperproliferative cells expressing the HPV16 E7 11-19 antigen, and optionally, 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 disease is associated with HPV infection, and optionally, the HPV infection is HPV16 infection; 20) The hyperproliferative disease is head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), oropharyngeal cancer, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, or penile cancer); 21) The subject is undergoing or has previously undergone hematopoietic cell transplantation (HCT), and optionally the HCT comprises cells that do not express the HPV16 E7 11-19 antigen, are not recognized by the binding protein of claim 1 or 2, are not serotype HLA-A*02, and / or do not express the HLA-A*02:01 allele; and optionally 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 lymphocyte-depleting chemotherapy, optionally wherein the lymphocyte-depleting chemotherapy comprises cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof; 23) the method further comprises administering to the subject at least one additional treatment for the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease, optionally the at least one additional treatment for the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease is administered simultaneously or sequentially with the composition; and / or 24) The method as described above, wherein the subject is an animal model of a disease characterized by HPV16 E7 11-19 expression and / or a mammal, and optionally 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, CD8β, DN-TGFβRII, and / or a selectable protein marker, optionally comprising: 1) the selectable protein marker is DHFR; 2) the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag such that the tag is fused to the CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker; 3) the nucleic acid encoding a tag is 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker such that the tag is fused to the N-terminus of CD8α, CD8β, the DN-TGFβRII, and / or the selectable protein marker; 4) the tag is a CD34 enrichment tag; 5) The vector further comprises a nucleic acid sequence encoding TCRα and / or TCRβ. 6) The TCRα, TCRβ, and / or the DN-TGFβRII comprises a mutated transmembrane domain and / or a mutated constant domain; 7) the mutated transmembrane domain and / or the mutated constant domain enhance cell surface expression of TCRα, TCRβ, and / or the DN-TGFβRII, while reducing expression of endogenous TCRα, TCRβ, and / or TGFβRII; 8) the nucleic acid sequences encoding CD8α, CD8β, the DN-TGFβRII, the selectable protein marker, the TCRα, and / or the TCRβ are interconnected with a nucleic acid sequence encoding an internal ribosome entry site or a self-cleaving peptide; 9) The self-cleaving peptide is P2A, E2A, F2A, or T2A; 10) 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, and optionally, the isolated nucleic acid molecule is 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) 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; and / or 11) The vector having or comprising a nucleic acid sequence set forth in Table 3, or a fragment thereof, optionally wherein the fragment encodes DN-TGFβRII.

20. A method for monitoring the progression of a non-malignant disease, a hyperproliferative disease, or a recurrence of a hyperproliferative disease in a subject characterized by expression of the HPV16 E7 11-19 antigen, comprising: a) detecting the level of the HPV16 E7 11-19 antigen or cells of interest expressing HPV16 E7 11-19 in a subject sample at a first time point according to claim 15; b) repeating step a) at a later time point; and c) comparing the levels of HPV16 E7 11-19 antigen or the cells of interest expressing HPV16 E7 11-19 detected in steps a) and b) to monitor the progression of a non-malignant disease, a hyperproliferative disease, or recurrence of a hyperproliferative disease characterized by expression of HPV16 E7 11-19 antigen in the subject; wherein the absence or reduced level of the HPV16 E7 11-19 antigen or the cells of interest expressing HPV16 E7 11-19 detected in step b) compared to step a) indicates inhibition of the progression of the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease in the subject characterized by expression of the HPV16 E7 11-19 antigen; Optionally, 1) between the first time point and the subsequent time point, the subject is undergoing treatment to treat the non-malignant disease, the hyperproliferative disease, or the recurrence of the hyperproliferative disease characterized by expression of an HPV16 E7 11-19 antigen; 2) the disease is associated with HPV infection, and optionally, the HPV infection is HPV16 infection; 3) the hyperproliferative disease is head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), oropharyngeal cancer, cervical cancer, anal cancer, vaginal cancer, vulvar cancer, or penile cancer); and / or 4) The method, wherein the subject is an animal model of a disease characterized by HPV16 E7 11-19 expression and / or a mammal, and optionally the mammal is a human, a primate, or a rodent.

Images