Antigen specific CD19-targeted car-t cells

Abstract

To provide a composition and method for targeted treatment of cancer such as blood cancer.SOLUTION: Disclosed are a CAR polypeptide including a target domain selectively binding to a B lymphocyte antigen (e.g., hematological malignant tumor, e.g., CD19, CD20, and / or CD22 antigen related to leukemia and / or lymphoma), and a chimeric antigen receptor (CAR) T-cell which expresses the polypeptide, and can be used together with an adoptive cell transfer. Also disclosed is a method for bringing antitumor immunity to a subject having blood cancer accompanied by adoptive transfer of a CAR T-cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 840,774, filed April 30, 2019, which is incorporated by reference in its entirety. [Background technology]

[0002] Hematological malignancies are some of the most common cancers occurring in both children and adults. For example, approximately 4,000 new cases of B-cell acute lymphoblastic leukemia (B-ALL), an aggressive B-cell lineage malignancy, are diagnosed each year in the United States, making it the most common malignancy in children. Genetic mutations that induce abnormal arrest of normal lymphocyte maturation, evasion of apoptosis, and uncontrolled cell proliferation result in the overproduction of B-cell lymphoblasts. In adults, more than 6,000 new cases of acute lymphoblastic leukemia (ALL) occur each year. (Hanahan, D. and Weinberg, R. (2000) The hallmarks of cancer. Cell 100: 57-70; Teitell, M. and Pandolfi, P. (2009) Molecular genetics of acute lymphoblastic leukemia. Annu Rev Pathol 4: 175-198) Furthermore, lymphomas (e.g., neoplasms of lymphoid tissues) account for approximately 5% of all cancer cases in the United States. The major class is malignant neoplasms (i.e., cancers) of lymphocytes, a cell type present in both lymph and blood. In this regard, lymphomas and leukemias are both malignant tumors (e.g., tumors) of hematopoietic and lymphoid tissues. As lymphoproliferative disorders, lymphomas and lymphocytic leukemias are so closely related that some of them are referred to by either name (e.g., adult T-cell leukemia / lymphoma). Summary of the Invention [Problem to be solved by the invention]

[0003] Preclinical and clinical research efforts are focused on investigating immunotherapy modalities, including antibody-based therapy and / or adoptive cell therapy, for high-risk cancers. Such strategies rely on tumor-associated antigens that enable specific targeting of cancerous cells and a small number of non-cancerous cells (i.e., on-target / off-tumor or bystander effects). Particular interest has been focused on adoptive immunotherapy approaches that involve genetic engineering of human immune effector cells with synthetic chimeric antigen receptors (CARs) that target tumor-associated antigens expressed on the cell surface in a major histocompatibility complex (MHC) antigen-independent manner. Without being bound by any particular theory, binding of a CAR expressed on a T cell to a cancer-associated antigen leads to intracellular signaling via the T cell costimulatory domain and subsequent expansion of CAR T cells, inducing further cancer cell killing. However, despite positive results from early trials, infusion of CD19 CAR T cells into patients still results in several "on-target / on-tumor" and "on-target / off-tumor" side effects of varying severity, such as tumor lysis syndrome (TLS), cytokine release syndrome (CRS) and macrophage activation syndrome, CNS trafficking, persistent B-cell aplasia, and immune escape. Thus, in view of the long-felt unmet need described herein, improved therapies for hematological malignancies are needed. [Means for solving the problem]

[0004] The present invention is based, at least in part, on the discovery that B lymphocyte antigens, such as CD19 (B lymphocyte antigen CD19), can be used for targeted treatment of hematological cancers (i.e., cancers of the hematopoietic and lymphatic tissues). In some aspects, provided herein are immune cells expressing chimeric antigen receptor (CAR) polypeptides that target B-lineage cells and cancer cells arising therefrom. In some embodiments, the CARs disclosed herein comprise a B lymphocyte antigen-targeting domain, e.g., a CD19, CD20, and / or CD22 binding domain, a transmembrane domain, and an intracellular signaling domain. In certain preferred embodiments, the B lymphocyte antigen-binding domain targets wild-type and / or mutant CD19 antigens.

[0005] In certain aspects, provided herein are bispecific chimeric antigen receptor (CAR) T cells that express a CAR polypeptide comprising a targeting domain that selectively binds a B lymphocyte antigen (e.g., CD19, CD20, and / or CD22 antigens associated with hematological malignancies, such as leukemia and / or lymphoma), and a CAR polypeptide comprising a targeting domain that selectively binds to another, different tumor-associated antigen. In some such embodiments, the targeting domain of the chimeric antigen receptor (e.g., the CD19 antigen-binding domain and / or another, different tumor-associated antigen-binding domain) comprises a functional antibody fragment. Preferably, the antigen-binding domain of the chimeric antigen receptor comprises a single-chain variable fragment (scFv). In a most preferred embodiment, the functional antibody fragment (e.g., scFv) is derived from the monoclonal antibody FMC63.

[0006] In some embodiments, the transmembrane domain of a CAR disclosed herein comprises at least one transmembrane domain of either CD28, 41BB, a variant thereof, or any combination thereof. In some preferred embodiments, the intracellular signaling domain of a CAR disclosed herein comprises at least one signaling domain of CD3ζ, a variant thereof, or any combination thereof. In some embodiments, a CAR disclosed herein further comprises at least one costimulatory signaling region, for example, a costimulatory signaling region comprising the signaling domain of any one of CD28 or a variant thereof, CD137 (41BB) or a variant thereof, or any combination thereof. In some embodiments, the costimulatory signaling region contains one, two, three, or four cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules. In some embodiments, the costimulatory signaling region contains one or more mutations in the cytoplasmic domain of CD28 and / or 4-1BB that attenuate or preferably enhance signal transduction. In certain embodiments, the CAR-expressing immune cells no longer express one or more immune checkpoint molecules. In some such embodiments, immune checkpoint molecules are blocked and / or inhibited by methods known in the art.

[0007] In some embodiments, the CAR polypeptide contains an incomplete endodomain. For example, the CAR polypeptide can contain either an intracellular signaling domain or a costimulatory domain, but not both. In these embodiments, the immune effector cell is not activated unless both the immune effector cell and a second CAR polypeptide (or endogenous T cell receptor) containing the missing domain bind to their respective antigens. Thus, in some embodiments, the CAR polypeptide contains a CD3 zeta (CD3ζ) signaling domain but does not contain a costimulatory signaling region (CSR). In other embodiments, the CAR polypeptide contains the cytoplasmic domain of CD28, 4-1BB, or a combination thereof, but does not contain the CD3ζ signaling domain (SD).

[0008] In some aspects, provided herein are methods of treating a B lymphocyte antigen (e.g., CD19)-associated cancer (e.g., hematological cancers, including leukemia and lymphoma) in a subject, comprising administering an effective amount of an adoptive immunotherapy composition comprising a CAR-expressing cell disclosed herein. In some embodiments, the CAR-expressing cells of the adoptive immunotherapy composition are derived from the subject (e.g., autologous). Preferably, the CAR-expressing cells of the adoptive immunotherapy composition are derived from a donor sample or a bank or library comprising immune cells not derived from the subject (e.g., allogeneic). For example, the methods disclosed herein can be used to treat allogeneic T cells (e.g., PBMC samples, CD4 + T cells and / or CD8 + and selecting T cells, e.g., CTLs, from a cell bank (e.g., a pre-generated third-party donor-derived epitope-specific CTL bank). In some embodiments, the method further comprises administering at least one immune checkpoint inhibitor.

[0009] In some aspects, disclosed herein are isolated nucleic acids encoding the disclosed CAR polypeptides, and nucleic acid vectors containing the isolated nucleic acids operably linked to expression control sequences. Additionally, disclosed herein are cells transfected with these vectors or otherwise comprising the disclosed nucleic acids, and the use of these cells to express and / or produce the disclosed CAR polypeptides. While not intended to be an exhaustive list, the cells can be immune effector cells, such as αβ T cells, γδ T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, or regulatory T cells. In some embodiments, when the antigen-binding domain of the CAR binds to a B lymphocyte antigen, such as CD19, CD20, and / or CD22, the cells exhibit anti-tumor immunity (e.g., mount an immune response against a tumor).

[0010] In a further aspect of the present invention, disclosed herein is a pharmaceutical composition comprising a molecule disclosed herein in a pharmaceutically acceptable carrier. Also disclosed herein is a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein. In some embodiments, the cancer can be, for example, any B lymphocyte antigen-expressing malignancy (e.g., expressing CD19, CD20, and / or CD22).

[0011] In some embodiments, the B lymphocyte antigen binding agents disclosed herein comprise antibody fragments that specifically bind to B lymphocyte surface peptides, e.g., CD19, CD20, and / or CD22. For example, without limitation, the antigen-binding domain can be a Fab or single-chain variable fragment (scFv) of an antibody that specifically binds to CD19, CD20, and / or CD22. In some such embodiments, the antigen binding agent is an aptamer that specifically binds to a B lymphocyte antigen, e.g., CD19, CD20, and / or CD22. For example, in certain embodiments, the antigen binding agent is or otherwise comprises a peptide aptamer selected from a random sequence pool based on its ability to bind to a B lymphocyte antigen, e.g., CD19, CD20, and / or CD22. In some embodiments, the B lymphocyte antigen binding agent can also comprise a natural ligand or variant and / or fragment thereof capable of binding to the B lymphocyte antigen.

[0012] The CAR (or CAR-related) polypeptides disclosed herein can also contain a transmembrane domain and an endodomain that can activate immune effector cells. For example, the endodomain can contain a signaling domain and one or more costimulatory signaling regions.

[0013] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. The present invention also relates to the following: [Item 1] A chimeric antigen receptor (CAR) polypeptide comprising a B lymphocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. [Item 2] 2. The CAR polypeptide of item 1, wherein the B lymphocyte antigen is a CD19 antigen, a CD20 antigen, or a CD22 antigen. [Item 3] 3. The CAR polypeptide of item 1 or 2, wherein the B lymphocyte antigen-binding domain is an anti-CD19 functional antibody fragment. [Item 4] 4. The CAR polypeptide of any one of items 1 to 3, wherein the B lymphocyte antigen-binding domain is an anti-CD19 single-chain variable fragment (scFv). [Item 5] 5. The CAR polypeptide of any one of items 1 to 4, wherein the B lymphocyte antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 9. [Item 6] 6. The CAR polypeptide of any one of items 1 to 5, wherein the transmembrane domain is derived from a transmembrane or membrane-associated polypeptide. [Item 7] The transmembrane domains of the polypeptides CD28, NKp30, CDS, DAP10, 41BB, DAP12, CD3C, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1, ICOS (CD278), 4 -1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL 7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, C 7. The CAR polypeptide of any one of items 1 to 6, comprising at least one transmembrane domain of any one of D11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, a variant thereof, or any combination thereof. [Item 8] 8. The CAR polypeptide of any one of items 1 to 7, wherein the transmembrane domain comprises the transmembrane domain of CD28 and / or 41BB. [Item 9] 9. The CAR polypeptide of any one of paragraphs 1 to 8, wherein the signaling domain comprises at least one signaling domain of any one of the polypeptides CD8, CD3zeta, CD3delta, CD3gamma, CD3epsilon, FcgammaRI-gamma, FcgammaRIII-gamma, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137(41BB), ICOS, CD27, CD28δ, CD80, NKp30, OX40, variants thereof, or any combination thereof. [Item 10] 10. The CAR polypeptide of any one of items 1 to 9, further comprising at least one costimulatory signaling region. [Item 11] 11. The CAR polypeptide of paragraph 10, wherein the costimulatory signaling region comprises a signaling domain of any one of the polypeptides CD8, CD3zeta, CD3delta, CD3gamma, CD3epsilon, FcγRI-γ, FcγRIII-γ, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137(41BB), ICOS, CD27, CD28δ, CD80, NKp30, OX40, a variant thereof, or any combination thereof. [Item 12] 11. The CAR polypeptide of item 10, wherein the costimulatory signaling region comprises the signaling domain of CD28 or a variant thereof. [Item 13] 13. The CAR polypeptide of item 12, wherein the CD28 signaling domain comprises at least one mutation in any one of the subdomains YMNM, PRRP, PYAP, or any combination thereof. [Item 14] 14. The CAR polypeptide of item 12 or 13, wherein the CD28 signaling domain lacks any one of the subdomains YMNM, PRRP, PYAP. [Item 15] 15. The CAR polypeptide of any one of items 12 to 14, wherein the CD28 signaling domain lacks any two of the subdomains selected from YMNM, PRRP, or PYAP. [Item 16] 12. The CAR polypeptide of item 10 or 11, wherein the costimulatory signaling region comprises the signaling domain of CD137(41BB) or a variant thereof. [Item 17] 17. The CAR polypeptide of any one of items 1 to 16, wherein at least one signaling domain comprises native CD3ζ or a variant thereof. [Item 18] 18. The CAR polypeptide of item 17, wherein the mutant CD3ζ lacks a C-terminal immunoreceptor tyrosine-based activation motif (ITAM). [Item 19] 18. The CAR polypeptide of item 17, wherein the mutant CD3ζ lacks two C-terminal immunoreceptor tyrosine-based activation motifs (ITAMs). [Item 20] 18. The CAR polypeptide of item 17, wherein the mutant CD3ζ contains only one immunoreceptor tyrosine-based activation motif (ITAM). [Item 21] 21. The CAR polypeptide of any one of items 17 to 20, wherein the mutant CD3ζ comprises the amino acid sequence set forth in SEQ ID NO: 11. [Item 22] 22. The CAR polypeptide of any one of items 1 to 21, further comprising a hinge sequence. [Item 23] 23. The CAR polypeptide of item 22, wherein the hinge sequence is derived from a CD8α molecule or a CD28 molecule. [Item 24] 24. The CAR polypeptide of any one of items 1 to 23, further comprising a signal peptide. [Item 25] 26. The CAR polypeptide of claim 24, wherein the signal peptide is derived from a CD8α leader sequence. 26. The CAR polypeptide of any one of items 1 to 25, comprising any one of the amino acid sequences set forth in SEQ ID NOs: 6 to 11, any fragment thereof, or any combination thereof. [Item 27] 26. The CAR polypeptide of any one of items 1 to 25, comprising the amino acid sequence set forth in SEQ ID NO: 7. [Item 28] 28. A nucleic acid encoding the CAR polypeptide of any one of items 1 to 27. [Item 29] 29. The nucleic acid according to Item 28, comprising any one of the nucleotide sequences set forth in SEQ ID NOs: 1 to 5, fragments thereof, or any combination thereof. [Item 30] 29. The nucleic acid of item 28, comprising the nucleotide sequence set forth in SEQ ID NO: 1. [Item 31] A vector comprising the nucleic acid of any one of items 28 to 30. [Item 32] 32. An immune cell comprising the nucleic acid of item 31. [Item 33] 33. An immune cell comprising the vector of item 32. [Item 34] 28. An immune cell expressing the CAR polypeptide of any one of items 1 to 27. [Item 35] 35. The immune cell of any one of items 32 to 34, which is a leukocyte. [Item 36] 36. The immune cell of any one of items 32 to 35, which is a lymphocyte, monocyte, macrophage, dendritic cell, mast cell, neutrophil, basophil, or eosinophil. [Item 37] 37. The immune cell of any one of items 32 to 36, wherein the immune cell is a lymphocyte selected from an αβ T cell, a γδ T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine-induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine-activated killer (LAK) cell, a regulatory T cell, or any combination thereof. [Item 38] 38. The immune cell according to item 37, which is a cytotoxic T lymphocyte (CTL). [Item 39] 39. The immune cell according to item 37 or 38, which is a viral antigen-sensitized CTL. [Item 40] 40. The immune cell of any one of items 37 to 39, which is a CTL sensitized to a viral antigen derived from any one of Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), picornavirus (e.g., hepatitis A virus), hepadnavirus (e.g., hepatitis B virus), hepacivirus (e.g., hepatitis C virus), deltavirus (e.g., hepatitis D virus), hepevirus (e.g., hepatitis E virus), or any combination thereof. [Item 41] 41. The immune cell of any one of items 37 to 40, which is an EBV-sensitized CTL. [Item 42] (a) a CAR polypeptide that selectively binds to a B lymphocyte antigen, and (b) a CAR polypeptide that selectively binds to a tumor-associated antigen; expressing bispecific chimeric antigen receptor (CAR) T cells. [Item 43] 43. The bispecific chimeric CAR T cell of item 42, wherein the B lymphocyte antigen is a CD19 antigen, a CD20 antigen, or a CD22 antigen. [Item 44] 41. The bispecific CAR T cell of item 39 or 40, wherein the tumor-associated antigen is GPC3, MAGE-A1, MAGE-A2, MAGE-C2, SSX-2, Ny-ESO-1, hTERT, or a viral hepatitis antigen. [Item 45] 45. The bispecific CAR T cell of any one of paragraphs 42 to 44, wherein the antigen-binding domains of the chimeric antigen receptors each comprise a functional antibody fragment. [Item 46] 46. ​​The bispecific CAR T cell of any one of paragraphs 42 to 45, wherein the antigen-binding domains of the chimeric antigen receptor each comprise a single-chain variable fragment (scFv). [Item 47] 47. The bispecific CAR T cell of any one of paragraphs 42 to 46, wherein the transmembrane domains of the chimeric antigen receptors each comprise at least one transmembrane domain of either CD28, 41BB, a variant thereof, or any combination thereof. [Item 48] 48. The bispecific CAR T cell of any one of paragraphs 42 to 47, wherein the signaling domain of the at least one chimeric antigen receptor comprises at least one signaling domain of CD3ζ, a variant thereof, or any combination thereof. [Item 49] 48. The bispecific CAR T cell of any one of paragraphs 42 to 47, wherein each of the signaling domains of the chimeric antigen receptors comprises the signaling domain of CD3ζ, a variant thereof, or any combination thereof. [Item 50] 50. The bispecific CAR T cell of item 48 or 49, wherein the mutant CD3ζ lacks a C-terminal immunoreceptor tyrosine-based activation motif (ITAM). [Item 51] 50. The bispecific CAR T cell of item 48 or 49, wherein the mutant CD3ζ lacks two C-terminal immunoreceptor tyrosine-based activation motifs (ITAMs). [Item 52] 50. The bispecific CAR T cell of item 48 or 49, wherein the mutant CD3ζ comprises only one immunoreceptor tyrosine-based activation motif (ITAM). [Item 53] 53. The bispecific CAR T cell of any one of paragraphs 42 to 52, wherein at least one of the chimeric antigen receptors comprises at least one costimulatory signaling region. [Item 54] 53. The bispecific CAR T cell of any one of paragraphs 42 to 52, wherein each chimeric antigen receptor comprises at least one costimulatory signaling region. [Item 55] 55. The bispecific CAR T cell of paragraph 53 or 54, wherein the costimulatory signaling region comprises the signaling domain of any one of CD28 or a variant thereof, CD137(41BB) or a variant thereof, or any combination thereof. [Item 56] 56. The CAR polypeptide of item 55, wherein the CD28 signaling domain comprises at least one mutation in any one of the subdomains YMNM, PRRP, PYAP, or any combination thereof. [Item 57] 57. The CAR polypeptide of item 55 or 56, wherein the CD28 signaling domain lacks the function of at least one subdomain selected from YMNM, PRRP, or PYAP. [Item 58] 59. The CAR polypeptide of any one of items 55 to 57, wherein the CD28 signaling domain lacks the function of any two of the subdomains selected from YMNM, PRRP, or PYAP. 59. The bispecific CAR T cell of any one of paragraphs 42 to 58, further comprising a hinge sequence derived from CD8a or CD28. [Item 60] 60. The CAR-expressing cell of any one of paragraphs 34 to 59, which has been genetically modified so that it no longer expresses one or more immune checkpoint molecules. [Item 61] 60. The CAR-expressing cell of any one of paragraphs 34 to 59, further comprising expression of one or more immune checkpoint molecules in a dominant negative form. [Item 62] 60. The CAR-expressing cell of any one of paragraphs 34 to 59, further comprising expression of a switch receptor specific for one or more immune checkpoint molecules. [Item 63] 60. The CAR-expressing cell of any one of paragraphs 34 to 59, further comprising expression of an antibody or functional fragment thereof capable of blocking signaling by one or more checkpoint molecules. [Item 64] 64. The CAR-expressing cell of any one of paragraphs 60 to 63, wherein the immune checkpoint molecule is selected from programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), cytotoxic T-lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4), CD160, and transforming growth factor beta (TGF-β) receptor. [Item 65] 65. The immune cell of any one of items 60 to 64, wherein the immune checkpoint molecule is PD-1 and / or CTLA-4. [Item 66] 66. A method of treating a B lymphocyte antigen-associated cancer in a subject, comprising administering an effective amount of an adoptive immunotherapy composition comprising the CAR-expressing cell of any one of paragraphs 32 to 65. [Item 67] 67. The method of item 66, wherein the B-lymphocyte antigen-associated cancer is a hematological cancer selected from acute leukemia, chronic leukemia, lymphocytic leukemia, myeloid leukemia, preleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, EBV-associated lymphoproliferative disease, mature B-cell neoplasm, mature T-cell and / or natural killer (NK)-cell neoplasm, precursor lymphoid neoplasm, and immunodeficiency-associated lymphoproliferative disorder. [Item 68] 68. The method of item 66 or 67, wherein the B lymphocyte antigen-associated cancer is an EBV-associated lymphoproliferative disease. [Item 69] 69. The method of any one of paragraphs 66 to 68, wherein the subject has received, is receiving, or will receive additional anti-cancer therapy. [Item 70] 70. The method of item 69, wherein the additional anti-cancer therapy comprises surgery, radiation, chemotherapy, immunotherapy, or hormone therapy. [Item 71] 71. The method of any one of paragraphs 66 to 70, wherein the adoptive immunotherapy composition is administered intrathoracically, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally, intracranially, or by direct administration to an organ. [Item 72] 72. The method of any one of items 66 to 71, wherein the subject is a human. [Item 73] 73. The method of claim 72, wherein the CAR-expressing cells of the adoptive immunotherapy composition are derived from the subject. [Item 74] 74. The method of claim 73, wherein the CAR-expressing cells of the adoptive immunotherapy composition are derived from a donor sample or a bank or library of donor samples. [Item 75] 75. The method of any one of items 66 to 74, further comprising administering at least one immune checkpoint inhibitor. [Item 76] 76. The method of item 75, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, or a combination thereof. [Item 77] 76. The method of item 75, wherein the immune checkpoint inhibitor comprises an RNAi molecule such as an antisense RNA molecule (asRNA), a microRNA molecule (miRNA), a short hairpin RNA molecule (shRNA), or a small interfering RNA molecule (siRNA). [Item 78] 79. The method of claim 75, wherein the immune checkpoint inhibitor comprises a CRISPR RNA (crRNA) molecule. 76. The method of item 75, wherein the immune checkpoint inhibitor comprises a dominant negative immune checkpoint molecule. [Item 80] 76. The method of item 75, wherein the immune checkpoint inhibitor comprises a recombinant switch receptor. [Item 81] 81. The method of any one of items 75 to 80, wherein the immune checkpoint inhibitor is expressed by a vector comprising a nucleic acid molecule encoding the immune checkpoint inhibitor, wherein the vector is selected from a DNA vector, an RNA vector, a plasmid, or a viral vector. [Item 82] 82. The method of claim 81, wherein a vector comprising a nucleic acid molecule encoding an immune checkpoint inhibitor is expressed in a CAR-expressing cell of the adoptive immunotherapy composition. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows characterization of CD19 protein expression in donor-derived target BLCLs. [Figure 2] Figure 1 shows data from an electrical impedance assay assessing targeted cytotoxicity and alloreactivity induced by donor-derived EBV-sensitized anti-CD19 CAR-T cells when cocultured with matched (autologous) or mismatched target BLCL. Cytolytic activity is induced by both non-transduced (NTD) EBV-CTL and CAR-expressing EBV-CTL (top panel). However, whereas CAR EBV-CTL can induce CD19-targeted TCR-independent cytolytic activity in response to contact with mismatched target BLCL, NTD EBV-CTL exhibit limited EBV-specific TCR-directed cytolytic activity. [Figure 3] Figure 1 shows the characterization of CD19 expression in each target cell line used in the luciferase assay. It should be understood that the K562 cell line expresses neither the CD19 antigen nor the EBV antigen and acts as a double negative control. [Figure 4]

[0023] Figure 1 shows luciferase assay data demonstrating targeted cytolytic activity induced by EBV-CAR-T cells. EBV-CAR-T cells exhibited CAR-directed cytolysis of the CD19-expressing cell lines NALM6 and RAJI, as well as K562 cells engineered to express CD19. Minimal cytolytic activity was observed in control K562 cells. [Figure 5]FIG. 1 is a graph showing donor-to-donor variability in nonspecific cytolysis induced by untransduced effector cells. [Figure 6] 1 is a graph showing exemplary cytokine release data for multiple target cell lines. [Figure 7] 1 is a graph showing TH1 pro-inflammatory cytokine release data for multiple target cell lines. [Figure 8] 1 is a graph showing TH2 pro-inflammatory cytokine release data for multiple target cell lines. [Figure 9] 1 is a graph showing TH17 pro-inflammatory cytokine release data for multiple target cell lines. [Figure 10] 1 is a graph showing regulatory inflammatory and cytolytic cytokine release data for multiple target cell lines. [Figure 11] 1 is a graph showing chemoattractive cytokine release data for multiple target cell lines. [Figure 12] 1 is a graph showing activation cytokine release data for multiple target cell lines. [Figure 13] Cytokine release heatmap for effector T cells generated from donor 014-18 and target K562-CD19 cells at various E:T ratios. [Figure 14] Cytokine release heatmap for effector T cells generated from donor 023-18 and target K562-CD19 cells at various E:T ratios. [Figure 15] Cytokine release heatmap for effector T cells generated from donor 009-19 and target K562-CD19 cells at various E:T ratios. [Figure 16] FIG. 1 illustrates the study design for the safety and efficacy evaluation of EBV-sensitized anti-CD19 CAR-expressing T cells in a systemic Nalm6-induced mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0015] detail As disclosed herein, the present invention relates, at least in part, to immune cells recombinantly expressing a chimeric antigen receptor (CAR) that targets a cancer-associated B lymphocyte antigen. Such B lymphocyte antigens include, but are not limited to, CD19, CD20, and CD22. In some such embodiments, the antigen is CD19, which is associated with hematological malignancies, such as leukemia and lymphoma. In a preferred embodiment, CD19 is targeted by immune effector cells (i.e., T cells or natural killer (NK) cells) engineered to express a chimeric antigen receptor (CAR) polypeptide that selectively binds to CD19.

[0016] A major advance in T cell therapy was the development of chimeric antigen receptors (CARs). First-generation CARs were developed as artificial receptors that, when expressed by T cells, can retarget T cells to a predetermined disease-associated antigen (e.g., tumor-associated antigen). Such CARs typically contain a single-chain variable fragment (scFv) derived from a target-specific antibody fused to a signaling domain derived from the T cell receptor (TCR), such as CD3ζ. Upon antigen binding, the CAR triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), initiating a signal cascade required for cell lysis, cytokine secretion, and proliferation that circumvents endogenous antigen processing pathways and MHC restriction. Second-generation CAR designs additionally include signaling domains, such as CD28 and / or 4-1BB, that enhance activation and costimulation. Compared to their predecessors, second-generation CARs have been observed to induce greater IL-2 secretion, enhance T cell proliferation and persistence, mediate greater tumor rejection, and prolong T cell survival. Third generation CARs are created by combining multiple signaling domains, such as CD3ζ-CD28-OC40 or CD3ζ-CD28-41BB, to increase potency in terms of stronger cytokine production and killing capacity.

[0017] In some embodiments, the CAR T cells described herein are engineered to counter any tolerogenic effects of the malignant cell microenvironment (e.g., tumor microenvironment), for example, but not limited to, by suppressing or inhibiting PD-1 signaling. In certain embodiments, the CARs described herein may be sensitized to or selectively target viral or non-viral antigens. An ideal target would not be expressed in any normal tissues / organs, or at least in vital normal tissues (heart, liver, CNS, lung, and other tissues that may be particularly susceptible to transient injury), nor in closely related normal cellular counterparts, e.g., stem and / or progenitor cells, to minimize side effects (e.g., on-target / off-tumor or bystander effects). Also disclosed herein are immune effector cells, e.g., T cells or natural killer (NK) cells, engineered to express a chimeric antigen receptor (CAR) polypeptide that selectively binds a B lymphocyte antigen (e.g., wild-type and / or mutant CD19). Accordingly, also disclosed are methods of providing targeted immunity (e.g., anti-tumor immunity) to a subject with a hematological malignancy involving adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CAR polypeptides.

[0018] In the tumor microenvironment, cancer cells and host immune cells interact, potentially promoting or inhibiting cancer progression. Ideally, the immune system identifies cancer cells and mobilizes an immune response to eliminate cancer. Unfortunately, at the T cell level, upregulation of inhibitory receptors, such as PD-1 and Tim-3, correlates with T cell dysfunction. This is due to the lack of HCV-specific CD8+ receptors in the circulation and liver of patients with chronic hepatitis C virus (HCV) infection. + T cells and HCV non-specific CD8 +T cell proliferation and IFN-γ secretion have been observed in both naive and naive T cells. Partial restoration of T cell proliferation and IFN-γ secretion can be achieved ex vivo by inhibiting the binding of PD-1 and Tim-3 to their respective ligands (i.e., B7-H1 and galectin-9, also known as PD-L1). Furthermore, recent reports have demonstrated that long-term administration of IFN-α, the standard treatment for persistent HCV infection, promoted telomere loss in naive T cells. Given the correlation between T cell telomere shortening and terminal differentiation (characterized by reduced proliferative potential), IFN-α-induced T cell "exhaustion" is likely to be a significant barrier to immunotherapy in HCV-infected patients. In certain embodiments disclosed herein, the present invention employs a checkpoint inhibition strategy. Checkpoint inhibition therapy targets key regulators of the immune system that stimulate or inhibit immune responses. Such immune checkpoints can be exploited in cancer disease states (e.g., by tumors) to evade attack by the immune system. Checkpoint inhibition studies have focused on the activity of PD-1 blockade therapy (El-Khoueiry et al., (2017). "Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1 / 2 dose escalation and expansion trial." Lancet 389 (10088): pp. 2492-2502), and the FDA has approved nivolumab as second-line treatment for HCC with a response rate of 20%.

[0019] definition The term "antibody" refers to immunoglobulins, derivatives thereof that retain specific binding ability, and proteins having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or may be partially or wholly synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class from any species, e.g., the human classes: IgG, IgM, IgA, IgD, and IgE. In an exemplary embodiment, the antibody used with the methods and compositions described herein is a derivative of the IgG class, e.g., clone FMC63, an anti-CD19 antibody. In addition to complete immunoglobulin molecules, chimeras, fragments, or polymers of immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind to target antigens, are also encompassed by the term "antibody."

[0020] The term "antibody fragment" refers to any derivative of an antibody that is less than full-length. In exemplary embodiments, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, Fc, and Fd fragments. Antibody fragments may be produced by any means. For example, antibody fragments may be enzymatically or chemically produced by fragmentation of a whole antibody, recombinantly produced from a gene encoding a partial antibody sequence, or synthetically produced in whole or in part. Optionally, an antibody fragment may be a single-chain antibody fragment. Alternatively, the fragment may comprise multiple chains linked together, for example, by disulfide linkages. Optionally, the fragment may also be a multimolecular complex. Functional antibody fragments typically contain at least about 50 amino acids, more typically at least about 200 amino acids.

[0021] The term "antigen-binding site" refers to the region of an antibody that specifically binds to an epitope of an antigen.

[0022] The term "aptamer" refers to an oligonucleic acid or peptide molecule that binds to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamer adopts a unique tertiary structure and can recognize the target molecule with high affinity and specificity. A "nucleic acid aptamer" is a DNA or RNA oligonucleic acid that binds to a target molecule through its three-dimensional structure, thereby inhibiting or suppressing the function of such a molecule. A nucleic acid aptamer can be composed of DNA, RNA, or a combination thereof. A "peptide aptamer" is a combinatorial protein molecule in which a variable peptide sequence is inserted into a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast two-hybrid conditions, which increases the probability that the selected peptide aptamer will be stably expressed and correctly folded in the cellular context.

[0023] The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or any other characteristic of the compound, or any degradation of the active ingredient, and minimizes any adverse side effects in a subject.

[0024] The term "chimeric molecule" refers to a single molecule created by joining two or more molecules that exist separately in nature. A single chimeric molecule possesses the desired functionality of all of its component molecules. One type of chimeric molecule is a fusion protein.

[0025] The term "engineered antibody" refers to a recombinant molecule comprising at least an antibody fragment comprising an antigen-binding site derived from the variable domain of the antibody heavy and / or light chain, and optionally comprising all or part of the variable and / or constant domains of an antibody from any of the Ig classes (e.g., IgA, IgD, IgE, IgG, IgM, and IgY).

[0026] The term "epitope" refers to the region of an antigen to which an antibody preferentially and specifically binds. A monoclonal antibody preferentially binds to a single, specific epitope of a molecule, which can be molecularly defined. In the present invention, multiple epitopes can be recognized by multispecific antibodies.

[0027] The term "fusion protein" refers to a polypeptide formed by the joining of two or more polypeptides via a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. Fusion proteins can be formed by chemical conjugation of the constituent polypeptides or can be expressed as a single polypeptide from a nucleic acid sequence encoding a single contiguous fusion protein. A single-chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared by joining two genes in frame into a single nucleic acid using conventional techniques in molecular biology, and then expressing the nucleic acid in a suitable host cell under conditions that produce the fusion protein.

[0028] The term "Fab fragment" refers to a fragment of an antibody containing the antigen-binding site produced by cleavage of the antibody with the enzyme papain, which cleaves at the N-terminal side of the intra-H chain disulfide bonds in the hinge region to produce two Fab fragments from one antibody molecule.

[0029] The term "F(ab')2 fragment" refers to a fragment of an antibody containing two antigen-binding sites produced by cleavage of the antibody molecule with the enzyme pepsin, which cleaves at the C-terminal side of the intra-H chain disulfide bond in the hinge region.

[0030] The term "Fc fragment" refers to a fragment of an antibody containing the constant domain of the antibody heavy chain.

[0031] The term "Fv fragment" refers to a fragment of an antibody that contains the variable domains of the antibody's heavy and light chains.

[0032] A "genetic construct" refers to a nucleic acid, e.g., a vector, plasmid, viral genome, etc., that comprises a "coding sequence" for a polypeptide or is otherwise transcribable into biologically active RNA (e.g., antisense, decoy, ribozyme, etc.), that can be transfected into a cell, e.g., a mammalian cell, and that can cause expression of the coding sequence in cells transfected with the construct. A genetic construct may include one or more regulatory elements operably linked to the coding sequence, as well as intron sequences, polyadenylation sites, origins of replication, marker genes, etc.

[0033] The term "linker" is art-recognized and refers to a molecule or group of molecules that connects two compounds, such as two polypeptides. A linker may consist of a single linking molecule or may include a linking molecule and a spacer molecule intended to separate the linking molecule and compounds by a specific distance.

[0034] The term "multivalent antibody" refers to an antibody or engineered antibody that contains two or more antigen recognition sites. For example, a "bivalent" antibody has two antigen recognition sites, and a "tetravalent" antibody has four antigen recognition sites. Terms such as "monospecific," "bispecific," "trispecific," and "tetraspecific" refer to the number of different antigen recognition site specificities (which are different from the number of antigen recognition sites) present in a multivalent antibody. For example, the antigen recognition sites of a "monospecific" antibody all bind to the same epitope. A "bispecific" antibody has at least one antigen recognition site that binds to a first epitope and at least one antigen recognition site that binds to a second epitope that is different from the first epitope. A "multivalent monospecific" antibody has multiple antigen recognition sites that all bind to the same epitope. A "multivalent bispecific" antibody has multiple antigen recognition sites, some of which bind to a first epitope and some of which bind to a second epitope that is different from the first epitope.

[0035] The term "nucleic acid" refers to a natural or synthetic molecule containing a single nucleotide or two or more nucleotides linked by a phosphate group between the 3' position of one nucleotide and the 5' end of another nucleotide. Nucleic acids are not limited by length and therefore can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0036] The term "operably linked to" refers to the functional relationship of a nucleic acid to another nucleic acid sequence. Promoters, enhancers, transcription and translation termination sites, and other signal sequences are examples of nucleic acid sequences that are operably linked to other sequences. For example, operably linking DNA to a transcriptional regulatory element refers to a physical and functional relationship between the DNA and the promoter such that transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0037] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0038] The term "polypeptide fragment" or "fragment," when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted relative to the reference polypeptide itself, but the remaining amino acid sequence is typically identical to that of the reference polypeptide. Such deletions may occur at the amino or carboxy terminus of the reference polypeptide, or both. Fragments are typically at least about 5, 6, 8, or 10 amino acids in length, at least about 14 amino acids in length, at least about 20, 30, 40, or 50 amino acids in length, at least about 75 amino acids in length, or at least about 100, 150, 200, 300, 500, or more amino acids in length. Fragments can retain one or more biological activities of the reference polypeptide. In various embodiments, fragments can include enzymatic activity and / or interaction sites of the reference polypeptide. In other embodiments, fragments can have immunogenic properties.

[0039] The term "single-chain variable fragment" or "scFv" refers to an Fv fragment in which the heavy and light chain domains are linked. One or more scFv fragments may be linked to other antibody fragments (e.g., heavy or light chain constant domains) to form antibody constructs with one or more antigen recognition sites.

[0040] "Spacer," as used herein, refers to a peptide that joins proteins, including fusion proteins. Generally, spacers have no specific biological activity other than joining proteins or preserving some minimum distance or other spatial relationship between the proteins. However, the constituent amino acids of a spacer may be selected to affect certain properties of the molecule, such as molecular folding, net charge, or hydrophobicity.

[0041] As used herein, the terms "specifically bind" or "specific binding," when referring to a polypeptide (including antibodies) or receptor, refer to a binding reaction that determines the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a given ligand or antibody "specifically binds" to its particular "target" (e.g., an antibody specifically binds to an endothelial antigen) if it does not bind in significant amounts to other proteins present in the sample or to other proteins with which the ligand or antibody may come into contact in an organism. Generally, a first molecule that "specifically binds" to a second molecule has a binding affinity of about 10 to the second molecule. 5 M -1 Over (e.g., 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , and 10 12 M -1 For example, in the case of the ability of a TCR to bind a peptide presented on an MHC (e.g., class I MHC or class II MHC), the TCR will typically specifically bind that peptide / MHC with an affinity of at least about 10-4 M or less, KD, and bind a given antigen / binding partner with an affinity (represented by KD) that is at least 10-, 100-, or 1000-fold less than the affinity for binding to a nonspecific and unrelated peptide / MHC complex (e.g., a complex containing BSA peptide or casein peptide).

[0042] The term "subject" refers to any individual who is the target of administration or treatment. A subject can be a vertebrate, e.g., a mammal. Thus, a subject can be a human or veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0043] In certain embodiments, the agents of the present invention may be used alone or in combination with another type of therapeutic agent. As used herein, the phrase "co-administration" or "co-administered" refers to any form of administration of two or more different therapeutic agents (e.g., a composition comprising a CAR T disclosed herein and an immune checkpoint inhibitor) such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., the two agents are effective in the subject simultaneously, which may include a synergistic effect of the two agents). For example, the different therapeutic agents can be administered simultaneously or sequentially in the same formulation or in separate formulations. In some preferred embodiments, the CAR T cells express (e.g., are present on the cell surface or secrete) the additional therapeutic agent. In certain embodiments, the different therapeutic agents (e.g., CAR T cells and an immune checkpoint blockade molecule) can be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 1 week of each other. Thus, a subject receiving such treatment can benefit from the combined effects of the different therapeutic agents.

[0044] The terms "transformation" and "transfection" refer to the introduction of a nucleic acid, eg, an expression vector, into a recipient cell, eg, the introduction of a nucleic acid into the chromosomal DNA of said cell.

[0045] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of a treated individual's clinical condition during its course. Desirable effects of treatment include slowing the rate of progression, improving or alleviating the pathological condition, and remission or improved prognosis of a particular disease, disorder, or condition. For example, an individual is successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are reduced or eliminated.

[0046] The term "variant" refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (e.g., degenerate variants), substitutions within the wobble positions of each codon encoding an amino acid (e.g., DNA and RNA), amino acids added to the C-terminus of a peptide, or peptides with 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.

[0047] The term "vector" refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which it has been linked. The term "expression vector" includes any vector (e.g., a plasmid, cosmid, or phage chromosome) that contains a genetic construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).

[0048] The terms "B lymphocyte antigen," and particularly "CD19," are intended to include fragments, variants (e.g., allelic variants), and derivatives of antigenic molecules, e.g., CD19 molecules. For example, without limitation, in some embodiments, CD19 is wild-type CD19 or a mutant CD19. In some such embodiments, the B lymphocyte antigen is expressed on the cell surface (e.g., on the surface of a precancerous or malignant cell).

[0049] Suitable anti-CD19 antibodies (and scFv formats thereof) for binding to CD19 are known in the art and include, for example, but are not limited to, antibodies FMC63, SJ25C1 (JCAR015), and HD37 (blinatumomab).

[0050] Chimeric antigen receptor (CAR) Disclosed herein are chimeric antigen receptor (CAR) polypeptides that can be expressed in immune effector cells to enhance activity against specific targets (e.g., anti-tumor activity against hematological cancers).

[0051] In some aspects, the CARs disclosed herein are composed of three domains: an ectodomain, a transmembrane domain, and an endodomain.

[0052] In certain embodiments, the ectodomain comprises a B lymphocyte antigen-binding region, such as a CD19-binding region, and is responsible for antigen recognition. CD19 may be wild-type CD19 or a mutant CD19. Optionally, the ectodomain also contains a signal peptide (SP) so that the CAR can be glycosylated and anchored to the cell membrane of immune effector cells.

[0053] In some embodiments, the transmembrane domain (TD) connects the ectodomain (i.e., extracellular domain) to the endodomain (i.e., intracellular domain) and resides within the cell membrane when expressed by a cell.

[0054] In some embodiments, the endodomain transmits an activation signal to immune effector cells after antigen recognition. In some such embodiments, the endodomain can contain an intracellular signaling domain (ISD) and, optionally, a costimulatory signaling region (CSR). A "signaling domain (SD)," e.g., an ISD, generally contains an immunoreceptor tyrosine-based activation motif (ITAM), which activates a signaling cascade when phosphorylated. The term "costimulatory signaling region (CSR)" refers to the intracellular signaling domain from costimulatory protein receptors, e.g., CD28, 41BB, and ICOS, which can enhance T cell activation by the T cell receptor.

[0055] In some embodiments, the endodomain contains either the SD or the CSR, but not both. In these embodiments, immune effector cells containing the disclosed CARs are activated only if another CAR (or T cell receptor) containing the missing domain also binds to the respective antigen.

[0056] In some embodiments, the disclosed CARs have the formula: SP-BCA-HG-TM-CSR-SD, or SP-BCA-HG-TM-SD-CSR where "SP" represents an optional signal peptide (e.g., from the CD8α leader sequence); "BCA" stands for B lymphocyte antigen binding domain (e.g., FMC63 and its derivatives); "HG" represents an optional hinge domain (spacer domain; e.g., from CD28); "TM" represents the transmembrane domain (e.g., from CD28); "CSR" refers to one or more costimulatory signaling domains (e.g., from CD28); "SD" represents the signaling domain (e.g., from CD3ζ and its variants); "-" represents a peptide bond or a linker) is defined by

[0057] Additional CAR constructs are described, for example, in Fresnak et al., Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer. 2016 Aug 23;16(9):566-81, which is incorporated by reference in its entirety for the teachings of these CAR models.

[0058] In certain embodiments, the CAR may be, for example (but not limited to), a TRUCK, a general-purpose CAR, a self-driving CAR, an armored CAR, a self-destructing CAR, a conditional CAR, a tagged CAR, a TenCAR, a dual CAR, or an sCAR.

[0059] TRUCK (T cells redirected for universal cytokine killing) co-express a chimeric antigen receptor (CAR) and an anti-tumor cytokine. Cytokine expression can be constitutive or induced by T cell activation. When targeted by CAR specificity, the localized production of pro-inflammatory cytokines can recruit endogenous immune cells to the tumor site, enhancing the anti-tumor response.

[0060] Universal allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0061] Self-driving CARs co-express CAR and chemokine receptors and bind to tumor ligands, thereby enhancing tumor homing.

[0062] CAR T cells engineered to be resistant to immunosuppression (armored CARs) may be genetically modified so that they no longer express various immune checkpoint molecules (e.g., cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD-1)). Exemplary "knockdown" and "knockout" techniques include, but are not limited to, RNA interference (RNAi) (e.g., asRNA, miRNA, shRNA, siRNA, etc.) and CRISPR interference (CRISPRi) (e.g., CRISPR-Cas9). In certain embodiments, CAR T cells are engineered to express a dominant-negative form of a checkpoint molecule. In some such embodiments, the extracellular ligand-binding domain (i.e., ectodomain) of the immune checkpoint molecule is fused to a transmembrane domain to compete for ligand binding. For example, the extracellular ligand-binding domain of PD-1 is fused to a CD8 transmembrane domain and can therefore compete for PD-1 ligand from target cells. In some embodiments, CAR T cells are engineered to express an immune checkpoint switch receptor that exploits inhibitory immune checkpoint ligands present on target cells. In such embodiments, the extracellular ligand-binding domain of the immune checkpoint molecule is fused to a signaling, stimulatory, and / or costimulatory domain. For example, the extracellular ligand-binding domain of PD-1 is fused to a CD28 domain, thus blocking PD-1 signaling while simultaneously achieving CD28 costimulation. In further embodiments, CAR T cells may be administered with an aptamer or monoclonal antibody that blocks immune checkpoint signaling. In some such embodiments, CAR T cells (e.g., CAR T cell therapy) are combined with PD-1 blockade, e.g., administration with a PD-1 / PD-L1 antagonist aptamer or an anti-PD-1 / PD-L1 antibody. In a preferred embodiment, CAR T cells and a PD-1 pathway-blocking antibody are administered concomitantly. In a further embodiment, the CAR T cells are engineered to express or express and secrete an immune checkpoint-blocking antibody, e.g., anti-PD-1 or anti-PD-L1, or a fragment thereof. In a further embodiment, the CAR T cells are engineered to express or express and secrete an immune checkpoint-blocking antibody, e.g., anti-PD-1 or anti-PD-L1, or a fragment thereof, as described herein. The vaccine is administered in conjunction with a vector (e.g., an engineered virus) expressing an immune checkpoint blockade molecule.

[0063] Self-destructing CARs can be designed using RNA encoding the CAR delivered by electroporation. Alternatively, inducible apoptosis of T cells can be achieved based on the activation of human caspase-9 by ganciclovir, which binds to thymidine kinase in genetically modified lymphocytes, or by the more recently described small molecule dimerizer.

[0064] Conditional CAR T cells are initially refractory or switched "off" until the CAR T cells are activated by the addition of a small molecule that completes the "circuit" (e.g., molecular pathway) that allows full transduction of both signal 1 and signal 2. Alternatively, T cells can be engineered to express an adaptor-specific receptor with affinity for a subsequently administered secondary antibody directed against the target antigen.

[0065] The tagged CAR T cells express both the CAR and the tumor epitope that existing monoclonal antibody agents bind to. In the setting of intolerable adverse effects, administration of a monoclonal antibody removes the CAR T cells, providing symptomatic relief without additional off-tumor effects.

[0066] Tandem CAR (TanCAR) T cells express a single CAR consisting of two linked single-chain variable fragments (scFv) with different affinities fused to an intracellular costimulatory domain and a CD3ζ domain. TanCAR T cell activation is achieved only when target cells co-express both targets.

[0067] Dual CAR T cells express two separate CARs with different ligand binding targets.For example, one CAR can only comprise the CD3ζ domain, and the other CAR can only comprise the costimulatory domain.In some such embodiments, dual CAR T cells are activated when both targets are expressed in tumors.

[0068] Safety CARs (sCARs) consist of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR are activated only when they encounter target cells bearing the standard CAR target but lacking the sCAR target.

[0069] In some embodiments, the antigen recognition domain of the disclosed CARs is an scFv. In further embodiments, the antigen recognition domain is derived from a natural T cell receptor (TCR) α and β single chain as described herein. Preferably, such an antigen recognition domain has a simple ectodomain (e.g., a CD4 ectodomain that recognizes HIV-infected cells). Alternatively, such an antigen recognition domain includes an exogenous recognition moiety, such as a linked cytokine (which can result in recognition of cells bearing cytokine receptors). Generally, for the methods disclosed herein, almost anything that binds with high affinity to a given target can be used as an antigen recognition region.

[0070] The intracellular domain transmits a signal to the CAR-expressing immune effector cell after antigen recognition, activating at least one of the normal effector functions of the immune effector cell. In certain embodiments, the effector function of the T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the intracellular domain can include the "intracellular signaling domain" of the T cell receptor (TCR) and an optional co-receptor. Typically, the entire intracellular signaling domain can be used, although in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the complete chain, so long as it transduces an effector function signal.

[0071] Cytoplasmic signaling sequences that regulate and stimulate primary activation of the TCR complex may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM-containing cytoplasmic signaling sequences include those derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (FcγRIIA), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG).

[0072] In certain embodiments, the intracellular signaling domain is derived from CD3 zeta (CD3ζ, e.g., TCR zeta; GenBank accession number BAG36664.1). The T cell surface glycoprotein CD3ζ chain, also known as the T cell receptor T3 zeta chain or CD247 (cluster of differentiation 247), is a protein encoded by the CD247 gene in humans. The intracellular tail of the CD3 molecule contains a single ITAM that is essential for the signaling ability of the TCR. The intracellular tail of the ζ chain (CD3ζ) contains three ITAMs. In some embodiments, the ζ chain is a mutant ζ chain. For example, a mutant ζ chain contains a mutation, e.g., a point mutation, in at least one ITAM that renders the ITAM nonfunctional. In some such embodiments, the membrane-proximal ITAM (ITAM1), the membrane-distal ITAM (the third ITAM at the C-terminus, ITAM3), or both are nonfunctional. In further embodiments, two membrane-proximal ITAMs (ITAM1 and ITAM2) or two membrane-distal ITAMs (ITAM2 and ITAM3) are non-functional. In further embodiments, only ITAM2 is non-functional. In some embodiments, the mutant ζ chain comprises a deletion (e.g., truncation) mutation such that at least one ITAM is missing. In some such embodiments, the ζ chain lacks the membrane-proximal ITAM (ITAM1), the membrane-distal ITAM (ITAM3), or both. In other embodiments, the ζ chain lacks two membrane-proximal ITAMs (ITAM1 and ITAM2) or two membrane-distal ITAMs (ITAM2 and ITAM3). In further embodiments, the ζ chain lacks ITAM2. Methods for producing mutant CD3ζ are known to those of skill in the art (Bridgeman JS et al., Clin Exp Immunol. 2014 Feb;175(2):258-67). Removing at least one ITAM from an introduced CAR can reduce CD3ζ-mediated apoptosis. Alternatively, removing at least one ITAM from an introduced CAR can reduce the size of the CAR without loss of function. CARs containing such altered CD3ζ domains are contemplated by the present invention.

[0073] Also contemplated are CARs that contain altered CD28 domains that confer unique functional properties to the CAR. In this regard, the native CD28 domain contains three intracellular subdomains consisting of the amino acid sequences YMNM, PRRP, and PYAP, which control signaling pathways following stimulation (see, e.g., International Publication No. 2019 / 010383, the teachings of which are incorporated herein by reference). The CAR constructs described herein may contain modified CD28 domains in which one or more of the YMNM, PRRP, and / or PYAP subdomains have been mutated or deleted to amplify, attenuate, or inactivate said subdomains, thereby modulating CAR-T function.

[0074] First-generation CARs typically contained an intracellular domain derived from the CD3ζ chain, the primary transmitter of signals from endogenous TCRs. Second-generation CARs added intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the CAR's internal domain to provide additional signals to T cells. For example, target-specific ScFvs were fused with the extracellular, transmembrane, and intracellular signaling domains of the costimulatory receptor CD28 and the cytoplasmic signaling domain of the T cell receptor-associated CD3ζ chain. Preclinical studies have shown that second-generation CAR designs improve the antitumor activity of T cells. More recent third-generation CARs combine multiple signaling domains to further enhance efficacy. T cells implanted with these CARs have demonstrated improved expansion, activation, persistence, and tumor eradication efficiency independent of costimulatory receptor / ligand interactions (Imai C et al., Leukemia 2004 18:676-84; Maher J et al., Nat Biotechnol 2002 20:70-5).

[0075] For example, the endodomain of a CAR can be designed to contain a CD3ζ signaling domain alone or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of a CAR can contain a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for efficient lymphocyte response to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, NKG2D, and variants thereof. Thus, although CARs are primarily exemplified with CD28 as the costimulatory signaling element, other costimulatory elements may be used alone or in combination with other costimulatory signaling elements.

[0076] In some embodiments, the CAR comprises a hinge sequence. A hinge sequence is a short amino acid sequence that increases antibody flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence may be located between the antigen recognition portion (e.g., anti-CD19, anti-CD20, anti-CD22, or scFv) and the transmembrane domain. The hinge sequence may be any suitable sequence derived from or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.

[0077] The transmembrane domain may be derived from natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from at least the alpha (α), beta (β), or zeta (ζ) chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7 , NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VL A-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, C D29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), C The transmembrane domain may be derived from (i.e., comprise) the transmembrane region of EACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​and PAG / Cbp. Alternatively, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues, such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. A short, e.g., 2-10 amino acid long, oligo- or polypeptide linker may form the link between the transmembrane domain and the endoplasmic domain of the CAR.

[0078] Thus, in a preferred embodiment of the invention disclosed herein, the CAR has the formula: SP-BCA-HG-TM-CSR-SD wherein the optional signal peptide / leader sequence is derived from the CD8α leader sequence; The B lymphocyte antigen-binding region is an scFv derived from the anti-CD19 antibody clone FMC63, the hinge domain is derived from human CD28 (e.g., SEQ ID NO: 12); the transmembrane domain is derived from human CD28 (e.g., SEQ ID NO: 13); the costimulatory signaling region is derived from human CD28 (e.g., SEQ ID NO: 14); The signaling domain is defined by the CD3 ζ chain (e.g., SEQ ID NO: 11), in which only the membrane-proximal ITAM (ITAM1) is functional. Optionally, the CAR may further comprise at least one molecular tag known in the art. For example, but not limited to, the CAR may be coupled to a labeled ligand, e.g., 124 The low affinity nerve growth factor receptor (LNGFR) may be included as a tag that binds to I-NGF, and such interactions, e.g. 124 I-NGF / LNGFR can preferably be monitored non-invasively (eg, by positron emission tomography).

[0079] In some embodiments, the CAR has two or more transmembrane domains, which may be repeats of the same transmembrane domain or different transmembrane domains.

[0080] In some embodiments, the CAR is a multi-chain CAR as described in International Publication No. 2015 / 039523, the teachings of which are incorporated by reference. A multi-chain CAR can comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble in a juxtamembrane position, forming a flexible structure that more closely resembles the natural receptor and provides optimal signaling. For example, a multi-chain CAR can comprise a portion of the FcεRIα chain and a portion of the FcεRIβ chain, such that these FcεRI chains naturally dimerize together to form a CAR.

[0081] In some embodiments, the CAR contains one signaling domain. In other embodiments, the CAR contains one or more signaling domains (co-stimulatory signaling domains). The one or more signaling domains can be a polypeptide selected from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, FcγRI-γ, FcγRIII-γ, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137(41BB), ICOS, CD27, CD28δ, CD80, NKp30, OX40, and variants thereof.

[0082] Tables 1, 2, and 3 below provide some exemplary combinations of target binding domains, costimulatory signaling domains, and intracellular signaling domains. Such examples are for illustrative purposes and are not intended to be an exhaustive list of combinations that may occur in the CARs disclosed herein.

[0083] [Table 1]

[0084] [Table 2-1] [Table 2-2]

Table 2-3

Table 2-4

[0085]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

Table 3-21

Table 3-22

Table 3-23

Table 3-24

Table 3-25

Table 3-26

Table 3-27

Table 3-28

Table 3-29

Table 3-30

Table 3-31

Table 3-32

Table 3-33

Table 3-34

Table 3-35

Table 3-36

Table 3-37

Table 3-38

Table 3-39

Table 3-40

Table 3-41

Table 3-42

Table 3-43

Table 3-44

Table 3-45

Table 3-46

Table 3-47

Table 3-48

Table 3-49

Table 3-50

Table 3-51

Table 3-52

Table 3-53

Table 3-54

Table 3-55

Table 3-56

Table 3-57

Table 3-58

Table 3-59

Table 3-60

Table 3-61

Table 3-62

Table 3-63

Table 3-64

Table 3-65

Table 3-66

Table 3-67

Table 3-68

Table 3-69

Table 3-70

Table 3-71

Table 3-72

Table 3-73

Table 3-74

Table 3-75

Table 3-76

Table 3-77

Table 3-78

Table 3-79

Table 3-80

Table 3-81

Table 3-82

Table 3-83

Table 3-84

Table 3-85

Table 3-86

Table 3-87

[0086]

Table 4

[0087]

Table 5-1

Table 5-2

[0088]

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

[0089] In some embodiments, the anti-B lymphocyte antigen binding agent is a single-chain variable fragment (scFv) antibody. Preferably, such an anti-B lymphocyte antigen binding agent is a single-chain variable fragment (scFv) anti-CD19 antibody. The affinity / specificity of the anti-CD19 scFv is largely determined by the variable fragment (V). H ) and light (V L ) strands are driven by specific sequences within the complementarity-determining regions (CDRs). H and V L The sequence has three CDRs (CDR1, CDR2, CDR3).

[0090] In some embodiments, the anti-CD19 binding agent is derived from a natural antibody, such as a monoclonal antibody. Optionally, the antibody is a human antibody. Optionally, the antibody has been modified to make it less immunogenic when administered to humans. For example, the modification includes one or more techniques selected from chimerization, humanization, CDR grafting, deimmunization, and mutation of framework amino acids corresponding to the most similar human germline sequence. Preferably, the antibody is FMC63.

[0091] In a preferred embodiment, the anti-CD19 binding agent is a single-chain variable fragment (scFv) antibody derived from the antibody FMC63.

[0092] Also disclosed are bispecific CARs that target an anti-B lymphocyte antigen, such as CD19, and at least one additional cancer-associated antigen (e.g., a tumor antigen). Also disclosed are CARs designed to work only in combination with another CAR that binds a different antigen, e.g., another cancer-associated antigen. For example, in these embodiments, the internal domain of the disclosed CAR can contain only a signaling domain (SD) or a costimulatory signaling region (CSR), but not both. The second CAR (or endogenous T cells) provides the missing signal when activated. For example, if a disclosed CAR contains an SD but not a CSR, immune effector cells containing this CAR will only be activated when another CAR (or T cells) containing a CSR binds to the respective antigen. Similarly, if a disclosed CAR contains a CSR but not an SD, immune effector cells containing this CAR will only be activated when another CAR (or T cells) containing an SD binds to the respective antigen.

[0093] The tumor antigen includes a protein produced by a tumor cell that induces an immune response, particularly a T-cell mediated immune response. The additional antigen-binding domain can be an antibody or a natural ligand of the tumor antigen. The selection of the additional antigen-binding domain will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, α-fetoprotein (AFP), ALK, alternative and / or specific CD19 epitopes, TIM3, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RUL, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAX5, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human telomerase reverse transcriptase, polysialic acid (physial acid), PLAC1, RUL, RU2(AS), intestinal carboxylesterase, Lewis Y, sLE, LY6K, HSP70, HSP27, mutated hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-1a, LMP2, NCAM, p53, p53 mutants, Ras mutants, gplOO, prostein, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-β, survivin and telomerase, legumain, HPV E6,E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, prostate cancer tumor antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAGE-A2, MAGE-C1, MAGE-C2, annexin-A2, MAD-CT-1, MAD-CT2, melanA / MART1, XAGE1, ELF2M, ERG (TMPRSS2) ETS fusion gene), NA17, neutrophil elastase, sarcoma translocation breakpoint, NY-BR-1, ephnnB2, CD20, CD22, CD24, CD30, TIM3, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGF II, IGF-I receptor, GD2, o-acetyl GD2, GD3, GM3, GPRC5D, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie2, TEM1, TEM7R, CLDN6, TSHR, UPK2, and mesothelin. In certain preferred embodiments, the tumor antigen is selected from folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, TIM3, BCMA, GD2, CLL-1, CA-IX, MUCl, HER2, and any combination thereof.

[0094] Further non-limiting examples of tumor antigens include differentiation antigens such as tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include SCCA, GP73, FC-GP73, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23H1, PSA, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, and 5T4. , 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, CA-50, CAM43, CD68\ P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCASl, SDCCAG1 6, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, TAG-72, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.

[0095] CAR ligand-binding domain The extracellular domain of a CAR disclosed herein generally comprises an antigen-recognition domain that binds to a target antigen. Such antigen-specific binding domains are typically derived from antibodies. In some embodiments, the antigen-binding domain is a functional antibody fragment or derivative thereof (e.g., an scFv or Fab, or any suitable antigen-binding fragment of an antibody). In preferred embodiments, the antigen-binding domain is a single-chain variable fragment (scFv). In some such embodiments, the scFv is derived from a monoclonal antibody (mAb). In certain preferred embodiments, the antigen-specific binding domain (e.g., an scFv) is fused to a transmembrane and / or signaling motif involved in lymphocyte activation, as disclosed in Sadelain et al., Nat Rev Cancer 2003 3:35-45, incorporated herein by reference in its entirety.

[0096] Anti-B lymphocyte antigen scFv In some embodiments, the anti-B lymphocyte antigen scFv comprises a heavy chain variable (V) having CDR1, CDR2, and CDR3 sequences. H ) domain, and a light chain variable (V) domain with CDR1, CDR2, and CDR3 sequences. L ) domain. In a preferred embodiment, the anti-B lymphocyte antigen scFv is an anti-CD19, anti-CD20, or anti-CD22 scFv. Most preferably, the scFv is an anti-CD19 scFv.

[0097] Some such antibodies are described, for example, in Zola et al., "Preparation and characterization of a chimeric CD19 monoclonal antibody," Immunology and Cell Biology 1991, 69, pp. 411-422, and Nicholson et al., "Construction and Characterization of a Functional CD19 Specific Single Chain Fv Fragment For Immunotherapy of B Lineage Leukemia and Lymphoma," Molecular Immunology 1997, (34) 16-17, pp. 1147-1165. These publications are incorporated herein by reference in their entirety, particularly with respect to the antibodies described therein.

[0098] Nucleic acids and vectors Also disclosed are polynucleotides and polynucleotide vectors encoding the disclosed B lymphocyte antigen-specific CARs that enable expression of the B lymphocyte antigen-specific CARs in the disclosed immune effector cells.

[0099] Nucleic acid sequences encoding the disclosed CARs and regions thereof can be obtained using recombinant methods known in the art, such as by screening libraries from cells expressing the gene, obtaining the gene from a vector known to contain it, or isolating it directly from cells and tissues containing it, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned. Exemplary nucleic acid sequences can encode CARs comprising each of a CD8 leader sequence, an FMC63 scFv targeting domain, a CD28 domain, a mutant CD3ζ domain (e.g., a CD3ζ domain lacking functionality in two C-terminal ITAM domains, i.e., ITAM2 and ITAM3), or any combination thereof. Preferably, such a CAR can be encoded by a nucleic acid sequence comprising the sequence set forth in SEQ ID NO: 1.

[0100] JPEG2025028959000104.jpg109170JPEG2025028959000105.jpg235170JPEG2025028959000106.jpg18170

[0101] Expression of a nucleic acid encoding a CAR is typically achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the desired nucleic acid sequence.

[0102] The disclosed nucleic acids can be cloned into several types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0103] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selection markers. In some embodiments, the polynucleotide vector is a lentivirus or retrovirus vector.

[0104] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector using techniques known in the art and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to cells of interest in vivo or ex vivo.

[0105] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences, such as, but not limited to, the simian virus 40 (SV40) early promoter, the MND (myeloproliferative sarcoma virus) promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate-early promoter, the Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter, can also be used. Alternatively, the promoter can be an inducible promoter. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0106] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although in recent years, some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved even when elements are inverted or moved relative to one another.

[0107] To assess the expression of a CAR polypeptide or portion thereof, the expression vector introduced into cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells attempted to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes.

[0108] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, but encodes a polypeptide whose expression is indicated by some easily detectable property, such as enzymatic activity, fluorescence, or specific binding to a detectable ligand. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene. For example, in some embodiments, the CAR disclosed herein and / or the nucleic acid sequence encoding the CAR may further comprise at least one molecular tag known in the art. Without wishing to be bound by any one particular theory or strategy, the tag may be used to identify a labeled ligand. 124 The low affinity nerve growth factor receptor (LNGFR) may be included as a transduction tag that binds I-NGF. 124The I-NGF / LNGFR interaction can be monitored noninvasively (e.g., by positron emission tomography). In some such embodiments, the nucleic acid sequence of the vector encoding the CAR described herein can include a nucleic acid sequence encoding a ribosomal skip sequence, such as a sequence encoding a 2A peptide. The 2A peptide, identified in the picornavirus foot-and-mouth disease virus subgroup, causes a ribosomal "skip" from one codon to the next, preventing the formation of a peptide bond between the two amino acids encoded by those codons. Thus, two polypeptides can be synthesized from a single, continuous open reading frame within an mRNA when separated by an in-frame 2A oligopeptide sequence (e.g., when the CAR and a reporter gene, such as a molecular tag, are separated by a 2A oligopeptide sequence). Such ribosomal "skip" or "self-cleavage" mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.

[0109] A secretory signal sequence (also known as a signal sequence, leader sequence, prepro sequence, or pre sequence) is provided in a polynucleotide or vector sequence to direct a polypeptide (e.g., a secreted, transmembrane, and / or cell surface polypeptide) into the secretory pathway of a host cell. The secretory signal sequence is operably linked to a nucleic acid sequence encoding a polypeptide of interest (e.g., a CAR). The two sequences are joined in the correct reading frame and are positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Such secretory signal sequences are typically located 5' to the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences may be located elsewhere in the nucleic acid sequence of interest. Thus, in preferred embodiments, the CAR disclosed herein and / or the nucleic acid sequence encoding said CAR may further comprise a signal sequence. Most preferably, the signal sequence comprises a CD8α leader sequence or a fragment thereof. It will be understood by those skilled in the art that such leader sequences can be removed from a CAR polypeptide displayed on the cell surface (i.e., a mature CAR polypeptide) by post-translational modification.

[0110] Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, a construct with the smallest 5'-flanking region that exhibits the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be linked to a reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription. Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, the expression vector can be transferred into host cells by physical, chemical, or biological means.

[0111] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle gun technology, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0112] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells.

[0113] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0114] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or be "collapsed." Alternatively, they may simply be dispersed in the solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include lipid droplets naturally occurring in the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.).

[0115] Immune effector cells Also disclosed are immune effector cells (also referred to herein as "CAR-T cells") engineered to express the disclosed CARs. These cells are preferably obtained from the subject to be treated (i.e., autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood drawn from a subject using many techniques known to those skilled in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood can be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. Specific subpopulations of immune effector cells can be further isolated by positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the cells undergoing positive selection, e.g., by incubation with antibody-conjugated beads for a time sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cell populations can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the cells undergoing negative selection.

[0116] In some embodiments, immune effector cells include any white blood cells involved in defending the body against infectious diseases and foreign substances. For example, immune effector cells can include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells can include T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).

[0117] T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on their surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with distinct functions.

[0118] Helper T cells (T H Helper T cells (HTCs) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. Helper T cells express the CD4 glycoprotein on their surface and are therefore CD4 + Helper T cells are also known as T cells. Helper T cells become activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Upon activation, helper T cells divide rapidly and secrete small proteins called cytokines that control or support active immune responses. Helper T cells are classified into T cells that secrete different cytokines to facilitate different types of immune responses. H 1. T H 2. T H 3. T H 17, T H 9 or T FH They can be differentiated into one of several subtypes, including:

[0119] Cytotoxic T cells (T C Cytotoxic T cells (CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. Cytotoxic T cells express the CD8 glycoprotein on their surface, and are therefore CD8 + Also known as T cells, these cells recognize targets by binding antigens in association with MHC class I molecules, which are present on the surface of all nucleated cells. They also recognize CD8 T cells through IL-10, adenosine, and other molecules secreted by regulatory T cells. + Cells can be inactivated to an anergic state, thereby preventing autoimmune disease.

[0120] Memory T cells are a subset of antigen-specific T cells that persist long after the infection has resolved. Upon re-exposure to the cognate antigen, memory T cells rapidly expand into large numbers of effector T cells, thus providing the immune system with a "memory" of the past infection. Memory cells are CD4 + or CD8 + Memory T cells typically express the cell surface protein CD45RO.

[0121] Regulatory T cells (T cells), formerly known as suppressor T cells, reg CD4 T cells are crucial for maintaining immunological tolerance. Their main role is to shut down T cell-mediated immunity at the end of an immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus. + T reg Two main classes of cells - naturally occurring T reg Cellular and adaptive T reg Cells - have been described.

[0122] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) act as a bridge between the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0123] In some embodiments, the T cells are CD4 + In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T cells are enriched for cytotoxic CD8 + In some embodiments, the T cells include γδ T cells, which have distinct T cell receptors (TCRs) with one γ chain and one δ chain instead of α and β chains.

[0124] Natural killer (NK) cells express CD56, which can kill virally infected and transformed cells. + CD3 - Large granular lymphocytes constitute a crucial cell subset of the innate immune system (Godfrey J et al., Leuk Lymphoma 2012 53:1666-1676). Cytotoxic CD8 + Unlike T lymphocytes, NK cells can initiate cytotoxicity against tumor cells without prior sensitization and can also eradicate MHC-I negative cells (Narni-Mancinelli E et al., Int Immunol 2011 23:427-431). NK cells are safer effector cells because they can avoid potentially fatal complications related to cytokine storm (Morgan RA et al., Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL et al., N Engl J Med 2011 365:725-733), and on-target and off-tumor effects. NK cells have a well-known role as cancer cell killers, and it is well documented that impairment of NK cells is crucial to the progression of multiple myeloma (MM) (Godfrey J et al., Leuk Lymphoma 2012 53:1666-1676; Fauriat C et al., Leukemia 2006 20:732-733), but means by which NK cell-mediated anti-MM activity could be enhanced had not been fully explored prior to the disclosed CARs.

[0125] Epstein-Barr virus (EBV)-induced lymphoproliferative disorders (EBV-LPD) and other EBV-associated cancers are significant causes of morbidity and mortality in recipients of allogeneic hematopoietic cell transplants (HCT) or solid organ transplants (SOT), particularly those who have received certain T cell-reactive Abs to prevent or treat graft-versus-host disease (GVHD). Prevention and treatment by adoptive transfer of autologous or allogeneic EBV-specific cytotoxic T cells and subsequent long-term restoration of immunity to EBV-associated lymphoproliferation have resulted in good outcomes in managing these uniformly fatal complications of allogeneic tissue transfer. Thus, in some embodiments, the disclosed immune effector cells comprising one or more CAR polypeptides of the invention are allogeneic or autologous EBV-specific cytotoxic T lymphocytes (CTLs). For example, generation of EBV-specific cytotoxic T cells can involve isolating PBMCs from EBV-seropositive autologous or allogeneic donors and enriching them for T cells by depletion of monocytes and NK cells. EBV-specific cytotoxic T cells can also be generated by contacting donor PBMCs or purified donor T cells with "stimulator" cells that express one or more EBV antigens and present the EBV antigens to unstimulated T cells, thereby stimulating and expanding EBV-specific CTLs. EBV antigens include, for example, latent membrane proteins (LMPs) and EBV nuclear antigen (EBNA) proteins, such as LMP-1, LMP-2A, and LMP-2B, as well as EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-LP. Cytotoxic T cells containing T cell receptors that recognize one or more EBV-specific antigens are considered to be "sensitized" to the EBV antigens and are therefore referred to herein as "EBV-sensitized cytotoxic T cells."Known methods for generating allogeneic or autologous EBV-specific cytotoxic T cell populations that may comprise one or more CAR polypeptides of the invention are described, for example, in Barker et al., Blood 2010 116(23):5045-49; Doubrovina et al., Blood 2012 119(11):2644-56; Koehne et al., Blood 2002 99(5):1730-40; and Smith et al., Cancer Res 2012 72(5):1116-25, which are incorporated by reference for their teachings. Similarly, cytotoxic T cells may be "sensitized" to other viral antigens, including cytomegalovirus (CMV), papillomavirus (e.g., HPV), adenovirus, polyomavirus (e.g., BKV, JCV, and Merkel cell virus), retrovirus (e.g., lentivirus, e.g., HTLV-I, also including HIV), picornavirus (e.g., hepatitis A virus), hepadnavirus (e.g., hepatitis B virus), hepacivirus (e.g., hepatitis C virus), deltavirus (e.g., hepatitis D virus), hepevirus (e.g., hepatitis E virus), and the like. In some preferred embodiments, the target antigen is derived from an oncovirus. In some such embodiments, the T cells used to generate the CAR-T cells of the invention are multifunctional T cells, i.e., T cells capable of inducing multiple immune effector functions, thereby achieving a more effective immune response against a pathogen than cells that produce only a single immune effector (e.g., a single biomarker, e.g., a cytokine or CD107a). Less functional, monofunctional, or even "exhausted" T cells can dominate the immune response during chronic infection, thus negatively impacting protection from virus-related complications. In a further preferred embodiment, the CAR-T cells of the invention are multifunctional. In certain embodiments, at least 50% of the T cells used to generate the CAR-T cells of the invention are CD4+. + In some such embodiments, the T cells are less than 50% CD4 + In a further embodiment, the T cells are predominantly CD4 +In some embodiments, at least 50% of the T cells used to generate the CAR-T cells of the present invention are CD8 + In some such embodiments, the T cells are less than 50% CD8 + In a further embodiment, the T cells are predominantly CD8 + In some embodiments, the T cells (e.g., primed T cells and / or CAR-T cells described herein) are stored in a cell library or bank before being administered to a subject. The methods disclosed herein (e.g., selection and / or preparation of immune effector cells disclosed herein, including CAR-T cells of the invention) can be performed using allogeneic immune effector cells (e.g., PBMCs, CD4 T cells, or CD4+ T cells) from a cell bank, e.g., a pre-generated third-party donor-derived cell bank. + T cells, CD8 + T cells, and / or CAR-T cells). Such a cell bank may comprise a donor PBMC sample. Preferably, the cell bank comprises a donor sample that is enriched for immune effector cells. Most preferably, the bank comprises donor CD4 + and / or CD8 + In some such embodiments, the donor-derived cell bank comprises antigen-specific immune effector cells (e.g., primed T cells and / or CAR-T cells described herein). In preferred embodiments, the HLA type of the donor-derived cells described herein is known. Thus, the methods disclosed herein further include selecting allogeneic immune effector cells (e.g., T cells and / or CAR-T cells described herein), because the allogeneic immune effector cells express TCRs restricted to class I MHC encoded by HLA alleles present in the subject. For example, allogeneic T cells (e.g., CD4 T cells and / or CAR-T cells described herein) can be selected. + T cells, CD8 +Pre-generated third-party donor-derived epitope-specific T cells (i.e., allogeneic T cells and / or CAR-T cells) are selected if the T cells and the recipient (e.g., the subject in need of treatment) share at least two (e.g., at least three, at least four, at least five, at least six) HLA alleles, and the cells are restricted by the shared HLA alleles. Preferably, such methods include testing the TCR repertoire of the pre-generated third-party donor-derived epitope-specific T cells (i.e., allogeneic T cells and / or CAR-T cells) by means known in the art, such as flow cytometry, tetramer assays, ELISA assays, Western blot assays, fluorescence microscopy, Edman degradation assays, and / or mass spectrometry assays (e.g., protein sequencing). In some embodiments, the TCR repertoire is analyzed using nucleic acid probes, nucleic acid amplification assays, and / or sequencing assays.

[0126] In some embodiments, engineered CAR-T cells expressing the disclosed CARs further express a dominant-negative mutation that results in immune checkpoint blockade (e.g., express a dominant-negative form of an immune checkpoint molecule, such as PD-1). While not intended to be an exhaustive list, the immune checkpoint molecule is selected from programmed death 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4), and CD160. The immune checkpoint molecule may also be a transforming growth factor beta (TGF-β) receptor. Preferably, the immune checkpoint molecule is CTLA-4. Most preferably, the immune checkpoint molecule is PD-1.

[0127] PCT application WO 2017 / 040945 describes a method for engineering CAR-T cells that express a dominant-negative cell-mediated immune response inhibitor. WO 2017 / 040945 application is incorporated herein by reference.

[0128] Treatment method Immune effector cells expressing the disclosed CARs can induce therapeutically beneficial immune responses against B lymphocyte antigen-expressing cancer cells (e.g., CD19-associated cancers). For example, the anti-tumor immune response induced by the disclosed CAR-modified immune effector cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy approach in which the CAR-modified immune effector cells induce an immune response specific to a B lymphocyte antigen, such as CD19.

[0129] Adoptive transfer of immune effector cells expressing chimeric antigen receptors is a promising anti-cancer treatment. After harvesting a patient's immune effector cells, the cells can be genetically engineered to express the disclosed B lymphocyte antigen-specific CAR and then infused back into the patient. Furthermore, immune effector cells obtained from a donor other than the patient (i.e., allogeneic to the patient) can be genetically engineered to express the disclosed B lymphocyte antigen-specific CAR, and the CAR-containing cells can then be infused back into the patient. In certain specific embodiments, the immune effector cells comprising the anti-B lymphocyte antigen CAR polypeptide are allogeneic EBV-specific cytotoxic T cells.

[0130] The disclosed CAR-modified immune effector cells may be administered alone or as a pharmaceutical composition in combination with diluents and / or other components, such as IL-2, IL-15, or other cytokines or cell populations. Briefly, pharmaceutical compositions can include the target cell populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, compositions for use in the disclosed methods are formulated for intravenous administration. The pharmaceutical compositions can be administered by any method appropriate for treating MM. The amount and frequency of administration will depend on factors such as the patient's condition and the severity of the patient's disease, but appropriate dosages may be determined through clinical trials.

[0131] Where an "immunologically effective amount," "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the invention to be administered can be determined by a physician taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). Generally, pharmaceutical compositions comprising the T cells described herein can be administered in amounts of 10 to 150 mg of cells, including all integer values ​​within the range. 4 ~10 9 cells / kg body weight, e.g., 10 cells 5 ~10 6It can be said that the T cell compositions can be administered at a dosage of 1000 cells / kg body weight. The T cell compositions can also be administered multiple times at these dosages. The cells can be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0132] In certain embodiments, it may be desirable to administer activated T cells to a subject, then subsequently draw blood again (or perform apheresis), activate the T cells therefrom according to the disclosed methods, and reinfuse these activated and expanded T cells into the patient. This process can be performed multiple times, every few weeks. In certain embodiments, T cells can be activated from a blood draw of 10 cc to 400 cc. In certain embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw / multiple reinfusion protocol can be useful for selecting for certain populations of T cells.

[0133] Administration of the disclosed compositions can be carried out by any convenient method, such as injection, infusion, or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously (iv) or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by iv injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0134] In certain embodiments, the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, concurrently with, or after) a number of relevant treatment modalities, including, but not limited to, thalidomide, dexamethasone, bortezomib, and lenalidomide. In further embodiments, the CAR-modified immune effector cells can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibody or other antibody therapy, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the CAR-modified immune effector cells are administered to the patient in conjunction with (e.g., before, concurrently with, or after) bone marrow transplantation, T-cell depletion therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In other embodiments, the cell compositions of the present invention are administered after B-cell depletion therapy, such as an agent that reacts with CD20, such as Rituxan. For example, in some embodiments, the subject may undergo standard treatment involving high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0135] The cancer of the disclosed methods can be any B lymphocyte antigen-expressing cell (e.g., any CD19-expressing cell) in a subject experiencing uncontrolled growth, invasion, or metastasis. Cancers expressing B lymphocyte antigens (e.g., CD19) include leukemias and lymphomas, such as acute leukemia, chronic leukemia, lymphocytic leukemia, myeloid leukemia, preleukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, EBV-associated lymphoproliferative disorders, mature B-cell neoplasms, mature T-cell and natural killer (NK) cell neoplasms, precursor lymphoid neoplasms, and immunodeficiency-associated lymphoproliferative disorders.

[0136] In some embodiments, the cancer may be any neoplasm or tumor of the hematopoietic and / or lymphoid tissues. Thus, the cancer may be any malignancy affecting the blood, bone marrow, lymph, and / or lymphatic system, as well as any such disease that results in uncontrolled myeloproliferation and / or lymphoproliferation. A representative, but non-limiting, list of cancers for which the disclosed compositions can be used to treat includes acute lymphoblastic leukemia (ALL), precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt's leukemia, acute mixed lymphoma, chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (PML), acute myeloblastic leukemia, acute megakaryoblastic leukemia, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, clonal eosinophilia, B-cell chronic lymphocytic leukemia, and leukemia. Lymphocytic leukemia / small cell lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasms such as plasma cell myeloma (multiple myeloma), plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal marginal zone B-cell lymphoma (MALT lymphoma), nodal marginal zone B-cell lymphoma, follicular lymphoma, primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (with or without chronic inflammation), Epstein-Barr virus-positive DLBCL in the elderly, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK +Large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Burkitt lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sézary syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorder, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma lymphoma, B lymphoblastic leukemia / lymphoma not otherwise specified, B lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, T lymphoblastic leukemia / lymphoma; classical Hodgkin lymphomas, e.g., nodular sclerosing, mixed cytology Hodgkin lymphoma, lymphocyte-rich Hodgkin lymphoma, lymphocyte-depleted or non-depleted Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma; and immunodeficiency-associated lymphoproliferative disorders associated with primary immune disorders, associated with human immunodeficiency virus (HIV), associated with methotrexate therapy, post-transplant, and primary central nervous system lymphoma.

[0137] The disclosed CARs can be used in combination with any compound, moiety, or group that has a cytotoxic or cytostatic effect, including chemotherapeutic agents that can function as microtubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators, particularly those used for cancer therapy.

[0138] The disclosed CARs can be used in combination with immune checkpoint inhibitors. Two known immune checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), are expressed on the surface of APCs, such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligand binds to PD-1, an inhibitory signal is transmitted to the T cell, reducing cytokine production and suppressing T cell proliferation. Checkpoint inhibitors include, but are not limited to, aptamers and antibodies that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016). Techniques for combining CARs with checkpoint inhibitors in immune effector cells and their use for the treatment of various disorders are described, for example, in WO 2017 / 040945, which is incorporated herein by reference.

[0139] Human monoclonal antibodies against programmed death 1 (PD-1) and methods of treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described for these antibodies in U.S. Patent No. 8,008,449, which is incorporated by reference. Anti-PD-L1 antibodies and their uses are described for these antibodies in U.S. Patent No. 8,552,154, which is incorporated by reference. Anti-cancer agents comprising anti-PD-1 antibodies or anti-PD-L1 antibodies are described for these antibodies in U.S. Patent No. 8,617,546, which is incorporated by reference.

[0140] In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds to PDL1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD-1 inhibitor comprises an antibody that specifically binds to PD-1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies against PD-1 and methods of treating cancer using anti-PD-1 antibodies, alone or in combination with other immunotherapeutics, are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and their uses are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anti-PD-1 antibodies or anti-cancer agents comprising anti-PD-L1 antibodies are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

[0141] The disclosed CARs can be used in combination with other cancer immunotherapies. There are two distinct types of immunotherapy: passive immunotherapy, which uses components of the immune system to direct targeted cytotoxic activity against cancer cells without necessarily initiating an immune response in the patient, and active immunotherapy, which actively elicits an endogenous immune response. Passive strategies involve the use of monoclonal antibodies (mAbs) produced by B cells in response to specific antigens. The development of hybridoma technology and the identification of tumor-specific antigens in the 1970s enabled the development of mAbs as medicines that can specifically target tumor cells for destruction by the immune system. Among these mAbs is rituximab (Rituxan, Genentech), which binds to the CD20 protein, which is highly expressed on the surface of B-cell malignancies, such as non-Hodgkin's lymphoma (NHL). Rituximab has been approved by the FDA for the treatment of NHL and chronic lymphocytic leukemia (CLL) in combination with chemotherapy. Another important mAb is trastuzumab (Herceptin, Genentech), which has revolutionized the treatment of HER2-positive breast cancer by targeting the expression of HER2 (human epidermal growth factor receptor 2).

[0142] Generating optimal "killer" CD8 T cell responses also requires T cell receptor activation and costimulation, which can be achieved by ligation of tumor necrosis factor receptor family members, including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because treatment with activating (agonist) anti-OX40 mAbs increases T cell differentiation and cytolytic function, resulting in enhanced antitumor immunity against a variety of tumors.

[0143] In some embodiments, such additional therapeutic agents may be selected from antimetabolites, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine.

[0144] In some embodiments, such additional therapeutic agents may be selected from alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin.

[0145] In some embodiments, such additional therapeutic agents may be selected from antimitotic agents, such as taxanes, eg, docetaxel and paclitaxel, and vinca alkaloids, eg, vindesine, vincristine, vinblastine, and vinorelbine.

[0146] In some embodiments, such additional therapeutic agents may be selected from topoisomerase inhibitors, such as topotecan or irinotecan, or cytostatics, such as etoposide and teniposide.

[0147] In some embodiments, such additional therapeutic agent may be selected from a growth factor inhibitor, e.g., an inhibitor of ErbB1 (EGFR) (e.g., an EGFR antibody, e.g., zalutumumab, cetuximab, panitumumab, or nimotuzumab, or another EGFR inhibitor, e.g., gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (e.g., a HER2 antibody, e.g., trastuzumab, trastuzumab-DM1, or pertuzumab), or an inhibitor of both EGFR and HER2, e.g., lapatinib).

[0148] In some embodiments, such additional therapeutic agents can be selected from tyrosine kinase inhibitors, such as imatinib (Glivec, Gleevec STI571) or lapatinib.

[0149] Thus, in some embodiments, the disclosed antibodies are used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and / or rituximab.

[0150] In some embodiments, therapeutic agents for use in combination with CARs to treat disorders such as those described above can be anti-cancer cytokines, chemokines, or combinations thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa. Suitable chemokines can include Glu-Leu-Arg (ELR)-negative chemokines from the human CXC and CC chemokine families, such as IP-10, MCP-3, MIG, and SDF-1a. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0151] In some embodiments, a therapeutic agent for use in combination with a CAR to treat a disorder such as those described above can be a cell cycle control / apoptosis regulator (or "regulator"). Cell cycle control / apoptosis regulators can include molecules that target and modulate cell cycle control / apoptosis regulators, such as (i) cdc-25 (e.g., NSC663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (e.g., flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (e.g., BIBR1532, SOT-095, GRN163, and compositions described, for example, in U.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limiting examples of molecules that interfere with the apoptotic pathway include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate the TRAIL receptor, IFN, and antisense Bcl-2.

[0152] In some embodiments, a therapeutic agent for use in combination with a CAR to treat a disorder such as those described above can be a hormone modulating agent, such as an agent useful in anti-androgen and anti-estrogen therapy. Examples of such hormone-modulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / estinyl, antiandrogens (e.g., flutamide / eulexin), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone / provera, megestrol acetate / megace), corticosteroids (e.g., hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and its analogs and other LHRH agonists, such as buserelin and goserelin), aromatase inhibitors (e.g., anastrazole / arimidex, aminoglutethimide / cytraden, exemestane), or hormone blockers (e.g., octreotide / sandostatin).

[0153] In some embodiments, a therapeutic agent for use in combination with a CAR to treat a disorder such as those described above can be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.

[0154] As noted above, administration of the combination may be simultaneous, separate, or sequential. In the case of simultaneous administration, the agents may be administered in one composition or in separate compositions, as appropriate.

[0155] In some embodiments, the disclosed CARs are administered in combination with radiation therapy. Radiation therapy can include irradiation, or the associated administration of a radiopharmaceutical to the patient is provided. The radiation source can be external or internal to the patient being treated (radiation treatment can be, for example, in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used when performing such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.

[0156] In some embodiments, the disclosed CARs are administered in combination with a surgical procedure.

[0157] CAR-T cells can be engineered in several ways to enhance tumor cytotoxicity and specificity, escape tumor immune suppression, avoid host rejection, and extend therapeutic half-life. TRUCK (T cells redirected for global cytokine killing) T cells, for example, carry a CAR but are also engineered to release cytokines, such as IL-12, that promote tumor killing. These CAR-T cells are sometimes referred to as "armored CARs" because they are designed to release a molecular payload upon localization to the tumor environment upon CAR activation. Several cytokines have been investigated preclinically and clinically as cancer therapies, and these may also prove useful if similarly incorporated into TRUCK forms of CAR-T therapy. These cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-α, IFN-γ, TNF-α, TRAIL, FLT3 ligand, lymphotactin, and TGF-β (Dranoff, (2004). "Cytokines in Cancer Pathogenesis and Cancer Therapy." Nat Rev Cancer, January, 4(1):11-22). "Self-driven" or "homing" CAR-T cells have been engineered to express chemokine receptors in addition to their CAR. Because certain chemokines can be upregulated in tumors, incorporation of chemokine receptors aids tumor trafficking and infiltration by adoptive T cells, thereby enhancing both the specificity and functionality of CAR-T (Moon (2011). "Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor." Clin Cancer Res. 2011 July 15; 17(14):4719-30).Universal CAR-T cells also carry a CAR but are engineered to express neither endogenous TCR (T cell receptor) nor MHC (major histocompatibility complex) proteins. Removal of these two proteins from the signaling repertoire of adoptive T cell therapy prevents graft-versus-host disease and rejection, respectively. Armored CAR-T cells are also so named for their ability to evade tumor immunosuppression and tumor-induced CAR-T dysfunction. These specific CAR-Ts carry a CAR and can be engineered not to express checkpoint inhibitors. Alternatively, these CAR-Ts can be co-administered with monoclonal antibodies (mAbs) that block checkpoint signaling. Administration of anti-PDL1 antibodies significantly restored the killing ability of CAR TILs (tumor-infiltrating lymphocytes). While the PD-1-PD-L1 and CTLA-4-CD80 / CD86 signaling pathways have been investigated, it is possible to target other immune checkpoint signaling molecules, including LAG-3, Tim-3, IDO-1, 2B4, and KIR, in the design of armored CAR-Ts. Other intracellular inhibitors of TILs include phosphatases (SHP1), ubiquitin ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinase). Armored CAR-Ts may also be engineered to express proteins or receptors that protect them from or render them resistant to the effects of tumor-secreted cytokines. For example, CTLs (cytotoxic T lymphocytes) transduced with a double-negative form of the TGF-β receptor are resistant to immunosuppression by lymphoma-secreted TGF-β. These transduced cells exhibited significantly increased antitumor activity in vivo compared with their control counterparts.

[0158] Tandem and dual CAR-T cells are unique in that they have two distinct antigen-binding domains. Tandem CARs contain two consecutive antigen-binding domains facing the extracellular environment, connected to an intracellular costimulatory and stimulatory domain. Dual CARs are engineered so that one extracellular antigen-binding domain is connected to an intracellular costimulatory domain, and a second distinct extracellular antigen-binding domain is connected to an intracellular stimulatory domain. Because the stimulatory and costimulatory domains are split between the two distinct antigen-binding domains, dual CARs are also referred to as "split CARs." In both tandem and dual CAR designs, the binding of both antigen-binding domains is necessary to enable signal transduction of the CAR circuit in T cells. These two CAR designs are also referred to as "bispecific" CARs because they have binding affinities for different distinct antigens.

[0159] One major concern regarding CAR-T cells as a form of "living therapeutic agent" is their in vivo operability and potential immunostimulatory side effects. To better control CAR-T therapy and prevent unwanted side effects, various features, including off-switches, safety mechanisms, and conditional control mechanisms, have been engineered. For example, both self-destructing CAR-T cells and labeled / tagged CAR-T cells are engineered to have an "off-switch" that facilitates clearance of CAR-expressing T cells. Self-destructing CAR-T cells contain a CAR but are also engineered to express a pro-apoptotic suicide gene or "elimination gene" that becomes inducible upon administration of an exogenous molecule. Various suicide genes can be used for this purpose, including HSV-TK (herpes simplex virus thymidine kinase), Fas, iCasp9 (inducible caspase 9), CD20, MYC TAG, and truncated EGFR (endothelial growth factor receptor). For example, HSV-TK can convert the prodrug ganciclovir (GCV) to GCV triphosphate, which is incorporated into replicating DNA and ultimately causes cell death. iCasp9 is a chimeric protein containing components of the FK506-binding protein that binds to the small molecule AP1903, which triggers caspase-9 dimerization and apoptosis. However, labeled / tagged CAR-T cells are cells that contain a CAR but are also engineered to express a selection marker. Administration of a mAb against this selection marker promotes CAR-T cell clearance. Truncated EGFR is one such antigen that can be targeted by anti-EGFR mAbs, and administration of cetuximab promotes CAR-T cell elimination. CARs engineered with these characteristics are also referred to as sCARs for "switchable CARs" and RCARs for "controllable CARs." "Safety CARs," also known as "inhibitory CARs" (iCARs), are engineered to express two antigen-binding domains. One of these ectodomains is directed against a tumor-associated antigen and binds to intracellular costimulatory and stimulatory domains. However, the second extracellular antigen-binding domain is specific for normal tissue and binds to an intracellular checkpoint domain, e.g., CTLA4, PD-1, or CD45. It is also possible to incorporate multiple intracellular inhibitory domains into iCARs. Some inhibitory molecules that can provide these inhibitory domains include B7-H1, B7-1, CD160, PIH, 2B4, CEACAM (CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, TIGIT, BTLA, LAIR1, and TGFβ-R. In the presence of normal tissue, stimulation of this second antigen-binding domain will inhibit the CAR. It should be noted that due to this dual antigen specificity, iCARs are also a form of bispecific CAR-T cells. Safety: CAR-T engineering enhances the specificity of CAR-T cells for tumor tissue, and in some situations, it is advantageous that certain normal tissues express very low levels of tumor-associated antigens that may result in off-target effects with standard CARs (Morgan (2010). "Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2." Molecular Therapy 2010;18(4):843-851). Conditional CAR-T cells express an extracellular antigen-binding domain connected to an intracellular costimulatory domain and a separate intracellular costimulatory factor. The costimulatory and stimulatory domain sequences are engineered so that upon administration of an exogenous molecule, the resulting multiple proteins enter the cell together and complete the CAR circuit. In this regard, CAR-T activation can be modulated and, in some cases, even "fine-tuned" or dedicated to a particular patient. Similar to the dual CAR design, the stimulatory and costimulatory domains are physically separated when inactive in conditional CARs; therefore, these are also referred to as "split CARs."

[0160] In some embodiments, two or more of these design features may be combined to create an enhanced, multifunctional CAR-T. For example, CAR-T cells can be created with either a dual CAR design or a conditional CAR design that also releases cytokines, such as TRUCK. In some embodiments, dual-conditional CAR-T cells can be engineered to express two CARs with two separate antigen-binding domains for two distinct cancer antigens, each binding to a respective costimulatory domain. The costimulatory domains, along with the stimulatory domains, can only become functional after an activating molecule is administered. For the CAR-T cells to be effective, the cancer must express both cancer antigens, and an activating molecule must be administered to the patient. This design thereby incorporates features of both dual CAR-T cells and conditional CAR-T cells.

[0161] Typically, CAR-T cells are generated using α-β T cells, although γ-δ T cells can also be used. In some embodiments, the described CAR constructs, domains, and engineered characteristics used to generate CAR-T cells can similarly be used in generating other types of CAR-expressing immune cells, including NK (natural killer) cells, B cells, mast cells, bone marrow-derived phagocytes, and NKT cells. Alternatively, CAR-expressing cells can be engineered to have properties of both T cells and NK cells. In additional embodiments, CAR-transduced cells can be autologous or allogeneic to the patient to whom they are administered.

[0162] Several different methods for CAR expression can be used, including retroviral transduction (including gamma-retrovirus), lentiviral transduction, transposon / transposase (Sleeping Beauty and PiggyBac systems), and messenger RNA transfer-mediated gene expression. Gene editing (gene insertion or gene deletion / disruption) is also becoming increasingly important for the possibility of engineering CAR-T cells. CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator-like effector nuclease) systems are three potential methods by which CAR-T cells can be generated.

[0163] [Example] [Example 1] CD19-CAR design Cytotoxic T cells (CTLs) sensitized to EBV antigens were used to engineer CAR T cells that selectively target at least one cancer-associated CD19 epitope. The CAR polypeptide was specifically designed to reduce CAR T cell exhaustion and enhance CAR T cell persistence in subjects. Briefly, the expressed CAR polypeptide contained a leader sequence (i.e., the amino acid sequence shown in SEQ ID NO: 8) that is N-terminal to the CD19 targeting domain (i.e., the FMC63 scFv amino acid sequence shown in SEQ ID NO: 9). Those skilled in the art will understand that when a CAR is expressed inside a cell, for example, an EBV-sensitized CTL, the nascent protein (i.e., a protein comprising the amino acid sequence shown in SEQ ID NO: 6) is processed, including the removal of the leader sequence. The resulting mature polypeptide (i.e., a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7) is translocated to the cell surface. Furthermore, the CAR domain was optimized by combining an intracellular domain including a hinge region, a transmembrane domain, and a costimulatory domain (i.e., the amino acid sequence shown in SEQ ID NO: 10, which includes the CD28 hinge region, transmembrane domain, and intracellular domain shown in SEQ ID NOs: 12, 13, and 14), and a signaling domain mutant (i.e., a 1XX CD3ζ mutant in which the CD3ζ domain lacks functionality in the two C-terminal ITAM domains, such as the amino acid sequence shown in SEQ ID NO: 11).

[0164] JPEG2025028959000107.jpg160160JPEG2025028959000108.jpg135161

[0165] Alternatively, the CAR may be coupled to a detectable ligand, e.g. 124 It may further comprise an LNGFR domain capable of binding I-NGF and thus acting as a molecular tag.

[0166] Optionally, CAR T cells can express inhibitors of immune checkpoint molecules (i.e., dominant-negative PD-1 polypeptides), and thus overcome the immunosuppressive microenvironment found in many tumors.

[0167] [Example 2] Electrical impedance assay for targeted cytotoxicity and alloreactivity Electrical impedance measurements of targeted cells were used to assess the specific cytotoxicity induced by EBV-sensitized anti-CD19 CAR-T cells (EBV-CAR-T). Briefly, donor-derived B lymphocyte (BLCL) target cells characterized for CD-19 expression (see Figure 1) were plated onto anti-CD40-coated 96-well impedance plates, effectively forming a monolayer of cells for analysis. The change in electrical impedance relative to the voltage applied to the antibody-bound cell monolayer in response to the addition of effector cells (e.g., EBV-CAR-T) was measured, thereby enabling measurements of cytotoxicity and / or alloreactivity.

[0168] result Donor-matched (autologous) BLCL exhibit lysis in both the presence of EBV-CAR-T cells and non-transduced EBV-sensitized T cells (NTD-EBV-T) (see Figure 2, top). In contrast, with mismatched donor target cells, EBV-CAR-T cells are capable of CD19 antigen-targeted cytolytic activity, whereas NTD-EBV-T cells are only capable of limited EBV-TCR-directed cytolytic activity against mismatched target cells (see Figure 2, bottom).

[0169] [Example 3] Luciferase assay for targeted cytolytic activity The cytolytic activity of antigen-specific T cells transduced with anti-CD19 CAR (i.e., EBV-CAR-T cells) was also determined by a standard luciferase-based assay. Cell lines endogenously expressing CD19, as well as K562 control cells (untransduced and transduced with a CD19 expression construct), were engineered to express luciferase and served as target cells, as summarized in Table 7 and Figure 3.

[0170] Effector and tumor target cells were plated at 5 x 10 in a total volume of 100 µl per well. 4 Black-walled 96-well plates with target cells were used to co-culture various effector:target ratios (1:4, 1:2, 1:1, 2:1, and 4:1). Target cells alone were plated at the same cell density to determine maximum luciferase expression (relative light units (RLU)). After 24 and 48 hours, 100 μl of luciferase substrate was added directly to each well. Luminescence was detected in a luminescence plate reader. Lysis is generally determined as (1 - (RLU sample / RLU max)) x 100.

[0171] [Table 7]

[0172] Briefly, luciferase-expressing target cells and effector mock-transduced or EBV-CAR-T cells were plated at the desired effector-to-target (E:T) ratio described above. Untransduced cells from the same donor were used to normalize the total cell count in all wells so that all wells containing T cells had the same total cell number. The co-culture plates were placed in an incubator at 37°C and 5% CO2 overnight. At 24 hours, 50 μl of supernatant was removed from the co-culture for use in a multiplex cytokine detection assay. To measure the cytotoxic activity of effector cells, 50 μl of culture medium was added to each well to compensate for the volume removed for cytokine / chemokine analysis. D-luciferin (luciferase substrate) was added to each well, and the plate was incubated at room temperature for 10 minutes. The plate was then read, RLU recorded, and specific lysis calculated. After the 24 hour luciferase read, the cells were returned to the incubator overnight and read again for a second time at 48 hours. The percentage of tumor lysis was calculated by the following formula: % Tumor lysis = (1-(RLU) 腫瘍細胞+T細胞 / RLU 腫瘍細胞 ))×100

[0173] result EBV-CAR-T cells exhibit CAR-directed cytolysis of CD19-expressing cell lines (i.e., NALM6, K562-CD19, and RAJI). (See Figure 4.) Notably, cytolysis was most efficient in the NALM6 cell line (CD19 positive, EBNA negative), followed by K562-CD19. RAJI cells (CD19 positive, EBNA positive) exhibited higher resistance to directed cytolysis.

[0174] Minimal nonspecific cytolysis was observed in the antigen-negative cell line (K562), and some cytolysis was observed when the E:T ratio of donor 001-19 EBV-CAR-T cells (001-19X) was increased. However, effector cells exhibited donor-to-donor variability in nonspecific cytolysis, demonstrating a lack of evidence of dose-dependent killing. Donor 001-19 exhibited the highest level of nonspecific cytolysis compared to the other two donors. (See Figure 5.) In subsequent assays, donor 001-19 exhibited the highest basal cytokine release and had much lower lysis compared to the positive target control.

[0175] [Example 4] Cytokine release profile of EBV-CD19 CAR-T cells Cytokine secretion of antigen-specific T cells transduced with anti-CD19 CAR (i.e., EBV-CAR-T cells) was determined using a bead-based multiplex cytokine detection assay (e.g., Luminex® Technology Multiplex Assay, ThermoFisher Scientific, USA). Cytokine profiling was performed using supernatants from the previously described co-cultures (i.e., the co-cultures of Example 3).

[0176] Briefly, cytokine profiling was performed using supernatants from the co-cultures of the luciferase assay described above, as indicated above. Supernatants (50 μL) from each well (24- or 48-hour co-cultures) were transferred to a 96-well storage plate sealed with a peelable foil seal and stored at -80°C until use. A 27-plex cytokine panel in a pre-assembled mixture of target-specific beads was used for quantitative analysis of select cytokines (see Table 8). For all test articles and untransduced controls, thawed samples, as well as T cells alone, co-cultures, and target cell lines alone, were added to the multiplex panel plate. Samples were measured and read on a microplate reader.

[0177] [Table 8]

[0178] result Analysis of supernatants from 48-hour co-cultures at a 1:1 E:T ratio showed that EBV-CAR-T cells induced elevated cytokine levels compared to NTD control T cells and the target cell line alone (see Table 9 and Figures 6-18).

[0179] [Table 9]

[0180] Although EBV-CAR-T cells exhibit a high degree of polyfunctionality in the presence of CD19-expressing cell lines, the cytokine release profile may be determined, in part, by the intrinsic properties of the cell line used, as observed in the cytolytic assays described above. Notably, K562-CD19 cells achieve the greatest amount of antigen-induced cytokine release compared to RAJI or NALM6 cells (see Figures 6-12).

[0181] For each T cell donor (001-19, 014-18, 023-18), analyses were also performed on K562-CD19 cells, including T cells alone, (E:F) 2:1, 1:1, 1:2, 1:4, and targets alone. Exemplary data for T cells (e.g., EBV-T cells and EBV-CAR-T cells) from donor 014-18 are provided in Tables 10-13.

[0182] [Table 10-1] [Table 10-2]

[0183] [Table 11-1] [Table 11-2]

[0184] [Table 12-1] [Table 12-2]

[0185] [Table 13-1] [Table 13-2]

[0186] EBV-CAR-T cells derived from all donors (i.e. EBV CAR-T cells and EBV CAR LNGFR -T cells) produced elevated cytokine levels (e.g., IFNγ, granzyme B, IL6, TNFα, MCP-1) compared to NTD controls and T cells alone across all E:F ratios (see Tables 10-13 and Figures 13-15).

[0187] [Example 5] In vivo safety and efficacy evaluation of EBV-CAR-T cells The efficacy of engineered EBV-specific anti-CD19-CAR-expressing T cells was evaluated in a NALM6-induced systemic B-cell acute lymphoblastic leukemia (B-ALL) mouse model. This study involved the administration of two to three doses of human donor-derived EBV-CAR-T cells (0.6 x 10 per dose in mice). 6 10 or more cells = at least 2 x 10 cells 6 This includes staging of tumor burden after treatment with 1000 mAbs (human equivalent of 1000 mAbs / kg).

[0188] Briefly, luciferase-expressing NALM6 cells were injected into immune-deficient mice (0.5 × 10 on day −3). 61000 IU / mouse), and tumor growth is allowed to progress for 3 days. Tumor staging is performed on day 0 by bioluminescence imaging, and treatment is administered intravenously or intraperitoneally. Animals are divided into treatment groups (8 per group) as described in Table 14. Recombinant human IL-2 (rhIL-2) is administered immediately after the treatment dose (2000 IU), and booster doses of rhIL-2 are administered on days 1 and 2 (2000 IU each). Blood samples are collected 48 hours after treatment and weekly thereafter (e.g., days 2, 10, 16, and 24). Tumor burden is measured twice weekly (e.g., days 4, 7, 10, 13, 16, 18, 21, and 24) by bioluminescence imaging. Clinical symptoms and body weight are assessed twice weekly (e.g., days 2, 4, 7, 10, 13, 16, 18, 21, and 24). On day 24, the animals are sacrificed and tissue samples are collected after necropsy (e.g., liver, bone marrow, and spleen). (See Figure 16)

[0189] [Table 14]

[0190] result Animals receiving EBV-CAR-T cells exhibit statistically significant reductions in clinical symptoms, including decreased tumor burden and increased survival. Additional analyses demonstrate that EBV-CAR-T cells persist in mice over the course of the study and exhibit a polyfunctional cytokine profile with minimal adverse effects while avoiding acute toxicity.

[0191] [Example 6] Safety of EBV-CAR-T cell therapy for relapsed / refractory aggressive B-cell non-Hodgkin lymphoma This study is a single-arm, open-label, multicenter study consisting of (a) dose escalation followed by (b) dose expansion of the biologically active maximum tolerated dose in human subjects treated with allogeneic (off-the-shelf) EBV-CAR-T cells to further define safety, tolerability, and preliminary clinical outcomes.

[0192] Briefly, the study group included: Diffuse large B-cell lymphoma not otherwise specified (DLBCL), e.g. Transformed low-grade non-Hodgkin's lymphoma (NHL, Grade 3B follicular lymphoma, T-cell / histiocytic-rich large B-cell lymphoma, EBV-positive DLBCL not otherwise specified, Primary mediastinal (thymic) large B-cell lymphoma, or High-grade B-cell lymphoma with DLBCL histology and MYC and BCL2 and / or BCL6 rearrangements (double / triple-hit lymphoma), Must be relapsed or refractory to at least two prior lines of systemic therapy for the disease under investigation Adult subjects (age 18 years or older) with histologically confirmed intermediate-grade B-cell NHL, defined as:

[0193] Previous therapy must have included a CD20-targeted agent and an anthracycline. Subjects treated with autologous anti-CD19-CAR-T therapy are permitted, but CD19 + It must be.

[0194] Dose levels explored include: ·Low dose: 1×10 cells 6 pieces / kg ·Medium dose: 3×10 cells 6 pieces / kg ·High dose: 6×10 cells 6 pieces / kg Includes:

[0195] Each dose escalation will use a sample size of at least three dose-limiting toxicity (DLT)-evaluable subjects per dose level. Dose expansion cohorts will begin at a dose level shown to be safe by at least six DLT-evaluable subjects who complete the DLT period. All subjects in dose escalation and dose expansion will be followed for disease status and treatment-related adverse events for 24 months after EBV-CAR-T cell infusion.

[0196] Further purposes include: Overall response rate at 1, 3, 6, 12, and 24 months, and the extent to which overall response rate is associated with HLA mismatches; and Safety in relation to HLA incompatibility and its relationship to cytokine release syndrome (CRS), neurotoxicity, and graft-versus-host disease (GVHD) Includes:

[0197] result The disclosed doses of EBV-CAR-T cells can induce and / or increase progression-free survival with minimal adverse effects. EBV-CAR-T cells are capable of both expansion and persistence in human subjects, exhibit a polyfunctional cytokine profile, and avoid CAR-T exhaustion.

[0198] Although several embodiments of the present invention have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0199] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are expressly incorporated by reference.

[0200] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0201] <110> ATARA BIOTHERAPEUTICS, INC. <120> ANTIGEN SPECIFIC CD19-TARGETED CAR-T CELLS <130> PA24-651 <150> US 62 / 840,774 <151> 2019-04-30 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 1455 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 1 atggctctcc cagtgactgc cctactgctt cccctagcgc ttctcctgca tgcagacatc 60 cagatgaccc agaccacaag cagcctgtct gccagcctgg gcgatagagt gaccatcagc 120 tgtagagcca gccaggacat cagcaagtac ctgaactggt atcagcagaa acccgacggc 180 accgtgaagc tgctgatcta ccacaccagc agactgcaca gcggcgtgcc aagcagattt 240 tctggcagcg gctctggcac cgactacagc ctgacaatca gcaacctgga acaagaggat 300 atcgctacct acttctgcca gcaaggcaac accctgcctt acacctttgg cggaggcacc 360 aagctggaaa tcaccggctc tacaagcggc agcggcaaac ctggatctgg cgagggatct 420 accaagggcg aagtgaaact gcaagagtct ggccctggac tggtggcccc atctcagtct 480 ctgagcgtga cctgtacagt cagcggagtg tccctgcctg attacggcgt gtcctggatc 540 agacagcctc ctcggaaagg cctggaatgg ctgggagtga tctggggcag cgagacaacc 600 tactacaaca gcgccctgaa gtcccggctg accatcatca aggacaactc caagagccag 660 gtgttcctga agatgaacag cctgcagacc gacgacaccg ccatctacta ttgcgccaag 720 cactact acggcggcag ctacgccatg gattattggg gccagggcac cagcgtgacc 780 gttcttctg cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag 840 agcaatggaa ccattatcca tgtgaaaggg aaacacctttt gtccaagtcc cctatttccc 900 ggaccttcta agccctttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 960 ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 1020 cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1080 ccctatgcc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1140 gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260 aagccgagaa ggaagaaccc tcaggaaggc ctgttcaatg aactgcagaa agataagatg 1320 gcggaggcct tcagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380 ggccttttcc aggggctcag tacagccacc aaggacacct tcgacgccct tcacatgcag 1440 gccctgcccc ctcgc 1455 <210> 2 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 atggctctcc cagtgactgc cctactgctt cccctagcgc ttctcctgca tgca 54 <210> 3 <211> 735 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 3 gacatccaga tgacccagac cacaagcagc ctgtctgcca gcctgggcga tagagtgacc 60 atcagctgta gagccagcca ggacatcagc aagtacctga actggtatca gcagaaaccc 120 gacggcaccg tgaagctgct gatctaccac accagcagac tgcacagcgg cgtgccaagc 180 agattttctg gcagcggctc tggcaccgac tacagcctga caatcagcaa cctggaacaa 240 gaggatatcg ctacctactt ctgccagcaa ggcaacaccc tgccttacac ctttggcgga 300 ggcaccaagc tggaaatcac cggctctaca agcggcagcg gcaaacctgg atctggcgag 360 ggatctacca agggcgaagt gaaactgcaa gagtctggcc ctggactggt ggccccatct 420 cagtctctga gcgtgacctg tacagtcagc ggagtgtccc tgcctgatta cggcgtgtcc 480 tggatcagac agcctcctcg gaaaggcctg gaatggctgg gagtgatctg gggcagcgag 540 acaacctact acaacagcgc cctgaagtcc cggctgacca tcatcaagga caactccaag 600 agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactattgc 660 gccaagcact actactacgg cggcagctac gccatggatt attggggcca gggcaccagc 720 gtgaccgttt cttct 735 <210> 4 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 4 attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60 catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120 tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 180 tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240 atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300 gacttcgcag cctatcgctc c 321 <210> 5 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 5 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgttcaat 180 gaactgcaga aagataagat ggcggaggcc ttcagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggccttttc caggggctca gtacagccac caaggacacc 300 ttcgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 6 <211> 485 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Put Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Wing Leu Leu Leu 1 5 10 15 His Ala Asp Ile Gln Put Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser 20 25 30 Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser 35 40 45 Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu 50 55 60 Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe 65 70 75 80 Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu 85 90 95 Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu 100 105 110 Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr 115 120 125 Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu 130 135 140 Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200 205 Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 210 215 220 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 225 230 235 240 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Ser Val Thr Val Ser Ser Ala Ala Ala Ile Glu Val Met Tyr Pro 260 265 270 Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val 275 280 285 Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys 290 295 300 Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 305 310 315 320 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 325 330 335 Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 340 345 350 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 355 360 365 Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 7 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140 Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 145 150 155 160 Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190 Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205 Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 225 230 235 240 Val Thr Val Ser Ser Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro 245 250 255 Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly 260 265 270 Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe 275 280 285 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 290 295 300 Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg 305 310 315 320 Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro 325 330 335 Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala 340 345 350 Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 355 360 365 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 370 375 380 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 385 390 395 400 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe Asn Glu 405 410 415 Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly Met Lys 420 425 430 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu 435 440 445 Ser Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu 450 455 460 Pro Pro Arg 465 <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 8 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala <210> 9 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140 Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 145 150 155 160 Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190 Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205 Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 225 230 235 240 Val Thr Val Ser Ser 245 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55 60 Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 65 70 75 80 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 85 90 95 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 100 105 <210> 11 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 12 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro 35 <210> 13 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 13 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 14 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 14 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 15 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 15 Tyr Met Asn Met 1 <210> 16 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 16 Pro Arg Arg Pro 1 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 17 Pro Tyr Ala Pro 1

Claims

1. A chimeric antigen receptor (CAR) polypeptide comprising a B lymphocyte antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

2. The CAR polypeptide of claim 1, wherein the B lymphocyte antigen is a CD19 antigen, a CD20 antigen, or a CD22 antigen.

3. The CAR polypeptide of claim 1 or 2, wherein the B lymphocyte antigen binding domain is an anti-CD19 functional antibody fragment.

4. 4. The CAR polypeptide of any one of claims 1 to 3, wherein the B lymphocyte antigen binding domain is an anti-CD19 single chain variable fragment (scFv).

5. 5. The CAR polypeptide of any one of claims 1 to 4, wherein the B lymphocyte antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO:

9.

6. 6. The CAR polypeptide of any one of claims 1 to 5, wherein the transmembrane domain is derived from a transmembrane or membrane-associated polypeptide.

7. The polypeptides CD28, NKp30, CDS, DAP10, 41BB, DAP12, CD3C, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1, ICOS (CD278), 4- 1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7 Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 7. The CAR polypeptide of any one of claims 1 to 6, comprising at least one transmembrane domain of any one of: 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, variants thereof, or any combination thereof.

8. 8. The CAR polypeptide of any one of claims 1 to 7, wherein the transmembrane domain comprises the transmembrane domain of CD28 and / or 41BB.

9. 9. The CAR polypeptide of any one of claims 1 to 8, wherein the signaling domain comprises at least one signaling domain of any one of the polypeptides CD8, CD3zeta, CD3delta, CD3gamma, CD3epsilon, FcgammaRI-gamma, FcgammaRIII-gamma, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137(41BB), ICOS, CD27, CD28δ, CD80, NKp30, OX40, variants thereof, or any combination thereof.

10. 10. The CAR polypeptide of any one of claims 1 to 9, further comprising at least one costimulatory signaling region.

11. 11. The CAR polypeptide of claim 10, wherein the costimulatory signaling region comprises a signaling domain of any one of the polypeptides CD8, CD3zeta, CD3delta, CD3gamma, CD3epsilon, FcgammaRI-gamma, FcgammaRIII-gamma, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137(41BB), ICOS, CD27, CD28δ, CD80, NKp30, OX40, variants thereof, or any combination thereof.

12. The CAR polypeptide of claim 10, wherein the costimulatory signaling region comprises the signaling domain of CD28 or a variant thereof.

13. The CAR polypeptide of claim 12, wherein the CD28 signaling domain comprises at least one or more mutations in any one of the subdomains YMNM, PRRP, PYAP, or any combination thereof.

14. 14. The CAR polypeptide of claim 12 or 13, wherein the CD28 signaling domain lacks any one of the subdomains YMNM, PRRP, or PYAP.

15. 15. The CAR polypeptide of any one of claims 12 to 14, wherein the CD28 signaling domain lacks any two of the subdomains selected from YMNM, PRRP, or PYAP.

16. The CAR polypeptide of claim 10 or 11, wherein the costimulatory signaling region comprises the signaling domain of CD137 (41BB) or a mutant thereof.

17. 17. The CAR polypeptide of any one of claims 1 to 16, wherein at least one signaling domain comprises native CD3zeta or a mutant thereof.

18. The CAR polypeptide of claim 17, wherein the mutant CD3ζ lacks a C-terminal immunoreceptor tyrosine-based activation motif (ITAM).

19. The CAR polypeptide of claim 17, wherein the mutant CD3ζ lacks two C-terminal immunoreceptor tyrosine-based activation motifs (ITAMs).

20. The CAR polypeptide of claim 17, wherein the mutant CD3ζ contains only one immunoreceptor tyrosine-based activation motif (ITAM).

21. 21. The CAR polypeptide of any one of claims 17 to 20, wherein the mutant CD3ζ comprises the amino acid sequence shown in SEQ ID NO:

11.

22. 22. The CAR polypeptide of any one of claims 1 to 21, further comprising a hinge sequence.

23. The CAR polypeptide of claim 22, wherein the hinge sequence is derived from a CD8α molecule or a CD28 molecule.

24. 24. The CAR polypeptide of any one of claims 1 to 23, further comprising a signal peptide.

25. The CAR polypeptide of claim 24, wherein the signal peptide is derived from the CD8α leader sequence.

26. 26. The CAR polypeptide of any one of claims 1 to 25, comprising any one of the amino acid sequences set forth in SEQ ID NOs: 6 to 11, any fragment thereof, or any combination thereof.

27. 26. A CAR polypeptide according to any one of claims 1 to 25, comprising the amino acid sequence set forth in SEQ ID NO:

7.

28. 28. A nucleic acid encoding a CAR polypeptide according to any one of claims 1 to 27.

29. 29. The nucleic acid of claim 28, comprising any one of the nucleotide sequences set forth in SEQ ID NOs: 1 to 5, fragments thereof, or any combination thereof.

30. 29. The nucleic acid of claim 28, comprising the nucleotide sequence set forth in SEQ ID NO:

1.

31. A vector comprising a nucleic acid according to any one of claims 28 to 30.

32. 32. An immune cell comprising the nucleic acid of claim 31.

33. 33. An immune cell comprising the vector of claim 32.

34. 28. An immune cell expressing a CAR polypeptide according to any one of claims 1 to 27.

35. 35. The immune cell of any one of claims 32 to 34, which is a white blood cell.

36. 36. The immune cell of any one of claims 32 to 35, which is a lymphocyte, monocyte, macrophage, dendritic cell, mast cell, neutrophil, basophil, or eosinophil.

37. 37. The immune cell of any one of claims 32 to 36, which is a lymphocyte selected from an αβ T cell, a γδ T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a B cell, an innate lymphoid cell (ILC), a cytokine-induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine-activated killer (LAK) cell, a regulatory T cell, or any combination thereof.

38. The immune cell of claim 37, which is a cytotoxic T lymphocyte (CTL).

39. The immune cell according to claim 37 or 38, which is a viral antigen-sensitized CTL.

40. The immune cell of any one of claims 37 to 39, which is a CTL sensitized to a viral antigen derived from any one of Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), picornavirus (e.g., hepatitis A virus), hepadnavirus (e.g., hepatitis B virus), hepacivirus (e.g., hepatitis C virus), delta virus (e.g., hepatitis D virus), hepevirus (e.g., hepatitis E virus), or any combination thereof.

41. The immune cell of any one of claims 37 to 40, which is an EBV-sensitized CTL.

42. (a) a CAR polypeptide that selectively binds to a B lymphocyte antigen, and (b) a CAR polypeptide that selectively binds to a tumor-associated antigen , bispecific chimeric antigen receptor (CAR) T cells expressing

43. The bispecific chimeric CAR T cell of claim 42, wherein the B lymphocyte antigen is a CD19 antigen, a CD20 antigen, or a CD22 antigen.

44. 41. The bispecific CAR T cell of claim 39 or 40, wherein the tumor associated antigen is GPC3, MAGE-A1, MAGE-A2, MAGE-C2, SSX-2, Ny-ESO-1, hTERT, or a viral hepatitis antigen.

45. 45. The bispecific CAR T cell of any one of claims 42 to 44, wherein the antigen binding domains of the chimeric antigen receptor each comprise a functional antibody fragment.

46. 46. ​​The bispecific CAR T cell of any one of claims 42 to 45, wherein the antigen binding domains of the chimeric antigen receptor each comprise a single chain variable fragment (scFv).

47. 47. The bispecific CAR T cell of any one of claims 42 to 46, wherein each of the transmembrane domains of the chimeric antigen receptors comprises at least one transmembrane domain of either CD28, 41BB, a variant thereof, or any combination thereof.

48. 48. The bispecific CAR T cell of any one of claims 42 to 47, wherein the signaling domain of at least one chimeric antigen receptor comprises at least one signaling domain of CD3zeta, a variant thereof, or any combination thereof.

49. 48. The bispecific CAR T cell of any one of claims 42 to 47, wherein each of the signaling domains of the chimeric antigen receptors comprises the signaling domain of CD3zeta, a variant thereof, or any combination thereof.

50. 50. The bispecific CAR T cell of claim 48 or 49, wherein the mutant CD3ζ lacks a C-terminal immunoreceptor tyrosine-based activation motif (ITAM).

51. 50. The bispecific CAR T cell of claim 48 or 49, wherein the mutant CD3ζ lacks two C-terminal immunoreceptor tyrosine-based activation motifs (ITAMs).

52. 50. The bispecific CAR T cell of claim 48 or 49, wherein the mutant CD3ζ comprises only one immunoreceptor tyrosine-based activation motif (ITAM).

53. The bispecific CAR T cell of any one of claims 42 to 52, wherein at least one of the chimeric antigen receptors comprises at least one costimulatory signaling region.

54. The bispecific CAR T cell of any one of claims 42 to 52, wherein each chimeric antigen receptor comprises at least one costimulatory signaling region.

55. 55. The bispecific CAR T cell of claim 53 or 54, wherein the costimulatory signaling region comprises the signaling domain of any one of CD28 or a variant thereof, CD137(41BB) or a variant thereof, or any combination thereof.

56. 56. The CAR polypeptide of claim 55, wherein the CD28 signaling domain comprises at least one or more mutations in any one of the subdomains YMNM, PRRP, PYAP, or any combination thereof.

57. 57. The CAR polypeptide of claim 55 or 56, wherein the CD28 signaling domain lacks the function of at least one subdomain selected from YMNM, PRRP, or PYAP.

58. 58. The CAR polypeptide of any one of claims 55 to 57, wherein the CD28 signaling domain lacks the function of any two of the subdomains selected from YMNM, PRRP, or PYAP.

59. 59. The bispecific CAR T cell of any one of claims 42 to 58, further comprising a hinge sequence derived from CD8a or CD28.

60. 60. The CAR-expressing cell of any one of Claims 34 to 59, which has been genetically modified so that it no longer expresses one or more immune checkpoint molecules.

61. 60. The CAR-expressing cell of any one of Claims 34 to 59, further comprising expression of one or more immune checkpoint molecules in a dominant negative form.

62. 60. The CAR-expressing cell of any one of Claims 34 to 59, further comprising expression of a switch receptor specific for one or more immune checkpoint molecules.

63. 60. The CAR-expressing cell of any one of Claims 34 to 59, further comprising expression of an antibody or functional fragment thereof capable of blocking signaling by one or more checkpoint molecules.

64. 64. The CAR-expressing cell of any one of claims 60 to 63, wherein the immune checkpoint molecule is selected from programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), cytotoxic T lymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4), CD160, and transforming growth factor beta (TGF-β) receptor.

65. The immune cell of any one of claims 60 to 64, wherein the immune checkpoint molecule is PD-1 and / or CTLA-4.

66. 66. A method of treating a B-lymphocyte antigen-associated cancer in a subject, comprising administering an effective amount of an adoptive immunotherapy composition comprising a CAR-expressing cell of any one of claims 32 to 65.

67. 67. The method of claim 66, wherein the B-lymphocyte antigen-associated cancer is a hematological cancer selected from acute leukemia, chronic leukemia, lymphocytic leukemia, myeloid leukemia, preleukemic conditions, Hodgkin's lymphoma, non-Hodgkin's lymphoma, EBV-associated lymphoproliferative disease, mature B-cell neoplasms, mature T-cell and / or natural killer (NK) cell neoplasms, precursor lymphoid neoplasms, and immunodeficiency-associated lymphoproliferative disorders.

68. The method of claim 66 or 67, wherein the B-lymphocyte antigen-associated cancer is an EBV-associated lymphoproliferative disease.

69. 69. The method of any one of claims 66 to 68, wherein the subject has undergone, is undergoing, or will undergo additional anti-cancer therapy.

70. 70. The method of claim 69, wherein the additional anti-cancer therapy comprises surgery, radiation, chemotherapy, immunotherapy, or hormonal therapy.

71. 71. The method of any one of claims 66 to 70, wherein the adoptive immunotherapy composition is administered intrathoracically, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally, intracranially, or by direct administration to an organ.

72. 72. The method of any one of claims 66 to 71, wherein the subject is a human.

73. The method of claim 72, wherein the CAR-expressing cells of the adoptive immunotherapy composition are derived from the subject.

74. 74. The method of claim 73, wherein the CAR-expressing cells of the adoptive immunotherapy composition are derived from a donor sample, or a bank or library of donor samples.

75. 75. The method of any one of Claims 66-74, further comprising administering at least one immune checkpoint inhibitor.

76. 76. The method of claim 75, wherein the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, or a combination thereof.

77. 76. The method of claim 75, wherein the immune checkpoint inhibitor comprises an RNAi molecule, such as an antisense RNA molecule (asRNA), a microRNA molecule (miRNA), a short hairpin RNA molecule (shRNA), or a small interfering RNA molecule (siRNA).

78. 76. The method of claim 75, wherein the immune checkpoint inhibitor comprises a CRISPR RNA (crRNA) molecule.

79. The method of claim 75, wherein the immune checkpoint inhibitor comprises a dominant negative immune checkpoint molecule.

80. 76. The method of claim 75, wherein the immune checkpoint inhibitor comprises a recombination switch receptor.

81. 81. The method of any one of claims 75 to 80, wherein the immune checkpoint inhibitor is expressed by a vector comprising a nucleic acid molecule encoding the immune checkpoint inhibitor, the vector being selected from a DNA vector, an RNA vector, a plasmid, or a viral vector.

82. The method of claim 81, wherein a vector comprising a nucleic acid molecule encoding an immune checkpoint inhibitor is expressed in a CAR-expressing cell of the adoptive immunotherapy composition.

Images