Binding proteins and engineered cells specific for neoantigens and uses thereof

Abstract

The present disclosure provides compositions and methods for targeting neoantigens, for example, to treat or prevent cancer. The disclosed embodiments include binding proteins, such as T cell receptors, that bind to the neoantigen: HLA complex. The binding protein further comprises a construct comprising a fusion protein of the CD95 extracellular domain and the CD137 intracellular signaling domain (Fas-41BB), and a CDS coreceptor alpha or beta chain. The disclosed binding proteins are highly sensitive to antigens and are capable of inducing activation of host T cells at low concentrations of peptide antigens. In certain embodiments, binding proteins of the present disclosure (i) have no alloreactivity, substantially no alloreactivity, and / or low risk of alloreactivity, to amino acid sequences from human proteomes and / or (ii) to 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 immunotherapy for the treatment of various cancers.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 344,965, filed on May 23, 2022; U.S. Provisional Application No. 63 / 380,527, filed on October 21, 2022; and U.S. Provisional Application No. 63 / 501,973, filed on May 12, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Development of T cell-based immunotherapy began when tumor-reactive T cells were discovered in a population of tumor-infiltrating lymphocytes (TILs). A strategy called donor T cell transfer involves, in some cases, isolation of tumor-infiltrating lymphocytes pre-selected for tumor reactivity, clonal expansion of tumor-reactive T cells induced by anti-CD3 and anti-CD28 antibodies in the presence of IL-2, and infusion of the expanded cell population back into the tumor-bearing patient. The isolation of tumor-reactive T cell clones has led to the development of another immunotherapy approach—the production of recombinant T cell receptors (TCRs) specific for specific antigens, 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 a major histocompatibility complex (MHC) molecule (called a human leukocyte antigen (HLA) molecule in humans) expressed on tumor cells. Summary of the invention

[0004] In some aspects, the present disclosure provides: a polynucleotide comprising a nucleic acid sequence encoding the following: (a) a binding protein, wherein the binding protein comprises: (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, wherein the fusion protein comprises: (i) an extracellular component comprising a CD95 ligand (FasL) binding domain, the CD95 ligand binding domain comprising a CD95 (Fas) extracellular domain 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 a nucleic acid sequence encoding the following: (c) a CD8 co-receptor α or β chain or a portion or variant thereof, wherein the sequence encoding the binding protein is located upstream of the sequence encoding the extracellular portion of the CD8 co-receptor α or β chain or a portion or variant thereof. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding the following: (c) CD8 co-receptor α and β chains or portions or variants thereof, wherein the sequence encoding the binding protein is located upstream of the sequence encoding the extracellular portion of the CD8 co-receptor α and β chains or portions or variants thereof. In some embodiments, the nucleic acid sequence encoding the fusion protein further encodes: a hydrophobic component between the extracellular component and the intracellular component of the fusion protein. In some embodiments, the binding protein comprises a binding domain that binds to a peptide:HLA complex, wherein the complex comprises a neoantigenic 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 extracellular domain 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) extracellular domain 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: 81. NO:82 has 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. 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) extracellular domain 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 coreceptor 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 neoantigenic peptide is a KRAS, HRAS, NRAS, p53 or PIK3CA mutant peptide. In some embodiments, the KRAS mutant peptide comprises xVGAxGxxK, wherein x represents any amino acid. In some embodiments, the KRAS mutant peptide is a KRAS G12V mutant peptide.In some embodiments, the KRAS G12V mutant 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 an 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, the self-cleaving peptide being between a nucleic acid sequence encoding a variable α (Vα) region of a TCR receptor and a nucleic acid sequence encoding a variable β (Vβ) region of a TCR receptor. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide, the self-cleaving peptide being disposed between (a) and (b) or disposed between (b) and (c) in the presence of (c). In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide, the nucleic acid sequence being between a sequence encoding a CD8 co-receptor α chain and a sequence encoding a CD8 co-receptor β chain. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide, the nucleic acid sequence being 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 coreceptor α chain; and / or 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 coreceptor β chain. In some embodiments, the polynucleotide further comprises the following operably linked in frame: (i) (pnBP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β)-(pnFP); (ii) (pnBP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α)-(pnFP); (iii) (pnBP)-(pnSCP1)-(pnFP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β); or (iv) (pnBP)-(pnSCP 1)-(pnFP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α); wherein pnCD8α is a nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, wherein pnCD8β is a nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, wherein pnBP is a nucleic acid sequence encoding a binding protein, wherein pnFP is a nucleic acid sequence encoding a fusion protein, and wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotide and / or the encoded self-cleaving peptide are optionally the same or different. 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 certain embodiments, the furin peptide comprises the amino acid sequence RAKR. In certain embodiments, the associated protein and the fusion protein are encoded in a single construct or a continuous genome segment. In certain embodiments, the associated protein, the fusion protein and CD8 α or CD8 β or both are encoded in a single construct or a continuous genome segment. In certain embodiments, the associated protein and the fusion protein are encoded in a single open reading frame. In certain embodiments, the associated protein and the fusion protein are operably connected to a single promoter. In certain embodiments, the associated protein and the fusion protein are operably connected to different promoters.

[0005] 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 gamma-retroviral vector.

[0006] In some aspects, the disclosure provides host cells comprising any polynucleotide or any vector described herein. In some embodiments, the host cell replicates for no more than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36, or 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 a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof. In some embodiments, the human immune cell comprises a T cell, and the T cell comprises a 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.

[0007] In some aspects, the present disclosure provides a method for treating a disease or condition associated with a KRAS G12V mutation or a NRAS G12V mutation or a HRAS G12V mutation in an individual, the method comprising administering to the individual an effective amount of any host cell described herein. In some embodiments, the disease or condition comprises 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; gallbladder cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally a myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma, such as non-Hodgkin's lymphoma (Non-Hodgkin lymphoma); chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestinal 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; unclassifiable myelodysplastic / myeloproliferative neoplasm; 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 carcinoma; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma. In some embodiments, an effective amount of host cells is administered parenterally or intravenously to an individual. In some embodiments, the effective amount comprises about 10 4 cells / kg to about 10 11 In some embodiments, the effective amount comprises CD4 + T cells and CD8 + In some embodiments, an effective amount comprises a large number of CD4 + T cells and CD8 +T cells. In some embodiments, the method further comprises administering a cytokine to the individual. In some embodiments, the cytokine comprises IL-2, IL-15, or IL-21. In some embodiments, the individual has received or is receiving an agonist of an immune checkpoint inhibitor and / or immune checkpoint stimulator. In some embodiments, the individual has received bone marrow ablation therapy. In some embodiments, within a period of time after administering an effective amount of host cells, the cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the time period comprises less than or equal to 120 days, less than or equal to 60 days, less than or equal to 50 days, less than or equal to 40 days, less than or equal to 30 days, or less than or equal to 20 days. In some embodiments, the method further comprises administering at least a second dose.

[0008] In some aspects, the present disclosure provides a method of eliciting an immune response against a cell expressing a neo-antigen, the method comprising contacting the cell with a cell comprising any of the polynucleotides or vectors described herein.

[0009] 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 host cell 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.

[0010] In some aspects, the present disclosure provides a method for genetically engineering immune cells, the method comprising contacting the cells with polynucleotides, the polynucleotides comprising a fusion protein nucleic acid sequence encoding a T cell receptor (TCR) or a functional fragment or variant thereof, a CD8α and / or CD8β co-receptor or a functional fragment or variant thereof, and a fusion protein comprising an extracellular domain of CD95 (Fas) or a functional fragment thereof and an intracellular component comprising an intracellular signaling domain of CD137 (4-1BB), and amplifying the immune cells. In some embodiments, the polynucleotide is any polynucleotide or any vector described herein.

[0011] In some aspects, 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, the CD95 ligand binding domain comprising a CD95 (Fas) extracellular domain 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; and (b) an exogenous CD8 co-receptor α or β chain or a portion or variant thereof. In some embodiments, the exogenous CD8 co-receptor α or β chain or a portion or variant thereof is expressed from a locus other than the native locus of the CD8 co-receptor α or β chain. In some embodiments, the host cell comprises an mRNA encoding an exogenous CD8 co-receptor α or β chain or a portion or variant thereof comprising a non-native 3' or 5' untranslated region (UTR). In some cases, the sequence encoding the exogenous CD8 co-receptor α or β chain or a portion 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 adenovirus UTR or a lentivirus UTR. In some embodiments, the host cell comprises a native TCR. An exogenous CD8 co-receptor alpha or beta chain or a portion or variant thereof. The fusion protein further encodes a hydrophobic component between the extracellular component and the intracellular component of the fusion protein. In some embodiments, the CD95 (Fas) ligand binding domain is a Fas extracellular domain 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) extracellular domain 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: 81. NO:82 has 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.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) extracellular domain 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 coreceptor 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, wherein the complex comprises a new antigenic peptide and an HLA protein. In some embodiments, the new antigenic peptide is a KRAS, HRAS, NRAS, p53 or PIK3CA mutant peptide. In some embodiments, the KRAS mutant peptide comprises xVGAxGxxK, wherein x represents any amino acid. In some embodiments, the new antigenic peptide is a KRAS mutant peptide, wherein the KRAS mutant peptide is a KRAS G12V mutant peptide. In some embodiments, the KRAS G12V mutant 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 an 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 continuous genomic segment. In some embodiments, the fusion protein and CD8α or CD8β or both are encoded in a single open reading frame. In some embodiments, the host cell replicates for no more than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36 or 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, wherein the human immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte or any combination thereof. In some embodiments, the human immune cell is a T cell, wherein the T cell comprises 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.

[0012] In some aspects, the present disclosure provides a method for treating cancer in an individual, the method comprising administering to the individual an effective amount of any host cell described herein. In some embodiments, the host cell further comprises a TCR for 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; gallbladder 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's lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally skin melanoma, anal melanoma or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytes and dendritic cells 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 tumors; mantle cell lymphoma; unclassifiable myelodysplastic / myeloproliferative neoplasms; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdomyosarcoma; neurothecoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; malignant thyroid neoplasms; thyroid cancer; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma. In some embodiments, an effective amount of host cells is administered parenterally or intravenously to an individual. In some embodiments, an effective amount comprises about 10 4 cells / kg to about 10 11 In some embodiments, the effective amount comprises CD4 + T cells and CD8 +T cells. In some embodiments, the method further comprises administering a cytokine to the individual. In some embodiments, the cytokine comprises IL-2, IL-15 or IL-21. In some embodiments, the individual has received or is receiving an agonist of an immune checkpoint inhibitor and / or immune checkpoint stimulator. In some embodiments, the individual has received bone marrow ablation therapy. In some embodiments, within a period of time after administering an effective amount of host cells, cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the time period comprises less than or equal to 120 days, less than or equal to 60 days, less than or equal to 50 days, less than or equal to 40 days, less than or equal to 30 days, or less than or equal to 20 days. In some embodiments, the method further comprises administering at least a second dose. In some embodiments, the host cell has been verified by any one of the methods described in Table 3.

[0013] In some aspects, the present disclosure provides a composition comprising a plurality of host cells, wherein the host cells comprise T cells specific for or against a new antigen (e.g., a mutant KRAS peptide), wherein the composition: (a) 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 peptide as assessed by flow cytometry; (b) comprises at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or more CD3-positive T cells as assessed by flow cytometry; (c) comprises at least 70%, 75%, 80%, 85%, 90% or more viable cells as assessed by automated cell counting. In some embodiments, the host cell is any one 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 assessed by flow cytometry. In some embodiments, the composition comprises at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or more CD3-positive T cells as assessed by flow cytometry. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0014] In some aspects, the disclosure provides any one of the host cells or any one of the vectors described herein and a pharmaceutically acceptable excipient.

[0015] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. It should be appreciated that the present disclosure is capable of other and different embodiments, and that several details thereof are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and descriptions are to be regarded as illustrative rather than restrictive in nature.

[0016] Incorporated by Reference

[0017] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference 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

[0018] The novel features of the present invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description of an illustrative embodiment and its accompanying drawings which illustrate the principles of the present invention:

[0019] Figures (FIG.) 1A, 1B, 1C, 1D and 1E relate to identification of KRAS G12V-specific T cell receptors (TCRs) from T cell lineages from healthy human donors. Figure 1A ) (Left) Schematic showing the method for identifying a T cell line specific for mutant KRAS (mKRAS) restricted to HLA-A11 from a donor sample and (right) TNFα production by CD8+ T cells expressing mKRAS-specific TCR in the absence (left) or presence (right) of mKRAS G12V peptide. ( Figure 1B ) (Top) Schematic diagram of the method for sorting and sequencing of mKRAS-reactive CD8+ T cells and (Bottom) engineering CD8+ T cells to heterologously express mKRAS-specific TCRs. Fifty-six mKRAS-specific TCRs (G12V-specific or G12D-specific) were isolated and sensitivity and cytotoxicity analyses were performed. ( Figure 1C )Fold enrichment of T cell clones in vitro in the presence and absence of KRAS G12V mutant peptide. ( Figure 1D ) Activation of TCR-transduced T cells in vitro in the presence of different concentrations of KRASG12V mutant peptide under the control of the Nur77 locus, as assessed by the percentage of T cells expressing GFP. T cells were transduced to express TCRs as indicated in the figure legend. ( Figure 1E )Log EC50 KRAS G12V 9-mer peptide value (representing the concentration of KRAS G12V peptide required for TCR-transduced T cells to generate a half-maximal response of their Nur77 expression).

[0020] FIG. 2A, 2B and 2C show the functional affinity of TCR 11NA4 (see Table 1) compared to TCR 220_21 (V domain amino acid sequences shown in SEQ ID NO: 61 (Vα) and 62 (Vβ)) and TCR "BNT" (Vα domain amino acid sequence shown in SEQ ID NO: 60 (with signal peptide); Vβ domain amino acid sequence shown in SEQ ID NO: 59 (with signal peptide)). Figure 2A ) The percentage of primary TCR-transduced CD8+ T cells expressing CD137 at the indicated concentrations of KRAS G12V peptide; ( Figure 2B ) log EC50 of TCR against KRAS G12V peptide; ( Figure 2C ) T cell activation, measured by the percentage of TCR-transduced primary CD8+ T cells expressing CD137 in the presence of the indicated concentrations of KRASG12V peptide. ( Figure 2D ) log EC50 of TCR against KRAS G12V after exposure to 9-mer and 10-mer peptides; ( Figure 2E ) T cell activation, measured by the percentage of primary TCR-transduced CD8+ T cells expressing CD137 after exposure to the indicated KRAS G12 peptides. Figure 2F ) Percentage of TCR-transduced primary CD8+ T cells expressing IFN-γ at the indicated concentrations of KRASG12V peptide.

[0021] Figures (FIG.) 3A and 3B show the activation of TCR-transduced T cells co-cultured with tumor cell lines expressing HLA-A11+KRAS G12V (assessed by the percentage of TCR-transduced T cells expressing CD137). ( Figure 3A ) shows activation of T cells expressing one of four different TCRs in multiple cell lines and in the presence of a KRAS peptide comprising the G12V mutation. "UT" = untransduced negative control. ( Figure 3B ) shows superior activation of T cells expressing TCR 11N4A relative to other TCRs. "UNTR" - untransduced negative control.

[0022] Figures (FIG.) 4A and 4B relate to CD8+ T cells expressing KRAS G12V-specific TCRs specifically killing tumor cell lines expressing HLA-A11+KRAS G12V in the Incuyte killing assay. In this assay, the red target area indicates the presence of tumor cells. ( Figure 4A ) Growth curves of mKRAS+ / HLA-A11+ tumor cells in killing assay. Test conditions were tumor cells alone, tumor cells + T cells transduced to express TCR 11N4A, and tumor cells transduced to express comparison TCR 220_21. Red target area on y axis shows tumor cell growth. Additional tumor cells were added at 72 hours. ( Figure 4B ) Data from another killing assay in which T cells and SW480 tumor cell line were co-cultured at the indicated effector:target ratios.

[0023] Figures (FIG.) 5A, 5B, and 5C relate to mutagenesis scanning experiments using KRAS G12 9-mer and 10-mer peptides to characterize the peptide binding motif of TCR 11N4A. Figure 5A ) The percentage of TCR-transduced T cells expressing Nur77-GFP in the presence of G12V peptide or G12V peptide variants with amino acids substituted with alanine, glycine, or threonine at the indicated positions, as indicated. Left: Mutation scanning results of KRAS G12 9-mer peptide. Right: Mutation scanning results of KRAS G12 10-mer peptide. ( Figure 5B ) Percentage of TCR 11N4A-transduced CD8+ T cells expressing the activation marker Nur77 (linked to a reporter gene) in the presence of the indicated 9-mer peptides. ( Figure 5C ) Results of searching the human proteome using ScanProsite (prosite.expasy.org / scanprosite / ) using the following search string: xVGAxGxxK (SEQ ID NO: 4). Peptides from the human proteome were scored for predicted binding to HLA-A11.

[0024] Figures (FIG.) 6A, 6B, 6C, 6D, 6E, 6F, 6G and 6H show that TCR 11N4A has a low risk of autoreactivity in humans. XScan analysis predicted a single peptide RAB7B with potential off-target reactivity in the genome. However, the RAB7B peptide failed to stimulate transduced CD4 / CD8 T cells at physiological concentrations, indicating a lack of autoreactivity. ( Fig. 6A , Figure 6B ) Reactivity of T cells transduced with TCR 11N4A to a panel of potentially cross-reactive peptides (see Figure 5B ). Figure 6C ) Peptide dose-response curves for cells transduced to express TCR 11N4A and exposed to KRAS G12V or RAB7B peptides and ( Fig.6D ) Calculated negative log EC50 of TCR 11NA4-transduced T cells against RAB7B peptide relative to the homologous KRAS G12V peptide. ( Fig. 6E ) Percentage of CD8+ T cells transduced with TCR 11N4A expressing CD137 in response to overnight incubation with a comprehensive positional scanning set of peptides containing substitutions of every possible amino acid at each position of the homologous KRAS G12V peptide (172 peptides). Peptides that elicited greater than 15% responses were considered positive in this analysis. ( Fig. 6F ) According to Fig. 6E Potential cross-reactive motifs identified by searching ScanProsite for potential cross-reactive peptides. Figure 6G ) CD137 expression (determined by flow cytometry) of sort-purified primary CD8+ T cells transduced to express TCR 11N4A or TCR 11N4A+CD8αβ and cultured overnight with 100 ng / ml of potentially cross-reactive peptides. ( Figure 6H ) is similar to Figure 6C Results shown in , CD8+ T cells lentivirally transduced with A11G12VTCR, CD8α / CD8β and FAS-41BB fusion protein were not stimulated after incubation with titrated RAB7B peptide (bottom line) and were stimulated after incubation with titrated KRAS mutant G12V peptide (top line).

[0025] Figures (FIG.) 7A and 7B relate to evaluating the potential alloreactivity of TCR 11N4A. Fig. 7A ) B lymphoblastoid cell lines (B-LCL) expressing different HLA alleles were cultured with CD8+ T cells transduced with TCR 11N4A and the responsiveness of T cells was assessed as measured by the expression of IFN-γ or CD137. ( Figure 7B ) Alloreactivity screening results: Percentage of CD137+ T cells transduced with TCR 11N4A against B-LCL expressing common HLA alleles in the presence (upper panel) or absence (lower panel) of CD8αβ.

[0026] FIG. 8 shows the killing activity of CD8+ and CD4+ T cells engineered to express TCR 11N4A and CD8αβ coreceptor (eg, exogenous CD8αβ coreceptor) against mKRAS:HLA-A11+ tumor cells.

[0027] Figures (FIG.) 9A, 9B, 9C, 9D, 9E, 9F, 9G and 9H show the nucleotides associated with TCR 11N4A ( Figure 9A-9E ) and amino acids ( Figure 9F-9H ) sequences and expression constructs encoding or comprising the same.

[0028] FIG. 10A, 10B, 10C, 10D, 10E and 10F show nucleotides associated with TCR 11N6 ( Figure 10A-10C ) and amino acids ( Figure 10D-10F ) sequences and expression constructs encoding or comprising the same.

[0029] It should be understood that Figures 9A-10F Not all sequences shown contain every sequence feature indicated in the legend. In the legend, CDR3 sequences are shown according to the IMGT junction definition.

[0030] Figure (FIG.) 11 proves that cells transduced by a single lentiviral construct carrying TCR 11N4A, CD8αβ co-receptors and FAS / 41BB fusions successfully express all three markers.Show the engineered TCR expression (G12V tetramer, top), FAS-41BB fusion protein (FAS, middle) and exogenous CD8 (CD8 is gated via CD4+, bottom) in the primary generation human CD4 / CD8T cells without transduction (left) or engineered to express A11G12V TCR+CD8αb+FAS-41BB (right) Representative flow cytometry diagram.Intracellular 2A staining (x-axis) identifies transduced cells via 2A elements that distinguish individual parameters in lentiviral constructs.CD8 analysis includes only CD4+T cells, therefore excluding endogenous CD8+T cells. T cells were activated with anti-CD3 / CD28 beads for 2 days, lentivirally transduced, and analyzed by flow cytometry after 3 days of expansion.

[0031] Figures (FIG.) 12A and 12B demonstrate that cells transduced with TCR 11N4A, CD8α / CD8β coreceptor, and FAS-41BB fusion protein are reactive to endogenous KRAS mutant peptides presented by class I MHC. Fig. 12A ) Bar graph showing CD137 expression on transduced CD4 T cells co-cultured with A11KRAS G12V mutant cell line. ( Fig. 12B ) shows a bar graph of CD137 expression on transduced CD8 T cells co-cultured with A11KRAS G12V mutant cell lines. Cell lines include SW527, SW620, CFPAC1, COR-L23, DAN-G, and NCI-H441 expressing HLA-A*11:01 and endogenous KRAS mutant G12V. The induced CD137 expression demonstrates responsiveness to endogenous KRAS mutant peptides presented by class I MHC.

[0032] Figure (FIG.) 13 confirms that FAS-41BB fusion protein improves the sensitivity of restimulated T cells to T cells engineered by KRAS. In this experiment, TCR 11N4A, CD8α and CD8β coreceptors for KRAS and FAS / 41BB fusion proteins (together with indicated controls) according to SEQ ID NO:80 are treated with increasing G12V peptide concentrations to stimulate TCR and assess the percentage of cells stimulated to express CD137 receptors. The extent of the stimulation response of G12V peptides is effectively increased by including FAS-41BB fusion proteins.

[0033] FIG. 14A-14E demonstrate that FAS-41BB fusion protein improves tumor killing of KRAS-engineered T cells in vitro (eg, in a cell line expressing Fas ligand). Fig.14A Shown is the confluence of SW527 after co-culture with untransduced T cells, primary CD4 and CD8 T cells transduced with TCRKRASG12V(11N4A)+CD8α / β coreceptors, or TCRKRASG12V, CD8α / β, and FAS-41BB at a 5:1 or 2:1 effector:target cell ratio. Fig. 14B is a graph summarizing the results of experiments in which untransduced T cells (UTD), T cells from donor 1 transduced with TCRKRASG12V+CD8α / β coreceptors, or T cells transduced with TCRKRASG12V, CD8α / β, and FAS-41BB were compared with 1×10 4 HLA-A*11:01SW620 tumor cells overexpressing FASLG and NucLight red fluorescent protein were co-cultured at a 5:1 effector cell:target cell ratio for up to 8 days. The cultures were restimulated with an equal number of tumor cells approximately every 72 hours to simulate chronic antigen stimulation (▲). Fig. 14C Results of the same experiment using T cells from different donors are shown. Fig.14D Shown are the results of the same experiment using T cells from donor 1 and co-culturing these cells with COR-L23 tumor cells. Fig.14E Demonstration of using T cells from different donors Fig.14D Results from the same experiment in . Two different donors were tested within the same study. Tumor confluence, measured by total NucLight Red target area, was reported as a measure of tumor cell growth / viability throughout the study.

[0034] Figure (FIG.) 15A confirms that FAS-41BB fusion protein improves the expansion of cells carrying KRAS TCR in in vitro re-attack analysis. The following process is shown in the left figure of the figure, wherein TCR 11N4A, CD8αβ coreceptor and FAS / 41BB fusion protein (together with the indicated control) for KRAS are included: 80 T cells and SW527 cells are co-cultured for 3-4 days, then counted and transferred to a fresh cell culture plate of SW527 cells; As indicated, the fresh culture plate of SW527 cells is repeatedly transferred. The right figure shows the figure of the transferred T cells amplified over time. As can be seen in the right figure, including FAS-41BB fusion protein and KRAS TCR can improve the replication of cells carrying KRAS TCR.

[0035] FIG. 15B demonstrates that expansion of cells harboring KRAS TCR, CD8α / CD8β, and FAS-41BB fusion proteins in an in vitro rechallenge assay is limited to cells containing CD4 + and CD8 + Figure 2 shows the cumulative expansion fold of CD4+ (triangles; middle line), CD8+ (squares; 2nd line from the bottom line), CD4+ / CD8+ mixture (circles; top line) or non-transduced corresponding control (bottom line) primary T cells co-cultured with SW527 cell line expressing HLA-A*11:01 and endogenous KRAS mutation G12V.

[0036] FIG. 15C shows TCR-engineered cells or untransduced donor T cells (UTD) from two different healthy donors (D1, D2) that were incubated with 1×10 4 Various HLA-A*11:01+KRASG12V+ tumor cells were co-cultured at a 5:1 effector cell: target cell ratio for 7 days, during which fresh tumor cells were added to the co-culture twice to restimulate T cells. On day 7, T cell proliferation was measured by flow cytometry propidium iodide (PI) staining of CD4+ and CD8+ T cells. PI negative T cell counts were plotted as viable lymphocyte counts / μL.

[0037] FIG. 16A demonstrates that the FAS-41BB fusion protein improves the efficacy of cells carrying the KRAS TCR in an in vivo xenograft tumor model with SW527 cells. In this experiment, T cells containing the TCR 11N4A for KRAS, a CD8αβ coreceptor (e.g., an exogenous CD8αβ coreceptor) and a FAS / 41BB fusion protein according to SEQ ID NO: 80 (together with the indicated controls) were cultured at 10×10 6A dose of 100 T cells was intravenously administered to immunodeficient mice bearing subcutaneous SW527 tumors and tumor volume was measured over time. As can be seen, including the Fas / 41BB fusion protein with the KRAS TCR enhanced the killing of SW527 tumors in vivo over cells lacking the Fas / 41BB fusion protein.

[0038] FIG. 16B demonstrates that tumor-bearing mice administered cells transduced with TCR 11N4A, CD8αβ coreceptor (e.g., exogenous CD8αβ coreceptor), and FAS / 41BB fusion protein have superior in vivo survival compared to cells transduced with TCR 11N4A and CD8αβ coreceptor (e.g., exogenous CD8αβ coreceptor) without FAS / 41BB fusion.

[0039] FIG. 16C demonstrates that complete responses were achieved in some mice inoculated subcutaneously with SW527 tumor cells, compared to mice that received approximately 1×10 7 A single intravenous administration of primary CD4 / CD8 T cells lentivirally transduced with A11G12V TCR, CD8αβ coreceptor (eg, exogenous CD8αβ coreceptor), and FAS-41BB (bottom line).

[0040] Figure (FIG.) 16D confirms that tumor-bearing mice using cells transduced by TCR 11N4A, CD8αβ co-receptors (e.g., exogenous CD8αβ co-receptors) and FAS / 41BB fusion proteins show enhanced survival rates relative to mice using untransduced cells. Kaplan-Meier survival curves of tumor-bearing mice after applying engineered CD4 / CD8T cells are shown. The survival rate of mice with SW527 xenografts expressing HLA-A*11:01 and endogenous KRAS mutation G12V is shown. Lines depict tumor-bearing mice that receive primary CD4 / CD8T cells that are not transduced (gray) or transduced with A11G12V TCR, CD8αβ co-receptors (e.g., exogenous CD8αβ co-receptors) and FAS-41BB (top flat line) without transduction (gray). Cells are amplified for 7 days with anti-CD3 / CD28 beads after transduction. To start the experiment, tumors were grown to approximately 100 mm 3 10 × 10 6 The T cells were cryopreserved and thawed prior to administration.

[0041] Figures 17A-17D demonstrate that cells carrying KRAS TCR, CD8α / CD8β, and FAS-41BB fusion proteins contain CD4+ and CD8 + Both cells showed improved anti-tumor activity. Fig.17A It is a confluence of SW527 tumor cell lines expressing red fluorescent protein, HLA-A*11:01 and endogenous KRAS mutation G12V, monitored in live tumor visual analysis, and quantified red fluorescent signals over time. Cultures contain SW527 monocultures (gray) or co-culture with untransduced CD4+ / CD8+ mixed T cells (black) or CD4+ (red), CD8+ (blue) or CD4+ / CD8+ mixed (green) T cells transduced with A11G12V TCR+CD8αβ+FAS-41BB lentivirus. Primary T cells were activated with anti-CD3 / CD28 beads, expanded for 5 days after transduction, co-cultured with SW527 cells at an initial ratio of 0.5:1, and additional fresh SW527 cells were added to the culture every 3 days (indicated by arrows). Fig. 17B The summary is as follows Fig.17A A graph showing the results of the same experiment performed in SW620 cells. Fig. 17C The summary is as follows Fig.17A Figure 3 is a graph of the results of the same experiment performed in , but performed in CFPAC1 cells. Fig.17D The summary is as follows Fig.17A A graph showing the results of the same experiment performed in COR-L23 cells.

[0042] Figure (FIG.) 18 confirms that cells transduced by TCR 11N4A, CD8αβ co-receptors (such as exogenous CD8αβ co-receptors) and FAS / 41BB fusion proteins cannot proliferate in the absence of exogenous cytokine support, thereby enhancing its safety profile.Show the persistence (measured by cell counting) of CD4+ / CD8+T cells monitored by quantitative cells every 2-4 days in the absence of exogenous cytokines.Show the primary T cells without transduction (grey line; Top line) or transduction (bottom line) of A11G12V TCR+CD8αb+FAS-41BB, which have been amplified 7-10 days with anti-CD3 / CD28 beads in the culture medium containing IL2 / IL7 / IL15 and transferred to the culture medium without cytokines.Supplement half of the culture medium (without cytokines) every 2-4 days.

[0043] FIG. 19 depicts several lentiviral vector designs comprising anti-KRAS TCR, FAS-41BB fusion protein and CD8α / CD8β. Most designs contemplate the expression of anti-KRAS TCR ("TCRb" or "TCRa"), CD8α / CD8β ("CD8a" or "CD8b") and FAS-41BB ("FasBB") under a single promoter ("MSCV" or murine stem cell virus promoter) on a single translating RNA, wherein the regions encoding the independent polypeptides are separated by in-frame sequences encoding self-cleaving peptides ("P2A", "T2A", "F-P2A"). Some (22992-8, 22992-9) involve the expression of Fas-41BB under an independent promoter ("PGK" or phosphoglycerate kinase promoter).

[0044] FIG. 20 demonstrates that T cells produced by a manufacturing strategy involving a single vector comprising an anti-KRAS TCR, a FAS-41BB fusion protein, and CD8α / CD8β exhibit superior TCR expression and surface activity relative to cells produced by a strategy involving anti-KRAS TCR and FAS-41BB fusion proteins on separate vectors. Fig. 20A Demonstrating alternative designs of lentiviral vectors. Fig. 20B Shown is FACS analysis of lentiviral vector-transduced T cells generated as previously described. Fig. 20C The percentage of cells expressing a cistron containing an anti-KRAS TCR ("2A+%"), the percentage of cells expressing a functional TCR and a cistron containing an anti-KRAS TCR ("Tet+2A+%"), total functional TCR expression ("Tet MFI"), FAS-41BB fusion protein expression ("Fas MFI"), and CD8α / CD8β coreceptor expression by CD4+ cells ("CD8 MFI below CD4+") are shown. FACS analysis showed that a single lentiviral strategy ("22992-4") was superior to a dual lentiviral strategy ("2 lentiviruses") for both TCR and CD8 expression.

[0045] FIG. 21A shows the activation of T cells generated by a manufacturing strategy involving a single vector comprising anti-KRAS TCR, FAS-41BB fusion protein and CD8α / CD8β or a dual vector system.

[0046] FIG. 21B shows the cytocidal activity of these cells against various tumor cell lines when administered as fresh TCR-T cells or after thawing.

[0047] FIG. 22A shows long-term re-stimulation of T cells and tumor cell killing generated by a manufacturing strategy involving a single vector comprising anti-KRAS G12V TCR, FAS-41BB fusion protein and CD8α / CD8β or a dual vector system.

[0048] FIG. 22B shows the changes in tumor cell volume after administration of these cells in an in vivo xenograft model.

[0049] FIG. 22C shows changes in tumor cell volume after administration of cells containing anti-KRAS G12D TCR, FAS-41BB fusion protein, and CD8α / CD8β in an in vivo xenograft model. DETAILED DESCRIPTION

[0050] Effective T cell activation generally requires simultaneous co-stimulatory signals or is enhanced by simultaneous co-stimulatory signals. In the tumor microenvironment, co-stimulatory molecules are generally downregulated. Therefore, it is necessary to configure cells for acceptability T cell therapy, which offset this downregulation of co-stimulatory molecules or generally enhance the effect of receptors of targeted antigens on such T cells in the tumor microenvironment. In addition, the tumor microenvironment may include heterogeneous cell types (e.g., stromal cells, endothelial cells, and tumor-associated macrophages, granulocytes, and inflammatory monocytes), which promote T cell inhibition via direct contact and secretion of soluble inhibitory factors.

[0051] Some aspects of the present disclosure generally relate to cells (e.g., immune effector cells, such as CD4+ and / or CD8+ T cells) expressing: 1) an exogenous binding protein that binds to a neoantigenic 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 neoantigenic 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).

[0052] Some aspects of the present disclosure generally relate to converting T cell inhibitory signals in tumor microenvironments into fusion proteins (e.g., fusion receptors or "switch" receptors) of T cell activation or proliferation signals. Therefore, some aspects of the present disclosure relate to fusion proteins, which include an extracellular domain connected to an intracellular domain, the extracellular domain is specific to a soluble or cell-anchored inhibitory ligand, and the intracellular domain contributes to T cell activation (e.g., 4-1BB intracellular signaling domain or CD28 intracellular signaling domain). In some cases, such proteins include an extracellular domain derived from a Fas receptor and an intracellular domain derived from a 4-1BB receptor (e.g., Fas-41BB fusion protein).

[0053] Without wishing to be bound by theory, such Fas-41BB fusion proteins can activate NF-κB, increase Bcl-2 expression and activate PI3K and MEK-1 / 2 signal transduction pathways via Fas ligand (FASLG) in response to the tumor microenvironment, inhibit T cell apoptosis, enhance IL-2 or IFN-γ secretion, promote memory T cell development, increase T cell metabolic capacity, and / or improve T cell proliferation, persistence and fitness. Alternatively or in addition, such Fas-41BB fusion proteins can work in a dominant negative manner, or isolate the expression of Fas ligands by stimulated T cells in tumors, endothelial cells, and tumor microenvironments, preventing the elimination of T cells or apoptosis during tumor infiltration. It has been shown that Fas ligands are expressed in the tumor microenvironment of many solid tumors, and it is considered that the presence of Fas ligands in the microenvironment of solid tumors can cause the limited efficacy of T cell acceptance cell therapy.

[0054] Some aspects of the present disclosure generally relate to binding proteins specific for Ras neoantigens, modified immune cells expressing the same, polynucleotides encoding the binding proteins, and related uses. Mutant Ras proteins (e.g., KRAS, NRAS, HRAS) can produce neoantigens, including a G→V mutation at position 12 of the full-length KRAS protein (SEQ ID NO: 1; UniProt KB P01116) or 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).

[0055] Some aspects of the present disclosure generally relate to binding proteins specific for p53 neoantigens, modified immune cells expressing the same, polynucleotides encoding binding proteins, and related uses. Mutant p53 proteins can potentially produce 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)).

[0056] 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. Mutated p53 proteins can potentially produce 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 about 70%-80% of PIK3CA mutations, and mutations in PIK3CA activity have been found in many human cancers (Ligresti et al., Cell Cycle, 8(9): 1352-58 (2009)).

[0057] In the present disclosure, a binding protein capable of binding to a new antigen is provided. In certain aspects, a binding protein comprising a TCR Vα domain and a TCR Vβ domain (and a host cell, such as an immune cell, comprising a heterologous polynucleotide encoding a binding protein of the present disclosure) is provided, wherein the binding protein is capable of binding to a new antigenic peptide: HLA complex.

[0058] For example, in the present disclosure, a binding protein capable of binding to a Ras neoantigen is provided. In certain aspects, a binding protein comprising a TCR Vα domain and a TCR Vβ domain (and a host cell, such as an immune cell, comprising a heterologous polynucleotide encoding a Ras-specific binding protein of the present disclosure) is provided, wherein the binding protein is capable of binding to a Ras peptide antigen:HLA complex, wherein the Ras peptide antigen comprises, is essentially composed of, or is composed of an amino acid sequence as shown in any one of SEQ ID NO: 2 or 3. In certain embodiments, HLA comprises HLA-A*11, such as HLA-A*11:01.

[0059] The disclosed binding proteins are highly sensitive to antigens, thereby being able to induce activation of host T cells at low concentrations of peptide antigens. In certain embodiments of (e.g., CD8+ and / or CD4+) T cell populations or samples expressing binding proteins, T cells have half-maximal expression of activation marker Nur77 in the presence of [LogEC50 is less than -9M (e.g., between -9M and -10M)] peptides. In certain embodiments of (e.g., CD8+ and / or CD4+) T cell populations or samples expressing binding proteins, T cells have half-maximal expression of CD137 in the presence of [LogEC50 is less than -10M (e.g., between -10M and -11M)]. In certain embodiments of (e.g., CD8+ and / or CD4+) T cell populations or samples expressing binding proteins, T cells have half-maximal expression of IFN-γ in the presence of [LogEC50 is less than -10M (e.g., between -10M and -11M)] peptides.

[0060] Host (e.g., T) cells expressing binding proteins according to the present disclosure are activated in the presence of a new antigen recognized by the binding protein (e.g., as determined by the expression of CD137). For example, binding proteins that recognize and bind to mutant KRAS are 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).

[0061] In some embodiments, host cells (e.g., T cells, e.g., CD4+ T cells or CD8+ T cells) expressing binding proteins according to the present disclosure are capable of specifically killing cells expressing a neoantigen (e.g., cells expressing mutant KRAS (e.g., SW480 cells, e.g., at an 8:1 effector cell: target cell ratio, a 4:1 effector cell: target cell ratio, or a 2:1 effector cell: target cell ratio)). In some embodiments, host cells expressing binding proteins according to the present disclosure are capable of specifically killing cells expressing a neoantigen (e.g., cells expressing mutant KRAS) in vitro within 144, including when additional tumor cells are added at 72 hours in a re-challenge setting.

[0062] In certain embodiments, the binding proteins of the present disclosure are non-alloreactive, substantially non-alloreactive, and / or have a low risk of alloreactivity (i) to amino acid sequences from the human proteome and / or (ii) to human HLA alleles.

[0063] In any of the embodiments disclosed herein, the binding protein may be human, humanized or chimeric. Polynucleotides encoding binding proteins, vectors comprising polynucleotides, and host cells comprising polynucleotides and / or vectors and / or expressing binding proteins are also provided. The binding proteins and host cells (e.g., T cells, NK cells, NK-T cells) disclosed in the present disclosure are suitable for treating diseases or conditions associated with KRAS neoantigens, such as cancer. The binding proteins disclosed in the present disclosure may also bind to the G12V antigen produced in human NRAS or human HRAS, and the protein contains a sequence consistent with KRAS in the region near residue G12. Therefore, the disclosed composition is suitable for treating diseases or conditions associated with KRAS neoantigens, NRAS neoantigens comprising G12V mutations, or HRAS neoantigens comprising G12V mutations, or any combination thereof.

[0064] Also provided are methods and uses of the binding proteins, polynucleotides, vectors, host cells and related compositions disclosed in the present disclosure for treating diseases or disorders associated with mutations in neoantigens (e.g., KRAS, NRAS, HRAS, p53 and / or PIK3CA) as provided herein.

[0065] Before describing the present disclosure in more detail, providing definitions of certain terms to be used herein may aid in understanding the present disclosure. Additional definitions are set forth throughout the present disclosure.

[0066] In this specification, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers) within the recited range. In addition, unless otherwise indicated, any numerical range related to any physical characteristic, such as polymer subunits, size, or thickness, recited herein should be understood to include any integer within the recited range. As used herein, the term "about" means ±20% of the specified range, value, or structure, unless otherwise indicated. It should be understood that the term "a" or "an" as used herein refers to "one or more" of the recited components. It should be understood that the use of alternatives (e.g., "or") means one, both, or any combination of the alternatives. As used herein, the terms "including," "having," and "comprising" are used synonymously, and the terms and variations thereof are intended to be understood as non-limiting.

[0067] In addition, it is understood that individual compounds or groups of compounds derived from various combinations of structures and substituents described herein are disclosed by this application to the same extent as if each compound or group of compounds were individually described. Therefore, the selection of a particular structure or a particular substituent is within the scope of this disclosure.

[0068] The term "consisting essentially of" is not equivalent to "comprising", and refers to the specified materials or steps of the technical solution, or materials or steps that do not significantly affect the basic characteristics of the claimed subject matter. For example, when a specific amino acid sequence of a domain, region, module or protein includes extensions, deletions, mutations or combinations thereof (e.g., amino acids located at the amino terminus or carboxyl terminus or between domains) and the combination thereof accounts for up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain, region, module or protein and does not substantially affect the activity of the domain, region, module or protein (e.g., the target binding affinity or avidity of the binding protein) (i.e., the activity is reduced by no more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%), the protein domain, region or module (e.g., binding domain, hinge region, linker module) or protein (which may have one or more domains, regions or modules) "consists essentially of the amino acid sequence".

[0069] As used herein, "protein" or "polypeptide" generally refers to a polymer of amino acid residues. Protein applies to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics 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 of about 8-10 amino acid residues in length.

[0070] As used herein, "hematopoietic progenitor cells" generally refer to cells that can be derived from hematopoietic stem cells or fetal tissue and are capable of further differentiation into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include those with CD24 Lo Lin - CD117 + Those cells that have a phenotype of thymocytes or those cells found in the thymus (called pioneer thymocytes).

[0071] As used herein, "immune system cell" generally refers to any cell of the immune system that is derived from bone marrow hematopoietic stem cells, which give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphocytes 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, gamma delta T cells, regulatory T cells, natural killer cells, natural killer T cells and dendritic cells. Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of the APCs complexed with peptides interact with the TCR on the surface of the T cells.

[0072] A "T cell" or "T lymphocyte" is a cell of the immune system that matures in the thymus and produces the T cell receptor (TCR). T cells can be naive ("T N "; not exposed to antigen; compared to T CM (described herein), increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased or absent expression of CD45RO); memory T cells (T M ) (antigen experienced and long-lived), including stem cell memory T cells; and effector cells (antigen experienced, cytotoxic). T M It can be further divided into central memory T cell subsets (T CM , expressing CD62L, CCR7, CD28, CD95, CD45RO and CD127) and effector memory T cell subsets (T EM , expressing CD45RO; expression of CD62L, CCR7, CD28 and CD45RA is reduced). Effector T cells (T E ) refers to CD8+ cytotoxic T lymphocytes that have encountered antigens and express CD45RA, which is higher than T CM Have reduced expression of CD62L, CCR7, and CD28, and are positive for granzymes and perforin. Helper T cells (T H ) is CD4 + Cells that influence the activity of other immune cells by releasing cytokines. + T cells can both activate and suppress adaptive immune responses, and which of those two functions is induced will depend on the presence of other cells and signals. T cells can be collected using known techniques, and various subpopulations or combinations thereof can be enriched or depleted using known techniques, such as by affinity binding with antibodies, flow cytometry, or immunomagnetic selection. Other example 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.

[0073] "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; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, p. 433, 1997), capable of specific binding to an antigenic peptide bound to an MHC receptor. TCRs can be found on the surface of cells or in soluble form and typically comprise heterodimers having α and β chains (also referred to as TCRα and TCRβ, respectively), or γ and δ chains (also referred to as TCRγ and TCRδ, respectively). In certain embodiments, polynucleotides encoding binding proteins (e.g., TCRs) of the present disclosure may be codon optimized to enhance expression in specific host cells, such as immune system cells, 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 that can express the binding proteins and TCRs of the present disclosure include CD4 + T cells, CD8 + T cells and their related subsets (e.g., naive, central memory, stem cell memory, effector memory).

[0074] Like other immunoglobulins (e.g., antibodies), the extracellular portion of a TCR chain (e.g., α-chain, β-chain) contains two immunoglobulin domains, an N-terminal variable domain (e.g., α-chain variable domain or V α , β-chain variable domain or V β based on Kabat numbering (Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th edition), usually amino acids 1 to 116) and one constant domain adjacent to the cell membrane (e.g., the α-chain constant domain or the C α , usually 5 amino acids 117 to 259 based on Kabat; the β-chain constant domain or C β, usually amino acids 117 to 295 based on Kabat). In addition, like 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; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The TCR used in the present disclosure can be derived from various animal species, such as humans, mice, rats, rabbits or other mammals.

[0075] The term "variable region" or "variable domain" generally refers to a domain of an immunoglobulin superfamily binding protein (e.g., a TCR α-chain or β-chain (or the γ chain and δ chain of a γδTCR)) that is involved in the binding of an immunoglobulin superfamily binding protein (e.g., a TCR) to an antigen. The variable domains of the α-chain and β-chain of a native TCR (Vα and Vβ, respectively) generally have similar structures, with each domain comprising 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 (VJ); the Vβ region is encoded by three separate DNA segments, a variable gene segment, a diversity gene segment, and a joining gene segment (VDJ). A single Vα or Vβ domain may be sufficient to confer antigen binding specificity. In addition, a TCR that binds to the antigen can be isolated by screening a library of complementary Vα or Vβ domains, respectively, from a TCR that binds to a specific antigen.

[0076] The terms "complementarity determining region" and "CDR" are generally synonymous with "hypervariable region" or "HVR" and generally refer to amino acid sequences within the variable region of an immunoglobulin (e.g., TCR). The CDRs confer antigen specificity and binding affinity and the primary amino acid sequences are separated from each other by framework regions. In general, there are three CDRs (αCDR1, αCDR2, αCDR3) in each TCR α-chain variable region and three CDRs (βCDR1, βCDR2, βCDR3) in each TCR β-chain variable region. CDR3 in TCR is considered to be the main CDR responsible for recognizing processed antigens. In general, CDR1 and CDR2 interact primarily or entirely with MHC.

[0077] CDR1 and CDR2 are encoded within the variable gene segment of the TCR variable region coding sequence, while CDR3 is encoded by a region spanning the variable and joining segments of Vα or by a region spanning the variable, diversity and joining segments of Vβ. Therefore, if the properties of the variable gene segments of Vα or Vβ are known, the sequences of their corresponding CDR1 and CDR2 can be derived; for example, according to the numbering scheme described herein. Due to the addition and loss of nucleotides during the recombination process, CDR3 (especially CDR3β) generally has significantly greater diversity than CDR1 and CDR2.

[0078] TCR variable domain sequences can be aligned according to numbering schemes (e.g., Kabat, Chothia, EU, IMGT, enhanced Chothia, and Aho), allowing annotation of equivalent residue positions and comparison of different molecules using, for example, the ANARCI software tool (2016, Bioinformatics 15: 298-300). The numbering scheme provides standardized delimitation for framework regions and CDRs in TCR variable domains. In certain embodiments, the CDRs of the present disclosure are identified according to the IMGT numbering scheme (Leverland et al., Development and Comparative Immunology, 27: 55, 2003; imgt.org / IMGTindex / V-QUEST.php). In some embodiments, CDRs (e.g., CDR3) are identified or defined according to the IMGT joint definition. In some embodiments, CDRs (e.g., CDR3) are identified or defined according to the IMGT definition. In some embodiments, CDRs of the present disclosure are identified or defined according to the Kabat numbering scheme or method. In some embodiments, 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 are identified or defined according to the Aho numbering scheme or method.

[0079] The TCR source used in the present disclosure may be from any of a variety of animal species, such as humans, mice, rats, rabbits or other mammals. The TCR constant domain sequence may be from, for example, humans, mice, marsupials (e.g., opossums, bandicoots, kangaroos), sharks or non-human primates. In certain embodiments, the TCR constant domain sequence is a human sequence or an engineered variant comprising a human sequence. The TCR constant domain may be engineered to improve pairing, expression, stability or any combination of these. See, for example, Cohen et al., Cancer Res, 2007; Kuball et al., Blood 2007; and Haga-Freidman et al., Journal of Immunology 2009. The engineering examples of TCR Cα and Cβ include mutating natural amino acids to cysteine ​​to form a disulfide bond between the introduced cysteine ​​of one TCR constant domain and the natural cysteine ​​of another TCR constant domain. Such mutations may include T48C in Cα, T57C in Cβ, or both. Mutations that improve stability may include a mutation in the Cα transmembrane domain from the sequence LSVIGF to the sequence LLVIVL ("LVL" mutation; see Hajia-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).

[0080] As used herein, the term "CD8 coreceptor" or "CD8" generally refers to the cell surface glycoprotein CD8, in the form of an α-α homodimer or an α-β heterodimer. The CD8 coreceptor helps cytotoxic T cells (CD8 + ) function, and exerts its effects by signaling 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 (5) human CD8 β chain isoforms (see UniProtKB identifier P10966) and a single human CD8 α chain isoform (see UniProtKB identifier P01732).

[0081] "CD4" generally refers to the immunoglobulin coreceptor glycoprotein that facilitates communication between the TCR and antigen presenting cells (see Campbell and Reece, biology ( Biology)909 (Benjamin Cummings, 6th edition, 2002)). CD4 is present on the surface of immune cells, such as T helper cells, monocytes, macrophages and dendritic cells, and includes four immunoglobulin domains (D1 to D4) expressed on the cell surface. During antigen presentation, CD4 is recruited together with the TCR complex to bind to different regions of the MHCII molecule (CD4 binds to MHCII β2, while the TCR complex binds to MHCII α1 / β1). Without wishing to be bound by theory, it is believed that the close proximity of the TCR complex allows the CD4-associated kinase molecules to phosphorylate the immunoreceptor tyrosine activation motif (ITAM) present on the cytoplasmic domain of CD3. This activity is believed to amplify the signal generated by the activated TCR in order to generate or recruit various types of immune system cells, including T helper cells, and immune responses.

[0082] In certain embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and associate with the CD3 complex. "CD3" is a six-chain multiprotein complex (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999) that is associated with antigen signaling in T cells. In mammals, the complex comprises a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ, CD3β, and CD3ε chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3β, and CD3ε chains are negatively charged, which is believed to allow these chains to associate 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 conserved motifs. Without wishing to be bound by theory, it is believed that ITAMs play an important role in the signaling ability of the TCR complex.CD3 as used in the present disclosure can be from a variety of animal species, including humans, mice, rats or other mammals.

[0083] As used herein, "TCR complex" generally refers to a complex formed by the association of CD3 and TCR. For example, the TCR complex may include a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, the TCR complex may include a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRβ chain.

[0084] As used herein, "component of a TCR complex" 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 sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ and two CD3ε chains).

[0085] A "chimeric antigen receptor" (CAR) generally refers to a fusion protein that has been engineered to contain two or more naturally occurring amino acid sequences, domains or motifs linked together in a manner that does not occur naturally or in a host cell, and which can function as a receptor when present on the surface of a cell. A CAR may include an extracellular portion comprising an antigen binding domain (e.g., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived from or derived from a TCR specific for a cancer antigen, a scFv derived from or derived from an antibody, or an antigen binding domain derived from or derived from a killer immune receptor from a NK cell) connected to a transmembrane domain and one or more intracellular signaling domains (optionally containing a co-stimulatory domain) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also 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. The CAR of the present invention specifically binds to the Ras antigen (e.g., in the case of a peptide:HLA complex) and comprises a TCR Vα region and a Vβ domain.

[0086] Any polypeptide disclosed herein may comprise a "signal peptide" (also referred to as a leader sequence, leader peptide or transition peptide) when encoded by a polynucleotide sequence. The signal peptide targets the newly synthesized polypeptide to its appropriate location inside or outside the cell. In some cases, 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., insertion into a membrane) or secretion or after localization or secretion is completed. A polypeptide with a signal peptide is referred to herein as a "preprotein", and a polypeptide with a signal peptide removed is referred to herein as a "mature" protein or polypeptide. In any embodiment disclosed herein, the binding protein or fusion protein comprises or is a mature protein, or is or comprises a preprotein.

[0087] "Linker" generally refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions or motifs, and can provide a spacing function compatible with the interaction of the two sub-binding domains, so that the resulting polypeptide maintains specific binding affinity to the target molecule (e.g., scTCR) or maintains signal transduction activity (e.g., TCR complex). In certain embodiments, the linker comprises, for example, about 2 to about 35 amino acids, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids. Example linkers include glycine-serine linkers.

[0088] As used herein, "antigen" or "Ag" refers to an immunogenic molecule that causes an immune response. This immune response may involve antibody production, activation of specific immune competent cells (e.g., T cells), or both. Antigens (immunogenic molecules) may be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, or their analogs. It is apparent that antigens may be synthesized, recombinantly produced, or derived from biological samples. Exemplary biological samples that may contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens may be produced by cells that are modified or genetically engineered to express antigens or endogenously (e.g., modified or genetically engineered without human intervention) express immunogenic mutations or polymorphisms.

[0089] As used herein, "neoantigen" generally refers to a host cell product containing a structural change, alteration or mutation that produces a new antigen or antigenic epitope that has not been previously observed in the individual's genome (i.e., in a healthy tissue sample from an individual) or has not been "discovered" or recognized by the host immune system, and the new antigen: (a) is 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) triggers an immune response, (e.g., a cellular (T cell) response). The new antigen can be derived from, for example, an encoding polynucleotide with an alteration (substitution, addition, deletion) that produces an altered or mutant product; or from the insertion of an exogenous nucleic acid molecule or protein into a cell, or from exposure to environmental factors that cause genetic changes (e.g., chemicals, radiation). The new antigen can be produced separately from a tumor antigen, or can be derived from or associated with a tumor antigen. "Tumor neoantigen" (or "tumor-specific neoantigen") refers to a protein containing a new antigenic determinant associated with, derived from, or produced in a tumor cell or multiple cells within a tumor. Tumor neoantigen determinants are found, for example, in antigenic tumor proteins or peptides containing one or more somatic mutations or chromosomal rearrangements encoded by tumor cell DNA (e.g., pancreatic cancer, lung cancer, colorectal cancer), and proteins or peptides from viral open reading frames associated with virus-associated tumors (e.g., cervical cancer, some head and neck cancers). When referring to Ras antigens comprising mutations as disclosed herein, the terms "antigen" and "neoantigen" are used interchangeably 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, a FGFR3 peptide, a FLT3 peptide, a GNA11 peptide, a GNAQ peptide, an IDH peptide, a MYD88 peptide, a p53 peptide, a PIK3CA peptide, or a SF3B1 peptide. In some embodiments, the neoantigen comprises an ALK peptide, an EGFR peptide, a HER2 peptide, a KIT peptide, a MET peptide, a NRG1 peptide, a 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 also contemplated. In some embodiments, the neoantigen comprises an oncogenic driver mutation. Without being bound by theory, it is believed that oncogenic driver mutations are responsible for the initiation and maintenance of cancer.

[0090] 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 (e.g., an immunoglobulin, a T cell receptor (TCR), a chimeric antigen receptor or other binding molecule, domain or protein). Epitope determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0091] 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 (or NRAS or HRAS) peptide" generally refers to a naturally occurring or synthetically produced peptide portion of a KRAS or NRAS or HRAS protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, up to about 20 amino acids, and comprising at least one amino acid change caused by a G12 (e.g., G12V) mutation (wherein position 12 is with reference to the full-length KRAS protein sequence shown in SEQ ID NO: 1; and also with reference to the full-length NRAS and HRAS protein sequences shown in SEQ ID NOs: 78 and 79, respectively), said peptide can form a complex with an MHC (e.g., HLA) molecule, and the binding proteins of the present disclosure that are specific for KRAS or NRAS or HRAS peptide:MHC (e.g., HLA) complexes can specifically bind to such complexes. An exemplary KRAS (or NRAS or HRAS) antigen comprises, consists essentially of, or consists of a peptide having the amino acid sequence of SEQ ID NO: 2 or 3.

[0092] "Major histocompatibility complex" (MHC) generally refers to glycoproteins that deliver peptide antigens to the cell surface of all nucleated cells. Class I MHC molecules are heterodimers that have a transmembrane α chain (with three α domains) and a non-covalently associated β2 microglobulin. Class II MHC molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain contains two domains. Class I MHC molecules deliver peptides derived from the cytosol to the cell surface, where the peptide:MHC complex is bound by CD8 + T cell recognition. Class II MHC molecules deliver peptides derived from the vesicle system to the cell surface where they are recognized by CD4 + T cell recognition. Human MHC is called human leukocyte antigen (HLA). HLA corresponding to "class I" MHC presents peptides from the inside of the cell and includes, for example, HLA-A, HLA-B and HLA-C. Alleles include, for example, HLA A*11, such as HLA-A*11:01. HLA corresponding to "class II" MHC presents peptides from the outside of the cell and includes, for example, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ and HLA-DR.

[0093] The principle of antigen processing by antigen presenting cells (APCs) (e.g., dendritic cells, macrophages, lymphocytes, or other cell types) and presentation of antigen by the APCs to T cells (including presentation restricted to the major histocompatibility complex (MHC) between APCs and T cells that is immunocompatible (e.g., sharing at least one allelic form of an MHC gene associated with antigen presentation)) is well established (see, e.g., Murphy, Janeway, Immunobiology (8th ed.) 2011 Garland Science, NY; Chapters 6, 9, and 16). For example, processed antigenic peptides derived from the cytosol (e.g., tumor antigens, intracellular pathogens) are generally about 7 to about 11 amino acids in length and will associate with class I MHC (HLA) molecules, while peptides processed in vesicular systems (e.g., bacteria, viruses) will vary in length from about 10 to about 25 amino acids and will associate with class II MHC (HLA) molecules.

[0094] As used herein, the term "KRAS-specific binding protein" generally refers to a protein or polypeptide, such as TCR, scTv, scTCR or CAR, that binds to a KRAS peptide antigen or a NRAS peptide antigen or a HRAS peptide antigen (or binds to, for example, a KRAS or NRAS or HRAS peptide antigen:HLA complex on the surface of a cell), and does not bind to a peptide that does not contain a KRAS or NRAS or HRAS peptide antigen and does not bind to an HLA complex containing such peptides.

[0095] The binding proteins disclosed herein, such as TCR, scTCR and CAR, contain a binding domain specific to the target. As used herein, a "binding domain" (also referred to as a "binding region" or "binding portion") refers to a molecule or a portion thereof (e.g., a peptide, oligopeptide, polypeptide, protein) that has the ability to specifically and non-covalently associate, combine or combine with a target (e.g., KRAS or NRAS or HRAS peptide, or KRAS or NRAS or HRAS peptide: MHC complex). The binding domain includes any naturally occurring, synthetic, semi-synthetic or recombinantly produced binding partner of a biomolecule, a molecular complex (i.e., a complex comprising two or more biomolecules) or other targets of interest. Example binding domains include immunoglobulin variable regions or single-chain constructs comprising them (e.g., single-chain TCR (scTCR) or scTv).

[0096] In certain embodiments, the Ras-specific binding protein is expressed in an amount 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 -13M of K d Or binds to a KRAS (or NRAS or HRAS) peptide (or KRAS (or NRAS or HRAS):HLA complex) with about the same affinity, at least about the same affinity, or greater than or about the affinity exhibited by an exemplary Ras-specific binding protein provided herein (e.g., any of the Ras-specific TCRs provided herein), e.g., as measured by the same assay. In certain embodiments, the Ras-specific binding protein comprises a Ras-specific immunoglobulin superfamily binding protein, or a binding portion thereof.

[0097] As used herein, "specific binding" or "specific for" generally refers to the binding of a binding protein (e.g., a TCR receptor) or a binding domain (or a fusion protein thereof) to a target molecule with a specificity of equal to or greater than 10 5 M -1 (which is equal to the association rate of this association reaction [k on ] and dissociation rate [k off ]) affinity or K a (i.e., the equilibrium association constant for a specific binding interaction, in units of 1 / M) while not significantly associating or associating with any other molecule or component in the sample. Binding proteins or binding domains (or their fusion proteins) can be classified as "high affinity" binding proteins or binding domains (or their fusion proteins) or "low affinity" binding proteins or binding domains (or their fusion proteins). "High affinity" binding proteins or binding domains refer to those with a K a For at least 10 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 "Low affinity" binding proteins or binding domains are those with a K a For up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 Alternatively, affinity can be defined as the equilibrium dissociation constant (K) for a specific binding interaction. d ), the unit is M (for example, 10 -5 M to 10 -13M).

[0098] 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 binds to a target antigen more strongly than a wild-type (or parent) binding domain. For example, enhanced affinity may be attributable to a K for the target antigen. a (equilibrium association constant) is higher than that of the wild-type binding domain, attributed to the K for the target antigen. d (dissociation constant) is smaller than that of the wild-type binding domain, due to the dissociation rate (k off ) is smaller than the wild-type binding domain, or a combination thereof.

[0099] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target and for determining the affinity of a binding domain or fusion protein, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance. Assay (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0100] In certain embodiments, the neoantigen (e.g., KRAS (or NRAS or HRAS), p53 and / or PIK3CA) specific binding domain alone (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 may be 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 dBinds to a neoantigen (e.g., KRAS (or NRAS) or HRAS), p53 and / or PIK3CA) (or a neoantigen (e.g., KRAS (or NRAS or HRAS), p53 and / or PIK3CA) peptide or a neoantigen (e.g., KRAS (or NRAS or HRAS), p53 and / or PIK3CA) peptide:HLA complex). In a specific embodiment, 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 αβTCR protein, such as Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vα or Vα-L-Vβ-Cβ, wherein Vα and Vβ are TCR α and β variable domains, respectively, Cα and Cβ are TCR α and β constant domains, respectively, and L is a linker, such as a linker described herein).

[0101] As used herein, the term "functional affinity" generally refers to a biological measure or activation threshold of the response of an in vitro immune cell (e.g., T cell, NK cell, NK-T cell) to a given concentration of a ligand, wherein the biological measure may include cytokine production (e.g., IFN-γ production, IL2 production, etc.), cytotoxic activity, activation markers (e.g., CD137, Nur77) and proliferation. For example, a biological (immunological) response to a low antigen dose in vitro, such as a T cell that produces a cytokine, exhibits cytotoxic activity, or proliferates, is considered to have a high functional affinity, while a T cell with a lower functional affinity requires a higher amount of antigen before inducing an immune response similar to a high-affinity T cell. It should be understood that functional affinity is different from affinity and avidity. Avidity refers to the strength of any specified bond between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind to a variety of antigens, in which case the total connection strength is avidity.

[0102] There are multiple correlations between functional avidity and the effectiveness of the immune response. Some in vitro studies have shown that different T cell functions (e.g., proliferation, cytokine production, etc.) can be triggered 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 may 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 level of the TCR and, in some embodiments, the CD4 or CD8 co-receptor on the host cell; (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273:104, 1996), as well as the expression levels of molecules that attenuate T cell function and TCR signaling.

[0103] After a specified exposure time, the antigen concentration required to induce a half-maximal response (e.g., production of cytokines or activation markers by host cells; fluorescence intensity when bound to labeled peptide:HLA multimers) between baseline and maximum response is called the "half-maximal effective concentration" or "EC50". EC50 values ​​are generally presented as molar concentrations (mol / L), but they are usually converted to logarithmic values ​​as follows -log 10 (EC50). For example, if EC50 is equal to 1 μM (10 -6 M), then 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 affinity of the binding proteins of the present disclosure will comprise a measure of the ability of the binding proteins to promote T cell activation and / or IFNγ production, which can be measured using assays known in the art and described herein. In certain embodiments, the functional affinity will comprise a measure of the ability of the binding proteins to activate host cells (e.g., T cells) after binding to an antigen.

[0104] The binding proteins disclosed herein may comprise high functional avidity, which may, for example, promote the initiation of immune cell effector functions (e.g., activation, proliferation, cytokine production and / or cytotoxicity) against even lower levels of presented neoantigenic peptides, such as the KRAS G12V mutant peptide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0105] In some embodiments, the binding protein has a log10EC50 for the neoantigenic 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 more 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.

[0106] In some embodiments, the host cells disclosed herein comprise a binding protein (e.g., a TCR) that binds to the binding protein's target neoantigen (e.g., a KRAS G12 mutant peptide, e ... G12V mutant peptide, e.g., present in a peptide:HLA complex): less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than 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 less than about 40pM, less than about 30pM, less than about 20pM, less than about 10nM, less than about 5nM, less than about 1nM, less than about 900pM, less than about 800pM, less than about 700pM, less than about 600pM, less than about 500pM, less than about 400pM, less than about 300pM, less than about 200pM, less than about 100pM, less than about 90pM, less than about 80pM, less than about 70pM, less than about 60pM, less than about 50pM, less than about 40pM, less than about 30pM, less than about 20pM, less than about 10pM, less than about 5pM, or less than about 1pM. EC50 can be determined by an assay to identify the peptide dose at which a T cell population achieves half-maximal activation, as reflected by the expression of activation markers (e.g., CD137, CD69, granzyme B, CD107a, IFN-γ, TNF-a, IL-12, cytokines, interleukins, interferons) following exposure of target cells to the presence of various concentrations of the mutant peptide.

[0107] In some embodiments, the host cells disclosed herein comprise a binding protein (e.g., a TCR) that binds to the binding protein's target neoantigen (e.g., a KRAS G12 mutant peptide, e ... G12V mutant peptide, e.g., present in a peptide:HLA complex): 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 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.

[0108] The host cell may comprise a transgenic polynucleotide encoding a chimeric fusion protein comprising an IL7R intracellular signaling domain. The chimeric fusion protein may comprise, for example, an intracellular portion of an interleukin 7 receptor A (IL7RA) polypeptide or a portion or variant thereof that is capable of contributing to an IL-7 signal in a host cell. The chimeric IL7R fusion protein may, for example, provide "signal 3" to increase STAT5 phosphorylation and host cell functionality, enhance host cell proliferation, increase host cell survival (e.g., in a tumor microenvironment), and / or enhance chemokine receptor expression.

[0109] Interleukin-7 receptor subunit alpha may also be referred to as IL7R-alpha, IL7RA, IL-7R-alpha, ILRA, interleukin-7 receptor-alpha, interleukin 7 receptor, cluster of differentiation 127, CD127, or CDW127.

[0110] The IL7R intracellular signaling domain may comprise 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.

[0111] 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) 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.

[0112] In some embodiments, the chimeric fusion protein comprises an IL7R transmembrane domain. The IL7R transmembrane domain may 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 with 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 one or more cysteines and / or one or more prolines inserted into the amino acid sequence of SEQ ID NO: 1042. In some embodiments, the mutation enables or promotes homodimerization of the receptor. In some embodiments, the mutation comprises inserting a trimeric peptide of cysteine, proline, threonine (CPT) into the transmembrane domain. In some embodiments, the threonine inserted by the CPT is not threonine but another amino acid, and in at least certain cases, the other amino acids are or are not cysteine ​​or proline. In some embodiments, the chimeric fusion protein comprises a transmembrane domain of IL7R, IL2RA, IL2RB, IL2RG, IL14R, IL15R, IL9R, IL21R, CD2, CD40L, CD58, CD80, or SIRPα.

[0113] In some embodiments, the chimeric fusion protein comprises an extracellular component, the extracellular component comprising: (i) an 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) an 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) an extracellular domain of a signal regulatory protein α (SIRPα) polypeptide, or a portion or variant thereof that can bind to a cluster of differentiation 47 (CD47) polypeptide; (iv) an extracellular domain of a cluster of differentiation 40L (CD40L) polypeptide, or a portion or variant thereof that can bind to a CD40 polypeptide; (v) an 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) an extracellular domain of a cluster of differentiation 34 (CD34) polypeptide.

[0114] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising an extracellular domain of a cluster of differentiation 80 (CD80) polypeptide or a portion or variant thereof capable of binding 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.

[0115] In some embodiments, the chimeric fusion protein comprises an extracellular component comprising an extracellular domain of a cluster of differentiation 58 (CD58) polypeptide or a portion or variant thereof capable of binding 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.

[0116] In some embodiments, the chimeric fusion protein includes 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.

[0117] In some embodiments, a host cell population comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or chimeric IL7R polypeptide disclosed herein) exhibits an increase in proliferation in response to a target cell (e.g., presenting a KRAS G12D peptide) 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 control cell population (e.g., a corresponding cell lacking a Fas-41BB fusion protein or a chimeric IL7R polypeptide). Proliferation can be determined, for example, by measuring the number of host cells in an in vitro lymphocyte proliferation assay or after co-cultivation. The host cell may comprise an extracellular binding protein (eg, a TCR comprising the Va and Vp regions and / or CDRs disclosed herein), and / or modifications that result in reduced expression of endogenous TRAC, TRBC1, and / or TRBC2.

[0118] In some embodiments, a host cell population comprising one or more modifications disclosed herein (e.g., expression of a Fas-41BB fusion protein or chimeric IL7R polypeptide disclosed herein) exhibits an increase in killing of target cells 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 control cell population (e.g., a corresponding cell lacking a 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 cell can comprise an extracellular binding protein (e.g., a TCR comprising a Vα and Vβ region and / or CDR disclosed herein), and / or a modification that causes reduced expression of endogenous TRAC, TRBC1, and / or TRBC2.

[0119] The nucleic acid encoding the polypeptide disclosed herein (e.g., an extracellular binding protein, a CD8 coreceptor chain or an extracellular portion thereof, a Fas-41BB fusion protein, or a chimeric IL7R fusion protein) may encode a signal peptide. In some cases, the polypeptide of the present disclosure comprises a signal peptide. The signal peptide may be cleaved during polypeptide processing, so in some cases the mature polypeptide disclosed herein does not contain a signal peptide.

[0120] The signal peptide at the N-terminus of the protein may be involved in transporting the protein to the membrane or transporting via the membrane, transporting to different membranous cell compartments, or secreting the protein from the cell. The nucleic acid encoding the protein of the present disclosure may encode a signal peptide to promote membrane insertion and surface localization of the protein. The signal peptide may be selected according to its ability to promote ER processing and cell surface localization of the protein. Any suitable signal peptide may be used. In some cases, the signal peptide comprises a G-CSF signal peptide or a CD8α signal peptide. The length of the signal peptide may be about 10 to about 40 amino acids. In some cases, the length of the signal peptide is at least about 10, 15, 16, 20, 21, 22, 25 or 30 amino acids or more. In some cases, the length of the signal peptide is at most about 15, 16, 20, 21, 22, 25 or 30 amino acids or less. In some cases, the length of the signal peptide is about 16-30 amino acids.

[0121] In some embodiments, the binding protein (e.g., TCR) is present at less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than 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 5 In some embodiments, the present invention relates to a target (e.g., a KRAS G12 mutant peptide, e.g., a KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex) with a KD of less than about 0 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.

[0122] Also contemplated are fusion proteins comprising a 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., a fragment that retains binding to one or more Fc receptors, C1q, protein A, protein G, or any combination thereof in some embodiments), and including immunoglobulin heavy chain monomers and multimers, such as Fc dimers; see, e.g., Wong et al., J. Immunol. 198:1 Supplement (2017). Variant Fc polypeptides comprising mutations that enhance, reduce, or eliminate binding to, or are bound by, e.g., FcRn or other Fc receptors are known and contemplated within the present disclosure.

[0123] In certain embodiments, the binding protein or fusion protein (e.g., TCR, scTCR, CAR) of the present disclosure is expressed by a host cell (e.g., a T cell, NK cell, or NK-T cell heterologously expressing the binding protein or fusion protein). Such host cells are to neoantigens (e.g., KRAS (or NRAS or HRAS), p53 and / or PIK3CA) peptide antigens or neoantigens (e.g., KRAS (or NRAS or HRAS), p53 and / or PIK3CA) peptide antigens: The affinity of the HLA complex can be determined by, for example, the following: the host cell is exposed to the peptide, or exposed to the peptide: HLA complex (e.g., organized as a tetramer), or exposed to an antigen presenting cell (APC) that presents the peptide to the host cell, optionally with a peptide: HLA complex, and then measuring the activity of the host cell, such as cytokines (e.g., IFN-γ; TNFα) production or secretion; increase host cell signaling or activation component (e.g., CD137 (4-1BB)) expression; host cell proliferation; or killing APC (e.g., using labeled chromium release analysis).

[0124] As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" generally refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, polynucleotides, fragments thereof, produced, for example, by polymerase chain reaction (PCR) or by in vitro translation, and also refers to fragments produced by any of ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids may be composed of monomers, which are naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-mirror image isomers of naturally occurring nucleotides), or a combination of the two. Modified nucleotides may have modifications in the sugar moiety, or in the pyrimidine or purine base moiety, or may have substitutions of the sugar moiety or the pyrimidine or purine base moiety. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such linkages. Similar bonds to the phosphodiester bond include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, anilide phosphorothioate, anilide phosphate, phosphoramidate, and the like. The nucleic acid molecule can be single-stranded or double-stranded.

[0125] The term "isolated" generally means that the material is 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 coexisting materials in the natural system is isolated. Such a nucleic acid can be part of a vector and / or such a nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate) and still be isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" means a segment of DNA involved in producing a polypeptide chain; it includes regions before and after the coding region ("leader and trailer sequences") and intervening sequences (introns) between individual coding segments (exons).

[0126] As used herein, the terms "recombinant", "engineered" and "modified" generally refer to cells, microorganisms, nucleic acid molecules, polypeptides, proteins, plasmids or vectors that have been modified by the introduction of exogenous nucleic acid molecules, or to cells or microorganisms that have been genetically engineered by human intervention, i.e., modified by the introduction of heterologous nucleic acid molecules, or to cells or microorganisms that have been altered so that the expression of endogenous nucleic acid molecules or genes is controlled, abnormally regulated or constitutive, wherein such changes or modifications can be introduced by genetic engineering. Human-generated genetic changes can include, for example, the introduction of nucleic acid molecules encoding one or more proteins or enzymes (which can include expression control elements, such as promoters), or other nucleic acid molecule additions, deletions, substitutions, or other functional disruptions or additions of cellular genetic material. Example modifications include modifications in the coding region or functional fragments thereof of heterologous or homologous polypeptides from a reference molecule or parent molecule.

[0127] As used herein, "mutation" generally refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutation can result in several different types of sequence changes, including substitution, insertion or deletion of nucleotides or amino acids. In certain embodiments, the mutation is a substitution of one or three codons or amino acids, a deletion of 1 to about 5 codons or amino acids, or a combination thereof.

[0128] "Conservative substitutions" generally refer to substitutions of one amino acid for another amino acid with similar properties. Such conservative substitutions are well known in the art (see, e.g., WO 97 / 09433, p. 10; Lehninger, Biochemistry, 2nd ed.; Worth Publishers, Inc. NY, NY, pp. 71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

[0129] In certain embodiments, proteins according to the present disclosure (e.g., binding proteins, immunogenic peptides) comprise variant sequences (e.g., variant TCR CDRs (e.g., CDR3β compared to reference TCR CDR3β disclosed herein) compared to a reference sequence. As used herein, a "variant" amino acid sequence, peptide, or polypeptide refers to an amino acid sequence (or peptide or polypeptide) having one, two, or three amino acid substitutions, deletions, and / or insertions compared to a reference amino acid sequence. In certain embodiments, the variant amino acid sequence, peptide, or polypeptide retains substantially the same function as the reference molecule (e.g., binding specificity and affinity for peptide:HLA complex); for example, a variant TCR fragment as disclosed herein retains 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.

[0130] An "altered domain" or "altered protein" generally refers to a motif, region, domain, peptide, polypeptide or protein that has a non-identical sequence identity of 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% or at least 99.9%) to a wild-type motif, region, domain, peptide, polypeptide or protein (e.g., a wild-type TCR alpha chain, TCR beta chain, TCR alpha constant domain, TCR beta constant domain).

[0131] The altered domain or altered protein or derivative may include those domains or proteins or derivatives based on all possible codon selections for the same amino acid and codon selections based on conservative amino acid substitutions. For example, the following six groups each contain amino acids that are conservative substitutions for each other: 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 (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, 2nd edition, Worth Publishers, Inc., NY, NY, pages 71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, page 8, 1990; Creighton, Proteins, WH 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."

[0132] The term "construct" generally refers to any polynucleotide containing a recombinant nucleic acid molecule. "Transgenic" or "transgenic construct" refers to a construct containing two or more genes operably linked according to an arrangement not found in nature. The term "operably linked (operably-linked)" (or "operably linked (operably linked)" herein) generally refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one nucleic acid molecule is affected by another nucleic acid molecule. For example, when a promoter can affect the expression of a coding sequence, the promoter is operably connected to that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Not connected" generally means that the related genetic elements are not closely related to each other, and the function of one does not affect the other. In some embodiments, the gene present in the transgene is operably connected to an expression control sequence (e.g., a promoter).

[0133] Constructs (e.g., transgenics) may be present in vectors (e.g., bacterial vectors, viral vectors) or may be integrated into a genome. "Vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid molecule. A vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule, which may include chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acid molecules. Example vectors are vectors (expression vectors) capable of autonomous replication (episomal vectors) or expression of attached nucleic acid molecules. Vectors suitable for use in the compositions and methods of the present disclosure are further described herein.

[0134] As used herein, the term "expression" generally refers to the process of producing a polypeptide based on the coding sequence of a nucleic acid molecule (e.g., a gene). The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0135] In the context of inserting a nucleic acid molecule into a cell, the term "introduced" 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 may be incorporated into the genome of the cell (e.g., chromosome, plasmid, chromatin or mitochondrial DNA), converted into an autonomous replicon or transiently expressed (e.g., transfected mRNA).

[0136] As used herein, "heterologous" or "exogenous" nucleic acid molecules, constructs or sequences generally refer to nucleic acid molecules or parts of nucleic acid molecules that are non-native to the host cell but can be homologous to nucleic acid molecules or parts of nucleic acid molecules from the host cell. The source of heterologous or exogenous nucleic acid molecules, constructs or sequences can be from different genera or species. In certain embodiments, heterologous or exogenous nucleic acid molecules are added (i.e., non-endogenous, or non-native) to the host cell or host genome by, for example, binding, transformation, transfection, transduction, electroporation or the like, wherein the added molecules can be integrated into the host genome or exist in the form of extrachromosomal genetic material (e.g., other forms of plasmids or self-replicating vectors) and can be present in multiple copies. In addition, "heterologous" refers to non-native enzymes, proteins or other activities encoded by exogenous nucleic acid molecules introduced into the host cell, even if the host cell encodes homologous proteins or activities. In addition, cells comprising "modified" or "heterologous" polynucleotides or binding proteins include the descendants of the cells, regardless of whether the descendants themselves are transduced, transfected or otherwise manipulated or changed.

[0137] As described herein, more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as multiple individually controlled genes, as a multicistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. For example, as disclosed herein, a host cell can be modified to express one or more heterologous or exogenous nucleic acid molecules encoding a desired TCR (e.g., TCRα and TCRβ) specific for a Ras antigenic peptide and, as disclosed herein, optionally also encoding a CD8 co-receptor polypeptide comprising an α chain, a β chain, or a portion thereof, such as an extracellular portion capable of binding to MHC. When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into a single site or multiple sites in the host chromosome, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities referred to refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of individual nucleic acid molecules introduced into the host cell.

[0138] As used herein, the term "endogenous" or "native" generally refers to a gene, protein, or activity that is normally present in a host cell. In addition, a gene, protein, or activity that is mutated, overexpressed, reorganized, duplicated, or otherwise altered relative to the parent gene, protein, or activity is still considered endogenous or native to the particular host cell. For example, an endogenous control sequence (e.g., a promoter, a translation attenuation sequence) from a first gene can be used to alter or regulate the expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule is different from the normal expression or regulation of the parent cell.

[0139] The term "homolog" or "homolog" generally refers 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 homologous to a native host cell gene, and can optionally have altered expression levels, a different sequence, an altered activity, or any combination thereof.

[0140] As used herein, "sequence identity" generally refers to the percentage of amino acid residues or nucleobases (respectively) in one sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage and not considering any conservative substitutions as part of the sequence identity. Percent sequence identity values ​​can be generated using NCBIBLAST 2.0 software as defined by Altschul et al. (1997), Nucl. Acids Res. 25:3389-3402, wherein each parameter is set to a preset value. Additionally or alternatively, the degree of sequence identity between two sequences can be determined, for example, using a computer program designed for this purpose, such as a global or local alignment algorithm to compare the two sequences. 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. For example, a global alignment algorithm of the Needleman and Wunsch algorithms can be used to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Preset settings can be used.

[0141] To generate a similarity score for two amino acid sequences, a scoring matrix can be used that assigns positive scores to some inconsistent amino acids (e.g., conservative amino acid substitutions, amino acids with similar physicochemical properties, and / or amino acids that exhibit frequent substitutions among orthologs, homologs or paralogs), non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0142] Variants of 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% identical to nucleic acid molecules of defined or reference polynucleotides as described herein, and may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identical, or hybridize to polynucleotides under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide at about 42°C. Nucleic acid molecule variants retain the ability to encode a binding protein or its binding domain that has a function as described herein, such as binding to a target molecule.

[0143] The term "isolated" generally means that the material is 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 coexisting materials in the natural system is isolated. Such a nucleic acid can be part of a vector and / or such a nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate) and still be isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" means a segment of DNA involved in producing a polypeptide chain; it includes regions before and after the coding region ("leader and trailer sequences") and intervening sequences (introns) between individual coding segments (exons).

[0144] In some cases, the term "variant" as used herein generally refers to at least one fragment of the full-length sequence mentioned, more specifically, one or more amino acid or nucleic acid sequences with one or more amino acids truncated at one or both ends relative 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 consecutive amino acids of the original sequence or its variant. The total length of the 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.

[0145] In some embodiments, the term "variant" refers not only to at least one fragment, but also to a polypeptide or fragment thereof comprising 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 a reference amino acid sequence or fragment thereof, wherein amino acids other than those required for the biological activity or folding or structure of the polypeptide are deleted or substituted, one or more such essential amino acids are substituted in a conservative manner, and / or amino acids are added in a manner that retains the biological activity of the polypeptide. Current state-of-the-art technology includes various methods that can be used to compare 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, third edition). In some embodiments, Clustal W software (Larkin, MA et al., (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) can be used using preset settings.

[0146] In certain embodiments, variants may further include chemical modifications, such as isotope labels or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, and the like. Methods for polypeptide modification are known and will generally be used to avoid eliminating or substantially reducing the desired activity of a polypeptide.

[0147] In one embodiment, the term "variant" of a nucleic acid molecule includes a complementary strand that hybridizes with a reference or wild-type nucleic acid, for example, under stringent conditions. The "stringency" of a hybridization reaction can be easily determined by a person of ordinary skill, and is generally empirically calculated, depending on the probe length, washing temperature, and salt concentration. In general, the longer the probe, the higher the temperature required for correct bonding, while shorter probes are not. Hybridization generally depends on the ability of denatured DNA to rebond complementary strands present in an environment below its melting temperature: the higher the degree of homology required between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Therefore, a higher relative temperature can make the reaction conditions more stringent, while a lower temperature is not the case. For other details and explanations on the stringency of hybridization reactions, see Ausubel, FM (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. In addition, regarding how to identify DNA sequences by hybridization, those skilled in the art can follow the instructions provided in the following manuals: Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and Liebl, W., Ehrmann, M., Ludwig, W. and Schleifer, KH (1991), International Journal of Systematic Bacteriology 41: 255-260. In one embodiment, any hybridization is applied with stringent conditions, i.e., hybridization occurs only when the probe is 70% or greater identical to the target sequence. Probes with a lower degree of identity to the target sequence can hybridize, but such hybrids are unstable and are removed in the wash steps under stringent conditions, such as reducing the salt concentration to 2×SSC or optionally and subsequently to 0.5×SSC, while the temperature is, for example, about 50° C.-68° C., about 52° C.-68° C., about 54° C.-68° C., about 56° C.-68° C., about 58° C.-68° C., about 60° C.-68° C., about 62° C.-68° C., about 64° C.-68° C., or about 66° C.-68° C. In one embodiment, the temperature is about 64° C.-68° C. or about 66° C.-68° C. The salt concentration can be adjusted to 0.2×SSC or even 0.1×SSC.Nucleic acid sequences having 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% identity to a reference or wild-type sequence can be isolated. In one embodiment, as used herein, the term nucleic acid sequence variant refers to any nucleic acid sequence encoding the same amino acid sequence and variants thereof as a reference nucleic acid sequence according to the degeneracy of the genetic code.

[0148] "Functional variants" generally refer to polypeptides or polynucleotides that are structurally similar or substantially structurally similar to a parent or reference compound of the present disclosure, but in some cases differ slightly in composition (e.g., one base, atom or functional group is different, one base, atom or functional group is added or removed; or one or more amino acids are mutated, inserted or deleted), such that the polypeptide or the encoded polypeptide is able to 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, when in a selected assay, such as in an assay for measuring binding affinity to a host cell (e.g., measuring association constant (Ka) or dissociation constant (KD) When the functional variant of the polypeptide or the encoded polypeptide of the present invention shows a decrease in potency of no more than 50% compared to the parent or reference polypeptide in an analysis of binding or tetramer staining), affinity or activation, the functional variant has "similar binding", "similar affinity" or "similar activity". As used herein, "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that only comprises a domain, motif, a part or fragment of a parent or reference compound, and the polypeptide or the encoded polypeptide retains at least 50% of the activity associated with the domain, part or fragment of the parent or reference compound, preferably retains 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, or provides a biological benefit (e.g., effector function).

[0149] When a "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure shows no more than a 50% decrease in potency (or, alternatively or additionally, no more than 20% or 10%, or no more than a logarithmic difference in affinity compared to a parent or reference) in a selected assay, such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release), the functional portion or fragment generally has "similar binding" or "similar activity." Functional variants of the binding proteins and polynucleotides specifically disclosed are contemplated.

[0150] An "altered domain" or "altered protein" generally refers to a motif, region, domain, peptide, polypeptide or protein that has a non-identical sequence identity of 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% or at least 99.9%) to a wild-type motif, region, domain, peptide, polypeptide or protein (e.g., a wild-type TCR alpha chain, TCR beta chain, TCR alpha constant domain or TCR beta constant domain).

[0151] The present disclosure includes variants of any binding protein described herein (e.g., a TCR α chain or a TCR β chain or a fragment thereof, such as a Vα or Vβ chain or a CDR1α, CDR2α, CDR3α, CDR1β, CDR2β or CDR3β) substituted with one or more conservative amino acids. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without destroying the three-dimensional structure or function of the polypeptide. Conservative substitutions can be achieved by replacing each other with amino acids having similar hydrophobicity, polarity and R chain length. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conservative residues that do not change the basic function of the encoded protein). Such conservatively substituted variants may include variants with 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 with any of the systems described herein. In some embodiments, such conservatively substituted variants are functional variants.

[0152] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd ed. (December 1993))). The following eight groups each contain amino acids that are conservative substitutions for each other:

[0153] a. Alanine (A), glycine (G);

[0154] b. Aspartic acid (D), glutamic acid (E);

[0155] c. Asparagine (N), glutamine (Q);

[0156] d. Arginine (R), lysine (K);

[0157] e. Isoleucine (I), leucine (L), methionine (M), valine (V);

[0158] f. Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0159] g. Serine (S), threonine (T); and

[0160] h. Cysteine ​​(C), methionine (M).

[0161] Binding protein

[0162] 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, wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3. In certain embodiments, HLA comprises HLA-A*11, optionally comprising HLA-A*11:01. In any embodiment disclosed in the present disclosure, the binding protein can be heterologously expressed by human immune system cells (e.g., T cells).

[0163] In certain embodiments, the Vα domain and / or the Vβ domain are each independently human, humanized or chimeric, and each is 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 their CDRs derived from human individuals, for example, via sequencing of isolated T cells or their populations from human individuals. Relative to variable domains and CDRs from other sources (e.g., single human HLA allele transgenic mice), TCR Vα domains, Vβ domains and their CDRs isolated from human individuals may have advantageous properties. For example, Vα domains, Vβ domains and CDRs derived from human individuals may be negatively selected for substantially complete human polypeptides presented by a set of complete human HLA molecules in vivo, which can reduce the possibility of cross-reaction of binding proteins with other human self-antigens. In some embodiments, the binding proteins disclosed herein are substantially unresponsive to human proteomes presented by one or more HLA alleles. Reactivity can be determined by any suitable method. In some embodiments, 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, no significant response of T cells transduced with the binding protein to the human proteome presented by one or more HLA alleles is observed or predicted.

[0164] In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from (e.g., identified therein) a T cell of an individual (e.g., a human individual) suffering from a disease (e.g., cancer). In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a human individual suffering from a cancer disclosed herein. In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of an individual (e.g., a human individual) suffering from a disease associated with a new antigen (e.g., KRAS (or NRAS or HRAS), p53 and / or PIK3CA) mutation, e.g., KRAS G12V or G12D mutation. In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of an individual (e.g., a human individual) having a cell comprising a new antigen (e.g., KRAS (or NRAS or HRAS), p53 and / or PIK3CA) mutation, e.g., KRAS G12V or G12D mutation.

[0165] In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a healthy individual (e.g., a healthy human individual). In some embodiments, the healthy individual lacks a specific pathological diagnosis (e.g., a disease diagnosis, such as a cancer diagnosis). In some embodiments, the healthy individual lacks a specific pathological diagnosis, but comprises a different pathological diagnosis, such as lacking a cancer diagnosis, but comprising a diagnosis of hypertension or type II diabetes.

[0166] The binding proteins disclosed in the present disclosure can be heterologously expressed by host cells (e.g., human immune cells, such as T cells). In addition, the expression of the binding proteins disclosed in the present disclosure can confer favorable properties on the host cells; for example, binding specificity to the new antigen: HLA complex of the present disclosure, increased activation, proliferation or killing activity in the presence of new antigen: HLA presenting tumor cells, or similar properties thereof.

[0167] For example, in certain embodiments, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell is capable of specifically killing an HLA-A*11:01 expressing a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 or 3. + Tumor cells. Killing of target cells can be measured, for example, using Bioimaging platform (Essen Bioscience) is used to measure killing. In certain embodiments, this platform uses activated apoptotic proteases and labeled (e.g., RapidRed or NucRed) tumor cell signals, where the overlap area is measured and increased overlap area is equivalent to tumor cell death due to apoptosis. Killing can also be measured using a 4-hour assay, where labeled chromium ( 51 Cr) were loaded on the target cells, and the supernatant was measured 51 Cr, for example, measured after 4 hours of incubation with immune cells expressing binding proteins of the present disclosure. In certain embodiments, killing assays can be performed using effector cell:target cell ratios of 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, or the like.

[0168] In any embodiment disclosed in the present disclosure, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell expresses a neoantigen peptide (e.g., a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 or 3) in a tumor cell (e.g., HLA-A11:01 +tumor cells), optionally further in the presence of exogenous IFN-γ, wherein the expression of Nur77 is increased compared to: (i) the expression of Nur77 of a reference immune cell that does not express the binding protein (i.e., the same cell type as the immune cell expressing the binding protein, and otherwise at least substantially identical or functionally equivalent to the immune cell expressing the binding protein in terms of phenotype and / or genotype), when the reference immune cell is in the presence of tumor cells; and / or (ii) the expression of Nur77 by an immune cell expressing the binding protein in the absence of tumor cells and / or in the absence of an antigen presenting cell expressing a neo-antigenic peptide:HLA complex (e.g., wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO: 2 or 3, and wherein the HLA is optionally HLA-A*11:01). Nur77 expression can be assayed, for example, using a transgenic expression construct comprising a Nur77 locus operably linked to a sequence encoding a reporter gene construct; for example, dTomato (see Ahsouri and Weiss, J Immunol 198(2):657-668 (2017)).

[0169] In any embodiment disclosed in the present disclosure, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell expresses a HLA-A*02 antigen peptide (e.g., a peptide comprising or consisting of an amino acid sequence shown in SEQ ID NO: 2 or 3). + In the presence of tumor cells, optionally further in the presence of exogenous IFN-γ, the expression of CD137 (also known as 41BB) is increased, wherein the CD137 expression is increased compared to the following: (i) the CD137 expression of a reference immune cell that does not express the binding protein, when the reference immune cell is in the presence of tumor cells; and / or (ii) the CD137 expression of an immune cell expressing the binding protein in the absence of tumor cells and / or in the absence of an antigen presenting cell expressing a new antigenic peptide: HLA complex (e.g., wherein the peptide comprises, is essentially composed of, or is composed of an amino acid sequence shown in SEQ ID NO: 2 or 3, and wherein HLA is optionally HLA-A*11:01). CD137 expression can be determined using, for example, flow cytometry, using a labeled anti-CD137 antibody. In certain embodiments, CD137 is measured using a 16-hour analysis, wherein immune cells are co-cultured with a peptide or a target cell expressing the peptide or stimulated with a peptide or a target cell expressing the peptide.

[0170] In any of the embodiments disclosed in the present disclosure: (i) the binding protein is encoded by a polynucleotide heterologous to the immune cell; (ii) the immune cell comprises a human CD8 + T cells, human CD4+ T cells, or both; (iii) the tumor cells expressing the neoantigenic peptide (e.g., a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 or 3) are HLA-A*11:01 + and / or (iv) the tumor cells comprise OVCAR5 (ovarian serous adenocarcinoma), DAN-G (pancreatic adenocarcinoma), CFPAC1 (pancreatic adenocarcinoma), SW480 (colon cancer), SW527 (breast cancer) or NCI-H441 (lung adenocarcinoma) cells.

[0171] In certain embodiments, the binding protein is capable of binding to the peptide:HLA complex independently of CD8 or in the absence of CD8. CD8-independent binding can be achieved by binding to CD8-negative cells (e.g., CD4 + In some embodiments, a binding protein is expressed in a T cell, a Jurkat cell or a similar cell and the binding of the cell to the target is determined. In some embodiments, a binding protein is provided, comprising: (a) a T cell receptor (TCR) α chain variable (Vα) domain comprising a complementarity determining region 3 (CDR3α) amino acid sequence as shown in any one of SEQ ID NOs: 16, 17, 42 and 43, or a variant thereof having one, two or three optionally conservative amino acid substitutions; and / or (b) a TCR β chain variable (Vβ) domain comprising a CDR3β amino acid sequence as shown in any one of SEQ ID NOs: 26, 27, 52 and 53, or a variant thereof having one, two or three, optionally conservative amino acid substitutions, wherein the binding protein is capable of binding to a peptide: HLA complex, wherein 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 wherein the HLA comprises HLA-A*11. In certain embodiments, the HLA comprises HLA-A*11:01.

[0172] The Vα domain and / or the Vβ domain may be human, humanized or chimeric, and may be human.

[0173] In certain embodiments, the binding protein comprises (i) the CDR3α and CDR3β amino acid sequences shown in SEQ ID NOs: 17 and 27, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; (ii) the CDR3α and CDR3β amino acid sequences shown in SEQ ID NOs: 16 and 26, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; (iii) the CDR3α and CDR3β amino acid sequences shown in SEQ ID NOs: 53 and 43, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; or (iv) the CDR3α and CDR3β amino acid sequences shown in SEQ ID NOs: 52 and 42, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions.

[0174] In some embodiments, the binding protein further: (i) comprises the CDR1α amino acid sequence shown in SEQ ID NO: 14 or 40 in the Vα domain, or a variant thereof having one or two optionally conservative amino acid substitutions; (ii) comprises the CDR2α amino acid sequence shown in SEQ ID NO: 15 or 41 in the Vα domain, or a variant thereof having one or two optionally conservative amino acid substitutions; (iii) comprises the CDR1β amino acid sequence shown in SEQ ID NO: 24 or 50 in the Vβ domain, or a variant thereof having one or two optionally conservative amino acid substitutions; (iv) comprises the CDR2β amino acid sequence shown in SEQ ID NO: 25 or 51 in the Vβ domain, or a variant thereof having one or two optionally conservative amino acid substitutions; or (v) any combination of (i)-(iv).

[0175] In certain embodiments, the binding protein comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences shown in SEQ ID NO: 14, 15, 16 or 17, 24, 25 and 26 or 27, respectively.

[0176] In certain embodiments, the binding protein comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences shown in SEQ ID NO: 40, 41, 42 or 43, 50, 51 and 52 or 52, respectively.

[0177] In some embodiments: (i) the Va domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO: 13 or 39; and / or (ii) the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO: 23 or 49.

[0178] In some embodiments, the Va 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:13, and wherein 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:23.

[0179] In some embodiments, the Va 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 wherein 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.

[0180] In certain embodiments, the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:13 and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:23.

[0181] In certain embodiments, the Va domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:39 and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:49.

[0182] 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.

[0183] For example, the variable domain can comprise 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 insertions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0184] 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 insertions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0185] 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 insertions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0186] One or more insertions may occur at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. One or more insertions may be contiguous, non-contiguous, or a combination thereof.

[0187] 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 deletions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0188] 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 deletions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0189] 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 deletions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0190] One or more deletions may occur at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. One or more deletions may be contiguous, non-contiguous, or a combination thereof.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] One or more substitutions may occur at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. One or more substitutions may be contiguous, non-contiguous, or a combination thereof.

[0195] The binding protein may further comprise a TCR α chain constant domain (Cα) and / or a TCR β chain constant domain (Cβ). The TCR α chain constant domain (Cα) and / or the TCR β chain constant domain (Cβ) may be human. The TCR α chain constant domain (Cα) and / or the TCR β chain constant domain (Cβ) may be mammalian. The TCR α chain constant domain (Cα) and / or the TCR β chain constant domain (Cβ) may be engineered.

[0196] In some embodiments, Ca 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.

[0197] In some embodiments, the 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 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 28, 29, 54, 55, and 70-73.

[0198] In certain embodiments, Ca and Cβ comprise or consist of an amino acid sequence that is 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% identical to, or comprises or consists of, an amino acid sequence as set forth in: (i) SEQ ID NOs: 18 and 28, respectively; (ii) SEQ ID NOs: 19 and 29, respectively; (iii) SEQ ID NOs: 44 and 54, respectively; or (iv) SEQ ID NOs: 45 and 55, respectively.

[0199] The binding protein may comprise (i) an extracellular domain of a TCR α chain, a TCR β chain, a TCR γ chain or a TCR δ chain; (ii) a transmembrane domain of a TCR α chain, a TCR β chain, a TCR γ chain or a TCR δ chain; and / or (iii) a cytoplasmic domain of a TCR α chain, a TCR β chain, a TCR γ chain or a TCR δ chain. The binding protein may comprise a full-length or substantially full-length TCR α chain, a TCR β chain, a TCR γ chain and / or a TCR δ chain.

[0200] 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.

[0201] For example, the binding protein can comprise 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 insertions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0202] In some embodiments, the binding protein 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 insertions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0203] 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 insertions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0204] One or more insertions may occur at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. One or more insertions may be contiguous, non-contiguous, or a combination thereof.

[0205] 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 deletions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0206] In some embodiments, the binding protein 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 deletions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0207] 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 deletions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0208] One or more deletions may occur at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. One or more deletions may be contiguous, non-contiguous, or a combination thereof.

[0209] 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.

[0210] In some embodiments, the binding protein 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: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0211] 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 of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0212] One or more substitutions may occur at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. One or more substitutions may be contiguous, non-contiguous, or a combination thereof.

[0213] In some embodiments, the binding protein comprises a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain 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 sequence shown in the following, or comprise or consist of the amino acid sequence shown in the following: (i) SEQ ID NOs: 12 and 22, respectively; (ii) SEQ ID NOs: 20 and 30, respectively; (iii) SEQ ID NOS: 12 and 30, respectively; (iv) SEQ ID NOs: 20 and 22, respectively; (v) SEQ ID NOs: 38 and 48, respectively; (vi) SEQ ID NOs: 46 and 56, respectively; (vii) SEQ ID NOs: 38 and 56, respectively; or (viii) SEQ ID NOs: 46 and 48, respectively.

[0214] In any embodiment disclosed in the present disclosure, the binding protein may include TCR, single-chain TCR (scTCR), scTv or chimeric antigen receptor (CAR). The method for producing engineered TCR is described in, for example, Bowerman et al., Molecular Immunology (Mol.Immunol.), 46 (15): 3000 (2009), and its technology is incorporated herein by reference. The method for preparing CAR is known in the art and described in, for example, U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO 2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al., 2007, Clinical Cancer Research (Clin.Cancer Res.) 13: 5426, the technology in the document is 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 a CD3 protein. See Elie Dolgin, Nature Biotechnology 40:441-449 (2022).

[0215] Some examples of binding proteins are included in Table 2. In some embodiments, the binding protein comprises an amino acid sequence in 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 a sequence in 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 a sequence in 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 a sequence in Table 2. In some embodiments, the binding protein comprises a sequence that is 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% identical to a sequence in Table 2. In some embodiments, the binding protein comprises a sequence that is 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% identical to a sequence in Table 2. In some embodiments, the binding protein comprises a sequence that is at most 90%, at most 85%, at most 80%, at most 75%, at most 70%, at most 65%, or at most 60% identical to a sequence in 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 with the sequence in Table 2, or a range defined by any two of the foregoing percentages. In some embodiments, the binding protein includes a fragment of any of the foregoing sequences. In some embodiments, the binding protein includes any combination of any of the foregoing sequences. Any of the foregoing binding proteins or binding protein sequences may be applicable to the methods or compositions described herein. For example, the binding protein can be included in a cell with a fusion protein that includes components of CD95 (Fas) and CD137 (4-1BB) and / or a CD8αβ coreceptor (eg, an exogenous CD8αβ coreceptor).

[0216] In any embodiment disclosed in the present disclosure, the polynucleotide encoding the binding protein may further comprise: (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, wherein optionally, 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, wherein optionally, the encoded polypeptide is or comprises the CD8 coreceptor β chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii). Without being bound by theory, in certain embodiments, co-expression or concurrent expression of the binding protein with a CD8 coreceptor protein or a portion thereof having the function of binding to an HLA molecule can improve host cells (e.g., immune cells, such as T cells, optionally CD4 + It should be understood that the polynucleotide encoding the binding protein and the polynucleotide encoding the CD8 co-receptor polypeptide may be present on a single nucleic acid molecule (e.g., in the same expression vector), or may be present on separate nucleic acid molecules in the host cell.

[0217] In any embodiment disclosed in 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.

[0218] In any embodiment disclosed in 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.

[0219] In certain other embodiments, the polynucleotide comprises: (a) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 coreceptor alpha chain; (b) a polynucleotide encoding a polypeptide comprising an extracellular portion of a 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 other embodiments, the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between: (1) a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 coreceptor alpha chain; and / or (2) a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 coreceptor beta chain.

[0220] In other embodiments, the polynucleotide may comprise the following operably linked in frame: (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)-(pn or (vi) (pnCD8β)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, wherein pnBP is a polynucleotide encoding a binding protein, and wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different (e.g., P2A, T2A, F2A, E2A).

[0221] It should be understood that the self-cleaving peptide may include a linker at its 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 the amino acid sequence of GSG and SEQ ID NO.: 75. In some embodiments, the GSG-P2A sequence comprises, consists essentially of, or consists of SEQ ID NO.: 74.

[0222] In certain embodiments, the encoded binding protein comprises a TCR α chain and a TCR β chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding the TCR α chain and the polynucleotide encoding the TCR β chain. In other embodiments, the polynucleotide comprises the following operably linked in frame: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα); (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα); (iii) (pnCD8α)-(pnSCP1)-(pn CD8β)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (iv)(pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRα)-(pn SCP3)-(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)-(pnCD 8α), wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, wherein pnTCRα is a polynucleotide encoding the TCRα chain, wherein pnTCRβ is a polynucleotide encoding the TCRβ chain, and wherein pnSCP1, pnSCP2 and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0223] In certain embodiments, the encoded polypeptides of the present disclosure comprise one or more junction amino acids. "Joint amino acids" or "junction amino acid residues" refer to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide (e.g., between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide). The junction amino acids may be generated by the design of the construct encoding the fusion protein (e.g., during the construction of the nucleic acid molecule encoding the fusion protein, amino acid residues generated by the use of restriction enzyme sites); or by cleavage of, for example, a self-cleaving peptide adjacent to one or more domains of the encoded binding protein of the present disclosure (e.g., a P2A peptide disposed between the TCR α chain and the TCR β chain, which self-cleavage may leave one or more junction amino acids in the α chain, the TCR β chain, or both).

[0224] In other embodiments, the binding protein is expressed as part of a transgenic construct, which encodes and / or the host cell of the present disclosure may encode: one or more other auxiliary proteins, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor β chain; a CD8 co-receptor α chain or both; or any combination thereof. Polynucleotides and transgenic constructs suitable for encoding and expressing binding proteins and auxiliary components (e.g., one or more of a safety switch protein, a selection marker, a CD8 co-receptor β chain, or a CD8 co-receptor α chain) are described in PCT application PCT / US2017 / 053112, wherein the polynucleotides, transgenic constructs, and auxiliary components (including nucleotide and amino acid sequences) are incorporated herein by reference. It should be understood that any or all binding proteins, safety switch proteins, tags, selection markers, CD8 co-receptor β chains, or CD8 co-receptor α chains disclosed herein may be encoded by a single nucleic acid molecule or may be encoded by a polynucleotide sequence located or present on a separate nucleic acid molecule.

[0225] Exemplary safety switch proteins include, for example, a truncated EGF receptor polypeptide (huEGFRt) lacking an extracellular N-terminal ligand binding domain and intracellular receptor tyrosine kinase activity but retaining its native amino acid sequence, having a type I transmembrane cell surface localization, and having a conformationally intact epitope that binds to the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011); apoptotic protease polypeptides (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; DiStasi et al., N. Engl. J. Med. 365:1673-1684, 2006). 3, 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 human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Sci. USA 105:623-628, 2008); and marker / safety switch polypeptides, such as RQR (CD20+CD34; Philip et al., 2014).

[0226] Other auxiliary components suitable for use in the modified host cells of the present disclosure include tags or selection markers that allow identification, sorting, separation, enrichment or tracking of cells. For example, labeled host cells with desired characteristics (e.g., antigen-specific TCRs and safety proteins) can be sorted out from a sample to separate from unlabeled cells and more efficiently activated and amplified for inclusion in a product of desired purity.

[0227] As used herein, the term "selection marker" includes nucleic acid constructs (and encoded gene products) that confer identifiable changes to cells, thereby allowing detection and positive selection of immune cells transduced by polynucleotides comprising selection markers. RQR is a selection marker comprising the main 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 co-receptor handle domain (Q8). In other embodiments, the CD34 minimal binding site sequence can be combined with the target epitope of CD20 to form a compact marker / suicide gene (RQR8) of T cells (Philip et al., 2014, which is incorporated herein by reference). This construct allows selection of host cells expressing the construct, for example using CD34-specific antibodies bound to magnetic beads (Miltenyi) and utilizing the clinically established pharmaceutical antibody rituximab to selectively delete engineered T cells expressing the transgene (Philip et al., 2014).

[0228] Other exemplary selection markers include several truncated type I transmembrane proteins that are not normally expressed on T cells: truncated low affinity nerve growth factor, truncated CD19, and truncated CD34 (see, e.g., 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 in its entirety). The useful features of CD19 and CD34 are readily available Miltenyi CliniMACs TMThe availability of the selection system, which can target these markers in clinical grade sorting. However, CD19 and CD34 are relatively large surface proteins, which can produce a heavy burden on the transcription efficiency of the vector packaging capacity and the integration vector. Surface markers containing extracellular non-signaling domains or various proteins (such as CD19, CD34, LNGFR) can also be used. Any selection marker (such as acceptable to Good Manufacturing Practices) can be used. In certain embodiments, the selection marker is expressed together with a polynucleotide encoding a gene product of interest (such as a binding protein of the present disclosure, such as TCR or CAR). Other examples of selection markers include, for example, reporter factors, such as GFP, EGFP, β-gal or chloramphenicol (chloramphenicol) acetyltransferase (CAT). In certain embodiments, selection markers (such as CD34) are expressed by cells and CD34 can be used to select, enrich or separate (such as by immunomagnetic selection) for transduced cells of interest in the methods described herein. As used herein, a CD34 marker is distinguished from an anti-CD34 antibody, or, for example, a scFv, TCR, or another antigen recognition moiety that binds to CD34.

[0229] In certain embodiments, the selection marker comprises an RQR polypeptide, truncated low affinity nerve growth factor (tNGFR), truncated CD19 (tCD19), truncated CD34 (tCD34), or any combination thereof.

[0230] With respect to the RQR polypeptide, without wishing to be bound by theory, it is believed that the distance from the host cell surface plays an important role for the RQR polypeptide to act as a selection marker / safety switch (Philip et al., 2010 (supra)). In some embodiments, the encoded RQR polypeptide is contained in the β chain, α chain, or both of the encoded CD8 coreceptor, or a fragment or variant of either or both. In specific embodiments, 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 an RQR polypeptide and further comprising a CD8 α chain.

[0231] In some embodiments, the encoded CD8 coreceptor includes an alpha chain or a fragment or variant thereof. The amino acid sequence of the human CD8 coreceptor alpha chain precursor is known and provided, for example, in UniProtKB-P30433 (see also UniProtKB-P31783; UniProtKB-P10732; and UniProtKB-P10731). In some embodiments, the encoded CD8 coreceptor includes a beta chain or a fragment or variant thereof. The amino acid sequence of the human CD8 coreceptor beta chain precursor is known and provided, for example, in UniProtKB-P10966 (see also UniProtKB-Q9UQ56; UniProtKB-E9PD41; UniProtKB-Q8TD28; and UniProtKB-P30434; and UniProtKB-P05541).

[0232] The isolated polynucleotides of the present disclosure may further comprise a polynucleotide encoding a safety switch protein, a selection marker, a CD8 coreceptor β chain, or a CD8 coreceptor α chain as disclosed herein, or may comprise a polynucleotide encoding any combination thereof.

[0233] In any of the embodiments disclosed herein, the polynucleotides may be codon optimized for expression in a host cell. In some embodiments, the host cell comprises a human immune system cell, such as a T cell, a NK cell, or a NK-T cell (Skolten et al., Clin. Immunol. 119:135, 2006). Codon optimization may be performed using known techniques and tools, such as using OptimumGene TM Tools or GeneArt (Life Technologies). Codon-optimized sequences include partially codon-optimized sequences (i.e., one or more codons are optimized for expression in a host cell) and fully codon-optimized sequences. It should be understood that in embodiments where the polynucleotide encodes more than one polypeptide (e.g., a TCR alpha chain, a TCR beta chain, a CD8 coreceptor alpha chain, a CD8 coreceptor beta chain, and one or more self-cleaving peptides), each polypeptide may be independently fully codon-optimized, partially codon-optimized, or not codon-optimized.

[0234] The amino acid and polynucleotide sequences of, for example, binding proteins "11N4A" and "11N6" are shown in Table 1.

[0235]

[0236]

[0237] Carrier

[0238] In another aspect, the present disclosure provides an expression vector comprising any polynucleotide as provided herein operably linked to an expression control sequence.

[0239] Also provided herein are vectors comprising polynucleotides or transgenic constructs disclosed herein.Some examples of vectors include plasmids, viral vectors, cosmids and other.Some vectors may be able to replicate autonomously in the host cell introduced therein (e.g., bacterial vectors and free mammalian vectors with bacterial replication origins), while other vectors may be integrated into the genome of the host cell, or promote the integration of polynucleotide inserts after being introduced into the host cell, thereby replicating with the host genome (e.g., slow virus vectors, retroviral vectors). In addition, some vectors can guide the expression of genes operably connected thereto (these vectors may be referred to as "expression vectors"). According to relevant embodiments, it should be further understood that if one or more agents (e.g., polynucleotides encoding polypeptides as described herein) are co-administered to an individual, each agent may reside in a separate or same vector, and multiple vectors (each containing different agents or the same agent) may be introduced into a cell or cell group or administered to an individual.

[0240] In certain embodiments, the polynucleotides of the present disclosure are operably linked to certain elements of the vector. For example, the polynucleotide sequences required for expression and processing of the linked coding sequence may be operably linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficacy (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when possible. If the expression control sequence is adjacent to the gene of interest and the expression control sequence acts on the opposite side or at a distance to control the gene of interest, the expression control sequence may be operably linked.

[0241] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from a lentiviral vector or a γ-retroviral vector). Viral vectors include retroviruses; adenoviruses; microviruses (e.g., adeno-associated viruses); coronaviruses; negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai); positive-strand RNA viruses, such as picornaviruses and alphaviruses; and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 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 virus, mammalian C-type virus, B-type virus, D-type virus, HTLV-BLV virus group, lentivirus and foamy virus (Coffin, JM, Retroviridae: The viruses and their replication, Fundamental Virology, 3rd edition, BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0242] "Retrovirus" is a virus with an RNA genome that is reverse transcribed into DNA using a reverse transcriptase, and the reverse transcribed DNA is subsequently incorporated into the host cell genome. "γ-retrovirus" refers to a genus of the Retroviridae family. Examples of γ-retroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendothelial cell proliferation virus. As used herein, "lentiviral vector" means a HIV-based lentiviral vector for gene delivery, which can be integrated or non-integrated, has a relatively large packaging capacity, and can transduce a range of different cell types. Slow virus vectors are usually produced after three (encapsulation, envelope, and transfer) or more plasmids are transiently transfected into production cells. Like HIV, slow virus vectors enter target cells via the interaction of viral surface glycoproteins with receptors on the cell surface. After entry, viral RNA is reversely transcribed, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is a substrate for viral integration into the DNA of infected cells.

[0243] In certain embodiments, the viral vector may be a gamma retrovirus, such as a Moloney murine leukemia virus (MLV) derived vector. In other embodiments, the viral vector may be a more complex retrovirus derived vector, such as a lentivirus derived vector. HIV-1 derived vectors belong to this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (sheep lentivirus). Methods of using retroviral and lentiviral vectors and packaging cells to transduce mammalian host cells with viral particles containing TCR or CAR transgenes are known in the art and have been previously described, for example, in: U.S. Pat. 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 can also be used for polynucleotide delivery including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0244] Other vectors developed for gene therapy use can also be used with the compositions and methods of the present disclosure. Such vectors include vectors derived from baculovirus and alpha-virus (Jolly, DJ. 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 (e.g., Sleeping Beauty or other transposon vectors).

[0245] When the viral vector genome comprises multiple polynucleotides to be expressed as separate transcripts in a host cell, the viral vector may also comprise additional sequences between the two (or more) transcripts that allow bicistronic or polycistronic expression. Examples of such sequences for viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0246] In certain embodiments, the vector is capable of delivering a polynucleotide or transgenic construct to a host cell (e.g., a hematopoietic progenitor cell or a human immune system cell). In certain embodiments, the vector is capable of delivering a polynucleotide or transgenic construct to a human immune system cell, such as a CD4 + T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, stem cell memory T cells, γδT cells, natural killer cells, dendritic cells or any combination thereof. In other embodiments, the vector can deliver the transgenic construct to initial T cells, central memory T cells, effector memory T cells or any combination thereof. In some embodiments, the vector encoding the polynucleotides of the present disclosure or the transgenic construct may further include a polynucleotide encoding a nuclease (e.g., CRISPR-Cas endonuclease or another endonuclease as disclosed herein) that can be used for chromosome elimination in host cells or encoding a nuclease that can be used for gene replacement therapy or gene repair therapy to deliver therapeutic polynucleotides or transgenes or a portion thereof to host cells. Alternatively, the nuclease for chromosome elimination or gene replacement or gene repair therapy can be delivered to the host cell independently of the vector encoding the polynucleotides of the present disclosure or the transgenic construct.

[0247] In certain embodiments, the vector is capable of delivering the polynucleotide to a host cell. In other embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In other embodiments, the human immune system cell is a CD4+T cell, a CD8+T cell, a CD4-CD8-double negative T cell, a γδT cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof. In other embodiments, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0248] In any embodiment disclosed in the present disclosure, the vector is a viral vector. In certain embodiments, the viral vector is a lentiviral vector or a γ-retroviral vector.

[0249] Host cells

[0250] Also provided herein are host cells encoding and / or expressing binding proteins (and optionally, one or more accessory proteins, such as transduction markers, CD8 co-receptor polypeptides or the like, as provided herein). In certain embodiments, host cells modified to contain polynucleotides and / or expression vectors of the present disclosure and / or to express binding proteins of the present disclosure are provided.

[0251] Any suitable host cell can be modified to include a heterologous polynucleotide encoding a binding protein of the present disclosure, including, for example, an immune cell, such as a T cell, a NK cell, or a NK-T cell. In some embodiments, the modified immune cell comprises a CD4 + T cells, CD8 + T cells or both. Methods for transfecting / transducing T cells with desired nucleic acids have been described (e.g., U.S. Patent Application Publication No. US2004 / 0087025) using a donor-acceptor transfer procedure of T cells with 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. 19:114, 2009; Wang et al., Hum. Gene Ther. 20:1261, 2008). Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011 / 0243972; US 2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), so that based on the teachings herein, it is considered to adapt these methods to the currently disclosed embodiments.

[0252] Any suitable method can be used for transfection or transduction of cells, such as T cells, or polynucleotides or compositions of the disclosed methods. Known methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercial products, such as IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-polydextrose, acoustic loading, lipid-mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, and similar methods thereof. Other methods for transfection or transduction of host cells are to use the carriers described in more detail herein.

[0253] In certain embodiments, the host cell or modified cell may be a peripheral blood mononuclear cell (PBMC). The host cell may be a lymphocyte. The host cell may be a lymphocyte. In certain embodiments, the host cell or modified cell may be a hematopoietic progenitor cell and / or a human immune cell. In certain embodiments, the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof. In certain embodiments, the host or modified cell is a mammalian cell (e.g., a human cell or a mouse cell). In other embodiments, the immune cell comprises a CD4+T cell, a CD8+T cell, a CD4-CD8-double negative T cell, a γδT cell, or any combination thereof. In certain other embodiments, the immune cell comprises a CD4+T cell and a CD8+T cell. In certain other embodiments, the CD4+ T cells, CD8+ T cells, or both comprise: (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 coreceptor α chain, wherein optionally, the encoded polypeptide is or comprises a CD8 coreceptor α chain; (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 coreceptor β chain, wherein optionally, the encoded polypeptide is or comprises a CD8 coreceptor β chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

[0254] In any of the foregoing embodiments, the host cell (e.g., immune cell) may be modified to reduce or eliminate the expression of one or more endogenous genes encoding polypeptides involved in immune signaling or other related activities. Example gene knockouts include those encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, HLA molecules, TCR molecules, or the like. Without wishing to be bound by theory, an allogeneic host receiving a modified immune cell may recognize certain endogenously expressed immune cell proteins as foreign matter, thereby causing the elimination of the modified immune cell (e.g., HLA allele), or may downregulate the immune activity of the modified immune cell (e.g., PD-1, LAG-3, CTLA4, FasL, TIGIT, TIM3), or may interfere with the binding activity of the heterologously expressed binding protein of the present disclosure (e.g., endogenous TCR of a modified T cell, which binds to a non-Ras antigen and thereby interferes with the binding of the modified immune cell to cells expressing, for example, Ras antigens).

[0255] Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerance or persistence of the modified cells in an autologous or allogeneic host environment, and can allow the administration of the cells to be universal (e.g., administration 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 present disclosure comprise a chromosomal knockout of one or more genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA component (e.g., a gene encoding α1 macroglobulin, α2 macroglobulin, α3 macroglobulin, β1 microglobulin, or β2 microglobulin), or a TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., 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 recipient cell therapies of which are incorporated herein by reference in their entireties).

[0256] As used herein, the term "chromosomal gene knockout" generally refers to a gene altering agent or an introduced inhibitor that prevents (e.g., reduces, delays, inhibits, or eliminates) the host cell from producing a functionally active endogenous polypeptide product. Changes that result in chromosomal gene knockout may include, for example, the introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and strand breaks, as well as heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in host cells.

[0257] In certain embodiments, chromosomal gene knockout or gene insertion is achieved by editing the chromosome of the host cell. Chromosome editing can be performed using, for example, an endonuclease. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of a phosphodiester bond in a polynucleotide chain. In certain embodiments, an endonuclease can cleave a target gene, thereby inactivating or "knocking out" the target gene. An endonuclease can be a naturally occurring, recombinant, genetically modified, or fused endonuclease. The nucleic acid chain breaks caused by an endonuclease are usually repaired via different mechanisms of homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, donor nucleic acid molecules can be used for "embedding" of donor genes, "knockout" of target genes, and optionally inactivating target genes via donor gene embedding or target gene knockout events. NHEJ is an error-prone repair process that usually results in changes in the DNA sequence of the cleavage site, such as 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.

[0258] 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 (e.g., FokI endonuclease). Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and the amino acids at certain residues can be changed to change the triple sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolf et al., J. Mol. Biol. 285:1917-1934, 1999). Multiple zinc finger motifs can be connected in series to produce binding specificity for a desired DNA sequence, e.g., a region ranging in length from about 9 to about 18 base pairs. As background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and promote targeted integration of transgenes containing flanking sequences homologous to the genome at the DSB site through homology-directed repair. Alternatively, DSBs generated by ZFNs can lead to gene knockout of the target gene via repair via non-homologous end joining (NHEJ), an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, gene knockout comprises insertions, deletions, mutations, or a combination thereof performed using ZFN molecules.

[0259] As used herein, "transcription activator-like effector nuclease" (TALEN) generally refers to a fusion protein comprising a TALE DNA binding domain and a DNA cleavage domain (e.g., FokI endonuclease). "TALE DNA binding domain" or "TALE" comprises one or more TALE repeat domains / units, each domain / unit generally having a highly conserved sequence of 33-35 amino acids, wherein the 12th and 13th amino acids are different. The TALE repeat domain participates in the binding of TALE to the target DNA sequence. The different amino acid residues, referred to as repeat variable di-residues (RVD), are associated with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined, so that the HD (histidine-aspartic acid) sequence at positions 12 and 13 of TALE causes TALE to bind to cytosine (C), NG (asparagine-glycine) to bind to T nucleotides, NI (asparagine-isoleucine) to bind to A, NN (asparagine-asparagine) to bind to G or A nucleotides, and NG (asparagine-glycine) to bind to T nucleotides. Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent 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 T cell genome. Non-homologous end joining (NHEJ) joins DNA from both sides of the double-strand break, with little or no sequence overlap for bonding, thereby introducing errors that knock out gene expression. Alternatively, homology-directed repair can introduce a transgene at the DSB site, subject to the presence of homologous flanking sequences in the transgene. In certain embodiments, gene knockout comprises insertion, deletion, mutation, or a combination thereof, and is performed using TALEN molecules.

[0260] As used herein, "Clustered Regularly Interspaced Short Palindromic Repeats / Cas" (CRISPR / Cas) nuclease system generally refers to a system that uses CRISPR RNA (crRNA)-guided Cas nucleases to recognize target sites (called protospacers) within the genome via base pairing complementarity, and then cleave DNA if a short conserved protospacer-associated motif (PAM) follows the 3' of the complementary target sequence. Based on the sequence and structure of the Cas nuclease, the CRISPR / Cas system is classified into three types (i.e., type I, type II, and type III). The crRNA-guided monitoring complexes of type I and type III require multiple Cas subunits. The most studied type II system comprises at least three components: RNA-guided Cas9 nuclease, crRNA, and lateral acting crRNA (tracrRNA). TracrRNA comprises a double helix forming region. CrRNA and tracrRNA form a double helix, which can interact with Cas9 nuclease, and through the Watson-Crick base-pairing between the prototype spacer on the crRNA and the PAM upstream of the target DNA, the Cas9 / crRNA:tracrRNA complex is guided to a specific site on the target DNA. Cas9 nuclease cleaves double-strand breaks in the region defined by the crRNA spacer. Repair by NHEJ leads to insertion and / or deletion, thereby destroying the expression of the targeted locus. Alternatively, a transgenic with a homologous flanking sequence can be introduced at the site of the DSB via homology-directed repair. CrRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, for example, Jinek et al., Science 337:816-21, 2012). In addition, the region of the guide RNA complementary to the target site can be altered or programmed to target the desired sequence (Xie et al., PLOS One 9: e100448, 2014; U.S. Patent Application Publication No. US2014 / 0068797, U.S. Patent Application Publication No. US2014 / 0186843; U.S. Patent No. 8,697,359 and PCT Publication No. WO 2015 / 071474; each of which is incorporated herein by reference). In certain embodiments, gene knockout comprises insertion, deletion, mutation, or a combination thereof, and is performed using a CRISPR / Cas nuclease system.

[0261] Example gRNA sequences and methods of using them to knock out endogenous genes encoding immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNA, CAS9 DNA, vectors, and gene knockout techniques therein are incorporated herein by reference in their entirety.

[0262] As used herein, "meganuclease" is also referred to as "homing endonuclease", and generally refers to deoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-Scel, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-ScelI, I-PpoI, I-ScelIII, I-Crel, I-TevI, I-TevII, and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Pat. 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. 34:116-117, 1989). 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).

[0263] In certain embodiments, naturally occurring meganucleases can be used to facilitate site-specific genomic modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, HLA encoding genes, or TCR component encoding genes. In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genome modification (see, e.g., Porteus et al., 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 Therapy 442:657-661, 2008; Ther.) 7: 49-66, 2007; U.S. Patent Publication Nos. US2007 / 0117128; US2006 / 0206949; US2006 / 0153826; US2006 / 0078552; and US2004 / 0002092). In other embodiments, chromosomal gene knockout is produced using homing endonucleases modified with the modular DNA binding domains of TALEN to produce fusion proteins called megaTALs. MegaTALs can be used not only to knock out one or more target genes, but also to introduce (embed) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.

[0264] In certain embodiments, the chromosomal gene knockout comprises introducing an inhibitory nucleic acid molecule into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor-associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression in the host cell (e.g., PD-1, TIM3, LAG3, CTLA4, TIGIT, FasL, an HLA component, or a TCR component, or any combination thereof).

[0265] In certain embodiments, the gene knockout comprises an insertion, a deletion, a mutation, or a combination thereof and is produced using a CRISPR / Cas nuclease system or a base editing system (Komor, AC; Kim, YB; Packer, MS; Zuris, JA; Liu, DR, Nature 533, 420-424 (2016). Briefly, base editing is a genome editing method that uses components of a CRISPR system and other enzymes to introduce point mutations directly into cellular DNA or RNA without generating double-stranded DNA breaks. Certain DNA base editors comprise a catalytically incapable nuclease fused to a nucleobase deaminase and in some cases a DNA glycosidase inhibitor. RNA base editors perform similar functions using components that target RNA. Base editors convert one base or base pair directly into another base or base pair, enabling efficient installation of point mutations in non-dividing cells without generating undesirable excess editing byproducts. See, e.g., Rees et al., Nature 533, 420-424 (2016). H) et al., Nature Reviews Genetics (2018).

[0266] After using a gene knockout procedure or agent, chromosomal gene knockout can be directly confirmed by DNA sequencing of host immune cells. Chromosomal gene knockout can also be inferred from the absence of gene expression (e.g., absence of mRNA or polypeptide product encoded by the gene) after gene knockout.

[0267] In certain embodiments, the chromosomal gene knockout comprises a knockout of an HLA component gene selected from the group consisting of an α1 macroglobulin gene, an α2 macroglobulin gene, an α3 macroglobulin gene, a β1 microglobulin gene, or a β2 microglobulin gene.

[0268] In certain embodiments, the chromosomal gene knockout comprises a knockout of a TCR component gene, wherein the TCR component gene is selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or a combination thereof.

[0269] In some embodiments, a population of host cells comprising a binding protein 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 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 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21-fold, at least 22-fold, at least 23-fold, at least 24-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold At least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1000 times, or at least 5000 times. The host cell may comprise a binding protein (e.g., a TCR comprising a Vα region and a Vβ region and / or CDRs disclosed herein) that binds to an antigen of interest (e.g., a neoantigen (e.g., p53PIK3CA, NRAS, HRAS, or KRAS (e.g., a KRAS G12 mutant peptide, e.g., a KRASG12V mutant peptide, e.g., present in a peptide:HLA complex)). The increase in avidity can be determined, for example, by an assay for determining expression of activation markers (e.g., CD137, CD69, granzyme B, CD107a, IFN-γ, TNF-α, IL-12, cytokines, interleukins, interferons) following exposure to a target cell expressing or presenting the antigen of interest; and / or an assay for determining EC50 (e.g., the peptide dose at which the T cell population achieves half-maximal activation).

[0270] Host cell compositions and unit dosages

[0271] In another aspect, provided herein are compositions and unit dosages comprising the modified host cells of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0272] In certain embodiments, the host cell composition or unit dose comprises (i) 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) 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 +

[0013] The invention also provides a composition of T cells 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 or substantially no naive T cells (i.e., having less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5% or less than about 1% of the population of naive T cells present in the unit dose compared to a patient sample having a comparable number of peripheral blood mononuclear cells (PBMCs).

[0273] In some embodiments, the host cell composition or unit dose comprises (i) a host cell comprising at least about 50% modified CD4 + T cells, and (ii) comprising at least about 50% modified CD8 + In other embodiments, the host cell composition or unit dose comprises (i) at least about 60% modified CD4 T cells. + T cells, and (ii) comprising at least about 60% modified CD8 + T cells 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 or substantially no naive T cells. In another embodiment, the host cell composition or unit dose comprises (i) at least about 70% engineered CD4 + T cells, and (ii) comprising at least about 70% engineered CD8 +T cells 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 or substantially no naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 80% modified CD4 + T cells, and (ii) comprising at least about 80% modified CD8 + T cells 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 host cell composition or unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 85% modified CD4 + T cells, and (ii) comprising at least about 85% modified CD8 + T cells 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 host cell composition or unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 90% modified CD4 + T cells, and (ii) comprising at least about 90% modified CD8 + T cells 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 host cell composition or unit dose contains reduced or substantially no naive T cells.

[0274] In some embodiments, the composition comprises a CD4+ cell population comprising (i) 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 (ii) 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.

[0275] In some embodiments, the host cell composition or unit dose comprises about a 1:1 ratio, about a 1:2 ratio, about a 1:3 ratio, about a 1:4 ratio, about a 1:5 ratio, about a 1:6 ratio, about a 1:7 ratio, about a 1:8 ratio, about a 1:9 ratio, about a 1:10 ratio, about a 2:1 ratio, about a 3:1 ratio, about a 4:1 ratio, about a 5:1 ratio, about a 6:1 ratio, about a 7:1 ratio, about an 8:1 ratio, about a 9:1 ratio, about a 10:1 ratio, about a 3:2 ratio, or about a 2:3 ratio of CD4+ to CD8+ T cells (e.g., CD4+ T cells modified to comprise or express a binding protein disclosed herein and CD8+ T cells modified to comprise or express a binding protein disclosed herein).

[0276] In some embodiments, the ratio of CD4+ to CD8+ T cells in a host cell composition or unit dose is 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.

[0277] In some embodiments, the ratio of CD4+ to CD8+ T cells in a host cell composition or unit dose 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.

[0278] In some embodiments, the ratio of CD4+ to CD8+ T cells in a host cell composition or unit dose is between about 1:10 and 10:1, 1:10 and 8:1, 1:10 and 7:1, 1:10 and 6:1, 1:10 and 5:1, 1:10 and 4:1, 1:10 and 3:1, 1:10 and 2:1, 1:10 and 1:1, 1:10 and 1:2, 1:10 and 1:3, 1:10 and 1:4, 1:10 and 1: 5, 1:10 and 1:7, 1:5 and 10:1, 1:5 and 8:1, 1:5 and 7:1, 1:5 and 6:1, 1:5 and 5:1, 1:5 and 4:1, 1:5 and 3:1, 1:5 and 2:1, 1:5 and 1:1, 1:5 and 1:2, 1:5 and 1:3, 1:5 and 1:4, 1:3 and 10:1, 1:3 and 8:1, 1:3 and 7:1, 1:3 and 6:1, 1:3 and 5:1, 1:3 and 4:1 , 1:3 and 3:1, 1:3 and 2:1, 1:3 and 1:1, 1:3 and 1:2, 1:2 and 10:1, 1:2 and 8:1, 1:2 and 7:1, 1:2 and 6:1, 1:2 and 5:1, 1:2 and 4:1, 1:2 and 3:1, 1:2 and 2:1, 1:2 and 1:1, 1:1 and 10:1, 1:1 and 8:1, 1:1 and 7:1, 1:1 and 6:1, 1:1 and 5:1, 1:1 and 4:1, 1 :1 and 3:1, 1:1 and 2:1, 2:1 and 10:1, 2:1 and 8:1, 2:1 and 7:1, 2:1 and 6:1, 2:1 and 5:1, 2:1 and 4:1, 2:1 and 3:1, 3:1 and 10:1, 3:1 and 8:1, 3:1 and 7:1, 3:1 and 6:1, 3:1 and 5:1, 3:1 and 4:1, 5:1 and 10:1, 5:1 and 8:1, 5:1 and 7:1 or 5:1 and 6:1.

[0279] The CD4+ T cells in the composition, host cell composition or unit dose can be CD4+ T cells that have been modified or engineered (eg, using a vector or polynucleotide disclosed herein) to express a CD8 coreceptor disclosed herein.

[0280] It should be understood that the host cell compositions or unit doses of the present disclosure may comprise any host cell or any combination of host cells as described herein. In certain embodiments, for example, the host cell composition or unit dose comprises modified CD8+ T cells, modified CD4+ T cells, or both, wherein 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 comprise any host cell or host cell combination as described herein, and may further comprise a modified cell (e.g., an immune cell, such as a T cell) expressing a binding protein specific for a different antigen (e.g., a different Ras antigen, or an antigen from a different protein or target, 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, Y), TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., including MAGE-A1, MAGE-A3 and MAGE-A4), mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor or pathogen associated peptides bound to HLA, hTERT peptide bound to HLA, tyrosine Acidase peptide, WT-1 peptide 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, Robol, α-fetoprotein (AFP), Frizzled, OX40, PRAME and SSX-2, or the like). In some embodiments, the binding protein binds to a peptide (e.g., a different antigen presented above) complexed with an HLA protein (e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, HLA-H, HLA-J, HLA-K or HLA-L). For example, a unit dose may include a modified CD8 + T cells and modified CD4 T cells expressing a binding protein (e.g., CAR) that specifically binds to the PSMA antigen + T cells (and / or modified CD8 + T cells). It will also be appreciated that any of the host cells disclosed herein can be administered in combination therapy.

[0281] 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 cell.

[0282] In any of the embodiments described herein, the host cell composition or unit dose comprises one or more cell populations (eg, CD4+ or CD8+ cells) that have undergone CD62L positive selection, eg, to improve in vivo persistence.

[0283] Host cells can be genetically engineered ex vivo, in vitro or in vivo to contain or express a binding protein.

[0284] use

[0285] In other aspects, the present disclosure provides methods for treating or preventing recurrence of a disease or condition associated with a KRAS G12V or NRAS G12V mutation or a HRAS G12V mutation in an individual. Such diseases or conditions include, for example, cancer, such as solid cancers and hematological malignancies. In certain exemplary embodiments, the disease or disorder comprises pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; gallbladder cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally a myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma, such as non-Hodgkin's 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; histiocyte and dendritic cell carcinoma blast cell neoplasm; 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; unclassifiable myelodysplastic / myeloproliferative neoplasm; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdomyosarcoma; neurothecoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma.

[0286] "Treat" or "treatment" or "ameliorate" generally refers to the medical management of a disease, disorder or condition in an individual (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse or rat). In general, an appropriate dose or treatment regimen comprising a composition of the present disclosure (e.g., comprising a binding protein, polynucleotide, vector, host cell, host cell composition, unit dose and / or immunogenic polypeptide) is administered in an amount sufficient to induce a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefits include improved clinical outcome; alleviation or alleviation of symptoms associated with the disease; reduction in the occurrence of symptoms; improved quality of life; longer disease-free state; attenuation of disease extent; stabilization of disease condition; delayed disease progression; remission; survival; prolonged survival; or any combination thereof.

[0287] As used herein, "therapeutically effective amount" or "effective amount" generally refers to an amount of a composition sufficient to produce a therapeutic effect, including a statistically significant improvement in clinical outcome; a reduction or alleviation of symptoms associated with the disease; a reduction in the occurrence of symptoms; an improvement in quality of life; a longer disease-free state; a reduction in the extent of the disease; a stabilization of the disease condition; a delay in disease progression; remission; survival; or an extension of survival. When referring to individual active ingredients administered alone or cells expressing a single active ingredient, a therapeutically effective amount refers to the effect of the ingredient alone or the cell expressing the ingredient. When referring to a combination, a therapeutically effective amount refers to the combined amount of the active ingredient or the combination auxiliary active ingredient and the cell expressing the active ingredient, whether administered consecutively or simultaneously, to produce a therapeutic effect. The combination can also be a cell expressing more than one active ingredient.

[0288] The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refers to a biologically compatible vehicle that is suitable for administration to humans or other non-human mammalian subjects and is generally considered safe or does not cause serious adverse events, such as physiological saline, which is described in more detail herein.

[0289] As used herein, "statistically significant" generally refers to a p-value of 0.050 or less when calculated using the Students t-test, or a value or indicator of statistical significance using another appropriate statistical test, and indicates that the particular event or result measured is unlikely to occur by chance.

[0290] In general, individuals that can be treated according to the present disclosure are generally humans and other primate individuals, such as monkeys and apes for veterinary purposes. In any of the aforementioned embodiments, the individual can be a human individual. The individual can be a mammal. The individual can be male or female and can be any suitable age, including infants, young children, youth, adults and elderly individuals. Compositions according to the present disclosure can be administered in a manner suitable for treating diseases, conditions or illnesses as determined by personnel skilled in medical technology. In any of the above embodiments, modified host cells, host cell compositions or unit doses as described herein are administered intravenously, intraperitoneally, intratumorally to bone marrow, lymph nodes or cerebrospinal fluid in order to encounter target cells (e.g., leukemia cells). The appropriate dose, suitable duration and frequency of administration of the composition will be determined based on factors such as: the patient's condition; the size, type and severity of the disease, condition or illness; the specific form of the active ingredient; and the method of administration.

[0291] As used herein, the term "recipient immunotherapy" or "recipient immunotherapy" generally refers to the administration of naturally occurring or genetically engineered disease or antigen-specific immune cells (e.g., T cells). Recipient cellular immunotherapy can be autologous (immune cells come from the recipient), allogeneic (immune cells come from a donor of the same species), or syngeneic (immune cells come from a donor that is genetically identical to the recipient).

[0292] In some embodiments, the individual expresses a Ras antigen comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-3 or consisting thereof.

[0293] In some embodiments, the individual is HLA-A + , HLA-B + or HLA-C + In some embodiments, the individual is HLA-A*11:01 + .

[0294] In certain embodiments, the methods comprise determining one or more HLA types of an individual and / or identifying the presence of neoantigens prior to administering a therapy according to the present disclosure.

[0295] The expression of HLA alleles can be determined, for example, by genetic 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 molecular methods in a clinical laboratory authorized to perform HLA typing. In some embodiments, HLA typing is performed using PCR amplification, followed by high-throughput NGS, and then HLA determination. Herein, the HLA haplotypes of the major HLA loci (e.g., HLA-A, HLA-B, HLA-C, etc.) can be determined.

[0296] HLA typing can be performed using any known method (including, for example, protein or nucleic acid testing). Examples of nucleic acid testing include sequence-based typing (SBT) and the use of sequence-specific oligonucleotide probes (SSOPs) or sequence-specific primers (SSPs). 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, in some embodiments, sequence typing is performed using a system that can be obtained via 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, those disclosed in Mayor et al., PLoS One 10 (5): e0127153 (2015), and the methods and reagents are incorporated herein by reference.

[0297] In specific 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.

[0298] In the case of a host cell composition or unit dose, the amount of cells is at least one cell (e.g., one modified CD8 + T cell subsets (e.g., optionally comprising memory and / or naive CD8 + T cells); a modified CD4 + T cell subsets (e.g., optionally comprising memory and / or naive CD4 + T cells), or more usually greater than 10 2 cells, for example up to 10 4 Up to 10 5 Up to 10 6 Up to 10 7 Up to 10 8 Up to 10 9 or more than 10 10 In certain embodiments, the cells are about 10 4 About 10 10 Cells / m 2 In the range of about 10 5 About 10 9 Cells / m 2 In some embodiments, the administered dose comprises up to about 3.3×10 5 In some embodiments, the administered dose comprises up to about 1×10 6 In some embodiments, the administered dose comprises up to about 3.3×10 6 In some embodiments, the administered dose comprises up to about 1×10 7 In certain embodiments, the modified immune cells are present in an amount comprising up to about 5×10 4 cells / kg, 5×10 5 cells / kg, 5×10 6 cells / kg or up to approximately 5 × 10 7 In certain embodiments, the modified immune cells are administered to a subject at a dose of at least about 5×10 4 cells / kg, 5×10 5 cells / kg, 5×10 6 cells / kg or up to approximately 5 × 10 7The number of cells is determined by the intended end use of the composition and the cell types included therein. For example, cells modified to contain binding proteins will include a cell population containing 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 purposes provided herein, the volume of the cells is generally one liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In embodiments, the density of the desired cells is generally greater than 10 4 cells / ml and generally greater than 10 7 cells / ml, usually 10 8 The cells may be administered as a single infusion or as multiple infusions over a period of time. A clinically relevant number of immune cells may be distributed over multiple infusions, the cumulative amount of which equals or exceeds 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 In certain embodiments, a unit dose of modified immune cells may be co-administered with hematopoietic stem cells from an allogeneic donor (e.g., simultaneously or concurrently). In some embodiments, one or more of the modified immune cells included in the unit dose are autologous to the individual.

[0299] In some embodiments, the individual receiving the modified immune cells has previously received lymphocyte-depleting chemotherapy. In other embodiments, the lymphocyte-depleting chemotherapy comprises cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof.

[0300] In some embodiments, the method further comprises administering to the individual an immune checkpoint molecule inhibitor as disclosed herein.

[0301] Also contemplated are pharmaceutical compositions (i.e., compositions) comprising a composition as disclosed herein (binding protein, polynucleotide, vector, host cell, host cell composition, unit dose and / or immunogenic polypeptide) and a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients include water, saline, dextrose, glycerol or its analogs and combinations thereof. In an embodiment, a composition comprising a fusion protein or host cell as disclosed herein further comprises a suitable infusion medium. Suitable infusion media can be any isotonic medium formulation, typically standard physiological saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water or Ringer's lactate. The infusion medium can be supplemented with human serum albumin or other human serum components.

[0302] The pharmaceutical composition can be administered in a manner suitable for the disease or condition to be treated (or prevented), as determined by a technician in the medical field. The appropriate dosage and suitable duration and frequency of administration of the composition will be determined based on factors such as the following: the patient's health, the patient's body type (i.e., weight, mass or body area), the type and severity of the patient's condition, the specific form of the active ingredient, and the method of administration. In general, appropriate dosages and treatment regimens provide compositions in an amount sufficient to provide therapeutic and / or preventive benefits (e.g., as described herein, including improved clinical outcomes, such as more frequent complete or partial remissions, or longer disease-free and / or total survival periods, or reduced symptom severity).

[0303] An effective amount of a pharmaceutical composition refers to an amount sufficient to achieve the desired clinical outcome or beneficial treatment as described herein, at the required dosage and time period. An effective amount can be delivered in one or more administrations. If an individual known or confirmed to suffer from a disease or disease state is administered, the term "therapeutic amount" can be used when referring to treatment, while a "prophylactically effective amount" can be used to describe the administration of an effective amount to an individual susceptible to or at risk of a disease or disease state (e.g., recurrence) as a preventive process.

[0304] The pharmaceutical compositions described herein may 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 prior to infusion into the patient. Dosages will vary, but administration of modified immune cells as described herein may be in a dose of 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 6Cells / 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 In certain embodiments, a unit dose comprises about 10 4 Cells / m 2 to about 10 11 Cells / m 2 The modified immune cells as described herein are developed to suitably administer and treat the specific compositions described herein in a variety of treatment regimens, including, for example, parenteral or intravenous administration or formulation.

[0305] If the subject composition is administered parenterally, the composition may also include a sterile aqueous or oily solution or suspension. Suitable parenteral acceptable nontoxic diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol mixed with water. The aqueous solution or suspension may further include one or more buffers, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, any material used to prepare any dosage unit formulation may be pharmaceutically pure and substantially nontoxic at the amount used. In addition, the active compound may be incorporated into sustained release formulations and formulations. As used herein, a unit dosage form refers to a discontinuous unit of a body suitable for an individual to be treated as a unit dose; each unit may contain a predetermined amount of engineered immune cells or active compounds and a suitable pharmaceutical carrier calculated to produce the desired effect.

[0306] In general, appropriate dosages and treatment regimens provide active molecules or cells in an amount sufficient to provide benefit. Such responses can be monitored by establishing improved clinical outcomes (e.g., more frequent complete or partial remissions, or longer disease-free survival) in treated individuals compared to untreated individuals. An increase in pre-existing immune responses to tumor proteins is generally associated with improved clinical outcomes. Such immune responses can generally be evaluated using routine standard proliferation, cytotoxicity, or cytokine assays.

[0307] For prophylactic use, the dose should be sufficient to prevent, delay the onset of, or lessen the severity of the disease or disorder associated with the disorder. The prophylactic benefit of the immunogenic composition administered according to the methods described herein can be determined by conducting preclinical (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 practiced by those skilled in the art.

[0308] As used herein, administration of a composition refers to its delivery to an individual, regardless of the route or mode of delivery. Administration may be performed continuously or intermittently and parenterally. The composition may be administered locally (e.g., intratumorally) or systemically (e.g., intravenously). Administration may be used to treat individuals who have been confirmed to have a recognized condition, disease, or disease state, or to treat individuals who are susceptible to or at risk of such a condition, disease, or disease state. Co-administration with adjuvant therapy may include simultaneous and / or sequential delivery of multiple agents (e.g., modified immune cells with one or more cytokines; immunosuppressive therapies, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low doses of mycophenolic acid prodrugs, or any combination thereof) in any order and at any dosing schedule.

[0309] In certain embodiments, multiple doses of a composition described herein are administered to a subject, which may be administered at an administration interval of between about two weeks to about four weeks.

[0310] Treatment or prevention methods disclosed herein can be used as a part of a treatment process or scheme for individual administration, and the treatment process or scheme may be included in the additional therapy before or after the unit dose, cell or composition disclosed in the present disclosure is administered. For example, in certain embodiments, the individual receiving the modified immune cell of the unit dose is receiving or has previously received hematopoietic cell transplantation (HCT; including myeloablative and non-myeloablative HCT). The techniques and schemes for performing HCT are known in the art and may include transplanting any suitable donor cell, such as cells derived from cord blood, bone marrow or peripheral blood, hematopoietic stem cells, mobilized stem cells or cells from amniotic fluid. Therefore, in certain embodiments, the modified immune cell of the present disclosure can be used together with or shortly thereafter with the improved HCT therapy using hematopoietic stem cells. In certain embodiments, HCT includes donor hematopoietic cells, and the donor hematopoietic cells include chromosome elimination of genes encoding HLA components, chromosome elimination of genes encoding TCR components, or both.

[0311] In other embodiments, the individual has previously received lymphocyte-depleting chemotherapy prior to receiving the composition or HCT. In certain embodiments, the lymphocyte-depleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof.

[0312] According to the method disclosed herein, one or more additional agents may be further included in the combination therapy to treat a disease or condition. For example, in certain embodiments, the combination therapy comprises administering the composition of the present disclosure in conjunction with an immune checkpoint inhibitor (in parallel, simultaneously or sequentially). In some embodiments, the combination therapy comprises administering the composition of the present disclosure in conjunction with an agonist of an immune checkpoint stimulator. In other embodiments, the combination therapy comprises administering the composition of the present disclosure in conjunction with a second therapy (e.g., chemotherapeutic agent, radiotherapy, surgery, antibody or any combination thereof).

[0313] As used herein, the term "immune suppression agent" refers to one or more cells, proteins, molecules, compounds or complexes that provide an inhibitory signal to help control or suppress an immune response. For example, immunosuppressants include those molecules that partially or completely block immune stimulation, reduce, prevent or delay immune activation, or increase, activate or upregulate immunosuppression. Example immunosuppressive agents that are targeted (e.g., using immune checkpoint inhibitors) include 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.

[0314] Immunosuppressants Inhibitors (also known as immune checkpoint inhibitors) can 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 embodiment disclosed herein, the method may include a composition of the present disclosure alone or in any combination with one or more inhibitors of any of the following immunosuppressive components.

[0315] In certain embodiments, the pharmaceutical 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 other 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 considered: cemiplimab; 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 hydrochloride) + pembrolizumab; pidilizumab; REGN-1979 + spartalizumab; ABBV-181; ADUS-100 + spartalizumab; AK-104; AK-105; AMP-224; BAT-1306; BI-754091; CC-90006; spartalizumab + REGN-3767; CS-1003; GLS-010; LZM-009; MEDI-5752; MGD-013; PF-06801591; Sym-021; Tislelizumab + pamipa pamiparib; XmAb-20717; AK-112; ALPN-202; AM-0001; antibodies against PD-1 in Alzheimer's disease; BH-2922; BH-2941; BH-2950; BH-2954; biological agents against CTLA-4 and PD-1 in solid tumors; bispecific monoclonal antibodies targeting PD-1 and LAG-3 in tumors; 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; monoclonal antibody that antagonizes tumor PDCD1; monoclonal antibody that antagonizes tumor PD-1; oncolytic virus that inhibits tumor PD-1; OT-2; PD-1 antagonist + ropeginterferon α-2b; PEGMP-7; PRS-332; RXI-762; STIA-1110; TSR-075; vaccines targeting HER2 and PD-1 in tumors vaccine; vaccine targeting PD-1 in tumors and autoimmune diseases; XmAb-23104; antisense oligonucleotides that inhibit PD-1 in tumors; AT-16201; bispecific monoclonal antibodies that inhibit PD-1 in tumors; IMM-1802; monoclonal antibodies that antagonize PD-1 and CTLA-4 in solid tumors and hematological tumors; nivolumab biosimilars; recombinant proteins that promote CD278 and CD28 in tumors and antagonize PD-1 in tumors; recombinant proteins that promote PD-1 in autoimmune and inflammatory diseases; SNA-01; SSI-361; YBL-006; AK- 103; JY-034; AUR-012; BGB-108; drugs that inhibit PD-1, Gal-9 and TIM-3 in solid tumors; ENUM-244C8; ENUM-388D4; MEDI-0680; monoclonal antibodies that antagonize PD-1 in metastatic melanoma and metastatic lung cancer; monoclonal antibodies that inhibit PD-1 in tumors; monoclonal antibodies that target CTLA-4 and PD-1 in tumors; monoclonal antibodies that antagonize PD-1 in NSCLC; monoclonal antibodies that inhibit PD-1 and TIM-3 in tumors; monoclonal antibodies that inhibit PD-1 in tumors; Recombinant proteins that inhibit PD-1 and VEGF-A in hematological malignancies and solid tumors; small molecules that antagonize PD-1 in tumors; Sym-016; inebilizumab + MEDI-0680; vaccines targeting PDL-1 and IDO in metastatic melanoma; anti-PD-1 monoclonal antibodies + cellular immunotherapy for glioblastoma; antibodies that antagonize PD-1 in tumors; monoclonal antibodies that inhibit PD-1 / PD-L1 in hematological malignancies and bacterial infections; monoclonal antibodies that inhibit PD-1 in HIV; or small molecules that inhibit PD-1 in solid tumors. ;

[0316] 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.

[0317] In certain embodiments, the compositions of the present disclosure are used in combination with CTLA4 inhibitors. In specific embodiments, the compositions of the present disclosure are used in combination with CTLA4-specific antibodies or binding fragments thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.

[0318] In certain embodiments, the compositions of the present disclosure are used in combination with: a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. The B7-H4 antibody binding fragment can be, for example, a scFv or fusion protein thereof as described in Dangaj et al., Cancer Res. 73:4820, 2013, and those described in U.S. Pat. No. 9,574,000 and PCT Patent Publication Nos. WO / 201640724A1 and WO2013 / 025779A1.

[0319] In certain embodiments, the compositions of the present disclosure are used in combination with a CD244 inhibitor.

[0320] 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. WO 2010 / 084158.

[0321] In certain embodiments, the compositions of the present disclosure are used in combination with a TIM3 inhibitor.

[0322] In certain embodiments, the compositions of the present disclosure are used in combination with a Gal9 inhibitor.

[0323] 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.

[0324] In certain embodiments, the compositions of the present disclosure are used in combination with an A2aR inhibitor.

[0325] In certain embodiments, the compositions of the present disclosure are used in combination with a KIR inhibitor, such as lirilumab (BMS-986015).

[0326] In certain embodiments, the compositions of the present disclosure are used in combination with suppressive cytokines (typically cytokines other than TGFβ) or inhibitors of Treg development or activity.

[0327] 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; April 6-10, 2013), 1-methyl-tryptophan (1-MT)-tira-pazamine, or any combination thereof.

[0328] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor, such as N(ω)-nitro-L-arginine methyl ester (L-NAME), N-ω-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-borohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0329] In certain embodiments, the compositions of the present disclosure are used in combination with a VISTA inhibitor, such as CA-170 (Curis, Lexington, Mass.).

[0330] In certain embodiments, the compositions of the present disclosure are used in combination with a TIGIT inhibitor, such as COM902 (Compugen, Toronto, Ontario Canada); a CD155 inhibitor, such as COM701 (Compugen); or both.

[0331] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO 2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO 2017 / 021526.

[0332] In certain embodiments, the pharmaceutical compositions of the present disclosure are used in combination with a LAIR1 inhibitor.

[0333] 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.

[0334] In certain embodiments, the compositions of the present disclosure are used in combination with agents that increase the activity of stimulatory immune checkpoint molecules (ie, agonists). For example, the compositions of the present disclosure can be used in combination with: CD137 (41BB) agonists (e.g., urelumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, CD27 agonists (e.g., CDX-1127), CD28 agonists (e.g., TGN1412, CD80, or CD86), CD40 agonists (e.g., CP-870,893, rhuCD40L, or SGN-40), CD122 agonists (e.g., IL-2), GITR agonists (e.g., humanized monoclonal antibodies described in PCT Patent Publication No. WO 2016 / 054638), ICOS (CD278) agonists (e.g., GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos314-8, or any combination thereof). In any embodiment disclosed herein, the method may comprise administering the composition of the present disclosure alone or in any combination with an agonist of one or more stimulatory immune checkpoint molecules, the agonist comprising any of the aforementioned agonists.

[0335] In certain embodiments, the combination therapy comprises a composition of the present disclosure and a second therapy comprising one or more of: an antibody or antigen-binding fragment thereof specific for a cancer antigen expressed by a non-inflamed solid tumor, radiation therapy, surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

[0336] In certain embodiments, the combination therapy method comprises administering the composition of the present disclosure and further administering radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma-radiation and radiopharmaceutical therapy. Surgery and surgical techniques suitable for treating a given cancer in an individual are well known to those of ordinary skill.

[0337] In certain embodiments, the combination therapy method comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitory drugs, DNA damaging agents, antimetabolites (e.g., folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactors (e.g., intercalators), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs; folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine);Microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactin omycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide, and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D D)), daunomycin, doxorubicin (adriamycin), idarubicin, anthracycline, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzyme (L-asparaginase, which metabolizes L-asparagine systemically and incapacitates cells from synthesizing their own asparagine); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimine and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine) BCNU) and analogs, streptozocin), triazene-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormones analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other inhibitors of thrombin); fibrinolytics (e.g., tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyrrolidone, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic 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 (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin, amsacrine, camptothecin, daunomycin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan) (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers; toxins, such as cholera toxin, ricin toxin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin, and apoptotic protease activators; and chromatin perturbants. ;

[0338] Cytokines can be used to manipulate the host immune response toward anticancer activity. See, for example, Floros and Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines suitable for promoting immune anticancer or antitumor responses 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, alone or in any combination with the compositions of the present disclosure.

[0339] Also provided herein are methods for regulating donor-acceptor immunotherapy, wherein the method comprises administering a homologous compound of a safety switch protein to an individual who has previously received a modified host cell of the present disclosure, comprising a heterologous polynucleotide encoding a safety switch protein, in an amount effective to eliminate the previously administered modified host cells in the individual.

[0340] In certain embodiments, the safety switch protein comprises tEGFR and the homologous compound is cetuximab, or the safety switch protein comprises iCasp9 and the homologous compound is AP1903 (e.g., dimeric AP1903), or the safety switch protein comprises an RQR polypeptide and the homologous compound is rituximab, or the safety switch protein comprises a myc binding domain and the homologous compound is an antibody specific to the myc binding domain.

[0341] In other aspects, methods of making compositions or unit doses of the present disclosure are provided. In certain embodiments, the methods comprise combining (i) an aliquot of a host cell transduced with a vector of the present disclosure with (ii) a pharmaceutically acceptable carrier. In certain embodiments, the vector of the present disclosure is used to transfect / transduce a host cell (e.g., T cell) for use in donor-recipient transfer therapy (e.g., targeting a cancer antigen).

[0342] In some embodiments, the method further comprises culturing the transduced host cells and selecting the transduced cells that have incorporated (i.e., expressed) the vector before aliquoting. In other embodiments, the method comprises amplifying the transduced host cells after culturing and selection and before aliquoting. In any embodiment of the disclosed method, the prepared composition or unit dose can be frozen (e.g., cryopreserved) for subsequent use. Any suitable host cell can be used to make a composition or unit dose according to the disclosed method, including, for example, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, or NK-T cells. In a specific embodiment, the host cell included in the method is a CD8 + T cells, CD4 + T cells or both.

[0343] Also provided are any of the binding proteins, polynucleotides, expression vectors, host cells, host cell compositions, unit doses, and immunogenic polypeptides, 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 a NRAS G12D mutation, or a NRAS G12V mutation, or a HRAS G12V mutation, or a HRAS G12D mutation in an individual.

[0344] Also provided are any of the binding proteins, polynucleotides, expression vectors, host cells, host cell compositions, unit doses, and immunogenic polypeptides, taken alone or in any combination, for use in the manufacture of a medicament for treating a disease or disorder in an individual, the disease or disorder being associated with a KRAS G12D mutation or a KRAS G12V mutation or a NRAS G12D mutation or a NRAS G12V mutation or a HRAS G12V mutation or a HRAS G12D mutation.

[0345] In certain embodiments, the disease or disorder comprises cancer. In some embodiments, the cancer is a solid cancer or a hematological malignancy. In certain embodiments, the disease or disorder is selected from pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; gallbladder 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's 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; histiocyte and dendritic cell carcinoma fibroblastic 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 tumors; mantle cell lymphoma; unclassifiable myelodysplastic / myeloproliferative neoplasms; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdomyosarcoma; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; malignant thyroid neoplasms; thyroid cancer; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma. In some embodiments, the method comprises parenteral or intravenous administration of a composition of the disclosure. In some embodiments, the methods comprise administering multiple doses of a binding protein, polynucleotide, expression vector, host cell, host cell composition, unit dose, and / or immunogenic polypeptide to a subject.

[0346] In certain embodiments, multiple doses are administered with administration intervals between about two weeks to about four weeks.

[0347] In certain embodiments, the composition comprises a modified host cell. In some embodiments, the method comprises exposing the modified host cell to about 10 4 cells / kg to about 10 11 A dose of 10 cells / kg is administered to an individual.

[0348] In certain embodiments, the method further comprises administering a cytokine to the individual. In some embodiments, the cytokine comprises IL-2, IL-15, or IL-21.

[0349] In certain embodiments, the individual has received or is currently receiving an immune checkpoint inhibitor and / or an agonist of an immune checkpoint stimulator.

[0350] Also provided are methods comprising introducing a polynucleotide encoding a binding protein of the disclosure into a host (eg, T) cell.

[0351] sequence

[0352] SEQ ID NO: 1-wt KRAS complete (UniProt: P01116)

[0353] MTEYKLVVVGAGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGETCLLDILDTAGQEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHHYREQIKRVKDSEDVP MVLVGNKCDL PSRTVDTKQAQDLARSYGIP FIETSAKTRQRVEDAFYTLV REIRQYRLKK ISKEEKTPGC VKIKKCIIM

[0354] SEQ ID NO:2-KRAS 7-16G12V

[0355] VVVGAVGVGK

[0356] SEQ ID NO:3-KRAS 8-16G12V

[0357] VVGAVGVGK

[0358] SEQ ID NO:4—KRAS 8-16G12V binding motif of TCR 11N4A

[0359] xVGAxGxxK

[0360] SEQ ID NO:5—TCR 11N4A alpha chain with signal peptide—original (WT) nucleotide sequence

[0361] atggccatgctcctgggggcatcagtgctgattctgtggcttcagccagactgggtaaacagtcaacagaagaatgatgaccagcaagttaagcaaaattcaccatccctgagcgtccaggaaggaagaatttctattctgaactgtgactatactaacagcatgtttgattatttcctatggtacaaaaaataccctgctgaaggtcctacattcctgatatctataagttccattaaggataaaaatgaagatggaagattcactgtcttcttaaacaaaagtgccaagcacctctctctgcacattgtgccctcccagcctggagactctgcagtgtacttctgtgcagcaagtggggtttcaggaaacacacctcttgtctttggaaagggcacaagactttctgtgattgcaaatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagctga

[0362] SEQ ID NO:6 - TCR 11N4 A beta chain with signal peptide - Original (WT) nucleotide sequence

[0363] atgggctccaggctgctctgttgggtgctgctttgtctcctgggagcaggcccagtaaaggctggagtcactcaaactccaagatatctgatcaaaacgagaggacagcaagtgacactgagctgctcccctatctctgggcataggagtgtatcctggtaccaacagaccccaggacagggccttcagttcctctttgaatacttcagtgagacacagagaaacaaaggaaacttccctggtcgattctcagggcgccagttctctaactctcgctctgagatgaatgtgagcaccttggagctgggggactcggccctttatctttgcgccagcagcgtcgggactgtggagcagtacttcgggccgggcaccaggctcacggtcacagaggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacctgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccagaggctag

[0364] SEQ ID NO:7-TCR 11N4A TCRβ-P2A-TCRα polynucleotide-codon optimized A

[0365]

[0366] SEQ ID NO:8-11N4A TCRβ-P2A-α polynucleotide codon optimized B

[0367]

[0368] SEQ ID NO:9-CD8α-T2A-CD8β-P2A-11N4A TCRβ-P2A-α polynucleotide codon optimized A

[0369]

[0370] SEQ ID NO:10-CD8α-T2A-CD8β-P2A-11N4A TCRβ-P2A-α polynucleotide codon optimized B

[0371]

[0372] SEQ ID NO:11 - 11N4A TCR α - chain - original protein, signal peptide is underlined

[0373] MAMLLGASVLILWLQPDWVNS QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0374] SEQ ID NO:12 - 11N4A TCR α - chain - original protein, without signal peptide

[0375] QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0376] SEQ ID NO:13 - 11N4A TCR α - chain variable domain, without signal peptide

[0377] QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIA

[0378] SEQ ID NO:14--11N4A TCR α - chain variable domain CDR1α

[0379] NSMFDY

[0380] SEQ ID NO:15---11N4A TCR α chain variable domain CDR2α

[0381] ISSIKDK

[0382] SEQ ID NO:16-11N4A TCR α chain variable domain CDR3α-IMGT junction

[0383] CAASGVSGNTPLVF

[0384] SEQ ID NO:17-11N4A TCR α chain variable domain CDR3α-IMGT

[0385] AASGVSGNTPLV

[0386] SEQ ID NO:18-11N4A TCR alpha chain constant domain (original protein)

[0387] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0388] SEQ ID NO:19-11N4A TCR alpha chain constant domain (cys-modified protein)

[0389] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0390] SEQ ID NO:20-11N4A TCR alpha chain, without signal peptide, cys modified

[0391] QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0392] SEQ ID NO:21 - 11N4A TCRβ-chain - original protein, signal peptide is underlined

[0393] MGSRLLCWVLLCLLGAGPVKA GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0394] SEQ ID NO:22 - 11N4A TCRβ-chain - original protein, without signal peptide

[0395] GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATG FYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0396] SEQ ID NO:23-11N4A TCR β chain variable domain, without signal peptide

[0397] GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFGPGTRLTVT

[0398] SEQ ID NO:24-11N4A TCRβ chain variable domain CDR1β

[0399] SGHRS

[0400] SEQ ID NO:25-11N4A TCRβ chain variable domain CDR2β

[0401] YF

[0402] SEQ ID NO:26-11N4A TCRβ chain variable domain CDR3β-IMGT junction

[0403] CASSVGTVEQYF

[0404] SEQ ID NO:27-11N4A TCRβ chain variable domain CDR3β-IMGT

[0405] ASSVGTVEQY

[0406] SEQ ID NO:28-11N4A TCR β chain constant domain (original protein)

[0407] EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*

[0408] SEQ ID NO:29-11N4A TCRβ chain constant domain (cys-modified protein)

[0409] EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0410] SEQ ID NO:30-11N4A TCR β chain, without signal peptide (cys-modified protein)

[0411] GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATG FYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0412] SEQ ID NO:31-11N4A TCRβ-P2A-α-protein, signal peptide is underlined

[0413] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGP MAMLLGASVLILWLQPDWVNS QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0414] SEQ ID NO:32 CD8α-T2A-CD8β-P2A-11N4A TCRβ-P2A-α-protein, with the signal peptide indicated by underlining

[0415] MALPVTALLLPLALLLHAARP SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLF

[0416] QPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYVGSGEGRGSLLTCGDVEENPGP MRPRLWLLLAAQLTVLHGNSV LQQTPAYIKVQTNKMV

[0417] MLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYKGSGATNFSLLKQAGDVEENPGP MGSRLLCWVLLCLLGAGPVKA GVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSVGTVEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGP MAMLLGASVLILWLQPDWVNS QQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGVSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0418] SEQ ID NO:33 - 11N6 TCRα - Original nucleotide sequence

[0419] Atggaaactctcctgggagtgtctttggtgattctatggcttcaactggctagggtgaacagtcaacagggagaagaggatcctcaggccttgagcatccaggagggtgaaaatgccaccatgaactgcagttacaaaactagtataaacaatttacagtggtatagacaaaattcaggtagaggccttgtccacctaattttaatacgttcaaatgaaagagagaaacacagtggaagattaagagtcacgcttgacacttccaagaaaagcagttccttgttgatcacggcttcccgggcagcagacactgcttcttacttctgtgctacggaccctatgaacaccaatgcaggcaaatcaacctttggggatgggactacgctcactgtgaagccaaatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagctga

[0420] SEQ ID NO:34 - 11N6 TCRβ - Original nucleotide sequence

[0421] atgggcaccaggctcctctgctgggcggccctctgtctcctgggagcagaactcacagaagctggagttgcccagtctcccagatataagattatagagaaaaggcagagtgtggctttttggtgcaatcctatatctggccatgctaccctttactggtaccagcagatcctgggacagggcccaaagcttctgattcagtttcagaataacggtgtagtggatgattcacagttgcctaaggatcgattttctgcagagaggctcaaaggagtagactccactctcaagatccaacctgcaaagcttgaggactcggccgtgtatctctgtgccagcagcccctacggggggagcgtctcctacgagcagtacttcgggccgggcaccaggctcacggtcacagaggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtgtgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacctgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccagaggctag

[0422] SEQ ID NO:35-11N6 TCRβ-P2A-α codon optimization

[0423]

[0424] SEQ ID NO:36-CD8α-T2A-CD8β-P2A-11N6 TCRβ-P2A-α codon optimization

[0425]

[0426] SEQ ID NO:37-11N6 TCR alpha chain - native protein, signal peptide is underlined

[0427] METLLGVSLVILWLQLARVNS QQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPMNTNAGKSTFGDGTTLTVKPNIQNPDPAVYQLRDS KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0428] SEQ ID NO:38-11N6 TCR alpha chain - native protein, without signal peptide

[0429] QQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPMNTNAGKSTFGDGTTLTVKPNIQNPDPAVYQLRDS KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0430] SEQ ID NO:39-11N6 TCR alpha chain variable domain, without signal peptide

[0431] QQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPMNTNAGKSTFGDGTTLTVKP

[0432] SEQ ID NO:40--11N6 TCR α chain variable domain CDR1α

[0433] TSINN

[0434] SEQ ID NO:41-11N6 TCR alpha chain variable domain CDR2 alpha

[0435] IRSNERE

[0436] SEQ ID NO:42--11N6 TCR α chain variable domain CDR3α-IMGT junction

[0437] CATDPMNTNAGKSTF

[0438] SEQ ID NO:43--11N6 TCR α chain variable domain CDR3α-IMGT

[0439] ATDPMNTNAGKST

[0440] SEQ ID NO:44--11N6 TCR alpha chain constant domain (original protein)

[0441] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0442] SEQ ID NO:45--11N6 TCR α chain constant domain (cys-modified protein)

[0443] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0444] SEQ ID NO:46-11N6 TCR alpha chain, without signal peptide, cys modified

[0445] QQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPMNTNAGKSTFGDGTTLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0446] SEQ ID NO: 47 - 11N6 TCRβ-chain original protein, with the signal peptide underlined

[0447] MGTRLLCWAALCLLGAELTEA GVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSPYGGSVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0448] SEQ ID NO: 48 - 11N6 TCRβ-chain original protein, without signal peptide

[0449] GVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSPYGGSVSYEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0450] SEQ ID NO:49-11N6 TCR β chain variable domain, without signal peptide

[0451] GVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSPYGGSVSYEQYFGPGTRLTVT

[0452] SEQ ID NO:50--11N6 TCRβ chain variable domain CDR1β

[0453] SGHAT

[0454] SEQ ID NO:51-11N6 TCRβ chain variable domain CDR2β

[0455] FWf

[0456] SEQ ID NO:52--11N6 TCRβ chain variable domain CDR3β-IMGT junction

[0457] CASSPYGGSVSYEQYF

[0458] SEQ ID NO:53--11N6 TCRβ chain variable domain CDR3β-IMGT

[0459] ASSPYGGSVSYEQY

[0460] SEQ ID NO:54 11N6 TCRβ chain constant domain (original protein)

[0461] EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0462] SEQ ID NO:55-11N6 TCRβ chain constant domain (cys-modified protein)

[0463] EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0464] SEQ ID NO:56-11N6 TCRβ chain (cys-modified protein)

[0465] GVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSPYGGSVSYEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL ATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0466] SEQ ID NO:57-11N6 TCRβ-P2A-α-protein, signal peptide is underlined

[0467] MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSPYGGSVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGP METLLGVSLVILWLQLARVNS QQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPMNTNAGKSTFGDGTTLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0468] SEQ ID NO:58 - CD8α - T2A - CD8β - P2A - 11N6 TCRβ - P2A - α - Protein, with the signal peptide underlined

[0469] MALPVTALLLPLALLLHAARP SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLF

[0470] QPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYVGSGEGRGSLLTCGDVEENPGP MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYKGSGATNFSLLKQAGDVEENPGP MGTRLLCWAALCLLGAEL TEA GVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSPYGGSVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGP METLLGVSLVILWLQLARVNS QQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDPMNTNAGKSTFGDGTTLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0471] SEQ ID NO:59 - TCR BNT Vβ, with signal peptide

[0472] MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHATLYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQPAKLEDSAVYLCASSLADIYEQYFGPGTRLTVT

[0473] SEQ ID NO:60 - TCR BNT Vα, with signal peptide

[0474] METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCATDRQSSGDKLTFGTGTRLAVRP

[0475] SEQ ID NO:61 - (TCR 220_21Vα)

[0476] GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEPIIGGNTPLVFGKGTRLSVIAN

[0477] SEQ ID NO:62(TCR 220_21Vβ)

[0478] GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSSEGLAGGPTAGELFFGEGSRLTVL

[0479] SEQ ID NO:63(TCR 129_5Vα)

[0480] AQSVTQPDIHITVSEGASLELRCNYSYGATPYLFWYVQSPGQGLQLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRKPSVHWSDAAEYFCAVGASGTYKYIFGTGTRLKVLAN

[0481] SEQ ID NO:64(TCR 129_5Vβ)

[0482] DAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLALSYEQYFGPGTRLTVT

[0483] SEQ ID NO:65—CD8α (Homo sapiens, UniProt accession P01732, signal peptide is underlined)

[0484] MALPVTALLLPLALLLHAARP SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHF VPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

[0485] SEQ ID NO:66—CD8β (Homo sapiens, UniProt accession P10966, signal peptide is underlined)

[0486] MRPRLWLLLAAQLTVLHGNSV LQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK

[0487] SEQ ID NO:67-[reserved]

[0488] SEQ ID NO:68-[reserved]

[0489] SEQ ID NO:69—TCR Cα amino acid sequence engineered to include threonine to cysteine ​​and LVL mutations

[0490] NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS

[0491] SEQ ID NO:70 - Amino acid sequence of TRBC1 (UniProt KB P01850)

[0492] EDLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE

[0493] VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY

[0494] GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSVSYQQG VLSATILYEI

[0495] LLGKATLYAV LVSALVLMAM VKRKDF

[0496] SEQ ID NO:71 - Amino acid sequence of TRBC1 (modified with cys)

[0497] EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0498] SEQ ID NO:72 - Amino acid sequence of TRBC2 (UniProt KB A0A5B9)

[0499] EDLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE

[0500] VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY

[0501] GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI

[0502] LLGKATLYAV LVSALVLMAM VKRKDSRG

[0503] SEQ ID NO:73—TRBC2 amino acid sequence (cys modified)

[0504] EDLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE

[0505] VHSGVCTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY

[0506] GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI

[0507] LLGKATLYAV LVSALVLMAM VKRKDSRG

[0508] (SEQ ID NO:74) Porcine Teschovirus-1 2A (P2A) self-cleaving peptide with N-terminal GSG linker

[0509] GSGATNFSLLKQAGDVEENPGP

[0510] (SEQ ID NO: 75) East Asian orchivirus 2A (T2A) self-cleaving peptide

[0511] LEGGGEGRGSLLTCGDVEENPGPR

[0512] (SEQ ID NO: 76) Equine rhinitis virus type A (ERAV) 2A (E2A) self-cleaving peptide

[0513] QCTNYALLKLAGDVESNPGP

[0514] (SEQ ID NO: 77) Foot-and-mouth disease virus 2A (F2A) self-cleaving peptide with N-terminal GSG linker

[0515] GSGVKQTLNFDLLKLAGDVESNPGP

[0516] (SEQ ID NO:78)NRAS (Uniprot KB P01111)

[0517] MTEYKLVVVGAGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET

[0518] CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNSKSF ADINLYREQI

[0519] KRVKDSDDVP MVLVGNKCDL PTRTVDTKQA HELAKSYGIP FIETSAKTRQ

[0520] GVEDAFYTLV REIRQYRMKK LNSSDDGTQG CMGLPCVVM

[0521] (SEQ ID NO:79)HRAS (Uniprot KB P01112)

[0522] MTEYKLVVVGAGGVGKSALT IQLIQNHFVD EYDPTIEDSY RKQVVIDGET

[0523] CLLDILDTAG QEEYSAMRDQ YMRTGEGFLC VFAINNTKSF EDIHQYREQI

[0524] KRVKDSDDVP MVLVGNKCDL AARTVESRQA QDLARSYGIP YIETSAKTRQ

[0525] GVEDAFYTLV REIRQHKLRK LNPPDESGPG CMSCKCVLS

[0526] (SEQ ID NO:80) Fas - 41BB fusion (amino acids)

[0527] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVE INCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0528] (SEQ ID NO: 81) Fragment containing the extracellular domain of FAS

[0529] MLGIWTTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN

[0530] (SEQ ID NO: 82) Fragment containing the intracellular domain of 41BB LCLLLLPIPLIVWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0531] (SEQ ID NO:83) FAS-41BB fusion (nucleotide coding sequence)

[0532] ATGCTGGGCATCTGGACCCTGCTGCCCTTGGTGCTGACTAGCGTGGCTAGACTGAGCAGCAAGAGCGTGAACGCCCAAGTGACCGACATCAACAGCAAGGGCCTGGAGCTGAGAAAGACCGTGACCACCGTGGAGACACAGAACCTGGAGGGCCTGCACCACGACGGGCAGTTCTGCCACAAGCCCTGCCCCCCCGGCGAGAGAAAGGCTAGAGACTGCACCGTGAACGGCGACGAGCCCGACTGCGTGCCCTGCCAAGAGGGCAAGGAGTACACCGACAAGGCCCACTTCAGCAGCAAGTGCAGAAGATGCAGACTGTGCGACGAGGGCCACGGCCTGGAGGTGGAGATCAACTGCACGCGTACGCAGAATACCAAATGCCGCTGCAAGCCCAACTTCTTCTGCAACAGCACCGTGTGCGAGCACTGCGACCCCTGCACCAAGTGCGAGCACGGCATCATCAAGGAGTGCACCCTGACAAGCAACACCAAGTGTAAGGAAGAGGGCTCACGGAGCAACCTGGGCTGGCTGTGCCTGCTGCTGCTGCCCATCCCCCTGATCGTGTGGGTGAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAAGAGGAGGACGGGTGCAGCTGTAGATTCCCCGAAGAAGAAGAAGGCGGCTGTGAGCTT

[0533] Table 2: Additional sequences of the polypeptide and nucleic acid components described herein

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617] SEQ ID NO:1039: Human p53 Amino Acid Sequence

[0618] MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPG

[0619] PDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD

[0620] SEQ ID NO:1040: Human PIK3CA Amino Acid Sequence

[0621]

[0622] SEQ ID NO: 1041: IL7R signaling domain

[0623] KKRIKPIVWPSPLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0624] SEQ ID NO: 1042: IL7R transmembrane domain

[0625] PILLTISILSFFSVALLVILACVLW

[0626] SEQ ID NO: 1043: CD80 extracellular domain

[0627] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKKQEHFPDN

[0628] SEQ ID NO: 1044: CD58 extracellular domain

[0629] MVAGSDAGRALGVLSVVCLLHCFGFISCFSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYNLTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCPMEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSIILTTCIPSSGHSRHR

[0630] SEQ ID NO: 1045: CD34 extracellular domain

[0631] MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSD LSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKT

[0632] Examples

[0633] Example 1

[0634] Identification of KRAS G12V-specific TCRs from the T cell lineage of a healthy donor

[0635] Dendritic cells derived from peripheral blood mononuclear cells (PBMCs) of HLA-A11-positive healthy donors were generated, irradiated, and expressed with KRAS-G12V 7-16 and KRAS-G12V 8-16 These cells were pulsed with autologous CD8 + T cells were cultured together for 8-10 days to induce activation / expansion of antigen-specific CD8+ T cells. These multiple T cell lines were then restimulated and expanded with peptide-pulsed and irradiated autologous PBMCs twice for 8 to 10 days to further expand antigen-specific clones. This method was performed on all ten CD8+ T cell lines 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 ).

[0636] In order to identify TCRs with strong binding to 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 CD137 upregulation was assessed by flow cytometry. Cells expressing CD137 were isolated by flow cytometry cell sorting and TCRβ lineage analysis was performed (Adaptive Biotechnologies, Seattle, WA). TCR clonotypes that were highly enriched in the CD137+ population and responsive to low concentrations of peptides were identified, and TCRα / β pairing was determined by 10x single cell RNAseq analysis (10x Genomics, Pleasanton, CA) of similarly sorted populations. Representative analysis of clonal enrichment in CD137+ sorted populations compared to all unsorted cells treated with low and high concentrations of peptides is shown in Figure 1B Paired TCR α / β sequences from identified clonotypes were assembled and synthesized as P2A-linked expression cassettes and transduced via lentiviral into reporter Jurkat cells expressing GFP under the control of the Nur77 locus (Nur77-GFP-Jurkat). Peptide dose-dependent responses of various TCRs were assessed by analyzing GFP expression after overnight incubation with A11 target cells pulsed with decreasing concentrations of the indicated peptides ( Figure 1C Dose-response curves were fitted by nonlinear regression, and EC50 values ​​were calculated using Graphpad Prism (Boston, MA). Figure 1D , 1E ).

[0637] Example 2

[0638] First generation CD8 + Functional avidity of KRAS-G12V-specific TCR expressed in T cells

[0639] Primary CD8+ T cells were transduced with polynucleotides encoding KRAS-G12V-specific TCRs, sorted and purified, and expanded. The sorted and purified T cells were then incubated with decreasing concentrations of KRAS-G12V 8-16 The peptides were cultured overnight and CD137 expression was assessed by flow cytometry. Dose response curves were fitted by nonlinear regression and EC50 values ​​were calculated using Graphpad Prism ( Figure 2A , 2B). In this experiment, TCR 11N4A was compared with KRAS G12V-specific TCR "220_21" (see SEQ ID N...

Claims

1. A polynucleotide comprising a nucleic acid sequence encoding: (a) a binding protein, wherein the binding protein comprises: (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, wherein the fusion protein comprises: (i) an extracellular component comprising a CD95 ligand (FasL) binding domain, wherein the CD95 ligand binding domain comprises an extracellular domain of CD95 (Fas) 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.

2. The polynucleotide according to claim 1, further comprising a nucleic acid sequence encoding: (c) CD8 coreceptor alpha or beta chain or a part or variant thereof, wherein said sequence encoding said binding protein is located upstream of said sequence encoding the extracellular part of CD8 coreceptor alpha or beta chain or said part or variant thereof.

3. The polynucleotide according to claim 1, further comprising a nucleic acid sequence encoding: (c) CD8 coreceptor α and β chains or parts or variants thereof, wherein said sequence encoding said binding protein is located upstream of said sequence encoding the extracellular portion of said CD8 coreceptor α and β chains or said parts or variants thereof.

4. The polynucleotide according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding the fusion protein further encodes: (d) a hydrophobic component between the extracellular component and the intracellular component of the fusion protein.

5. The polynucleotide of any one of claims 1 to 4, wherein the binding protein comprises a binding domain that binds to a peptide:HLA complex, wherein the complex comprises a neoantigenic peptide and an HLA protein.

6. The polynucleotide of any one of claims 1 to 5, wherein the binding protein comprises a single-chain TCR (scTCR) or a single-chain T-cell receptor variable fragment (scTv).

7. The polynucleotide according to any one of claims 1 to 5, wherein the binding protein comprises a TCR α chain variable (Vα) domain or a TCR β chain variable (Vβ) domain.

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

9. The polynucleotide according to any one of claims 1 to 8, wherein the CD95 (Fas) ligand binding domain is the Fas extracellular domain 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 polynucleotide according to any one of claims 1 to 10, wherein the CD95 (Fas) extracellular domain 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:

81. NO:82 has 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.

12. The polynucleotide of 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 of 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) extracellular domain 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 portion or variant thereof comprises the amino acid sequence of SEQ ID NO:

82.

16. The polynucleotide of claim 2 or 3, wherein the CD8 coreceptor 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.

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

18. The polynucleotide of claim 17, wherein the KRAS mutant peptide comprises xVGAxGxxK, wherein x represents any amino acid.

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

20. The polynucleotide of claim 19, wherein the KRAS G12V mutant peptide comprises the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGAVGVGK (SEQ ID NO: 3).

21. The polynucleotide of any one of claims 5 to 20, wherein the HLA protein is encoded by an HLA-A*11 or 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, wherein the nucleic acid sequence is 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.

23. The polynucleotide of claim 2 or 3, further comprising a nucleic acid sequence encoding a self-cleaving peptide disposed between (a) and (b) or disposed between (b) and (c) in the presence of (c).

24. The polynucleotide of claim 2 or 3, further comprising a nucleic acid sequence encoding a self-cleaving peptide, the nucleic acid sequence being interposed between the sequence encoding the CD8 coreceptor α chain and the sequence encoding the CD8 coreceptor 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, wherein the nucleic acid sequence is disposed 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 α chain; and / or disposed 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 β chain.

26. The polynucleotide of any one of claims 23 to 25, further comprising the following operably linked in frame: (iii) (pnBP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β)-(pnFP); or (iv) (pnBP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α)-(pnFP); (iii) (pnBP)-(pnSCP1)-(pnFP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β); or (iv) (pnBP)-(pnSCP1)-(pnFP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α); wherein pnCD8α is said nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the α chain of the CD8 coreceptor, wherein pnCD8β is said nucleic acid sequence encoding a polypeptide comprising the extracellular portion of the α chain of the CD8 coreceptor, wherein pnBP is said nucleic acid sequence encoding a binding protein, wherein pnFP is the nucleic acid sequence encoding the fusion protein, and Wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotide and / or the self-cleaving peptide encoded are optionally the same or different.

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

28. The polynucleotide of 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 of claim 27, wherein the furin peptide comprises the amino acid sequence RAKR.

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

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

32. The polynucleotide of 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 of 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 of 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 of claim 35, wherein the vector is a viral vector.

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

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

39. The host cell of claim 38, wherein the host cell replicates in the absence of exogenous cytokines for no more than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36, or 48 hours.

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

41. The host cell of claim 40, wherein the host cell is a human immune cell, wherein the human immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof.

42. The host cell of claim 41, wherein the human immune cell comprises a T cell, wherein the T cell comprises a 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.

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

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

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

46. ​​The method of claim 44 or 45, wherein the cancer is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; gallbladder cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; Liver cancer; Myeloid leukemia, optionally a myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma, such as non-Hodgkin's lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally a 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 carcinoma lung cancer; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-bomb lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; unclassifiable myelodysplastic / myeloproliferative neoplasm; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; neurothelial carcinoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; malignant thyroid neoplasm; thyroid carcinoma; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma.

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

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

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

50. The method of any one of claims 43-49, wherein the method further comprises administering a cytokine to the individual.

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

52. The method of any one of claims 43 to 51, wherein the individual has received or is receiving an immune checkpoint inhibitor and / or an agonist of an immune checkpoint stimulator.

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

54. The method of any one of claims 44-52, wherein the cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% within a period of time following administration of the effective amount of the host cells.

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

56. The method of any one of claims 43-56, 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 a polynucleotide according to any one of claims 1 to 35 or a 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 a host cell according to any one of claims 38 to 42.

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

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

61. The method of claim 58, wherein the pancreatic cancer cells are pancreatic ductal adenocarcinoma cells.

62. The method of claim 60, wherein the lung cancer cells are non-small cell lung cancer cells.

63. A method for genetically engineering immune cells, the method comprising contacting the cells with a polynucleotide comprising a nucleic acid sequence encoding a fusion protein of 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 an extracellular domain of CD95 (Fas) or a functional fragment thereof and an intracellular component comprising an intracellular signaling domain of CD137 (4-1BB), and amplifying the immune cells.

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

65. A host cell comprising: (a) a fusion protein, wherein the fusion protein comprises: (i) an extracellular component comprising a CD95 ligand (FasL) binding domain, wherein the CD95 ligand binding domain comprises an extracellular domain of CD95 (Fas) 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; and (b) exogenous CD8 coreceptor α or β chain or a portion or variant thereof.

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

67. The host cell of claim 65 or 66, wherein the host cell comprises mRNA encoding the exogenous CD8 coreceptor alpha or beta chain or a portion or variant thereof comprising a non-native 3' or 5' untranslated region (UTR).

68. The host cell of claim 67, wherein the non-native 3' or 5' UTR is a viral UTR, an adenoviral UTR, or a lentiviral UTR.

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

70. The host cell of 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 the Fas extracellular domain or a functional fragment thereof.

72. A 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 host cell according to any one of claims 65 to 72, wherein the CD95 (Fas) extracellular domain 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:

81. NO:82 has 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.

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

82.

77. The host cell of any one of claims 65 to 76, wherein the CD8 coreceptor alpha or beta 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.

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

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

80. The host cell of claim 79, wherein the neoantigenic peptide is a KRAS, HRAS, NRAS, p53 or PIK3CA mutant peptide.

81. The host cell of claim 80, wherein the KRAS mutant peptide comprises xVGAxGxxK, wherein x represents any amino acid.

82. The host cell of claim 80 or 81, wherein the neoantigenic peptide is a KRAS mutant peptide, wherein the KRAS mutant peptide is a KRAS G12V mutant peptide.

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

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

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

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

87. The host cell of any one of claims 65 to 86, wherein the host cell replicates in the absence of exogenous cytokines for no more than 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 36, or 48 hours.

88. The host cell of 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 of claim 88, wherein the host cell is a human immune cell, wherein the human immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof.

90. The host cell of claim 41, wherein the human immune cell is a T cell, wherein the T cell comprises a 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.

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

92. The method of claim 91, wherein the host cell further comprises a TCR directed against an antigen presented by the cancer.

93. The method of any one of claims 91 to 92, wherein the cancer is pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; gallbladder cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; Liver cancer; Myeloid leukemia, optionally a myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma, such as non-Hodgkin's lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally a 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-strand lymphoma; head and neck cancer; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; unclassifiable myelodysplasia / myeloproliferative neoplasm; 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 carcinoma; malignant thyroid neoplasm; thyroid carcinoma; thyroid adenocarcinoma; urothelial carcinoma; or papillary thyroid carcinoma.

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

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

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

97. The method of any one of claims 91-96, wherein the method further comprises administering a cytokine to the individual.

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

99. The method of any one of claims 91 to 98, wherein the individual has received or is receiving an agonist of an immune checkpoint inhibitor and / or immune checkpoint stimulator.

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

101. The method of any one of claims 91-100, wherein the cancer is reduced by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% within a period of time after administration of the effective amount of the host cells.

102. The method of claim 101, wherein the time period comprises less than or equal to 120 days, less than or equal to 60 days, less than or equal to 50 days, less than or equal to 40 days, less than or equal to 30 days, or less than or equal to 20 days.

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

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

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

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

107. The composition of 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 of CD3+ cells stained with a dextramer specific for a mutant KRAS G12V peptide as assessed by flow cytometry.

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

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

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

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

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