Non-HLA-restricted T cell receptors and their use

Abstract

To provide non-HLA restricted T cell receptors and uses thereof.SOLUTION: The present disclosed subject matter provides methods and compositions for enhancing the immune response to cancers and pathogens. It relates to novel designs of T cell receptors (TCRs) and engineered immunoresponsive cells comprising the same. The novel TCR binds to an antigen in an HLA-independent manner. In certain embodiments, the novel TCR provides enhanced sensitivity for a target gene having a low expression level.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 629,072, filed on 11 February 2018, which is incorporated in its entirety by reference and for which priority is claimed.

[0002] Introduction The subject matter of this disclosure provides methods and compositions for enhancing immune responses to cancer and pathogens. It relates to novel designs of T cell receptors (TCRs) and engineered immune-responsive cells comprising them. Engineered immune-responsive cells comprising novel TCRs are antigen-targeting. [Background technology]

[0003] Adoptive immunotherapy using antigen-recognition receptors (e.g., chimeric antigen receptors (CARs)) has shown remarkable clinical results in the treatment of leukemia and is one of the most promising new strategies for treating cancer. To generate CAR therapies, current clinical protocols use autologous T cells as well as randomly integrated vectors containing gamma-retroviruses, lentiviruses, and transposons, all of which undergo transgene variegation. This results in semi-random incorporation and variable expression of CARs. In short, the collaboration of self-cell procurement and random vector incorporation tends to produce cell products with variable potency. Therefore, there is a need for the novel design of antigen-recognition receptors with consistent potency and increased ability to detect low levels of target antigens. [Overview of the project] [Means for solving the problem]

[0004] The subject matter of this disclosure generally provides HLA-independent (or non-HLA-restricted) T cell receptors (referred to as "HI-TCRs") that bind to an antigen of interest in an HLA-independent manner, and immune-responsive cells comprising such receptors. The subject matter of this disclosure also provides methods of using such cells to induce and / or enhance an immune response to a target antigen, and / or to treat and / or prevent a neoplasm or other disease / disorder, wherein an increase in antigen-specific immune response is desirable.

[0005] The subject of this disclosure is a recombinant T cell receptor (TCR) comprising an antigen-binding chain including an extracellular antigen-binding domain and a constant domain, which binds to an antigen in an HLA-independent manner.

[0006] In certain embodiments, the constant domain comprises a natural or modified TRAC peptide and / or a natural or modified TRBC peptide. In certain embodiments, the constant domain can form homodimers or heterodimers with another constant domain.

[0007] In certain embodiments, recombinant TCRs are expressed from expression cassettes positioned at the endogenous TRAC and / or TRBC loci of immune-responsive cells. In certain embodiments, the positioning of the recombinant TCR expression cassette disrupts or inactivates the endogenous expression of TCRs, including the native TCRα and / or native TCRβ chains, in immune-responsive cells. In certain embodiments, the positioning of the recombinant TCR expression cassette prevents or eliminates mispairing between recombinant TCRs and the native TCRα and / or native TCRβ chains in immune-responsive cells. In certain embodiments, an antigen-binding chain can associate with a CD3ζ polypeptide. Upon binding to an antigen, the antigen-binding chain can activate the CD3ζ polypeptide associated with it. Activation of the CD3ζ polypeptide can activate immune-responsive cells. The CD3ζ polypeptide may be endogenous or exogenous. In certain embodiments, the CD3ζ polypeptide is endogenous and integrates with the native CD3 complex. In certain embodiments, the CD3ζ polypeptide is The recombinant TCR is exogenous and optionally integrated with a costimulatory molecule selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, and any combination thereof. In certain embodiments, the antigen-binding chain further comprises a costimulatory region, and the recombinant TCR can stimulate immune-responsive cells upon binding to the antigen. The costimulatory region may contain a costimulatory molecule selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, and any combination thereof. In certain embodiments, the costimulatory region comprises a CD28 polypeptide.

[0008] In certain embodiments, the recombinant TCR can associate with the CD3 complex. In certain embodiments, the recombinant TCR can integrate with the CD3 complex and effect HLA-independent antigen recognition. In certain embodiments, the CD3 complex is endogenous. In certain embodiments, the recombinant TCR replaces the endogenous TCR in the CD3 / TCR complex.

[0009] In certain embodiments, the extracellular antigen-binding domain can dimerize with another extracellular antigen-binding domain. The extracellular antigen-binding domain can include a ligand of a cell surface receptor, a receptor of a cell surface ligand, an antigen-binding portion or fragment thereof of an antibody, or an antigen-binding portion of a TCR. In certain embodiments, the extracellular antigen-binding domain includes the variable heavy chain region (V H , ) of an antibody, or the VHH derived only from camelid antibodies and / or the variable light chain region (V H ) of an antibody. In certain embodiments, the extracellular antigen-binding domain can dimerize with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain includes the V L of an antibody, and the V H of human, mouse or camelid origin can dimerize with another extracellular antigen-binding domain including the V H of an antibody to form a variable fragment (Fv). In certain embodiments, the extracellular antigen-binding dom ain includes the V L of an antibody, and the V L can dimerize with another extracellular antigen-binding domain including the V H of an antibody to form a variable fragment (Fv).

[0010] ​​In certain embodiments, recombinant TCRs bind to tumor antigens. These tumor antigens include CD19, MUC16, MUC1, CAlX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, and ER. The group can be selected from BB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB. In certain embodiments, the tumor antigen is CD19.

[0011] In certain embodiments, recombinant TCRs exhibit higher antigen sensitivity than CARs targeting the same antigen. In certain embodiments, recombinant TCRs can induce an immune response by binding to antigens present at low densities on the surface of tumor cells. In certain embodiments, the antigen present at low densities on the cell surface is less than approximately 2,000 molecules per cell.

[0012] The subject matter of this disclosure further provides immune-responsive cells comprising recombinant TCRs as described herein. In certain embodiments, an expression cassette of at least one antigen-binding chain of the recombinant TCR is located at an endogenous locus of the immune-responsive cell. In certain embodiments, an expression cassette of two antigen-binding chains of the recombinant TCR is located at an endogenous locus of the immune-responsive cell, and the two antigen-binding chains can be dimerized. The arrangement of the recombinant TCR expression cassette disrupts or inactivates the endogenous expression of the TCR, including the native TCRα chain and / or native TCRβ chain, thereby preventing or eliminating mispairing between the recombinant TCR and the native TCRα chain and / or native TCRβ chain in the immune-responsive cell. The endogenous locus may be the CD3δ locus, CD3ε locus, CD247 locus, B2M locus, TRAC locus, TRBC locus, TRGC locus, or TRDC locus. In certain embodiments, the endogenous locus is the TRAC locus and / or the TRBC locus. The endogenous locus may include a modified transcriptional terminator region. In certain embodiments, the modified transcriptional terminator region includes a genomic element selected from the group consisting of the TK transcriptional terminator, GCSF transcriptional terminator, TCRA transcriptional terminator, HBB transcriptional terminator, bovine growth hormone transcriptional terminator, SV40 transcriptional terminator, and P2A element; the P2A element enables the use of the endogenous transcriptional terminator of the targeted gene. In certain embodiments, if one endogenous T cell receptor locus in the cell is modified to express at least one antigen-binding chain of a recombinant TCR, one or more other endogenous T cell receptor loci in the cell are modified to exclude the expression of an endogenous TCR chain. In certain embodiments, one or more other endogenous T cell receptor loci are further modified to express the gene of interest. The target gene may be an antitumor cytokine, a costimulatory molecule ligand, a tracking gene, or a suicide gene.In certain embodiments, one or more endogenous TCR loci are further modified to incorporate sequences encoding co-stimulatory signaling domains to generate TCR chains containing such signaling domains at their carboxyl termini.

[0013] In certain embodiments, the immune-responsive cells are selected from a group consisting of T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, human embryonic stem cells, and pluripotent stem cells capable of differentiating into lymphoid cells. In certain embodiments, the immune-responsive cells are autologous.

[0014] In certain embodiments, the immune-responsive cells further comprise at least one exogenous costimulatory ligand. In certain embodiments, the costimulatory ligand is selected from the group consisting of CD80, CD86, 41BBL, CD275, CD40L, OX40L, and any combination thereof. In certain embodiments, the cells further comprise or comprise one exogenous costimulatory ligand. In certain embodiments, the exogenous costimulatory ligand is CD80 or 4-1BBL. In certain embodiments, the cells further comprise or comprise two exogenous costimulatory ligands. In certain embodiments, the two exogenous costimulatory ligands are CD80 and 4-1BBL.

[0015] In certain embodiments, the immune-responsive cell further comprises at least one chimeric costimulatory receptor (CCR). In certain embodiments, the CCR comprises a costimulatory molecule selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, and any combination thereof.

[0016] The subject matter of this disclosure also provides a pharmaceutical composition comprising immune-responsive cells disclosed herein and pharmaceutically acceptable excipients. The pharmaceutical composition can be used for the treatment of neoplasms.

[0017] Further methods for reducing tumor burden in subjects are provided. In addition, the subject matter of this disclosure provides methods for extending the survival of subjects having neoplasms (e.g., cancer). In certain embodiments, the method comprises administering to a subject an effective amount of immune-responsive cells or pharmaceutical compositions described herein.

[0018] The subject matter of this disclosure also provides methods for treating or preventing neoplasms. In certain embodiments, the method comprises administering to a subject an effective amount of the immunoresponsive cells or pharmaceutical composition described herein. The neoplasm can be selected from the group consisting of hematological malignancies, B-cell leukemias, multiple myelomas, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemias, non-Hodgkin lymphomas, and adenocarcinomas. In certain embodiments, the neoplasm is a B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the recombinant TCR binds to CD19. In certain embodiments, the neoplasm is B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, adenocarcinoma, or non-Hodgkin lymphoma, and the recombinant TCR is CD19, MUC16, MUC1, CAlX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor-α, GD2, GD3, HER-2, hTERT, IL-13 It binds to R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB. In certain embodiments, the neoplasm is CD19+ALL.

[0019] The subject matter of this disclosure further provides methods for producing antigen-specific immune-responsive cells. In certain embodiments, the method comprises introducing a nucleic acid sequence encoding a recombinant TCR described herein into immune-responsive cells. The nucleic acid sequence may be contained in a vector. In certain embodiments, an expression cassette of at least one antigen-binding chain of the recombinant TCR is located at an endogenous locus of the immune-responsive cell. In certain embodiments, an expression cassette of two antigen-binding chains of the recombinant TCR is located at an endogenous locus of the immune-responsive cell, and the two antigen-binding chains can be dimerized. The endogenous locus may be the CD3δ locus, CD3ε locus, CD247 locus, B2M locus, TRAC locus, TRBC locus, TRDC locus and / or TRGC locus. In certain embodiments, the endogenous locus is the TRAC locus or the TRBC locus. In certain embodiments, the arrangement of the recombinant TCR expression cassette disrupts or inactivates the endogenous expression of the TCR, including the native TCRα chain and / or native TCRβ chain, in immune-responsive cells, thereby preventing or eliminating mispairing between the recombinant TCR and the native TCRα chain and / or native TCRβ chain in immune-responsive cells. In certain embodiments, the endogenous locus includes a modified transcriptional terminator region. In certain embodiments, the modified transcriptional terminator region includes a genomic element selected from the group consisting of the TK transcriptional terminator, GCSF transcriptional terminator, TCRA transcriptional terminator, HBB transcriptional terminator, bovine growth hormone transcriptional terminator, SV40 transcriptional terminator, and P2A element. In certain embodiments, if one endogenous T cell receptor locus in a cell is modified to express at least one antigen-binding chain of the recombinant TCR, one or more other endogenous T cell receptor loci in the cell are modified to exclude the expression of the endogenous TCR chain. In certain embodiments, one or more other endogenous T cell receptor loci are further modified to express a gene of interest. In certain embodiments, the gene of interest is an antitumor cytokine, a costimulatory molecule ligand, a tracking gene, or a suicide gene.

[0020] The subject matter of this disclosure further provides nucleic acids (nucleotide acids) encoding recombinant TCRs as described herein, and nucleic acid compositions comprising recombinant TCRs as described herein. In certain embodiments, the nucleic acid sequence is contained in a vector. The subject matter of this disclosure also provides vectors comprising nucleic acid compositions as described herein. Kits comprising recombinant TCRs as described herein, immune-responsive cells as described herein, pharmaceutical compositions as described herein, nucleic acid compositions as described herein, or vectors as described herein are further provided. In certain embodiments, the kit further includes written instructions for treating and / or preventing neoplasms, pathogen infections, autoimmune disorders, or allogeneic transplants.

[0021] Exemplary neoplasms that can be used as subject matter of this disclosure include leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Valdenström macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, intralymphatic sarcoma). (lymphangioendotheliosarcoma), synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, transverse Examples of cancers that can be classified as cerebrospinal carcinoma include, but are not limited to, monosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma (nileduct carcinoma), choriocarcinoma, seminomas, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendrocyte, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0022] The subject matter of this disclosure further provides the use of any recombinant TCR, any pharmaceutical composition, or any immune-responsive cell disclosed herein for therapeutic use.

[0023] The following detailed descriptions are provided as examples, but are not intended to limit the subject matter of this disclosure to the specific embodiments described herein and should be understood in conjunction with the accompanying drawings. In embodiments of the present invention, for example, the following items are provided. (Item 1) A recombinant T cell receptor (TCR) comprising an antigen-binding chain including an extracellular antigen-binding domain and a constant domain, wherein the recombinant TCR binds to an antigen in an HLA-independent manner, and the constant domain comprises a natural or modified TRAC peptide and / or a natural or modified TRBC peptide. (Item 2) Recombinant TCRs as described in item 1, expressed from expression cassettes located at the endogenous TRAC locus and / or TRBC locus of immune-responsive cells. (Item 3) The recombinant TCR according to item 2, wherein the arrangement of the recombinant TCR expression cassette disrupts or inactivates the endogenous expression of the TCR, including the native TCRα chain and / or native TCRβ chain, in the immune-responsive cells. (Item 4) The recombinant TCR according to item 2, wherein the arrangement of the recombinant TCR expression cassette prevents or eliminates mispairing between the recombinant TCR and the native TCRα chain and / or native TCRβ chain in the immune-responsive cells. (Item 5) The recombinant TCR according to any one of items 1 to 4, wherein the constant domain can form a homodimer or heterodimer with another constant domain. (Item 6) The recombinant TCR according to any one of items 1 to 5, wherein the antigen-binding chain can associate with a CD3ζ polypeptide. (Item 7) The recombinant TCR according to item 6, wherein the antigen-binding chain can activate the CD3ζ polypeptide associated with the antigen-binding chain when it binds to the antigen. (Item 8) The recombinant TCR described in item 7, wherein activation of the CD3ζ polypeptide can activate immune-responsive cells. (Item 9) The recombinant TCR described in item 6, wherein the CD3ζ polypeptide is endogenous and integrated into the natural CD3 complex. (Item 10) The recombinant TCR described in item 6, wherein the CD3ζ polypeptide is exogenous and optionally integrated with a co-stimulatory molecule selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, and any combination thereof. (Item 11) The recombinant TCR according to any one of items 1 to 10, wherein the antigen-binding chain further comprises a costimulatory region, and the recombinant TCR can stimulate immune-responsive cells when it binds to an antigen. (Item 12) The recombinant TCR according to item 11, wherein the co-stimulatory region comprises a co-stimulatory molecule selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, and any combination thereof. (Item 13) The recombinant TCR according to item 12, wherein the aforementioned co-stimulatory region contains the CD28 polypeptide. (Item 14) A recombinant TCR described in any one of items 1 to 13, which can integrate with the CD3 complex and result in HLA-independent antigen recognition. (Item 15) The aforementioned CD3 complex is endogenous, and is a recombinant TCR as described in item 14. (Item 16) Recombinant TCRs, as described in item 15, that replace endogenous TCRs in the CD3 / TCR complex. (Item 17) The recombinant TCR according to any one of items 1 to 16, wherein the extracellular antigen-binding domain can be dimerized with another extracellular antigen-binding domain. (Item 18) The recombinant TCR according to any one of items 1 to 17, wherein the extracellular antigen-binding domain comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding portion or fragment thereof of an antibody, or an antigen-binding portion of a TCR. (Item 19) The recombinant TCR according to any one of items 1 to 18, wherein the extracellular antigen-binding domain comprises one or two immunoglobulin variable regions. (Item 20) The extracellular antigen-binding domain is the variable region of the antibody's heavy chain (V H Recombinant TCRs as listed in item 18, including ) (Item 21) The extracellular antigen-binding domain is the variable region of the antibody's light chain (V L Recombinant TCRs as listed in item 18, including ) (Item 22) The recombinant TCR described in item 18, wherein the extracellular antigen-binding domain can be dimerized with another extracellular antigen-binding domain. (Item 23) The extracellular antigen-binding domain is the V of the antibody. H Including the above V H However, the V of the antibody LA recombinant TCR as described in item 20, which can dimerize with another extracellular antigen-binding domain containing a variable fragment (Fv). (Item 24) The extracellular antigen-binding domain is the V of the antibody. L Including the above V L However, the V of the antibody H A recombinant TCR as described in item 21, which can dimerize with another extracellular antigen-binding domain containing a variable fragment (Fv). (Item 25) A recombinant TCR that binds to a tumor antigen, as described in any one of items 1 through 24. (Item 26) The aforementioned tumor antigens include CD19, MUC16, MUC1, CAlX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2, 3, 4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, mesothelin, ERBB2, Recombinant TCRs as described in item 25, selected from the group consisting of MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB. (Item 27) The recombinant TCR described in item 25 or 26, wherein the tumor antigen is CD19. (Item 28) A recombinant TCR described in any one of items 1 through 27, which exhibits greater antigen sensitivity than a CAR targeting the same antigen. (Item 29) A recombinant TCR described in any one of items 1 to 28, which can induce an immune response by binding to an antigen present at low density on the surface of tumor cells. (Item 30) The recombinant TCR according to any one of items 1 to 28, wherein the antigen present at a low density on the cell surface has a density of less than approximately 10,000 molecules per cell. (Item 31) Immune-responsive cells containing recombinant TCRs as described in any one of items 1 through 30. (Item 32) The immune-responsive cell according to item 31, wherein the expression cassette of at least one antigen-binding chain of the recombinant TCR is located at the endogenous gene locus of the immune-responsive cell. (Item 33) The immune-responsive cell according to item 32, wherein the expression cassette of the two antigen-binding chains of the recombinant TCR is located at the endogenous gene locus of the immune-responsive cell, and the two antigen-binding chains can be dimerized. (Item 34) An immune-responsive cell according to any one of items 31 to 33, wherein the endogenous gene locus is the CD3δ locus, CD3ε locus, CD247 locus, B2M locus, TRAC locus, TRBC locus, TRDC locus and / or TRGC locus. (Item 35) The immune-responsive cells described in item 34, wherein the endogenous gene locus is the TRAC locus and / or the TRBC locus. (Item 36) The immune-responsive cell according to item 35, wherein the arrangement of the expression cassette of the recombinant TCR disrupts or inactivates the endogenous expression of the TCR, including the native TCRα chain and / or native TCRβ chain, in the immune-responsive cell, thereby preventing or eliminating mispairing between the recombinant TCR and the native TCRα chain and / or native TCRβ chain in the immune-responsive cell. (Item 37) An immune-responsive cell according to any one of items 32 to 36, wherein the endogenous gene locus includes a modified transcriptional terminator region. (Item 38) The immune-responsive cell described in item 37, wherein the modified transcriptional terminator region comprises a genomic element selected from the group consisting of the TK transcriptional terminator, GCSF transcriptional terminator, TCRA transcriptional terminator, HBB transcriptional terminator, bovine growth hormone transcriptional terminator, SV40 transcriptional terminator, and P2A element. (Item 39) An immune-responsive cell according to any one of items 32 to 38, wherein one endogenous T cell receptor locus in the cell is modified to express at least one antigen-binding chain of the recombinant TCR, and one or more other endogenous T cell receptor loci in the cell are modified to exclude the expression of the endogenous TCR chain. (Item 40) The immune-responsive cells described in item 39, wherein one or more of the aforementioned endogenous T cell receptor gene loci are further modified to express the gene of interest. (Item 41) The immune-responsive cells described in item 40, wherein the target gene is an antitumor cytokine, a costimulatory molecule ligand, a tracking gene, or a suicide gene. (Item 42) Immune-responsive cells as described in any one of items 31 to 41, selected from the group consisting of T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, human embryonic stem cells, and pluripotent stem cells capable of differentiating into lymphoid cells. (Item 43) An immune-responsive cell belonging to the individual, as described in any one of items 31 through 42. (Item 44) An immune-responsive cell according to any one of items 31 to 43, further comprising at least one exogenous costimulatory ligand. (Item 45) The immune-responsive cells described in item 44, wherein the exogenous costimulatory ligand is selected from the group consisting of CD80, CD86, 41BBL, CD275, CD40L, OX40L, and any combination thereof. (Item 46) An immune-responsive cell as described in item 44 or 45, further comprising or consisting of one exogenous costimulatory ligand. (Item 47) The immune-responsive cells described in item 46, wherein the aforementioned exogenous costimulatory ligand is CD80 or 4-1BBL. (Item 48) An immune-responsive cell as described in item 44 or 45, further comprising or consisting of two exogenous costimulatory ligands. (Item 49) The immune-responsive cells described in item 48, wherein the two exogenous costimulatory ligands are CD80 and 4-1BBL. (Item 50) An immune-responsive cell as described in any one of items 31 to 49, further comprising at least one chimeric costimulatory receptor (CCR). (Item 51) The immune-responsive cell described in item 50, wherein the CCR comprises a costimulatory molecule selected from the group consisting of CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, and any combination thereof. (Item 52) A pharmaceutical composition comprising an effective amount of immune-responsive cells as described in any one of items 31 to 47, and a pharmaceutically acceptable excipient. (Item 53) A pharmaceutical composition as described in item 52 for treating neoplasms. (Item 54) A method for reducing tumor burden in a subject, comprising administering to the subject an effective amount of immune-responsive cells described in any one of items 31 to 51 or a pharmaceutical composition described in item 52 or 53. (Item 55) A method for treating or preventing a neoplasm, comprising administering to a subject an effective amount of immune-responsive cells according to any one of items 31 to 51 or a pharmaceutical composition according to item 52 or 53. (Item 56) The method according to item 55, wherein the neoplasm is selected from the group consisting of hematological cancer, B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, non-Hodgkin lymphoma, and adenocarcinoma. (Item 57) The method according to item 55 or 56, wherein the neoplasm is B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the recombinant TCR binds to CD19. (Item 58) The method according to item 55 or 56, wherein the neoplasm is B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, or non-Hodgkin lymphoma, and the recombinant TCR binds to BCMA, ADGRE2, CCR1, CD22, CD70 or a combination thereof. (Item 59) The aforementioned new organism is CD19 + The method described in any one of items 55 to 58, which is ALL. (Item 60) A method for producing antigen-specific immune-responsive cells, comprising introducing a nucleic acid sequence encoding a recombinant TCR as described in any one of items 1 to 30 into immune-responsive cells. (Item 61) The method according to item 60, wherein the nucleic acid sequence is contained in the vector. (Item 62) The method according to item 60 or 61, wherein the expression cassette of at least one antigen-binding chain of the recombinant TCR is located at the endogenous gene locus of the immune-responsive cell. (Item 63) The method according to item 62, wherein the expression cassette of the two antigen-binding chains of the recombinant TCR is located at the endogenous gene locus of the immune-responsive cell, and the two antigen-binding chains can be dimerized. (Item 64) The method according to item 62 or 63, wherein the endogenous locus is the CD3δ locus, the CD3ε locus, the CD247 locus, the B2M locus, the TRAC locus, the TRBC locus, the TRDC locus and / or the TRGC locus. (Item 65) The method according to item 64, wherein the endogenous locus is the TRAC locus and / or the TRBC locus. (Item 66) The method according to any one of items 63 to 65, wherein the arrangement of the expression cassette of the recombinant TCR disrupts or inactivates the endogenous expression of the TCR, including the native TCRα chain and / or native TCRβ chain, in the immune-responsive cells, thereby preventing or eliminating mispairing between the recombinant TCR and the native TCRα chain and / or native TCRβ chain in the immune-responsive cells. (Item 67) The method according to any one of items 62 to 66, wherein the endogenous gene locus includes a modified transcriptional terminator region. (Item 68) The method according to item 67, wherein the modified transcription terminator region includes a genomic element selected from the group consisting of a TK transcription terminator, a GCSF transcription terminator, a TCRA transcription terminator, an HBB transcription terminator, a bovine growth hormone transcription terminator, an SV40 transcription terminator, and a P2A element. (Item 69) The method according to any one of items 62 to 68, wherein if one endogenous T cell receptor locus in a cell is modified to express the at least one antigen-binding chain of the recombinant TCR, then one or more other endogenous T cell receptor loci in the cell are modified to exclude the expression of the endogenous TCR chain. (Item 70) The method according to item 69, wherein one or more other endogenous T cell receptor loci are further modified to express the gene of interest. (Item 71) The method according to item 70, wherein the target gene is an antitumor cytokine, a costimulatory molecule ligand, a tracking gene, or a suicide gene. (Item 72) A method for extending the survival of a subject having a neoplasm, comprising the step of administering to the subject an effective amount of immune-responsive cells according to any one of items 31 to 51 or a pharmaceutical composition according to item 52 or 53. (Item 73) A nucleic acid encoding a recombinant TCR as described in any one of items 1 through 30. (Item 74) A nucleic acid composition comprising a recombinant TCR as described in any one of items 1 to 30. (Item 75) The nucleic acid composition according to item 74, wherein the nucleic acid sequence is contained in the vector. (Item 76) A vector comprising the nucleic acid composition described in item 74 or 75. (Item 77) A kit comprising a recombinant TCR as described in any one of items 1 to 30, an immune-responsive cell as described in any one of items 31 to 51, a pharmaceutical composition as described in item 52 or 53, a nucleic acid composition as described in item 74 or 75, or a vector as described in item 76. (Item 78) The kit described in item 77, further including written instructions for treating and / or preventing neoplasms, pathogen infections, autoimmune disorders, or allogeneic transplantation. (Item 79) A recombinant TCR as described in any one of items 1 to 30, for use in the treatment of therapies. (Item 80) A pharmaceutical composition as described in item 51 or 52 for use in the treatment of medicine. (Item 81) Immune-responsive cells as described in any one of items 31 to 51, for use in therapy. (Item 82) Use of immune-responsive cells as described in any one of items 31 to 51 or a pharmaceutical composition as described in item 52 or 53 to reduce tumor burden in a subject. (Item 83) Use of immune-responsive cells as described in any one of items 31 to 51 or a pharmaceutical composition as described in item 52 or 53 for treating or preventing neoplasms. (Item 84) Use of immune-responsive cells as described in any one of items 31 to 51 or a pharmaceutical composition as described in item 52 or 53 to extend the survival of a subject having a neoplasm (e.g., cancer). [Brief explanation of the drawing]

[0024] [Figure 1-1] Figures 1A–1E illustrate HLA-independent TCR-based chimeric antigen receptor HIT (HIT-CAR, i.e., HI-TCR or HIT) and gene targeting strategies at the TRAC locus in human T cells. (A) Schematic diagram of T cell receptor (TCR), B cell receptor (BCR), chimeric antigen receptor (CAR), and HLA-independent TCR-based chimeric antigen receptor (HIT-CAR, i.e., HI-TCR or HIT). (B) CRISPR / Cas9-targeted integration of three receptors into the TRAC locus. Top: TRAC locus; Center: rAAV6 containing different receptor cassettes flanked by homology arms. (C) Representative TCR / mouse F(ab')2 flow cytometry plot 4 days after TRAC targeting. TCR antibody epitopes recognize the constant chains of TCR alpha and beta. (D) Cytotoxic activity (n=3) using an 18-hour bioluminescence assay with firefly luciferase (FFL)-expressing NALM-6 as the target cells. (E) Relative CAR MFI (MFI at 1=0 hours) of CAR T cells after 1, 2, or 4 (arrow) stimulations in CD19-positive target cells. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0025] [Figure 2]Figures 2A and 2B show HI-TCR expression and therapeutic efficacy. (A) Representative TCR / mouse F(ab')2 flow cytometry plot 4 days after TRAC targeting. (B) Kaplan-Meier analysis of mouse survival when mice with NALM-6 were treated with 5 × 10⁵ CAR T cells.

[0026] [Figure 3-1] Figures 3A to 3E show the gene targeting strategies and expression of NYESO TCR. (A) Schematic diagram of the NYESO TCR gene incorporated into the TCR alpha or beta chain. (B) Representative TCR-V-beta-1 flow cytometry plot 4 days after TRAC or TRBC targeting. (C) Cytotoxic activity (n=3) using an 18-hour bioluminescence assay with firefly luciferase (FFL) expressing PC3 as target cells. (D) Schematic diagram of simultaneous targeting of both TCR alpha and TCR beta. (E) Representative TCR-V-beta-1 / 4-1BBL flow cytometry plot 4 days after simultaneous targeting of TRAC and TRBC. [Figure 3-2] Same as above.

[0027] [Figure 4-1] Figures 4A–4C illustrate the CAR integration strategies into the TRAC locus and the modulation of expression by various transcription termination signals / 3' untranslated regions (3'UTRs). (A) Schematic diagram of the 1928z CAR gene integrated into the TRAC locus. PolyA (black box) corresponds to segments of the CAR cassette modified to examine different viral and mammalian 3'UTRs. (B) Representative CAR flow plot (left panel) after 3 days of TRAC, as well as geometric mean fluorescence intensity (gMFI), MFI, and median (right panel) of the CAR expression population. The original 3'UTR sequence of bovine growth hormone polyA is boxed. (C) Absolute (top) and relative (bottom) CAR MFI (MFI at 1=0 hours) of CAR T cells after 1 and 2 stimulations in CD19-positive target cells (as shown in Figure 1). [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0028] [Figure 5-1] Figures 5A and 5B demonstrate the efficacy of genetically integrated CARs with different 3'UTR sequences. (A) Mice with FFL-NALM-6 were treated with 1 × 10⁵ CAR T cells, in which case tumor burden is shown as a bioluminescent signal quantified per animal 14 days post-T cell injection. n=6 mice per group. (B) Tumor burden (mean radiance) of NALM-6 mice treated with 1 × 10⁵ CAR T cells, quantified at 7, 14, and 21 days post-T cell injection (n=6; line=1 mouse). [Figure 5-2] Same as above.

[0029] [Figure 6] Figures 6A–6C show HIT gene targeting at the TRAC locus in human T cells. (A) CRISPR / Cas9-targeted CAR or HIT gene insertion into the TRAC locus. Top: TRAC locus; Center: rAAV6 containing a CAR cassette flanked by homologous arms; Bottom: rAAV6 containing a HIT cassette flanked by homologous arms. (B) Representative CAR / HIT flow plots 4 days after transfection of T cells with Cas9 mRNA and TRAC gRNA, and addition of AAV6. CAR and HIT surface proteins were detected using goat anti-mouse IgG. (C) Mean CAR / HIT mean fluorescence intensity (MFI) analyzed by FACS 4 days after transduction (n=6 independent experiments).

[0030] [Figure 7]Figures 7A and 7B demonstrate that HIT T cells are superior to CAR T cells in killing target cells expressing low antigen levels. Nalm6 cell lines (expressing firefly luciferase) were gene-edited at the CD19 locus using CRISPR / Cas9 to generate clones expressing different CD19 levels. (A) FACS analysis of representative Nalm6 clones for each CD19 expression level group (Neg = negative). (B) Cytotoxic activity using a 4-hour bioluminescence assay with NALM6 and CAR (red square) or HIT (blue circle) T cells as target cells expressing different CD19 levels in a 1:1 effector (E):target (T) ratio.

[0031] [Figure 8] Figures 8A–8C show that HIT T cells are superior to CAR T cells in killing target cells expressing low antigen levels. Cytotoxic activity using an 18-hour bioluminescence assay with NALM6 as target cells expressing different CD19 levels (shown on the right), incubated with untransduced T cells (A), CAR T cells (B), and HIT T cells (C) in different effector (E):target (T) ratios.

[0032] [Figure 9]Figures 9A–9D show that HIT T cells expressing a costimulatory ligand are superior to CAR T cells in controlling established B-ALL tumors with very low CD19 levels. Mice with NALM-6 were treated with 4 × 10⁵ untransduced (NT), CAR, or HIT T cells. Tumor load was quantified weekly over a 54-day period using BLI. Quantification is the mean photon counts obtained ventrally and dorsally per animal at all time points. Each line represents one mouse, with n=5 mice per group. (A) Untransduced (black) vs. CAR (red) T cells. (B) CAR (red) vs. HIT (green) T cells. (C) T cells expressing HIT alone (green), HIT + CD80 costimulatory ligand (orange), HIT + 41BBL costimulatory ligand (pink), or HIT + CD80 + 41BBL costimulatory ligand (blue). (D) Mouse survival analysis.

[0033] [Figure 10] Figures 10A–10C demonstrate that baseline TRAC-CAR expression can be controlled by distinct 3'UTR sequences without affecting cell surface replenishment dynamics after antigen encounter. (A) CRISPR / Cas9-targeted CAR gene integration into the TRAC locus. The targeted construct (AAV) contains a 1928z CAR coding sequence flanked by homologous sequences to the TRAC locus (LHA□ and RHA, left and right homology arms), followed by a 3'UTR sequence. (B) Each 3'UTR sequence results in different CAR surface levels (measured by FACS). TK: Thymidine kinase (short version); GCSF: Human GCSF derived from pEF-BOS plasmid; TCR: TCR alpha, exon 4; HBB: Human B-globin; RBG: Rabbit B-globin; SV40: Monkey virus 40 polyA; P2A: Porcine staghorn virus-1 autocleavage 2A sequence; This allowed the use of the endogenous TRAC polyA sequence. (C)CAR T cells were stimulated once with CD19-expressing 3T3 cells (indicated by the red arrows), and CAR MFI was measured every 24 hours over a 3-day period. All CAR T cells showed similar regulation of CAR expression. [Modes for carrying out the invention]

[0034] The subject matter of this disclosure provides an HLA-independent (or non-HLA-restricted) T cell receptor ("HI-TCR") that binds to an antigen of interest in an HLA-independent manner. In certain embodiments, the HI-TCR is a TCR molecule in which the TCR variable domain is replaced by an antibody-derived variable domain (Fv) to result in an FvTCR. In certain non-limiting embodiments, the HI-TCR can bind to a tumor antigen or a pathogen antigen. The subject matter of this disclosure also provides cells comprising genetically modified immune-responsive cells (e.g., T cells, NKT cells, or CTL cells) containing the HI-TCR of this disclosure. In certain non-limiting embodiments, antigen binding by the HI-TCR can activate the immune-responsive cells. The subject matter of this disclosure also provides methods of using such cells to induce and / or enhance an immune response to a target antigen, and / or to treat and / or prevent neoplasms or other diseases / disorders in which an increased antigen-specific immune response is desired.

[0035] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The following references provide general definitions of many terms used in the subject matter of this disclosure: Singletonet al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise specified, the following terms have the meanings of the following:

[0036] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, and will depend in part on how that value is measured or determined, i.e., on the limits of the measurement system. For example, “about” may mean within three standard deviations or more than three standard deviations per practice in the art. Alternatively, “about” may mean a range of up to about 20% of a given value, for example, up to about 10%, up to about 5%, or up to about 1%. Or, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of a given value, for example, up to about five times or up to about two times.

[0037] "Activating immune-responsive cells" means inducing signaling or protein changes within cells that lead to the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor-suppressive tyrosine motifs (ITAMs), a signaling cascade is produced. In certain embodiments, when an endogenous TCR or exogenous CAR binds to an antigen, immunological synapse formation occurs near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.), involving the clustering of many molecules. This clustering of membrane-bound signaling molecules leads to phosphorylation of the ITAM motif contained within the CD3 chain. This phosphorylation then initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce overall gene expression in T cells, increasing IL-2 production for the proliferation and expression of master regulatory T cell proteins, and initiating a T cell-mediated immune response.

[0038] "Stimulating immune-responsive cells" refers to signals that elicit a robust and sustained immune response. In various embodiments, this occurs after the activation of immune cells (e.g., T cells) or is simultaneously mediated via receptors including CD28, CD137(4-1BB), OX40, CD40, and ICOS, but is not limited to these. Receiving multiple stimulating signals may be crucial for initiating a robust and long-lasting T-cell-mediated immune response. T cells can be rapidly inhibited and become unresponsive to antigens. While the effects of these co-stimulatory signals may vary, they generally result in increased gene expression to produce long-lasting, proliferating, and anti-apoptotic T cells that respond robustly to antigens for complete and sustained eradication.

[0039] As used herein, the term “antigen-recognizing receptor” refers to a receptor that can activate an immune or immune-responsive cell (e.g., a T cell) in response to its binding to an antigen. Non-limiting examples of antigen-recognizing receptors include innate or endogenous T cell receptors ("TCRs") and chimeric antigen receptors ("CARs").

[0040] As used herein, the term “antibody” means not only intact antibody molecules but also fragments of antibody molecules that retain immunogenic binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')2 and Fab. Fab fragments lacking F(ab')2 and the Fc fragment of the intact antibody may disappear more rapidly from circulation and have lower nonspecific tissue binding of the intact antibody (Wahlet al., J. Nucl. Med. 24:316-325 (1983)). As used herein, antibody includes whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single-chain V-region fragments (scFv), fusion polypeptides, and atypical antibodies. In certain embodiments, the antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (V in this specification). H (and abbreviated) and heavy chain steady state (C H The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region (V in this specification). L (and abbreviated) and light chain steady C L It is composed of regions. The light chain constant region is one domain, C L It consists of V. H and V L The region can be further subdivided into hypervariable regions called complementary determination regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). H and V LIt consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0041] As used herein, "CDR" is defined as the amino acid sequence of the complementarity-determining region of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabatet al., Sequences of Proteins of Immunological Interest, 4th U.S. Department of Health and Human Services, National Institutes of Health (1987). Generally, antibodies It comprises three heavy chains and three light chain CDRs or CDR regions within the variable region. The CDRs provide the majority of the contact residues for the antibody's binding to the antigen or epitope. In certain embodiments, the CDR regions are described using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242).

[0042] As used herein, the terms “single-chain variable fragment” or “scFv” mean V H ::V L A heavy chain of immunoglobulin covalently linked to form a heterodimer (V H ) and light chain (V L It is a fusion protein of the variable region of ). H and V LThey are either directly concatenated or V H The N-terminus of V L Connect to the C terminal of, or V H The C-terminus of V L The N-terminus of the protein is linked by a peptide-encoding linker (e.g., 10, 15, 20, or 25 amino acids). The linker is typically glycine-rich for flexibility and serine or threonine-rich for solubility. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are used to test for V, as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). H and V L It can be expressed from nucleic acids containing the sequence encoding it. See also U.S. Patents 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publications 20050196754 and 20050196754. Inhibitory antagonist scFv has been described (e.g., Zhaoetal., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al.). al., J CachexiaSarcopeniaMuscle 2012 August 12;Shieh et al., J Imunol2009183(4):2277-85;Giomarellietal., Thromb Haemost 2007 97(6):955-63;Fife eta., JClin See Invst2006116(8):2252-61;Brocks et al., Immunotechnology 1997 3(3):173-84;Moosmayer et al.,Ther Immunol 1995 2(10:31-40). Stimulating jaw Nist scFv is described (see, for example, Peteretal., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho etal., BioChim Biophys Acta 2003 1638(3):257-66).

[0043] As used herein, the term “affinity” means a measure of binding strength. Affinity may depend on the closeness of stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term “affinity” also includes “avidity,” which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody to an antigen are known in the art and are not limited to, but include various antigen-binding experiments, such as functional assays (e.g., flow cytometry assays).

[0044] As used herein, the term “chimeric antigen receptor” or “CAR” refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating immune-responsive cells, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an scFv. The scFv can be derived from fusing the variable heavy and light chain regions of an antibody. Alternatively, or further, the scFv may be derived from Fab' (obtained, for example, from a Fab library, instead of being derived from an antibody). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain. In certain embodiments, the CAR has high binding affinity or avidity to the antigen.

[0045] As used herein, the term “nucleic acid molecule” includes any nucleic acid molecule encoding the polypeptide of interest or a fragment thereof. Such a nucleic acid molecule does not need to be 100% homologous or identical to the endogenous nucleic acid sequence, but may exhibit substantial identity. A polynucleotide having “substantial identity” or “substantial homology” to the endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. “Hybridize” means a pair that, under various stringency conditions, forms a double-stranded molecule with a complementary polynucleotide sequence (e.g., a gene described herein), or a portion thereof. (See, for example, Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0046] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than 75 mM trisodium citrate, e.g., less than about 500 mM NaCl and less than 50 mM trisodium citrate, or less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low-stringency hybridization is obtained in the absence of organic solvents, e.g., formamide, while high-stringency hybridization is obtained in the presence of at least about 35% formamide, e.g., at least about 50% formamide. Stringent temperature conditions will typically include temperatures of at least about 30°C, at least about 37°C, or at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of surfactants, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various conditions as needed, various levels of stringency can be achieved. In certain embodiments, hybridization is carried out at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In certain embodiments, hybridization is carried out at 500 mM The hybridization is carried out at 37°C in NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In certain embodiments, hybridization is carried out at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

[0047] For many applications, the stringency will also change during the washing step after hybridization. Washing stringency conditions can be defined by salt concentration and temperature. As mentioned above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, a stringent salt concentration for the washing step may be less than about 30 mM NaCl and less than 3 mM trisodium citrate, e.g., less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step will typically include temperatures of at least about 25°C, at least about 42°C, or at least about 68°C. In certain embodiments, the washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the washing step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In certain embodiments, the washing step is carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guideto Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrooketal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0048] "Substantially identical" or "substantially homologous" means a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homology or identity with the amino acid or nucleic acid sequence used for comparison.

[0049] Sequence identity can be verified using sequence analysis software (e.g., Sequence Analysis). This can be measured by using the Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 (BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine substitutions. An exemplary method for determining the degree of identity is to use the BLAST program with probability scores from e-3 to e-100 indicating closely related sequences.

[0050] "Analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0051] As used herein, the term "ligand" refers to a molecule that binds to a receptor. In certain embodiments, a ligand binds to a receptor on another cell, enabling intercellular recognition and / or interaction.

[0052] As used herein, the terms “constitutive expression” or “to be constitutively expressed” mean expression or being expressed under any physiological conditions.

[0053] "Disease" means any condition, disease, or disorder that damages or interferes with the normal function of a cell, tissue, or organ, such as neoplasms and pathogenic infections of cells.

[0054] "Effective dose" means an amount sufficient to have a therapeutic effect. In certain embodiments, the "effective dose" is an amount sufficient to stop, improve, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0055] "Forcing tolerance" means preventing the activation of autoreactive or immune-responsive cells that target the transplanted organ or tissue.

[0056] "Endogenous" refers to nucleic acid molecules or polypeptides that are normally expressed in cells or tissues.

[0057] "Exogenous" means a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or is not present in sufficient levels to achieve the functional effect obtained when overexpressed. Therefore, the term "exogenous" encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, including foreign, heterologous, overexpressed nucleic acid molecules and polypeptides. "Exogenous" nucleic acid means a nucleic acid that is not present in natural wild-type cells; for example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, location / situation, or both. For clarity, an exogenous nucleic acid may have the same or different sequences compared to its natural endogenous counterpart; it may be introduced into the cell itself or its precursor cells by genetic engineering and, if necessary, ligated to alternative regulatory sequences, such as non-natural promoters or secretory sequences.

[0058] "Heterogeneous nucleic acid molecules or polypeptides" means nucleic acid molecules (e.g., cDNA, DNA, or RNA molecules) or polypeptides that are not normally present in cells or samples obtained from cells. These nucleic acids may originate from another organism, or they may be, for example, mRNA molecules that are not normally expressed in cells or samples.

[0059] "Immune-responsive cells" refer to cells that function in the immune response, or their progenitor cells or offspring.

[0060] "Modulating" means changing something in a positive or negative direction. Examples of modulation include changes of approximately 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0061] "Increase" means a positive change of at least approximately 5%. The change may be approximately 5%, 10%, 25%, 30%, 50%, 75%, 100%, or higher.

[0062] "Decrease" means a negative change of at least about 5%. The change may be about 5%, 10%, 25%, 30%, 50%, 75%, or even 100%.

[0063] "Isolated cells" refers to cells that have been separated from the molecules and / or cellular components that naturally accompany them.

[0064] The terms “isolated,” “purified,” or “biologically pure” refer to material that contains, to the extent that it alters the components normally associated with it as found in its natural state. “Isolating” indicates the degree of separation from the original source or its surroundings. “Purifying” indicates a higher degree of separation than isolation. A “purified” or “biologically pure” protein is free from other material to such an extent that impurities do not substantially affect the protein’s biological properties or cause other harmful consequences. That is, a nucleic acid or peptide is purified if it is substantially free from cellular material, viral material, or culture medium if produced by recombinant DNA technology, or from chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, e.g., polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” may also indicate that the nucleic acid or protein produces essentially one band in the electrophoretic gel. For proteins that can be modified, e.g., phosphorylated or glycosylated, different modifications may produce different isolated proteins that can be purified separately.

[0065] As used herein, the term "antigen-binding domain" refers to a domain that can specifically bind to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0066] As used herein, “linker” should mean a functional group (e.g., a chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids so that they are linked to one another. As used herein, “peptide linker” means a functional group (e.g., a peptide linker) that couples two proteins together (e.g., a peptide linker). H and V L This refers to one or more amino acids used to couple domains. In certain embodiments, the linker includes the sequence described in GGGGSGGGGSGGGGS (SEQ ID NO: 31).

[0067] "Neoplasm" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplasmic proliferation is typically uncontrolled, progressive, and occurs under conditions that do not induce or cause the cessation of replication of normal cells. Neoplasms can affect a variety of cell types, tissues, or organs, including, but are not limited to, organs selected from the group consisting of the bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestines, kidneys, liver, lungs, lymph nodes, nerve tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureters, urethra, uterus, and vagina, or their tissues or cell types. Neoplasms include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In certain embodiments, a neoplasm is a solid tumor.

[0068] Exemplary neoplasms that can be used as subject matter of this disclosure include leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Valdenström macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endosarcoma, lymphangiosarcoma, lymphangiosarcoma, lymphangiothelial endothelial sarcoma). Examples of cancers that can be classified as cancer include, but are not limited to, sarcomas, synoviomas, mesotheliomas, Ewing's tumors, leiomyosarcomas, rhabdomyosarcomas, colon cancers, pancreatic cancers, breast cancers, ovarian cancers, prostate cancers, squamous cell carcinomas, basal cell carcinomas, adenocarcinomas, sweat gland cancers, sebaceous gland cancers, papillary carcinomas, papillary adenocarcinomas, cystadenocarcinomas, medullary carcinomas, bronchogenic carcinomas, renal cell carcinomas, liver cancers, cholangiocarcinomas, choriocarcinomas, seminomas, embryonal carcinomas, Wilms' tumors, cervical cancers, uterine cancers, testicular cancers, lung cancers, small cell lung cancers, bladder cancers, epithelial carcinomas, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal glandomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, Schwannomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas.

[0069] A "receptor" refers to a polypeptide, or a part thereof, located on the cell membrane that selectively binds to one or more ligands.

[0070] "Recognizing" means selectively binding to a target. T cells that recognize tumors can express receptors (e.g., TCRs or CARs) that bind to tumor antigens.

[0071] "Reference" or "control" refers to the standard of comparison. For example, the level of scFv-antigen binding in cells expressing both CAR and scFv can be compared to the level of scFv-antigen binding in corresponding cells expressing only CAR.

[0072] "Secreted" refers to polypeptides that are released from a cell via secretory pathways through the endoplasmic reticulum and Golgi apparatus, and as vesicles that transiently fuse at the cell plasma membrane, releasing proteins to the outside of the cell.

[0073] A "signal sequence" or "leader sequence" refers to a peptide sequence (e.g., 5, 10, 15, 20, 25, or 30 amino acids) located at the N-terminus of a newly synthesized protein that directs its entry into the secretory pathway. Examples of suitable leader sequences include, but are not limited to, the IL-2 signal sequences: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 12) (human), MYSMQLASCVTLTLVLLVNS (SEQ ID NO: 13) (mouse); Kappa leader sequences: METPAQLLFLLLLWLPDTTG (SEQ ID NO: 14) (human), METDTLLLWVLLLWVPGSTG (SEQ ID NO: 15) (mouse); CD8 leader sequence: MALPVTALLLPLALLLHAARP (SEQ ID NO: 16) (human); truncated human CD8 signal peptide: MALPVTALLLPLALLLHA (SEQ ID NO: 28) (human); Albumin signal sequence: MKWVTFISLLFSSAYS (SEQ ID NO: 29) (human); and prolactin signal sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 30) (human). "Soluble" means a polypeptide that can freely diffuse in an aqueous environment (e.g., not bound to a membrane).

[0074] "Specifically binding" means a polypeptide or fragment thereof that recognizes and binds to a target biomolecule (e.g., a polypeptide), but substantially does not recognize or bind to other molecules in a sample, e.g., a biological sample naturally containing the polypeptide of this disclosure.

[0075] As used herein, the term “tumor antigen” refers to an antigen (e.g., polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-IS neoplastic cells. In certain embodiments, the tumor antigen includes any polypeptide expressed by a tumor that can activate or induce an immune response via an antigen-recognition receptor (e.g., CD19, MUC-16) or suppress an immune response via receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0076] The terms "comprises" and "comprising" are intended to have a broad meaning in U.S. patent law, and may also mean "includes" and "including."

[0077] As used herein, “treatment” refers to a clinical intervention in an attempt to alter the course of a disease in an individual or cell being treated, and may be performed for preventive purposes or during the course of a clinicopathological condition. The therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of the rate of disease progression, improvement or mitigation of the disease state, and remission or improvement of prognosis. By preventing the progression of disease or disability, treatment can not only prevent the exacerbation caused by the disability in a subject who is affected or diagnosed with the disability or suspected to have the disability, but also prevent the onset of the disability or symptoms of the disability in a subject who is at risk of disability or suspected to have the disability.

[0078] In this specification, “individual” or “subject” refers to a human or a non-human animal, such as a vertebrate, including mammals. Mammals include, but are not limited to, humans, primates, livestock, sport animals, rodents, and pets. Non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, and non-human primates such as apes and monkeys.

[0079] The term “immunely vulnerable” as used herein refers to a subject who is immunocompromised. This subject is highly vulnerable to opportunistic infections, which are infections caused by organisms that do not normally cause disease in individuals with a healthy immune system but can affect individuals with a poorly functioning or suppressed immune system.

[0080] Other aspects of the subject matter of this disclosure are described in the following disclosures and are within the scope of the subject matter of this disclosure.

[0081] 2.HLA-independent T cell receptor (HI-TCR) This disclosure provides HI-TCRs that bind to an antigen of interest in an HLA-independent manner. In certain non-limiting embodiments, antigen binding can activate immune-responsive cells containing the HI-TCR. In certain non-limiting embodiments, the HI-TCR comprises an antigen-binding chain. In certain embodiments, the antigen-binding chain comprises an extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain is derived from scFv, Fab, or an antibody of mouse, human, or camelid (e.g., llama) origin. In certain embodiments, the antigen-binding chain further comprises a constant domain.

[0082] 2.1. Antigen In certain embodiments, HI-TCR binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, oncoproteins. The antigen can be expressed as a peptide, or as part of a intact protein. The intact protein or part thereof may be native or mutagenic.Non-limiting examples of tumor antigens include carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, antigens of cells infected with cytomegalovirus (CMV) (e.g., cell surface antigens), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), and epithelial cell adhesion Molecules (EpCAM), receptor tyrosine protein kinases erb-B2, 3, 4 (erb-B2, 3, 4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), κ light chain, kinase insertion domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), mucin 16 (MUC16), mucin 1 (MUC1), mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, cancer testicular antigen NY-ES0-1, cancer fetal antigen (h5 Examples include T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms oncoprotein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB.

[0083] In certain embodiments, the HI-TCR binds to CD19. In certain embodiments, the HI-TCR binds to a human CD19 polypeptide. In certain embodiments, the human CD19 polypeptide contains the amino acid sequence described in SEQ ID NO: 11.

[0084] [ka]

[0085] In a particular embodiment, the HI-TCR binds to the extracellular domain of the CD19 protein.

[0086] In certain embodiments, HI-TCRs bind to pathogenic antigens for use, for example, in the treatment and / or prevention of pathogenic infections or other infectious diseases in immunocompromised subjects. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites, and protozoa that can cause disease.

[0087] Non-exclusive examples of viruses include Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also known as HDTV-III, LAVE, or HTLV-III / LAV) or HIV-III, and other isolated strains such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flavi idae (e.g., dengue virus, encephalitis virus, yellow fever virus), Coronaviridae (e.g., coronavirus), Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus), Filoviridae (e.g., Ebola virus), Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, polynuclear respiratory virus), Orthomyxoviridae (e.g., influenza virus), Bunyaviridae (e.g., Hantan virus, Bunya virus, Fre Boviruses and Nairo (Naira) viruses, Arenaviridae (hemorrhagic fever viruses) Reoviruses, orbiviruses (for example, reoviruses, orbiviruses) Examples include virulence factors for hepatitis delta (and rotavirus), Birnaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae (parvovirus), Papovaviridae (papillomavirus, polyomavirus), Adenoviridae (most adenoviruses), Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus), Poxviridae (smallpox virus, vaccinia virus, poxvirus), and Iridoviridae (e.g., African swine fever virus), as well as unclassified viruses (e.g., virulence factors for hepatitis delta (considered to be a deficient satellite of hepatitis B virus), virulence factors for non-A, non-B hepatitis (Class 1 = internally transmitted, Class 2 = parenterally transmitted (i.e., hepatitis C), Norwalk and related viruses, and astroviruses)).

[0088] Non-specific examples of bacteria include Pasteurella, Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria, though not limited to these, include Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), and Streptococcus coccus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelli.

[0089] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus.

[0090] 2.2. Extracellular antigen-binding domain In certain embodiments, the antigen-binding chain includes an extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen, such as a tumor antigen or a pathogen antigen, such as those disclosed in Section 2.1. In certain embodiments, the extracellular antigen-binding domain can dimerize with another extracellular antigen-binding domain (e.g., to form a variable fragment (Fv)), and the dimerized antigen-binding domain (e.g., Fv) specifically binds to an antigen, such as a tumor antigen or a pathogen antigen.

[0091] In certain embodiments, the extracellular antigen-binding domain includes a ligand for a cell surface receptor.

[0092] In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen, such as a tumor antigen or pathogen antigen. In certain embodiments, the antigen-binding chain can dimerize with another antigen-binding chain. In certain embodiments, the HI-TCR comprises a heterodimer containing two different antigen-binding chains. In certain embodiments, the HI-TCR comprises a homodimer containing two identical antigen-binding chains. In certain embodiments, the antigen-binding chain dimerizes via one or more disulfide linkages. In certain embodiments, the antigen-binding chain can trimer or oligomerize with one or more identical or different antigen-binding chains. In certain embodiments, the extracellular antigen-binding domain can dimerize with another extracellular antigen-binding domain (e.g., forming a variable fragment (Fv)), and the dimerized antigen-binding domain (e.g., Fv) specifically binds to an antigen, such as a tumor antigen or pathogen antigen.

[0093] In certain non-limiting embodiments, the extracellular antigen-binding domain of the HI-TCR (e.g., Fv or its analogue) is approximately 2 × 10⁻⁶ -7 M or a dissociation constant less than (K) d ) binds to the antigen. In a particular embodiment, K d It is approximately 2 x 10-7 M or less, approximately 1 x 10 -7 M or smaller, approximately 9 x 10 -8 M or less, approximately 1 x 10 -8 M or smaller, approximately 9 x 10 -9 M or less, approximately 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, approximately 3 x 10 -9 M or less, approximately 2 x 10 -9 M or less, or approximately 1 × 10 -9 M or less. In certain non-limiting embodiments, K d It is approximately 3 x 10 -9 M or less. In certain non-limiting embodiments, K d It is approximately 1 x 10 -9 M ~ approx. 3×10 -7 M is M. In certain non-limiting embodiments, K d It is approximately 1.5 × 10 -9 M ~ approx. 3×10 -7 M is M. In certain non-limiting embodiments, K d It is approximately 1.5 × 10 -9 M ~ approx. 2.7×10 -7 It is M.

[0094] The binding of the extracellular antigen-binding domain (e.g., in Fv or its analogues) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays typically detects the presence of a specific target protein-antibody complex by using a reagent (e.g., antibody, or Fv) that is specifically labeled for the complex of interest. For example, Fv can be radiolabeled and used in radioimmunoassay (RIA) (e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on, incorporated herein by reference). See Radioligand Assay Techniques, The Endocrine Society, March, 1986. Radioactive isotopes can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0095] In certain embodiments, the extracellular antigen-binding domain includes the antigen-binding portion of the TCR.

[0096] In certain embodiments, the extracellular antigen-binding domain includes the antigen-binding portion or a fragment thereof of the antibody. In certain embodiments, the extracellular antigen-binding domain includes the heavy chain variable region (V) of the antibody. H ) and / or light chain variable region (V L ) includes. In certain embodiments, the extracellular antigen-binding domain includes a single-chain variable fragment (scFv). In certain embodiments, the extracellular antigen-binding domain includes a heavy-chain-only antibody (VHH). In certain embodiments, the extracellular antigen-binding domain includes Fab which is crosslinked as needed. In certain embodiments, the extracellular antigen-binding domain includes F(ab)2. In certain embodiments, any of the above molecules can be included in a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain.

[0097] In certain embodiments, the extracellular antigen-binding domain is the variable region of the antibody's heavy chain (V H ) and / or light chain variable region (V L ) including, V H or V LIt can dimerize with another extracellular antigen-binding domain containing VL or VH (for example, to form a variable fragment (Fv)). In certain embodiments, Fv is human Fv. In certain embodiments, Fv is humanized Fv. In certain embodiments, Fv is mouse Fv. In certain embodiments, Fv is identified by screening an Fv phage library with an antigen-Fc fusion protein.

[0098] Additional extracellular antigen-binding domains targeting antigens of interest can be obtained by sequencing existing scFvs or the Fab region of existing antibodies that target the same antigen.

[0099] In certain embodiments, the dimerized extracellular antigen-binding domain of the HI-TCR of this disclosure is a mouse Fv. In certain embodiments, the dimerized extracellular antigen-binding domain is an Fv that binds to a human CD19 polypeptide. In certain embodiments, the extracellular antigen-binding domain is an Fv comprising the amino acid sequence of SEQ ID NO: 9 and specifically binding to a human CD19 polypeptide (e.g., a human CD19 polypeptide comprising the amino acid sequence described in SEQ ID NO: 11). In certain embodiments, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 is described in SEQ ID NO: 10.

[0100] In a particular embodiment, Fv is a heavy chain variable region (V) containing the amino acid sequence described in SEQ ID NO: 7. H ) includes. In certain embodiments, Fv is a light chain variable region (V) containing the amino acid sequence described in SEQ ID NO: 8. L ) includes. In a particular embodiment, Fv is V containing the amino acid sequence described in SEQ ID NO: 7. H and V containing the amino acid sequence described in Sequence ID No. 8 L This includes. In certain embodiments, the extracellular antigen-binding domain is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 7, or contains the same amino acid sequence as V. HFor example, the extracellular antigen-binding domain contains an amino acid sequence that is at least approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% homologous or identical to SEQ ID NO: 7. H Includes. In certain embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 7. H In certain embodiments, the extracellular antigen-binding domain includes an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least 90%, or at least about 95%) homologous to SEQ ID NO: 8. L For example, the extracellular antigen-binding domain contains an amino acid sequence that is at least approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% homologous or identical to SEQ ID NO: 8. L Includes. In certain embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 8. L Includes. In certain embodiments, the extracellular antigen-binding domain includes an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 7. H V containing an amino acid sequence that is at least approximately 80% (e.g., at least approximately 85%, at least approximately 90%, or at least approximately 95%) homologous or identical to SEQ ID NO: 8. L Includes. In certain embodiments, the extracellular antigen-binding domain includes the amino acid sequence described in SEQ ID NO: 7. H , and V containing the amino acid sequence described in Sequence ID No. 8 L Includes.

[0101] In a particular embodiment, Fv is a heavy chain variable region (V) containing the amino acid sequence described in SEQ ID NO: 44. H) and includes. In certain embodiments, the Fv includes a variable light chain region (V L ) and includes. In certain embodiments, the Fv includes a V H having the amino acid sequence set forth in SEQ ID NO: 44, and a V L having the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the extracellular antigen-binding domain includes a V H having an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 44. For example, the extracellular antigen-binding domain includes a V H having an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ ID NO: 44. In certain embodiments, the extracellular antigen-binding domain includes a V H having the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the extracellular antigen-binding domain includes a V L having an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 45. For example, the extracellular antigen-binding domain includes a V L having an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ ID NO: 45. In certain embodiments, the extracellular antigen-binding domain includes a V L having the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the extracellular antigen-binding domain includes a V Hand a V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 45 L is included. In certain embodiments, the extracellular antigen-binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 44 H and a V comprising the amino acid sequence set forth in SEQ ID NO: 45 L is included.

[0102] In certain embodiments, the extracellular antigen-binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 1 or a conservative modification thereof H CDR1, a V comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof H CDR2, and a V comprising the amino acid sequence set forth in SEQ ID NO: 3, a conservative modification thereof H CDR3 is included. In certain embodiments, the extracellular antigen-binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 1 H CDR1, a V comprising the amino acid sequence set forth in SEQ ID NO: 2 H CDR2, and a V comprising the amino acid sequence set forth in SEQ ID NO: 3 H CDR3 is included. In certain embodiments, the extracellular antigen-binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof L CDR1, a V comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof L CDR2, and a V comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof L CDR3 is included. In certain embodiments, the extracellular antigen-binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 4 L CDR1, a V comprising the amino acid sequence set forth in SEQ ID NO: 5 <00001?10> CDR2, and a V comprising the amino acid sequence set forth in SEQ ID NO: 6 L CDR3 is included. In certain embodiments, the extracellular antigen-binding domain comprises a V comprising the amino acid sequence set forth in SEQ ID NO: 1 H CDR1 or a conservative modification thereof, a V comprising the amino acid sequence set forth in SEQ ID NO: 2 H CDR2 or a conservative modification thereof, a V comprising the amino acid sequence set forth in SEQ ID NO: 3H CDR3 or its conservative modification, V containing the amino acid sequence described in SEQ ID NO: 4 L CDR1 or its conservative modification, V containing the amino acid sequence described in SEQ ID NO: 5 L CDR2 or its conservative modifications, and V containing the amino acid sequence described in SEQ ID NO: 6 L Includes CDR3 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain includes an amino acid having the sequence described in SEQ ID NO: 1. H V containing the amino acid sequence described in CDR1, SEQ ID NO: 2 H V containing the amino acid sequence described in CDR2, SEQ ID NO: 3 H V containing the amino acid sequence described in CDR3, SEQ ID NO: 4 L V containing the amino acid sequence described in CDR1, SEQ ID NO. 5 L V containing the amino acid sequence described in CDR2 and SEQ ID NO: 6 L Includes CDR3. [Table 1]

[0103] As used herein, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding properties of the HI-TCR of this disclosure, including the amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into the Fv of the HI-TCR of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acids in the CDR region can be replaced with other amino acid residues from the same group, and the modified antibody can be tested for retained function (i.e., the function described in (c) to (l) above) using the functional assay described herein. In certain embodiments, one or fewer, two or fewer, three or fewer, four or five or fewer residues in a particular sequence or CDR region are modified.

[0104] V has at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology to a specific sequence (e.g., sequence number 7 and sequence number 8). H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a particular sequence, but retain the ability to bind to a target antigen (e.g., CD19). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in a particular sequence (e.g., SEQ ID NOs. 7 and SEQ ID NOs. 8). In certain embodiments, the substitutions, insertions, or deletions are located in a region outside the CDR of the extracellular antigen-binding domain (e.g., in the FR). In certain embodiments, the extracellular antigen-binding domain is a V selected from the group consisting of SEQ ID NOs. 7 and SEQ ID NOs. 8, including post-translational modifications of its sequence (SEQ ID NOs. 7 and SEQ ID NOs. 8). H and / or V L Includes arrays.

[0105] As used herein, the percentage of homology between two amino acid sequences is equivalent to the percentage of identity between two sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap (i.e., homology % = number of identical positions / total number of positions × 100). The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms.

[0106] The percentage of homology between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput.Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 residue weighting table, 12 gap length penalties, and 4 gap penalties. Furthermore, the homology percentage between two amino acid sequences was calculated using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, using the Needleman and Wunsch algorithm (J.Mol.Biol.48:444-453 (1970)). It can be determined using this method.

[0107] Furthermore, or alternatively, the amino acid sequences of the subject matter of this disclosure can be further used as “query sequences” for performing searches against public databases, for example, to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) from Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A BLAST protein search can be performed using the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to specific sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison, Gapped BLAST can be used from Altschuletal., (1997) Nucleic Acids Res. It can be used as described in 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0108] 2.3. Steady-state domain In certain embodiments, the antigen-binding chain further comprises a constant domain. In certain embodiments, the constant domain comprises a hinge / spacer region and a transmembrane domain. In certain embodiments, the constant domain can form homodimers or heterodimers with another constant domain. In certain embodiments, the constant domain dimerizes via one or more disulfide linkages. In certain embodiments, the antigen-binding chain can form trimers or oligomers with one or more identical or different constant domains.

[0109] In certain non-limiting embodiments, the constant domain includes T cell receptor constant regions, such as the T cell receptor alpha constant region (TRAC), the T cell receptor beta constant region (TRBC, e.g., TRBC1 or TRBC2), the T cell receptor gamma constant region (TRGC, e.g., TRGC1 or TRGC2), the T cell receptor delta constant region (TRDC), or any variant or functional fragment thereof.

[0110] In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRAC peptide. In certain embodiments, the TRAC polypeptide comprises an amino acid sequence or fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence described in SEQ ID NO: 38 provided below, and / or optionally comprises up to one, two, or three conservative amino acid substitutions. [ka]

[0111] In certain embodiments, the TRAC polypeptide has an amino acid sequence or fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence encoded by the transcript expressed by the gene of NCBI Genbank ID:28755, NG_001332.3, range 925603~930229 (Sequence ID 29 provided below), and / or may optionally include up to one, two, or three conserved amino acid substitutions. [ka] [ka]

[0112] According to the subject matter of this disclosure, “TRAC nucleic acid molecule” refers to a polynucleotide that encodes a TRAC polypeptide.

[0113] In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRBC peptide. In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRBC2 peptide. In certain embodiments, the TRBC2 polypeptide comprises an amino acid sequence or fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence described in SEQ ID NO: 39 provided below, and / or optionally comprises up to one, two, or three conservative amino acid substitutions. [ka]

[0114] In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRBC1 peptide. In certain embodiments, the TRBC1 polypeptide comprises an amino acid sequence or fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence described in SEQ ID NO: 40 provided below, and / or optionally comprises up to one, two, or three conservative amino acid substitutions. [ka]

[0115] In certain embodiments, the TRBC polypeptide has an amino acid sequence or fragment thereof that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence encoded by the transcript expressed by the genes NCBI Genbank ID:28639, NG_001333.2, range 645749~647196 (TRBC1, SEQ ID NO: 30) and / or may optionally include up to one, two, or three conserved amino acid substitutions. [ka] [ka]

[0116] According to the subject matter of this disclosure, “TRBC nucleic acid molecule” refers to a polynucleotide that encodes a TRBC polypeptide.

[0117] In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRGC peptide. In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRGC1 peptide. In certain embodiments, the TRGC1 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence described in SEQ ID NO: 42, provided below. [ka]

[0118] In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRGC2 peptide. In certain embodiments, the TRGC2 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence described in SEQ ID NO: 43, provided below. [ka]

[0119] In certain embodiments, the TRGC polypeptide has an amino acid sequence, or a fragment thereof, that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence encoded by the transcript expressed by the genes NCBI Genbank ID:6966,NG_001336.2, range 108270~113860 (TRGC1, SEQ ID NO: 32), NCBI Genbank ID:6967,NG_001336.2, range 124376~133924 (TRGC2, SEQ ID NO: 33), and / or may optionally include up to one, two, or three conserved amino acid substitutions. [ka] [ka] [ka] [ka] [ka]

[0120] According to the subject matter of this disclosure, “TRGC nucleic acid molecule” refers to a polynucleotide that encodes a TRGC polypeptide.

[0121] In certain embodiments, the constant domain of the HI-TCR of the Disclosure comprises a natural or modified TRDC peptide. In certain embodiments, the TRDC polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence described in SEQ ID NO: 41 provided below. [ka]

[0122] In certain non-limiting embodiments, the T cell receptor constant domain includes a hinge / spacer region that ligates an extracellular antigen-binding domain to the constant domain. The hinge / spacer region may be flexible enough to orient the antigen-binding domain to different orientations to facilitate antigen recognition. In certain non-limiting embodiments, the hinge / spacer region may be a hinge region derived from the CH2CH3 region of IgG1 or immunoglobulin, and a portion of the CD3, a portion of the CD28 polypeptide, a portion of the CD8 polypeptide, any of the aforementioned variants that are at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical thereto, or a synthetic spacer sequence. In certain non-limiting embodiments, the hinge / spacer region of the CAR may include a native or modified hinge region of a CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 peptide, CD166 peptide, CD8a peptide, CD8b peptide, ICOS polypeptide, ICAM-1 peptide, CTLA-4 peptide, synthetic peptide (not based on an immune response-related protein), or a combination thereof.

[0123] 2.4 Intracellular signal transduction domains In certain non-limiting embodiments, the HI-TCR of this disclosure comprises an antigen-binding chain that does not include an intracellular domain. In certain embodiments, the antigen-binding chain can associate with a CD3ζ polypeptide. In certain embodiments, the antigen-binding chain comprises a constant domain that can associate with a CD3ζ polypeptide. In certain embodiments, the CD3ζ polypeptide is endogenous. In certain embodiments, the CD3ζ polypeptide is exogenous. In certain embodiments, binding of the antigen-binding chain to an antigen can activate the CD3ζ polypeptide associated with the antigen-binding chain. In certain embodiments, the exogenous CD3ζ polypeptide is fused to or integrated with a co-stimulatory molecule disclosed herein.

[0124] In certain non-limiting embodiments, the HI-TCR of this disclosure comprises an antigen-binding chain including an intracellular domain. In certain embodiments, the intracellular domain comprises a CD3ζ polypeptide. In certain embodiments, binding of the antigen-binding chain to an antigen can activate the CD3ζ polypeptide of the antigen-binding chain.

[0125] Activated CD3ζ polypeptides can activate and / or stimulate immune-responsive cells (e.g., lymphoid cells, e.g., T cells). CD3ζ comprises three immunoreceptor-activating tyrosine motifs (ITAM1, ITAM2, and ITAM3) and three basic-rich stretch (BRS) regions (BRS1, BRS2, ​​and BRS3), which transmit activation signals to cells (e.g., lymphoid cells, e.g., T cells) after an antigen has bound to the antigen-binding chain. The intracellular signaling domain of the CD3ζ chain is the primary signaling molecule derived from the endogenous TCR. In certain embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the sequence having NCBI reference number: NP_932170 (SEQ ID NO: 17), NCBI reference number: NP_000725.1 (SEQ ID NO: 18), and / or may optionally contain up to one, two, or three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3ζ polypeptide comprises or has an amino acid sequence that is a contiguous portion of SEQ ID NO: 17, having at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acid lengths. Alternatively, or further, in various non-limiting embodiments, the CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 1-164, 1-50, 50-100, 100-150, or 150-164 of SEQ ID NO: 17. In certain embodiments, the CD3ζ polypeptide comprises or has the amino acid sequence of amino acids 52-164 of SEQ ID NO: 17. Sequence ID 17 is provided below: [ka]

[0126] In certain embodiments, the intracellular signaling domain comprises a human CD3ζ polypeptide. The human CD3ζ polypeptide comprises, or may contain, an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO: 18, and / or may optionally contain up to one, two, or up to three conserved amino acid substitutions. SEQ ID NO: 18 is provided below:

[0127] [ka]

[0128] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18 is described in SEQ ID NO: 19, provided below.

[0129] [ka]

[0130] 2.4.1. Co-stimulation area In certain non-limiting embodiments, the HI-TCR of the Disclosure comprises an antigen-binding chain including an intracellular domain, the intracellular domain including a costimulatory region. In certain embodiments, the intracellular domain includes a costimulatory region and a CD3ζ polypeptide. In certain embodiments, the intracellular domain includes a costimulatory region but does not include a CD3ζ polypeptide.

[0131] In certain embodiments, the co-stimulatory region includes at least one costimulatory molecule that can result in optimal lymphocyte activation. As used herein, “co-stimulatory molecule” refers to a cell surface molecule other than the antigen receptor or its ligand required for an efficient lymphocyte response to an antigen. The at least one costimulatory signaling region may include CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, or a combination thereof. The costimulatory molecule can bind to a costimulatory ligand, which is a protein expressed on the cell surface that generates a costimulatory response, i.e., an intracellular response that results in the stimulus provided when the antigen binds to its CAR molecule. Examples of costimulatory ligands, but not limited to, include CD80, CD86, CD70, OX40L, and 4-1BBL. As an example, the 4-1BB ligand (i.e., 4-1BBL) can, along with the CAR signal, + It can bind to 4-1BB (also known as "CD137") to provide intracellular signals that induce effector cell function of T cells. CARs comprising an intracellular signaling domain including a costimulatory signaling region containing 4-1BB, ICOS, or DAP-10 are disclosed in U.S. Patent No. 7,446,190, which is incorporated herein by reference in its entirety.

[0132] In certain embodiments, the co-stimulatory region of the HI-TCR antigen-binding chain comprises a co-stimulatory signaling region that includes a CD28 polypeptide. The CD28 polypeptide may comprise, or have, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the sequence having NCBI reference number: P10747 or NP_006130 (SEQ ID NO: 20), or a fragment thereof, and / or may optionally include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises, or has, an amino acid sequence that is a continuous portion of SEQ ID NO: 20 that is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively, or additionally, in various non-limiting embodiments, the CD28 polypeptide comprises, or has, the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, or 200-220 of SEQ ID NO: 20. In certain embodiments, the co-stimulatory region comprises a co-stimulatory signaling region that includes a CD28 polypeptide that comprises, or has, the amino acid sequence of amino acids 180-220 of SEQ ID NO: 20.

Chemical formula

[0133] In certain embodiments, the co-stimulatory region includes the human intracellular signaling domain of CD28. The human intracellular signaling domain of CD28 may comprise, or have, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the sequence provided below, or a fragment thereof, and / or may optionally include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. SEQ ID NO: 21 is provided below:

[0134]

Chemical formula

[0135] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21 is set forth in SEQ ID NO: 22 provided below.

Chemical Structure

[0136] In certain embodiments, the co-stimulatory region comprises a co-stimulatory signaling region that includes two co-stimulatory molecules, such as the co-stimulatory signaling regions of CD28 and 4-1BB, or the co-stimulatory signaling regions of CD28 and OX40.

[0137] 4-1BB can act as a tumor necrosis factor (TNF) ligand and has stimulatory activity. The 4-1BB polypeptide may comprise, or have, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the sequence having NCBI Reference No.: P41273 or NP_001552 (SEQ ID NO: 23), or a fragment thereof, and / or may optionally contain up to 1, or up to 2, or up to 3 conservative amino acid substitutions as necessary. SEQ ID NO: 23 is provided below:

Chemical Structure

[0138] According to the subject matter of the present disclosure, a "4-1BB nucleic acid molecule" refers to a polynucleotide encoding a 4-IBB polypeptide.

[0139] In certain embodiments, the co-stimulatory region includes an intracellular signaling domain at 4-1BB. The intracellular signaling domain at 4-1BB includes, or may have, an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO: 24, and / or may include, optionally, up to one, two, or up to three conserved amino acid substitutions. SEQ ID NO: 24 is provided below:

[0140] [ka]

[0141] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 is described in SEQ ID NO: 27, provided below. [ka]

[0142] The OX40 polypeptide contains, or may contain, an amino acid sequence or fragment thereof that is at least approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% homologous or identical to the sequence having NCBI reference number: P43489 or NP_003318 (SEQ ID NO: 25), and / or may contain, as necessary, up to one, two, or three conservative amino acid substitutions. Sequence ID 25 is provided below: [ka]

[0143] According to the subject matter of this disclosure, “OX40 nucleic acid molecule” refers to a polynucleotide that encodes an OX40 polypeptide.

[0144] The ICOS polypeptide may comprise, or have, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the sequence having NCBI Reference Number: NP_036224 (SEQ ID NO: 26), or a fragment thereof, and / or may optionally include up to 1, or up to 2, or up to 3 conservative amino acid substitutions. SEQ ID NO: 26 is provided below:

Chemical formula

[0145] According to the subject matter of the present disclosure, an "ICOS nucleic acid molecule" refers to a polynucleotide encoding an ICOS polypeptide.

[0146] In certain embodiments, mutation sites and / or junctions between domains / motifs / regions of CARs derived from different proteins are deimmunized. The immunogenicity of junctions between different CAR moieties can be predicted using the NetMHC4.0 server. For each peptide containing at least 1 aa derived from the following portions, the binding affinity for HLA A, B, and C for all alleles can be predicted. An immunogenicity score can be assigned to each peptide. The immunogenicity score can be calculated using the formula: Immunogenicity score = [(50 - binding affinity) * HLA frequency] n where n is the number of predictions for each peptide.

[0147] 2.5. CD3 Complex In certain embodiments, the HI-TCR of this disclosure can associate with the CD3 complex (also known as the “T cell coreceptor”). In certain embodiments, the HI-TCR and CD3 complex form an antigen-recognizing receptor complex similar to the native TCR / CD3 complex. In certain embodiments, the CD3 complex is endogenous. In certain embodiments, the CD3 complex is exogenous. In certain embodiments, the HI-TCR of this disclosure replaces the native and / or endogenous TCR in the CD3 / TCR complex. In certain embodiments, the CD3 complex comprises a CD3γ chain, a CD3δ chain, and two CD3ε chains.

[0148] In certain embodiments, the CD3γ chain may contain, or have contained, an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to NCBI reference number: NP_000064.1 (Sequence ID 34 provided below), and / or may contain, as necessary, up to one, two, or three conservative amino acid substitutions. [ka]

[0149] In certain embodiments, the CD3δ chain contains, or may contain, an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to NCBI reference number: NP_000723.1 (Sequence ID 35 provided below), NP_001035741.1 (Sequence ID 36 provided below), and / or may contain, as necessary, up to one, two, or three conservative amino acid substitutions. [ka]

[0150] In certain embodiments, the CD3ε chain contains or has an amino acid sequence or fragment thereof that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to NCBI reference number: NP_000724.1 (Sequence ID 37 provided below), and / or may contain, as necessary, up to one, two, or three conservative amino acid substitutions. [ka]

[0151] In certain embodiments, recombinant TCRs exhibit greater antigen sensitivity than CARs targeting the same antigen. In certain embodiments, recombinant TCRs can induce an immune response by binding to antigens present at low densities on the surface of tumor cells. In certain embodiments, immune-responsive cells containing the HI-TCRs of this disclosure can be used to treat subjects having tumor cells with low levels of surface antigen expression due to disease recurrence, for example, the subjects having undergone treatment resulting in residual tumor cells. In certain embodiments, tumor cells have low densities of target molecules on their surface. In certain embodiments, the low densities of target molecules present on the cell surface have densities of less than approximately 5,000 molecules per cell, less than approximately 4,000 molecules per cell, less than approximately 3,000 molecules per cell, less than approximately 2,000 molecules per cell, less than approximately 1,500 molecules per cell, less than approximately 1,000 molecules per cell, less than approximately 500 molecules per cell, less than approximately 200 molecules per cell, or less than approximately 100 molecules per cell. In certain embodiments, the target molecules present at low density on the cell surface have a density of less than approximately 2,000 molecules per cell. In certain embodiments, the target molecules present at low density on the cell surface have a density of less than approximately 1,500 molecules per cell. In certain embodiments, the target molecules present at low density on the cell surface have a density of less than approximately 1,000 molecules per cell. In certain embodiments, the target molecules present at low density on the cell surface have densities between approximately 4,000 molecules and approximately 2,000 molecules per cell, between approximately 2,000 molecules and approximately 1,000 molecules per cell, between approximately 1,500 molecules and approximately 1,000 molecules per cell, between approximately 2,000 molecules and approximately 500 molecules per cell, between approximately 1,000 molecules and approximately 200 molecules per cell, or between approximately 1,000 molecules and approximately 100 molecules per cell.

[0152] 2.6.HI-TCR 19 In certain embodiments, the HI-TCR of the Disclosure comprises two dimerizable antigen-binding chains, e.g., VL-TRAC and VH-TRBC, where the HI-TCR binds to CD19 (e.g., human CD19).

[0153] VL-TRAC In certain embodiments, the HI-TCR of the present disclosure is the V of the antibody. L The antigen-binding chain includes an extracellular antigen-binding domain of the domain and a constant domain of the TRAC. In certain embodiments, the antibody binds to CD19 (e.g., human CD19). In certain embodiments, the antigen-binding chain is named "VL-TRAC".

[0154] VH-TRBC In certain embodiments, the HI-TCR of the present disclosure is the V of the antibody. H The antigen-binding chain includes an extracellular antigen-binding domain of the domain and a constant domain of the TRBC. In certain embodiments, the antibody binds to CD19 (e.g., human CD19). In certain embodiments, the antigen-binding chain is named "VH-TRBC".

[0155] 3. Immune-responsive cells The subject matter of this disclosure provides immune-responsive cells comprising HI-TCRs. In certain embodiments, HI-TCRs can activate immune-responsive cells. By binding to an antigen, immune-responsive cells exhibit a cytolytic effect against cells possessing the antigen. In certain embodiments, immune-responsive cells comprising HI-TCRs exhibit equivalent or better therapeutic efficacy compared to cells comprising chimeric antigen receptors (CARs) targeting the same antigen. In certain embodiments, immune-responsive cells comprising HI-TCRs secrete antitumor cytokines. Cytokines secreted by immune-responsive cells include, but are not limited to, TNFα, IFNγ, and IL2.

[0156] The immune-responsive cells of the subject matter of this disclosure may be lymphoid cells. The lymphoid system, including B, T, and natural killer (NK) cells, enables antibody production, regulation of the cellular immune system, detection of exogenous substances in the blood, detection of cells foreign to the host, etc. Non-limiting examples of immune-responsive cells of the lymphoid system include T cells, natural killer (NKT) cells, and their progenitor cells, including embryonic stem cells and pluripotent stem cells (e.g., those capable of differentiating lymphoid cells). T cells may be lymphocytes that mature in the thymus and primarily play a role in cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of this disclosure include helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells) and two types of effector memory T cells: e.g., T EM Cells and T EMRA The T cells may be any type of T cell, including, but not limited to, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient's own T cells can be genetically modified to target specific antigens through the introduction of HI-TCRs. In certain embodiments, the immune-responsive cells are T cells. T cells are CD4 + T cells or CD8 + It may be a T cell. In a particular embodiment, the T cell is a CD4 + These are T cells. In certain embodiments, T cells are CD8 + These are T cells.

[0157] In certain embodiments, immune-responsive cells contain at least one exogenous or recombinant costimulatory ligand (e.g., cells are transduced to it). In certain embodiments, immune-responsive cells co-express HI-TCR and at least one exogenous costimulatory ligand. The interaction between HI-TCR and at least one exogenous costimulatory ligand results in a non-antigen-specific signal crucial for the full activation of immune-responsive cells (e.g., T cells). Examples of costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its primary role is in regulating immune cells. Members of the TNF superfamily share numerous common characteristics. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. The TNF superfamily includes, but is not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFβ) / lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257 / B-cell activator (BAFF) / Blys / THANK / Tall-1, glucocorticoid-inducible TNF receptor ligand (GITRL) and TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural characteristics with immunoglobulins and possess an immunoglobulin domain (fold). Examples of immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both of which are ligands for CD28.In certain embodiments, at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD275, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof. In certain embodiments, the immune-responsive cell contains or consists of one exogenous or recombinant co-stimulatory ligand. In certain embodiments, the exogenous or recombinant co-stimulatory ligand is 4-1BBL or CD80. In certain embodiments, the exogenous or recombinant co-stimulatory ligand is 4-1BBL. In certain embodiments, the immune-responsive cell contains or consists of two exogenous or recombinant co-stimulatory ligands. In certain embodiments, the two exogenous or recombinant co-stimulatory ligands are 4-1BBL and CD80.

[0158] In certain embodiments, immune-responsive cells may contain or be transduced of at least one chimeric costimulatory receptor (CCR). As used herein, the term “chimeric costimulatory receptor” or “CCR” refers to a chimeric receptor that binds to an antigen and, by binding to the antigen, brings a costimulatory signal to a cell containing the CCR (e.g., a T cell), but does not bring an activation signal to the cell on its own. CCRs are described in their entirety in Krause, et al., J. Exp. Med. (1998);188(4):619-626 and US20020018783, which are incorporated herein by reference. CCRs mimic costimulatory signals but, unlike CARs, do not bring a T cell activation signal; for example, CCRs lack the CD3ζ polypeptide. CCRs bring a costimulatory signal, e.g., a CD28-like signal, in the absence of a native costimulatory ligand in antigen-presenting cells. Combinatorial antigen recognition, i.e., the use of CCR in combination with CAR, can increase T cell responsiveness to dual-antigen expressing T cells, thereby improving selective tumor targeting. See WO2014 / 055668, which is incorporated herein by reference in its entirety. Kloss et al. describe a strategy that integrates combinatorial antigen recognition, separate signaling, and, importantly, balanced intensities of T cell activation and co-stimulation to generate T cells that eliminate target cells expressing the combination of antigens while preserving cells that express each antigen individually (see Kloss et al., Nature Biotechnology (2013);31(1):71-75, which is incorporated herein by reference in its entirety). This approach allows T cells Activation requires CAR-mediated recognition of one antigen, while co-stimulation is independently mediated by a CCR specific to a second antigen. To achieve tumor selectivity, the combinatorial antigen recognition approach reduces the efficiency of T cell activation to a level that becomes ineffective without rescue brought about by simultaneous CCR recognition of the second antigen.

[0159] In certain embodiments, the CCR includes an extracellular antigen-binding domain that binds to a second antigen, a transmembrane domain, and a costimulatory signaling region containing at least one costimulatory molecule. In certain embodiments, the CCR alone does not deliver an activation signal to the cell. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, and any combination thereof. In certain embodiments, the costimulatory signaling region of the CCR contains one costimulatory signaling molecule. In certain embodiments, the one costimulatory signaling molecule is CD28. In certain embodiments, the one costimulatory signaling molecule is 4-1BB. In certain embodiments, the costimulatory signaling region of the CCR contains two costimulatory signaling molecules. In certain embodiments, the two costimulatory signaling molecules are CD28 and 4-1BB. The second antigen is selected such that the expression of both the first and second antigens is restricted to targeted cells (e.g., cancerous tissue or cancerous cells). Similar to CARs, the extracellular antigen-binding domain can be scFv, Fab, F(ab)2, or a fusion protein having heterologous sequences to form the extracellular antigen-binding domain.

[0160] In a particular embodiment, the CCR is co-expressed with a HI-TCR that binds to a different antigen than the antigen to which the CCR binds, for example, the HI-TCR binds to a first antigen and the CCR binds to a second antigen.

[0161] The types of human lymphocytes that are the subject of this disclosure include, but are not limited to, peripheral donor lymphocytes, e.g., Sadelain, M., et al. 2003 Nat RevCancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), Morgan, RA, et al. 2006 Science 314:126-129 (expressing full-length tumor antigen-recognizing T cell receptor complexes containing α and β heterodimers). (Disclosing genetically modified peripheral donor lymphocytes), Panelli, MC, et al. 2000 J Immunol 164:495-504; Panelli, MC, et al. 2000 JImmunol164:4382-4392 (Discloses lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA, et al. 2003 Blood102:2498-2505 (Selection using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells) This includes disclosing antigen-specific peripheral blood leukocytes selectively enlarged in vitro. Immune-responsive cells (e.g., T cells) may be self, non-self (e.g., allogeneic), or can be induced in vitro from engineered progenitor cells or stem cells.

[0162] The immune-responsive cells of this disclosure are capable of modulating the tumor microenvironment. Tumors have a microenvironment that is adversarial to the host immune response, including a set of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This “adversarial tumor microenvironment” is influenced by invasion-modulating CD4 + This includes the expression of various immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines such as TGF-β, and ligands that target immunosuppressive receptors expressed by activated T cells (CTLA-4 and PD-1). While these mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inadequate immune responses, within the tumor microenvironment, these mechanisms interfere with effective anti-tumor immune responses. In summary, these immunosuppressive factors can induce significant anergy or apoptosis of CAR-modified T cells that are adopted upon encounter with targeted tumor cells.

[0163] The unpurified source of CTLs may be any known in the art, e.g., bone marrow, fetal, neonatal or adult or other hematopoietic cell sources, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be used to isolate the cells. For example, negative selection methods can remove non-CTLs first. mAbs are particularly useful for identifying markers associated with specific cell lineages and / or differentiation stages for both positive and negative selection.

[0164] Relatively coarse separation allows for the initial removal of a large proportion of terminally differentiated cells. For example, magnetic bead separation can be used first to remove a large number of irrelevant cells. In certain embodiments, at least about 80%, and usually at least about 70%, of all hematopoietic cells are removed before cell isolation.

[0165] The separation procedure may include, but is not limited to, density gradient centrifugation; recoagulation (resetting); and cell density. Coupling with modified particles; magnetic separation by antibody-coated magnetic beads; affinity chromatography; cytotoxic agents, including, but not limited to, complement and cytotoxins, linked to or used with mAbs; as well as panning, elatrillation, or any other convenient techniques using antibodies attached to solid matrices, such as plates or chips.

[0166] The separation and analysis techniques include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, for example, multiple color channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels.

[0167] Cells can be selected for dead cells by using dead cell-associated dyes such as propidium iodide (PI). In certain embodiments, cells are collected in a medium containing 2% fetal bovine serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, for example, sterile, isotonic medium.

[0168] 4. Vector Genetic modification of immune-responsive cells (e.g., T cells or NKT cells) can be achieved by transducing a substantially homogeneous cell composition with recombinant DNA constructs. In certain embodiments, retroviral vectors (either gamma-retroviruses or lentiviruses) are used to introduce DNA constructs into cells. For example, a polynucleotide encoding HI-TCR can be cloned into a retroviral vector, and expression can be induced from its endogenous promoter, from the terminal repeat sequence of the retrovirus, or from a promoter specific to the target cell type of interest. Non-viral vectors can be used in a similar manner.

[0169] When initially genetically modifying immune-responsive cells to include HI-TCRs, retroviral vectors are commonly used for transduction, but any other suitable viral vector or nonviral delivery system can be used. HI-TCRs can be constructed in a single multi-cistronic expression cassette, in multiple expression cassettes of a single vector, or in multiple vectors together with auxiliary molecules (e.g., cytokines). Examples of elements for constructing polycistronic expression cassettes include, but are not limited to, various viral and nonviral intra-sequence ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aftvirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). A combination of a retroviral vector and an appropriate packaging system is also suitable if the capsid protein is functional for infecting human cells. Various amphitrophic virus-producing cell lines are known, including but not limited to PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphitrophic particles, such as pseudotyped particles with VSVG, RD114, or GALV envelopes, and any other particles known in the art are also suitable.

[0170] Possible methods of transduction include, for example, direct co-culture of cells with producing cells by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culture with viral supernatant alone or with or without enriched vector stocks containing appropriate growth factors and polycations, by the methods of Xu, et al. (1994) Exp. Hemat. 22:223-230 and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0171] Other transdextrin viral vectors can be used to modify immune-responsive cells. In certain embodiments, the selected vectors exhibit highly efficient infection and stable incorporation and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996 and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus or herpesvirus, such as Epstein-Barr virus (e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitisetal., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322). 1987;Anderson,Science226:401-409,1984;Moen, Blood Cells 17:407-416, 1991;Miller et al.,Biotechnology7:980-990,1989;LeGal La Salle et al., Science 259:988-990, 1993 and Johnson, Chest 107:77S-83S, 1995. (See also ctor). Retroviral vectors are particularly well developed and used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0172] Non-viral approaches can also be used for genetic modification of immune-responsive cells. For example, by administering nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Onoetal., NeuroscienceLetters 17:259, 1990; Brigham et al., Am. J. Med.Sci. 298:278, 1989; Staubinger et al., Methods Nucleic acid molecules can be introduced into immune-responsive cells by means of (in Enzymology 101:512, 1983), asialorosomucoid-polylysine conjugation (Wuetal., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for DNA delivery to cells. Transplantation of normal genes into the affected tissue of a subject can also be achieved by ex vivo introduction of normal nucleic acids into a cultureable cell type (e.g., autologous or xenogeneic primary cells or their progeny), which are then injected into the targeted tissue or administered systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation. In certain embodiments, recombinant receptors can be introduced by transposon-based vectors. In certain embodiments, the transposon-based vectors contain transposons (also known as transposable elements). In certain embodiments, the transposons can be recognized by transposases. In certain embodiments, the transposase is a Sleeping Beauty transposase.

[0173] The clustered, regularly arranged short palindromic repeat (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (a crRNA containing a region that binds to tracrRNA (generally in the form of a hairpin loop) to form an active complex with Cas9, which is used by Cas9 to guide it to the correct part of the host DNA), transactivating crRNA (tracrRNA that binds to crRNA and forms an active complex with Cas9), and a DNA repair template (DNA that induces a cellular repair process that allows for the insertion of a specific DNA sequence), as needed. CRISPR / Cas9 often uses plasmids to transfect target cells. The crRNA is the sequence that Cas9 uses to identify the target DNA in the cell and bind to it directly, so it needs to be designed for each application. The repair template with a CAR expression cassette also needs to be designed for each application, as it must overlap with the sequence on either side of the cleavage and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can then be ligated together with the Cas9 gene to form a plasmid for transfection into cells.

[0174] Zinc finger nucleases (ZFNs) are artificial restriction enzymes produced by combining a zinc finger DNA-binding domain with a DNA-cleaving domain. The zinc finger domain can be manipulated to target specific DNA sequences, enabling the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of individual ZFNs typically contains multiple individual zinc finger repeat sequences, each capable of recognizing multiple base pairs. The most common method for generating novel zinc finger domains is by combining known zinc finger "modules" with less specificity. The most common cleavage domain in ZFNs is the nonspecific cleavage domain from the type II restriction endonuclease FokI. ZFNs can be used to insert CAR expression cassettes into the genome using homologous DNA templates with endogenous homologous recombination (HR) mechanisms and CAR expression cassettes. When a target sequence is cleaved by a ZFN, the HR mechanism searches for homology between the damaged chromosome and a homologous DNA template, then copies the template sequence between the two cleaved ends of the chromosome, thereby incorporating the homologous DNA template into the genome.

[0175] Activator-like effector nucleases (TALENs) are restriction enzymes that can be manipulated to cleave specific sequences of DNA. The TALEN system operates on almost the same principle as ZFNs. They are generated by combining an activator-like effector DNA-binding domain with a DNA-cleaving domain. Activator-like effectors (TALEs) consist of a repeating motif of 33-34 amino acids with two variable positions that have strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be manipulated to bind to a desired DNA sequence and thereby induce a nuclease to cleave at a specific site in the genome. cDNA expression for use in polynucleotide therapy can be induced from any suitable promoter (e.g., human cytomegalovirus (CMV), Simian virus 40 (SV40), or metallothionein promoter) and can be regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, enhancers known to preferentially induce gene expression in specific cell types may be used to induce nucleic acid expression, if desired. The enhancers used may include, but are not limited to, those characterized as tissue or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation may be mediated by a congeneral regulatory sequence, or optionally by a regulatory sequence derived from a heterologous source containing any of the promoters or regulatory elements described above.

[0176] The resulting cells can be grown under conditions similar to those for unmodified cells, thereby allowing the modified cells to be enlarged and used for various purposes.

[0177] 6. Genome editing methods In certain non-limiting embodiments, the HI-TCR, costimulatory ligand, CCR, or any other molecule / transgene disclosed herein is expressed by immune-responsive cells via a modified genomic locus. In certain embodiments, the transgene expression cassette is incorporated into a targeted genomic locus of immune-responsive cells by a targeted genome editing method. In certain embodiments, the targeted genomic locus may be the CD3δ, CD3ε, CD247, B2M, TRAC, TRBC1, TRBC2, TRGC1, and / or TRGC2 loci.

[0178] 6.1. Manipulation of T cell receptor gene loci In certain embodiments, HI-TCR is expressed by immune-responsive cells via a modified endogenous T cell receptor locus. In certain embodiments, the HI-TCR expression cassette is integrated into the endogenous T cell receptor locus. In certain embodiments, the HI-TCR expression cassette is integrated into the T cell receptor alpha locus (TRA, GenBank) In certain embodiments, the HI-TCR expression cassette is incorporated into the T cell receptor beta locus (TRB, GenBank ID:6957). In certain embodiments, the HI-TCR expression cassette is incorporated into the T cell receptor gamma locus (TRG, GenBank ID:6965).

[0179] In certain embodiments, the HI-TCR expression cassette includes an extracellular antigen-binding domain incorporated into the first exon of the TCR constant domain locus, such that the extracellular antigen-binding domain and the TCR constant domain are included in one antigen-binding chain of the HI-TCR. In certain embodiments, the TCR constant domain locus may be TRAC, TRBC1, TRBC2, TRGC1, or TRGC2. In certain embodiments, the HI-TCR expression cassette includes an extracellular antigen-binding domain incorporated into the first exon of the TRAC locus, such that the extracellular antigen-binding domain and the TRAC peptide are included in the first antigen-binding chain of the HI-TCR. In certain embodiments, the HI-TCR expression cassette further includes a second antigen-binding chain optionally comprising an extracellular antigen-binding domain and the TRBC peptide. In certain embodiments, the HI-TCR expression cassette includes an extracellular antigen-binding domain incorporated into the first exon of the TRBC locus, such that the extracellular antigen-binding domain and the TRBC peptide are included in the first antigen-binding chain of the HI-TCR. In certain embodiments, the HI-TCR expression cassette further comprises a second antigen-binding chain optionally comprising an extracellular antigen-binding domain and a TRAC peptide. In certain embodiments, the expression cassette comprises elements that make up a polycistronic expression cassette, e.g., a cleavable peptide, e.g., a 2A peptide.

[0180] In certain embodiments, recombinant TCRs are expressed from expression cassettes located at the endogenous TRAC locus and / or TRBC locus of immune-responsive cells. In certain embodiments, the placement of the recombinant TCR expression cassette disrupts or inactivates the endogenous expression of TCRs, including the native TCRα and / or native TCRβ chains, in immune-responsive cells. In certain embodiments, the placement of the recombinant TCR expression cassette prevents or eliminates mispairing between recombinant TCRs and the native TCRα and / or native TCRβ chains in immune-responsive cells.

[0181] Any suitable genetic editing method and system can be used to modify the endogenous T cell receptor locus. The genome editing method disclosed in Section 4 can be used to modify the endogenous T cell receptor locus. In certain embodiments, the CRISPR system is used to modify the T cell receptor locus. In certain embodiments, the CRISPR system targets exon 1 of the human TRAC locus. In certain embodiments, the CRISPR system includes a guide RNA (gRNA) that targets exon 1 of the human TRAC locus.

[0182] In certain embodiments, zinc finger nucleases are used to modify the endogenous T cell receptor locus. In certain embodiments, TALEN systems are used to modify the endogenous T cell receptor locus.

[0183] In certain embodiments, if one endogenous T cell receptor locus in a cell is modified to express one or more antigen-binding chains of the HI-TCR, one or more other endogenous T cell receptor loci in the cell are modified to exclude the endogenous expression of the endogenous TCR chain. In certain embodiments, one or more other endogenous T cell receptor loci are further modified to express a gene of interest, such as an antitumor cytokine (e.g., IL-2, IL-12, TNFα, and INFγ), a costimulatory molecule ligand (e.g., 4-1BBL), a tracking gene (e.g., eGFP), or a suicide gene.

[0184] 6.2 Modification of gene expression by genome editing of promoters or transcriptional terminators In certain non-limiting embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is driven by the locus's endogenous promoter / enhancer. In certain embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is driven by a modified promoter / enhancer introduced into the locus. Using any targeted genome editing method, the promoter / enhancer region of a targeted genomic locus can be modified to enhance or modify HI-TCR expression in immune-responsive cells. In certain embodiments, the modification includes replacing the endogenous promoter with a constitutive or inductive promoter, or inserting a constitutive or inductive promoter into the promoter region of the targeted genomic locus. In certain embodiments, a constitutive promoter is placed in the targeted genomic locus to drive HI-TCR gene expression. Eligible constitutive promoters include, but are not limited to, the CMV promoter, EF1a promoter, SV40 promoter, PGK1 promoter, Ubc promoter, beta-actin promoter, and CAG promoter. Alternatively, or furthermore, a conditional or inducible promoter may target They are positioned at targeted genomic loci and drive the gene expression of HI-TCRs. Non-limiting examples of conditional promoters include tetracycline response element (TRE) promoters and estrogen response element (ERE) promoters. In addition, enhancer elements can be positioned in regions other than the promoter region.

[0185] In certain non-limiting embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is regulated by the endogenous transcriptional terminator of the genomic locus. In certain embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is regulated by a modified transcriptional terminator introduced into the genomic locus. Using any targeted genome editing method, the transcriptional terminator region of a targeted genomic locus can be modified, thereby altering the expression of HI-TCR in immune-responsive cells. In certain embodiments, the modification includes replacing the endogenous transcriptional terminator with an alternative transcriptional terminator, or inserting an alternative transcriptional terminator into the transcriptional terminator region of a targeted genomic locus. In certain embodiments, the alternative transcriptional terminator includes the 3'UTR region or poly(ploy)A region of the gene. In certain embodiments, the alternative transcriptional terminator is endogenous. In certain embodiments, the alternative transcriptional terminator is exogenous. In certain embodiments, alternative transcription terminators include, but are not limited to, the TK transcription terminator, GCSF transcription terminator, TCRA transcription terminator, HBB transcription terminator, bovine growth hormone transcription terminator, SV40 transcription terminator, and P2A element.

[0186] Any of the targeted genome editing methods can be used to modify the promoter / enhancer region and / or transcriptional terminator region of a targeted genomic locus. In certain embodiments, the CRISPR system is used to modify the promoter / enhancer region and / or transcriptional terminator region of a targeted genomic locus. In certain embodiments, zinc finger nucleases are used to modify the promoter / enhancer region and / or transcriptional terminator region of a targeted genomic locus. In certain embodiments, the TALEN system is used to modify the promoter / enhancer region and / or transcriptional terminator region of a targeted genomic locus.

[0187] The method for delivering genome editing agents / systems may vary depending on the need. In certain embodiments, components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, components are delivered through a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene guns, impalefection, hydrostatic pressure, serial injection, sonication, magnetofection, adeno-associated viruses, envelope protein pseudotyping of viral vectors, cis and trans-acting elements of replicating vectors, herpes simplex viruses, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-permeable peptides).

[0188] Modifications can be made at any location within the targeted genomic locus, or at any location that may affect gene expression at the targeted genomic locus. In certain embodiments, the modification occurs upstream of the transcription start site of the targeted genomic locus. In certain embodiments, the modification occurs between the transcription start site and the protein-coding region of the targeted genomic locus. In certain embodiments, the modification occurs downstream of the protein-coding region of the targeted genomic locus. In certain embodiments, the modification occurs upstream of the transcription start site of the targeted genomic locus, in which case the modification produces a new transcription start site.

[0189] 7. Polypeptides and Analogues Polypeptides (e.g., CD19, CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, CD3γ, CD3δ, CD3ε, and CTLA-4) or fragments thereof that have been modified to enhance their antineoplastic activity when expressed in immune-responsive cells are also included in the subject matter of this disclosure. The subject matter of this disclosure provides methods for optimizing amino acid sequences or nucleic acid sequences by introducing changes in the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of this disclosure further includes analogs of any naturally occurring polypeptide disclosed herein (including, but not limited to, CD19, CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, CD3γ, CD3δ, CD3ε, and CTLA-4). The analogs may differ from the naturally occurring polypeptides disclosed herein by differences in amino acid sequence, by post-translational modification, or both. The analogs may exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher homology to all or part of the naturally occurring amino acid sequences of the subject matter of this disclosure. The length of the sequence comparison is at least 5, 10, 15, or 20 amino acid residues, for example, at least 25, 50, or 75 amino acid residues, or longer than 100 amino acid residues. In this case as well, in an exemplary approach to determining the degree of identity, the BLAST program is used to show closely related sequences. -3 from e -100The probability score between these may be used. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation, and such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modified enzymes. Analogues may also differ from naturally occurring polypeptides due to changes in the primary sequence. These include both native and derived gene variants (e.g., resulting from random mutagenesis due to irradiation or exposure to ethanemethyl sulfate, or Sambrook, Fritsch This includes and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSHPress, 1989 or Ausubelet al. (by site-directed mutagenesis as described above). It also includes cyclized peptides, molecules, and analogs containing residues other than L-amino acids, such as D-amino acids or naturally occurring or synthetic amino acids, such as β- or γ-amino acids.

[0190] In addition to full-length polypeptides, the subject matter of this disclosure also provides fragments of either polypeptides or peptide domains disclosed herein. As used herein, the term “fragment” means at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 consecutive amino acids, at least 30 consecutive amino acids, or at least 50 consecutive amino acids. In certain embodiments, a fragment comprises at least 60–80, 100, 200, 300, or more consecutive amino acids. Fragments can be generated by methods known to those skilled in the art, or by conventional protein processing (e.g., removal of amino acids not required for biological activity from a nascent polypeptide, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0191] Non-protein analogs have a chemical structure designed to mimic the functional activity of the proteins disclosed herein. Such analogs may exceed the physiological activity of the original polypeptide. Methods for designing analogs are well known in the art, and the synthesis of analogs can be carried out according to such methods by modifying the chemical structure of the original polypeptide so that the resulting analog increases the antineoplastic activity when expressed in immune-responsive cells. These chemical modifications include, but are not limited to, substituting alternative R groups and altering the degree of saturation of specific carbon atoms of the reference polypeptide. In certain embodiments, protein analogs are relatively resistant to degradation in vivo and, when administered, result in a more sustained therapeutic effect. Assays for measuring functional activity include, but are not limited to, the assays described in the following examples.

[0192] 8. Administration Compositions comprising the immune-responsive cells of this disclosure can be delivered systemically or directly to a subject to induce and / or enhance an immune response to an antigen, and / or to treat and / or prevent neoplasms, pathogen infections, or infectious diseases. In certain embodiments, the immune-responsive cells or compositions comprising the same of this disclosure are injected directly into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the immune-responsive cells or compositions comprising the same of this disclosure are delivered indirectly to an organ of interest by administration, for example, to the circulatory system (e.g., the vascular system of a tumor). Growth and differentiation agents can be provided before, during, or after administration of the cells or compositions to increase the production of T cells, NKT cells, or CTL cells in vitro or in vivo.

[0193] The immune-responsive cells of this disclosure can typically be administered intravascularly in any physiologically acceptable vehicle, but they may also be introduced into bone or other convenient sites (e.g., the thymus) where the cells can find suitable sites for regeneration and differentiation. Typically, at least about 1 × 10⁻⁶ cells are present. 5Individual cells are administered, ultimately resulting in approximately 1 x 10⁶ cells. 10 The number of cells may reach one or more. The immune-responsive cells of this disclosure may include a purified cell population. Those skilled in the art can easily determine the percentage of immune-responsive cells of this disclosure in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable purity ranges for a population containing immune-responsive cells of this disclosure are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be easily adjusted by those skilled in the art (for example, a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc.

[0194] The compositions of this disclosure may be pharmaceutical compositions comprising the immune-responsive cells or precursors of this disclosure and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, immune-responsive cells or precursors may be obtained from one subject and administered to the same subject or to subjects of different compatibility. Immune-responsive cells or their progeny (e.g., derived from peripheral blood, ex vivo, or in vitro) may be administered via local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a therapeutic composition of the subject matter of this disclosure (e.g., a pharmaceutical composition comprising the immune-responsive cells of this disclosure) is administered, it may be formulated as a unit dose injection form (solution, suspension, emulsion).

[0195] 9. Formulation The compositions comprising immune-responsive cells of this disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions that can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range and can provide a longer contact period with specific tissues. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline solution, phosphate-buffered saline solution, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0196] Sterile injectable solutions can be prepared by incorporating genetically modified immune-responsive cells in the required amount of appropriate solvent, along with various amounts of other components, if desired. Such compositions can be mixed with suitable carriers, diluents, or excipients, such as sterile water, saline, glucose, or dextrose. The compositions can also be lyophilized. Depending on the desired administration route and preparation, the compositions may contain auxiliary substances, such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling or viscosity-enhancing additives, preservatives, flavoring agents, colorants, etc. To prepare suitable preparations without excessive experimental procedures, one may refer to standard texts, such as "REMINGTON'SPHARMACEUTICAL SCIENCE", 17th edition, 1985, which is incorporated herein by reference.

[0197] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Sustained absorption of the injectable pharmaceutical form can be achieved by the use of absorption retarders, such as aluminum monostearate and gelatin. However, according to the subject matter of this disclosure, any vehicle, diluent, or additive used must be compatible with genetically modified immune-responsive cells or their offspring.

[0198] The compositions may be isotonic, meaning they may have the same osmotic pressure as blood and tears. The desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable active ingredients, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride may be particularly useful for buffers containing sodium ions.

[0199] The viscosity of the composition can, if desired, be maintained at a selected level using a pharmaceutically acceptable thickener. For example, methylcellulose is readily and inexpensively available and easy to work with. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and carbomer. The concentration of the thickener may depend on the selected agent. The important point is to use an amount that achieves the desired viscosity. Obviously, the selection of a suitable carrier and other additives depends on the precise route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether to formulate the composition as a solution, suspension, gel, or another liquid form, e.g., time-release or liquid-filled form).

[0200] The amount of cells administered varies with respect to the subject being treated. In one embodiment, about 10 4 ~about 10 10 pieces, about 10 5 ~about 10 910, or about 10 6 ~about 10 8 A number of immune-responsive cells of this disclosure are administered to a human subject. More effective cells may be administered in even smaller quantities. In certain embodiments, at least about 1 × 10⁶ cells are administered. 8 , about 2×10 8 , about 3×10 8 , about 4×10 8 , or approximately 5 x 10 8 Individual immunoresponsive cells of this disclosure are administered to human subjects. The precise determination of the amount considered effective may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. The dosage can be readily determined by those skilled in the art from this disclosure and knowledge in the art.

[0201] Those skilled in the art can easily determine the amounts of cells and, as necessary, additives, vehicles, and / or carriers administered in the composition and by the method. Typically, any additives (in addition to active cells and / or drugs) are present in phosphate-buffered saline in amounts of 0.001 to 50% (by weight) solution, and the active ingredients are present in amounts on the order of micrograms to milligrams, for example, about 0.0001 to about 5% by weight, about 0.0001 to about 1% by weight, about 0.0001 to about 0.05% by weight, or about 0.001 to about 20% by weight, about 0.01 to about 10% by weight, or about 0.05 to about 5% by weight. With respect to any composition administered to animals or humans, the following can be determined: toxicity by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., rodents such as mice; the dosage of the composition that elicits a suitable response, the concentration of its components, and the timing of administration of the composition. Such a determination does not require excessive experimentation, based on the knowledge of those skilled in the art, this disclosure, and the documents referenced herein. The timing of sequential dosing can be determined without excessive experimentation.

[0202] 10. Treatment Method The subject matter of this disclosure provides methods for inducing and / or increasing an immune response in a subject in need. The immune-responsive cells and compositions comprising the same of this disclosure can be used to treat and / or prevent neoplasms in a subject. The immune-responsive cells and compositions comprising the same of this disclosure can be used to prolong the survival of a subject suffering from a neoplasm. The immune-responsive cells and compositions comprising the same of this disclosure can also be used to treat and / or prevent pathogen infection or other infectious diseases in a subject, for example, an immunodeficient human subject. Such methods involve administering an effective amount of the immune-responsive cells or compositions comprising the same (e.g., a pharmaceutical composition) of this disclosure to achieve the desired effect, whether it be mitigation of an existing condition or prevention of recurrence. In the case of treatment, the amount administered is an effective amount to produce the desired effect. The effective amount may be provided in one or a series of doses. The effective amount may be provided in a bolus or by serial infusion.

[0203] In certain embodiments, immune-responsive cells containing HI-TCRs disclosed herein can be used to treat subjects having tumor cells with low levels of surface antigen expression due to disease recurrence, for example, the subject having undergone treatment resulting in residual tumor cells. In certain embodiments, the tumor cells have a low density of target molecules on their surface. In certain embodiments, the low density of target molecules on the cell surface has densities of less than about 5,000 molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In certain embodiments, the low density of target molecules on the cell surface has a density of less than about 2,000 molecules per cell. In certain embodiments, the target molecules present at low density on the cell surface have a density of less than approximately 1,500 molecules per cell. In certain embodiments, the target molecules present at low density on the cell surface have a density of less than approximately 1,000 molecules per cell. In certain embodiments, the target molecules present at low density on the cell surface have densities of approximately 4,000 molecules to approximately 2,000 molecules per cell, approximately 2,000 molecules to approximately 1,000 molecules per cell, approximately 1,500 molecules to approximately 1,000 molecules per cell, approximately 2,000 molecules to approximately 500 molecules per cell, approximately 1,000 molecules to approximately 200 molecules per cell, or approximately 1,000 molecules to approximately 100 molecules per cell. In certain embodiments, an immune-responsive cell comprising HI-TCR disclosed herein can be used to treat a subject with disease relapse, the subject having received immune-responsive cells (e.g., T cells) comprising an intracellular signaling domain (e.g., CAR) comprising a costimulatory signaling domain comprising a 4-1BB polypeptide. In certain embodiments, tumor cells have a low density of tumor-specific antigens on the surface of the tumor cells. In certain embodiments, the disease is CD19 +ALL. In certain embodiments, tumor cells have a low density of CD19 on the tumor cells. Such methods include administering an effective amount of the immune-responsive cells of the present disclosure or a composition containing them (e.g., a pharmaceutical composition) to achieve a desired effect, mitigation of an existing condition, or prevention of recurrence.

[0204] An effective dose (or therapeutic effective dose) is the amount sufficient to produce a beneficial or desired clinical outcome through a treatment. An effective dose may be administered to a subject in one or more doses. With respect to a treatment, an effective dose is the amount sufficient to alleviate, improve, stabilize, reverse, or slow the progression of a disease, or otherwise reduce the pathological outcome of the disease. The effective dose is generally determined by a physician on a case-by-case basis and is within the scope of the skill of a person skilled in the art. When determining the appropriate dosage to achieve an effective dose, several factors are typically taken into consideration. These factors include the subject's age, sex, and weight, the condition being treated, the severity of the condition, and the morphology and effective concentration of the immune-responsive cells being administered.

[0205] In adoptive immunotherapy using antigen-specific T cells, approximately 10 6 ~10 10 pieces (for example, about 10) 9 Typically, cell doses in the range of (1) cells are injected. When the cells of this disclosure are administered to a host and subsequently differentiated, T cells specifically directed to a particular antigen are induced. The modified cells can be administered by any method known in the art, including but not limited to intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus.

[0206] The subject matter of this disclosure provides a method for treating and / or preventing neoplasms in a subject. The method may include administering an effective amount of the immune-responsive cells of this disclosure or a composition comprising them to a subject having a neoplasm.

[0207] Non-exclusive examples of neoplasms include blood cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, pharyngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate and small cell lung cancer). Suitable carcinomas include astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, undifferentiated neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangiosarcoma, hepatocellular carcinoma, cholangiocarcinoma, synoviomas, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminomas, embryonal carcinoma, and Wilms' tumor. This includes, but is not limited to, any carcinoma known in the field of oncology, including, but also including, ulcers, testicular tumors, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease, breast tumors such as ductal adenocarcinoma and lobular adenocarcinoma, squamous and adenocarcinoma of the cervix, epithelial carcinoma of the uterus and ovaries, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma. In certain embodiments, the neoplasm is selected from the group consisting of hematological cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, and pharyngeal cancer. In certain embodiments, the immune-responsive cells of the Disclosure and compositions comprising them can be used to treat and / or prevent hematological cancers (e.g., leukemia, lymphoma, and myeloma) or ovarian cancers that cannot be treated with conventional therapeutic interventions. In certain embodiments, the neoplasm is a solid tumor.

[0208] The subject may have an advanced form of the disease, in which case the objective of treatment may include mitigating or reversing disease progression and / or improving side effects. The subject may have a history of being treated for the same condition, in which case the objective of treatment typically includes reducing or delaying the risk of recurrence.

[0209] Human subjects suitable for treatment typically include two treatment groups that can be distinguished by clinical criteria. Subjects with “progressive disease” or “high tumor burden” are subjects with clinically measurable tumors. Clinically measurable tumors are those that can be detected based on tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or X-ray; positivity of biochemical or histopathological markers alone is insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to induce an antitumor response with the aim of alleviating their condition. Ideally, a reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduction in the risk or rate of tumor progression, or a reduction in its pathological outcomes.

[0210] A second group of suitable subjects is known in the art as the “adjuvant group.” These are individuals with a history of neoplasms but who have responded to other modes of treatment. Previous treatments may include, but are not limited to, surgical resection, radiotherapy, and conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk of disease progression near the original tumor site or due to metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on features observed before or after initial treatment. These features are known in the clinical art and are appropriately defined for each different neoplasm. Typical features of the high-risk subgroup include tumors that invade adjacent tissues or show lymph node involvement.

[0211] Another group has a genetic predisposition to neoplasms but has not yet shown clinical signs of neoplasms. For example, a woman who tests positive for a gene mutation associated with breast cancer and is still of childbearing age may, if so desire, receive one or more of the immune-responsive cells described herein in a prophylactic treatment to prevent the development of neoplasms until it is appropriate to perform prophylactic surgery.

[0212] In addition, the subject matter of this disclosure provides a method for treating and / or preventing a pathogen infection (e.g., viral infection, bacterial infection, fungal infection, parasitic infection, or protist infection) in a subject, for example, an immunodeficient subject. The method involves administering an effective amount of the immune-responsive cells of this disclosure or a composition comprising them to a subject having a pathogen infection. This may include, but is not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infections.

[0213] Further modifications can be introduced into the immune-responsive cells (e.g., T cells) of this disclosure to prevent or minimize the risk of immunological complications (known as “malignant T cell transformation”), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigens as tumor cells, leading to outcomes similar to GvHD. A possible solution to this problem is to manipulate suicide genes into the immune-responsive cells of this disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase-9 suicide gene (iCasp-9), and cleaved human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is the EGFRt polypeptide. The EGFRt polypeptide can enable the elimination of T cells by administration of an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently conjugated upstream of the antigen-recognition receptor of the CAR of this disclosure. The suicide gene can be incorporated into a vector containing the nucleic acid encoding the CAR of this disclosure. Thus, administration of a prodrug designed to activate the suicide gene during malignant T cell transformation (e.g., GVHD) (e.g., AP1903, which can activate iCasp-9) induces apoptosis in suicide gene-activated CAR-expressing T cells. Incorporating the suicide gene into the CAR of this disclosure allows for the elimination of the majority of CAR T cells in a very short time, providing an additional level of safety. The immune-responsive cells (e.g., T cells) of this disclosure incorporating the suicide gene can be preemptively eliminated at a given point in time after CAR T cell injection, or eradicated at the earliest signs of toxicity.

[0214] 11. Kit The subject matter of this disclosure provides kits for inducing and / or enhancing an immune response in a subject, and / or treating and / or preventing neoplasm or pathogen infection. In certain embodiments, the kit comprises an effective amount of the immune-responsive cells of this disclosure or a pharmaceutical composition comprising them. In certain embodiments, the kit comprises a sterile container; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metallic foil, or other materials suitable for holding pharmaceuticals. In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule encoding the HI-TCR disclosed herein against an antigen of interest in an expessible form, which may optionally be contained in one or more vectors.

[0215] If desired, immune-responsive cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to subjects who have or are at risk of developing a neoplasm or pathogen or immune disorder. The instructions generally include information on the use of the composition for the treatment and / or prevention of neoplasm or pathogen infection. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosing schedule and administration for the treatment or prevention of neoplasm, pathogen infection or immune disorder or its symptoms; precautions; warnings; indications; contraindications; information on overdose; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if any), or as a label affixed to the container, or as a separate sheet, brochure, card, or folder supplied inside or with the container. [Examples]

[0216] The implementation of this disclosure will use the usual techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of a person skilled in the art, unless otherwise indicated. Such techniques are well described in literature such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "GeneTransfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and can therefore be considered in the preparation and implementation of the subject matter of this disclosure. Techniques particularly useful with respect to specific embodiments will be discussed in the following sections.

[0217] The following examples are provided to give a complete disclosure and explanation of the methods for preparing and using the cells and compositions of this disclosure to those skilled in the art, and are not intended to limit the scope that the inventors consider to be part of the present invention.

[0218] (Example 1: Targeting of T cells by endogenous non-HLA-restricted T cell receptor / CD3 complex) Introduction We performed specific modifications at the TCR locus designed to alter the antigen specificity of human T cells without disrupting or bypassing the CD3 complex that physiologically controls and regulates T cell activation. In this approach, T cells lost their endogenous T cell receptor and gained the ability to recognize antigens independently of HLA by leveraging their CD3 complex, which is bypassed in other cases when using CARs. This was achieved by targeted disruption of the endogenous TCR, combined with the introduction of an antigen-binding domain to the TRAC locus in a manner that preserves all components of the native CD3 complex in its native structure. Recombinant TCR-like molecules (i.e., HI-TCR, FvTCR, or HIT-CAR) may have a variable domain derived from an immunoglobulin gene or an alternative ligand to direct antigen recognition. The recombinant receptor signaling via the CD3 complex may be intact (i.e., in its physiological composition) or amplified by the non-covalent incorporation of the costimulatory domain.

[0219] result We developed a novel strategy for one-step generation of TCRs and TCR-like T cells by incorporating TCR or TCR-like genes that have distinct variable domains for specific antigens. This resulted in the expression of new TCR / TCR-like genes under the control of endogenous TCR promoters (alpha, beta, or both) by concomitant disruption of endogenous TCR surface expression.

[0220] We developed a one-step strategy for the generation of universal CAR T cells by targeting the integration of a promoter-less CAR gene cassette in the first exon of the TCR alpha constant chain (TRAC). This resulted in CAR expression under the control of the endogenous TCR alpha promoter, due to the concomitant disruption of TCR alpha gene expression, which leads to the absence of TCR expression on the cell surface. CAR gene targeting at the TRAC locus was achieved by homologous recombination (HR) using site-specific nucleases (e.g., CRISPR / Cas9) and AAV donor templates. Using HR-based gene targeting, this strategy enabled the generation of T cells with specific specificity encoded by chimeric TCR receptors containing specific antigen-binding domains. The antigen-binding domains were derived from either immunoglobulins (i.e., Fv fragments), cell surface receptor ligands, or TCRs (αβ or γδ). Hybrid immunoglobulin-TCR chimeric antigen receptors (i.e., HIT-CAR, FvTCR, or HI-TCR) or γδTCRs have enabled T cells to recognize target cells in an HLA-independent manner. Insertion of novel αβTCRs could be achieved in a similar manner, but would result in HLA restriction antigen recognition.

[0221] For example, Figure 1A shows schematic diagrams of T cell receptors (TCRs), B cell receptors (BCRs), chimeric antigen receptors (CARs), and HLA-independent TCR-based chimeric antigen receptors (i.e., HIT-CARs, FvTCRs, or HI-TCRs). Figure 1B shows CRISPR / Cas9-targeted integration of the three receptors into the TRAC locus. The manipulated HIT-CAR targets CD19. Figure 1C shows cell surface expression of HIT-CARs (i.e., HI-TCRs) from the TRAC locus. Figures 1D and 1E show the cytolytic effect and proliferation of HIT-CAR (i.e., HI-TCRs or FvTCRs) T cells, respectively.

[0222] As shown in Figure 2A, a combination of single-step targeted gene disruption and HIT-CAR (i.e., HI-TCR or FvTCR) targeted insertion established conditions for obtaining up to 65% HIT-CAR (i.e., HI-TCR or FvTCR) T cells. These T cells, as shown in Figure 2B, exhibited similar in vitro and in vivo oncolytic activity to previously characterized TRAC-CAR T cells expressing 1928z CAR. In addition, antigen interactions induced downregulation of HIT-CAR (i.e., HI-TCR or FvTCR) T cells, which was dependent on the number of antigen-dependent stimuli. Since endogenous TCR expression is eliminated on the cell surface, these HIT-CAR (i.e., HI-TCR or FvTCR) T cells were found in off-the-shelf immunotherapy. It was useful in the development of [the product / service].

[0223] Other targets besides CD19 can be used in immunotherapy. A schematic diagram of the NYESO TCR gene incorporated into the TCR alpha or beta chain is shown in Figure 3A. Cell surface expression of TRAC-NYESO-TCR and TRBC-NYESO-TCR (i.e., HI-TCR or FvTCR) is shown in Figure 3B. A representative TCR-V-beta-1 flow cytometry plot 4 days after TRAC or TRBC targeting is shown in Figure 3B. Cytotoxic activity of manipulated T cells is shown in Figure 3C. A schematic diagram of an alternative design for simultaneous targeting of both TCR alpha and TCR beta is shown in Figure 3D, in which the NYESO-HI-TCR is located at the TRAC locus and the 4-1BBL expression cassette is located at the TRBC locus. Cell surface expression of TRAC-NYESO-TCR and TRBC-4-1BBL is shown in Figure 3E.

[0224] A variant approach was also provided in which a costimulatory domain, resulting in hyperphysiological antigen sensing and activation, was inserted non-covalently into the CD3 complex. This is achieved by fusing the costimulatory domain to one or both TCR chains. Furthermore, two different costimulatory dosages are possible by fusing the costimulatory domain to either one or both modified TCR chains (i.e., antigen-binding chains).

[0225] Furthermore, HI-TCRs showed superior sensitivity compared to CARs. Human T cells edited to replace endogenous TCRs with HI-TCRs gained the ability to engage with lower antigen densities and kill such cells. For example, Table 2 shows the in vitro cytotoxic activity of HI-TCR cells. HI-TCRs or CD19-CARs were introduced into the endogenous TRAC locus of human peripheral T cells using CRISPR / Cas9-mediated gene editing and an AAV6 donor vector. Five days after gene targeting, T cells (6,000, 30,000, or 150,000) were incubated with 250,000 Nalm6 leukemia cells expressing different CD19 levels (very low to high). Cells were incubated for 22 hours in 500 μl of X-Vivo medium containing serum (without IL-2). The total number of Nalm6 cells was determined by analyzing the co-culture by FACS in the presence of counting beads. Cytotoxic activity is shown as the percentage of Nalm6 cells killed. E / T: Ratio of effector (T cell) to target (Nalm6). The data clearly demonstrate that HI-TCR can detect lower levels of CD19 antigen than CAR. This feature may be very useful for antigens with moderate or low expression, such as CD22, BCMA, CCR1, and CD70, and may be useful in treating relapse after CAR therapy for any of these antigens, as recurrent tumor cells frequently show reduced antigen density on their surface. [Table 2]

[0226] Furthermore, a panel of exogenous and endogenous 3' untranslated regions (3'UTRs) was found to be able to modulate accurate and predictable gene expression at TCR loci. For example, Figure 4A shows a schematic diagram of the CD28z CAR gene integrated into the TRAC locus. PolyA (black boxes) correspond to segments of the CAR cassette modified to examine different viral and mammalian 3'UTRs. Using TRAC-CAR T cells expressing CD28z CAR as a model, it was shown that certain 3'UTRs reduced CAR expression compared to bovine growth hormone (bGH) polyA sequences (Figures 4B and 4C), resulting in CAR-T cells with impaired in vivo cytotoxic activity (Figures 5A and 5B). Certain other 3'UTRs, including endogenous TRAC 3'UTRs, were shown to increase CAR surface expression levels (Figures 4B and 4C), resulting in CAR-T cells with improved in vivo cytotoxic activity (Figures 5A and 5B). These 3'UTRs can also be used to modulate the precise and predictable gene expression of any of the HI-TCRs disclosed herein.

[0227] The aforementioned genetic modifications at the TRAC locus enable the generation of T cells expressing an optimal level of HI-TCR, similar to physiological TCRs but distinct from CARs, and leverage endogenous T cell activation mechanisms (CD3 complex and downstream signaling elements). This approach can advance both autologous and allogeneic T cell therapies.

[0228] Another relevant field where TCR gene editing has relevant applications is T-iPS-derived T cells. T-iPS cells are pluripotent stem cells obtained by reprogramming peripheral T lymphocytes. Therefore, these T-iPS cells contain a rearranged T cell receptor, which is either αβ or γδ TCR, that can be modified using gene editing techniques. T cells obtained from T-iPS cells by directed differentiation express a rearranged TCR that can be detected and sequenced. This sequence can be used to determine the precise location of the rearranged variable domain in the T-iPS cell genome. The endogenous variable domain can be replaced with the new variable domain using a nuclease that specifically targets this rearranged variable domain and donor DNA containing a TCR variable domain of known specificity. This approach requires two-step gene editing: one targeting the α (or γ) chain and the other targeting the β (or δ) chain. The approach can be carried out in a single step, as only two alleles need to be modified. In addition, the TCR chain can be modified to express any of the HI-TCRs disclosed herein using strategies similar to those described for primary human T cells.

[0229] (Example 2) Figure 1A shows the CRISPR / Cas9-targeted integration of three receptors into the TRAC locus. The engineered HIT-CAR targets CD19. Cell surface expression of HIT-CAR (i.e., HI-TCR) from the TRAC locus is shown in Figures 6B and 6C. The cytolytic effect and proliferation of HIT-CAR (i.e., HI-TCR or FvTCR) T cells are shown in Figures 7A, 7B, and 8A-8C. In particular, these data show that HIT T cells were superior to CAR-T cells in killing target cells expressing low antigen levels.

[0230] Furthermore, as shown in Figures 9A–9D, HI-TCR T cells were engineered to co-express costimulatory ligands. In this specific experiment, the day after targeting the TRAC locus with HI-TCR, T cells were transfused with retroviral SFG vectors encoding CD80, 41BBL, or both. Five days after these cells were grown ex vivo, 4e5 TRAC-CAR or TRAC-HI-TCR-positive T cells were injected into NSG mice with NALM6 cells expressing very low levels of CD19. The TRAC-HI-TCR T cells expressed CD80, 41BBL, or both ligands, or neither. Bioluminescence was used to assess tumor burden weekly and track mouse survival.

[0231] TRAC-CAR T cells were unable to control tumors with very low levels of CD19, and all mice were observed to be in the terminal stage by day 30. At this low dose, HI-TCR T cells initially controlled the tumor load, but mice relapsed by day 10 and were in the terminal stage by day 40. When HI-TCR T cells co-expressed CD80 and 4-1BBL, the addition of a costimulatory ligand to HI-TCR T cells improved antitumor activity with an optimal response. This improved activity resulted in long-term control of NALM6 expressing very low levels of CD19.

[0232] Figures 10A–10C further demonstrate that baseline TRAC-CAR expression can be controlled by distinct 3'UTR sequences without affecting cell surface replenishment dynamics after antigen encounter. All constructs were engineered in the same manner as previously described, so that they were under the transcriptional control of the TRAC endogenous promoter. Modulation of baseline expression levels was observed by modifying the 3'UTR (containing the poly(A) signaling pathway) (Figure 10B). When these different TRAC-CAR T cells were cultured on CD19-expressing tumor cells, a reduction in CAR cell surface expression, followed by replenishment, was observed by flow cytometry (Figure 10C). Different 3'UTRs altered baseline expression levels but maintained the same replenishment dynamics, with final expression levels similar to baseline.

[0233] Furthermore, HI-TCRs targeting CD70 and CD22 were also created. By sequencing the existing scFv or Fab region of existing antibodies targeting the same antigen, an extracellular antigen-binding domain can be obtained, thereby creating HI-TCRs targeting antigens of interest.

[0234] Subjective embodiments of this disclosure From the foregoing description, it is clear that the subject matter of this disclosure may be modified and altered to be adopted for various uses and conditions. Such embodiments are also within the scope of the following claims.

[0235] Any enumeration of elements in any definition of a variable herein includes the definition of that variable as any single element or as a combination (or subcombination) of the elements described. Any enumeration of embodiments herein includes that embodiment as any single embodiment or as a combination with any other embodiment or part thereof.

[0236] All patents and publications referenced herein are incorporated herein by reference to the same extent that each individual patent and publication is specifically indicated as being incorporated herein by reference.

Claims

1. i) A first antigen-binding chain comprising an antigen-binding fragment of the heavy chain variable region (VH) of an antibody and ii) A second antigen-binding chain containing the antigen-binding fragment of the variable region (VL) of the antibody light chain. Recombinant T cell receptors (TCRs) including, Each of the first and second antigen-binding chains is, TRAC polypeptide or TRBC polypeptide Recombinant TCR comprising, wherein at least one of the TRAC polypeptide and the TRBC polypeptide is natural.

2. (a) The first antigen-binding chain comprises an antigen-binding fragment of the heavy chain variable region (VH) of the antibody and a TRAC polypeptide; (b) The second antigen-binding chain comprises an antigen-binding fragment of the variable region (VL) of the antibody light chain and a TRBC polypeptide, The recombinant TCR according to claim 1.

3. (a) The first antigen-binding chain comprises an antigen-binding fragment of the heavy chain variable region (VH) of the antibody and a TRBC polypeptide; (b) The second antigen-binding chain comprises an antigen-binding fragment of the variable region (VL) of the antibody light chain and a TRAC polypeptide, The recombinant TCR according to claim 1.

4. The recombinant TCR according to any one of claims 1 to 3, wherein the first antigen-binding chain or the second antigen-binding chain further comprises a costimulatory region.

5. The first antigen-binding chain and the second antigen-binding chain are 1 × 10 －８ Dissociation constant (K) M or less Ｄ A recombinant TCR according to any one of claims 1 to 4, which binds to the antigen.

6. The recombinant TCR binds to the tumor antigen, and the tumor antigen, (a) CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, Fetal Acetylcholine Receptor, Folate Receptor-α, GD2, GD3, HER-2, hTERT, IL-13R-α2, κ-Light Chain, KDR, LeY, L1 Cell Adhesion Molecule, MAGE-A 1. Selected from mesothelin, ERBB2, MAGEA3, p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivorbin, hTERT, EphA2, NKG2D ligand, NY-ES0-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB; (b) Selected from CD22, BCMA, CCR1 and CD70; (c) CD70; or (d) CD19, Recombinant TCR according to any one of claims 1 to 5.

7. The recombinant TCR according to any one of claims 1 to 6, wherein the antigen has a density of less than 10,000 molecules per cell.

8. The aforementioned tumor, a) It is cancer; b) It is a recurrent tumor; c) It is a solid tumor or a blood cancer; d) It is a hematological malignancy; and / or e) Select from B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), and adenocarcinoma. The recombinant TCR according to claim 7.

9. An immune-responsive cell comprising a recombinant TCR according to any one of claims 1 to 8.

10. The immune-responsive cell according to claim 9, wherein the recombinant TCR is expressed by a transgene incorporated into the endogenous gene locus of the immune-responsive cell.

11. The immune-responsive cell according to claim 10, wherein the endogenous gene locus is the TRAC gene locus and / or the TRBC gene locus.

12. The immune-responsive cell according to claim 11, wherein in the immune-responsive cell, the endogenous expression of TCRs including native TCRα chains and / or native TCRβ chains is disrupted or inactivated, thereby preventing or eliminating mispairing between the recombinant TCRs and native TCRα chains and / or native TCRβ chains in the immune-responsive cell.

13. An immune-responsive cell according to any one of claims 10 to 12, comprising a transcriptional terminator region in which the endogenous gene locus is modified.

14. The immune-responsive cell according to any one of claims 10 to 13, wherein the endogenous gene locus is a first endogenous T cell receptor (TCR) locus, and a second endogenous TCR locus, different from the first endogenous TCR locus, is modified to exclude the expression of the endogenous TCR chain encoded by the second endogenous TCR locus.

15. The immune-responsive cells according to any one of claims 9 to 14, wherein the cells are selected from pluripotent stem cells that can differentiate into T cells, natural killer (NK) cells, and lymphoid cells.

16. (a) further comprising at least one exogenous costimulatory ligand; (b) further comprising at least one chimeric costimulatory receptor (CCR); and / or (c) further comprising at least one chimeric antigen receptor (CAR), An immune-responsive cell according to any one of claims 9 to 15.

17. A pharmaceutical composition comprising an effective amount of immune-responsive cells according to any one of claims 9 to 16 and a pharmaceutically acceptable excipient.

18. A composition comprising immune-responsive cells according to any one of claims 9 to 16 or a pharmaceutical composition according to claim 17, for use in reducing tumor burden, treating or preventing tumors, and / or extending the survival of subjects having tumors.

19. A method for producing antigen-specific immune-responsive cells, comprising introducing a nucleic acid encoding a recombinant TCR according to any one of claims 1 to 8 into immune-responsive cells.

20. A nucleic acid encoding a recombinant TCR according to any one of claims 1 to 8.

21. A vector comprising the nucleic acid described in claim 20.

22. A kit comprising a recombinant TCR according to any one of claims 1 to 8, an immune-responsive cell according to any one of claims 9 to 16, a pharmaceutical composition according to claim 17, a nucleic acid according to claim 20, or a vector according to claim 21.

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