Binding protein specific for neoantigen, engineered cell, and use thereof

Abstract

The present disclosure provides compositions and methods for targeting neoantigens, for example, to treat or prevent cancer. Embodiments disclosed include binding proteins that bind to neoantigen:HLA complexes, such as T cell receptors. The binding proteins further include a fusion protein of a CD95 ectodomain and a CD137 intracellular signaling domain (Fas-41BB), and a construct comprising a CDS coreceptor α or β chain. The disclosed binding proteins are sensitive to antigens and can induce activation of host T cells with low concentrations of peptide antigens. In certain embodiments, the binding proteins of the present disclosure are (i) non-alloreactive, substantially non-alloreactive, and / or have a low risk of alloreactivity against amino acid sequences derived from the human proteome and / or (ii) human HLA alleles. Polynucleotides encoding such binding proteins can be introduced into host cells, such as T cells, and the cells can be used in immunotherapies for treating various cancers.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 344,965, filed May 23, 2022; U.S. Provisional Application No. 63 / 380,527, filed Oct. 21, 2022; and U.S. Provisional Application No. 63 / 501,973, filed May 12, 2023, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] T cell-based immunotherapy began to be developed when tumor-reactive T cells were found within a population of tumor-infiltrating lymphocytes (TILs). In some situations, one strategy known as adoptive T cell transfer involves the isolation of tumor-infiltrating lymphocytes preselected for tumor reactivity, the clonal expansion of tumor-reactive T cells induced by anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and the injection back into the tumor-bearing patient of the expanded cell population. The isolation of tumor-reactive T cell clones has led to the development of another immunotherapy approach, namely the generation of recombinant T cell receptors (TCRs) specific for a particular antigen, which can be introduced into T cells, for example, using a vector delivery system, to confer specificity for a desired target such as a tumor-associated peptide presented by major histocompatibility complex (MHC) molecules (known as human leukocyte antigen (HLA) molecules in humans) expressed on tumor cells.

Summary of the Invention

[0003] In some embodiments, the present disclosure provides a polynucleotide encoding: (a) a binding protein comprising (i) a T cell receptor (TCR) or a functional derivative thereof, or (ii) a chimeric antigen receptor (CAR) or a functional derivative thereof; and (b) a fusion protein comprising (i) an extracellular component comprising a CD95 ligand (FasL) binding domain comprising a CD95 (Fas) ectodomain or a functional fragment thereof, and (ii) an intracellular component comprising a CD137 (4-1BB) intracellular signaling domain, wherein the nucleic acid sequence encoding the binding protein is located upstream of the nucleic acid sequence encoding the fusion polypeptide. In some embodiments, the polynucleotide further comprises (c) a nucleic acid sequence encoding a CD8 coreceptor α or β chain or a portion or variant thereof, and the sequence encoding the binding protein is located upstream of the sequence encoding the extracellular portion of the CD8 coreceptor α or β chain or a portion or variant thereof. In some embodiments, the polynucleotide further comprises (c) a CD8 coreceptor α and β chain or a portion or variant thereof, and the sequence encoding the binding protein is located upstream of the sequence encoding the extracellular portion of the CD8 coreceptor α and β chain or a portion or variant thereof. In some embodiments, the nucleic acid sequence encoding the fusion protein further encodes a hydrophobic component between the extracellular and intracellular components of the fusion protein. In some embodiments, the binding protein comprises a binding domain that binds to a peptide:HLA complex, the complex comprising a neoantigen peptide and an HLA protein. In some embodiments, the binding protein comprises a single-chain TCR (scTCR) or a single-chain T cell receptor variable fragment (scTv). In some embodiments, the binding protein comprises a TCR α-chain variable (Vα) domain or a TCR β-chain variable (Vβ) domain. In some embodiments, the binding protein comprises a TCR α-chain variable (Vα) domain and a TCR β-chain variable (Vβ) domain. In some embodiments, the CD95 (Fas) ligand binding domain is a Fas ectodomain or a functional fragment thereof.In some embodiments, the intracellular component is the CD137 (4-1BB) transmembrane domain or a functional fragment thereof. In some embodiments, the CD95 (Fas) ectodomain or a functional fragment thereof comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81, or the CD137 (4-1BB) intracellular signaling domain comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 82. In some embodiments, the fusion protein comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 80.In some embodiments, the nucleic acid sequence encoding the fusion protein comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 83. In some embodiments, the CD95 (Fas) ectodomain or a functional fragment thereof comprises at least one of residues R68, F97, K100, R102, R103, L106, F133, H142 of SEQ ID NO: 81. In some embodiments, the CD137 (4-1BB) intracellular signaling domain, or a portion or variant thereof, comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the CD8 co-receptor α or β chain, or a portion or variant thereof, comprises the amino acid sequence of SEQ ID NO: 65 or the amino acid sequence of SEQ ID NO: 66. In some embodiments, the neoantigen peptide is a KRAS, HRAS, NRAS, p53, or PIK3CA variant peptide. In some embodiments, the KRAS variant peptide comprises x-V-G-A-x-G-x-x-K, where x represents any amino acid. In some embodiments, the KRAS variant peptide is a KRAS G12V variant peptide. In some embodiments, the KRAS G12V variant peptide comprises the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGAVGVGK (SEQ ID NO: 3). In some embodiments, the HLA protein is encoded by the HLA-A*11 or HLA-A*11:01 allele. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide between the nucleic acid sequence encoding the TCR receptor variable α (Vα) region and the nucleic acid sequence encoding the TCR receptor variable β (Vβ) region. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide disposed between (a) and (b), or between (b) and (c) if (c) is present.In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide between the sequence encoding the CD8 coreceptor alpha chain and the sequence encoding the CD8 coreceptor beta chain. In some embodiments, the polynucleotide is between the nucleic acid sequence encoding the binding protein and the nucleic acid sequence encoding the polypeptide comprising the extracellular portion of the CD8 coreceptor alpha chain and / or between the nucleic acid sequence encoding the binding protein and the nucleic acid sequence encoding the polypeptide comprising the extracellular portion of the CD8 coreceptor beta chain, and further comprises a nucleic acid sequence encoding a self-cleaving peptide. In some embodiments, the polynucleotide is operably linked in-frame to (i) (pnBP)-(pnSCP. 1 )-(pnCD8α)-(pnSCP 2 )-(pnCD8β)-(pnFP); (ii) (pnBP)-(pnSCP 1 )-(pnCD8β)-(pnSCP 2 )-(pnCD8α)-(pnFP); (iii) (pnBP)-(pnSCP 1 )-(pnFP)-(pnSCP 1 ))-(pnCD8α)-(pnSCP 2 )-(pnCD8β); or (iv) (pnBP)-(pnSCP 1 )-(pnFP)-(pnSCP 1 )-(pnCD8β)-(pnSCP 2 )-(pnCD8α), wherein pnCD8α is a nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor alpha chain, pnCD8β is a nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor alpha chain, pnBP is a nucleic acid sequence encoding a binding protein, pnFP is a nucleic acid sequence encoding a fusion protein, pnSCP 1 and pnSCP 2Each is independently a polynucleotide encoding a self-cleaving peptide, and the polynucleotide and / or the encoded self-cleaving peptide are the same or different, as necessary. In some embodiments, the self-cleaving peptide is a P2A, T2A, E2A, or furin peptide. In some embodiments, the P2A, T2A, or E2A peptide comprises the amino acid sequence of SEQ ID NO: 74, 75, or 76, respectively. In some embodiments, the furin peptide comprises the amino acid sequence RAKR. In some embodiments, the binding protein and the fusion protein are encoded in a single construct or contiguous genomic segment. In some embodiments, the binding protein, the fusion protein, and CD8α or CD8β or both are encoded in a single construct or contiguous genomic segment. In some embodiments, the binding protein and the fusion protein are encoded in a single open reading frame. In some embodiments, the binding protein and the fusion protein are operably linked to a single promoter. In some embodiments, the binding protein and the fusion protein are operably linked to different promoters.

[0004] In some aspects, the disclosure provides a vector comprising any of the polynucleotides described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector or a γ-retroviral vector.

[0005] In some embodiments, the disclosure provides a host cell comprising any of the polynucleotides described herein or any of the vectors. In some embodiments, the host cell does not replicate for 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36, or more than 48 hours in the absence of an exogenous cytokine. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune cell. In some embodiments, the host cell is a human immune cell, and the human immune cell comprises T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof. In some embodiments, the human immune cell comprises T cells, and the T cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - double-negative T cells, γδ T cells, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof.

[0006] In one aspect, the present disclosure provides a method for treating a disease or disorder associated with a KRAS G12V mutation, an NRAS G12V mutation, or an HRAS G12V mutation in a subject, the method comprising administering to the subject an effective amount of any of the host cells described herein. In some embodiments, the disease or disorder includes cancer. In some embodiments, the cancer is a solid cancer or a hematological malignancy. In some embodiments, the cancer is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; anaplastic thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell carcinoma of the lung; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; squamous cell carcinoma of the head and neck; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic cancer; follicular thyroid cancer; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial cancer; or papillary thyroid cancer. In some embodiments, the effective amount of host cells is administered to the subject parenterally or intravenously. In some embodiments, the effective amount includes from about 10 4 cells / kg to about 10 11 cells / kg. In some embodiments, the effective amount includes CD4 + T cells and CD8 + T cells. In some embodiments, the effective amount includes a substantial amount of CD4 + T cells and CD8 +It includes T cells. In some embodiments, the method further includes administering a cytokine to the subject. In some embodiments, the cytokine includes IL-2, IL-15, or IL-21. In some embodiments, the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent. In some embodiments, the subject has received a myeloablative therapy. In some embodiments, the cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% during a period after administering an effective amount of the host cell. In some embodiments, the period includes 120 days or less, 60 days or less, 50 days or less, 40 days or less, 30 days or less, or 20 days or less. In some embodiments, the method further includes administering at least a second dose.

[0007] In some aspects, the present disclosure provides a method of eliciting an immune response against a cell expressing a neoantigen, the method comprising contacting the cell with a cell comprising any of the polynucleotides or vectors described herein.

[0008] In some aspects, the present disclosure provides a method of eliciting an immune response against a cell expressing a neoantigen, the method comprising contacting the cell with any of the host cells described herein. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is a pancreatic cancer cell, a lung cancer cell, or a colorectal cancer cell. In some embodiments, the pancreatic cancer cell is a pancreatic ductal adenocarcinoma cell. In some embodiments, the lung cancer cell is a non-small cell lung cancer cell.

[0009] In some aspects, the present disclosure provides a method of genetically engineering immune cells, the method comprising contacting the cells with a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a T cell receptor (TCR) or a functional fragment or variant thereof, a CD8α and / or CD8β coreceptor or a functional fragment or variant thereof, and an intracellular component comprising a CD95 (Fas) ectodomain or a functional fragment thereof and a CD137 (4-1BB) intracellular signaling domain, and expanding the immune cells. In some embodiments, the polynucleotide is any of the polynucleotides described herein or any of the vectors.

[0010] In one aspect, the present disclosure provides a host cell comprising: (a) a fusion protein, wherein the fusion protein comprises: (i) an extracellular component comprising a CD95 ligand (FasL) binding domain comprising a CD95 (Fas) ectodomain or a functional fragment thereof, and (ii) an intracellular component comprising a CD137 (4-1BB) intracellular signaling domain, and a nucleic acid sequence encoding a binding protein is located upstream of the nucleic acid sequence encoding the fusion polypeptide; and (b) an exogenous CD8 coreceptor α or β chain or a part or variant thereof. In some embodiments, the exogenous CD8 coreceptor α or β chain or a part or variant thereof is expressed from a locus other than the native locus of the CD8 coreceptor α or β chain. In some embodiments, the host cell comprises an mRNA encoding an exogenous CD8 coreceptor α or β chain or a part or variant thereof, which comprises a non-native 3' or 5' untranslated region (UTR). In some cases, the sequence encoding the exogenous CD8 coreceptor α or β chain or a part or variant thereof is on the same mRNA as the sequence encoding the fusion polypeptide. In some embodiments, the non-native 3' or 5' UTR is a viral UTR, an adenoviral UTR, or a lentiviral UTR. In some embodiments, the host cell comprises a native TCR. The exogenous CD8 coreceptor α or β chain or a part or variant thereof fusion protein further encodes a hydrophobic component between the extracellular and intracellular components of the fusion protein. In some embodiments, the CD95 (Fas) ligand binding domain is a Fas ectodomain or a functional fragment thereof. In some embodiments, the intracellular component is a CD137 (4-1BB) transmembrane domain or a functional fragment thereof.In some embodiments, the CD95 (Fas) ectodomain or a functional fragment thereof comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81, or the CD137 (4-1BB) intracellular signaling domain comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 82. In some embodiments, the fusion protein comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 80. In some embodiments, the CD95 (Fas) ectodomain or a functional fragment thereof comprises at least one of residues R68, F97, K100, R102, R103, L106, F133, H142 of SEQ ID NO: 81. In some embodiments, the CD137 (4-1BB) intracellular signaling domain, or a portion or variant thereof, comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the CD8 co-receptor α or β chain, or a portion or variant thereof, comprises the amino acid sequence of SEQ ID NO: 65 or the amino acid sequence of SEQ ID NO: 66.In some embodiments, the host cell further comprises a binding protein comprising an exogenous TCR. In some embodiments, the binding protein comprises a binding domain that binds to a peptide:HLA complex, the complex comprising a neoantigen peptide and an HLA protein. In some embodiments, the neoantigen peptide is a KRAS, HRAS, NRAS, p53, or PIK3CA variant peptide. In some embodiments, the KRAS variant peptide comprises x-V-G-A-x-G-x-x-K, where x represents any amino acid. In some embodiments, the neoantigen peptide is a KRAS variant peptide, and the KRAS variant peptide is a KRAS G12V variant peptide. In some embodiments, the KRAS G12V variant peptide comprises the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGAVGVGK (SEQ ID NO: 3). In some embodiments, the HLA protein is encoded by the HLA-A*11 or HLA-A*11:01 allele. In some embodiments, the fusion protein and CD8α or CD8β or both are encoded in a single construct or contiguous genomic segment. In some embodiments, the fusion protein and CD8α or CD8β or both are all encoded in a single open reading frame. In some embodiments, the host cell does not replicate for 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36, or more than 48 hours in the absence of exogenous cytokines. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune cell. In some embodiments, the host cell is a human immune cell, and the human immune cell comprises T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof. In some embodiments, the human immune cell is a T cell, and the T cell is CD4. + T cells, CD8 + T cells, CD4 - CD8 - including double-negative T cells, γδ T cells, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof.

[0011] In some embodiments, the present disclosure provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of any of the host cells described herein. In some embodiments, the host cell further comprises a TCR targeting an antigen presented by the cancer. In some embodiments, the cancer is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; cholangiocarcinoma; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell lung cancer; anaplastic astrocytoma; chronic myelogenous leukemia; diffuse large B cell lymphoma; double hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; thyroid follicular carcinoma; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial cancer; or papillary thyroid cancer. In some embodiments, the effective amount of host cells is administered parenterally or intravenously to the subject. In some embodiments, the effective amount comprises from about 10 4 cells / kg to about 10 11 cells / kg. In some embodiments, the effective amount is CD4 + T cells and CD8 +It includes T cells. In some embodiments, the method further includes administering a cytokine to the subject. In some embodiments, the cytokine includes IL-2, IL-15, or IL-21. In some embodiments, the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent. In some embodiments, the subject has received a myeloablative therapy. In some embodiments, the cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% during a period after administering an effective amount of the host cells. In some embodiments, the period includes 120 days or less, 60 days or less, 50 days or less, 40 days or less, 30 days or less, or 20 days or less. In some embodiments, the method further includes administering at least a second dose. In some embodiments, the host cells have been verified by any of the methods described in Table 3.

[0012] In some embodiments, the present disclosure provides a composition comprising a plurality of host cells, wherein the host cells are targeted to neoantigens (e.g., mutant KRAS peptides) or contain T cells specific thereto, and the composition comprises (a) at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more CD3+ cells stained with a dextramer specific for a mutant KRAS peptide as evaluated by flow cytometry, (b) at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or more T cells that are CD3 positive as evaluated by flow cytometry, and (c) at least 70%, 75%, 80%, 85%, 90%, or more viable cells as evaluated by automated cell counting. In some embodiments, the host cells are any of the host cells described herein. In some embodiments, the composition comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more CD3+ cells stained with a dextramer specific for a mutant KRAS G12V peptide as evaluated by flow cytometry. In some embodiments, the composition comprises at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or more T cells that are CD3 positive as evaluated by flow cytometry. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0013] In some embodiments, the present disclosure provides any of the host cells or any of the vectors described herein and a pharmaceutically acceptable excipient.

[0014] Additional aspects and advantages of the present disclosure will be readily apparent to those of ordinary skill in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other and different embodiments, and some of the details thereof are capable of various obvious modifications all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0015] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Brief Description of the Drawings

[0016] The novel features of the invention are particularly pointed out in the appended claims. A better understanding of the features and advantages of the present invention will be obtained from the following detailed description of the exemplary embodiments that utilize the principles of the present invention and the accompanying drawings.

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Mode for Carrying Out the Invention

[0017] Effective T cell activation often requires or is enhanced by co-stimulatory signals. In the tumor microenvironment, co-stimulatory molecules are generally downregulated. Therefore, a composition of cells used in adoptive T cell therapy that counteracts this downregulation of co-stimulatory molecules or generally enhances the effect of antigen-targeting receptors on such T cells in the tumor microenvironment is needed. Additionally, the tumor microenvironment can contain heterogeneous cell types (e.g., stromal cells, endothelial cells, and tumor-associated macrophages, granulocytes, and inflammatory monocytes) that contribute to T cell suppression through direct contact and secretion of soluble inhibitory factors.

[0018] Some aspects of the present disclosure generally relate to cells (e.g., immune effector cells such as CD4+ and / or CD8+ T cells) that express 1) an exogenous binding protein that binds to a neoantigen peptide:HLA complex, 2) a fusion protein (e.g., a Fas-41BB fusion protein), and 3) a CD8αβ coreceptor (e.g., an exogenous CD8αβ coreceptor). Some aspects of the present disclosure generally relate to one or more constructs encoding 1) an exogenous binding protein that binds to a neoantigen peptide:HLA complex, 2) a fusion protein (e.g., a Fas-41BB fusion protein), and 3) a CD8αβ coreceptor (e.g., an exogenous CD8αβ coreceptor).

[0019] Some aspects of the present disclosure generally relate to fusion proteins (e.g., chimeric receptors or "switch" receptors) that convert T cell inhibitory signals in the tumor microenvironment into T cell activation or proliferation signals. Accordingly, some aspects of the present disclosure relate to fusion proteins that include an extracellular domain specific for a soluble or cell-anchored inhibitory ligand linked to an intracellular domain that contributes to T cell activation (e.g., the 4-1BB intracellular signaling domain, or the CD28 intracellular signaling domain). In some cases, such proteins include an extracellular domain derived from the Fas receptor and an intracellular domain derived from the 4-1BB receptor (e.g., a Fas-41BB fusion protein).

[0020] Although not wishing to be bound by theory, such Fas-41BB fusion proteins can inhibit T cell apoptosis, enhance IL-2 or IFN-γ secretion, promote the development of memory T cells, increase T cell metabolic capacity, and / or improve T cell proliferation, persistence, and fitness by activating NF-κB, increasing Bcl-2 expression, and activating the PI3K and MEK-1 / 2 signaling pathways in response to Fas ligand (FASLG) in the tumor microenvironment. Alternatively or additionally, such Fas-41BB fusion proteins can act in a dominant inhibitory manner or sequester Fas ligand expression by tumors, endothelium, and activated T cells in the tumor microenvironment, preventing the elimination or apoptosis of T cells during tumor infiltration. It is documented that Fas ligand is expressed in the tumor microenvironment of many solid tumors, and it is contemplated that the presence of Fas ligand in the microenvironment of solid tumors can contribute to the limited efficacy of adoptive T cell therapy.

[0021] Some aspects of the present disclosure generally relate to binding proteins specific for Ras neoantigens, modified immune cells that express them, polynucleotides encoding the binding proteins, and related uses. Mutated Ras proteins (e.g., KRAS, NRAS, HRAS) can produce neoantigens containing a G→V mutation at position 12 of the full-length KRAS protein (SEQ ID NO: 1; UniProt KB P01116), position 12 of the full-length NRAS protein (SEQ ID NO: 78; Uniprot KB P01111), or position 12 of the full-length HRAS protein (SEQ ID NO: 79; Uniprot KB P01112).

[0022] Some aspects of the present disclosure generally relate to binding proteins specific for p53 neoantigens, modified immune cells expressing the same, polynucleotides encoding the binding proteins, and related uses. Mutant p53 proteins can potentially generate neoantigens, for example, at positions R175, G245, R248, R249, R273, and R282 (relative to SEQ ID NO: 1039 (wild-type p53)). Missense mutations account for approximately 70% - 80% of p53 mutations, and downregulation of wild-type p53 activity occurs in most, if not all, human malignancies (Duffy et al., Seminars Cancer Bio., 79:58 - 67 (2022)).

[0023] Some aspects of the present disclosure generally relate to binding proteins specific for PIK3CA neoantigens, modified immune cells expressing the same, polynucleotides encoding the binding proteins, and related uses. Mutant p53 proteins can potentially generate neoantigens, for example, at positions R38, G106, C420, E453, E542, E545, M1043, and H1047 (relative to SEQ ID NO: 1040 (wild-type PIK3CA)). Missense mutations account for approximately 70% - 80% of PIK3CA mutations, and mutated PIK3CA activity has been found in many human cancers (Ligresti et al., Cell Cycle, 8(9):1352 - 58 (2009)).

[0024] The present disclosure provides binding proteins capable of binding to neoantigens. In certain aspects, binding proteins (and host cells such as immune cells comprising heterologous polynucleotides encoding the binding proteins of the present disclosure) comprising a TCR Vα domain and a TCR Vβ domain are provided, and the binding proteins can bind to a neoantigen peptide:HLA complex.

[0025] For example, in the present disclosure, a binding protein capable of binding to a Ras neoantigen is provided. In certain embodiments, a binding protein (and a host cell such as an immune cell comprising a heterologous polynucleotide encoding the Ras-specific binding protein of the present disclosure) comprising a TCR Vα domain and a TCR Vβ domain is provided, the binding protein being capable of specifically binding to a Ras peptide antigen:HLA complex, the Ras peptide antigen comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NO: 2 or 3. In certain embodiments, the HLA comprises HLA-A*11 such as HLA-A*11:01.

[0026] The disclosed binding protein is highly sensitive to the antigen and can induce activation of host T cells with a low concentration of peptide antigen. In certain embodiments, a population or sample of T cells (e.g., CD8+ and / or CD4+) expressing the binding protein has a maximum half-maximal expression of the activation marker Nur77 in the presence of a peptide with a [LogEC50 of less than -9 M (e.g., -9 M to -10 M)]. In certain embodiments, among a population or sample of T cells (e.g., CD8+ and / or CD4+) expressing the binding protein, the T cells have a maximum half-maximal expression of CD137 in the presence of a [LogEC50 of less than -10 M (e.g., -10 M to -11 M)]. In certain embodiments, among a population or sample of T cells (e.g., CD8+ and / or CD4+) expressing the binding protein, the T cells have a maximum half-maximal expression of IFN-γ in the presence of a [LogEC50 of less than -10 M (e.g., -10 M to -11 M)] peptide.

[0027] Host (e.g., T) cells expressing the binding protein according to the present disclosure are activated (e.g., as determined by the expression of CD137) in the presence of neoantigens recognized by the binding protein. For example, a binding protein that recognizes and binds to mutant KRAS is activated in the presence of cancer cell lines expressing mutant KRAS (e.g., OVCAR5 (ovarian serous adenocarcinoma), DAN-G (pancreatic adenocarcinoma), CFPAC1 (pancreatic adenocarcinoma), SW480 (colon cancer), SW527 (breast cancer), and NCI-H441 (lung adenocarcinoma) cell lines).

[0028] In some embodiments, host cells (e.g., T cells such as CD4+ T cells or CD8+ T cells) expressing the binding protein according to the present disclosure can specifically kill cells expressing neoantigens (e.g., mutant KRAS-expressing cells (e.g., SW480 cells such as an effector:target ratio of 8:1, an effector:target ratio of 4:1, or an effector:target ratio of 2:1)). In some embodiments, host cells expressing the binding protein according to the present disclosure can specifically kill cells expressing neoantigens (e.g., mutant KRAS-expressing cells) in vitro for at least 144 hours, including cases where additional tumor cells are added at 72 hours in a re-challenge setting.

[0029] In certain embodiments, the binding protein of the present disclosure is (i) an amino acid sequence derived from the human proteome and / or (ii) non-alloreactive, substantially non-alloreactive, and / or has a low risk of alloreactivity against human HLA alleles.

[0030] In any of the embodiments disclosed herein, the binding protein can be human, humanized, or chimeric. Also provided are polynucleotides encoding the binding protein, vectors comprising the polynucleotide, and host cells comprising the polynucleotide and / or vector and / or expressing the binding protein. The binding proteins and host cells (e.g., T cells, NK cells, NK-T cells) disclosed herein are useful for treating diseases or disorders associated with KRAS neoantigens, such as cancer. The binding proteins disclosed herein can also bind to the G12V antigen that occurs in human NRAS or human HRAS, and this protein contains the same sequence as KRAS in the region near residue G12. Accordingly, the disclosed compositions are useful in treating diseases or disorders associated with KRAS neoantigens, NRAS neoantigens containing a G12V mutation, or HRAS neoantigens containing a G12V mutation, or any combination thereof.

[0031] Also provided are methods and uses of the binding proteins, polynucleotides, vectors, host cells, and related compositions disclosed herein for the treatment of diseases or disorders associated with neoantigen (e.g., KRAS, NRAS, HRAS, p53, and / or PIK3CA) mutations provided herein.

[0032] Before describing the present disclosure in more detail, it may be helpful to provide definitions of certain terms used herein. Additional definitions are set forth throughout the present disclosure.

[0033] In this specification, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated. Also, any number range recited herein with respect to any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ±20% of the recited range, value, or structure, unless otherwise indicated. The terms "a" and "an" as used herein should be understood to refer to "one or more" of the recited components. The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination thereof of the alternatives. As used herein, the terms "include", "have", and "comprise" are used synonymously, and these terms and their variations are intended to be construed as non-limiting.

[0034] In addition, individual compounds or groups of compounds derived from various combinations of the structures and substituents described herein should be understood to be disclosed by this application to the same extent as if each compound or group of compounds were individually recited. Thus, the selection of a particular structure or particular substituents is within the scope of the present disclosure.

[0035] The term "consisting essentially of" is not equivalent to "comprising" and refers to those that do not substantially affect the specified materials or steps of the claim or the basic characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., binding domain, hinge region, linker module), or a protein (which may have one or more domains, regions, or modules) has an amino acid sequence of the domain, region, module, or protein that contributes at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein in combination, including extensions, deletions, mutations, or combinations thereof (e.g., amino acids at the amino terminus or carboxy terminus or between domains), and does not substantially affect the activity of the domain(s), region(s), module(s), or protein (i.e., does not reduce the activity by more than 50%, such as 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% or less), then a particular amino acid sequence "consists essentially of".

[0036] As used herein, "protein" or "polypeptide" generally refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids and non-naturally occurring amino acid polymers. In some embodiments, "peptide" (e.g., peptide antigen) refers to a polymer having a length of about 8 to 10 amino acid residues.

[0037] "Hematopoietic progenitor cells", as used herein, generally refers to cells that may be derived from hematopoietic stem cells or fetal tissues and are capable of further differentiation into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include CD24 Lo Lin - CD117 +Those having a phenotype, or those found in the thymus (referred to as progenitor thymocytes).

[0038] As used herein, "immune system cells" generally refers to any cells of the immune system that arise from hematopoietic stem cells in the bone marrow, which give rise to two major lineages, namely, myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes), and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 - CD8 - double-negative T cells, γδ T cells, regulatory T cells, natural killer cells, natural killer T cells, and dendritic cells. Macrophages and dendritic cells can also be referred to as "antigen-presenting cells" or "APCs", which are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with the TCR on the surface of the T cell.

[0039] "T cells" or "T lymphocytes" generally refers to immune system cells that mature in the thymus and produce a T cell receptor (TCR). T cells are naive ("T N”; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased or no expression of CD45RO compared to TCM (described herein), and may include memory T cells (TM) including stem cell memory T cells (experienced antigen, long-lived), and effector cells (experienced antigen, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM expressing CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (TEM expressing CD45RO and decreased expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (TE) refer to antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 compared to TCM, and are positive for granzyme and perforin. Helper T cells (T H ) are CD4 + cells that affect the activity of other immune cells by releasing cytokines. CD4 + T cells can activate and suppress the adaptive immune response, and which of these two functions is induced depends on the presence of other cells and signals. T cells can be collected using known techniques, and various subsets or combinations thereof can be enriched or depleted by known techniques, such as binding affinity to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4 + CD25 + (Foxp3 + ) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8 + CD28 - , and Qa-1 restricted T cells.

[0040] The "T cell receptor" (TCR) generally refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail) that can specifically bind to an antigen peptide bound to an MHC receptor; see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997). The TCR may be found on the surface of a cell or in a soluble form and is generally composed of a heterodimer having an α chain and a β chain (also known as TCRα and TCRβ, respectively) or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively). In certain embodiments, the binding proteins of the present disclosure, for example, polynucleotides encoding a TCR, may be codon-optimized to enhance expression in certain host cells such as, for example, cells of the immune system, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, or natural killer T cells (Scholten et al., Clin. Immunol. 119:135, 2006). Exemplary T cells capable of expressing the binding proteins and TCRs of the present disclosure include CD4 + T cells, CD8 + T cells, and related subsets thereof (e.g., naive, central memory, stem cell memory, effector memory).

[0041] Similar to other immunoglobulins (e.g., antibodies), the extracellular portion of the TCR chains (e.g., α-chain, β-chain) contains two immunoglobulin domains at the N-terminus, a variable domain (e.g., α-chain variable domain or Vα, β-chain variable domain or Vβ, typically amino acids 1-116 based on Kabat numbering (Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.)), and one constant domain adjacent to the cell membrane (e.g., α-chain constant domain or Cα, typically 5 amino acids 117-259 based on Kabat, β-chain constant domain or Cβ, typically amino acids 117-295 based on Kabat). Also, similar to immunoglobulins, the variable domain contains complementarity-determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores, et al., Proc. Nat’l Acad. Sci. USA 87:9138, 1990, Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The source of the TCR used in the present disclosure may be derived from various animal species such as human, mouse, rat, rabbit, or other mammals.

[0042] The terms "variable region" or "variable domain" generally refer to the domain of an immunoglobulin superfamily binding protein (e.g., TCR) involved in binding to an antigen, such as the TCR α-chain or β-chain (or for γδ TCR, the γ-chain and δ-chain). The variable domains of the α-chain and β-chain of a native TCR (Vα and Vβ, respectively) generally have a similar structure, and each domain contains four generally conserved framework regions (FRs) and three CDRs. The Vα domain is encoded by two separate DNA segments, a variable gene segment and a joining gene segment (V-J); the Vβ domain is encoded by three separate DNA segments, a variable gene segment, a diversity gene segment, and a joining gene segment (V-D-J). A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Further, a TCR that binds to a particular antigen can be isolated using the Vα or Vβ domain from the TCR that binds to the antigen to screen a library of complementary Vα or Vβ domains, respectively.

[0043] The terms "complementary determining region" and "CDR" are generally synonymous with "hypervariable region" or "HVR" and generally refer to the amino acid sequences within the variable region of an immunoglobulin (e.g., TCR). CDRs confer antigen specificity and binding affinity and are separated from each other by framework regions in the primary amino acid sequence. Generally, each TCR α-chain variable region has three CDRs (αCDR1, αCDR2, αCDR3), and each TCR β-chain variable region has three CDRs (βCDR1, βCDR2, βCDR3). In TCRs, CDR3 is considered the major CDR involved in recognizing processed antigen. Generally, CDR1 and CDR2 interact primarily or exclusively with MHC.

[0044] CDR1 and CDR2 are encoded within the variable gene segments of the TCR variable region coding sequences, while CDR3 is encoded by the region spanning the variable and joining segments for Vα, or the region spanning the variable, diversity, and joining segments for Vβ. Thus, when the identities of the variable gene segments of Vα or Vβ are known, the sequences of their corresponding CDR1 and CDR2 can be inferred, for example, according to the numbering scheme described herein. Compared to CDR1 and CDR2, CDR3, particularly CDR3β, is typically significantly more diverse, due to nucleotide addition or loss during the recombination process.

[0045] The TCR variable domain sequences can be aligned according to numbering schemes (e.g., Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho), which enable, for example, annotating equivalent residue positions and comparing different molecules using, e.g., the ANARCI software tool (2016, Bioinformatics 15:298-300). The numbering schemes provide a standard delineation of the framework regions and CDRs in the TCR variable domains. In certain embodiments, the CDRs of the present disclosure are identified according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003, imgt.org / IMGTindex / V-QUEST.php). In some embodiments, a CDR (e.g., CDR3) is identified or defined according to the IMGT junction definition. In some embodiments, a CDR (e.g., CDR3) is identified or defined according to the IMGT definition. In some embodiments, the CDRs of the present disclosure are identified or defined according to the Kabat numbering scheme or method. In some embodiments, the CDRs of the present disclosure are identified or defined according to the Chothia numbering scheme or method. In some embodiments, the CDRs of the present disclosure are identified or defined according to the EU numbering scheme or method. In some embodiments, the CDRs of the present disclosure are identified or defined according to the Enhanced Chothia numbering scheme or method. In some embodiments, the CDRs of the present disclosure or the defined CDRs are identified according to the Aho numbering scheme or method.

[0046] The source of the TCRs used in the present disclosure can be derived from any of a variety of animal species, such as human, mouse, rat, rabbit, or other mammals. The TCR constant domain sequences can be derived from, for example, human, mouse, marsupials (such as opossum, bandicoot, wallaby), shark, or non-human primates. In certain embodiments, the TCR constant domain sequences are human or engineered variants of human sequences. The TCR constant domains can be engineered to improve pairing, expression, stability, or any combination thereof. See, for example, Cohen et al., Cancer Res, 2007, Kuball et al., Blood 2007, and Haga-Freidman et al., Journal of Immunology 2009. Examples of engineering in TCR Cα and Cβ include mutations of natural amino acids to cysteine such that a disulfide bond is formed between an introduced cysteine in one TCR constant domain and a native cysteine in the other TCR constant domain. Such mutations can include T48C in Cα, T57C in Cβ, or both. Mutations to improve stability can include mutations in the Cα transmembrane domain from the sequence LSVIGF to the sequence LLVIVL (the “L-V-L” mutation; see Haga-Friedman et al., J Immunol 188:5538-5546 (2012), the TCR mutations and mutant TCR constant domain sequences of which are incorporated herein by reference).

[0047] As used herein, the term "CD8 coreceptor" or "CD8" generally refers to the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer. The CD8 coreceptor supports the function of cytotoxic T cells (CD8+), and functions via signal transduction through its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1:81-88, 2004). There are five human CD8 beta chain isoforms (see UniProtKB identifier P10966) and a single human CD8 alpha chain isoform (see UniProtKB identifier P01732).

[0048] "CD4" generally refers to the immunoglobulin coreceptor glycoprotein that supports the TCR in its interaction with antigen-presenting cells (see Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002)). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and contains four immunoglobulin domains (D1-D4) expressed on the cell surface. During antigen presentation, CD4 is recruited together with the TCR complex and binds to different regions of the MHCII molecule (CD4 binds to MHCIIβ2, while the TCR complex binds to MHCIIα1 / β1). Without wishing to be bound by theory, it is thought that proximity to the TCR complex enables CD4-associated kinase molecules to phosphorylate immunoreceptor tyrosine activation motifs (ITAMs) present on the cytoplasmic domain of CD3. This activity is thought to amplify signals generated by the activated TCR and the immune response, in order to generate or recruit various types of immune system cells, including T helper cells.

[0049] In certain embodiments, the TCR is found on the surface of a T cell (or T lymphocyte) and associates with the CD3 complex. "CD3" is a six-chain multi-protein complex associated with antigen signaling in T cells (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999). In mammals, the complex contains one CD3γ chain, one CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. The CD3γ, CD3β, and CD3ε chains are related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3β, and CD3ε chains are negatively charged, which is thought to associate these chains with the positively charged regions of the T cell receptor chains. The intracellular tails of the CD3γ, CD3β, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, while each CD3ζ chain has three. Without wishing to be bound by theory, ITAMs are thought to be important for the signaling ability of the TCR complex. The CD3 used in the present disclosure may be derived from various animal species, including humans, mice, rats, or other mammals.

[0050] As used herein, "TCR complex" generally refers to the complex formed by the association of CD3 and TCR. For example, the TCR complex may be composed of a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRα chain, and a TCRβ chain. Alternatively, the TCR complex may be composed of a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRγ chain, and a TCRβ chain.

[0051] "Components of the TCR complex", as used herein, generally 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 partial TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0052] A "chimeric antigen receptor" (CAR) generally refers to a fusion protein engineered to contain two or more naturally occurring amino acid sequences, domains, or motifs that are not naturally present or are linked together in a way that is not naturally present in the host cell, and which, when present on the surface of a cell, can function as a receptor. A CAR can include an extracellular portion that includes an antigen-binding domain (e.g., a TCR-binding domain derived from or obtained from a TCR specific for a cancer antigen, a scFv derived from or obtained from an antibody, or an antigen-binding domain derived from or obtained from a killer immunoglobulin receptor derived from NK cells, etc., derived from or obtained from an immunoglobulin or immunoglobulin-like molecule) linked to a transmembrane domain, and one or more intracellular signaling domains (optionally including a co-stimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013), and also see Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014), and Walseng et al., Scientific Reports 7:10713 (2017), the CAR constructs and methods of making the same are incorporated herein by reference). The CARs of the present disclosure that specifically bind to a Ras antigen (e.g., in the context of a peptide:HLA complex) include a TCR Vα domain and a Vβ domain.

[0053] Any polypeptide of the present disclosure, when encoded by a polynucleotide sequence, may include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). The signal peptide can target a newly synthesized polypeptide to its appropriate location inside or outside the cell. In some contexts, the signal peptide is about 15 to about 22 amino acids in length. The signal peptide may be removed from the polypeptide during localization (e.g., membrane insertion) or secretion, or after localization (e.g., membrane insertion) or secretion is complete. A polypeptide having a signal peptide is referred to herein as a "preprotein", and a polypeptide from which its signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In any of the embodiments disclosed herein, a binding protein or fusion protein may comprise or be a mature protein, or may be a preprotein or comprise a preprotein.

[0054] A "linker" generally refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and provides a spacer function compatible with the interaction of two sub-binding domains such that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., scTCR) or retains signaling activity (e.g., TCR complex). In certain embodiments, the linker is composed of about 2 to about 35 amino acids, such as, or about 4 to about 20 amino acids or about 8 to about 15 amino acids or about 15 to about 25 amino acids. An exemplary linker includes a glycine-serine linker.

[0055] "Antigen" or "Ag", as used herein, generally refers to an immunogenic molecule that elicits an immune response. This immune response may be accompanied by antibody production, activation of specific immune cells (e.g., T cells), or both. The antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, or lipid. It is readily apparent that the antigen may be synthetic, produced recombinantly, or derived from a biological sample. Exemplary biological samples that may contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. The antigen may be produced by cells that have been modified or genetically engineered to express the antigen, or by cells that endogenously express a mutation or polymorphism that is immunogenic (e.g., without any modification or genetic engineering by human intervention).

[0056] "Neoantigen," as used herein, generally refers to a host cell product containing a structural change, alteration, or mutation that creates a new antigen or antigenic epitope that was not previously observed in the subject's genome (i.e., in a sample of healthy tissue from the subject) or recognized or seen by the host immune system, which is (a) processed by the cell's antigen processing and transport machinery and presented on the cell surface in association with MHC (e.g., HLA) molecules; and (b) elicits an immune response (e.g., a cellular (T cell) response). Neoantigens can originate, for example, from coding polynucleotides having alterations (substitutions, additions, deletions) that result in an altered or mutated product, or from the insertion of exogenous nucleic acid molecules or proteins into the cell, or from exposure to environmental factors (e.g., chemical factors, radiological factors) that cause genetic changes. Neoantigens can arise separately from, or result from or be associated with, tumor antigens. "Tumor neoantigen" (or "tumor-specific neoantigen") refers to a protein containing neoantigenic determinants that are associated with, result from, or occur in tumor cells or multiple cells within a tumor. Tumor neoantigenic determinants are found, for example, on antigenic tumor proteins or peptides containing one or more somatic mutations or chromosomal rearrangements encoded by the DNA of tumor cells (e.g., pancreatic cancer, lung cancer, colorectal cancer), and on proteins or peptides derived from viral open reading frames associated with virus-related tumors (e.g., cervical cancer, some head and neck cancers). The terms "antigen" and "neoantigen" are used synonymously herein when referring to Ras antigens containing the mutations disclosed herein. In some embodiments, the neoantigen comprises a RAS peptide (e.g., KRAS, HRAS, or NRAS), a BRAF peptide, a CALR peptide, a DNMT3A peptide, an EGFR peptide, an ERBB2 peptide, an ESR1 peptide, an FGFR3 peptide, a FLT3 peptide, a GNA11 peptide, a GNAQ peptide, an IDH peptide, a MYD88 peptide, a p53 peptide, a PIK3CA peptide, or an SF3B1 peptide.In some embodiments, the neoantigen comprises an ALK peptide, an EGFR peptide, a HER2 peptide, a KIT peptide, a MET peptide, an NRG1 peptide, an NTRK peptide, a PDGFRα peptide, a RAF peptide, a RET peptide, or a ROS1 peptide. [WH1]This list is not exhaustive as other neoantigens are contemplated. In some embodiments, the neoantigen comprises an oncogenic driver mutation. Without being bound by theory, oncogenic driver mutations are thought to be involved in cancer initiation and maintenance.

[0057] The term "epitope" or "antigenic epitope" generally includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule such as an immunoglobulin, a T cell receptor (TCR), a chimeric antigen receptor, or other binding molecule, domain, or protein. Epitope determinants generally contain surface groups of chemically active molecules such as amino acids or sugar side chains and can have specific three-dimensional structural features as well as specific charge features.

[0058] As used herein, the term "KRAS (or NRAS or HRAS) antigen (or neoantigen)" or "KRAS (or NRAS or HRAS) peptide antigen (or neoantigen)" or "KRAS (NRAS or HRAS) peptide" generally refers to a peptide moiety of a KRAS or NRAS or HRAS protein that is naturally or synthetically produced, having a length in the range of from about 7 amino acids to about 20 amino acids, such as about 8 amino acids, about 9 amino acids, about 10 amino acids, and including at least one amino acid change caused by a G12 (e.g., G12V) mutation (position 12 conforms to the full-length KRAS protein sequence shown in SEQ ID NO: 1; also conforms to the full-length NRAS and HRAS protein sequences shown in SEQ ID NO: 78 and 79, respectively), and this peptide can form a complex with an MHC (e.g., HLA) molecule, and the binding proteins of the present disclosure specific for the KRAS or NRAS or HRAS peptide:MHC (e.g., HLA) complex can specifically bind to such a complex. Exemplary KRAS (or NRAS or HRAS) antigens include, consist essentially of, or consist of a peptide having the amino acid sequence of SEQ ID NO: 2 or 3.

[0059] The "major histocompatibility complex" (MHC) generally refers to glycoproteins that deliver peptide antigens to the cell surface of all nucleated cells. MHC class I molecules are heterodimers having a transmembrane α chain (with three α domains) and non-covalently associated β2-microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain contains two domains. MHC class I molecules deliver peptides of cytosolic origin to the cell surface, where the peptide:MHC complex is recognized by CD8 + T cells. MHC class II molecules deliver peptides of vesicular origin to the cell surface, where the peptide is CD4 +Recognized by T cells. Human MHC is called human leukocyte antigen (HLA). HLAs corresponding to "class I" MHC present peptides derived from inside the cell, and such HLAs include, for example, HLA-A, HLA-B, and HLA-C. Alleles include, for example, HLA A*11 such as HLA-A*11:01. HLAs corresponding to "class II" MHC present peptides derived from outside the cell, and such HLAs include, for example, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0060] The principle of antigen processing by antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, lymphocytes or other cell types), and major histocompatibility complex (MHC)-restricted presentation between APCs and T cells that are immunocompatible (e.g., share at least one allelic form of an MHC gene related to antigen presentation), the principle of antigen presentation by APCs to T cells is well established (e.g., see Murphy, Janeway’s Immunobiology (8 th Ed.) 2011 Garland Science, NY; chapters 6, 9 and 16). For example, processed antigen peptides originating from the cytosolic sol (e.g., tumor antigens, intracellular pathogens) generally have a length of about 7 amino acids to about 11 amino acids and associate with class I MHC (HLA) molecules, while peptides processed in the vesicular system (e.g., bacteria, viruses) vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC (HLA) molecules.

[0061] As used herein, the term "KRAS-specific binding protein" generally refers to a protein or polypeptide such as a TCR, scTv, scTCR, or CAR that binds to a KRAS peptide antigen or an NRAS peptide antigen or an HRAS peptide antigen (or, for example, to a KRAS or NRAS or HRAS peptide antigen:HLA complex on the cell surface), does not bind to a peptide that does not contain a KRAS peptide antigen, an NRAS peptide antigen, or an HRAS peptide antigen, and does not bind to an HLA complex containing such a peptide.

[0062] The binding proteins of the present disclosure, such as TCRs, scTCRs, and CARs, contain a binding domain specific for the target. A "binding domain" (also referred to as a "binding region" or "binding moiety") as used herein refers to a molecule or a portion thereof (e.g., a peptide, oligopeptide, polypeptide, protein) that has the ability to specifically associate, integrate, or react with a target (e.g., a KRAS or NRAS or HRAS peptide or a KRAS or NRAS or HRAS peptide:MHC complex) by non-covalent bonds. Binding domains include biological molecules, molecular complexes (i.e., complexes containing two or more biological molecules), or any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for another target of interest. Exemplary binding domains include immunoglobulin variable regions or single-chain constructs containing them (e.g., single-chain TCR (scTCR) or scTv).

[0063] In certain embodiments, the Ras-specific binding protein has a K of less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M of K dor binds to a KRAS (or NRAS or HRAS) peptide (or KRAS (or NRAS or HRAS):HLA complex) with an affinity that is about the same as, at least about the same as, or greater than the affinity presented by any of the exemplary Ras-specific binding proteins provided herein, e.g., any of the Ras-specific TCRs provided herein, when measured by, for example, the same assay. In certain embodiments, the Ras-specific binding protein comprises a Ras-specific immunoglobulin superfamily binding protein or a binding portion thereof.

[0064] "Specifically binds to" or "specific for" as used herein generally means that a binding protein (e.g., a TCR receptor) or binding domain (or a fusion protein thereof) associates or integrates with a target molecule with an affinity that is equal to or greater than the ratio of the on-rate [k 5 M -1 (which is equal to the ratio of the on-rate [k on to the off-rate [k off ) for this association reaction and does not significantly associate or integrate with any other molecule or component in the sample. The binding protein or binding domain (or a fusion protein thereof) can be classified as a "high affinity" binding protein or binding domain (or a fusion protein thereof) or a "low affinity" binding protein or binding domain (or a fusion protein thereof). A "high affinity" binding protein or binding domain is at least 10 a M 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M-1 refers to a binding protein or binding domain having a K a . A "low affinity" binding protein or binding domain has a maximum of 10 7 M -1 , a maximum of 10 6 M -1 , a maximum of 10 5 M -1 for K a . Alternatively, affinity can be defined as the equilibrium dissociation constant (K d ) of a particular binding interaction in units of M (e.g., 10 -5 M to 10 -13 M).

[0065] In certain embodiments, a receptor or binding domain may have "enhanced affinity," which generally refers to a selected or engineered receptor or binding domain that has stronger binding to a target antigen than the wild-type (or parental) binding domain. For example, enhanced affinity can result from a higher K a (equilibrium association constant) for the target antigen than that of the wild-type binding domain, a lower K d (dissociation constant) for the target antigen than that of the wild-type binding domain, a lower off-rate (k off ) for the target antigen than that of the wild-type binding domain, or a combination thereof.

[0066] To identify the binding domains of the present disclosure that specifically bind to a particular target, as well as to determine the affinity of a binding domain or fusion protein, various assays are known, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents).

[0067] In certain embodiments, only the neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA)-specific binding domain (i.e., without any other portion of the neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA)-specific binding protein) may be soluble and be about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, or about 10 -13 M or less of K d and can bind to the neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA) (or neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA) peptide, or neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA) peptide:HLA complex). In certain embodiments, the neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA)-specific binding domain comprises a neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA)-specific scTCR (e.g., a single-chain αβ TCR protein such as Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vα, or Vα-L-Vβ-Cβ, where Vα and Vβ are the TCRα and β variable domains, respectively, Cα and Cβ are the TCRα and β constant domains, respectively, and L is a linker such as the linker described herein).

[0068] As used herein, the term "functional avidity" generally refers to a biological measure of the response of immune cells (e.g., T cells, NK cells, NK-T cells) in vitro or the activation threshold to a given ligand concentration, and the biological measures can include cytokine production (e.g., IFN-γ production, IL-2 production, etc.), cytotoxic activity, activation markers (e.g., CD137, Nur77), and proliferation. For example, T cells that respond biologically (immunologically) in vitro to a low antigen dose by producing cytokines, exhibiting cytotoxic activity, or proliferating are considered to have high functional avidity, while T cells with lower functional avidity require a greater amount of antigen before an immune response similar to that of high-avidity T cells is induced. It will be understood that functional avidity is different from affinity and avidity. Affinity refers to the strength of any given binding between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind to multiple antigens, in which case the overall strength of the linkage is avidity.

[0069] There are numerous correlations between functional avidity and the effectiveness of the immune response. Some ex vivo studies have shown that the functions of individual T cells (e.g., proliferation, cytokine production, etc.) can be induced at different thresholds (see, e.g., Betts et al., J. Immunol. 172:6407, 2004; Langenkamp et al., Eur. J. Immunol. 32:2046, 2002). Factors that affect functional avidity can include (a) the affinity of the TCR for the pMHC complex, i.e., the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 167:2577, 2001), (b) the expression levels of the TCR on host cells and, in some embodiments, the CD4 or CD8 coreceptor, and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273:104, 1996), and additionally, the expression levels of molecules that weaken T cell function and TCR signaling.

[0070] The concentration of antigen required to induce the maximum half-maximal response between the baseline response and the maximum response after a specified exposure time (e.g., cytokine production or activation marker by host cells; fluorescence intensity when a labeled peptide binds to an HLA multimer) is referred to as the "maximum half-maximal effective concentration" or "EC50". EC50 values are generally expressed as an amount of molar concentration (moles / liter), but are often converted to logarithmic values as follows: -log 10 (EC50). For example, if the EC50 is equal to 1 μM (10 -6 M), the log 10 (EC50) value is -6. Another value used is pEC50, which is defined as the negative logarithm of EC50 (-log 10 (EC50)). In the above example, an EC50 equal to 1 μM has a pEC50 value of 6. In certain embodiments, the functional avidity of the binding proteins disclosed herein includes a measure of the ability of the binding proteins to promote T cell activation and / or IFNγ production, which is known in the art and can be measured using the assays described herein. In certain embodiments, the functional avidity includes a measure of the ability of the binding protein to activate host cells, such as T cells, upon binding to the antigen.

[0071] The binding proteins disclosed herein can include high functional avidity, capable of promoting the induction of immune cell effector functions (e.g., activation, proliferation, cytokine production, and / or cytotoxicity) against neoantigen peptides presented at even lower levels, such as the KRAS G12V mutant peptide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0072] In some embodiments, the binding protein has a log10EC50 for a neoantigen peptide of about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, about -9.2 or less, about -9.3 or less, about -9.4 or less, about -9.5 or less, about -9.6 or less, about -9.7 or less, about -9.8 or less, about -9.9 or less, or about -10 or less.

[0073] In some embodiments, the host cells disclosed herein bind to a target neoantigen of a binding protein (e.g., a TCR) with an EC50 (e.g., the peptide dose that reaches half-maximal activation of T cell proliferation) of less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM. The target neoantigen is, for example, a KRAS G12 variant peptide (such as a KRAS G12V variant peptide) present in a peptide:HLA complex. The EC50 can be determined, for example, by an assay to identify the peptide dose that reaches half-maximal activation of a T cell population, as reflected by the expression of activation markers (e.g., CD137, CD69, granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, cytokines, interleukins, interferons) upon exposure of target cells to various concentrations of the variant peptide.

[0074] In some embodiments, the host cells disclosed herein comprise a binding protein (e.g., a TCR) that binds to a target neoantigen of the binding protein (e.g., a KRAS G12 variant peptide such as a KRAS G12V variant peptide present in a peptide:HLA complex) with an EC50 (e.g., the peptide dose that reaches the maximum half-maximal activation of T cell proliferation) of at least about 100 mM, at least about 10 mM, at least about 1 mM, at least about 500 μM, at least about 100 μM, at least about 50 μM, at least about 10 μM, at least about 5 μM, at least about 4 μM, at least about 3 μM, at least about 2 μM, at least about 1 μM, at least about 900 nM, at least about 800 nM, at least about 700 nM, at least about 600 nM, at least about 500 nM, at least about 400 nM, at least about 300 nM, at least about 200 nM, at least about 100 nM, at least about 90 nM, at least about 80 nM, at least about 70 nM, at least about 60 nM, at least about 50 nM, at least about 40 nM, at least about 30 nM, at least about 20 nM, at least about 10 nM, at least about 5 nM, at least about 1 nM, at least about 900 pM, at least about 800 pM, at least about 700 pM, at least about 600 pM, at least about 500 pM, at least about 400 pM, at least about 300 pM, at least about 200 pM, at least about 100 pM, at least about 90 pM, at least about 80 pM, at least about 70 pM, at least about 60 pM, at least about 50 pM, at least about 40 pM, at least about 30 pM, at least about 20 pM, at least about 10 pM, at least about 5 pM, or at least about 1 pM.

[0075] A host cell can contain a transgenic polynucleotide encoding a chimeric fusion protein comprising an IL7R intracellular signaling domain. The chimeric fusion protein can include, for example, the intracellular portion of the interleukin 7 receptor A (IL7RA) polypeptide, or a portion or variant thereof that can contribute to IL-7 signaling in the host cell. The chimeric IL7R fusion protein can, for example, provide "signal 3" to increase STAT5 phosphorylation and host cell function, enhance host cell proliferation, increase host cell survival (e.g., in the tumor microenvironment), and / or enhance chemokine receptor expression.

[0076] Interleukin-7 receptor subunit alpha can also be referred to as IL7R-α, IL7RA, IL-7R-alpha, ILRA, interleukin-7 receptor-alpha, interleukin 7 receptor, cluster of differentiation 127 as CD127, or CDW127.

[0077] The IL7R intracellular signaling domain can include an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to SEQ ID NO: 1041.

[0078] In some embodiments, the IL7R intracellular signaling domain comprises (a) one or more residues of the BOX1 motif corresponding to residues 8-15 (VWPSLPDH) relative to SEQ ID NO: 1041 when optimally aligned, or (b) includes Y185 relative to SEQ ID NO: 1041 when optimally aligned. In some embodiments, the IL7R intracellular signaling domain comprises one or more residues of the FERM domain corresponding to residues 1-6 (KKRIKPI) or residues 16-28 (KKTLEHLCKKPRK) relative to SEQ ID NO: 1041 when optimally aligned.

[0079] In some embodiments, the chimeric fusion protein comprises the IL7R transmembrane domain. The IL7R transmembrane domain can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity relative to SEQ ID NO: 1042. In some embodiments, the IL7R transmembrane domain comprises a mutation relative to SEQ ID NO: 1042. In some embodiments, the mutation is or comprises the insertion of one or more cysteines and / or one or more prolines into the amino acid sequence of SEQ ID NO: 1042. In some embodiments, the mutation enables or facilitates receptor homodimerization. In some embodiments, the mutation comprises inserting a trimeric peptide of cysteine, proline, threonine (CPT) into the transmembrane domain. In some embodiments, the threonine of the CPT insertion is not threonine but another amino acid, and in at least certain cases, the other amino acid is cysteine or proline or neither cysteine nor proline.

[0080] In some embodiments, the chimeric fusion protein comprises the transmembrane domain of IL7R, IL2RA, IL2RB, IL2RG, IL14R, IL15R, IL9R, IL21R, CD2, CD40L, CD58, CD80, or SIRPα.

[0081] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising (i) the extracellular domain of a Cluster of Differentiation 80 (CD80) polypeptide, or a portion or variant thereof that can bind to a CD28 or CTLA-4 polypeptide; (ii) the extracellular domain of a Cluster of Differentiation 58 (CD58) polypeptide, or a portion or variant thereof that can bind to a Cluster of Differentiation 2 (CD2) polypeptide; (iii) the extracellular domain of a Signal Regulatory Protein Alpha (SIRPα) polypeptide, or a portion or variant thereof that can bind to a Cluster of Differentiation 47 (CD47) polypeptide; (iv) the extracellular domain of a Cluster of Differentiation 40 Ligand (CD40L) polypeptide, or a portion or variant thereof that can bind to a CD40 polypeptide; (v) the extracellular domain of a Cluster of Differentiation 2 (CD2) receptor, or a portion or variant thereof that can bind to a CD58 polypeptide; or (vi) the extracellular domain of a Cluster of Differentiation 34 (CD34) polypeptide.

[0082] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising the extracellular domain of a Cluster of Differentiation 80 (CD80) polypeptide, or a portion or variant thereof that can bind to a CD28 or CTLA-4 polypeptide. In some embodiments, the extracellular domain of CD80 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1043.

[0083] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising the extracellular domain of a Cluster of Differentiation 58 (CD58) polypeptide, or a portion or variant thereof that can bind to a CD28 or CTLA-4 polypeptide. In some embodiments, the extracellular domain of CD80 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1044.

[0084] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising the extracellular domain of CD34. In some embodiments, the extracellular domain of CD34 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1045.

[0085] In some embodiments, a population of host cells comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or chimeric IL7R polypeptide disclosed herein) exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold, at least 500-fold, or at least 1000-fold increased proliferation in response to a target cell (e.g., presenting a KRAS G12D peptide), compared to a population of control cells (e.g., corresponding cells lacking the Fas-41BB fusion protein or chimeric IL7R polypeptide). Proliferation can be determined, for example, by an in vitro lymphoproliferation assay or measurement of the number of host cells after co-incubation. The host cells can comprise modifications that result in a decrease in the expression of an extracellular binding protein (e.g., a TCR comprising Vα and Vβ regions and / or CDRs disclosed herein), and / or endogenous TRAC, TRBC1, and / or TRBC2.

[0086] In some embodiments, a population of host cells comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or chimeric IL7R polypeptide disclosed herein) exhibits killing of target cells that is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold, at least 500-fold, or at least 1000-fold compared to a population of control cells (e.g., corresponding cells lacking the Fas-41BB fusion protein or chimeric IL7R polypeptide). Killing of target cells can be determined, for example, by an in vitro cytotoxicity assay. The host cells can comprise modifications that result in a decrease in the expression of an extracellular binding protein (e.g., a TCR comprising Vα and Vβ regions and / or CDRs disclosed herein), and / or endogenous TRAC, TRBC1, and / or TRBC2.

[0087] Nucleic acids encoding the polypeptides disclosed herein (e.g., extracellular binding proteins, CD8 coreceptor chains or extracellular portions thereof, Fas-41BB fusion proteins, or chimeric IL7R fusion proteins) can encode a signal peptide. Optionally, the polypeptides of the present disclosure comprise a signal peptide. Since the signal peptide can be cleaved during processing of the polypeptide, optionally, the mature polypeptides disclosed herein do not contain the signal peptide.

[0088] The signal peptide at the N-terminus of a protein can be involved in the transport of the protein to or through the membrane, transport to different membrane-bound cell compartments, or secretion of the protein from the cell. The nucleic acid encoding the protein of the present disclosure can encode a signal peptide to facilitate membrane insertion and surface localization of the protein. The signal peptide can be selected for its ability to facilitate ER processing and cell surface localization of the protein. Any suitable signal peptide can be used. In some cases, the signal peptide can include the G-CSF signal peptide or the CD8α signal peptide. The signal peptide can be about 10 to about 40 amino acids in length. In some cases, the signal peptide is at least about 10, 15, 16, 20, 21, 22, 25, or 30 amino acids in length. In some cases, the signal peptide is at most about 15, 16, 20, 21, 22, 25, or 30 amino acids in length, or less. In some cases, the signal peptide is about 16 to 30 amino acids in length.

[0089] In some embodiments, the binding protein (e.g., TCR) binds to a target (e.g., a KRAS G12 variant peptide such as a KRAS G12V variant peptide present in a peptide:HLA complex) with a KD of less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM.

[0090] Also contemplated are fusion proteins comprising the scTCR or scTv of the present disclosure linked to a constant domain of an antibody (e.g., IgG(1, 2, 3, 4), IgE, IgD, IgA, IgM, and variants thereof) or a fragment thereof (e.g., in some embodiments, a fragment that maintains binding to one or more Fc receptors, to C1q, to protein A, to protein G, or any combination thereof), including immunoglobulin heavy chain monomers and multimers, such as Fc dimers; see, e.g., Wong et al., J. Immunol. 198:1 Supp. (2017). Variants of the Fc polypeptide that contain mutations that enhance, reduce, or block binding to or by FcRn or other Fc receptors are known and contemplated within the present disclosure.

[0091] In certain embodiments, the binding protein or fusion protein of the present disclosure (e.g., TCR, scTCR, CAR) is expressed by a host cell (e.g., by a T cell, NK cell, or NK-T cell that heterologously expresses the binding protein or fusion protein). Neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA) peptide antigen or neoantigen (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA) peptide antigen: The avidity of such a host cell for the peptide:HLA complex can be determined, for example, by exposing the host cell to the peptide, or to the peptide:HLA complex (e.g., organized as a tetramer), or to an antigen-presenting cell (APC) that presents the peptide to the host cell as needed in a peptide:HLA complex, and then measuring the activity of the host cell, e.g., production or secretion of cytokines (e.g., IFN-γ; TNFα); increased expression of host cell signaling or activation components (e.g., CD137 (4-1BB)); proliferation of the host cell; or killing of the APC, etc. (e.g., using a labeled chromium release assay).

[0092] "Nucleic acid", "nucleic acid molecule", or "polynucleotide", as used herein, generally refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, polynucleotides, such as any of their fragments generated by polymerase chain reaction (PCR) or in vitro translation, and also fragments generated by any of ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modifications in the sugar moiety, or in the pyrimidine or purine base moiety, or can have replacements of the sugar moiety, or of the pyrimidine or purine base moiety. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphororoselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphororanilidate, phosphoramidate, and the like. Nucleic acid molecules can be either single-stranded or double-stranded.

[0093] The term "isolated" generally means that a material has been removed from its original environment (e.g., its natural environment if it occurs naturally). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the materials that naturally accompany it in a natural system is isolated. Such nucleic acids can be part of a vector, and / or such nucleic acids or polypeptides can be part of a composition (e.g., a cell lysate), and such a vector or composition is still considered isolated in that it is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" means a segment of DNA involved in the production of a polypeptide chain, including regions before and after the coding region ("leader and trailer"), as well as intervening sequences (introns) between individual coding segments (exons).

[0094] As used herein, the terms "recombinant," "engineered," and "modified" generally refer to a cell, microorganism, nucleic acid molecule, polypeptide, protein, plasmid, or vector that has been modified by the introduction of an exogenous nucleic acid molecule, or to a cell or microorganism that has been genetically engineered by human intervention, i.e., modified by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism in which the expression of an endogenous nucleic acid molecule or gene has been altered such that it is controlled, deregulated, or constitutive, and such changes or modifications can be introduced by genetic engineering. Examples of artificially generated genetic changes include, for example, modifications that introduce a nucleic acid molecule (which may contain expression control elements such as a promoter) encoding one or more proteins or enzymes, or the addition, deletion, substitution, or other functional disruption or other functional addition to the genetic material of a cell. Examples of modifications include modifications in the coding region or a functional fragment thereof of a heterologous or homologous polypeptide from a reference or parental molecule.

[0095] "Mutation", as used herein, generally refers to a change in the sequence of a nucleic acid molecule or a polypeptide molecule as compared to a respective reference or wild-type nucleic acid or polypeptide molecule. Mutations can result in several different types of changes in the sequence, including substitutions, insertions, or deletions of nucleotides or amino acids. In certain embodiments, a mutation is a substitution of 1 or 3 codons or amino acids, a deletion of from 1 to about 5 codons or amino acids, or a combination thereof.

[0096] "Conservative substitution" generally refers to the substitution of one amino acid with another amino acid having similar properties. Exemplary conservative substitutions are well known in the art (see, for example, page 10 of WO97 / 09433; Lehninger, Biochemistry, 2 nd nd Edition, Worth Publishers, Inc. NY, NY, pp. 71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

[0097] In certain embodiments, a protein according to the present disclosure (e.g., a binding protein, an immunogenic peptide) has a variant sequence (e.g., a variant TCR CDR (e.g., including CDR3β) compared to a reference TCR CDR3β disclosed herein) compared to a reference sequence. A "variant" amino acid sequence, peptide, or polypeptide, as used herein, refers to an amino acid sequence (or peptide or polypeptide) having one, two, or three amino acid substitutions, deletions, or insertions compared to a reference amino acid sequence. In certain embodiments, the variant amino acid sequence, peptide, or polypeptide maintains substantially the same functionality (e.g., binding specificity and affinity for a peptide:HLA complex) as the reference molecule; e.g., a variant TCR fragment disclosed herein maintains about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the antigen binding specificity and affinity compared to a reference TCR binding fragment.

[0098] A "modified domain" or "modified protein" generally refers to a motif, region, domain, peptide, polypeptide, or protein (e.g., a wild-type TCRα chain, TCRβ chain, TCRα constant domain, TCRβ constant domain) having at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%) non-identical sequence identity to a wild-type motif, region, domain, peptide, polypeptide, or protein.

[0099] Examples of modified domains or modified proteins or derivatives include those based on all possible codon selections for the same amino acids and codon selections based on conservative amino acid substitutions. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (ala; A), serine (ser; S), threonine (thr; T); 2) aspartic acid (asp; D), glutamic acid (glu; E); 3) asparagine (asn; N), glutamine (gln; Q); 4) arginine (arg; R), lysine (lys; K); 5) isoleucine (ile; I), leucine (leu; L), methionine (met; M), valine (val; V); and 6) phenylalanine (phe; F), tyrosine (tyr; Y), tryptophan (trp; W). (See also WO97 / 09433, page 10, Lehninger, Biochemistry, 2 nd nd Edition, Worth Publishers, Inc., NY, NY, pp. 71-77, 1975, Lewin Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990, Creighton, Proteins, W.H. Freeman and Company 1984). In addition, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence are also "conservative substitutions".

[0100] The term "construct" generally refers to any polynucleotide containing a recombinant nucleic acid molecule. A "transgene" or "transgene construct" refers to a construct containing two or more genes operably linked in an arrangement not found in nature. The term "operably-linked" (or "operably linked" herein) generally refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it can affect the expression of that coding sequence (i.e., if the coding sequence is under the transcriptional control of the promoter). "Unlinked" generally means that the relevant genetic elements are not closely associated with each other and that the function of one does not affect the other. In some embodiments, the genes present in the transgene are operably linked to an expression control sequence (e.g., a promoter).

[0101] A construct (e.g., a transgene) may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into the genome. A "vector" generally is a nucleic acid molecule capable of transporting another nucleic acid molecule. Vectors can include chromosomal, extrachromosomal, semisynthetic or synthetic nucleic acid molecules, e.g., plasmids, cosmids, viruses, RNA vectors or linear or circular DNA or RNA molecules. Exemplary vectors are those capable of autonomous replication (episomal vectors) or those capable of expressing the nucleic acid molecules linked thereto (expression vectors). Vectors useful in the compositions and methods of the present disclosure are further described herein.

[0102] The term "expression" as used herein generally refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule such as a gene. This process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0103] The term "introduced", in the context of inserting a nucleic acid molecule into a cell, generally means "transfection", or "transformation", or "transduction", and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule can be incorporated into the genome of the cell (e.g., chromosomal, plasmid, plastid, or mitochondrial DNA), can be converted into an autonomous replicon, or can be transiently expressed (e.g., transfected mRNA).

[0104] A "heterologous" or "exogenous" nucleic acid molecule, construct or sequence, as used herein, generally refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to the host cell but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule derived from the host cell. The source of the heterologous or exogenous nucleic acid molecule, construct or sequence may be from a different genus or species. In certain embodiments, the heterologous or exogenous nucleic acid molecule is added to the host cell or host genome (i.e., is neither endogenous nor native), for example, by conjugation, transformation, transfection, transduction, electroporation, etc., and the added molecule may be incorporated into the host genome, or may exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies. In addition, "heterologous" refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell is homologous to the protein or activity encoded. Further, cells containing "modified" or "heterologous" polynucleotides or binding proteins include the progeny of such cells, whether or not the progeny themselves have been transduced, transfected or otherwise manipulated or changed by other means.

[0105] As described herein, more than one heterologous or exogenous nucleic acid molecule may be introduced into a host cell as separate nucleic acid molecules, as multiple individually regulated genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or as any combination thereof. For example, as disclosed herein, a host cell may be modified to express one or more heterologous or exogenous nucleic acid molecules that encode a desired TCR (e.g., TCRα and TCRβ) specific for a Ras antigen peptide and, optionally, also encode a CD8 coreceptor polypeptide, as disclosed herein, that includes an extracellular portion capable of binding to MHC, such as an α-chain, a β-chain, or portions thereof. When two or more exogenous nucleic acid molecules are introduced into a host cell, the two or more exogenous nucleic acid molecules may be introduced as a single nucleic acid molecule (e.g., in a single vector), by separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. It is understood that the number of heterologous nucleic acid molecules or protein activities referred to is the number of coding nucleic acid molecules or protein activities, not the number of separate nucleic acid molecules introduced into the host cell.

[0106] As used herein, the terms "endogenous" or "native" generally refer to a gene, protein, or activity that is normally present in a host cell. Further, a gene, protein, or activity that has been mutated, overexpressed, shuffled, duplicated, or otherwise altered compared to a parental gene, protein, or activity is still considered endogenous or native to that particular host cell. For example, an endogenous regulatory sequence (e.g., a promoter, a translational attenuation sequence) from a first gene may be used to alter or regulate the expression of a second native gene or nucleic acid molecule, and the expression or regulation of the second native gene or nucleic acid molecule may be different from the normal expression or regulation in the parental cell.

[0107] The terms "identical" or "identity" generally refer to a molecule or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous nucleic acid molecule can be identical to a native host cell gene and, if desired, can have an altered expression level, a different sequence, an altered activity, or any combination thereof.

[0108] "Sequence identity", as used herein, generally refers to the percentage of amino acid residues or nucleic acid bases in one sequence that are identical to the amino acid residues or nucleic acid bases, respectively, in a reference sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as part of sequence identity. Percentage sequence identity values can be generated using the NCBI BLAST 2.0 software defined by Altschul et al. (1997), Nucl. Acids Res. 25:3389-3402, using parameters set to default values. Additionally or alternatively, the degree of sequence identity between two sequences can be determined by comparing the two sequences using a computer program designed for this purpose, such as a global or local alignment algorithm. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm. Global alignment algorithms, such as the Needleman and Wunsch algorithm, can be used to align two sequences over their entire lengths, maximizing the number of matches and minimizing the number of gaps. Default settings can be used.

[0109] To generate a similarity score for two amino acid sequences, a scoring matrix can be used that assigns positive scores to some non-identical amino acids (e.g., conservative amino acid substitutions, amino acids with similar physiochemical properties, and / or amino acids that frequently show substitutions in orthologs, homologs, or paralogs). Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0110] Variants of the nucleic acid molecules of the present disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and up to at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identical to the defined polynucleotide or the nucleic acid molecule of the reference polynucleotide described herein, or hybridize to the polynucleotide under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65 - 68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the ability to encode a binding protein or a binding domain thereof having a function described herein, such as binding to a target molecule.

[0111] The term "isolated" generally means that a material has been removed from its original environment (e.g., the natural environment if it occurs naturally). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the materials that naturally accompany it in the natural system is isolated. Such a nucleic acid may be part of a vector, and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), and such a vector or composition is still considered isolated in that it is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" means a segment of DNA involved in the production of a polypeptide chain, including regions before and after the coding region ("leader and trailer"), as well as intervening sequences (introns) between individual coding segments (exons).

[0112] In some contexts, the term "variant", as used herein, generally refers to at least one fragment of a referenced full-length sequence, and more specifically, to one or more amino acid or nucleic acid sequences that are truncated at one or both ends by one or more amino acids as compared to the full-length sequence. Such fragments include, or encode, peptides having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, or 200 contiguous amino acids of the original sequence or a variant thereof. The full length of a variant can be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids.

[0113] In some embodiments, the term "variant" refers to a polypeptide or a fragment thereof that includes not only at least one fragment but also an amino acid sequence that is at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the reference amino acid sequence or a fragment thereof that is recited, wherein amino acids other than those essential for biological activity or for the folding or structure of the polypeptide are deleted or substituted, one or more such essential amino acids are replaced in a conservative manner, and / or amino acids are added such that the biological activity of the polypeptide is preserved. The state of the art includes various methods that can be used to align two given nucleic acid or amino acid sequences and calculate the degree of identity (see, for example, Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition). In some embodiments, Clustal W software can be used with default settings (Larkin, M.A., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948).

[0114] In certain embodiments, a variant can additionally include chemical modifications, such as covalent modifications, for example, isotope labeling or glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, and the like. Methods for modifying polypeptides are known and are generally used such that they do not abolish or substantially decrease the desired activity of the polypeptide.

[0115] In one embodiment, the term "variant" of a nucleic acid molecule includes, for example, a nucleic acid of a complementary strand that hybridizes to a reference nucleic acid or a wild-type nucleic acid under stringent conditions. The stringency of a hybridization reaction is readily determinable by one of ordinary skill in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes do not. Hybridization generally depends on the ability of denatured DNA to re-anneal to a complementary strand that is present in an environment below their melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, the higher the relative temperature, the more stringent the reaction conditions can be, while lower temperatures are not. For further details and explanations of the stringency of hybridization reactions, see Ausubel, F.M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Further, one of ordinary skill in the art can follow the descriptions set forth in the manual Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer, K.H. (1991) International Journal of Systematic Bacteriology 41:255-260 with respect to methods for identifying DNA sequences by hybridization. In one embodiment, stringent conditions are applied to any hybridization. That is, hybridization occurs only when the probe is at least 70% identical to the target sequence.Probes with a lower degree of identity with the target array can hybridize, but such hybrids are unstable and will be removed in a stringent washing step, for example, by reducing the salt concentration to 2×SSC or, if necessary and subsequently, to 0.5×SSC, while the temperature during that time is, for example, about 50°C to 68°C, about 52°C to 68°C, about 54°C to 68°C, about 56°C to 68°C, about 58°C to 68°C, about 60°C to 68°C, about 62°C to 68°C, about 64°C to 68°C, or about 66°C to 68°C. In one embodiment, the temperature is about 64°C to 68°C or about 66°C to 68°C. The salt concentration can be adjusted to 0.2×SSC or even 0.1×SSC. Nucleic acid sequences having a degree of identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% with respect to a reference or wild-type sequence can be isolated. In one embodiment, as used herein, the term variant of a nucleic acid sequence refers to any nucleic acid sequence encoding the same amino acid sequence as the reference nucleic acid sequence and variants thereof, along the degeneracy of the genetic code.

[0116] A "functional variant" generally refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of the present disclosure, but in some contexts has a slightly different composition (e.g., one base, atom, or functional group is different, added, or removed, or one or more amino acids are mutated, inserted, or deleted), such that the polypeptide or encoded polypeptide can perform at least one function of the encoded parent polypeptide with at least 50% efficiency, or at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% of the activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of the present disclosure has "similar binding", "similar affinity", or "similar activity" if the functional variant shows a reduction in performance in a selected assay of 50% or less compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining to measure the association (Ka) or dissociation (KD) constant), avidity, or activation of host cells. As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that contains only a domain, motif, portion, or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, portion, or fragment of the parent or reference compound, or at least 55, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% of the level of the activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).

[0117] A "functional portion" or "functional fragment" of a polypeptide of the present disclosure or the encoded polypeptide generally has a reduction in performance in an assay (alternatively or additionally, 20% or less, or 10% or less, or a logarithmic difference or less compared to a parent or reference with respect to affinity) of 50% or less compared to the parent or reference polypeptide in an assay selected to measure binding affinity or to measure an effector function (e.g., cytokine release). Functional variants of specifically disclosed binding proteins and polynucleotides are contemplated.

[0118] A "modified domain" or "modified protein" generally refers to a motif, region, domain, peptide, polypeptide, or protein having at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%) non-identical sequence identity to a wild-type motif, region, domain, peptide, polypeptide, or protein (e.g., wild-type TCRα chain, TCRβ chain, TCRα constant domain, or TCRβ constant domain).

[0119] The present disclosure includes variants of the binding proteins described herein (e.g., TCRα chain or TCRβ chain, or fragments thereof, such as Vα or Vβ chains, or CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, or CDR3β) having one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of the polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by substituting amino acids having similar hydrophobicity, polarity, and R chain length for each other. Additionally or alternatively, by comparing the aligned sequences of homologous proteins from different species, conservative substitutions can be identified by positioning amino acid residues that vary between species (e.g., non-conservative residues that do not alter the basic function of the encoded protein). Such conservatively substituted variants can include variants having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of the systems described herein. In some embodiments, such conservatively substituted variants are functional variants.

[0120] Tables of conservative substitutions that provide functionally similar amino acids are available from a variety of references (see, e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd Edition (December 1993))). The following eight groups each contain amino acids that are conservative substitutions for each other: a. Alanine (A), Glycine (G), b. Aspartic acid (D), Glutamic acid (E), c. Asparagine (N), Glutamine (Q), d. Arginine (R), Lysine (K), e. Isoleucine (I), leucine (L), methionine (M), valine (V), f. Phenylalanine (F), tyrosine (Y), tryptophan (W), g. Serine (S), threonine (T), and h. Cysteine (C), methionine (M).

[0121] Binding protein In one aspect, the present disclosure provides a binding protein comprising a T cell receptor (TCR) α-chain variable (Vα) domain and a TCR β-chain variable (Vβ) domain, wherein the binding protein is capable of binding to a peptide:HLA complex, and the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In certain embodiments, the HLA comprises HLA-A*11, optionally HLA-A*11:01. In any of the embodiments disclosed herein, the binding protein can be heterologously expressed by human immune system cells, such as T cells.

[0122] In certain embodiments, the Vα domain and / or the Vβ domain are each independently human, humanized, or chimeric, and each can be human. In some embodiments, the Vα domain is human and the Vβ domain is human. The binding proteins, compositions, and methods disclosed herein can utilize Vα domains, Vβ domains, or CDRs derived therefrom from a human subject, e.g., CDRs derived from the sequencing of isolated T cells or populations thereof from a human subject. TCR Vα domains, Vβ domains, and CDRs isolated from a human subject can have advantageous properties over CDRs from other sources, such as transgenic mice that are transgenic for the variable domains and a single human HLA allele. For example, Vα domains, Vβ domains, and CDRs derived from a human subject can undergo negative thymic selection against substantially the entire human peptidome presented by the complete set of human HLA molecules in vivo, which can reduce the likelihood that the binding protein is cross-reactive with other human self-antigens. In some embodiments, the binding proteins disclosed herein are substantially non-reactive against the human proteome presented by one or more HLA alleles. Reactivity can be determined by any suitable method. In some embodiments, a significant response by protein-transduced T cells to the human proteome presented by one or more HLA alleles is not observed or predicted at a peptide concentration of 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less.

[0123] In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from (e.g., identified in) T cells of a subject (e.g., a human subject) having a disease such as cancer. In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of a human subject having a cancer disclosed herein. In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of a subject (e.g., a human subject) having a disease associated with neoantigens such as KRAS G12V or G12D mutations (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA mutations). In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of a subject (e.g., a human subject) having cells comprising neoantigens such as KRAS G12V or G12D mutations (e.g., KRAS (or NRAS, or HRAS), p53, and / or PIK3CA mutations).

[0124] In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of a healthy subject (e.g., a healthy human subject). In some embodiments, the healthy subject lacks a specific pathological diagnosis (e.g., a disease diagnosis such as a cancer diagnosis). In some embodiments, the healthy subject lacks a specific pathological diagnosis but includes a different pathological diagnosis; e.g., lacks a cancer diagnosis but includes a diagnosis of hypertension or type II diabetes.

[0125] The binding proteins disclosed herein can be heterologously expressed by a host cell, e.g., a human immune cell, e.g., a T cell. Furthermore, expression of the binding proteins disclosed herein can confer advantageous properties on the host cell; e.g., having binding specificity for the neoantigen:HLA complex, improved activation, proliferation, or killing activity in the presence of neoantigen:HLA-presenting tumor cells, etc., of the present disclosure.

[0126] For example, in certain embodiments, when the binding protein is expressed by immune cells (e.g., human T cells, CD8+ and / or CD4+ T cells, NK cells, or NK-T cells as needed), the immune cells express HLA-A*11:01 that expresses a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 or 3 + can specifically kill tumor cells. In certain embodiments, this platform uses activated caspase and labeled (e.g., RapidRed or NucRed) tumor cell signals, where overlap is measured and increased overlap area is equal to tumor cell death by apoptosis. Killing is determined using an assay where target cells are loaded with labeled chromium ( 51 Cr) and 51 Cr in the supernatant is measured after 4 hours of co-incubation with immune cells expressing the binding protein of the present disclosure. In certain embodiments, the killing assay can be performed using effector:target cell ratios such as 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 25:1, 50:1, or 100:1.

[0127] In any of the embodiments disclosed herein, when the binding protein is expressed by immune cells (e.g., human T cells, CD8+ and / or CD4+ T cells, NK cells, or NK-T cells as needed), the immune cells express tumor cells (e.g., HLA-A11:01) that express neoantigen peptides (e.g., peptides comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 or 3) +In the presence of tumor cells, and optionally in the further presence of exogenous IFN-γ, having increased expression of Nur77, wherein Nur77 expression is (i) Nur77 expression by reference immune cells (i.e., of the same cell type as the immune cells expressing the binding protein and at least substantially phenotypically and / or genotypically identical or functionally equivalent in other respects to the immune cells expressing the binding protein) when the reference immune cells that do not express the binding protein are in the presence of tumor cells; and / or (ii) Nur77 expression by immune cells expressing the binding protein when not in the presence of tumor cells and / or not in the presence of antigen-presenting cells expressing neoantigen peptide:HLA complexes (e.g., the peptide comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 2 or 3, and the HLA is HLA-A*11:01 if desired), which is increased as compared to. Nur77 expression can be determined, for example, using a transgenic expression construct comprising a Nur77 locus operably linked to a sequence encoding a reporter construct; for example, dTomato (see Ahsouri and Weiss, J Immunol 198(2):657-668 (2017)).

[0128] In any of the embodiments disclosed herein, when the binding protein is expressed by immune cells (e.g., human T cells, optionally CD8+ and / or CD4+ T cells, NK cells, or NK-T cells), the immune cells express HLA-A*02 for a neoantigen peptide (e.g., a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 or 3). +In the presence of tumor cells, and optionally in the further presence of exogenous IFN-γ, there is an elevated expression of CD137 (also known as 41BB), and the CD137 expression is increased compared to (i) the CD137 expression by reference immune cells in the presence of tumor cells, where the reference immune cells do not express the binding protein; and / or (ii) the CD137 expression by immune cells that express the binding protein in the absence of tumor cells and / or in the absence of antigen-presenting cells expressing the neoantigen peptide:HLA complex (e.g., where the peptide comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 2 or 3, and the HLA is HLA-A*11:01 as appropriate). The CD137 expression can be determined, for example, using flow cytometry with a labeled anti-CD137 antibody. In certain embodiments, CD137 is measured after a 16-hour assay in which immune cells are incubated with or stimulated by a peptide or target cells expressing the peptide.

[0129] In any of the embodiments disclosed herein, (i) the binding protein is encoded by a polynucleotide that is heterologous to the immune cell; (ii) the immune cells include human CD8 + T cells, human CD4+ T cells, or both; (iii) the tumor cells express a neoantigen peptide (e.g., a peptide comprising or consisting of the amino acid sequence set shown in SEQ ID NO: 2 or 3 is HLA-A*11:01 + ); and / or (iv) the tumor cells include OVCAR5 (ovarian serous adenocarcinoma), DAN-G (pancreatic adenocarcinoma), CFPAC1 (pancreatic adenocarcinoma), SW480 (colon cancer), SW527 (breast cancer), or NCI-H441 (lung adenocarcinoma) cells.

[0130] In certain embodiments, the binding protein is capable of binding to the peptide:HLA complex independently of CD8 or in the absence of CD8. Binding independent of CD8 is demonstrated by binding to CD8-negative cells (e.g., CD4 +It can be determined by expressing a binding protein in T cells, Jurkat cells, etc. and identifying the binding of cells to the target. In some embodiments, (a) a T cell receptor (TCR) α-chain variable (Vα) domain comprising a complementarity-determining region 3 (CDR3α) amino acid sequence set forth in any one of SEQ ID NOs: 16, 17, 42, and 43, or a variant thereof having one, two, or three conservative amino acid substitutions as needed, and / or (b) a CDR3β amino acid sequence set forth in any one of SEQ ID NOs: 26, 27, 52, and 53, or a variant thereof having one, two, or three conservative amino acid substitutions as needed, are provided. The binding protein can bind to a peptide:HLA complex, where the peptide comprises, consists essentially of, or consists of the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGAVGVGK (SEQ ID NO: 3), and the HLA comprises HLA-A*11. In certain embodiments, the HLA comprises HLA-A*11:01.

[0131] The Vα domain and / or the Vβ domain can be human, humanized, or chimeric and can be human.

[0132] In certain embodiments, the binding protein comprises (i) the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: 17 and 27, respectively, or a variant thereof having one, two, or three conserved amino acid substitutions as needed; (ii) the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: 16 and 26, respectively, or a variant thereof having one, two, or three conserved amino acid substitutions as needed; (iii) the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: 53 and 43, respectively, or a variant thereof having one, two, or three conserved amino acid substitutions as needed; or (iv) the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: 52 and 42, respectively, or a variant thereof having one, two, or three conserved amino acid substitutions as needed.

[0133] In some embodiments, the binding protein comprises (i) in the Vα domain, the CDR1α amino acid sequence set forth in SEQ ID NO: 14 or 40, or a variant thereof having 1 or 2 conservative amino acid substitutions as needed, (ii) in the Vα domain, the CDR2α amino acid sequence set forth in SEQ ID NO: 15 or 41, or a variant thereof having 1 or 2 conservative amino acid substitutions as needed, (iii) in the Vβ domain, the CDR1β amino acid sequence set forth in SEQ ID NO: 24 or 50, or a variant thereof having 1 or 2 conservative amino acid substitutions as needed, (iv) in the Vβ domain, the CDR2β amino acid sequence set forth in SEQ ID NO: 25 or 51, or a variant thereof having 1 or 2 conservative amino acid substitutions as needed, or (v) further comprises any combination of (i)-(iv).

[0134] In certain embodiments, the binding protein comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 14, 15, 16 or 17, 24, 25, and 26 or 27, respectively.

[0135] In other embodiments, the binding protein comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 40, 41, 42 or 43, 50, 51, and 52 or 53, respectively.

[0136] In some embodiments, (i) the Vα domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 13 or 39; and / or (ii) the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 23 or 49.

[0137] In some embodiments, the Vα domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 13, and the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 23.

[0138] In some embodiments, the Vα domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 39, and the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO: 49.

[0139] In certain embodiments, the Vα domain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 13, and the Vβ domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 23.

[0140] In certain embodiments, the Vα domain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 39, and the Vβ domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 49.

[0141] In some embodiments, the variable domain comprises an amino acid sequence having one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0142] For example, the variable domain can include an amino acid sequence having an insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acids with respect to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0143] In some embodiments, the variable domain includes an amino acid sequence having an insertion of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acids with respect to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0144] In some embodiments, the variable domain includes an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids with respect to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0145] One or more insertions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. One or more insertions can be continuous, discontinuous, or a combination thereof.

[0146] In some embodiments, the variable domain comprises an amino acid sequence having a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0147] In some embodiments, the variable domain comprises an amino acid sequence having a deletion of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0148] In some embodiments, the variable domain comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0149] One or more deletions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. One or more deletions can be continuous, discontinuous, or a combination thereof.

[0150] In some embodiments, the variable domain comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid substitutions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0151] In some embodiments, the variable domain comprises an amino acid sequence having at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0152] In some embodiments, the variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0153] One or more substitutions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. One or more substitutions can be continuous, discontinuous, or a combination thereof.

[0154] The binding protein may further comprise a TCRα chain constant domain (Cα) and / or a TCRβ chain constant domain (Cβ). The TCRα chain constant domain (Cα) and / or the TCRβ chain constant domain (Cβ) can be human. The TCRα chain constant domain (Cα) and / or the TCRβ chain constant domain (Cβ) can be mammalian. The TCRα chain constant domain (Cα) and / or the TCRβ chain constant domain (Cβ) can be engineered.

[0155] In some embodiments, Cα comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 44, 45, and 69, or an amino acid sequence comprising or consisting of the same.

[0156] In some embodiments, Cβ comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 28, 29, 54, 55, and 70 - 73, or an amino acid sequence comprising or consisting of the same.

[0157] In certain embodiments, Cα and Cβ comprise, or consist of, an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences set forth in each of (i) SEQ ID NOs: 18 and 28; (ii) SEQ ID NOs: 19 and 29; (iii) SEQ ID NOs: 44 and 54; or (iv) SEQ ID NOs: 45 and 55, or an amino acid sequence that includes or consists of the same.

[0158] The binding protein can comprise (i) the extracellular domain of a TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain; (ii) the transmembrane domain of a TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain; and / or (iii) the cytoplasmic domain of a TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain. The binding protein can comprise a full-length or substantially full-length TCR alpha chain, TCR beta chain, TCR gamma chain, and / or TCR delta chain.

[0159] In some embodiments, the binding protein comprises an amino acid sequence having one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0160] For example, the binding protein can include an amino acid sequence having an insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acids with respect to any one of SEQ ID NOs: 12, 18 - 22, 28 - 30, 38, 44 - 46, 48, 54 - 56, and 69.

[0161] In some embodiments, the binding protein can include an amino acid sequence having an insertion of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acids with respect to any one of SEQ ID NOs: 12, 18 - 22, 28 - 30, 38, 44 - 46, 48, 54 - 56, and 69.

[0162] In some embodiments, the binding protein includes an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids with respect to any one of SEQ ID NOs: 12, 18 - 22, 28 - 30, 38, 44 - 46, 48, 54 - 56, and 69.

[0163] One or more insertions can be at the N - terminus, C - terminus, within the amino acid sequence, or combinations thereof. One or more insertions can be continuous, discontinuous, or combinations thereof.

[0164] In some embodiments, the binding protein comprises an amino acid sequence having a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acids with respect to any one of SEQ ID NOs: 12, 18 - 22, 28 - 30, 38, 44 - 46, 48, 54 - 56, and 69.

[0165] In some embodiments, the binding protein comprises an amino acid sequence having a deletion of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acids with respect to any one of SEQ ID NOs: 12, 18 - 22, 28 - 30, 38, 44 - 46, 48, 54 - 56, and 69.

[0166] In some embodiments, the binding protein comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids with respect to any one of SEQ ID NOs: 12, 18 - 22, 28 - 30, 38, 44 - 46, 48, 54 - 56, and 69.

[0167] One or more deletions can be at the N - terminus, C - terminus, within the amino acid sequence, or a combination thereof. One or more deletions can be continuous, discontinuous, or a combination thereof.

[0168] In some embodiments, the binding protein comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid substitutions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0169] In some embodiments, the binding protein comprises an amino acid sequence having up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acid substitutions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0170] In some embodiments, the binding protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0171] One or more substitutions can be at the N-terminus, C-terminus, within the amino acid sequence, or combinations thereof. One or more substitutions can be continuous, discontinuous, or combinations thereof.

[0172] In some embodiments, the binding protein comprises a TCR α-chain and a TCR β-chain, and the TCR α-chain and TCR β-chain are (i) each of SEQ ID NO: 12 and 22; (ii) each of SEQ ID NO: 20 and 30; (iii) each of SEQ ID NO: 12 and 30; (iv) each of SEQ ID NO: 20 and 22; (v) each of SEQ ID NO: 38 and 48; (vi) each of SEQ ID NO: 46 and 56; (vii) each of SEQ ID NO: 38 and 56; or (viii) each of SEQ ID NO: 46 and 48, or a set of amino acid sequences comprising the same, or consisting of the same, or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0173] In any of the embodiments disclosed herein, the binding protein can comprise a TCR, a single-chain TCR (scTCR), an scTv, or a chimeric antigen receptor (CAR). Methods for generating engineered TCRs are described, for example, in Bowerman et al., Mol. Immunol., 46(15):3000 (2009), the techniques of which are incorporated herein by reference. Methods for generating CARs are known in the art and are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Application 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. 13:5426, the techniques of which are incorporated herein by reference. In some embodiments, the binding protein comprises a soluble TCR optionally fused to a binding domain (e.g., scFv) specific for the CD3 protein. See Elie Dolgin, Nature Biotechnology 40:441-449 (2022).

[0174] Some examples of the binding protein are included in Table 2. In some embodiments, the binding protein comprises the amino acid sequence of Table 2. In some embodiments, the binding protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% sequence identity to the sequence of Table 2. In some embodiments, the binding protein comprises an amino acid sequence having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of Table 2. 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the sequence of Table 2. In some embodiments, the binding protein comprises a sequence having at most 99.9%, at most 99.8%, at most 99.7%, at most 99.6%, at most 99.5%, at most 99.4%, at most 99.3%, at most 99.2%, or at most 99.1% sequence identity to the sequence of Table 2. In some embodiments, the binding protein comprises a sequence having at most 99%, at most 98%, at most 97%, at most 96%, at most 95%, at most 94%, at most 93%, at most 92%, or at most 91% sequence identity to the sequence of Table 2. In some embodiments, the binding protein comprises a sequence having at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, or at most 60% sequence identity to the sequence of Table 2. In some embodiments, the binding protein comprises a sequence having about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or about 99.9% sequence identity, or a sequence identity within a range defined by any two of the foregoing percentages, to the sequence of Table 2. In some embodiments, the binding protein comprises a fragment of any of the foregoing sequences.In some embodiments, the binding protein comprises any combination of any of the foregoing sequences. Either the foregoing binding protein or any of the binding protein sequences may be useful in the methods or compositions described herein. For example, the binding protein may be included in a cell having a fusion protein comprising components of CD95 (Fas) and CD137 (4-1BB), and / or the CD8αβ coreceptor (e.g., an exogenous CD8αβ coreceptor).

[0175] In any of the embodiments disclosed herein, the polynucleotide encoding the binding protein is: (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor alpha chain, optionally wherein the encoded polypeptide is or comprises the CD8 coreceptor alpha chain; (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor beta chain, optionally wherein the encoded polypeptide is or comprises the CD8 coreceptor beta chain; or (iii) may further comprise the polynucleotide of (i) and the polynucleotide of (ii). Without being bound by theory, in certain embodiments, co-expression or co-occurring expression of the binding protein and the CD8 coreceptor protein or those portions thereof that are functional to bind to the HLA molecule may improve one or more desired activities of the host cell (e.g., an immune cell, e.g., a T cell, optionally a CD4 + T cell) as compared to expression of the binding protein alone. It will be understood that the polynucleotide encoding the binding protein and the polynucleotide encoding the CD8 coreceptor polypeptide may be present on a single nucleic acid molecule (e.g., in the same expression vector) or on separate nucleic acid molecules in the host cell.

[0176] In any of the embodiments of the present disclosure, the CD8 coreceptor alpha chain may comprise, consist essentially of, or consist of SEQ ID NO: 87, or SEQ ID NO: 87 with the signal peptide removed. An example of a polynucleotide encoding SEQ ID NO: 87 is provided in SEQ ID NO: 88. In some embodiments, the CD8 coreceptor alpha chain comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 87, or SEQ ID NO: 87 with the signal peptide removed.

[0177] In any of the embodiments of the present disclosure, the CD8 coreceptor beta chain may comprise, consist essentially of, or consist of SEQ ID NO: 89, or SEQ ID NO: 89 with the signal peptide removed. An example of a polynucleotide encoding SEQ ID NO: 89 is provided in SEQ ID NO: 90. In some embodiments, the CD8 coreceptor beta chain comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 89, or SEQ ID NO: 89 with the signal peptide removed.

[0178] In certain further embodiments, the polynucleotide comprises: (a) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor alpha chain; (b) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor beta chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b). In further embodiments, the polynucleotide encodes a self-cleaving peptide and comprises a polynucleotide disposed (1) between a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor alpha chain; and / or (2) between a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor beta chain.

[0179] In still further embodiments, the polynucleotide is operably linked in-frame to: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnBP); (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnBP); (iii) (pnBP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β); (iv) (pnBP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α); (v) (pnCD8α)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8β); or (vi) (pnCD8β)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnBP is a polynucleotide encoding a binding protein, and pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, and these polynucleotides and / or the encoded self-cleaving peptides are the same or different (e.g., P2A, T2A, F2A, E2A) as appropriate.

[0180] It will be understood that the self-cleaving peptide can include its linker N-terminus and / or C-terminus. An example of a linker is GSG. In some embodiments, a T2A peptide comprising an N-terminal GSG linker is provided. In some embodiments, the GSG-T2A sequence comprises, consists essentially of, or consists of GSG and the amino acid sequence of SEQ ID NO: 75. In some embodiments, the GSG-P2A sequence comprises, consists essentially of, or consists of SEQ ID NO: 74.

[0181] In certain embodiments, the encoded binding protein comprises a TCRα chain and a TCRβ chain, and the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between a polynucleotide encoding a TCRα chain and a polynucleotide encoding a TCRβ chain. In further embodiments, the polynucleotide is operably linked in-frame to (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα); (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα); (iii) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (iv) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (v) (pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β); (vi) (pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α); (vii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β); (viii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α), where pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnTCRα is a polynucleotide encoding a TCRα chain, pnTCRβ is a polynucleotide encoding a TCRβ chain, and pnSCP1, pnSCP2, and pnSCP3 are each independently polynucleotides encoding self-cleaving peptides, and the polynucleotides and / or the encoded self-cleaving peptides are the same or different as necessary.

[0182] In certain embodiments, the encoded polypeptides of the present disclosure include 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, for example, between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can result from the design of constructs encoding fusion proteins (e.g., amino acid residues resulting from the use of restriction enzyme sites during the construction of nucleic acid molecules encoding fusion proteins), or, for example, from the cleavage of a self-cleaving peptide adjacent to one or more domains of an encoded binding protein of the present disclosure (e.g., a P2A peptide disposed between a TCRα chain and a TCRβ chain. Its self-cleavage can leave one or more junction amino acids in the α chain, the TCRβ chain, or both).

[0183] In further embodiments, the binding protein is expressed as part of the encoding transgene construct and / or the host cells of the present disclosure may further encode one or more additional accessory proteins such as safety switch proteins; tags, selectable markers; CD8 co-receptor beta chain; CD8 co-receptor alpha chain or both; or any combination thereof. Polynucleotides and transgene constructs and accessory components (e.g., one or more of a safety switch protein, selectable marker, CD8 co-receptor beta chain, or CD8 co-receptor alpha chain) useful for encoding and expressing the binding protein are described in PCT application PCT / US2017 / 053112, and the polynucleotides, transgene constructs, and accessory components are incorporated herein by reference, including nucleotide and amino acid sequences. Any or all of the binding proteins, safety switch proteins, tags, selectable markers, CD8 co-receptor beta chain, or CD8 co-receptor alpha chain of the present disclosure may be encoded by a single nucleic acid molecule or may be separate nucleic acid molecules or encoded by polynucleotide sequences present in separate nucleic acid molecules.

[0184] Examples of safety switch proteins include, for example, those that lack an extracellular N-terminal ligand binding domain and an intracellular receptor tyrosine kinase activity, but retain their native amino acid sequence, have a type I transmembrane cell surface localization, a pharmaceutical grade anti-EGFR monoclonal antibody, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011), caspase polypeptide (e.g., iCasp9, Straathof et al, Blood 105:4247-4254, 2005, Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011, Zhou and Brenner, Exp. Hematol. pii: S0301-472X(16)30513-6.doi:10.1016 / j.exphem.2016.07.011), RQR8 (Philip et al., Blood 124:1277-1287, 2014), a 10-amino acid tag derived from the human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Sci. USA 105:623-628, 2008), and a cleaved EGF receptor polypeptide (huEGFRt) having a structurally intact binding epitope for marker / safety switch polypeptides such as RQR (CD20+CD34; Philip et al., 2014).

[0185] Other accessory components useful in the modified host cells of the present disclosure include tags or selectable markers that enable the identification, sorting, isolation, enrichment, or tracking of cells. For example, labeled host cells having desired characteristics (e.g., antigen-specific TCR and safety switch proteins) can be sorted from unlabeled cells in a sample and more efficiently activated and expanded for inclusion in a product of desired purity.

[0186] As used herein, the term "selectable marker" includes a nucleic acid construct (and the encoded gene product) that confers an identifiable change on a cell that enables the detection and positive selection of immune cells transfected with a polynucleotide containing the selectable marker. RQR is a selectable marker that includes 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, the CD34 minimal epitope is incorporated at the amino-terminal position of the CD8 coreceptor stalk domain (Q8). In further embodiments, the CD34 minimal binding site sequence can combine with the target epitope for CD20 to form a compact marker / suicide gene for T cells (RQR8) (Philip et al., 2014, which is incorporated herein by reference). This construct uses the clinically approved pharmaceutical antibody rituximab and enables the selective deletion of genetically engineered T cells that express the transgene, for example, using a CD34-specific antibody conjugated to magnetic beads (Miltenyi) to enable the selection of host cells that express this construct (Philip et al., 2014).

[0187] Additional exemplary selectable markers typically include several truncated type I transmembrane proteins that are not expressed on T cells, namely, truncated low affinity nerve growth factor, truncated CD19, and truncated CD34 (see, for example, Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011; Mavilio et al., Blood 83:1988-1997, 1994; Fehse et al., Mol. Ther. 1:448-456, 2000, each of which is incorporated herein by reference in its entirety). A useful feature of CD19 and CD34 is that a ready-to-use Miltenyi CliniMACs™ selection system that can target such markers for clinical grade sorting is available. However, CD19 and CD34 are relatively large surface proteins that can burden the vector packaging capacity and the transcriptional efficiency of the integrating vector. Surface markers containing extracellular non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR) can also be used. Any selectable marker can be used (e.g., those compliant with Good Manufacturing Practices). In certain embodiments, the selectable marker is expressed together with a polynucleotide encoding a gene product of interest (e.g., a binding protein of the present disclosure such as a TCR or CAR). Further examples of selectable markers include, for example, reporters such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In certain embodiments, a selectable marker such as CD34 is expressed in cells and CD34 can be used to selectively enrich or isolate the transduced cells of interest for use in the methods described herein (e.g., by immunomagnetic selection). As used herein, the CD34 marker is distinguished from an anti-CD34 antibody or, for example, an scFv, TCR, or other antigen recognition moiety that binds to CD34.

[0188] In certain 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.

[0189] With respect to the RQR polypeptide, without wishing to be bound by theory, the distance from the host cell surface is thought to be important for the RQR polypeptide to function as a selectable marker / safety switch (Philip et al., 2010 (supra)). In some embodiments, the encoded RQR polypeptide is contained within the β-chain, α-chain, or both, of the encoded CD8 coreceptor, or in any fragment or variant of either or both. In a specific embodiment, the modified host cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 coreceptor protein comprising a β-chain containing the RQR polypeptide and further comprising a CD8α-chain.

[0190] The encoded CD8 coreceptor, in some embodiments, comprises an α-chain or a fragment or variant thereof. The amino acid sequence of the human CD8 coreceptor α-chain precursor is known and is provided, for example, in UniProtKB - P30433 (see also UniProtKB - P31783; - P10732; and - P10731). The encoded CD8 coreceptor, in some embodiments, comprises a β-chain or a fragment or variant thereof. The amino acid sequence of the human CD8 coreceptor β-chain precursor is known and is provided, for example, in UniProtKB - P10966 (see also UniProtKB - Q9UQ56; - E9PD41; Q8TD28; and - P30434; and - P05541).

[0191] The isolated polynucleotides of the present disclosure may further comprise a polynucleotide encoding a safety switch protein, a selectable marker, a CD8 coreceptor beta chain, or a CD8 coreceptor alpha chain disclosed herein, or a polynucleotide encoding any combination thereof.

[0192] In any of the embodiments disclosed herein, the polynucleotide can be codon-optimized for expression in a host cell. In some embodiments, the host cell includes human immune system cells such as T cells, NK cells, or NK-T cells (Scholten et al., Clin. Immunol. 119:135, 2006). Codon optimization can be performed using known techniques and tools, for example, using the GenScript® OptimumGene™ tool or GeneArt (Life Technologies). Codon-optimized sequences include partially codon-optimized sequences (i.e., one or more codons are optimized for expression in the host cell) and fully codon-optimized sequences. In embodiments where the polynucleotide encodes more than one polypeptide (e.g., TCRα chain, TCRβ chain, CD8 coreceptor α chain, CD8 coreceptor β chain, and one or more self-cleaving peptides), it will be understood that each polypeptide can independently be fully codon-optimized, partially codon-optimized, or not codon-optimized.

[0193] Amino acid and polynucleotide sequences, such as the binding proteins “11N4A” and “11N6,” are shown in Table 1.

Table 1-1

Table 1-2

[0194] Vector In another aspect, the disclosure provides an expression vector comprising any polynucleotide provided herein operably linked to an expression control sequence.

[0195] Vectors containing the polynucleotides or transgenes of the present disclosure are also provided herein. Some examples of vectors include plasmids, viral vectors, cosmids, and the like. Some vectors may be capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), while other vectors may integrate into the host cell genome or facilitate the integration of polynucleotide inserts upon introduction into the host cell, thereby being replicated along with the host genome (e.g., lentiviral vectors, retroviral vectors). Additionally, some vectors are capable of directing the expression of genes to which they are operably linked (such vectors may be referred to as "expression vectors"). According to related embodiments, when co-administering one or more agents (e.g., polynucleotides encoding polypeptides as described herein), each agent may be present in a separate or the same vector, and multiple vectors (each containing a different or the same agent) may be introduced into a cell or cell population or administered to a subject.

[0196] In certain embodiments, the polynucleotides of the present disclosure may be operably linked to certain elements of a vector. For example, polynucleotide sequences necessary to effect the expression and processing of the coding sequences to which they are ligated may be operably linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and perhaps sequences that enhance protein secretion. Expression control sequences may be operably linked when they are adjacent to the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0197] In certain embodiments, the vector includes a plasmid vector or a viral vector (e.g., a vector selected from a lentiviral vector or a γ-retroviral vector). Examples of viral vectors include: retroviruses; adenoviruses; parvoviruses (e.g., adeno-associated viruses); coronaviruses; ortho-myxoviruses (e.g., influenza viruses), negative-strand RNA viruses such as rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai); positive-strand RNA viruses such as picornaviruses and alphaviruses; and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian type C, type B virus, type D virus, HTLV-BLV group, lentivirus, and spumavirus (Coffin, J.M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B.N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0198] A "retrovirus" is a virus having an RNA genome, which is reverse transcribed into DNA using reverse transcriptase, and then the reverse transcribed DNA is integrated into the host cell genome. "Gamma-retrovirus" refers to a genus of the Retroviridae family. Examples of gamma-retroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. A "lentiviral vector", as used herein, means an HIV-based lentiviral vector for gene delivery, which may be integrative or non-integrative, may have a relatively large packaging capacity, and can transduce a series of different cell types. Lentiviral vectors are usually generated after transiently transfecting producer cells with three (packaging, envelope, and transfer) or more plasmids. Similar to HIV, lentiviral vectors enter target cells through the interaction of the viral surface glycoprotein with a receptor on the cell surface. Once inside, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of the infected cell.

[0199] In certain embodiments, the viral vector may be a gammaretrovirus, such as a vector derived from Moloney murine leukemia virus (MLV). In other embodiments, the viral vector may be a more complex retrovirus-derived vector, such as a lentivirus-derived vector. Vectors derived from HIV-1 fall within this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and maedi-visna virus (ovine lentivirus). Methods for using retroviral and lentiviral viral vectors and packaging cells to transduce mammalian host cells with virus particles containing a TCR or CAR transgene are known in the art and have been previously described in the following references: for example, U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors, such as DNA viral vectors including adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; and vectors derived from herpes simplex virus (HSV) including amplicon vectors, replication-deficient HSV, and attenuated HSV, can also be used for polynucleotide delivery (Krisky et al., Gene Ther. 5:1517, 1998).

[0200] Other vectors developed for use in gene therapy can also be used with the compositions and methods of the present disclosure. Such vectors include those derived from baculovirus and α-virus. (Jolly, D J. 1999. Emerging Viral Vectors. pp 209-40 Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0201] If the viral vector genome contains multiple polynucleotides that are expressed as separate transcripts in the host cell, the viral vector may also contain additional sequences between two (or more) transcripts that allow for bicistronic or polycistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0202] In certain embodiments, the vector is capable of delivering a polynucleotide or transgene construct to a host cell (e.g., a hematopoietic progenitor cell or a human immune system cell). In specific embodiments, the vector delivers a polynucleotide or transgene construct to, for example, CD4 + T cells, CD8 + T cells, CD4 - CD8 -It is possible to deliver to human immune system cells such as double-negative T cells, stem cell memory T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof. In further embodiments, the vector is capable of delivering the transgene construct to naive T cells, central memory T cells, effector memory T cells, or any combination thereof. In some embodiments, the polynucleotide or vector encoding the transgene construct of the present disclosure can be used to perform chromosomal knockout in a host cell (e.g., CRISPR-Cas endonuclease or another endonuclease as disclosed herein), or can further comprise a polynucleotide encoding a nuclease that can be used to deliver a therapeutic polynucleotide or transgene or a portion thereof to a host cell in gene therapy replacement or gene repair therapy. Alternatively, the nuclease used in chromosomal knockout or gene replacement or gene repair therapy may be delivered to the host cell independently of the vector encoding the polynucleotide or transgene construct of the present disclosure.

[0203] In certain embodiments, the vector is capable of delivering a polynucleotide to a host cell. In further embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In still further embodiments, the human immune system cell is a CD4+ T cell, CD8+ T cell, CD4-CD8-double negative T cell, γδ T cell, natural killer cell, natural killer T cell, macrophage, monocyte, dendritic cell, or any combination thereof. In still further embodiments, the T cell is a naive T cell, central memory T cell, effector memory T cell, or any combination thereof.

[0204] In any of the embodiments disclosed herein, the vector is a viral vector. In certain embodiments, the viral vector is a lentiviral vector or a γ-retroviral vector.

[0205] Host cell Also provided herein are host cells that encode and / or express a binding protein (and, optionally, one or more accessory proteins provided herein, such as a transduction marker, a CD8 co-receptor polypeptide, etc.). In certain embodiments, provided are host cells that are modified to contain the polynucleotides and / or expression vectors of the present disclosure and / or to express the binding proteins of the present disclosure.

[0206] For example, any suitable host cell, including immune cells such as T cells, NK cells, or NK-T cells, can be modified to contain a heterologous polynucleotide encoding a binding protein of the present disclosure. In some embodiments, the modified immune cell comprises CD4 + T cells, CD8 + T cells, or both. Methods for transfecting / transducing T cells with a desired nucleic acid (e.g., US Patent Application Publication No. 2004 / 0087025) have been described as having adoptive transfer procedures using T cells having the desired target specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US2011 / 0243972; US2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), and adaptation of these methodologies to the embodiments disclosed herein is contemplated based on the teachings herein.

[0207] Any suitable method can be used to transfect or transduce cells, such as T cells, or to administer the polynucleotides or compositions of the methods of the invention. Known 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-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonoporation, liposome-mediated transfection, receptor-mediated transduction, particle gun, transposon-mediated transfer, and the like. Still further methods of transfecting or transducing host cells use vectors described in more detail herein.

[0208] In certain embodiments, the host cell or modified cell can be a peripheral blood mononuclear cell (PBMC). The host cell can be a lymphoid cell. The host cell can be a lymphocyte. In some embodiments, the host cell or modified cell can be a hematopoietic progenitor cell and / or a human immune cell. In some embodiments, the immune cells include T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof. In some embodiments, the host or modified cell is a mammalian cell (e.g., a human cell or a mouse cell). In further embodiments, the immune cells include CD4+ T cells, CD8+ T cells, CD4-CD8-double negative T cells, γδ T cells, or any combination thereof. In certain further embodiments, the immune cells include CD4+ T cells and CD8+ T cells. In certain still further embodiments, the CD4+ T cells, CD8+ T cells, or both are: (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, optionally wherein the encoded polypeptide is or comprises the CD8 coreceptor α chain; (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor β chain, optionally wherein the encoded polypeptide is or comprises the CD8 coreceptor β chain; or (iii) a polynucleotide comprising the polynucleotide of (i) and the polynucleotide of (ii).

[0209] In any of the above embodiments, a host cell (e.g., an immune cell) can be modified to reduce or eliminate the expression of one or more endogenous genes encoding polypeptides involved in immune signaling or other related activities. Examples of gene knockouts include those encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, HLA molecules, TCR molecules, and the like. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins can be recognized as foreign by allogeneic hosts receiving the modified immune cells, which can result in the elimination of the modified immune cells (e.g., HLA alleles), or can downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, FasL, TIGIT, TIM3), or can interfere with the binding activity of the heterologously expressed binding proteins of the present disclosure (e.g., endogenous TCRs of modified T cells that bind to non-Ras antigens and thereby interfere with modified immune cells that bind to, for example, cells expressing Ras antigens).

[0210] Accordingly, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerance, or persistence of modified cells in autologous or allogeneic host settings and enable the universal administration of cells (e.g., to any recipient regardless of HLA type). In certain embodiments, the modified cells are donor cells (e.g., allogeneic) or autologous cells. In certain embodiments, the modified cells of the disclosure comprise chromosomal gene knockouts of one or more genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, HLA components (e.g., genes encoding α1 macroglobulin, α2 macroglobulin, α3 macroglobulin, β1 microglobulin, or β2 microglobulin), or TCR components (e.g., genes encoding TCR variable regions or TCR constant regions) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119(24):5697 (2012); and Torikai et al., Blood 122(8):1341 (2013). The gene editing techniques, compositions, and adoptive cell therapies are hereby incorporated by reference in their entirety).

[0211] As used herein, the term "chromosomal gene knockout" generally refers to a genetic alteration or an inhibitory agent introduced into a host cell that prevents (e.g., reduces, delays, suppresses, or blocks) the production of a functionally active endogenous polypeptide product by the host cell. Alterations that result in a chromosomal gene knockout can include, for example, the introduction of a nonsense mutation (including the formation of a premature stop codon), a missense mutation, a gene deletion, and a strand break, as well as the heterologous expression of an inhibitory nucleic acid molecule that inhibits endogenous gene expression in the host cell.

[0212] In certain embodiments, chromosomal gene knockout or gene knock-in is performed by chromosomal editing of a host cell. Chromosomal editing can be carried out, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme capable of catalytically cleaving a phosphodiester bond within a polynucleotide chain. In certain embodiments, the endonuclease is capable of cleaving a target gene, thereby inactivating or "knocking out" the target gene. The endonuclease may be naturally occurring, recombinant, genetically modified, or a fusion endonuclease. The nucleic acid strand breaks caused by the endonuclease are generally repaired by different mechanisms of homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, donor nucleic acid molecules can be used for donor gene "knock-in", for target gene "knock-out", and, if necessary, to inactivate the target gene by a donor gene knock-in event or a target gene knock-out event. NHEJ is an error-prone repair process that often results in changes in the DNA sequence at the cleavage site (e.g., substitution, deletion, or addition of at least one nucleotide). NHEJ can be used to "knock out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0213] As used herein, "zinc finger nuclease" (ZFN) generally refers to a fusion protein comprising a zinc finger DNA binding domain fused to a non-specific DNA cleavage domain such as FokI endonuclease. Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA and can alter triplet sequence specificity by changing the amino acids of certain residues (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285:1917-1934, 1999). Multiple zinc finger motifs can be linked in tandem to create binding specificity to a desired DNA sequence, such as a region having a length in the range of about 9 to about 18 base pairs. Background, ZFN mediates genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and targeted integration of a transgene containing flanking sequences homologous to the genome at the DSB site is facilitated by homologous recombination repair. Alternatively, DSBs generated by ZFNs can result in knockout of the target gene via repair by non-homologous end joining (NHEJ). This is an error-prone cellular repair pathway that results in nucleotide insertions or deletions at the cleavage site. In certain embodiments, gene knockout includes insertions, deletions, mutations, or combinations thereof that are performed using ZFN molecules.

[0214] As used herein, "transcription activator-like effector nuclease" (TALEN) generally refers to a fusion protein that includes a TALE DNA-binding domain and a DNA cleavage domain such as FokI endonuclease. A "TALE DNA-binding domain" or "TALE" is generally composed of one or more TALE repeat domains / units, each having a highly conserved sequence of 33-35 amino acids where the 12th and 13th amino acids vary widely. TALE repeat domains are involved in the binding of TALE to the target DNA sequence. Such widely varying amino acid residues are called repeat variable diresidues (RVDs) and are associated with specific nucleotide recognition. The natural (standard) code for such TALE DNA recognition is such that the HD (histidine-aspartic acid) sequence at positions 12 and 13 of TALE results in TALE binding 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-standard (atypical) RVDs are also known (see, e.g., U.S. Patent Application Publication No. US2011 / 0301073, the atypical RVDs of which are incorporated herein by reference in their entirety). TALEN can be used to direct site-specific double-strand breaks (DSBs) in the genome of T cells. Non-homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break with little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homologous recombination repair can introduce a transgene into the DSB site if homologous flanking sequences are present in the transgene. In certain embodiments, gene knockout involves insertions, deletions, mutations, or combinations thereof, performed using TALEN molecules.

[0215] As used herein, the “clustered regularly interspaced short palindromic repeat / Cas” (CRISPR / Cas) nuclease system generally 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 by base complementarity and cleave DNA when a short conserved protospacer adjacent motif (PAM) follows immediately downstream of the complementary target sequence. The CRISPR / Cas system is classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nuclease. The type I and type III crRNA-guided surveillance complexes require multiple Cas subunits. The most studied type II system contains at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-activating crRNA (tracrRNA). The tracrRNA contains a duplex-forming region. The crRNA and tracrRNA interact with the Cas9 nuclease to form a duplex capable of guiding the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA by Watson-Crick base pairing between the spacer of the crRNA and the protospacer of the labeled DNA upstream of the PAM. The Cas9 nuclease cleaves a double-stranded break within the region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions that disrupt the expression of the target locus. Alternatively, a transgene with homologous flanking sequences can be introduced into the DSB site by homologous recombination repair. The crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012).Furthermore, the region of the guide RNA complementary to the target site can be altered or programmed to target a desired sequence (Xie et al., PLOS One 9:e100448, 2014; US Patent Application Publication No. US2014 / 0068797, US Patent Application Publication No. US2014 / 0186843, US Patent No. 8,697,359, and PCT Publication No. WO2015 / 071474; each of which is incorporated by reference). In certain embodiments, gene knockout is performed using the CRISPR / Cas nuclease system and includes insertions, deletions, mutations, or combinations thereof.

[0216] Examples of gRNA sequences and methods of using them to knockout endogenous genes encoding immune cell proteins are described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNA, CAS9 DNA, vectors, and gene knockout techniques of which are incorporated herein by reference in their entirety.

[0217] As used herein, "meganuclease" is also referred to as "homing endonuclease" and generally refers to an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Meganucleases can be divided into five families, LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK, based on sequence and structural motifs. Examples of meganucleases include: I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII, and their recognition sequences are known (see, for example, U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res. 25:3379-3388, 1997, Dujon et al., Gene 82:115-118, 1989, Perler et al., Nucleic Acids Res. 22:1125-1127, 1994, Jasin, Trends Genet. 12:224-228, 1996, Gimble et al., J. Mol. Biol. 263:163-180, 1996, Argast et al., J. Mol. Biol. 280:345-353, 1998).

[0218] In certain embodiments, naturally occurring meganucleases can be used to facilitate site-specific genomic modification of a target selected from genes encoding PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, HLA, or genes encoding TCR components. In other embodiments, engineered meganucleases having novel binding specificities for target genes are used for site-specific genomic modification (see, for example, Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952-62, 2003; Chevalier et al., Molec. Cell 10:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49-66, 2007; U.S. Patent Application Publication Nos. US 2007 / 0117128, US 2006 / 0206949, US 2006 / 0153826, US 2006 / 0078552, and US 2004 / 0002092). In further embodiments, chromosomal gene knockouts are generated using a homing endonuclease modified with the modular DNA binding domain of TALENs to create a fusion protein known as megaTAL. MegaTAL can be used not only to knockout 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.

[0219] In certain embodiments, chromosomal gene knockout involves an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) that contains a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor-associated antigen, the inhibitory nucleic acid molecule encoding a target-specific inhibitor, and the encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of PD-1, TIM3, LAG3, CTLA4, TIGIT, FasL, HLA components, or TCR components, or any combination thereof) in the host cell.

[0220] In certain embodiments, gene knockout involves insertions, deletions, mutations, or combinations thereof, and is generated using a CRISPR / Cas nuclease system or a base editing system (Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., Liu, D.R. Nature 533, 420-424 (2016)). Briefly, base editing is a genome editing approach that uses components from the CRISPR system together with other enzymes to directly introduce point mutations into cellular DNA or RNA without making double-stranded DNA breaks. Certain DNA base editors contain a catalytically inactive nuclease fused to a nucleic acid base deaminase enzyme, and in some cases, a DNA glycosylase inhibitor. RNA base editors function similarly using components that target RNA. Base editors can directly convert one base or base pair to another, enabling the efficient incorporation of point mutations into non-dividing cells without generating excessive unwanted editing by-products. See, for example, Rees H et al. Nature Reviews Genetics (2018).

[0221] Chromosomal gene knockout can be directly confirmed by sequencing the DNA of the host immune cell after use of the knockout procedure or agent. Chromosomal gene knockout can also be inferred from the absence of gene expression (e.g., the absence of the mRNA or polypeptide product encoded by the gene) after knockout.

[0222] In certain embodiments, chromosomal gene knockout comprises knockout of an HLA component gene selected from the α1 macroglobulin gene, α2 macroglobulin gene, α3 macroglobulin gene, β1 microglobulin gene, or β2 microglobulin gene.

[0223] In certain embodiments, chromosomal gene knockout comprises knockout of a TCR component gene selected from the TCRα variable region gene, TCRβ variable region gene, TCR constant region gene, or combinations thereof.

[0224] In some embodiments, a population of host cells comprising the binding proteins disclosed herein exhibits a functional avidity that is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, or at least 5000-fold compared to a population of control cells (e.g., cells expressing a control binding protein specific for the same target antigen). The host cells can comprise a binding protein (e.g., a TCR comprising the Vα and Vβ regions and / or CDRs disclosed herein) that binds to a target antigen (e.g., a neoantigen (e.g., p53, PIK3CA, NRAS, HRAS, or KRAS (e.g., a KRAS G12 variant peptide such as the KRAS G12V variant peptide present in a peptide:HLA complex))). The increase in avidity can be determined, for example, by an assay for determining the expression of activation markers (e.g., CD137, CD69, granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, cytokines, interleukins, interferons) upon exposure to target cells expressing or presenting the target antigen, or and / or by an assay for determining the EC50 (e.g., the peptide dose at which half-maximal activation of a T cell population is reached).

[0225] Host cell compositions and unit doses In another aspect, provided herein are compositions and unit doses comprising a modified host cell of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0226] In certain embodiments, the host cell composition or unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4 + T cells, and (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8 + T cells, in a combination in a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, 1:0.1, 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, wherein the unit dose contains a reduced amount of naive T cells or substantially no naive T cells (i.e., less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% naive T cell population is present in the unit dose as compared to a patient sample having an equivalent number of peripheral blood mononuclear cells (PBMCs)).

[0227] In some embodiments, the host cell composition or unit dose comprises (i) a composition comprising at least about 50% modified CD4 + T cells, and (ii) a composition comprising at least about 50% modified CD8 +A composition comprising T cells, in a combination at a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, 1:0.1, 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or substantially no naive T cells. In a further embodiment, the host cell composition or unit dose comprises (i) at least about 60% modified CD4 + A composition comprising T cells, and (ii) at least about 60% modified CD8 + A composition comprising T cells, in a combination at a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, 1:0.1, 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, wherein the unit dose contains a reduced amount of naive T cells or substantially no naive T cells. In yet a further embodiment, the host cell composition or unit dose comprises (i) at least about 70% engineered CD4 + A composition comprising T cells, and (ii) at least about 70% engineered CD8 + A composition comprising T cells, in a combination at a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, 1:0.1, 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, wherein the unit dose contains a reduced amount of naive T cells or substantially no naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 80% modified CD4 + A composition comprising T cells, and (ii) at least about 80% modified CD8 +A composition comprising T cells, in a combination of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, 1:0.1, 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 ratio, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or substantially no naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 85% modified CD4 + A composition comprising T cells, and (ii) at least about 85% modified CD8 + A composition comprising T cells, in a combination of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, 1:0.1, 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 ratio, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or substantially no naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 90% modified CD4 + A composition comprising T cells, and (ii) at least about 90% modified CD8 + A composition comprising T cells, in a combination of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0.5:1, 0.1:1, 1:0.1, 1:0.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 ratio, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or substantially no naive T cells.

[0228] In some embodiments, the composition comprises a CD4+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition further comprises a CD8+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells.

[0229] In some embodiments, the host cell composition or unit dose comprises a CD4+ to CD8+ T cell ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 3:2, or about 2:3 (e.g., CD4+ T cells modified to contain or express a binding protein disclosed herein versus CD8+ T cells modified to contain or express a binding protein disclosed herein).

[0230] In some embodiments, the host cell composition or unit dose comprises a CD4+ to CD8+ T cell ratio of at least 1:1, at least 1:2, at least 1:3, at least 1:4, at least 1:5, at least 1:6, at least 1:7, at least 1:8, at least 1:9, at least 1:10, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 3:2, or at least 2:3.

[0231] In some embodiments, the host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells that is at most 1:1, at most 1:2, at most 1:3, at most 1:4, at most 1:5, at most 1:6, at most 1:7, at most 1:8, at most 1:9, at most 1:10, at most 2:1, at most 3:1, at most 4:1, at most 5:1, at most 6:1, at most 7:1, at most 8:1, at most 9:1, at most 10:1, at most 3:2, or at most 2:3.

[0232] In some embodiments, the host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells that is about 1:10 to 10:1, 1:10 to 8:1, 1:10 to 7:1, 1:10 to 6:1, 1:10 to 5:1, 1:10 to 4:1, 1:10 to 3:1, 1:10 to 2:1, 1:10 to 1:1, 1:10 to 1:2, 1:10 to 1:3, 1:10 to 1:4, 1:10 to 1:5, 1:10 to 1:7, 1:5 to 10:1, 1:5 to 8:1, 1:5 to 7:1, 1:5 to 6:1, 1:5 to 5:1, 1:5 to 4:1, 1:5 to 3:1, 1:5 to 2:1, 1:5 to 1:1, 1:5 to 1:2, 1:5 to 1:3, 1:5 to 1:4, 1:3 to 10:1, 1:3 to 8:1, 1:3 to 7:1, 1:3 to 6:1, 1:3 to 5:1, 1:3 to 4:1, 1:3 to 3:1, 1:3 to 2:1, 1:3 to 1:1, 1:3 to 1:2, 1:2 to 10:1, 1:2 to 8:1, 1:2 to 7:1, 1:2 to 6:1, 1:2 to 5:1, 1:2 to 4:1, 1:2 to 3:1, 1:2 to 2:1, 1:2 to 1:1, 1:1 to 10:1, 1:1 to 8:1, 1:1 to 7:1, 1:1 to 6:1, 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, 1:1 to 2:1, 2:1 to 10:1, 2:1 to 8:1, 2:1 to 7:1, 2:1 to 6:1, 2:1 to 5:1, 2:1 to 4:1, 2:1 to 3:1, 3:1 to 10:1, 3:1 to 8:1, 3:1 to 7:1, 3:1 to 6:1, 3:1 to 5:1, 3:1 to 4:1, 5:1 to 10:1, 5:1 to 8:1, 5:1 to 7:1, or 5:1 to 6:1.

[0233] The CD4+ T cells in the composition, host cell composition, or unit dose can be, for example, CD4+ T cells that are modified or engineered to express the CD8 coreceptor disclosed herein using the vectors or polynucleotides disclosed herein.

[0234] It will be understood that the host cell compositions or unit doses of the present disclosure may include any of the host cells described herein, or any combination of host cells. In certain embodiments, for example, the host cell composition or unit dose includes modified CD8+ T cells, modified CD4+ T cells, or both, and these T cells are modified to encode a binding protein specific for the Ras peptide:HLA-A*11:01 complex. Additionally or alternatively, the host cell compositions or unit doses of the present disclosure may include any host cell or combination of host cells described herein, and may have different antigens (e.g., different Ras antigens, or different proteins or targets such as BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., MAGE-A1, MAGE-A3, and MAGE-A4, etc.), mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor- or pathogen-associated peptides bound to HLA, hTERT peptides bound to HLA, tyrosinase peptides bound to HLA, WT-1 peptides bound to HLA, LTβR, LIFRβ, LRP5, MUC1, OSMRβ, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, HA 1-H, Robo1, alpha-fetoprotein (AFP), Frizzled, OX40, PRAME, and SSX-2, etc.) antigens), and may further comprise modified cells (e.g., immune cells, e.g., T cells) that express a binding protein specific for the antigen. In some embodiments, the binding protein binds to a peptide (e.g., the different antigens described above) complexed with an HLA protein, e.g., HLA-A, -B, -C, E, -G, -H, -J, -K, or -L. For example, a unit dose is a modified CD8 that expresses a binding protein that specifically binds to a Ras-HLA complex + T cells and modified CD4 that express a binding protein (e.g., a CAR) that specifically binds to the PSMA antigen + T cells (and / or modified CD8 + T cells). It will also be understood that any of the host cells disclosed herein can be administered in combination therapy.

[0235] In any of the embodiments described herein, the host cell composition or unit dose comprises an equal or approximately equal number of engineered CD45RA - CD3 + CD8 + and modified CD45RA - CD3 + CD4 + T M cells.

[0236] In any of the embodiments described herein, the host cell composition or unit dose comprises, for example, one or more populations of cells (e.g., CD4+ or CD8+ cells) that have undergone CD62L positive selection to improve persistence in vitro.

[0237] The host cell can be genetically engineered to contain or express the binding protein ex vivo, in vitro, or in vivo.

[0238] Use In an additional aspect, the present disclosure provides a method for treating or preventing relapse of a disease or disorder associated with a KRAS G12V mutation or an NRAS G12V mutation or an HRAS G12V mutation in a subject. Such diseases or disorders include, for example, cancer, such as solid tumors and hematological malignancies. In certain embodiments, the disease or disorder is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; anaplastic thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell lung cancer; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid cancer; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial cancer; or papillary thyroid cancer.

[0239] "To treat" or "treatment" or "alleviate" generally refers to the medical management of a disease, disorder, or condition in a subject (e.g., a human or non-human mammal such as a primate, horse, cat, dog, goat, mouse, or rat). Generally, an appropriate dose or treatment regimen comprising a composition of the present disclosure (e.g., a binding protein, polynucleotide, vector, host cell, host cell composition, unit dose, and / or an immunogenic polypeptide) is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefits include improvement in clinical outcome; reduction or alleviation of symptoms associated with the disease; decrease in the occurrence of symptoms; improvement in quality of life; longer disease-free state; reduction in the extent of the disease; stabilization of the disease state; delay in disease progression; remission; survival; long-term survival; or any combination thereof.

[0240] As used herein, "therapeutically effective amount" or "effective amount" generally refers to an amount of a composition sufficient to produce a therapeutic effect, such as improvement in clinical outcome; reduction or alleviation of symptoms associated with the disease; decrease in the occurrence of symptoms; improvement in quality of life; longer disease-free state; reduction in the extent of the disease, stabilization of the disease state; delay in disease progression; remission; survival; or long-term survival, in a statistically significant manner. A therapeutically effective amount, when referring to an individual active ingredient administered alone or a cell expressing a single active ingredient, refers to the action of that ingredient alone or the cell expressing that ingredient. A therapeutically effective amount, when referring to a combination, refers to the combined amount of the active ingredient or combined auxiliary active ingredients and the cells expressing the active ingredient that produces a therapeutic effect, whether administered sequentially or simultaneously. The combination may also be cells expressing more than one active ingredient.

[0241] The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" generally refers to a biocompatible medium, such as physiological saline, which is generally recognized as being suitable for administration to a human or other non-human mammalian subject and is safe or does not cause serious adverse events, and these are described in more detail herein.

[0242] As used herein, "statistically significant" generally refers to a p-value of 0.050 or less when calculated using a Student's t-test, or a value or indicator of statistical significance using another appropriate statistical test, indicating that the particular event or result being measured is unlikely to have occurred by chance.

[0243] Subjects that can be treated by the present disclosure are generally human and other primate subjects, such as monkeys and apes for veterinary purposes. In any of the foregoing embodiments, the subject can be a human subject. The subject can be a mammal. The subject can be male or female and can be of any suitable age, including infant, juvenile, young adult, adult, and elderly subjects. The compositions according to the present disclosure may be administered in a manner appropriate for the disease, condition, or disorder being treated, as determined by one of ordinary skill in the art. In any of the above embodiments, the modified host cells, host cell compositions, or unit doses described herein are administered intravenously, intraperitoneally, intratumorally, intramedullary, intranodally, or into the cerebrospinal fluid so as to encounter target cells (e.g., leukemia cells). The appropriate dose, suitable duration, and frequency of administration of the composition are determined by factors such as the patient's condition; the size, type, and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.

[0244] As used herein, the term "adoptive immune therapy" or "adoptive immunotherapy" generally refers to the administration of naturally occurring or genetically engineered disease-specific or antigen-specific immune cells (e.g., T cells). Adoptive cell immunotherapy can be autologous (the immune cells are derived from the recipient), allogeneic (the immune cells are derived from a donor of the same species), or syngeneic (the immune cells are derived from a donor genetically identical to the recipient).

[0245] In some embodiments, the subject expresses a Ras antigen comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2-3.

[0246] In some embodiments, the subject is HLA-A + , HLA-B + , or HLA-C + . In some embodiments, the subject is HLA-A*11:01 + .

[0247] In certain embodiments, the method includes determining the HLA type(s) of the subject and / or identifying the presence of neoantigens prior to administering a therapy according to the present disclosure.

[0248] The expression of HLA alleles can be determined, for example, by gene sequencing (e.g., high-throughput next-generation sequencing (NGS)). This genetic determination of HLA expression is referred to herein as "HLA typing" and can be determined via a molecular approach in a clinical laboratory licensed for HLA typing. In some embodiments, HLA typing is performed using PCR amplification, followed by high-throughput NGS, and subsequent HLA determination. As used herein, HLA haplotypes can be determined at the major HLA loci (e.g., HLA-A, HLA-B, HLA-C, etc.).

[0249] HLA typing can be performed using any known method, including, for example, protein or nucleic acid assays. Examples of nucleic acid assays include sequence-based typing (SBT), as well as the use of sequence-specific oligonucleotide probes (SSOP) or sequence-specific primers (SSP). In certain embodiments, HLA typing is performed using PCR amplification, followed by high-throughput next-generation sequencing (NGS), and subsequent HLA determination. In some embodiments, sequence typing is performed using a system available through Scisco Genetics (sciscogenetics.com / pages / technology.html, the contents of which are incorporated herein by reference in their entirety). Other methods for HLA typing include, for example, the methods disclosed in Mayor et al. PLoS One 10(5):e0127153 (2015), and these methods and reagents are incorporated herein by reference.

[0250] In certain embodiments, the method comprises administering a composition comprising modified CD8+ and / or modified CD4+ T cells comprising a heterologous polynucleotide encoding a second binding protein, as provided herein.

[0251] In the case of a host cell composition or unit dose, the amount of cells therein is at least one cell (e.g., one modified CD8 + T cell subset (e.g., memory and / or naive CD8 + T cells, if desired); one modified CD4 + T cell subset (e.g., memory and / or naive CD4 + T cells, if desired)), or more typically more than 10 2 cells, e.g., up to 10 4 to the maximum of 10 5 to the maximum of 10 6 to the maximum of 10 7 to the maximum of 10 8 to the maximum of 10 9 to the maximum of 1010 more than one cell. In certain embodiments, the cells are about 10 4 ~ about 10 10 cells / m 2 and are administered in the range of about 10 5 ~ about 10 9 cells / m 2 In some embodiments, the dose administered contains up to about 3.3×10 5 cells / kg. In some embodiments, the dose administered contains up to about 1×10 6 cells / kg. In some embodiments, the dose administered contains up to about 3.3×10 6 cells / kg. In some embodiments, the dose administered contains up to about 1×10 7 cells / kg. In certain embodiments, the modified immune cells are up to about 5×10 4 cells / kg, 5×10 5 cells / kg, 5×10 6 cells / kg, or up to about 5×10 7 cells / kg and are administered to a subject in a dose containing. In certain embodiments, the modified immune cells are at least about 5×10 4 cells / kg, 5×10 5 cells / kg, 5×10 6 cells / kg, or up to about 5×10 7 cells / kg and are administered to a subject in a dose containing. The number of cells depends on the intended final use of the composition and the type of cells contained therein. For example, cells modified to contain a binding protein contain a cell population that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of such cells. For the uses provided herein, the cells are generally in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In embodiments, the desired cell density is typically 10 4Greater than cells / ml, generally 10 7 Greater than cells / ml, generally 10 8 cells / ml or greater. The cells may be administered as a single injection or as multiple injections over a period of time. A clinically relevant number of immune cells may be distributed over multiple injections of 10 6 10 7 10 8 10 9 10 10 10 11 cells or more per injection. In certain embodiments, a unit dose of modified immune cells may be co-administered with hematopoietic stem cells derived from an allogeneic donor (e.g., simultaneously or contemporaneously). In some embodiments, one or more of the modified immune cells included in the unit dose are autologous to the subject.

[0252] In some embodiments, the subject receiving the modified immune cells has previously received lymphodepleting chemotherapy. In further embodiments, the lymphodepleting chemotherapy includes cyclophosphamide, fludarabine, antithymocyte globulin, or combinations thereof.

[0253] In some embodiments, the method further includes administering to the subject an inhibitor of an immune checkpoint molecule disclosed herein.

[0254] Also contemplated are pharmaceutical compositions (i.e., compositions) comprising the compositions (binding proteins, polynucleotides, vectors, host cells, host cell compositions, unit doses, and / or immunogenic polypeptides) disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include, for example, water, saline, dextrose, or glycerol, and combinations thereof. In embodiments, compositions comprising a fusion protein or host cell as disclosed herein further comprise a suitable infusion medium. Suitable infusion media may be any isotonic media formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water, lactated Ringer's solution. The infusion medium may be supplemented with human serum albumin or other human serum components.

[0255] The pharmaceutical compositions can be administered in a manner suitable for the disease or condition to be treated (or prevented) as determined by one of ordinary skill in the medical arts. The appropriate dosage of the composition, as well as the suitable dosing period and frequency of administration, will be determined by factors such as the patient's health status, the patient's size (i.e., weight, bulk, or body surface area), the type and severity of the patient's condition, the particular form of the active ingredient, and the method of administration. Generally, the appropriate dosage and treatment regimen will provide a therapeutically and / or prophylactically beneficial amount of the composition(s) (including those described herein) to provide an improvement in clinical outcome (including, for example, more frequent complete or partial remissions, or longer disease-free and / or overall survival periods, or reduction in the severity of symptoms).

[0256] An effective amount of a pharmaceutical composition refers to an amount sufficient to achieve the desired clinical result or beneficial treatment described herein at the required dosage and over such a period. The effective amount can be delivered in one or more administrations. When the administration is to a subject already known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used with reference to treatment, while the "prophylactically effective amount" can be used to describe administering an effective amount to a subject at risk of developing or likely to develop a disease or disease state (e.g., recurrence) as a prophylactic process.

[0257] The pharmaceutical compositions described herein can be provided in unit dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to maintain the stability of the formulation until it is injected into the patient. The dosage can vary, but the dosage for administration of the modified immune cells described herein is about 10 4 cells / m 2 about 5×10 4 cells / m 2 about 10 5 cells / m 2 about 5×10 5 cells / m 2 about 10 6 cells / m 2 about 5×10 6 cells / m 2 about 10 7 cells / m 2 about 5×10 7 cells / m 2 about 10 8 cells / m 2 about 5×10 8 cells / m 2 about 10 9 cells / m 2 about 5×10 9 cells / m 2 about 10 10 cells / m 2 about 5×10 10 cells / m 2 or about 10 11 cells / m 2 and can be. In certain embodiments, the unit dose is about 10 4 cells / m2 ~about 10 11 cells / m 2 at a dosage of, and includes the modified immune cells described herein. For example, the development of suitable dosages and treatment regimens for using the specific compositions described herein in a variety of treatment regimens including parenteral or intravenous administration or formulations.

[0258] When the compositions of the subject are administered parenterally, the compositions can also include sterile aqueous or oily solutions or suspensions. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol in mixtures with water. Aqueous solutions or suspensions may further include one or more buffering agents such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any materials used in preparing any dosage unit formulation are pharmaceutically pure and can be substantially non-toxic in the amounts used. In addition, the active compounds may be incorporated into sustained release preparations and formulations. Dosage unit forms, as used herein, refer to physically discrete units suitable for one unit dosage for a subject to be treated, each unit containing a predetermined quantity of the engineered immune cells or active compound calculated to produce the desired effect, together with a suitable pharmaceutical carrier.

[0259] Generally, suitable dosages and treatment regimens provide an amount of the active molecule or cell sufficient to provide a benefit. Such responses can be monitored by establishing an improvement in the clinical outcome of the treated subject (e.g., more frequent remissions, complete or partial or longer disease-free survival periods) compared to untreated subjects. An increase in the existing immune response to tumor proteins generally correlates with an improvement in clinical outcome. Such immune responses can generally be evaluated using standard proliferation, cytotoxicity, or cytokine assays that are routine.

[0260] In the case of prophylactic use, the dosage may be sufficient to prevent, delay the onset of, or attenuate the severity of a disease associated with a disorder. The prophylactic benefit of an immunogenic composition administered according to the methods described herein can be determined by conducting preclinical studies (including in vitro and in vivo animal studies) and clinical studies and analyzing the data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can be readily performed by those of ordinary skill in the art.

[0261] As used herein, administration of a composition refers to delivering it to a subject, regardless of the route or mode of delivery. Administration can be continuous or intermittent and parenteral. The composition can be administered locally (e.g., intratumorally) or systemically (e.g., intravenously). Administration can be for treating a subject that already has a recognized condition, disease, or disease state, or for treating a subject that is at risk of developing or is likely to develop such a condition, disease, or disease state. Co-administration with adjuvant therapies can include simultaneous and / or sequential delivery of multiple agents in any order and any dosing schedule (e.g., modified immune cells with one or more cytokines; immunosuppressive therapy agents such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolic acid prodrugs, or any combination thereof).

[0262] In certain embodiments, multiple doses of the composition described herein are administered to a subject, and the composition may be administered to the subject at dosing intervals of about 2 to about 4 weeks.

[0263] The treatment or prevention methods of the present disclosure can be administered to a subject as part of a treatment process or regimen, and may include additional treatments before or after administration of the unit dose, cells, or composition of the present disclosure. For example, in certain embodiments, a subject receiving a unit dose of modified immune cells has received or has previously received a hematopoietic cell transplantation (HCT; including myeloablative and non-myeloablative HCT). Techniques and regimens for performing HCT are known in the art and may include transplantation of any suitable donor cells, such as cells derived from umbilical cord blood, bone marrow, or peripheral blood, hematopoietic stem cells, mobilized stem cells, or cells derived from amniotic fluid. Thus, in certain embodiments, one modified immune cell of the present disclosure can be administered with or immediately after hematopoietic stem cells in a modified HCT therapy. In some embodiments, the HCT includes donor hematopoietic cells that include chromosomal knockout of a gene encoding an HLA component, chromosomal knockout of a gene encoding a TCR component, or both.

[0264] In further embodiments, the subject may have previously received lymphodepleting chemotherapy prior to receiving the composition or HCT. In certain embodiments, the lymphodepleting chemotherapy includes a conditioning regimen that includes cyclophosphamide, fludarabine, antithymocyte globulin, or combinations thereof.

[0265] The methods according to the present disclosure may further include administering one or more additional agents to treat a disease or disorder in a combination therapy. For example, in certain embodiments, the combination therapy includes administering the composition of the present disclosure together with (in combination, simultaneously, or sequentially) an immune checkpoint inhibitor. In some embodiments, the combination therapy includes administering the composition of the present disclosure together with an agonist of a stimulatory immune checkpoint agent. In further embodiments, the combination therapy includes administering the composition of the present disclosure together with a secondary therapy, such as a chemotherapeutic agent, radiation therapy, surgery, antibody, or any combination thereof.

[0266] As used herein, the term "immune suppression agent" or "immunosuppression agent" refers to one or more cells, proteins, molecules, compounds, or complexes that provide inhibitory signals to assist in the control or suppression of the immune response. For example, immunosuppressive agents include molecules that partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immunosuppression. Examples of immunosuppressive agents to be targeted (e.g., using immune checkpoint inhibitors) include the following: PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244 / 2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

[0267] Immunosuppressant inhibitors (also referred to as immune checkpoint inhibitors) may be compounds, antibodies, antibody fragments, or fusion polypeptides (e.g., Fc fusions such as CTLA4-Fc or LAG3-Fc), antisense molecules, ribozymes, or RNAi molecules, or low molecular weight organic molecules. In any of the embodiments disclosed herein, the method may include a composition of the present disclosure comprising one or more inhibitors of any one of the following immunosuppressive components, alone or in any combination.

[0268] In certain embodiments, the compositions of the present disclosure are used in combination with a PD-1 inhibitor, such as a PD-1 specific antibody or a binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558, or any combination thereof. In further embodiments, the compositions of the present disclosure are used in combination with a PD-L1 specific antibody or a binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof. Also contemplated are semiprimab; IBI-308; nivolumab + relatlimab; BCD-100; camrelizumab; JS-001; spartalizumab; tislelizumab; AGEN-2034; BGBA-333 + tislelizumab; CBT-501; dostarlimab; durvalumab + MEDI-0680; JNJ-3283; pazopanib hydrochloride + pembrolizumab; pidilizumab; REGN-1979 + semiprimab; ABBV-181; ADUS-100 + spartalizumab; AK-104; AK-105; AMP-224; BAT-1306; BI-754091; CC-90006; semiprimab + REGN-3767; CS-1003; GLS-010; LZM-009; MEDI-5752; MGD-013; PF-06801591; Sym-021; tislelizumab + pamiparib; XmAb-20717; AK-112; ALPN-202; AM-0001; an antibody that antagonizes PD-1 with respect to Alzheimer's disease; BH-2922; BH-2941; BH-2950; BH-2954; a biologic agent that antagonizes CTLA-4 and PD-1 with respect to solid tumors; a bispecific monoclonal antibody that targets PD-1 and LAG-3 with respect to oncology; BLSM-101; CB-201; CB-213; CBT-103; CBT-107; cellular immunotherapy + PD-1 inhibitor; CX-188; HAB-21; HEISCOIII-003; IKT-202; JTX-4014; MCLA-134; MD-402; mDX-400; MGD-019; a monoclonal antibody that antagonizes PDCD1 with respect to oncology;Monoclonal antibodies that antagonize PD-1 with respect to oncology; Oncolytic viruses that inhibit PD-1 with respect to oncology; OT-2; PD-1 antagonist + pegylated interferon alpha-2b; PEGMP-7; PRS-332; RXI-762; STIA-1110; TSR-075; Vaccines that target HER2 and PD-1 with respect to oncology; Vaccines that target PD-1 with respect to oncology and autoimmune disorders; XmAb-23104; Antisense oligonucleotides that inhibit PD-1 with respect to oncology; AT-16201; Bispecific monoclonal antibodies that inhibit PD-1 with respect to oncology; IMM-1802; Monoclonal antibodies that antagonize PD-1 and CTLA-4 with respect to solid tumors and hematological tumors; Nivolumab biosimilar; Recombinant proteins that agonize CD278 and CD28 and antagonize PD-1 with respect to oncology; Recombinant proteins that agonize PD-1 with respect to autoimmune and inflammatory disorders; SNA-01; SSI-361; YBL-006; AK-103; JY-034; AUR-012; BGB-108; Drugs that inhibit PD-1, Gal-9, and TIM-3 with respect to solid tumors; ENUM-244C8; ENUM-388D4; MEDI-0680; Monoclonal antibodies that antagonize PD-1 with respect to metastatic melanoma and metastatic lung cancer; Monoclonal antibodies that inhibit PD-1 with respect to oncology; Monoclonal antibodies that target CTLA-4 and PD-1 with respect to oncology; Monoclonal antibodies that antagonize PD-1 with respect to NSCLC; Monoclonal antibodies that inhibit PD-1 and TIM-3 with respect to oncology; Monoclonal antibodies that inhibit PD-1 with respect to oncology; Recombinant proteins that inhibit PD-1 and VEGF-A with respect to hematological malignancies and solid tumors; Small molecules that antagonize PD-1 with respect to oncology; Sym-016; Inebilizumab + MEDI-0680; Vaccines that target PDL-1 and IDO with respect to metastatic melanoma; Anti-PD-1 monoclonal antibody plus cellular immunotherapy for glioblastoma; Antibodies that antagonize PD-1 with respect to oncology; Monoclonal antibodies that inhibit PD-1 / PD-L1 with respect to hematological malignancies and bacterial infections; Monoclonal antibodies that inhibit PD-1 with respect to HIV;Or a small molecule that inhibits PD-1 with respect to solid tumors;

[0269] In certain embodiments, the compositions of the present disclosure are used in combination with a LAG3 inhibitor such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0270] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of CTLA4. In certain embodiments, the compositions of the present disclosure are used in combination with a CTLA4-specific antibody or a binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (e.g., abatacept, belatacept), or any combination thereof.

[0271] In certain embodiments, the compositions of the present disclosure are used in combination with a B7-H3-specific antibody or a binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. B7-H4 antibody binding fragments may be, for example, scFv or fusion proteins thereof as described in Dangaj et al., Cancer Res. 73:4820, 2013, and those described in U.S. Patent No. 9,574,000 and PCT Patent Publications WO / 201640724A1 and WO2013 / 025779A1.

[0272] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of CD244.

[0273] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication No. WO2010 / 084158.

[0274] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of TIM3.

[0275] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of Gal9.

[0276] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor.

[0277] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of A2aR.

[0278] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015).

[0279] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitory cytokine (typically a cytokine other than TGFβ) or an inhibitor of Treg generation or activity.

[0280] In certain embodiments, the compositions of the present disclosure are used in combination with an IDO inhibitor, such as levo-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (1-MT)-tir- pazamine, or any combination thereof.

[0281] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor such as N(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-L-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.

[0282] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0283] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of TIGIT, such as COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as COM701 (Compugen), or both.

[0284] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO2017 / 021526.

[0285] In certain embodiments, the compositions of the present disclosure are used in combination with an LAIR1 inhibitor.

[0286] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0287] In certain embodiments, the compositions of the present disclosure are used in combination with an agent that increases the activity of stimulatory immune checkpoint molecules (i.e., is an agonist). For example, the compositions of the present disclosure can be used in combination with: a CD137 (41BB) agonist (e.g., urelumab, etc.), a CD134 (OX-40) agonist (e.g., MEDI6469, MEDI6383, or MEDI0562, etc.), lenalidomide, pomalidomide, a CD27 agonist (e.g., CDX-1127, etc.), a CD28 agonist (e.g., TGN1412, CD80, or CD86, etc.), a CD40 agonist (e.g., CP-870,893, rhuCD40L, or SGN-40, etc.), a CD122 agonist (e.g., IL-2, etc.), an agonist of GITR (e.g., a humanized monoclonal antibody described in PCT Patent Publication No. WO2016 / 054638, etc.), an agonist of ICOS (CD278) (e.g., GSK3359609, mAb88.2, JTX-2011, Icos145-1, Icos314-8, or any combination thereof, etc.). In any of the embodiments disclosed herein, the method may comprise administering the composition of the present disclosure together with one or more agonists of stimulatory immune checkpoint molecules, including any of the foregoing alone or in any combination.

[0288] In certain embodiments, the combination therapy comprises the composition of the present disclosure and a secondary therapy comprising one or more of an antibody or antigen-binding fragment thereof specific for a cancer antigen expressed by a non-inflammatory solid tumor, radiation therapy, surgery, chemotherapeutic agents, cytokines, RNAi, or any combination thereof.

[0289] In certain embodiments, the method of combination therapy comprises administering the composition of the present disclosure and further performing radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy such as gamma irradiation and radiopharmaceutical therapy. Appropriate surgeries and surgical techniques for treating a given cancer in a subject are well known to those of skill in the art.

[0290] In certain embodiments, the method of combination therapy comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, the following: inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactants (such as intercalating agents), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine), and purine analogs, folic acid antagonists, and related inhibitors (such as antimetabolite / anticancer agents like mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), taxanes (paclitaxel, docetaxel), microtubule disrupting agents such as vincristine, vinblastine, nocodazole, epothilone, and navelbine, epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide, and etoposide (VP16)) including antiproliferative / antimitotic agents; antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin;Enzymes (L-asparaginase that systemically metabolizes L-asparagine and depletes cells lacking the ability to synthesize their own asparagine); antiplatelet agents; nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes - dacarbazinine (DTIC), and other antineoplastic / antimitotic alkylating agents; antineoplastic / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migration agents; anti-secretory agents (breveldin); immunosuppressive agents (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (retinoic acid);mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11), and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disrupting factors;

[0291] Cytokines can be used to manipulate the host immune response towards anti-cancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting an immune anti-cancer or anti-tumor response, which are used alone or in any combination with the compositions of the present disclosure, include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF.

[0292] Methods for modulating adoptive immunotherapy are also provided herein, the method comprising administering to a subject previously administered a modified host cell an amount of a cognate compound of a safety switch protein effective to remove the modified host cell previously received by the subject, the modified host cell comprising a heterologous polynucleotide encoding the safety switch protein.

[0293] In certain embodiments, the safety switch protein comprises tEGFR and the cognate compound is cetuximab, or the safety switch protein comprises iCasp9 and the cognate compound is AP1903 (e.g., dimerized AP1903), or the safety switch protein comprises an RQR polypeptide and the cognate compound is rituximab, or the safety switch protein comprises a myc binding domain and the cognate compound is an antibody specific for the myc binding domain.

[0294] In yet further embodiments, provided are compositions of the present disclosure, or methods for manufacturing unit doses. In certain embodiments, the method comprises (i) aliquoting host cells transfected with a vector of the present disclosure and (ii) combining with a pharmaceutically acceptable carrier. In certain embodiments, the vector of the present disclosure is used to transfect / transduce host cells (e.g., T cells) for use in adoptive cell therapy (e.g., targeting cancer antigens).

[0295] In some embodiments, the method further comprises culturing the transfected host cells and selecting the transfected cells in which the vector has integrated (i.e., expressing the vector) prior to aliquoting. In further embodiments, the method comprises expanding the transfected host cells after culturing and selection and prior to aliquoting. In any of the embodiments of the methods of the invention, the manufactured composition or unit dose may be frozen (e.g., cryopreserved) for later use. For example, any suitable host cell, such as hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, or NK-T cells, may be used to manufacture a composition or unit dose according to the methods of the invention. In a specific embodiment, the method is CD8 + T cells, CD4 + T cells, or host cells that are both.

[0296] Also provided are a binding protein, a polynucleotide, an expression vector, a host cell, a host cell composition, a unit dose, and an immunogenic polypeptide, which are taken alone or in any combination for use in treating a disease or disorder associated with a KRAS G12D mutation or a KRAS G12V or NRAS G12D mutation or a NRAS G12V mutation or a HRAS G12V mutation or a HRAS G12D mutation in a subject.

[0297] Also provided are a binding protein, a polynucleotide, an expression vector, a host cell, a host cell composition, a unit dose, and an immunogenic polypeptide, which are taken alone or in any combination for use in the manufacture of a medicament for treating a disease or disorder associated with a KRAS G12D mutation or a KRAS G12V or NRAS G12D mutation or a NRAS G12V mutation or a HRAS G12V mutation or a HRAS G12D mutation in a subject.

[0298] In certain embodiments, the disease or disorder includes cancer. In some embodiments, the cancer is a solid cancer or a hematological malignancy. In certain embodiments, the disease or disorder is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell lung cancer; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic cancer; follicular thyroid cancer; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial cancer; or papillary thyroid cancer. In some embodiments, the method includes parenteral or intravenous administration of the subject composition. In some embodiments, the method includes administering to the subject a plurality of doses of a binding protein, polynucleotide, expression vector, host cell, host cell composition, unit dose, and / or immunogenic polypeptide.

[0299] In certain embodiments, the plurality of doses are administered at an administration interval of about 2 to about 4 weeks.

[0300] In certain embodiments, the composition includes a modified host cell. In some embodiments, the method includes administering the modified host cell to the subject at a dose of about 10 4 cells / kg to about 10 11 cells / kg.

[0301] In certain embodiments, the method further comprises administering a cytokine. In some embodiments, the cytokine comprises IL-2, IL-15, or IL-21.

[0302] In certain embodiments, the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agonist.

[0303] Also provided is a method comprising introducing into a host (e.g., T) cell a polynucleotide encoding a binding protein of the present disclosure.

[0304] Sequence SEQ ID NO:1 - wt KRAS full length (UniProt:P01116) MTEYKLVVVG AGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQI KRVKDSEDVP MVLVGNKCDL PSRTVDTKQA QDLARSYGIP FIETSAKTRQ RVEDAFYTLV REIRQYRLKK ISKEEKTPGC VKIKKCIIM SEQ ID NO:2 - KRAS 7-16 G12V VVVGAVGVGK SEQ ID NO:3 - KRAS 8-16 G12V VVGAVGVGK SEQ ID NO:4 - KRAS 8-16 G12V binding motif for TCR 11N4A x-V-G-A-x-G-x-x-K SEQ ID NO:5 - TCR 11N4A alpha chain with signal peptide - original (WT) nucleotide sequence atggccatgctcctgggggcatcagtgctgattctgtggcttcagccagactgggtaaacagtcaacagaagaatgatgaccagcaagttaagcaaaattcaccatccctgagcgtccaggaaggaagaatttctattctgaactgtgactatactaacagcatgtttgattatttcctatggtacaaaaaataccctgctgaaggtcctacattcctgatatctataagttccattaaggataaaaatgaagatggaagattcactgtcttcttaaacaaaagtgccaagcacctctctctgcacattgtgccctcccagcctggagactctgcagtgtacttctgtgcagcaagtggggtttcaggaaacacacctcttgtctttggaaagggcacaagactttctgtgattgcaaatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagctga TCR 11N4A beta chain with SEQ ID NO: 6 - signal peptide - original (WT) nucleotide sequence atgggctccaggctgctctgttgggtgctgctttgtctcctgggagcaggcccagtaaaggctggagtcactcaaactccaagatatctgatcaaaacgagaggacagcaagtgacactgagctgctcccctatctctgggcataggagtgtatcctggtaccaacagaccccaggacagggccttcagttcctctttgaatacttcagtgagacacagagaaacaaaggaaacttccctggtcgattctcagggcgccagttctctaactctcgctctgagatgaatgtgagcaccttggagctgggggactcggccctttatctttgcgccagcagcgtcgggactgtggagcagtacttcgggccgggcaccaggctcacggtcacagaggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacctgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccagaggctag SEQ ID NO: 7 - TCR 11N4A TCR beta - P2A - TCR alpha polynucleotide - codon optimized A Array numbers 8 to 11 N4A TCR beta-P2A-alpha polynucleotide codon optimization B SEQ ID NO:9 - CD8 alpha - T2A - CD8 beta - P2A - 11N4A TCR beta - P2A - alpha polynucleotide codon optimized A Array number 10 - CD8 alpha - T2A - CD8 beta - P2A - 11N4A TCR beta - P2A - alpha polynucleotide codon optimization B Original protein with the signal peptide of SEQ ID NO: 11-11N4A TCR alpha chain underlined [Chemical Formula] Original protein without the signal peptide of SEQ ID NO: 12-11N4A TCR alpha chain QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS* Variable domain of 11N4A TCR alpha chain without the signal peptide of SEQ ID NO: 13 QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIA CDR1α of variable domain of 11N4A TCR alpha chain of SEQ ID NO: 14 NSMFDY CDR2α of variable domain of 11N4A TCR alpha chain of SEQ ID NO: 15 ISSIKDK CDR3α-IMGT junction of variable domain of 11N4A TCR alpha chain of SEQ ID NO: 16 CAASGVSGNTPLVF CDR3α-IMGT of variable domain of 11N4A TCR alpha chain of SEQ ID NO: 17 AASGVSGNTPLV Constant domain of 11N4A TCR alpha chain (original protein) of SEQ ID NO: 18 NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 19 - 11N4A TCR alpha chain constant domain (cys - modified protein) [Chemistry] SEQ ID NO: 20 - cys - modified, 11N4A TCR alpha chain without signal peptide [Chemistry] SEQ ID NO: 21 - Original protein of 11N4A TCR beta chain - signal peptide underlined [Chemistry] SEQ ID NO: 22 - Original protein of 11N4A TCR beta chain without signal peptide GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 23 - 11N4A TCR beta chain variable domain without signal peptide GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFGPGTRLTVT Variable domain CDR1β of the array number 24 - 11N4A TCR beta chain SGHRS Variable domain CDR2β of the array number 25 - 11N4A TCR beta chain YFSETQ Junction of variable domain CDR3β - IMGT of the array number 26 - 11N4A TCR beta chain CASSVGTVEQYF Variable domain CDR3β - IMGT of the array number 27 - 11N4A TCR beta chain ASSVGTVEQY Constant domain of the array number 28 - 11N4A TCR beta chain (original protein) EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG* Constant domain of the array number 29 - 11N4A TCR beta chain (cys - modified protein)

Chem.

Chem.

Chem.

Chem.

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Table 2-1

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[0305] Array number 1039: Human p53 amino acid sequence MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD Array number 1040: Human PIK3CA amino acid sequence Accession No. 1041: IL7R signal transduction domain

Chem.

Example

[0306] Example 1 Identification of KRAS G12V-specific TCRs from the T cell repertoire of healthy donors Dendritic cells were generated from peripheral blood mononuclear cells (PBMCs) of healthy HLA-A11-positive donors, irradiated, and pulsed with KRAS-G12V 7-16 and KRAS-G12V 8-16 peptides. These were incubated with autologous CD8 + T cells for 8 - 10 days to induce activation / expansion of antigen-specific CD8+ T cells. These polyclonal T cell lines were then restimulated and expanded twice over 8 - 10 days using peptide-pulsed irradiated autologous PBMCs to further expand antigen-specific clones. This process was carried out for 10 lines of CD8+ T cells from each of 15 HLA-matched donors. (Ho WY et al., J Immunol Methods. 2006;310(1):40 - 52. doi:10.1016 / j.jim.2005.11.023) (Figure 1A).

[0307] To identify TCRs that have strong binding to those cognate peptides (i.e., KRAS peptides) presented in the context of HLA-A11, T cells were stimulated overnight with titrated concentrations of cognate KRAS G12V peptides, and upregulation of CD137 was evaluated by flow cytometry. Cells expressing CD137 were isolated by flow cytometry cell sorting and TCR beta repertoire analysis was performed (Adaptive Biotechnologies, Seattle, WA). TCR clone types that were highly enriched in the CD137+ population and responded to low concentrations of peptide were identified, and TCR alpha / beta pairing was determined by 10x single cell RNAseq analysis in a similarly sorted population (10x Genomics, Pleasanton, CA). A representative analysis of clonotype enrichment in the CD137+ sorted population compared to all unsorted cells treated with low and high peptide concentrations is shown in Figure 1B. Paired TCR alpha / beta sequences from the identified clone types were assembled and synthesized as P2A-linked expression cassettes and transduced into reporter Jurkat cells that express GFP under the control of the Nur77 locus (Nur77-GFP-Jurkats) by lentivirus. The peptide dose-dependent response for each TCR was evaluated by analyzing GFP expression after overnight culture with A11 target cells pulsed with decreasing concentrations of peptide as shown (Figure 1C). Dose-response curves were fit by non-linear regression and EC50 values were calculated using Graphpad Prism (Boston, MA) (Figures 1D, 1E).

[0308] Example 2 Primary CD8 + Functional Affinity of KRAS-G12V-Specific TCR Expressed in T Cells Primary CD8+ T cells were transduced with a polynucleotide encoding a KRAS-G12V-specific TCR, sorted, purified, and expanded. Then, KRAS-G12V 8-16Sorted and purified T cells with reduced peptide concentration were cultured overnight, and the expression of CD137 was evaluated by flow cytometry. The dose-response curve was fitted by non-linear regression, and the EC50 value was calculated using Graphpad Prism (Figures 2A, 2B). In this experiment, TCR 11N4A was compared with the KRAS G12V-specific TCR "220_21" (see SEQ ID NOs: 61 and 62 herein) and the TCR "BNT" with variable domains encoded by SEQ ID NOs:...

Claims

Claim 1 (a) A binding protein, wherein the binding protein is (i) a T cell receptor (TCR) or a functional derivative thereof, or (ii) a chimeric antigen receptor (CAR) or a functional derivative thereof, and the binding protein (b) A fusion protein, wherein the fusion protein is (i) an extracellular component containing a CD95 (Fas) ectodomain or a functional fragment thereof and containing a CD95 ligand (FasL) binding domain, and (ii) an intracellular component containing a CD137 (4-1BB) intracellular signaling domain, and a nucleic acid sequence encoding the binding protein is located upstream of a nucleic acid sequence encoding a fusion polypeptide, and the fusion protein, a polynucleotide comprising a nucleic acid sequence encoding the fusion protein. Claim 2 (c) CD8 coreceptor α or β chain or a part or variant thereof, and a sequence encoding the binding protein is located upstream of a sequence encoding an extracellular part of CD8 coreceptor α or β chain or a part or variant thereof, and the polynucleotide according to claim 1, further comprising a nucleic acid sequence encoding CD8 coreceptor α or β chain or a part or variant thereof. Claim 3 (c) CD8 coreceptor α and β chains or a part or variant thereof, and the sequence encoding the binding protein is located upstream of the sequence encoding the extracellular part of CD8 coreceptor α and β chains or a part or variant thereof, and the polynucleotide according to claim 1, further comprising a nucleic acid sequence encoding CD8 coreceptor α and β chains or a part or variant thereof. Claim 4 The nucleic acid sequence encoding the fusion protein is (d) further encoding a hydrophobic component between the extracellular component and the intracellular component of the fusion protein, and the polynucleotide according to any one of claims 1 to 3. Claim 5 The binding protein contains a binding domain that binds to a peptide:HLA complex, and the complex contains a neoantigen peptide and an HLA protein, and the polynucleotide according to any one of claims 1 to 4. Claim 6 The binding protein contains a single-chain TCR (scTCR) or a single-chain T cell receptor variable fragment (scTv), and the polynucleotide according to any one of claims 1 to 5. Claim 7 The polynucleotide according to any one of claims 1 to 5, wherein the binding protein comprises a TCRα chain variable (Vα) domain or a TCRβ chain variable (Vβ) domain.

8. The polynucleotide according to any one of claims 1 to 6, wherein the binding protein comprises a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain.

9. The polynucleotide according to any one of claims 1 to 8, wherein the CD95 (Fas) ligand binding domain is a Fas ectodomain or a functional fragment thereof.

10. The polynucleotide according to any one of claims 1 to 9, wherein the intracellular component is a CD137 (4-1BB) transmembrane domain or a functional fragment thereof.

11. The CD95 (Fas) ectodomain or a functional fragment thereof comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81, or the CD137 (4-1BB) intracellular signaling domain comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 82, the polynucleotide according to any one of claims 1 to 10.

12. The polynucleotide according to any one of claims 1 to 10, wherein the fusion protein comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

80.

13. The polynucleotide according to any one of claims 1 to 10, wherein the nucleic acid sequence encoding the fusion protein comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

83.

14. The polynucleotide according to any one of claims 1 to 13, wherein the CD95 (Fas) ectodomain or a functional fragment thereof comprises at least one of residues R68, F97, K100, R102, R103, L106, F133, H142 of SEQ ID NO:

81.

15. The polynucleotide according to any one of claims 1 to 14, wherein the CD137 (4-1BB) intracellular signaling domain or a part or variant thereof comprises the amino acid sequence of SEQ ID NO:

82.

16. The polynucleotide according to claim 2 or 3, wherein the CD8 coreceptor α or β chain or a part or variant thereof comprises the amino acid sequence of SEQ ID NO: 65 or the amino acid sequence of SEQ ID NO:

66.

17. The polynucleotide according to any one of claims 5 to 16, wherein the neoantigen peptide is a KRAS, HRAS, NRAS, p53, or PIK3CA variant peptide.

18. The polynucleotide according to claim 17, wherein the KRAS variant peptide comprises x-V-G-A-x-G-x-x-K, wherein x represents any amino acid.

19. The polynucleotide according to claim 17 or 18, wherein the KRAS variant peptide is a KRAS G12V variant peptide.

20. The polynucleotide according to claim 19, wherein the KRAS G12V variant peptide comprises the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGA VGVGK (SEQ ID NO: 3).

21. The polynucleotide according to any one of claims 5 to 20, wherein the HLA protein is encoded by HLA-A*11 or the HLA-A*11:01 allele.

22. The polynucleotide according to any one of claims 7 to 21, further comprising a nucleic acid sequence encoding a self-cleaving peptide between a nucleic acid sequence encoding a TCR receptor variable α (Vα) region and a nucleic acid sequence encoding a TCR receptor variable β (Vβ) region.

23. The polynucleotide according to claim 2 or 3, further comprising a nucleic acid sequence encoding a self-cleaving peptide disposed between (a) and (b), or, if (c) is present, between (b) and (c).

24. The polynucleotide according to claim 2 or 3, further comprising a nucleic acid sequence encoding a self-cleaving peptide between the sequence encoding the CD8 co-receptor α chain and the sequence encoding the CD8 co-receptor β chain.

25. The polynucleotide according to any one of claims 2 to 24, further comprising a nucleic acid sequence encoding a self-cleaving peptide disposed between the nucleic acid sequence encoding the binding protein and the nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, and / or between the nucleic acid sequence encoding the binding protein and the nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain.

26. Operably linked in-frame, (iii) (pnBP)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnCD8β)-(pnFP), or (iv) (pnBP)-(pnSCP １ )-(pnCD8β)-(pnSCP ２ )-(pnCD8α)-(pnFP), (iii) (pnBP)-(pnSCP １ )-(pnFP)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnCD8β), or (iv) (pnBP) - (pnSCP １ ) - (pnFP) - (pnSCP １ ) - (pnCD8β) - (pnSCP ２ ) - (pnCD8α) further comprises, pnCD8α is the nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnCD8β is the nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnBP is the nucleic acid sequence encoding the binding protein, pnFP is the nucleic acid sequence encoding the fusion protein, pnSCP １ and pnSCP ２ each independently is a polynucleotide encoding a self-cleaving peptide, and the polynucleotide and / or the encoded self-cleaving peptide are, if necessary, the same or different polynucleotides according to any one of claims 23 to 25.

27. The polynucleotide according to any one of claims 22 to 26, wherein the self-cleaving peptide is P2A, T2A, E2A, or a furin peptide.

28. The polynucleotide according to claim 27, wherein the P2A, T2A, or E2A peptide comprises the amino acid sequence of SEQ ID NO: 74, 75, or 76, respectively.

29. The polynucleotide according to claim 27, wherein the furin peptide comprises the amino acid sequence RAKR.

30. The polynucleotide according to any one of claims 22 to 29, wherein the binding protein and the fusion protein are encoded in a single construct or a continuous genomic segment.

31. The polynucleotide according to any one of claims 2 to 30, wherein the binding protein, the fusion protein, and the CD8α or the CD8β or both are encoded in a single construct or a continuous genomic segment.

32. The polynucleotide according to any one of claims 1 to 31, wherein the binding protein and the fusion protein are encoded in a single open reading frame.

33. The polynucleotide according to any one of claims 1 to 32, wherein the binding protein and the fusion protein are operably linked to a single promoter.

34. The polynucleotide according to any one of claims 1 to 32, wherein the binding protein and the fusion protein are operably linked to different promoters.

35. A vector comprising the polynucleotide according to any one of claims 1 to 34.

36. The vector according to claim 35, wherein the vector is a viral vector.

37. The vector according to claim 36, wherein the viral vector is a lentiviral vector or a γ-retroviral vector.

38. A host cell comprising the polynucleotide according to any one of claims 1 to 34 or the vector according to any one of claims 35 to 37.

39. The host cell according to claim 39, which does not replicate for a period longer than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36, or 48 hours in the absence of exogenous cytokines.

40. The host cell according to claim 38 or 39, wherein the host cell is a hematopoietic progenitor cell or a human immune cell.

41. The host cell according to claim 40, wherein the host cell is a human immune cell, and the human immune cell comprises T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof.

42. The human immune cells include T cells, and the T cells include CD4 ＋ T cells, CD8 ＋ T cells, CD4 － CD8 － The host cell according to claim 41, comprising double-negative T cells, γδ T cells, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof.

43. A method for treating a disease or disorder associated with a KRAS G12V mutation or an NRAS G12V mutation or an HRAS G12V mutation in a subject, the method comprising administering to the subject an effective amount of the host cell according to any one of claims 38 to 42.

44. The method according to claim 43, wherein the disease or disorder comprises cancer.

45. The method according to claim 44, wherein the cancer is a solid cancer or a hematological malignancy.

46. The cancer is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell lung cancer; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid cancer; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial cancer; or papillary thyroid cancer, according to claim 44 or 45.

47. The method according to any one of claims 43 to 46, wherein the effective amount of the host cell is administered to the subject parenterally or intravenously.

48. wherein the effective amount comprises from about 10 ４ cells / kg to about 10 １１ cells / kg, the method according to any one of claims 43 to 47.

49. the effective amount being CD4 ＋ T cells and CD8 ＋ T cells, the method according to any one of claims 43 to 48.

50. The method according to any one of claims 43 to 49, further comprising administering a cytokine to the subject.

51. The method according to claim 50, wherein the cytokine comprises IL-2, IL-15, or IL-21.

52. The method according to any one of claims 43 to 51, wherein the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

53. The method according to any one of claims 43 to 52, wherein the subject has received a myeloablative therapy.

54. The method according to any one of claims 44 to 52, wherein the cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% during the period after administration of the effective amount of the host cell.

55. The method according to claim 54, wherein the period comprises 120 days or less, 60 days or less, 50 days or less, 40 days or less, 30 days or less, or 20 days or less.

56. The method according to any one of claims 43 to 56, further comprising administering at least a second dose.

57. A method of eliciting an immune response against a cell expressing a neoantigen, the method comprising contacting the cell with a cell comprising the polynucleotide according to any one of claims 1 to 35 or the vector according to any one of claims 35 to 37.

58. A method of eliciting an immune response against a cell expressing a neoantigen, the method comprising contacting the cell with the host cell according to any one of claims 38 to 42.

59. The method according to claim 57 or 58, wherein the cell is a cancer cell.

60. The method according to claim 58, wherein the cancer cell is a pancreatic cancer cell, a lung cancer cell, or a colorectal cancer cell.

61. The method according to claim 58, wherein the pancreatic cancer cell is a pancreatic ductal adenocarcinoma cell.

62. The method according to claim 60, wherein the lung cancer cell is a non-small cell lung cancer cell.

63. A method of genetically engineering immune cells, the method comprising contacting the cells with a polynucleotide comprising a nucleic acid sequence encoding a fusion protein comprising a T cell receptor (TCR) or a functional fragment or variant thereof, a CD8α and / or CD8β coreceptor or a functional fragment or variant thereof, and an intracellular component comprising a CD95 (Fas) ectodomain or a functional fragment thereof and a CD137 (4-1BB) intracellular signaling domain, and expanding the immune cells.

64. The method according to claim 63, wherein the polynucleotide is the polynucleotide according to any one of claims 1 to 34, or the vector according to any one of claims 35 to 37.

65. (a) A fusion protein, wherein the fusion protein comprises (i) an extracellular component comprising a CD95 ligand (FasL) binding domain comprising a CD95 (Fas) ectodomain or a functional fragment thereof, and (ii) an intracellular component comprising a CD137 (4-1BB) intracellular signaling domain, and a nucleic acid sequence encoding a binding protein is located upstream of the nucleic acid sequence encoding the fusion polypeptide, the fusion protein; (b) A host cell comprising an exogenous CD8 coreceptor α or β chain or a part or variant thereof.

66. The host cell according to claim 65, wherein the exogenous CD8 coreceptor α or β chain or a part or variant thereof is expressed from a locus other than the natural locus of the CD8 coreceptor α or β chain.

67. The host cell according to claim 65 or 66, wherein the host cell comprises an mRNA encoding an exogenous CD8 coreceptor α or β chain or a part or variant thereof, the mRNA comprising an unnatural 3' or 5' untranslated region (UTR).

68. The host cell according to claim 67, wherein the unnatural 3' or 5' UTR is a viral UTR, an adenoviral UTR, or a lentiviral UTR.

69. The host cell according to any one of claims 65 to 68, wherein the host cell comprises a natural TCR.

70. The host cell according to any one of claims 65 to 69, wherein the fusion protein further encodes a hydrophobic component between the extracellular component and the intracellular component of the fusion protein.

71. The host cell according to any one of claims 65 to 70, wherein the CD95 (Fas) ligand binding domain is a Fas ectodomain or a functional fragment thereof.

72. The host cell according to any one of claims 65 to 71, wherein the intracellular component is a CD137 (4-1BB) transmembrane domain or a functional fragment thereof.

73. The CD95 (Fas) ectodomain or a functional fragment thereof comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 81, or the CD137 (4-1BB) intracellular signaling domain comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

82. The host cell according to any one of claims 65 to 72.

74. The host cell according to any one of claims 65 to 73, wherein the fusion protein comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

80.

75. The host cell according to any one of claims 65 to 74, wherein the CD95 (Fas) ectodomain or a functional fragment thereof comprises at least one of residues R68, F97, K100, R102, R103, L106, F133, H142 of SEQ ID NO:

81.

76. The host cell according to any one of claims 65 to 75, wherein the CD137 (4-1BB) intracellular signaling domain or a part or variant thereof comprises the amino acid sequence of SEQ ID NO:

82.

77. The host cell according to any one of claims 65 to 76, wherein the CD8 coreceptor α or β chain or a part or variant thereof comprises the amino acid sequence of SEQ ID NO: 65 or the amino acid sequence of SEQ ID NO:

66.

78. The host cell according to any one of claims 65 to 77, wherein the host cell further comprises a binding protein comprising an exogenous TCR.

79. The host cell according to claim 78, wherein the binding protein comprises a binding domain that binds to a peptide:HLA complex, and the complex comprises a neoantigen peptide and an HLA protein.

80. The host cell according to claim 79, wherein the neoantigen peptide is a KRAS, HRAS, NRAS, p53, or PIK3CA variant peptide.

81. The host cell according to claim 80, wherein the KRAS variant peptide comprises x-V-G-A-x-G-x-x-K, wherein x represents any amino acid.

82. The host cell according to claim 80 or 81, wherein the neoantigen peptide is a KRAS variant peptide, and the KRAS variant peptide is a KRAS G12V variant peptide.

83. The host cell according to claim 82, wherein the KRAS G12V variant peptide comprises the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGAVGVGK (SEQ ID NO: 3).

84. The host cell according to any one of claims 79 to 83, wherein the HLA protein is encoded by HLA-A*11 or the HLA-A*11:01 allele.

85. The host cell according to any one of claims 65 to 77, wherein the fusion protein and CD8α or CD8β or both are encoded in a single construct or contiguous genomic segment.

86. The host cell according to any one of claims 65 to 78, wherein the fusion protein and CD8α or CD8β or both are all encoded in a single open reading frame.

87. The host cell according to any one of claims 65 to 86, wherein the host cell does not replicate in the absence of an exogenous cytokine for a period longer than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36, or 48 hours.

88. The host cell according to any one of claims 65 to 87, wherein the host cell is a hematopoietic progenitor cell or a human immune cell.

89. The host cell according to claim 88, wherein the host cell is a human immune cell, and the human immune cell comprises T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof.

90. The human immune cell is a T cell, and the T cell is CD4 ＋ T cell, CD8 ＋ T cell, CD4 － CD8 － The host cell according to claim 41, comprising double-negative T cells, γδ T cells, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof.

91. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of the host cell according to any one of claims 65 to 90.

92. The method according to claim 91, wherein the host cell further comprises a TCR targeting an antigen presented by the cancer.

93. wherein the cancer is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung carcinoma; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell lung cancer; anaplastic astrocytoma; chronic myelogenous leukemia; diffuse large B-cell lymphoma; double hit lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic cancer; follicular thyroid cancer; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial cancer; or papillary thyroid cancer, the method according to claim 91 or 92.

94. The method according to any one of claims 91 to 93, wherein the effective amount of the host cells is administered parenterally or intravenously to the subject.

95. wherein the effective amount comprises from about 10 ４ cells / kg to about 10 １１ cells / kg, the method according to any one of claims 91 to 94.

96. wherein the effective amount is CD4 ＋ T cells and CD8 ＋ T cells, and the method according to any one of claims 91 to 95.

97. The method according to any one of claims 91 to 96, further comprising administering a cytokine to the subject.

98. The method according to claim 97, wherein the cytokine comprises IL-2, IL-15, or IL-21.

99. The method according to any one of claims 91 to 98, wherein the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

100. The method according to any one of claims 91 to 99, wherein the subject has received a myeloablative therapy.

101. The method according to any one of claims 91 to 100, wherein the cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% during the period after administration of the effective amount of the host cell.

102. The method according to claim 101, wherein the period includes 120 days or less, 60 days or less, 50 days or less, 40 days or less, 30 days or less, or 20 days or less.

103. The method according to any one of claims 91 to 102, further comprising administering at least a second dose.

104. The method according to any one of claims 91 to 103, wherein the host cell has been verified by any of the methods described in Table 3.

105. A composition comprising a plurality of host cells, wherein the host cells comprise T cells targeting a mutant KRAS peptide, and the composition (a) at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more CD3+ cells stained with a dextramer specific for the mutant KRAS peptide, as evaluated by flow cytometry; (b) at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or more T cells that are CD3 positive, as evaluated by flow cytometry; (c) the composition comprising at least 70%, 75%, 80%, 85%, 90%, or more viable cells, as evaluated by automated cell counting.

106. The composition according to claim 105, comprising the host cell according to any one of claims 38 to 42 or 65 to 90.

107. The composition according to claim 105 or 106, wherein the composition comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more CD3+ cells stained with a dextramer specific for the mutant KRAS G12V peptide, as evaluated by flow cytometry.

108. The composition according to any one of claims 105 to 107, wherein the composition contains at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or more CD3-positive T cells as evaluated by flow cytometry.

109. The composition according to any one of claims 105 to 108, further comprising a pharmaceutically acceptable excipient.

110. A composition comprising the host cell according to any one of claims 38 to 42 or 65 to 90 and a pharmaceutically acceptable excipient.

111. A composition comprising the polynucleotide according to any one of claims 1 to 34 or the vector according to any one of claims 35 to 37 and a pharmaceutically acceptable excipient.

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