IN VITRO OR EX VIVO METHOD FOR MANUFACTURING T CELLS, COMPOSITION UNDERSTANDING A CRYOPROTECTION AGE

专利摘要:
abstract? improved t cell compositions? The present invention provides improved t-cell compositions and methods for making t-cells. more particularly, the present invention provides t-cell manufacturing methods that result in adoptive t-cell immunotherapies with improved survival, expansion and persistence in vivo.
公开号:BR112016028644B1
申请号:R112016028644
申请日:2015-06-05
公开日:2019-12-03
发明作者:friedman Kevin
申请人:Bluebird Bio Inc；
IPC主号:

专利说明:

“IN VITRO OR EX VIVO METHOD FOR MANUFACTURING T CELLS, COMPOSITION UNDERSTANDING A CRYOPROTECTION AGENT AND A POPULATION OF IMMUNE EFFECTIVE CELLS AND THERAPEUTIC USES OF THAT COMPOSITION” CROSS REFERENCE TO RELATED REQUESTS
[001] This application claims the benefit under 35 U.S.C. § 119 (e) of U.S. Provisional Application No. 62 / 008,957, filed on June 6, 2014, which is hereby incorporated by reference here in its entirety.
BACKGROUND
Technical Field [002] The present invention relates to improved T cell compositions and methods for making T cells. More particularly, the present invention relates to methods of making T cell that result in adoptive T cell immunotherapies with improved survival, expansion and persistence in vivo.
Description of the Related Technique [003] Adoptive immunotherapy is the transfer of T lymphocytes to an individual for disease therapy. Adoptive immunotherapy has not yet realized the potential to treat a wide variety of diseases including cancer, infectious disease, autoimmune disease, inflammatory disease and immunodeficiency. However, most, if not all adoptive immunotherapy strategies require that the T cell activation and expansion steps generate a clinically effective therapeutic dose of T cells. Current technologies for generating therapeutic doses of T cells, which include cells T manipulated by genetic engineering, remain constrained by embarrassing T cell manufacturing processes. For example, T cell expansion often requires intensive and uneconomical cloning work, and / or multiple rounds of activation / expansion to reach cell numbers Therapeutically relevant tees. In addition, existing T cell activation / expansion methods are usually coupled with substantial T cell differentiation and generally result in short-term effects, which include short-term survival and the lack of persistence and lack of in vivo expansion of transferred T cells. Thus, existing T cell manufacturing processes produce an inferior T cell product that is prone to exhaustion and loss of effector immune cell function.
[004] Currently, the clinical efficacy of adoptive T-cell immunotherapies manipulated by genetic engineering is limited by poor T-cell expansion and persistence after infusion in patients. Therefore, these therapies are not suitable for widespread clinical use. Thus, there is a persistent need, unfulfilled by improvements in T cell manufacturing and therapeutic T cell compositions that survive, expand and persist in vivo.
BRIEF SUMMARY
[005] The present invention in general provides adoptive T cell immunotherapies comprising persistent and potent anti-tumor T cell compositions and methods of producing them.
[006] The present invention relates to improved T cell compositions and methods for making T cells. More particularly, the present invention relates to methods of making T cell which results in improved in vivo survival, expansion and persistence.
[007] In several embodiments, a method for making T cells comprising: (a) isolating the T cell population, for example, tumor infiltrating cytotoxic T lymphocytes (TIL), from an individual; (b) activate the T cell population and stimulate the T cell population to proliferate, in which the activation and stimulation steps are performed in the presence of an AKT / mTOR pathway inhibitor; (c) cultivating the T cells to proliferate; in which the steps of activating and stimulating performed in the presence of the PI3K / AKT / mTOR pathway inhibitor results in the proliferation of T cells compared to the proliferation of T cells that were activated and stimulated in the absence of the PI3K / AKT pathway inhibitor / mTOR is provided.
[008] In several modalities, a method for making T cells that comprises: (a) activating the T cell population and stimulating the T cell population to proliferate, in which the stages of activation and stimulation are carried out in the presence of a AKT / mTOR path inhibitor; (b) transducing T cells with a viral vector comprising a genetically engineered T cell receptor (TCR) or a chimeric antigen (CAR) receptor; (c) cultivating the transduced T cells to proliferate; where the steps of activating and stimulating performed in the presence of the PI3K / AKT / mTOR pathway inhibitor results in the proliferation of transduced T cells compared to the proliferation of transduced T cells that were activated and stimulated in the absence of the PI3K pathway inhibitor / AKT / mTOR is provided.
[009] In particular modalities, the methods contemplated here comprise isolating mononuclear cells from peripheral blood as the source of T cells.
[010] In certain embodiments, T cell activation involves contacting T cells with an anti-CD3 antibody or CD3-binding fragment thereof.
[011] In additional embodiments, stimulation of T cells comprises contacting T cells with an anti-CD28 antibody or an anti-CD28 antibody fragment thereof, B7-1 or an anti-CD28 antibody fragment thereof, or B7-2 or an anti-CD28 antibody fragment thereof.
[012] In some embodiments, cells are transduced with the viral vector before T cell proliferation.
[013] In certain embodiments, cells are transduced with the viral vector after T cell proliferation.
[014] In particular modalities, the vector is a retroviral vector.
[015] In additional modalities, the vector is a lentiviral vector.
[016] In other particular embodiments, cells comprise a chimeric antigen receptor (CAR).
[017] In particular modalities, the CAR comprises: an extracellular domain that binds to an antigen selected from the group consisting of: alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX , CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, CMV, EBV, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA- A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, HPV, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs and VEGFR2; a transmembrane domain derived from a polypeptide selected from the group consisting of: CD8a; CD4, CD28, CD45, PD-1 and CD152; one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1) and CD278 (ICOS); and a CD3Z signaling domain.
[018] In additional embodiments, the extracellular domain comprises an antibody or antigen-binding fragment that binds to the antigen.
[019] In certain embodiments, the transmembrane domain is derived from CD8a or CD28.
[020] In additional modalities, the one or more co-stimulatory signaling domains are selected from the group consisting of: CD28, CD134 and CD137.
[021] In additional modalities, the CAR comprises a polypeptide from the articulation region.
[022] In particular embodiments, the hinge region polypeptide comprises the hinge region of IgG1 or CD8a.
[023] In particular embodiments, the CAR comprises a signal peptide.
[024] In some embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8a signal polypeptide.
[025] In some modalities, the PI3K / AKT / mTOR path inhibitor is selected from the group consisting of: BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI -145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103 , NU7026, PIK-75, ZSTK474 and PP-121.
[026] In particular modalities, the PI3K / AKT / mTOR pathway inhibitor is a pan-PI3K inhibitor selected from the group consisting of: BEZ235, LY294002 and GDC-0941.
[027] In other particular modalities, the PI3K / AKT / mTOR pathway inhibitor is a selective PI3K inhibitor selected from the group consisting of: BYL719, GSK2636771, TGX-221, AS25242, CAL-101 and IPI-145 .
[028] In other particular embodiments, the PI3K / AKT / mTOR pathway inhibitor is the PI3K ZSTK474 inhibitor.
[029] In particular modalities, the PI3K / AKT / mTOR pathway inhibitor is a pan-AKT inhibitor selected from the group consisting of: MK-2206, GSK690693 and GDC-0068.
[030] In additional embodiments, the PI3K / AKT / mTOR pathway inhibitor is the selective AKT1 inhibitor A-674563.
[031] In certain embodiments, the PI3K / AKT / mTOR pathway inhibitor is the selective AKT2 inhibitor CCT128930.
[032] In certain embodiments, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT DNA-PK activation.
[033] In additional modalities, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT PDK-1 activation.
[034] In particular modalities, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT mTORC2 activation.
[035] In additional modalities, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT HSP activation.
[036] In other particular embodiments, the PI3K / AKT / mTOR pathway inhibitor is a pan-mTOR inhibitor selected from the group consisting of: AZD8055, INK128 and rapamycin.
[037] In certain embodiments, the PI3K / AKT / mTOR pathway inhibitor is a selective mTORC1 inhibitor selected from the group consisting of: PF-04691502 and everolimus.
[038] In particular embodiments, the PI3K / AKT / mTOR pathway inhibitor is a s6 kinase inhibitor selected from the group consisting of: BI-D1870, H89, PF-4708671, FMK and AT7867.
[039] In particular embodiments, the PI3K / AKT / mTOR pathway inhibitor is a DNA-PK inhibitor selected from the group consisting of: NU7441, PI-103, NU7026, PIK-75 and PP-121.
[040] In some embodiments, the population of activated and stimulated T cells in the presence of a PI3K / AKT / mTOR pathway inhibitor has a greater number of T cells that express one or more markers selected from the group consisting of: CD62L, CCR7, CD28, CD27, CD122 and CD127 compared to the population of activated and stimulated T cells in the absence of the PI3K / AKT / mTOR pathway inhibitor.
[041] In additional modalities, the population of activated and stimulated T cells in the presence of a PI3K / AKT / mTOR pathway inhibitor does not express CD57 or KLRG1 or expresses less CD57 or KLRG1 compared to the population of activated and stimulated T cells in the absence of the PI3K / AKT / mTOR path inhibitor.
[042] In several modalities, a method of maintaining proliferation and reducing differentiation of restimulated T cells that express a modified TCR or CAR by genetic engineering comprising: (a) bringing into contact all or a portion of a population of proliferated T cells which comprises a TCR or CAR modified by genetic engineering with an anti-CD3 antibody or CD3 binding fragment thereof and an anti-CD28 antibody or CD28 binding fragment thereof, which stimulates the accessory CD28 molecule on the surface of cells T, thereby stimulating activated T cells to proliferate; wherein the restimulated T cells maintained proliferation and reduced differentiation compared to the proliferation of T cells that were stimulated or restimulated in the absence of the PI3K / AKT / mTOR pathway inhibitor is provided.
[043] In particular modalities, cells comprise a TCR modified by genetic engineering.
[044] In certain embodiments, cells comprise CAR.
[045] In other particular embodiments, the cells comprise a viral vector that encodes a TCR or CAR modified by genetic engineering.
[046] In additional modalities, the vector is a retroviral vector.
[047] In additional modalities, the vector is a lentiviral vector.
[048] In particular modalities, the CAR comprises: an extracellular domain that binds to an antigen selected from the group consisting of: alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX , CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, 'Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY -ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivina, TAG72, TEMs and VEGFR2; a transmembrane domain derived from a polypeptide selected from the group consisting of: CD8a; CD4, CD28, CD45, PD-1 and CD152; one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1) and CD278 (ICOS); and a CD3Z signaling domain.
[049] In some embodiments, the extracellular domain comprises an antibody or antigen-binding fragment that binds to the antigen.
[050] In certain embodiments, the transmembrane domain is derived from CD8a or CD28.
[051] In additional modalities, the one or more co-stimulatory signaling domains are selected from the group consisting of: CD28, CD134 and CD137.
[052] In particular modalities, the CAR comprises a polypeptide from the articulation region.
[053] In additional embodiments, the hinge region polypeptide comprises the hinge region of IgG1 or CD8a.
[054] In additional embodiments, the CAR comprises a signal peptide.
[055] In other particular embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8a signal polypeptide.
[056] In particular modalities, the PI3K / AKT / mTOR path inhibitor is selected from the group consisting of: BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI -145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103 , NU7026, PIK-75, ZSTK474 and PP-121.
[057] In additional modalities, the PI3K / AKT / mTOR path inhibitor is a pan-PI3K inhibitor selected from the group consisting of: BEZ235, LY294002 and GDC-0941.
[058] In other particular modalities, the PI3K / AKT / mTOR pathway inhibitor is a selective PI3K inhibitor selected from the group consisting of: BYL719, GSK2636771, TGX-221, AS25242, CAL-101 and IPI-145 .
[059] In other particular embodiments, the PI3K / AKT / mTOR pathway inhibitor is the PI3K ZSTK474 inhibitor.
[060] In certain embodiments, the PI3K / AKT / mTOR path inhibitor is a pan-AKT inhibitor selected from the group consisting of: MK-2206, GSK690693 and GDC-0068.
[061] In particular modalities, where the PI3K / AKT / mTOR pathway inhibitor is the selective AKT1 inhibitor A-674563.
[062] In additional embodiments, the PI3K / AKT / mTOR pathway inhibitor is the selective AKT2 inhibitor CCT128930.
[063] In some embodiments, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT DNA-PK activation.
[064] In particular embodiments, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT PDK-1 activation.
[065] In certain embodiments, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT mTORC2 activation.
[066] In particular embodiments, the PI3K / AKT / mTOR pathway inhibitor inhibits AKT HSP activation.
[067] In additional embodiments, the PI3K / AKT / mTOR pathway inhibitor is a pan-mTOR inhibitor selected from the group consisting of: AZD8055, INK128 and rapamycin.
[068] In additional embodiments, the PI3K / AKT / mTOR pathway inhibitor is a selective mTORC1 inhibitor selected from the group consisting of: PF-04691502 and everolimus.
[069] In some embodiments, the PI3K / AKT / mTOR pathway inhibitor is a s6 kinase inhibitor selected from the group consisting of: BI-D1870, H89, PF-4708671, FMK and AT7867.
[070] In other particular embodiments, the PI3K / AKT / mTOR pathway inhibitor is a DNA-PK inhibitor selected from the group consisting of: NU7441, PI-103, NU7026, PIK-75 and PP-121.
[071] In particular modalities, the population of activated T cells restimulated in the presence of a PI3K / AKT / mTOR pathway inhibitor has a greater number of T cells that express one or more markers selected from the group consisting of: CD62L , CCR7, CD28, CD27, CD122 and CD127 compared to the population of activated and stimulated T cells in the absence of the PI3K / AKT / mTOR pathway inhibitor.
[072] In additional modalities, the population of activated T cells restimulated in the presence of a PI3K / AKT / mTOR pathway inhibitor does not express CD57 or KLRG1 or expresses less CD57 or KLRG1 compared to the population of activated and stimulated T cells in the absence of pathway inhibitor of PI3K / AKT / mTOR.
[073] In various modalities, the population of T cells comprising a vector comprising a TCR or CAR modified by genetic engineering, in which the cells were activated and stimulated to proliferate in the presence of a PI3K / AKT / mTOR pathway inhibitor is provided.
[074] In several particular modalities, the population of T cells comprising a vector comprising a TCR or CAR modified by genetic engineering, in which the cells were activated and stimulated to proliferate in the presence of a PI3K / AKT / pathway inhibitor mTOR and were stimulated by contacting all or a portion of a population of proliferated immune effector cells with an anti-CD3 antibody or CD3 binding fragment thereof and an anti-CD28 antibody or CD28 binding fragment thereof, which stimulates the accessory CD28 molecule on the surface of immune effector cells is provided [075] In certain embodiments, immune effector cells comprise T cells.
[076] In one embodiment, the immune effector cells are TILs.
[077] In various embodiments, a composition comprising the population of immune effector cells contemplated here and a physiologically acceptable excipient is provided.
[078] In several certain embodiments, a method of treating cancer in an individual in need thereof, which comprises administering to the individual a therapeutically effective amount of the contemplated T cell composition is provided herein.
[079] In particular modalities, cancer is selected from the group consisting of Wilms' tumor, Ewing's sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, breast cancer colon, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkirís lymphoma, non-Hodgkin's lymphoma and urinary bladder cancer.
[080] In one embodiment, cancer is associated with or caused by a viral infection, for example, CMV, HPV or EBV infection.
[081] In additional modalities, the cancer is pancreatic cancer and the extracellular binding domain binds to a PSCA or MUC1 epitope [082] In some modalities, the cancer is bladder cancer and the extracellular binding domain binds to a epitope of PSCA or MUC1 [083] In additional modalities, the cancer is glioblastoma multiforme and the extracellular binding domain binds to an epitope of EFA2, EGFRvIII or CSPG4.
[084] In particular modalities, cancer is lung cancer and the extracellular binding domain binds to a PSCA or GD2 epitope.
[085] In certain modalities, cancer is breast cancer and the extracellular binding domain binds to an epitope of CSPG4 or HER2.
[086] In additional modalities, the cancer is melanoma and the extracellular binding domain binds to an epitope of CSPG4 or GD2.
[087] In particular modalities, cancer is a B cell malignancy and the binding domain binds to a BCMA epitope.
[088] In various embodiments, a method of treating a hematological malignancy in an individual in need thereof, which comprises administering to the individual a therapeutically effective amount of the contemplated T cell composition is provided herein.
[089] In certain modalities, hematological malignancy is a B cell malignancy selected from the group consisting of: multiple myeloma (MM), chronic lymphocytic leukemia (CL), or non-Hodgkin's lymphoma (NHL).
[090] In particular modalities, MM is selected from the group consisting of: evident multiple myeloma, burning multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma and extramedullary plasmacytoma .
[091] In certain modalities, NHL is selected from the group consisting of: Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CL / SL), diffuse large B-cell lymphoma, follicular lymphoma, large cell lymphoma immunoblastic, precursor B lymphoblastic lymphoma and mantle cell lymphoma.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[092] Figure 1 shows a representative example of maintaining T cell proliferation in T cells treated with an AKT inhibitor. T cells were cultured with various concentrations of the AKT inhibitor, MK-22067 for up to seven days. The rightmost panels show the percentage of T cells divided from T cell cultures initiated from six PBMC samples from normal donors. Each symbol on the rightmost panel represents a single culture performed in parallel with the titrated MK-2206 dose. The leftmost panels show a representative example from these experiments. A) After three days of culture with MK-2206, T cell proliferation was only relatively reduced when compared to the control without treatment. B) After 7 days of culture with MK-2206, T cell proliferation was not substantially different compared to the untreated control.
[093] Figure 2 shows a representative example of CD62L expression from T cells treated with an AKT inhibitor. T cells were cultured with various concentrations of the AKT inhibitor, MK-22067 for up to seven days. The rightmost panels show the CD62L percentage expressing T cells from T cell cultures initiated from six normal donor PBMC samples. Each symbol on the rightmost panel represents a single culture performed in parallel with the titrated MK-2206 dose. The leftmost panels show a representative example from these experiments. A) After three days of culture with MK-2206, the expression of CD62L in T cells treated with MK-2206 was not substantially different when compared to the control without treatment. B) After 7 days of culture, MK-2206-treated T cells showed significantly higher levels of CD62L expression compared to the untreated control.
[094] Figure 3 shows the expression of CD62L in the anti-BCMA CAR T cells assessed by flow cytometry at the end of the culture with MK-2206, TCN, or ZSTK474. MK-2206 and ZSTK474 showed significantly higher CD62L expression compared to cultures of T-CAR cells treated with IL-2 alone or with TCN.
[095] Figure 4 shows mean tumor volume of multiple myeloma tumors in mice treated with anti-BCMA CAR T cells cultured with IL-2, IL-7 and IL-15, MK-2206, ZST747, or TCN. Anti-BCMA T CAR cells cultured with IL-7 and IL-15, MK-2206, or ZST747 showed similar levels of anti-tumor activity compared to anti-BCMA T T cells cultured with standard IL-2. Anti-BCMA CAR T cells cultured with TCN do not show an anti-tumor response.
[096] Figure 5 shows the anti-tumor activity of anti-BCMA T CAR cells treated with IL-2, IL-7/15, MK-2206, TCN, or ZSTK474 in a Daudi tumor model. Daudi's tumor progression was not affected after treatment with anti-BCMA T-CAR cells cultured with IL-2- or IL7 / 15. Anti-BCMA T CAR cells cultured with either MK-2206 or ZST474 caused complete tumor regression.
[097] Figure 6 shows the persistence of anti-BCMA T CAR cells cultured with ZSTK474 in animals treated with anti-BCMA T CAR cells cultured with IL-2-, MK-2206-, or ZSTK474 that completely regressed a 100mm3 tumor. RPMI-8226. The animals were challenged again 13 days later with RPMI-8226 on the opposite flank. Animals treated with T-CAR cells cultured with IL-2 were unable to prevent tumor development. None of the animals treated with anti-BCMA T-CAR cells cultured with ZSTK474 showed any evidence of tumor grafting.
DETAILED DESCRIPTION
Overview [098] The present invention relates in general to improved methods for the manufacture of T cell compositions. Without wishing to be tied to any particular theory, the methods of the present invention contemplated here disassociate T cell proliferation from differentiation to produce T cells having superior properties, for example, greater survival, expansion and persistence in vivo along with a concomitant reduction in differentiation, compared to T cell compositions existing in the art. Thus, the T cell compositions contemplated herein comprise potent T cells, which have characteristics of natural or young T cell populations, which are capable of multiple rounds of expansion with little differentiation. In addition, expanded cells are able to subsequently differentiate and provide immune effector cell functions.
[099] In various embodiments, a method for making T cells is provided that maintains or minimally reduces T cell proliferation and reduces, decreases, or mitigates T cell differentiation during T cell expansion.
In particular preferred embodiments, a T cell composition engineered by genetic engineering is manufactured by the methods contemplated herein, which can additionally increase the effectiveness of a adoptive T cell immunotherapy. The manufactured T-cell compositions contemplated herein are useful in treating or preventing numerous conditions that include, but are not limited to, cancer, infectious disease, autoimmune disease, inflammatory disease and immunodeficiency. Without wishing to be tied to any particular theory, the present inventor has surprisingly and unexpectedly discovered that modulation of cell signaling pathways in T cells, whose pathways are normally associated with cancer cell proliferation, results in substantially or not substantially maintaining reduce T cell proliferation and reduce T cell differentiation during T cell expansion compared to T cells where cell signaling paths are not modulated.
[0100] In one embodiment, a method of making T cells manipulated by genetic engineering comprises putting T cells in contact with an agent that inhibits a PI3K / AKT / mTOR pathway in the cells. The cells can be contacted before, during, and / or after activation and expansion. Genetically engineered T cell compositions retain sufficient T cell potency so that they can undergo multiple rounds of expansion without a substantial increase in differentiation.
[0101] Thus, the methods and compositions contemplated here represent a quantum of improvement compared to existing adoptive cell immunotherapies.
[0102] The practice of the present invention will employ, unless specifically indicated otherwise, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology that are within technique of those skilled in the art, many of which are described below for the purpose of illustration. These techniques are explained widely in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Tanrslação (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in newspapers such as Advances in Immunology.
[0103] All publications, payers and payers' requests cited here are hereby incorporated by reference in their entirety.
Definitions [0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by those skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred embodiments of compositions, methods and materials are described here. For the purposes of the present invention, the following terms are defined below.
[0105] Articles "a", "o", "one" and "one" are used here to refer to one or more than one (that is, at least one) of the grammatical object of the article. For example, "an element" means an element or more than one element.
[0106] As used here, the term “about” or “approximately” refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies by as much as 30, 25 , 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% for a reference of quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length . In particular modalities, the terms "about" or "approximately" when preceding a numerical value indicate the value more or less in a range of 15%, 10%, 5%, or 1%.
[0107] As used here, the term "substantially" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that is 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater of the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length . In one modality, “substantially the same” refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that produces an effect, for example, a physiological effect, which is approximately same as the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length.
[0108] Throughout this specification, unless the context otherwise indicates, the terms “comprising”, “understands” and “that understands” will be understood to imply the inclusion of a particular step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "that is to say that includes and limited to what follows the phrase" that consists of. Thus, the phrase “which consists of indicates that the elements listed are necessary or mandatory and that no other elements can be present. By "which essentially consists of" is meant to include any elements listed after the sentence and limited to other elements that do not interfere with or contribute to the activity or action specified in the description for the listed elements. Thus, the phrase “which essentially consists of” indicates that the elements listed are necessary or mandatory, but that no other elements are optional and may or may not be present depending on whether or not it affects the activity or action of the listed elements [0109 ] Reference through the present specification to “a modality”, “the modality”, “a particular modality”, “a related modality”, “a particular modality”, “an additional modality”, or “another modality” or combinations of same means that a particular feature, structure or features described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the following phrases in various places throughout the present specification are not necessarily all referring to the same modality. In addition, the particular features, structures, or features can be combined in any suitable mode in one or more embodiments.
[0110] As used herein, the terms "T cell manufacturing" or "methods of making T cells" or comparable terms refer to the process of producing a therapeutic T cell composition, whose manufacturing methods may comprise one or more of, or all of the following steps: collect, stimulate, activate and expand.
[0111] The terms "T cell" or "T lymphocyte" are recognized in the art and are intended to include thymocytes, pure T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell can be an auxiliary T cell (Th), for example an auxiliary T cell 1 (Th1) or an auxiliary T cell 2 (Th2). The T cell can be an auxiliary T cell (HTL; CD4 + T cell) CD4 + T cell, a cytotoxic T cell (CTL; CD8 + T cell), a cytotoxic T cell that infiltrates a tumor (TIL; CD8 + T cell), T cell CD4 + CD8 +, CD4-CD8- T cell, or any other subset of T cells. Other illustrative T cell populations suitable for use in particular embodiments include natural T cells and memory T cells.
[0112] "Potent T cells" and "young T cells", are used interchangeably in particular modalities and refer to T cell phenotypes in which the T cell is capable of proliferation and a concomitant reduction in differentiation. In particular modalities, the young T cell has the phenotype of a “natural T cell”. In various modalities, the manufacturing methods contemplated here produce young T cells; cells in which T cell proliferation has been disassociated from T cell differentiation during T cell stimulation, activation and expansion. Without intending to be tied to any particular theory, the potent T cells manufactured with the compositions and methods contemplate having greater anti-tumor efficacy after adoptive transfer. In particular embodiments, young T cells comprise one or more of or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122 and CD127. In one embodiment, young T cells comprise one or more of, or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122 and CD127 and CD57, CD244, CD160, PD-1, CTLA4 is missing, TIM3 and LAG3.
[0113] As used here, the term "proliferation" refers to an increase in cell division, whether symmetric or asymmetric cell division. In particular modalities, “proliferation” refers to the symmetrical or asymmetric division of T cells. “Greater proliferation” occurs when there is an increase in the number of cells in a treated sample compared to cells in an untreated sample.
[0114] As used here, the term "differentiation" refers to a method of reducing the potency or proliferation of a cell or moving the cell to a more restricted state of development. In particular modalities, differentiated T cells acquire immune effector cell functions.
[0115] An "immune effector cell" is any cell in the immune system that has one or more effector functions (for example, cytotoxic cell extermination activity, cytokine secretion, ADCC induction and / or CDC). The illustrative immune effector cells contemplated here are T lymphocytes, in particular cytotoxic T cells (CTLs; CD8 + T cells), TILs and helper T cells (HTLs; CD4 + T cells).
[0116] "Modified T cells" refers to T cells that have been modified by the introduction of a polynucleotide that encodes a TCR or CAR modified by genetic engineering contemplated here. The modified T cells include not only genetic modifications but also non-genetic modifications (for example, episomal or extrachromosomal).
[0117] As used here, the term "modified by genetic engineering" or "genetically modified" refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in the cell.
[0118] The terms, “genetically modified cells”, “modified cells”, and, “redirected cells”, are used interchangeably.
[0119] As used here, the term “gene therapy” refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects, or modifies the expression of a gene, or for the purpose of expressing a therapeutic polypeptide, for example, TCR or CAR and / or one or more cytokines. In particular embodiments, T cells are modified to express a TCR or CAR modified by genetic engineering without modifying the cell's genome, for example, by introducing an episomal vector that expresses the TCR or CAR into the cell.
[0120] The term "ex vivo" generally refers to activities that take place outside an organism, such as experimentation or measurements carried out on or in living tissue in an artificial environment outside the organism, preferably with minimal change in natural conditions. In particular modalities, "ex vivo" procedures involve living cells or tissues obtained from an organism and cultured or modulated in a laboratory apparatus, usually under sterile conditions and typically for a few hours or even about 24 hours, but which includes up to 48 or 72 hours, depending on the circumstances. In certain embodiments, said tissues or cells can be collected and frozen and then thawed for ex vivo treatment. Tissue culture experiments or procedures that last longer than a few days using cells or living tissue are typically considered to be "in vitro", although in certain embodiments, the term can be used interchangeably with ex vivo.
[0121] The term "in vivo" generally refers to activities that occur within an organism, such as cell self-renewal and cell expansion. In one embodiment, the term "in vivo expansion" refers to the ability of a cell population to increase in number in vivo.
[0122] The term "stimulation" refers to the main response induced by the binding of a stimulatory molecule (for example, a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event that includes, but is not limited to signal transduction via the TCR / CD3 complex.
[0123] The "stimulatory molecule", refers to a molecule in the T cell that specifically binds to a stimulating cognate ligand.
[0124] A "stimulatory ligand", as used here, means a ligand that when present in a cell that has an antigen (for example, an aAPC, a dendritic cell, a B cell and the like) can specifically bind with a cognate binding partner (referred to herein as a "stimulatory molecule") in the T cell, thereby mediating a major response to the T cell, which includes, but is not limited to, activation of an immune response, proliferation and the like. Stimulatory ligands include, but are not limited to, CD3 ligands, for example, an anti-CD3 antibody and CD2 ligands, for example, anti-CD2 antibody and peptides, for example, CMV, HPV, EBV peptides.
[0125] The term, "activation" refers to the state of the T cell that has been sufficiently stimulated to induce detectable cell proliferation. In particular modalities, activation can also be associated with the production of induced cytokine and detectable effector functions. The term "activated T cells" refers to, among other things, the T cells that are proliferating. The signals generated through the TCR alone are insufficient for complete T cell activation and one or more secondary or co-stimulatory signals are also required. Thus, T cell activation comprises a major stimulation signal through the TCR / CD3 complex and one or more secondary co-stimulatory signals. Co-stimulation can be evidenced by proliferation and / or cytokine production by T cells that have received a major activation signal, such as stimulation through the CD3 / TCR complex or through CD2.
[0126] A “co-stimulatory signal” refers to a signal, which in combination with a main signal, such as TCR / CD3 binding, leads to T cell proliferation, cytokine production, and / or regulation for more or down regulation of particular molecules (for example, CD28).
[0127] A "co-stimulatory ligand" refers to a molecule that binds to a co-stimulatory molecule. A co-stimulatory binder can be soluble or provided on the surface. The "co-stimulatory molecule" refers to the cognate binding partner in the T cell that specifically binds to a co-stimulatory ligand (for example, anti-CD28 antibody).
[0128] "Autologous", as used here, refers to cells from the same individual.
[0129] "Allogeneic", as used here, refers to cells of the same species that differ genetically from a cell in comparison.
[0130] “Singeneic”, as used here, refers to cells from a different individual that are genetically identical to the cell in comparison.
[0131] “Xenogenetic”, as used here, refers to cells of a different species for the cell in comparison. In preferred embodiments, the cells of the present invention are allogeneic.
[0132] As used here, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits a cancer symptom that can be treated with gene therapy vectors, procedures cell-based therapies and methods described at some point here. Suitable individuals (for example, patients) include laboratory animals (such as mice, rats, rabbits, or Guinea pigs), farm animals and domestic animals or pets (such as a dog or cat). Non-human primates and, preferably, human patients, are included. Typical individuals include human patients who have cancer, who have been diagnosed with cancer, or who are at risk for or who have cancer.
[0133] As used here, the term "patient" refers to an individual who has been diagnosed with a particular indication that can be treated with gene therapy vectors, cell-based therapeutic procedures and methods described elsewhere here.
[0134] As used here, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may even include minimal reductions in one or more measurable markers of the disease or condition being treated, for example, cancer. Treatment may optionally involve either reducing or ameliorating the symptoms of the disease or condition, or delaying the progression of the disease or condition. "Treatment" does not necessarily indicate the complete eradication or cure of the disease or condition, or associated symptoms.
[0135] As used here, “avoid” and similar terms such as “avoided”, “avoid” etc., indicate an approach to avoid, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition, for example, cancer. The same also refers to the delay in the onset or recurrence of a disease or condition or the delay in the occurrence or recurrence of symptoms of a disease or condition. As used here, "prevention" and similar terms also include reducing the intensity, effect, symptoms and / or burden of a disease or condition before the onset or recurrence of the disease or condition.
[0136] As used here, the term "amount" refers to "an effective amount" or "an effective amount" of a genetically modified therapeutic cell, for example, T cell, to achieve a beneficial or desired prophylactic or therapeutic result, which includes clinical results.
[0137] A "prophylactically effective amount" refers to an amount of an effective genetically modified therapeutic cell to achieve the desired prophylactic result. Typically but not necessarily, since the prophylactic dose is used in individuals before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.
[0138] The "therapeutically effective amount" of a genetically modified therapeutic cell can vary according to factors such as an individual's disease stage, age, sex and weight and the ability of T cells to elicit a desired response in the individual. The therapeutically effective amount is also one in which any toxic or harmful effects of the virus or the transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective for "treating" an individual (for example, a patient). When the therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration for individual differences in age, weight, tumor size, extent of infection or metastasis and condition of the patient (individual) .
[0139] As used here, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.
[0140] As used here, the term "malignant" refers to cancer in which a group of tumor cells exhibits one or more of uncontrolled development (ie, division beyond normal limits), invasion (ie, intrusion into and destruction of adjacent tissues) and metastasis (ie, spread to other locations in the body via lymph or blood). As used here, the term "metastasis" refers to the spread of cancer from one part of the body to the other. A tumor formed by cells that have spread is called a "metastatic tumor" or a "metastasis". The metastatic tumor contains cells that are similar to those of the original (main) tumor.
[0141] As used here, the term "benign" or "non-malignant" refers to tumors that can develop and grow but cannot spread to other parts of the body. Benign tumors are self-limiting and typically do not invade or metastasize.
[0142] A "cancer cell" or "tumor cell" refers to an individual cell of a cancerous development or tissue. A tumor generally refers to swelling or injury formed by abnormal cell development, which can be benign, pre-malignant, or malignant. Most cancers form tumors, but some, for example, leukemia, do not necessarily form tumors. For said cancers that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably. The amount of a tumor in an individual is the "tumor burden" that can be measured as the number, volume, or weight of the tumor.
[0143] An "infectious disease" refers to a disease that can be transmitted from person to person or from organism to organism and is caused by a microbial agent (for example, a common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV / AIDS, diphtheria, hepatitis B, hepatitis C, cholera and influenza.
[0144] An "autoimmune disease" refers to a disease in which the body produces an immunogenic response (ie, immune system) to some constituent of its own tissue. In other words, the immune system loses its ability to recognize some tissue or system within the body as "self" and aims and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which an organ is predominantly affected (for example, hemolytic anemia and anti-immune thyroiditis) and those in which the autoimmune disease process is spread through many tissues (for example, systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells that attack the sheaths that surround nerve fibers in the brain and spine. This results in loss of coordination, weakness and blurred vision. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis and the like.
[0145] An "immunodeficiency" means the condition of a patient whose immune system has been compromised by the disease or by the administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, syndrome hyper-IgM and diabetes.
[0146] By "intensifying" or "promoting", or "increasing" or "expanding" refers in general to the ability of a composition contemplated here to produce, elicit, or cause a greater physiological response (that is, effects downstream) compared to the response caused either by vehicle or by a control molecule / composition. A measurable physiological response may include an increase in T cell expansion, activation, persistence, and / or an increase in cancer cell extermination capacity, among others apparent from the understanding of the technique and the description here. A "greater" or "intensified" amount is typically an "statistically significant" amount and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 , 20, 30 or more times (for example, 500, 1000 times) (which includes all integers and decimal points between them and above 1, for example, 1.5, 1.6, 1.7. 1.8, etc.) the answer produced by the vehicle or the control composition.
[0147] By “decreasing” or “lowering”, or “attenuating”, or “reducing”, or “abating” refers in general to the ability of the composition contemplated here to produce, elicit, or cause a lesser physiological response (ie , downstream effects) compared to the response caused either by the vehicle or by the control molecule / composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (for example, 500, 1000 times) (which includes all integers and decimal points between them and above 1, for example, 1.5, 1.6, 1.7. 1.8, etc.) the answer ( reference response) produced by vehicle, the control composition, or the response in a particular cell line.
[0148] By "maintain", or "preserve", or "maintenance", or "no change", or "no substantial change", or "no substantial decrease" refers in general to the ability of a composition contemplated here to produce , elicit, or cause a minor physiological response (ie, downstream effects) in a cell, compared to responses caused either by the vehicle, the control molecule / composition, that is, the response in a particular cell line. A comparable response is one that is not significantly different or measurably different from the reference response.
[0149] The terms "specific binding affinity" or "specifically binding to" or "specifically binding" or "specific binding" or "specific targets" as used here, describe the binding of one molecule to another with greater binding affinity than the bottom link. A binding domain (or a CAR that comprises a binding domain or a fusion protein that contains a binding domain) "specifically binds" to a target molecule if it binds to or associates with a target molecule with a affinity or Ka (that is, an equilibrium association constant of a particular bonding interaction with 1 / M units) of, for example, greater than or equal to about 105 M-1. In certain embodiments, a binding domain (or a fusion protein thereof) binds to a target with a Ka greater than or equal to about 106 M-1, 107 M-1, 108 M-1, 109 M -1, 1010 M-1, 1011 M-1, 1012 M-1, or 1013 M-1. “High affinity” binding domains (or single-chain fusion proteins therein) refers to Ka binding domains of at least 107 M'1, at least 108 M'1, at least 109 M-1, at least 1010 M-1, at least 1011 M-1, at least 1012 M-1, at least 1013 M-1, or greater.
[0150] Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular bonding interaction with units of M (for example, 10-5 M to 10-13 M, or less). Binding domain affinities of polypeptides and CAR proteins according to the present description can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme linked immunosorbent assay), or by binding association, or displacement tests using labeled ligands, or using a surface plasmon resonance device such as the Biacore T100, which is offered by Biacore, Inc., Piscatem Clearance, NJ, or an optical biosensor technology such as the EPIC or EnSpire system that are offered by Corning and Perkin Elmer respectively (see also, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51: 660; and Paying US Nos. 5,283,173; 5,468,614, or the equivalent).
[0151] In one embodiment, the affinity of a specific bond is about 2 times greater than the bottom bond, about 5 times greater than the bottom bond, about 10 times greater than the bottom bond, about 20 times greater than bottom bond, about 50 times greater than bottom bond, about 100 times greater than bottom bond, or about 1000 times greater than bottom bond or more.
[0152] An "antigen (Ag)" refers to a compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, which includes compositions (such as one that includes a specific protein tumor) that are injected or absorbed into an animal. An antigen reacts with specific humoral or cellular immunity products, which include those induced by heterologous antigens, such as the described antigens. A "target antigen" or "target or interest antigen" is an antigen that a CAR binding domain contemplated here is designed to bind to.
[0153] An "epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds.
[0154] An "isolated peptide" or "isolated polypeptide" and the like, as used herein, refers to the isolation and / or in vitro purification of a peptide or polypeptide molecule from a cellular environment and from association with other components of the cell, that is, it is not significantly associated with substances in vivo. Similarly, an "isolated cell" refers to a cell that was obtained from a tissue or organ in vivo and is substantially free of extracellular matrix.
[0155] As used here, "isolated polynucleotide" refers to a polynucleotide that has been purified from the sequences that border it in a naturally occurring state, for example, a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. An "isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or another polynucleotide that does not exist in nature and was produced by the hand of man.
T-Cell Manufacturing Methods
[0156] T cells manufactured by the methods contemplated here provide improved adoptive immunotherapy compositions. Without wishing to be tied to any particular theory, it is believed that the T cell compositions manufactured by the methods contemplated here are imbued with superior properties, which include greater survival, expansion in the relative absence of differentiation and persistence in vivo. In one embodiment, a method of making T cells comprises contacting the cells with one or more agents that modulate the PI3K / Akt / mTOR cell signaling path. In various modalities, T cells can be obtained from any source and put in contact with the agent during the activation and / or expansion phases of the manufacturing process. The resulting T cell compositions are enriched in potent developed T cells that have the ability to proliferate and express one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122 and CD127.
[0157] In one embodiment, modified T cells that comprise maintained levels of proliferation and that have reduced differentiation are manufactured. In a particular embodiment, T cells are manufactured by stimulating T cells to become activated and to proliferate in the presence of one or more stimulatory signals and an agent that is an inhibitor of a PI3K / Akt / mTOR cell signaling pathway.
[0158] T cells can then be modified to express one or more TCRs or CARs modified by genetic engineering. In one embodiment, T cells are modified by transducing T cells with a viral vector that comprises a TCR or CAR modified by genetic engineering. In a given embodiment, T cells are modified before stimulation and activation in the presence of an inhibitor of a PI3K / Akt / mTOR cell signaling pathway. In another embodiment, T cells are modified after stimulation and activation in the presence of an inhibitor of a PI3K / Akt / mTOR cell signaling pathway. In a particular embodiment, T cells are modified within 12 hours, 24 hours, 36 hours, or 48 hours of stimulation and activation in the presence of an inhibitor of a PI3K / Akt / mTOR cell signaling pathway.
[0159] After T cells are activated, cells are cultured to proliferate. T cells can be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more expansion rounds.
[0160] In various embodiments, T cell compositions are manufactured in the presence of one or more inhibitors of the PI3K / AKT / mTOR pathway. Inhibitors can target one or more activities on the route or a single activity. Without wishing to be tied to any particular theory, it is contemplated that the treatment or contact of T cells with one or more inhibitors of the PI3K / AKT / mTOR pathway during the stimulation, activation, and / or expansion phases of the manufacturing process preferably increases young T cells, thereby producing superior therapeutic T cell compositions.
[0161] In a particular embodiment, a method for increasing the proliferation of T cells expressing a genetically engineered modified T cell receptor is provided. Said methods may comprise, for example, collecting the T cell source from an individual, stimulating and activating T cells in the presence of one or more inhibitors of the PI3K / AKT / mTOR pathway, modification of T cells to express a TCR or CAR modified by genetic engineering and expanding T cells in culture.
[0162] In a given embodiment, a method for producing enriched T cell populations for the expression of one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122 and CD127. In a related embodiment, a method for augmenting T cells that express CD62L, CCR7, CD28, CD27, CD122 and CD127 and that do not express or express low levels of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 is provided. As discussed at some point here, the expression levels of young T cell biomarkers is relative to the expression levels of said markers in more differentiated T cells or in populations of immune effector cells.
[0163] In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as the source of T cells in the T cell manufacturing methods contemplated here. PBMCs form a heterogeneous population of T lymphocytes that can be CD4 +, CD8 +, or CD4 + and CD8 + and can include other mononuclear cells such as monocytes, B cells, NK cells and NKT cells. An expression vector comprising a polynucleotide encoding a genetically engineered TCR or CAR contemplated herein can be introduced into a population of human donor T cells, NK cells or NKT cells. Successfully transduced T cells carrying an expression vector can be classified using flow cytometry to isolate CD3 positive T cells and then further propagated to increase the number of T cells further modified for cell activation using anti-CD3 antibody and or anti-CD28s and IL-2, IL-7, and / or IL-15 antibodies or any other methods known in the art as described elsewhere here.
[0164] The manufacturing methods contemplated herein may additionally comprise the cryopreservation of modified T cells for storage and / or preparation for use in a human subject. T cells are cryopreserved so that the cells remain viable with thawing. When necessary, transformed and cryopreserved immune effector cells can be thawed, developed and expanded to more of said cells. As used here, “cryopreserve” refers to the preservation of cells by cooling to sub-zero temperatures, such as (typically) 77 K or -196 ° C. (the boiling point of liquid nitrogen). Cryoprotective agents are often used at sub-zero temperatures to prevent cells from being preserved from damage due to freezing at low temperatures or heating to room temperature. Cryopreservation agents and optimal cooling coefficients can protect against cell damage. Cryoprotective agents that can be used include but are not limited to dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol , polyvinylpyrrolidine (Rinfret, Ann. NY
Acad. Sci., 1960; 85: 576) and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling coefficient is 1 ° to 3 ° C / minute. After at least two hours, T cells have reached a temperature of -80 ° C. and can be disposed directly into liquid nitrogen (-196 ° C.) for permanent storage such as in a long-term cryogenic storage vessel.
T cells
[0165] The present invention contemplates the manufacture of improved T cell compositions. T cells can be autologous / autogenous ("self") or non-autologous ("non-self", for example, allogeneic, syngeneic or xenogenetic). In preferred embodiments, T cells are obtained from a mammalian individual. In a more preferred embodiment, T cells are obtained from a primate individual. In a more preferred embodiment, T cells are obtained from a human subject.
[0166] T cells can be obtained from a number of sources that include, but are not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes, cord blood, thymus tissue, tissue from a field of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from an individual using any number of techniques known to those skilled in the art, such as sedimentation, for example, FICOLTM separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, which include T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for further processing. The cells can be washed with PBS or another suitable solution that is devoid of calcium, magnesium and most, if not all, divalent cations. As can be seen by those skilled in the art, a washing step can be performed by methods known to those skilled in the art, such as by using a semi-automated direct flow centrifuge. For example, the Cobe 2991 cell processor, the Baxter CitoMato, or the like. After washing, cells can be resuspended in a variety of biocompatible buffers or another saline solution with or without a buffer. In certain embodiments, the undesirable components of the apheresis sample can be removed in a cell directly resuspended in culture medium.
[0167] In particular embodiments, the cell population comprising T cells, for example, PBMCs, is used in the manufacturing methods contemplated here. In other embodiments, an isolated or purified T cell population is used in the manufacturing methods contemplated here. The cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLTM gradient. In some modalities, after isolation of PBMC, not only cytotoxic lymphocytes but also helper T lymphocytes can be classified into subpopulations of natural, memory and effector T cells either before or after activation, expansion, and / or genetic modification.
[0168] A specific subpopulation of T cells, which express one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127 and HLA-DR can be further isolated by positive or negative selection techniques . In one embodiment, a specific subpopulation of T cells, which express one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122 and CD127 is further isolated by positive or negative selection techniques. In various embodiments, compositions made of T cells do not express or do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3.
[0169] In one embodiment, expression of one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122 and CD127 is greater than at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, or more compared to the activated T cell population and expanded without a PI3K / AKT / mTOR inhibitor.
[0170] In one embodiment, the expression of one or more of the markers selected from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3 and LAG3 is reduced by at least 1.5 times, at least 2 times at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, or more compared to population of T cells activated and expanded with a PI3K / AKT / mTOR inhibitor.
[0171] In one embodiment, the manufacturing methods contemplated here increase the number of T cells comprising one or more natural cell markers or potent developed T cells. Without wishing to be tied to any particular theory, the present inventors believe that treating the cell population comprising T cells with one or more PI3K / AKT / mTOR inhibitors disassociates T cell proliferation and differentiation signals and thereby results in an increase in the expansion of potent developed T cells and provides a more robust and effective adoptive immunotherapy than existing T cell therapies.
[0172] Illustrative examples of natural cell markers or larger potent developed T cells in T cells manufactured using the methods contemplated herein include, but are not limited to CD62L, CCR7, CD28, CD27, CD95, CD122 and CD127. In particular embodiments, natural T cells do not express or substantially do not express one or more of the following markers: CD57, CD244, CD160, PD-1, BTLA, CD45RA, CTLA4, TIM3 and LAG3.
[0173] With regard to T cells, the T cell populations that result from the various expansion methodologies contemplated here may have a variety of specific phenotypic properties, depending on the conditions employed. In various embodiments, the expanded T cell populations comprise one or more of the following phenotypic markers: CCR7, CD3, CD4, CD8, CD27, CD28, CD62L, CD95, CD122, CD127 and HLA-DR.
[0174] In one embodiment, said phenotypic markers include the enhanced expression of one or more of, or all of CD62L, CCR7, CD28, CD27, CD122 and CD127. In particular embodiments, CD8 + T lymphocytes characterized by the expression of phenotypic markers of natural T cells including CD62L, CCR7, CD28, CD27, CD122 and CD127 are expanded.
[0175] In particular embodiments, T cells characterized by the expression of phenotypic markers of central memory T cells that include CD45RO, CD62L, CCR7, CD28, CD27, CD122 and CD127 and negative for granzyme B are expanded. In some embodiments, the central memory T cells are CD45RO +, CD62L +, CD8 + T cells.
[0176] In certain embodiments, CD4 + T lymphocytes characterized by the expression of phenotypic markers of natural CD4 + cells that include CD62L and negative for CD45RA and / or CD45RO expression are expanded. In some embodiments, CD4 + cells characterized by the expression of phenotypic markers of central memory CD4 + cells that include CD62L and CD45RO positive. In some embodiments, effector CD4 + cells are CD62L positive and CD45RO negative.
[0177] In certain embodiments, T cells are isolated from an individual and modified without further manipulation ex vivo or in vitro. Said cells can then be directly re-administered to the individual. In additional embodiments, T cells are first activated and stimulated to proliferate in vitro before being genetically modified to express a TCR or CAR modified by genetic engineering. In this regard, T cells can be cultured before and / or after being genetically modified (ie, transduced or transfected to express a TCR or CAR modified by genetic engineering contemplated here).
Activation and Expansion [0178] In order to achieve sufficient therapeutic doses of T cell compositions, T cells are often subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be activated and expanded in general using methods as described, for example, in US Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety. T cells modified to express a TCR or CAR modified by genetic engineering can be activated and expanded before and / or after T cells are modified. In addition, T cells can be brought into contact with one or more agents that modulate the PI3K / Akt / mTOR cell signaling path before, during, and / or after activation and / or expansion. In one embodiment, the T cells manufactured by the methods contemplated here undergo one, two, three, four, or five or more rounds of activation and expansion, each of which may include one or more agents that modulate the cell signaling path. PI3K / Akt / mTOR.
[0179] In one embodiment, a co-stimulatory ligand is presented in a cell that presents an antigen (for example, aAPC, dendritic cell, B cell and the like) that specifically binds to a cognate co-stimulatory molecule in the T cell , thereby providing a signal which, in addition to the main signal provided, for example, by binding a TCR / CD3 complex, mediates the desired T cell response. Suitable ligand co-stimulators include, but are not limited to, CD7 , B7-1 (CD80), B7-2 (CD86), PD-L 1, PD-L2, 4-1BBL, OX40L, inducible ligand co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM) , CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, beta lymphotoxin receptor, ILT3, ILT4, an agonist or antibody that binds to a Tol ligand receptor and a ligand that specifically binds to B7-H3.
[0180] In a particular embodiment, a co-stimulatory ligand comprises an antibody or antigen binding fragment thereof that specifically binds to a co-stimulatory molecule present in the T cell, which includes but is not limited to CD27, CD28, 4- IBB, OX40, CD30, CD40, PD-1, 1COS, antigen associated with lymphocyte-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds to with CD83.
[0181] Suitable co-stimulatory ligands additionally include target antigens, which can be provided in soluble form or expressed in APCs or aAPCs, which bind modified TCRs or CARs by genetic engineering expressed in modified T cells.
[0182] In several embodiments, a method for making the T cells contemplated here comprises activating the cell population that comprises T cells and expanding the T cell population. T cell activation can be accomplished by providing a primary stimulation signal through of the TCR / CD3 complex of the T cell or via the stimulation of the surface protein CD2 and by providing a secondary signal of costimulation through an accessory molecule, for example, CD28.
[0183] The TCR / CD3 complex can be stimulated by contacting the T cell with a suitable CD3 binding agent, for example, a CD3 ligand or anti-CD3 monoclonal antibody. Illustrative examples of CD3 antibodies include, but are not limited to OKT3, G19-4, BC3 and 64.1.
[0184] In another embodiment, a CD2 binding agent can be used to provide a major stimulation signal to T cells. Illustrative examples of CD2 binding agents include, but are not limited to, CD2 ligands and anti-CD2 antibody, for example , the T11.3 antibody in combination with the T11.1 or T11.2 antibody (Meuer, SC et al. (1984) Cel 36: 897-906) and the 9.6 antibody (which recognizes the same epitope as TI 1.1) in combination with the 9-1 antibody (Yang, SY et al. (1986) J. Immunol. 137: 1097-1100). Other antibodies that bind to the same epitopes as any of the antibodies described above can also be used. Additional antibodies, or combinations of antibodies, can be prepared and identified by standard techniques as described elsewhere here.
[0185] In addition to the main stimulation signal provided through the TCR / CD3 complex, or via CD2, induction of Ts cell response requires a second, co-stimulatory signal. In particular embodiments, a CD28 binding agent can be used to provide a costimulatory signal. Illustrative examples of CD28s binding agent include but are not limited to: natural CD28 linkers, for example, a natural linker for CD28 (for example, a member of the B7 family of proteins, such as B7-1 (CD80) and B7- 2 (CD86) and anti-CD28 monoclonal antibody or a fragment thereof capable of cross-linking the CD28 molecule, for example, monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248, 23.2 and EX5.3D10.
[0186] In one embodiment, the molecule providing the main stimulation signal, for example the molecule providing stimulation through the TCR / CD3 or CD2 complex and the co-stimulatory molecule are coupled to the same surface.
[0187] In certain embodiments, the binding agents that provide stimulatory and co-stimulatory signals are located on the cell surface. This can be accomplished by transfecting or transducing the cell with a nucleic acid that encodes the binding agent in a form suitable for its expression on the cell surface or alternatively by coupling a binding agent to the cell surface.
[0188] In another embodiment, the molecule providing the main stimulation signal, for example, the molecule that provides stimulation through the TCR / CD3 or CD2 complex and the co-stimulatory molecule are displaced in the cell that presents the antigens.
[0189] In one embodiment, the molecule providing the main stimulation signal, for example, the molecule providing stimulation through the TCR / CD3 or CD2 complex and the co-stimulatory molecule are provided on separate surfaces.
[0190] In a given embodiment, one of the binding agents that provide stimulatory and co-stimulatory signals is soluble (provided in solution) and the other agent (s) is provided on one or more surfaces.
[0191] In a particular embodiment, the binding agents that provide stimulatory and co-stimulatory signals are both provided in a soluble form (provided in solution).
[0192] In several embodiments, the methods for making the T cells contemplated here comprise activating the T cells with anti-CD3 and anti-CD28 antibody.
[0193] T cell compositions manufactured by the methods contemplated herein comprise activated and / or expanded T cells in the presence of one or more agents that inhibit a PI3K / Akt / mTOR cell signaling path. T cells modified to express a TCR or CAR modified by genetic engineering can be activated and expanded before and / or after T cells are modified. In particular embodiments, the T cell population is activated, modified to express a TCR or CAR modified by genetic engineering and then cultivated by expansion.
[0194] In one embodiment, T cells manufactured by the methods contemplated here comprise a greater number of T cells that express markers of high proliferative potential and the ability to self-regenerate but that do not express or substantially express cell differentiation markers Undetectable tees. Said T cells can be repeatedly activated and expanded in a robust manner and thereby provide an enhanced therapeutic T cell composition.
[0195] In one embodiment, the population of activated and expanded T cells in the presence of one or more agents that inhibit a PI3K / Akt / mTOR cell signaling pathway is expanded at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 250 times, at least 500 times, at least 1000 times, or more compared to the population of activated and expanded T cells without a PI3K / AKT / mTOR inhibitor.
[0196] In one embodiment, the T cell population characterized by the expression of young T cell markers is activated and expanded in the presence of one or more agents that inhibit a PI3K / Akt / mTOR cell signaling pathway is expanded at least 1.5 times at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times at least 50 times, at least 100 times, at least 250 times, at least 500 times, at least 1000 times, or more compared to the population of activated and expanded T cells without a PI3K / AKT / mTOR inhibitor.
[0197] In one embodiment, expanding T cells activated by the methods contemplated here further comprises cultivating the cell population comprising T cells for several hours (about 3 hours) to about 7 days to about 28 days or any number value hour between them. In another embodiment, the T cell composition can be cultured for 14 days. In a particular embodiment, T cells are cultured for about 21 days. In another embodiment, T cell compositions are grown for about 2-3 days. Several stimulation / activation / expansion cycles can also be desired so that the T cell culture time can be 60 days or more.
[0198] In particular embodiments, suitable conditions for T cell culture include a suitable medium (for example, Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability that include, but are not limited to, serum (eg, fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL- 21, GM-CSF, IL-10, IL-12, IL-15, TGFp and TNF-α or any other additives suitable for the development of cells known to those skilled in the art.
[0199] Additional illustrative examples of cell culture medium include, but are not limited to RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, with the addition of amino acids, sodium pyruvate and vitamins, either free of serum or supplemented with an adequate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine (s) sufficient to the development and expansion of T cells.
[0200] Illustrative examples of other additives for T cell expansion include, but are not limited to, surfactant, piasmanate, pH buffers such as HEPES and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol [0201] Antibiotics , for example, penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that have to be infused into an individual. The target cells are maintained under conditions necessary to support development, for example, at an appropriate temperature (for example, 37 ° C) and atmosphere (for example, air plus 5% CO2).
[0202] In particular modalities, PBMCs or isolated T cells are brought into contact with a stimulatory agent and costimulatory agent, such as anti-CD3 and anti-CD28s antibody, usually attached to a bead or other surface, in a medium culture with suitable cytokines, such as IL-2, IL-7, and / or IL-15.
[0203] In other modalities, artificial APC (aAPC) produced by genetic manipulation K562, U937, 721.221, T2 and C1R cells to direct the stable expression and secretion of a variety of costimulatory molecules and cytokines. In a particular embodiment K32 or U32 aAPCs are used to direct the display of one or more antibody-based stimulatory molecules on the cell's AAPC surface. T cell populations can be expanded by aAPCs that express a variety of costimulatory molecules that include, but are not limited to, CD137L (4-1BBL), CD134L (OX40L), and / or CD80 or CD86. Finally, aAPCs provide an efficient platform to genetically expand modified T cells and to maintain the expression of CD28 and CD28 T cells. The aAPCs provided in WO 03/057171 and US2003 / 0147869 are hereby incorporated by reference in their entirety.
Agents [0204] In various embodiments, a method for making T cells is provided that expands non-differentiated cells or potent developed T cells that comprises contacting T cells with an agent that modulates a PI3K / AKT / mTOR pathway in the cells. The cells can be brought into contact before, during, and / or after activation and expansion. T cell compositions retain sufficient T cell power so that they can undergo multiple rounds of expansion without a substantial increase in differentiation.
[0205] As used here, the terms “modulate”, “modulator”, or “modulation agent” or comparable term refer to an agent's ability to elicit a change in a signaling cell path. A modulator can increase or decrease an amount, activity of a path component, or increase or decrease the desired effect or output of the signal cell path. In one embodiment, the modulator is an inhibitor. In another modality, the modulator is an activator.
[0206] An "agent" refers to a compound, small molecule, for example, small organic molecule, nucleic acid, polypeptide, or a fragment, isoform, variant, analog, or derivative thereof used in modulating a PI3K path / AKT / mTOR.
[0207] A "small molecule" refers to a composition that has a molecular weight of less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD , less than about 1 kD, or less than about .5 kD. Small molecules can comprise nucleic acids, peptides, polypeptides, peptidomimetics, peptides, carbohydrates, lipids, components of the same or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungi, bacteria, or seaweed extracts, are known in the art and can be searched with any of the tests of the present invention. Examples of methods for the synthesis of molecular libraries can be found at: (Carel et al., 1994a; Carel et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Galop et al., 1994;
Zuckermann et al., 1994).
[0208] An "analogue" refers to a small organic compound, a nucleotide, a protein, or a polypeptide that has an activity or function (S) similar or identical to that of the compound, nucleotide, protein or polypeptide or compound having the desired activity of the present invention, but need not necessarily understand the sequence or structure that is similar or identical to the sequence or structure of the preferred embodiment.
[0209] A "derivative" refers to either a compound, a protein or polypeptide that comprises an amino acid sequence of a parent protein or polypeptide that has been altered by the introduction of substitutions, deletions or additions of amino acid residue, or a nucleic acid or nucleotide that has been modified either by introducing nucleotide substitutions or deletions, additions or mutations. The nucleic acid, nucleotide, protein or polypeptide derivative has a similar or identical function to that of the parent polypeptide.
[0210] In several modalities, the agent that modulates a PI3K / AKT / mTOR path activates a component of the path. An "activator", or "agonist" refers to an agent that promotes, increases, or induces one or more activities of a molecule in a PI3K / AKT / mTOR pathway that includes, without limitation, the molecule that inhibits one or more activities of a PI3K, an Akt, or an mTOR (or mTORC1, mTORC2 complex).
[0211] In several modalities, the agent that modulates a PI3K / AKT / mTOR path inhibits a component of the path. An "inhibitor" or "antagonist" refers to an agent that inhibits, decreases, or reduces one or more activities of a molecule in a PI3K / AKT / mTOR path that includes, without limitation, a PI3K, an Akt, or a mTOR (or mTORC1, mTORC2 complex). In one embodiment, the inhibitor is a double molecule inhibitor. In one embodiment, the inhibitor prevents the formation of protein complexes such as mTORCl or related complexes. In a particular embodiment, the inhibitor can inhibit a class of molecules that have the same or substantially similar activities (a pan-inhibitor) or can specifically inhibit the activity of the molecule (a selective or specific inhibitor). The inhibition can also be irreversible or reversible.
[0212] In one embodiment, the inhibitor has an IC 50 of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 10 pM, at least 50 pM, or at least 100 pM. IC50 determinations can be performed using any conventional techniques known in the art. For example, an IC50 can be determined by measuring the activity of a particular enzyme in the presence of a range of concentrations of the inhibitor under study. The values obtained in an experimental way of enzymatic activity are then plotted against the concentrations of the inhibitor used. The concentration of the inhibitor that shows 50% enzyme activity (as compared to the activity in the absence of any inhibitor) is obtained as the “IC50” value. Similarly, other inhibitory concentrations can be defined through appropriate activity determinations.
[0213] In various modalities, T cells are contacted or treated or cultured with one or more modulators of a PI3K / AKT / mTOR pathway at a concentration of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 pM, at least 10 pM, at least 50 pM, at least 100 pM, or at least 1 M .
[0214] In particular modalities, T cells can be contacted or treated or cultured with one or more modulators of a PI3K / AKT / mTOR path for at least 12 hours, 18 hours, at least 1,2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1,2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9 , or 10 or more expansion rounds.
PI3K / AKT / mTOR path
[0215] The phosphatidyl-inositol-3 kinase / Akt / rapamycin target mammal (PI3K / Akt / mTOR) pathway serves as a conduit for integrating signaling development factor with cell proliferation, differentiation, metabolism and survival. PI3Ks are the family of highly conserved intracellular lipid kinases. Class IA PI3Ks are activated by receptor tyrosine development factor kinases (RTKs), either directly or through interaction with the insulin receptor substrate family of the adapter molecules. Said activity results in the production of phosphatidyl-inositol-3,4,5-triphosphate (PIP3) a serine / threonine kinase Akt regulator. mTOR acts through the canonical PI3K pathway through 2 distinct complexes, each of which is characterized by different liaison partners that confer different activities. mTORC1 (mTOR in the complex with PRAS40, raptor and mLST8 / GbL) acts as an effector downstream of signaling PI3K / Akt, linking the development factor signals with the protein translation, cell development, proliferation and survival. mTORC2 (mTOR in complex with rictor, mSIN1, protor and mLST8) acts as an activator upstream of Akt.
[0216] With PI3K activation mediated by a development factor receptor, Akt is recruited to the membrane by interacting its pleckstrin homology domain with PIP3, thereby exposing its activation cycle and allowing phosphorylation in threonine 308 (Thr308) by constructively active phosphoinositide-dependent protein kinase 1 (PDK1). For maximum activation, Akt is also phosphorylated by mTORC2, in serine 473 (Ser473) from its C-terminal hydrophobic motif. DNA-PK and HSP have also been shown to be important in regulating Akt activity. Akt activates mTORC1 through inhibitory phosphorylation of TSC2, which together with TSC1, downregulates mTORC1 by inhibiting Rheb GTPase, a positive regulator of mTORCI. mTORCI has 2 well-defined substrates, p70S6K (referred to here after as S6K1) and 4E-BP1, both of which critically regulate protein synthesis. Thus, mTORC1 is an important effector downstream of PI3K, which links the signaling development factor with protein translation and cell proliferation.
b) mTOR inhibitors
[0217] The terms "mTOR inhibitor" or "agent that inhibits mTOR" refer to a nucleic acid, peptide, compound, or small organic molecule that inhibits at least one activity of a mTOR protein, such as, for example, serine / threonine protein kinase activity on at least one of its substrates (for example, p70S6 kinase 1,4E-BP1, AKT / PKB and eEF2). MTOR inhibitors are able to bind directly to and inhibit mTORC1, mTORC2 or both mTORC1 and mTORC2.
[0218] The inhibition of mTORC1 and / or mTORC2 activity can be determined by the reduction in signal transduction of the PI3K / AKT / mTOR path. A wide variety of readings can be used to establish the reduction in the output of that signaling path. Some exemplary, non-limiting readings include (1) a decrease in Akt phosphorylation in residues, which include but are not limited to 5473 and T308; (2) a decrease in Akt activation as evidenced, for example, by the reduction in phosphorylation of Akt substrates that include but are not limited to Fox01 / O3a T24 / 32, GSK3a / p; S21 / 9 and TSC2 T1462; (3) a decrease in the phosphorylation of signaling molecules downstream of mTOR, which include but are not limited to ribosomal S6 S240 / 244, 70S6K T389 and 4EBP1 T37 / 46; and (4) inhibition of cancer cell proliferation.
[0219] In one embodiment, mTOR inhibitors are active field inhibitors. Said are mTOR inhibitors that bind to the ATP binding field (also referred to as ATP binding pocket) of mTOR and the inhibition of the catalytic activity of both mTORCI and mTORC2. A class of active field inhibitors suitable for use in the T cell manufacturing methods contemplated here are dual specificity inhibitors that aim to and directly inhibit both PI3K and mTOR. Dual specificity inhibitors bind to both the ATP binding field of mTOR and PI3K. Illustrative examples of said inhibitors include, but are not limited to: imidazoquinazolines, wortmanin, LY294002, PI-103 (Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765 (Exelixis) and NVP-BEZ235 (Novartis).
[0220] Another class of active field mTOR inhibitors suitable for use in the methods contemplated here selectively inhibit the activity of mTORC1 and mTORC2 with respect to one or more type I phosphatidylinositol 3-kinases, for example, PI3 kinase α, β, γ , or δ. Said active field inhibitors bind to the mTOR active field but not to PI3K. Illustrative examples of said inhibitors include, but are not limited to: pyrazolopyrimidines, Torin1 (Guertin and Sabatini), PP242 (2- (4-Amino-1-isopropyl-1H-pyrazolo [3,4-d] pyrimidin-3-yl ) -1H-indol-5-ol), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth) and AZD8055 (Liu et al., Nature Review, 8, 627-644, 2009). I
[0221] In one embodiment, a selective mTOR inhibitor refers to an agent that exhibits a 50% inhibitory concentration (IC50) with respect to mTORC1 and / or mTORC2, which is at least 10 times, at least 20 times, at least at least 50 times, at least 100 times, at least 1000 times, or more, below the IC50 of the inhibitor with respect to one, two, three, or more types I PI3-kinases or all types of I PI3-kinases .
[0222] Another class of mTOR inhibitors for use in the present invention is referred to here as "rapalogs". As used herein the term "rapalogs" refers to compounds that specifically bind to the FRB domain of mTOR (FKBP rapamycin binding domain), are structurally related to rapamycin and retain the mTOR inhibiting properties. The term rapalogs excludes rapamycin. Rapalogs include rapamycin esters, ethers, oximes, hydrazones and hydroxylamines, as well as compounds in which the functional groups in the rapamycin core structure have been modified, for example, by reduction or oxidation. Pharmaceutically acceptable salts of said compounds are also considered to be derived from rapamycin. Illustrative examples of rapalogs suitable for use in the methods contemplated herein include, without limitation, temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca) and OSI-027 (OSI).
[0223] In one embodiment, the agent is mTOR-inhibiting rapamycin (sirolimus).
[0224] In a particular embodiment, examples of mTOR inhibitors for use in the present invention that inhibit be either mTORC1, mTORC2 or both mTORC1 and mTORC2 with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM, or less. In one aspect, an mTOR inhibitor for use in the present invention inhibits either mTORC1, mTORC2 or both mTORC1 and mTORC2 with an IC 50 from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.
[0225] In one embodiment, examples of mTOR inhibitors inhibit either PI3K and mTORC1 or mTORC2 or both mTORC1 and mTORC2 and PI3K with an IC50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM, or less. In one aspect, an mTOR inhibitor for use in the present invention inhibits PI3K and mTORCI or mTORC2 or both mTORCI and mTORC2 and PI3K with an IC 50 from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.
[0226] Additional illustrative examples of mTOR inhibitors suitable for use in particular embodiments contemplated herein include, but are not limited to AZD8055, INK128, rapamycin, PF-04691502 and everolimus.
[0227] mTOR demonstrated robust and specific catalytic activity towards the proteins of the physiological substrate, p70 S6 ribosomal protein kinase I (p70S6K1) and eIF4E 1 binding protein (4EBP1) as measured by phosphorus-specific antibodies in Western blotting.
[0228] In one embodiment, the PI3K / AKT / mTOR pathway inhibitor is a s6 kinase inhibitor selected from the group consisting of: BI-D1870, H89, PF-4708671, FMK and AT7867.
c) PI3K inhibitors
[0229] As used herein, the term "PI3K inhibitor" refers to a nucleic acid, peptide, compound, or small organic molecule that binds to and inhibits at least one PI3K activity. PI3K proteins can be divided into three classes, class 1 PI3Ks, class 2 PI3Ks and class 3 PI3Ks. Class 1 PI3Ks exist as heterodimers that consists of one of four catalytic subunits p110 (p110α, p110β, p110õ and p110γ) and one of two families of regulatory subunits. A PI3K inhibitor of the present invention preferably targets class 1 PI3K inhibitors. In one embodiment, a PI3K inhibitor will exhibit selectivity for one or more isoforms of class 1 PI3K inhibitors (i.e., selectivity for p110a, p110p, p110õ and p110Y or one or more of p110a, p110p, p110õ and p110Y). In another aspect, a PI3K inhibitor will not exhibit selectivity for isoforms and will be considered a "pan-PI3K inhibitor". In one embodiment, a PI3K inhibitor will compete for binding with ATP for the catalytic domain of PI3K.
[0230] In certain embodiments, a PI3K inhibitor can, for example, target PI3K as well as additional proteins in the PI3K-AKT-mTOR path. In particular embodiments, a PI3K inhibitor that targets both mTOR and PI3K can be referred to as either an mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that targets only PI3K can be referred to as a selective PI3K inhibitor. In one embodiment, a selective PI3K inhibitor can be understood to refer to an agent that exhibits a 50% inhibitory concentration with respect to a PI3K that is at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times, or more, lower than the IC50 of the inhibitor with respect to mTOR and / or other proteins in the path.
[0231] In a particular embodiment, examples of PI3K inhibitors inhibit PI3K with an IC 50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM, or less. In one embodiment, a PI3K inhibitor inhibits PI3K with an IC 50 from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.
[0232] Illustrative examples of PI3K inhibitors suitable for use in the T cell manufacturing methods contemplated here include, but are not limited to BKM120 (class 1 PI3K inhibitor, Novartis), XL147 (class 1 PI3K inhibitor, Exelixis), (pan-PI3K inhibitor, GlaxoSmithKline) and PX-866 (class 1 PI3K inhibitor; p110α, p110p and p110γ isoforms, Oncothyreon).
[0233] Other illustrative examples of selective PI3K inhibitors include, but are not limited to BIL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474 and IPI-145.
[0234] Additional illustrative examples of pan-PI3K inhibitors include, but are not limited to BEZ235, LY294002, GSK1059615 and GDC-0941.
d) AKT inhibitors
[0235] As used here, the term "AKT inhibitor" refers to a nucleic acid, peptide, compound, or small organic molecule that inhibits at least one AKT activity. AKT inhibitors can be grouped into several classes, which include lipid-based inhibitors (for example, inhibitors that target the AKT domain of pleckstrin homology that prevents AKT from being located in plasma membranes), competitive ATP inhibitors and inhibitors allosteric. In one embodiment, AKT Inhibitors act by binding to the AKT catalytic field. In a particular embodiment, AKT inhibitors act by inhibiting phosphorylation of downstream AKT targets such as mTOR. In another embodiment, AKT activity is inhibited by inhibiting input signals to activate Akt by inhibiting, for example, AKT DNA-PK activation, AKT PDK-1 activation, and / or mTORC2 activation Akt.
[0236] AKT inhibitors can target all three isoforms of AKT, AKT1, AKT2, AKT3 or can be selective isoforms and target only one or two of the AKT isoforms. In one embodiment, an AKT inhibitor can target AKT as well as additional proteins in the PI3K-AKT-mTOR path. An AKT inhibitor that only targets AKT can be referred to as a selective AKT inhibitor. In one embodiment, a selective AKT inhibitor can be understood to refer to an agent that exhibits a 50% inhibitory concentration with respect to AKT that is at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 1000 times, or lower than the IC50 of the inhibitor with respect to other proteins in the path.
[0237] In a particular embodiment, examples of AKT Inhibitors inhibit AKT with an IC 50 (concentration that inhibits 50% of activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM, or less. In one embodiment, an Akt inhibits AKT with an IC 50 from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.
[0238] Illustrative examples of AKT inhibitors for use in combination with drug-antibody conjugates based on auristatin include, for example, perifosine (Keryx), MK2206 (Merck), VQD-002 (VioQuest), XL418 (Exelixis), GSK690693, GDC-0068 and PX316 (PROLX Pharmaceuticals).
[0239] An illustrative non-limiting example of a selective Akt1 inhibitor is A-674563.
[0240] An illustrative non-limiting example of a selective Akt2 inhibitor is CCT128930.
[0241] In particular embodiments, the AKT inhibitor the activation of Akt's DNA-PK, the activation of Akt's PDK-1, the activation of Akt's mTORC2, or the activation of Akt's HSP.
[0242] Illustrative examples of DNA-PK inhibitors include, but are not limited to, NU7441, PI-103, NU7026, PIK-75 and PP-121.
Genetically modified T cell receptors and chimeric antigen receptors [0243] The contemplated T cell manufacturing methods are particularly useful for expanding T cells modified to express with high affinity T cell receptors (genetically modified TCRs) or chimeric antigen receptor ( CARs) without a concomitant increase in the differentiation of said modified T cells. In one embodiment, the T cell is genetically modified to express one or more TCRs or CARs modified by genetic engineering. As used here, T cells modified to express a genetically engineered TCR or CAR contemplated here can be referred to as, "antigen-specific redirected T cells." 1. Genetically modified TCRs [0244] Naturally occurring T cell receptors comprise two subunits, an α subunit and a β subunit, each of which is a unique protein produced by a recombination event in each T cell genome. of TCRs can be screened for their selectivity to particular target antigens. In this way, natural TCRs, which have a high avidity and reactivity towards target antigens can be selected, cloned and subsequently introduced into the population of T cells used for adoptive immunotherapy.
[0245] In one embodiment, T cells are modified by introducing a polynucleotide that encodes the subunit of a TCR that has the ability to form TCRs that confer specificity to T cells for tumor cells that express a target antigen. In particular embodiments, the subunits have one or more substitutions, deletions, insertions, or modifications of amino acids compared to the naturally occurring subunit, as long as the subunits retain the ability to form TCRs that confer on the transfected T cells the ability to orient themselves to target cells and participate in immunologically relevant signaling cytokine. Genetically modified TCRs preferably also bind to target cells that exhibit the relevant tumor-associated peptide with high avidity and optionally mediate the efficient extermination of target cells that display the relevant peptide in vivo.
[0246] Nucleic acids encoding genetically modified TCRs are preferably isolated from their natural context on a (naturally occurring) T cell chromosome and can be incorporated into suitable vectors as described elsewhere here. Not only the nucleic acids but also the vectors that comprise them can be usefully transferred into the cell, the cell of which is preferably a T cell. The modified T cells are then capable of expressing one or more TCR chains (and preferably two strands) terminated by the transduced nucleic acid or nucleic acids. In preferred embodiments, the genetically modified TCR is an exogenous TCR because it is introduced into T cells that do not normally express the particular TCR. The essential aspect of genetically modified TCRs is that they are highly avid for a tumor antigen presented by a large histocompatibility complex (MHC) or similar immune component. Unlike genetically modified TCRs, CARs are genetically engineered to bind to target antigens in an MHC-independent way.
[0247] The protein encoded by the nucleic acids of the present invention can be expressed with additional polypeptides attached to an aminoterminal or carboxyl-terminal portion of the α or β chain of the present invention of a TCR provided that the additional fixed polypeptide does not interfere with the ability the α chain or the β chain to form a functional T cell receptor and MHC-dependent antigen recognition.
[0248] Antigens that are recognized by the genetically modified TCRs contemplated here include, but are not limited to, cancer antigens, which include the antigens in both hematological cancers and solid tumors. Illustrative antigens include, but are not limited to, alpha folate receptor, 5T4, integrin ave6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70 , CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, Fetal AchR, FRa, GD2, GD3, Glipican -3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY- ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivina, TAG72 , TEMs and VEGFR2. 2. Chimeric Antigen Receptors (CARs) [0249] The T cell manufacturing methods contemplated here include modifying T cells to express one or more CARs as contemplated here. In various embodiments, the present invention provides T cells modified by genetic engineering with vectors designed to express CARs that redirect cytotoxicity towards tumor cells. CARs are molecules that combine antibody-based specificity for a target antigen (e.g., tumor antigen) with an intracellular T cell receptor activation domain to generate a chimeric protein that exhibits specific anti-tumor cellular immune activity. As used here, the term, "chimeric", describes being composed of parts of different proteins or DNAs from different origins.
[0250] The CARs contemplated here comprise an extracellular domain that binds to a specific target antigen (also referred to as an antigen-specific binding domain or binding domain), a transmembrane domain and an intracellular signaling domain. The main characteristic of CARs is their ability to redirect the specificity of the immune effector cell, thereby triggering proliferation, cytokine production, phagocytosis or the production of molecules that can mediate the cell death of the cell that expresses the target antigen in a independent (MHC) mode of great histocompatibility, exploring the capabilities of specifically targeting the cell for monoclonal antibodies, soluble ligands or specific cell co-receptors.
[0251] In particular embodiments, a CAR comprises an extracellular binding domain that includes but is not limited to an antibody or antigen binding fragment thereof, a bound ligand, or the extracellular domain of a co-receptor, which specifically binds to a target antigen selected from the group consisting of: alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, AchR fetal, FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA -A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivina, TAG72, TEMs and VEGFR2; one or more articulation domains or spacer domains; a transmembrane domain that includes, but is not limited to, transmembrane domains from CD8a, CD4, CD45, PD-1 and CD152; one or more intracellular co-stimulatory signaling domains that include but are not limited to intracellular co-stimulatory signaling domains from CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4) , CD273 (PD-L2), CD274 (PD-L1) and CD278 (ICOS); and a major signaling domain from CD3Z or FcRy.
Binding domain [0252] In particular embodiments, CARs contemplated herein comprise an extracellular binding domain that specifically binds to a target polypeptide, for example, target antigen, expressed in the tumor cell. As used here, the terms, "binding domain", "extracellular domain", "extracellular binding domain", "antigen specific binding domain" and "extracellular antigen specific binding domain", are used interchangeably and provide a CAR with the ability to specifically bind to the target antigen of interest. A binding domain can comprise any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biological molecule (for example, a cell surface receptor or tumor protein, lipid, polysaccharide, or other molecule cell surface target, or a component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for the biological molecule of interest.
[0253] In particular embodiments, the extracellular binding domain of a CAR comprises an antibody or antigen binding fragment thereof. An "antibody" refers to a binding agent that is a polypeptide that comprises at least one variable region of light or heavy chain immunoglobulin that specifically recognizes and binds to an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid that contains an antigenic determinant, such as those recognized by an immune cell. Antibodies include antigen-binding fragments thereof. The term also includes forms engineered by genetic engineering such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as, bi-specific antibodies) and antigen-binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.
[0254] In particular embodiments, the target antigen is an epitope of an alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6 , CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, fetal AchR, FRa , GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1 , SSX, Survivin, TAG72, TEMs, or VEGFR2 polypeptide.
[0255] Variable regions of light chain and heavy chain contain a "structure" region interrupted by three hypervariable regions, also called "complementarity determination regions" or "CDRs". CDRs can be defined or identified by conventional methods, such as by sequence according to Kabat et al (Wu, TT and Kabat, EA, J Exp Med. 132 (2): 211-50, (1970); Borden, P and Kabat EA, PNAS, 84: 2440-2443 (1987); (see, Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is incorporated herein by reference), or peka structure according to Chotia et al (Choitia, C. and Lesk, AM, J Mol. Biol., 196 (4): 901-917 (1987), Choitia, C. et al, Nature, 342: 877 - 883 (1989)).
[0256] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The structure regions of an antibody, which are the combined structure regions of the light and heavy constituent chains, serve to position and align the CDRs in a three-dimensional space. CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each chain are typically referred to as CDR1, CDR2 and CDR3, numbered in sequence starting from the N-terminus and are also typically identified by the chain in which the particular CDR is located. Thus, CDRs located in the variable domain of the antibody heavy chain are referred to as CDRH1, CDRH2 and CDRH3, while CDRs located in the variable domain of the antibody light chain are referred to as CDRL1, CDRL2 and CDRL3. Antibodies with different specificities (that is, different combination fields for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determination residues (SDRs).
[0257] References to "VH" or "VH" refers to the variable region of an immunoglobulin heavy chain, which includes that of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment as described here. References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, which includes that of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment as described here.
[0258] A "monoclonal antibody" is an antibody produced by a single B lymphocyte clone or by the cell into which the light chain and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by producing hybrid antibody-forming cells from the fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.
[0259] A "chimeric antibody" has structure residues from one species, such as human and CDRs (which generally confer antigen binding) from another species, such as a mouse. In a particularly preferred embodiment, a CAR contemplated herein comprises an antigen-specific binding domain which is a chimeric antibody-binding fragment or antigen thereof.
[0260] In certain preferred embodiments, the antibody is a humanized antibody (such as a humanized monoclonal antibody) that specifically binds to the surface protein in the tumor cell. A "humanized" antibody is an immunoglobulin that includes a human frame region and one or more CDRs from a non-human immunoglobulin (for example, a mouse, rat, or synthetic). Humanized antibodies can be constructed by means of genetic manipulation (see for example, US patent No. 5,585,089).
[0261] In particular embodiments, the extracellular binding domain of a CAR comprises an antibody or antigen binding fragment thereof, which include but are not limited to a Camel Ig (camelid antibody (VHH)), NAR Ig, fragments Fab, fragments of Fab ', fragments of F (ab)' 2, fragments of F (ab) '3, Fv, single chain Fv antibody (“scFv”), bis-scFv, (scFv) 2, minibody, diabody, triacorp, tetribody, disulfide stabilized Fv protein (“dsFv”) and single domain antibody (sdAb, Nanocorp).
[0262] "Camel Ig" or "camelid VHH" as used here refers to the smallest known antigen binding unit of a heavy chain antibody (Koch-Nolte, et al, FASEB J., 21: 3490-3498 (2007 )). A "heavy chain antibody" or a "camelid antibody" refers to an antibody that contains two VH domains and no light chain (Riechmann L. et al, J. Immunol. Methods 231: 25-38 (1999); WO94 / 04678; WO94 / 25591; US Patent No. 6,005,079).
[0263] "New immunoglobulin antigen receptor" IgNAR "refers to the class of antibodies from a shark immune repertoire consisting of homodimers of a new antigen receptor (VNAR) variable domain and five receptor domains of constant new antigen (CNAR).
[0264] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding field and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. The Fab fragment contains the heavy and light chain variable dominoes and also contains the light chain constant domain and the first heavy chain constant domain (CH1). Fab 'fragments differ from Fab fragments by the addition of a few residues at the CARboxi end of the CH1 heavy chain domino which includes one or more cysteines from the antibody hinge region. Fab'-SH is the designation here for Fab 'in which the cysteine residue (s) of the constant domains support a free thio group. The F (ab ') 2 fragment of antibodies was originally produced as pairs of Fab' fragments that have articulating cysteines between them. Other chemical couplings of antibody fragments are also known.
[0265] "Fv" is the minimum antibody fragment that contains a complete antigen-binding field. In a single Fv (scFv) species, a variable domain of a heavy chain and a light chain can be covalently linked by the flexible peptide linker so that the light and heavy chains can associate in an analogous "dimeric" structure to that in a kind of two Fv chains.
[0266] The term "diabody" refers to antibody fragments with two antigen-binding fields, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding fields. Diabodies can be bivalent or bispecific. Diabodies are described more widely in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Holinger et al., PNAS USA 90: 6444-6448 (1993). Tribodies and tetribodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).
[0267] "Single antibody domain" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of a heavy chain antibody (VH domain) or the variable region of a light chain antibody ( VL domain) (Holt, L., et al, Trends in Biotechnology, 21 (11): 484-490).
[0268] "Single chain Fv" or "scFv" antibody fragments comprise the VH and VL antibody domains, where said domains are present in a single polypeptide chain and in any orientation (for example, VL-VH or VH-VL). In general, the scFv polypeptide additionally comprises a polypeptide linker between the VH and VL dominoes that allow scFv to form the desired structure for antigen binding. For a review of scFv, see, for example, Pluckthün, in The Farmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.
[0269] In preferred embodiments, a CAR contemplated here comprises an antigen-specific binding domain that is a scFv (a murine, human or humanized scFv) that binds to an antigen expressed in a cancer cell. In a given embodiment, scFv binds to an alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8 , CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3 , 'Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivina, TAG72, TEMs and VEGFR2. b) Ligands [0270] In certain modalities, the CARs contemplated here may comprise residues of ligands between the various domains, for example, between the Vh and Vl domains, added for adequate spacing and conformation of the molecule. the CARs contemplated here may comprise one, two, three, four, or five or more ligands. In particular embodiments, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intermediate amino acid length. In some embodiments, the ligand is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22 , 23, 24, 25, or more amino acids in length.
[0271] Illustrative examples of binders include polymers of glycine (G) n; glycine-serine polymers (G1-5S1-5) n, where n is an integer of at least one, two, three, four or five; glycine-alanine polymers; alanine-serine polymers; and other flexible binders known in the art. The glycine and glycine-serine polymers are relatively unstructured and therefore may be able to serve as a neutral tie between domains of fusion proteins such as the CARs described herein. Glycine accesses significantly more fi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art may recognize that the design of a CAR in particular embodiments may include binders that are all or partially flexible, so that the binder may include a flexible binder as well as one or more portions that provide less flexible structure to provide the desired CAR structure.
[0272] Other examples of linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: X); TGEKP (SEQ ID NO: X) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: X) (Pomerantz et al. 1995, supra); (GGGGS) n where = 1, 2, 3, 4 or 5 (SEQ ID NO: X) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: X) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: X) (Bird et al., 1988, Science 242: 423-426), GGRRGGGS (SEQ ID NO: X); LRQRDGERP (SEQ ID NO: X); LRQKDGGGSERP (SEQ ID NO: X); LRQKd (GGGS) 2 ERP (SEQ ID NO: X). Alternatively, flexible binders can be rationally designed using a computer program able to model both DNA binding fields and the peptides themselves (Desjarlais & Berg, PNAS 90: 2256-2260 (1993), PNAS 91: 11099-11103 (1994) or by fage display methods.
[0273] In particular embodiments, a CAR comprises a scFV which additionally comprises a variable region binding sequence. The “variable region binding sequence” is an amino acid sequence that connects the heavy chain variable region to a light chain variable region and provides a spacer function compatible with the interaction of the two binding subdomains so that the The resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same variable regions of light and heavy chain. In one embodiment, the variable region binding sequence is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more long amino acids. In a particular embodiment, the variable region binding sequence comprises a glycine-serine polymer (G1-5S1-5) n, where n is an integer of at least 1, 2, 3, 4, or 5. In another embodiment, the variable region binding sequence comprises the (G4S) 3 amino acid linker. c) Spacer domain [0274] In particular modalities, the CAR binding domain is followed by one or more “spacer domains”, which refers to the region that moves the antigen binding domain away from the surface of the effector cell to allow adequate cell / cell contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The spacer domain can be derived either from a natural, synthetic, semi-synthetic or recombinant source. In certain embodiments, a spacer domain is a portion of an immunoglobulin, which includes, but is not limited to, one or more heavy chain constant regions, for example, CH2 and CH3. The spacer domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
[0275] In one embodiment, the spacer domain comprises CH2 and CH3 of IgG1. d) Articulation domain [0276] The CAR binding domain is generally followed by one or more “articulation domains”, which plays a role in positioning the antigen-binding domain away from the surface of the effector cell to allow adequate cell / cell contact, antigen binding and activation. A CAR in general comprises one or more domains of articulation between the binding domain and the transmembrane (TM) domain. The articulation domain can be derived either from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain can include an amino acid sequence from a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
[0277] Articulation domains suitable for use in the CARs described here include the articulation region derived from extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28 and CD7, which can be wild type articulation regions from said molecules or can be changed. In another modality, the articulation domain comprises the CD8a articulation region. e) Transmembrane Domain (TM) [0278] The “transmembrane domain” is the portion of the CAR that merges with the extracellular binding and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain can be derived either from a natural, synthetic, semi-synthetic or recombinant source.
[0279] Illustrative TM domains can be derived from (i.e., comprise at least the transmembrane region (s) of) the alpha, beta, or zeta chain of the T cell receptor, epsilon CD3 , zeta CD3, CD4, CD5, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152 and CD154.
[0280] In one embodiment, the CARs contemplated here comprise a TM domain derived from CD8a. In another embodiment, a CAR contemplated herein comprises a TM domain derived from CD8a and a short oligo- or polypeptide linker, preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that bind to the TM domain and the intracellular CAR signaling domain. The glycine-serine binder provides a particularly suitable binder. f) Intracellular Signaling Domain [0281] In particular modalities, the CARs contemplated here comprise an intracellular signaling domain. An “intracellular signaling domain” refers to the part of a CAR that participates in transducing the CAR effective binding message to a target antigen within the effector cell immune to elicit the effector cell's function, for example, activation, cytokine production, proliferation and cytotoxic activity, which includes the release of cytotoxic factors to the target cell linked to CAR, or other cellular responses elicited with antigen binding to the extracellular domino of CAR.
[0282] The term "effector function" refers to a specialized function of the cell. Effective T cell function, for example, can be cytolytic or helping activity or activity that includes the secretion of a cytokine. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces the signal of the effector function and directs the cell to perform a specialized function. Although in general the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. In the sense that a truncated portion of an intracellular signaling domain is used, said truncated portion can be used in place of the entire domain as long as it transduces the signal effector function. The term intracellular signaling domain means that it includes any truncated portion of the intracellular signaling domain sufficient to transduce the signal of the effector function.
[0283] It is known that the signals generated through the TCR alone are insufficient for the complete activation of the T cell and that secondary signals or co-stimulatory signals are also necessary. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: major signaling of domains that initiates major antigen-dependent activation via the TCR (for example, a TCR / CD3 complex) and domains of signaling co-stimulators that act in an antigen-independent way to provide secondary signals or co-stimulatory signals. In preferred embodiments, a CAR contemplated here comprises an intracellular signaling domain comprising one or more "co-stimulatory signaling domain" and a "major signaling domain".
[0284] Main signaling domains regulate the main activation of the TCR complex either in a stimulatory mode, or in an inhibitory mode. The major signaling domains that act in a stimulatory mode may contain the signaling motifs that are known as tyrosine immunoreceptor-based activation motifs or ITAMs.
[0285] Illustrative examples of ITAMs containing the major signaling domains that are of particular use in the present invention include those derived from TCRZ, FcRy, FcRp, CD3y, CD3, CD3s, CD3Z, CD22, CD79a, CD79b and CD66d. In a particularly preferred embodiment, a CAR comprises a major CD3Z signaling domain and one or more co-stimulatory signaling domains. The main intracellular signaling domain and the co-stimulatory signaling domain can be linked in any cantilevered order to the CARboxyl terminus of the transmembrane domain.
[0286] CARs contemplated here comprise one or more co-stimulatory signaling domains to enhance the efficacy and expansion of T cells that express CAR receptors. As used here, the term, "co-stimulatory signaling domain", or "co-stimulatory domain", refers to an intracellular signaling domain of a co-stimulatory molecule.
[0287] Illustrative examples of said co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), CTLA4, LFA-1, CD2, CD7 , LIGHT, TRIM, LCK3, SLAM, DAP10, LAG3, HVEM and NKD2C and CD83. In one embodiment, a CAR comprises one or more co-stimulatory signaling domains that are selected from the group consisting of CD28, CD137 and CD134 and a major CD3Z signaling domain.
[0288] In one embodiment, a CAR comprises a scFv that binds to an alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44 , CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, AchRal , FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA , ROR1, SSX, Survivina, TAG72, TEMs, or VEGFR2 polypeptide; a transmembrane domain derived from a polypeptide selected from the group consisting of: CD8a; CD4, CD45, PD-1 and CD152; and one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD54, CD134, CD137, CD152, CD273, CD274 and CD278; and a CD3Z main signaling domain.
[0289] In another embodiment, a CAR comprises a scFv that binds to an alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44 , CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, AchRal , FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO- 1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA , ROR1, SSX, Survivina, TAG72, TEMs, or VEGFR2; an articulation domain selected from the group consisting of: IgG1 / CH2 / CH3 and CD8a and CD8a articulation; a transmembrane domain derived from a polypeptide selected from the group consisting of: CD8a; CD4, CD45, PD-1 and CD152; and one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD134 and CD137; and a CD3Z main signaling domain.
[0290] In yet another embodiment, a CAR comprises an scFv, which additionally comprises a ligand, which binds to an alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20 , CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO -1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, or VEGFR2 polypeptide; an articulation domain selected from the group consisting of: IgG1 / CH2 / CH3 and CD8a and CD8a articulation; a transmembrane domain comprising a TM domain derived from a polypeptide selected from the group consisting of: CD8a; CD4, CD45, PD-1 and CD152 and a short oligo- or polypeptide linker, preferably between 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length that links the TM domain the intracellular CAR signaling domain; and one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD134 and CD137; and a CD3Z main signaling domain.
[0291] In a particular embodiment, a CAR comprises a scFv that binds to an alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family that includes ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, AchR fetal, FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO -1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs and VEGFR2 polypeptide; a hinge domain comprising the CD8a polypeptide; the CD8a transmembrane domain which comprises a polypeptide linker of about 3 amino acids; one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD134 and CD137; and a CD3Z main signaling domain.
Polypeptides [0292] The present invention contemplates, in part, genetically modified TCR and CAR polypeptides and fragments thereof, cells and compositions comprising the same and vectors that express the polypeptides. "Polypeptide", "polypeptide fragment", "peptide" and "protein" are used interchangeably, unless otherwise specified and according to a conventional meaning, that is, as the sequence of amino acids. Polypeptides are not limited to a specific length, for example, they may comprise a full-length protein sequence or a fragment of a protein length and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and similar, as well as other known modifications in the art, both naturally occurring and non-naturally occurring. In various embodiments, the polypeptides contemplated herein comprise a signal sequence (or leader) at the N-terminal end of the protein, which directs the transfer of the protein in co-translational or post-translational mode. Illustrative examples of suitable useful signal sequences described herein include, but are not limited to, the IgG1 heavy chain signal sequence and the CD8a signal sequence. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides contemplated herein specifically encompass the CARs of the present description, or sequences that have deletions from, additions to, and / or substitutions of one or more amino acids of a polypeptide as contemplated herein.
[0293] Polypeptides include "polypeptide variants". Polypeptide variants can differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and / or insertions. Said variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences. For example, in particular modalities, it may be desirable to improve the binding affinity and / or other biological properties of TCRs or CARs modified by genetic engineering by introducing one or more substitutions, deletions, additions and / or insertions. Preferably, the polypeptides of the present invention include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity thereto.
[0294] Polypeptides include "polypeptide fragments". Polypeptide fragments refer to a polypeptide, which can be monomeric or multimeric, which has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment may comprise an amino acid chain of at least 5 to about 500 amino acids in length. It will be noted that in certain embodiments, the fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.
[0295] The polypeptide can also be fused in-frame or conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance the binding of the polypeptide to a solid support.
[0296] As noted above, the polypeptides of the present invention can be altered in a number of ways that include amino acid substitutions, deletions, truncations and insertions. Methods for said manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and changes in the nucleotide sequence are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (Molecular Biology of the Gene, quatroth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. The guidelines for adequate amino acid substitutions that do not affect the biological activity of the protein of interest can be observed in the model by Dayhoff et al., (1978) Atlas of Protein Sequence and structure (Natl. Biomed. Res. Found., Lavagemton , DC).
[0297] In certain modalities, the variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced by another amino acid that has similar properties, so those skilled in the art of peptide chemistry can expect the secondary structures and hydropathic nature of the polypeptide to be substantially unchanged. Modifications can be made in the polynucleotide and polypeptide structure of the present invention and still obtain a functional molecule that encodes a variant or polypeptide derivative with desirable characteristics.
[0298] Polypeptide variants additionally include glycosylated forms, aggregation conjugates with other molecules and covalent conjugates with unrelated chemical fractions (eg, pegylated molecules). Covalent variants can be prepared by group-linking functionalities that are found on the amino acid chain or on the N- or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants and muteins. Truncations or deletions of regions that do not affect the functional activity of proteins are also variants.
[0299] In one embodiment, where the expression of two or more polypeptides is desired, the polynucleotide sequences that encode it can be separated by and the IRES sequence as discussed at some point here. In another embodiment, two or more polypeptides can be expressed as a fusion protein that comprises one or more autocleaving polypeptide sequences.
[0300] The polypeptides of the present invention include the fusion polypeptides. In preferred embodiments, fusion polypeptides and polynucleotides that encode fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments. Fusion polypeptides are typically linked from C-terminus to N-terminus, although they can also be linked from C-terminus to C-terminus, from N-terminus to N-terminus, or from N-terminus to C-terminus . The polypeptides of the fusion protein can be in any order or in a specified order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences and interspecies homologs, as long as the desired transcription activity of the fusion polypeptide is preserved. Fusion polypeptides can be produced by synthetic chemical methods or by chemical bonding between the two fractions or they can generally be prepared using other standard techniques. The linked DNA sequences that comprise the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as discussed elsewhere here.
[0301] In one embodiment, a fusion partner comprises a sequence that helps to express the protein (an expression enhancer) in greater yield than the native recombinant protein. Other fusion partners can be selected to increase the solubility of the protein or to allow the protein to target the desired intracellular compartments or to facilitate the transport of the fusion protein across the cell membrane.
[0302] The fusion polypeptides may additionally comprise a peptide cleavage signal between each of the polypeptide domains described herein. In addition, the polypeptide field can be placed in any peptide linker sequence. Examples of polypeptide cleavage signals include polypeptide cleavage recognition fields such as protease cleavage fields, nuclease cleavage fields (for example, rare restriction enzyme recognition fields, ribozyme recognition fields of self-cleavage) and viral self-cleavage oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5 (8); 616-26).
[0303] Suitable protease cleavage fields and self-cleaving peptides are known to those skilled in the art (see, for example, in Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scimczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage fields include, but are not limited to, polyvirus Nla protease cleavage fields (e.g. tobacco etch virus protease), HC potivirus proteases, P1 potivirus proteases (P35), NIa proteases from biovirus, RNA-2-encoded biovirus proteases, L aftovirus proteases, 2A enterovirus proteases, 2A rhinovirus proteases, 3C protease, 24K comovirus proteases, 24K nepovirus proteases, RTSV (spherical tree tungro virus) 3C -like protease, PIVF (turnip yellow spot virus) protease such as 3C, heparin, thrombin, factor Xa and enterokinase. Because of their high cleavage stringency, TEV (tobacco etch virus) protease cleavage fields are preferred in one embodiment, for example, EXXYXQ (G / S) (SEQ ID NO :), for example, ENLYFQG (SEQ ID NO :) and ENLYFQS (SEQ ID NO :), where X represents any amino acid (cleavage by TEV occurs between Q and G or Q and S).
[0304] In a particular embodiment, the self-cleaving peptides include the polypeptide sequences obtained from potivirus and cardiovascular 2A peptides, FMDV (foot-and-mouth disease virus), equine rhinitis virus A, Thosea virus asigna and porcine teschovirus.
[0305] In certain embodiments, the self-cleaving polypeptide field comprises a 2A field or as a 2A, sequence or domain (Donnely et al., 2001. J. Gen. Virol. 82: 1027-1041).
Polynucleotides [0306] In particular embodiments, polynucleotides that encode one or more genetically modified TCR or CAR polypeptides contemplated herein are provided. As used here, the terms "polynucleotide" or "nucleic acid" refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), strand RNA plus (RNA (+)), strand minus RNA (- )), Genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single-stranded or double-stranded polynucleotides. Preferably, the polynucleotides of the present invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% or 100% sequence identity for any of the reference sequences described here (see, for example, Sequence Listing), typically where the variant maintains at least one biological activity of the reference sequence . In various illustrative embodiments, the present invention contemplates, in part, polynucleotides that comprise expression vectors, viral vectors and transfer plasmids and compositions and cells that comprise them.
[0307] In particular embodiments, polynucleotides are provided by the present invention that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more contiguous amino acid residues of a polypeptide of the present invention, as well as all intermediate lengths. It will be readily understood that “intermediate lengths”, in this context, means any length between the quoted values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc .; 151, 152, 153, etc .; 201, 202, 203, etc.
[0308] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity with the reference polynucleotide sequence or polynucleotides that hybridize to the reference sequence under strict conditions that are defined here later. Said terms include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides compared to the reference polynucleotide. In this regard, it is well understood in the art that certain changes including mutations, additions, deletions and substitutions can be produced to the reference polynucleotide whereby the altered polynucleotide retains the biological polynucleotide function or activity.
[0309] The recitations “sequence identity” or, for example, comprising a “sequence 50% identical to”, as used here, refer to the extent to which the sequences are identical on a nucleotide-by-nucleotide basis or in amino acid-by-amino acid basis over a comparison window. Thus, a “percent sequence identity” can be calculated by comparing two sequences optimally aligned over the comparison window, determining the number of positions in which the identical nucleic acid base (for example, A, T, C, G, I) or the identical amino acid residue (eg Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Fe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cis and Met) occurs in both sequences to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (that is, the size of the window) and multiplying the result by 100 to produce the percentage string identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% sequence identity for any of the reference sequences described here, typically where the polypeptide variant maintains at least one biological activity of the reference polypeptide.
[0310] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "sequence identity percentage" and "substantial identity" . The “reference sequence” is at least 12 but often 15 to 18 and often at least 25 monomer units, including nucleotides and amino acid residues, in length. Because two polynucleotides can each comprise (1) the sequence (that is, only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides and (2) the sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides over the "comparison window" to identify and compare local regions of sequence similarity. The “comparison window” refers to a conceptual segment of at least 6 contiguous positions, generally about 50 to about 100, more generally about 100 to about 150 in which the sequence is compared to the reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can comprise additions or deletions (ie spaces) of about 20% or less compared to the reference strings (which do not comprise additions or deletions) for optimal alignment of the two sequences. The optimal alignment of the strings to align the comparison window can be driven by computerized implementations of algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (that is, resulting in the highest percentage of homology over the comparison window) generated by any of the several selected methods. Reference can also be made to the BLAST family of programs as, for example, described by Altschul et al., 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 by Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 19941998, Chapter 15.
[0311] The polynucleotides of the present invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (RTUs), Kozak sequences, polyadenylation signals , additional restriction enzyme fields, multiple cloning fields, internal ribosome entry fields (IRES), recombinase recognition fields (for example, LoxP, FRT and Att fields), termination codons, transcription termination signals and polynucleotides encoding self-cleaving polypeptides, epitope tags, as described at some point here or as known in the art, so that their overall length can vary considerably. Therefore, it is contemplated that a polynucleotide fragment of almost any length can be employed, with the total length being preferably limited by the ease of preparation and use of the intended recombinant DNA protocol.
[0312] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of known and well established techniques and offered in the art. In order to express the desired polypeptide, a nucleotide sequence that encodes the polypeptide, can be inserted into the appropriate vector. Examples of vectors are plasmids, autonomous replication sequences and transposable elements. Additional examples of vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as the yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1 derivative artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retrovirus (which includes lentivirus), adenovirus, adeno-associated virus, herpes virus (for example, herpes simplex virus), poxvirus, baculovirus, papillomavirus and papovavirus (e.g. SV40). Examples of expression vectors are the pClneo vectors (Promega) for expression in mammalian cells; pLenti4 / V5-DEST ™, pLenti6 / V5-DEST ™ and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the chimeric protein coding sequences described herein can be linked within said expression vectors for the expression of the chimeric protein in mammalian cells.
[0313] The “control elements” or “regulatory sequences” present in an expression vector are the untranslated regions of the vector — origin of replication, selection cassettes, promoters, intensifiers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5 'and 3' untranslated regions — that interact with host cell proteins to perform transcription and translation. These elements may vary in strength and specificity. Depending on the vector system and the host used, any number of suitable elements of transcription and translation, including ubiquitous promoters and induction promoters, can be used.
[0314] In particular embodiments, a vector for use in the practice of the present invention that includes, but is not limited to expression vectors and viral vectors, includes exogenous, endogenous control sequences, or heterologous control sequences, such as promoters and / or intensifiers. An “endogenous” control sequence is one that is naturally linked to a particular gene in the genome. An "exogenous" control sequence is one that is arranged in juxtaposition to a gene by means of genetic manipulation (ie, molecular biology techniques) so that the transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" control sequence is an exogenous sequence that is from a different species than the cell being genetically manipulated.
[0315] The term "promoter" as used here refers to a polynucleotide (DNA or RNA) recognition field to which an RNA polymerase binds. An RNA polymerase initiates and transcribes the polynucleotides operably linked to the promoter. In particular embodiments, mammalian cell operational promoters comprise an AT-rich region located approximately 25 to 30 bases upstream from the field where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the beginning of transcription, a CNCAAT region where N can be any nucleotide.
[0316] The term "enhancer" refers to a segment of DNA that contains sequences capable of providing enhanced transcription and in some cases may work regardless of its orientation relative to another control sequence. An intensifier can work in cooperation mode or in addition mode with promoters and / or other intensifying elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.
[0317] The term "operationally linked" refers to the juxtaposition in which the components described are in a relationship that allows them to function in their intended ways. In one embodiment, the term refers to a functional link between a nucleic acid expression control sequence (such as a promoter, and / or an enhancer) and a second polynucleotide sequence, for example, a polynucleotide of interest. , where the expression control sequence directs the transcription of the nucleic acid that corresponds to the second sequence.
[0318] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that continuously or continuously permits transcription of an operably linked sequence. The constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a "specific cell", "specific cell type", " specific cell line ”, or“ specific tissue ”of a promoter, enhancer, or promoter / enhancer that allows expression in a restricted variety of cell and tissue types, respectively.
[0319] Illustrative ubiquitous expression control sequences suitable for use in particular embodiments of the present invention include, but are not limited to an immediate cytomegalovirus (CMV) early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) promoter (thymidine kinase), H5, P7.5 and P11 from vaccinia virus, a factor 1-alpha (EF1 a) elongation promoter, early development response (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH ), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock beta 90kDa protein, member 1 (HSP90B1), heat shock 70kDa protein (HSP70), β- kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechn ology 25, 1477 - 1482 (2007)), a Ubiquitin C (UBC) promoter, a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirus / chicken β-actin (CAG) promoter, a β- actin and a myeloproliferative sarcoma virus enhancer, deleted negative controllable region, substituted dl587rev primer-binding field promoter (MND) (Chalita et al., J Virol. 69 (2): 748-55 (1995)).
[0320] In a particular embodiment, it may be desirable to express a polynucleotide that comprises a genetically engineered TCR or CAR from a promoter that provides stable, long-term expression in T cells and at levels sufficient to redirect T cells in cells that express the target antigen. In a preferred embodiment, the promoter is an EF1 α promoter or an MND promoter.
[0321] As used here, "conditional expression" can refer to any type of conditional expression that includes, but is not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state, etc. the said definition is not intended to exclude the expression of specific cell or tissue type. Certain embodiments of the present invention provide for the conditional expression of a polynucleotide of interest, for example, expression is controlled by subjecting the cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or which increases or decreases the expression of the polynucleotide encoded by the polynucleotide of interest.
[0322] Illustrative examples of promoters / inducible systems include, but are not limited to, steroid-inducible promoters such as promoters for the genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallohionine promoter (inducible by treatment with several heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” system adjustable by mifepristone (Sirin et al., 2003, Gene, 323: 67), the cumato inducible gene key (WO 2002/088346), tetracycline-dependent regulatory systems, etc.
[0323] Conditional expression can also be achieved by using field-specific DNA recombinase. According to certain embodiments of the present invention the vector comprises at least one (typically two) field (s) for field-specific recombinase-mediated recombination. As used here, the terms "recombinase" or "field-specific recombinase" include proteins, enzymes, excision or integrative cofactors or associated proteins that are involved in recombination reactions that involve one or more recombination fields (for example, two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which can be wild type proteins (see Landy, Current Opinion in Biotechnology 3: 699-707 (1993)), or mutants, derivatives (e.g., fusion proteins that contain the recombination protein sequences or fragments thereof), fragments and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, 0C31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1 and ParA.
Viral vectors [0324] In particular modalities, the cell (for example, T cell) is transduced with a retroviral vector, for example, a lentiviral vector, which encodes a TCR or CAR modified by genetic engineering as contemplated here. Transduced T cells elicit a stable, long-term and persistent T cell response.
[0325] As used here, the term “retrovirus” refers to a virus RNA that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA within a host genome. Illustrative retroviruses suitable for use in particular modalities, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), virus murine breast tumor (MuMTV), gibbon monkey leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend murine leukemia virus, murine stem cell virus (MSCV) and sarcoma virus Rous (RSV)) and lentivirus.
[0326] As used here, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; which includes HIV type 1 and HIV type 2); visna-maedi virus (VMV); goat arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); equine immunodeficiency virus (IVF); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector structures (i.e., HIV cis action sequence elements) are preferred.
[0327] The term "vector" is used here to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, for example, inserted into, the vector of the nucleic acid molecule. A vector can include sequences that direct autonomous replication in a cell, or it can include enough sequences to allow integration into the host cell's DNA. Useful vectors include, for example, plasmids (for example, DNA plasmids or RNA plasmids), transposons, cosmids, artificial bacterial chromosomes and viral vectors. Useful viral vectors include, for example, replication defective retroviruses and lentiviruses.
[0328] As will be apparent to those skilled in the art, the term "viral vector" is widely used to refer to either a nucleic acid molecule (for example, a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate the transfer of the nucleic acid molecule or integration within the cell genome or into a viral particle that mediates the transfer of the nucleic acid. Viral particles will typically include several viral components and sometimes also host cell components in addition to the nucleic acid (s).
[0329] The term viral vector can refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are mainly derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and / or functional genetic elements, or portions thereof, which are mainly derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and / or functional genetic elements, or portions thereof, that includes LTRs that are mainly derived from a lentivirus. The term "hybrid vector" refers to a vector, LTR or other nucleic acid that contains not only retroviral sequences, for example, lentiviral, but also non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a transfer vector or plasmid that comprises retroviral sequences, for example, lentiviral, for reverse transcription, replication, integration and / or packaging.
[0330] In particular modalities, the terms "lentiviral vector", "lentiviral expression vector" can be used to refer to infectious lentiviral transfer plasmids and / or lentiviral particles. Where reference is made here to elements such as cloning fields, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of said elements are present in the form of RNA in the lentiviral particles of the present invention and are present in the form of DNA in the DNA plasmids of the present invention.
[0331] At each end of the provirus are structures called “long terminal repetitions” or “LTRs”. The term "long terminal repeat (LTR)" refers to pairs of base domains located at the ends of retroviral DNAs that, in their natural sequence context, are direct repetitions and contain U3, R and U5 regions. LTRs in general provide fundamental functions for the expression of retroviral genes (for example, promotion, initiation and polyadenylation of gene transcriptions) and viral replication. The LTR contains numerous regulatory signals that include the elements of transcription control, polyadenylation signals and sequences necessary for replication and integration of the viral genome. Viral LTR is divided into three regions called U3, R and U5. The U3 region contains the intensifier and the promoting elements. The U5 region is the sequence between the primer binding field and the R region and contains the polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. The LTR consists of U3, R and U5 regions and appears at both the 5 'and 3' ends of the viral genome. Adjacent to the 5 'LTR are the sequences necessary for reverse transcription of the genome (the binding field to the tRNA primer) and for efficient packaging of the viral RNA into particles (the Psi field).
[0332] As used here, the term "packaging signal" or "packaging sequence" refers to sequences located within the retroviral genome that are necessary for the insertion of the viral RNA into the viral caspid or particle, see for example, Clever et al., 1995. J. de Virology, Vol. 69, No. 4; pp. 21012109. Several retroviral vectors use the minimal packaging signal (also referred to as the psi [Ψ] sequence) necessary for the encapsidation of the viral genome.
Thus, as used here, the terms "packaging sequence", "packaging signal", "psi" and the symbol "Ψ", are used in reference to the non-coding sequence required for the encapsidation of retroviral RNA strands during the formation of the viral particle.
[0333] In various embodiments, the vectors comprise modified LTR 5 'and / or LTR 3'. Either or both of the LTRs may comprise one or more modifications that include, but are not limited to, one or more deletions, insertions or substitutions. Modifications to the LTR 3 are often made to improve the security of lentiviral or retroviral systems by rendering replication viruses defective. As used here, the term "defective replication" refers to viruses that are unable to complete, effective replication so that infection virions are not produced (for example, defective replication of lentiviral progeny). The term "replication component" refers to a wild-type virus or mutant virus that is capable of replication, so that viral replication of the virus is capable of producing infectious virions (for example, lentiviral progeny replication component).
[0334] "Self-inactivating" (SIN) vectors refer to defective replication vectors, for example, retroviral or lentiviral vectors, in which the right region of the (3 ') intensifier-promoter LTR, known as the U3 region, has been modified (for example, by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is due to the fact that the right (3 ') LTR U3 region is used as the matrix for the left (5') LTR U3 region during viral replication and thus viral transcription cannot be produced without the intensifier- U3 district Attorney. In a further embodiment of the present invention, the 3 'LTR is modified so that the U5 region is replaced, for example, with an ideal poly (A) sequence. It should be noted that modifications to the LTRs such as modifications to the 3 'LTR, the 5' LTR, or both 3 'and 5' LTRs, are also included in the present invention.
[0335] Additional security enhancement is provided by replacing the U3 region of the 5 'LTR with a heterologous promoter to target transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, promoters of viral simian virus 40 (SV40) (for example, early or late), cytomegalovirus (CMV) (for example, immediate early), Murine leukemia virus Moloney (MoMLV), Rous sarcoma virus (RSV) and herpes simplex virus (HSV) (thymidine kinase). Typical promoters are able to trigger high levels of transcription in a Tat-independent mode. This substitution reduces the possibility of recombination to generate a competent replicating virus due to the fact that there is no complete U3 sequence in the virus production system. In certain embodiments, the heterologous promoter has additional advantages in controlling and in the way in which the viral genome is transcribed. For example, the heterologous promoter may be inducible, so that transcription of all or part of the viral genome will occur only when the inducing factors are present. Induction factors include, but are not limited to, one or more chemical compounds or physiological conditions such as temperature or pH, in which host cells are grown.
[0336] In some embodiments, viral vectors comprise a TAR element. The term "TAR" refers to the genetic element of "trans-activation response" located in the R region of lentiviral LTRs (for example, HIV). Said element interacts with the lentiviral trans-activator (tat) genetic element to enhance viral replication. However, said element is not necessary in modalities in which the U3 region of the 5 'LTR is replaced by the heterologous promoter.
[0337] The "R region" refers to the region within the retroviral LTRs that begins at the beginning of the coverage group (ie, the beginning of transcription) and ends immediately before the start of the poly A tract. The R region is also defined as being flanked by the U3 and U5 regions. The R region plays a role during reverse transcription to allow the transfer of nascent DNA from one end of the genome to the other.
[0338] As used here, the term "FLAP element" refers to a nucleic acid whose sequence includes the central polipurin tract and the central termination sequences (cPPT and CTS) of a retrovirus, for example, HIV-1 or HIV -2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and in Zennou, et al., 2000, Cel, 101: 173. During HIV-1 reverse transcription, the central initiation of strand DNA further into the central polipurin tract (cPPT) and central termination in the central termination sequence (CTS) leads to the formation of a three-stranded DNA structure: the flap of central HIV-1 DNA. Although one does not want to be tied to any theory, the DNA flap can act as an active cis determinant of the nuclear lentiviral genome that matters and / or can increase the virus titer. In particular embodiments, the retroviral structures or the lentiviral vector comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors. For example, in particular embodiments, a transfer plasmid includes a FLAP element. In one embodiment, a vector of the present invention comprises a FLAP element isolated from HIV-1.
[0339] In one embodiment, retroviral or lentiviral transfer vectors comprise one or more elements of export. The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of an RNA transcription from the nucleus to the cell's cytoplasm. Examples of elements of exporting RNA include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see for example, Culen et al., 1991. J. Virol. 65: 1053; and Culen et al., 1991. Cell 58: 423) and the post-transcriptional regulatory element of the hepatitis B virus (HPRE). In general, the RNA element of exporting the RNA is arranged within the 3 'UTR of a gene and can be inserted as one or multiple copies.
[0340] In particular modalities, the expression of heterologous sequences in viral vectors is greater for incorporating post-transcriptional regulatory elements, efficient polyadenylation fields and optionally, transcription termination signals within the vectors. A variety of post-transcriptional regulatory elements can increase the expression of a heterologous nucleic acid in the protein, for example, post-transcriptional regulatory element of the groundhog hepatitis virus (WPRE; Zufferey et al., 1999, J. Virol., 73 : 2886); the post transcriptional regulatory element present in the hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5: 3864); and the like (Liu et al., 1995, Genes Dev., 9: 1766). In particular embodiments, vectors of the present invention comprise a post-transcriptional regulatory element such as WPRE or HPRE
[0341] In particular embodiments, vectors of the present invention are devoid of or do not comprise a post transcriptional regulatory element such as WPRE or HPRE due to the fact that in some cases the said elements increase the risk of cell transformation and / or not substantially or significantly increase the amount of mRNA transcription or increase mRNA stability. Therefore, in some embodiments, the vectors of the present invention are devoid of or do not understand WPRE or HPRE as an added security measure.
[0342] Elements that guide the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase the expression of the heterologous gene. Transcription termination signals are generally found downstream of the polyadenylation signal. In particular embodiments, the vectors comprise a 3 'polyadenylation frequency of a polynucleotide that encodes a polypeptide to be expressed. The term "polyA field" or "polyA sequence" as used here denotes a DNA sequence that directs not only the termination but also the polyadenylation of the transcription of nascent RNA by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA extension to the 3 'end of the coding sequence and thus contribute to greater translation efficiency. Efficient polyadenylation of recombinant transcription is desirable as transcripts lacking a polyA extension are unstable and are rapidly degraded. Illustrative examples of polyA signals that can be used in a vector of the present invention include an ideal polyA sequence (for example, AATAAA, ATTAAA, AGTAAA), a bovine growth hormone polyA sequence (BGHpA), a sequence of rabbit β-globin polyA (rPgpA), or other suitable heterologous or endogenous polyA sequences known in the art.
[0343] In several embodiments, the vectors of the present invention comprise a promoter operably linked to a polynucleotide encoding a TCR or CAR modified by genetic engineering polypeptide. Vectors can have one or more LTRs, where any LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vectors may additionally comprise one or more accessory elements to increase the efficiency of transduction (for example, cPPT / FLAP), viral packaging (for example, Psi (Ψ) packaging signal, RRE), and / or other elements that increase therapeutic gene expression (e.g., poly (A) sequences) and can optionally comprise WPRE or HPRE. Those skilled in the art can see that many other different modalities can be implemented from the existing modalities of the present invention.
[0344] A "host cell" includes cells transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the present invention. Host cells can include packaging cells, producer cells and cells infected with viral vectors. In particular embodiments, the host cells infected with the viral vector of the present invention are administered to an individual in need of therapy. In certain embodiments, the term "target cell" is used interchangeably with the host cell and refers to cells transfected, infected, or transduced from a desired type of cell. In preferred embodiments, the target cell is the T cell.
[0345] Large-scale production of viral particles is often necessary to achieve reasonable viral titration. Viral particles are produced by transfecting a transfer vector into a packaging cell line that comprises viral structural and / or accessory genes, for example, gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or genes nef or other retroviral genes.
[0346] As used here, the term "packaging vector" refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide that encodes one, two, three, four or more structural viral genes and / or accessories. Typically, packaging vectors are included in a packaging cell and are introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known to those skilled in the art. A retroviral / lentiviral transfer vector of the present invention can be introduced into a packaging cell line, via transfection, transduction or infection, to generate a producing cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines by standard methods which include, for example, calcium phosphate transfection, lipofection or electroporation. In some embodiments, packaging vectors are introduced into cells along with a selectable dominant marker, such as neomycin, hygromycin, puromycin, blastocidine, zeocin, thymidine kinase, DHFR, Gln syntase or ADA, followed by selection in the presence of a drug isolation of clones. A selectable marker gene can be physically linked to genes encoding the packaging vector, for example, by IRES or self-cleaving viral peptides.
[0347] Viral envelope proteins (env) determine the range of host cells that can actually be infected and transformed by recombinant retroviruses generated from cell lines. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by the packaging cells of the present invention are encoded in a separate vector from the viral gag and pol genes, as previously described.
[0348] Illustrative examples of retroviral derived env genes that can be employed in the present invention include, but are not limited to: MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (virus of avian plague) and envelopes of influenza viruses. Similarly, the genes that encode envelopes from RNA viruses (for example, RNA virus families from Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramixoviridae, Rhabdoviridae, Filoviridae, Orthomixirida Reoviridae, Birnaviridae, Retroviridae) as well as from DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxiiridae and Iridoviridae) can be used. Representative examples include, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10 and EIAV.
[0349] In other embodiments, envelope proteins for the pseudotyping of the virus of the present invention include, but are not limited to any of the following viruses: Influenza A such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), Influenza B, Influenza C, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus in the Norwalk virus group, enteric adenovirus, parvovirus, fever virus Dengue fever, monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European merchant virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, vesicular stomatitis virus ( VSV), Herpesvirus such as Herpes simplex virus types 1 and 2, chickenpox zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpes virus (HHV), human herpes virus types 6 and 8, immunodeficiency human (HIV), papilloma virus, murine herpes gamma virus, Arenavirus such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, virus Lassa fever, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, virus that causes renal hemorrhagic fever syndrome, Rift Valey fever virus, Filoviridae (filovirus) that includes Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae which includes Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, virus that causes Tick and Paramixoviridae encephalitis such as Hendra virus and Nipah virus, major smallpox and minor smallpox (smallpox), alphavirus such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus al, SARS-associated coronavirus (SARS-CoV), West Nile virus, any viruses that cause encephalitis.
[0350] In one embodiment, the present invention provides packaging cells that produce recombinant retroviruses, for example, lentiviruses, pseudotyped with the VSV-G glycoprotein.
[0351] The terms "pseudotype" or "pseudotyping" as used here, refer to a virus whose viral envelope proteins have been replaced with those of other viruses that have preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus (VSV-G) G-protein envelope proteins, which allows HIV to infect a wider range of cells due to the fact that HIV envelope proteins (encoded) by the env gene) normally target the virus in cells that have CD4 +. In the preferred embodiment of the present invention, lentiviral envelope proteins are pseudotyped with VSV-G. In one embodiment, the present invention provides packaging cells that produce recombinant retroviruses, for example, lentiviruses, pseudotyped with the VSV-G envelope glycoprotein.
[0352] As used here, the term “packaging cell line” is used in reference to cell lines that do not contain a packaging signal, but which stable or transiently express structural viral proteins and replication enzymes ( for example, gag, pol and env) which are necessary for the correct packaging of viral particles. Any suitable cell lines can be employed to prepare the packaging cells of the present invention. In general, the cells are mammalian cells. In a particular embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, cells BSC 1, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, cells Huh7, HeLa cells, W163 cells, 211 cells and 21A cells. In preferred embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.
[0353] As used here, the term "producing cell line" refers to a cell line that is capable of producing recombinant retroviral particles, which comprise the packaging cell line and a transfer vector construct that comprises a signal packaging. The production of infectious viral particles and viral shock solutions can be performed using conventional techniques. Methods of preparing viral shock solutions are known in the art and are illustrated, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23: 628-633 and N. R. Landau et al. (1992) J. Virol. 66: 51105113. Infectious viral particles can be collected from the packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis, or the collection of cell culture supernatant, as is known in the art. Optionally, the collected virus particles can be purified if desired. Suitable purification techniques are well known to those skilled in the art.
[0354] Sending a gene (s) or other polynucleotide sequence using a retroviral vector or lentiviral vector by means of viral infection rather than by transfection is referred to as "transduction". In one embodiment, retroviral vectors are transduced in a cell through infection and provirus integration. In certain embodiments, a target cell, for example, the T cell, is "transduced" if it comprises a gene or other polynucleotide sequence sent to the cell by infection using a viral or retroviral vector. In particular embodiments, the transduced cell comprises one or more genes or other polynucleotide sequences sent by the retroviral or lentiviral vector in its cell genome.
[0355] In particular embodiments, host cells transduced with the viral vector of the present invention that express one or more polypeptides, are administered to an individual to treat and / or prevent a B cell malignancy. Other methods that refer to the use of vectors viruses in gene therapy, which can be used according to certain embodiments of the present invention, can be found in, for example, Kay, MA (1997) Chest 111 (6 Supp.): 138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9: 1975-81; Shiratory, Y. et al. (1999) Liver 19: 265-74; Oka, K. et al. (2000) Curr. Opin. Lipidol. 11: 179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7: 1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12: 335-56; Alt, M. (1995) J. Hepatol. 23: 746-58; Brody, S. L. and Crystal, R. G. (1994) Ann. N.Y. Acad. Sci. 716: 90-101; Strayer, D. S. (1999) Expert Opin. Investig. Drugs 8: 2159-2172; Smith-Arica, J. R. and Bartlett, J. S. (2001) Curr. Cardiol. Rep. 3: 43-49; and Lee, H. C. et al. (2000) Nature 408: 483-8.
Compositions and Formulations [0356] The compositions contemplated here may comprise one or more polypeptides, polynucleotides, vectors comprising them and T cell compositions, as contemplated here. The compositions include, but are not limited to pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the present invention can be administered in combination with other agents as well, such as, for example, cytokines, development factors, hormones, small molecules, chemotherapeutics, pre-drugs, drugs, antibodies , or other pharmaceutically active agents. There is in fact no limit to other components that can also be included in the compositions, as long as the additional agents do not adversely affect the composition's ability to deliver the intended therapy.
[0357] The phrase “pharmaceutically acceptable” is used here to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of medical judgment, suitable for use in contact with human tissues and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable risk-benefit ratio.
[0358] As used herein, pharmaceutically acceptable "vehicle, diluent or excipient" includes without limitation any adjuvant, vehicle, excipient, glidant, sweetening agent, diluent, preservative, ink / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent , suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier that has been approved by the United States Food and Drug Administration as being acceptable for use in humans or pets. Examples of pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; Starches, such as corn starch and potato starch; Cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; Tragacanth; malt; Gelatine; baby powder; Cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; Oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; Glycols, such as propylene glycol; Polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; Esters, such as ethyl oleate and ethyl laurate; Agar; Buffering agents, such as magnesium hydroxide and aluminum hydroxide; Alginic acid; Pyrogen-free water;
Isotonic saline solution; Ringer's solution; Ethyl alcohol; Phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations.
[0359] In particular embodiments, the compositions of the present invention comprise a number of modified T cells manufactured by the methods contemplated herein. In preferred embodiments, pharmaceutical T cell compositions comprise potent T cells having one or more of, or all of the following markers: CD62L, CCR7, CD28, CD27, CD122 and CD127.
[0360] It can generally be determined that pharmaceutical compositions comprising T cells manufactured by the methods contemplated herein can be administered in a dosage of 102 to 1010 cells / kg body weight, 105 to 109 cells / kg body weight, 105 to 108 cells / kg body weight, 105 to 107 cells / kg body weight, 107 to 109 cells / kg body weight, or 107 to 108 cells / kg body weight, which includes all integer values within those tracks. The number of cells will depend on the use for which the composition is intended and on the type of cells included in the composition. For the uses provided here, the cells are generally in a volume of one liter or less, it can be 500 mls or less, even 250 mls or 100 mls or less. Thus the density of the desired cells is typically greater than 106 cells / ml and in general is greater than 107 cells / ml, in general 108 cells / ml or greater. The number of clinically relevant immune cells can be divided into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. In some aspects of the present invention, particularly as long as all infused cells are redirected to a particular target antigen (eg, BCMA), fewer cells, in the range of 106 / kg (106-1011 per patient) can be administered . T cells modified to express a TCR or CAR modified by genetic engineering can be administered multiple times in dosages within said ranges.
The cells can be allogeneic, syngeneic, xenogenetic, or autologous for the patient undergoing therapy. If desired, treatment may also include administration of mitogens (for example, FA) or lymphokines, cytokines, and / or chemokines (for example, IFN-γ, IL-2, IL-7, IL-15, IL-12 , TNF-alpha, IL-18 and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1a, etc.) as described here to enhance the grafting and function of infused T cells .
[0361] In general, compositions comprising the activated and expanded cells as described here can be used in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the compositions comprising the modified T cells manufactured by the methods contemplated herein are used in the treatment of cancer. The modified T cells of the present invention can be administered either alone, or as a pharmaceutical composition in combination with vehicles, diluents, excipients, and / or with other components such as IL-2, IL-7, and / or IL-15 or other cytokines or cell populations. In particular embodiments, the pharmaceutical compositions contemplated herein comprise a quantity of genetically modified T cells, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
[0362] Pharmaceutical compositions comprising modified T cells contemplated herein may additionally comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.
[0363] Liquid pharmaceutical compositions, whether the same in solutions, suspensions or other similar forms, may include one or more of the following: a sterile diluent such as water for injection, saline, preferably saline, Ringer's solution, chloride isotonic sodium, fixed oils such as synthetic mono- or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetra acetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be included in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.
[0364] In a particular embodiment, the compositions contemplated herein comprise an effective amount of a modified expanded T cell composition, alone or in combination with one or more therapeutic agents. Thus, T cell compositions can be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions can also be administered in combination with antibiotics. Said therapeutic agents can be accepted in the art as a standard treatment for a particular disease state as described herein, such as a particular cancer. Examples of contemplated therapeutic agents include cytokines, development factors, steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active and auxiliary agents.
[0365] In certain embodiments, the compositions comprising T cells contemplated herein can be administered in conjunction with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CITOXAN ™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines that include altretamine, triethylenomelamine, triethylenophosphoramide, triethylenethiophosphoramide and trimethylolomelamine summary; nitrogen mustards such as chlorambucil, chlornafazine, cholophosphamide, estramustine, ifosfamide, meclorethamine, meclorethamide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, photemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-lorubin, iduborubin, iduborubin, idorubin, , marcelomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutetimide, mitotane, trilostane; replenishing folic acid such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcine; diaziquone; elformitine; eliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofirana; spirogermanium; tenuazonic acid; triaziquone; 2, 2 ', 2 "-trichlorotriethylamine; urethane; vindesina; dacarbazine; Mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, for example, paclitaxel (TAXOL®, Bristol-Miers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®., Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; new chair; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomitine (DMFO); derivatives of retinoic acid such as Targretin ™ (bexarotene), Panretin ™ (alitretinoin); ONTAK ™ (denileucine diftitox); speramycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit the action of the hormone in tumors such as anti-estrogens that include, for example, tamoxifen, raloxifene, aromatase inhibitor 4 (5) -imidazoles, 4 -hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and the pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0366] A variety of other therapeutic agents can be used in conjunction with the compositions described here. In one embodiment, the composition comprising T cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to steroids and glucocorticoids (which include betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs. , ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.
[0367] Other examples of NSAIDs are chosen from the group consisting of ibuprofen, naproxen, sodium naproxen, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib) and siallylates. Examples of painkillers are chosen from the group consisting of acetaminophen, oxycodone, tramadol, propxifene hydrochloride. Examples of glucocorticoids are chosen from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Examples of biological response modifiers include molecules directed against cell surface markers (eg, CD4, CD5, etc.), cytokine inhibitors, such as TNF antagonists (eg, etanercept (ENBREL®), adalimumab ( HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and molecule adhesion inhibitors Biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules Examples of DMARD include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine , leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.
[0368] Illustrative examples of therapeutic antibodies suitable for combination with the modified T-cell CAR contemplated herein, include but are not limited to abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bectumizumab, bectumizomab, bectumizomab, bectumizomab, bectumizomab bivatuzumab, blinatumomab, vedotin, mertansine, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab, n ofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumoma, robin, rumabuma, rumabuma, rumabuma taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8.
[0369] In certain embodiments, the compositions described here are administered in conjunction with a cytokine. By "cytokine" as used here is meant a generic term for proteins released by a population of cells that act on other cells as intercellular mediators. Examples of such cytokines are lymphokines, monocines, chemokines and traditional polypeptide hormones. Included among cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); liver development factor; fibroblast development factor; prolactin; placental lactogen; tumor necrosis factor -alpha and -beta; mulerian inhibiting substance; rat gonadotropin-associated peptide; inhibin; activin; vascular endothelial development factor; integrin; thrombopoietin (TPO); nerve development factors such as NGF-beta; platelet development factor; transformation development factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like development factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta and gamma; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors that include LIF and linker kit (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native cytokine sequences. Target cells and Antigens [0370] The present invention contemplates, in part, genetically modified immune effector cells redirected to a target cell, for example, a tumor or cancer cell and comprising genetically modified T cell receptors or CARs having a domain of binding that binds to target antigens on cells. Cancer cells can also spread to other parts of the body through the blood and lymphatic systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover the internal organs. Sarcoma is a cancer that begins in the bones, cartilage, fat, muscle, blood vessels, or other connective or supporting tissue. Leukemia is a cancer that begins in the blood-forming tissue such as the bone marrow and causes a large number of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that start in the cells of the immune system. Cancers of the central nervous system are cancers that start in the tissues of the brain and spine.
[0371] In one embodiment, the target cells express an antigen, for example, target antigen, which is not substantially found on the surface of other normal (desired) cells. In one embodiment, the target cell is a target cell is a pancreatic parenchymal cell, a pancreatic duct cell, a liver cell, a cardiac muscle cell, a skeletal muscle cell, an osteoblast, a skeletal myoblast, a neuron, a vascular endothelial cell, pigment cell, smooth muscle cell, glial cell, fat cell, bone cell, chondrocyte, pancreatic islet cell, CNS cell, SNP cell, liver cell, fat cell, kidney cell, lung cell, skin cell, ovarian cell, follicular cell, epithelial cell, immune cell or endothelial cell.
[0372] In certain embodiments, the target cell is part of pancreatic tissue, neural tissue, cardiac tissue, bone marrow, muscle tissue, bone tissue, skin tissue, liver tissue, hair follicles, vascular tissue, adipose tissue, lung tissue and kidney tissue.
[0373] In a particular embodiment, the target cell is the tumor cell. In another particular embodiment, the target cell is a cancer cell, just like the cell in a cancer patient. Examples of cells that can be exterminated by the methods described include cells from the following tumors: a liquid tumor such as leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute and myeloblastic myeloid leukemia, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic leukemia (granulocytic) and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease).
[0374] In another embodiment, the cell is a solid tumor cell, such as sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, carcinoma colon, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer carcinoma, hepatocellular cancer, lung cancer, rectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (e.g. pancreatic adenocarcinoma, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), carcinoma of the sweat glands, carcinoma of the sebaceous glands, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choroid Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, CNS tumors (such as glioma, astrocytoma, medullobl astoma, craniofariogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).
[0375] In one embodiment, the cancer is selected from the group consisting of: Method, according to claim 1, characterized by the fact that the cancer is selected from the group consisting of Wilms tumor, sarcoma of Ewing, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, cancer biliary, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, leukemia chronic myelogenous, Hodgkirís lymphoma, non-Hodgkin's lymphoma and urinary bladder cancer.
[0376] In one embodiment, the target cell is a malignant cell in the liver, pancreas, lung, breast, bladder, brain, bone, thyroid, kidney, skin and hematopoietic system. In another embodiment, the target cell is a liver cancer cell, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, skin cancer or hematological cancer.
[0377] In one embodiment, the target cell is the cell, for example, a cancer cell infected by the virus, which includes but is not limited to CMV, HPV and EBV.
[0378] In one embodiment, the target antigen is an epitope of alpha folate receptor, 5T4, integrin avp6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, CMV, EBV, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, Efa2, EpCAM, FAP, AchR fetal, FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO -1, HLA-A3 + NY-ESO-1, HPV, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivina, TAG72, TEMs, or VEGFR2. Therapeutic Methods [0379] The modified T cells manufactured by the methods contemplated here provide enhanced adoptive immunotherapy for use in the treatment of various conditions that include, without limitation, cancer, infectious disease, autoimmune disease, inflammatory disease and immunodeficiency. In particular embodiments, the specificity of a primary T cell is redirected to tumor or cancer cells by genetically modifying the primary T cell with a TCR or CAR modified by genetic engineering contemplated here. In one embodiment, the present invention includes a type of cellular therapy where T cells are modified to express a TCR or CAR modified by genetic engineering that targets cancer cells that express a target antigen and the modified T cell is infused into a recipient in need of the same. The infused cell is able to exterminate the tumor cells in the recipient. Unlike antibody therapies, TCR or CAR of genetically modified T cells are able to replicate in vivo; thus, contributing to the long-term persistence that can lead to sustained cancer therapy.
[0380] In one embodiment, the genetically modified TCR and T CAR cells of the present invention can undergo robust in vivo T cell expansion and can persist for an extended period of time. In another embodiment, the genetically modified TCR or CAR T cells of the present invention evolve into specific memory T cells that can be reactivated to inhibit any tumor formation or growth.
[0381] In particular embodiments, compositions comprising a genetically modified immune effector cell with a vector comprising a promoter operably linked to a polynucleotide encoding a CAR are used in the treatment of solid tumors or cancers that include, without limitation, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer.
[0382] In particular embodiments, compositions comprising a genetically modified immune effector cell with a vector comprising a promoter operably linked to a polynucleotide encoding a genetic engineered TCR or CAR comprising an antigen-specific binding domain that binds to a PSCA or MUC1 epitope are used to treat various cancers that include but are not limited to pancreatic, bladder and lung.
[0383] In particular embodiments, compositions comprising a genetically modified immune effector cell with a vector comprising a promoter operably linked to a polynucleotide encoding a TCR or CAR modified by genetic engineering are used in the treatment of liquid tumors, which include but leukemia, which includes acute leukemia (for example, AL, AML and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (eg, CL, SL, CML, HCL), polycythemia vera, lymphoma, Hodgkin, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease.
[0384] In particular embodiments, compositions comprising a genetically modified immune effector cell with a vector comprising a promoter operably linked to a polynucleotide encoding a genetically engineered TCR or CAR are used to treat B cell malignancies , which include but are not limited to multiple myeloma (MM), non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CL).
[0385] Multiple myeloma is a B cell malignancy of mature plasma cell morphology characterized by the neoplastic transformation of a single clone of said cell types. Said plasma cells proliferate in BM and can invade adjacent bone and sometimes blood. The variant forms of multiple myeloma include evident multiple myeloma, burning multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma and extramedullary plasmacytoma (see, for example, Braunwald, et al. (eds), Harrison's Principies of Internai Medicine, 15th Edition (McGraw-Hil 2001)).
[0386] Non-Hodgkin's lymphoma encompasses a large group of lymphocyte cancers (white blood cells). Non-Hodgkin's lymphomas can occur at any age and are often marked by lymph nodes that are larger than normal, fever and weight loss. There are many different types of non-Hodgkin's lymphoma.
For example, non-Hodgkin's lymphoma can be divided into aggressive (rapid development) and indolent (slow development) types. Although non-Hodgkin's lymphomas can be derived from B cells and T cells, as used here, the term "non-Hodgkin's lymphoma" and "non-Hodgkin's B-cell lymphoma" are used interchangeably. Non-Hodgkin's B-cell lymphomas (NHL) include Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CL / SL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma and mantle cell lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin's lymphomas.
[0387] Chronic lymphocytic leukemia (CL) is an indolent (slow developing) cancer that causes a slow increase in immature white blood cells called B lymphocytes, or B cells. Cancer cells spread through the blood and bone marrow and can also affect lymph nodes or other organs such as the liver and spleen. CL eventually causes the spinal cord to fail. Sometimes, in later stages of the disease, the disease is called small lymphocytic lymphoma.
[0388] In particular embodiments, methods that comprise administering a therapeutically effective amount of modified T cells contemplated herein or a composition that comprises them, to a patient in need of them, alone or in combination with one or more therapeutic agents, are proportionate. In certain embodiments, the cells of the present invention are used to treat patients at risk of developing cancer. Thus, the present invention provides methods for the treatment or prevention of a cancer which comprises administering to a subject in need thereof, a therapeutically effective amount of the modified T cells of the present invention.
[0389] In one embodiment, a method of treating cancer in an individual in need of it comprises administering an effective amount, for example, therapeutically effective amount of a composition comprising genetically modified immune effector cells contemplated here. The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's illness, although appropriate dosages can be determined by clinical tests.
[0390] In one embodiment, the amount of T cells in a composition administered to an individual is at least 0.1 x 105 cells, at least 0.5 x 105 cells, at least 1 x 105 cells, at least 5 x 105 cells, at least 1 x 106 cells, at least 0.5 x 107 cells, at least 1 x 107 cells, at least 0.5 x 108 cells, at least 1 x 108 cells, at least 0.5 x 109 cells, at least 1 x 109 cells, at least 2 x 109 cells, at least 3 x 109 cells, at least 4 x 109 cells, at least 5 x 109 cells, or at least 1 x 1010 cells. In particular embodiments, about 1 x 107 CAR T cells to about 1 x 109 CAR T cells, about 2 x 107 CAR T cells to about 0.9 x 109 CAR T cells, about 3 x 107 CAR T cells to about of 0.8 x 109 CAR T cells, about 4 x 107 CAR T cells at about 0.7 x 109 CAR T cells, about 5 x 107 CAR T cells at about 0.6 x 109 CAR T cells, or about 5 x 107 CAR T cells at about 0.5 x 109 CAR T cells are administered to an individual.
[0391] In one embodiment, the amount of T cells in a composition administered to an individual is at least 0.1 x 104 cells / kg body weight, at least 0.5 x 104 cells / kg body weight, at least 1 x 104 cells / kg body weight, at least 5 x 104 cells / kg body weight, at least 1 x 105 cells / kg body weight, at least 0.5 x 106 cells / kg body weight, at least 1 x 106 cells / kg body weight, at least 0.5 x 107 cells / kg body weight, at least 1 x 107 cells / kg body weight, at least 0.5 x 108 cells / kg body weight, at least 1 x 108 cells / kg body weight body weight, at least 2 x 108 cells / kg body weight, at least 3 x 108 cells / kg body weight, at least 4 x 108 cells / kg body weight, at least 5 x 108 cells / kg body weight, or at least 1 x 109 cells / kg of body weight. In particular embodiments, about 1 x 106 CAR T cells / kg body weight at about 1 x 108 CAR T cells / kg body weight, about 2 x 106 CAR T cells / kg body weight at about 0.9 x 108 CAR T cells / kg body weight, about 3 x 106 CAR T cells / kg body weight at about 0.8 x 108 CAR T cells / kg body weight, about 4 x 106 CAR T cells / kg body weight about 0.7 x 108 CAR T cells / kg body weight, about 5 x 106 CAR T cells / kg body weight about 0.6 x 108 CAR T cells / kg body weight, or about 5 x 106 CAR T cells / kg body weight at about 0.5 x 108 CAR T cells / kg body weight are administered to an individual.
[0392] Those skilled in the art may recognize that multiple administrations of the compositions of the present invention may be necessary for the desired effect of therapy. For example a composition can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.
[0393] In certain embodiments, it may be desirable to administer activated T cells to an individual and then subsequently draw blood (or have an apheresis performed), activate T cells from there according to the present invention and reinfuse the patient with said activated cells and expanded T cells. This process can be performed multiple times every few weeks. In certain embodiments, T cells can be activated by drawing blood from 10cc to 400cc. In certain modalities, T cells are activated from blood drawn from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, 100cc, 150cc, 200cc, 250cc, 300cc, 350cc, or 400cc or more. Not tied to a theory, using this multiple blood draw / multiple reinfusion protocol can serve to select certain populations of T cells.
[0394] The administration of the compositions contemplated here can be carried out in any convenient way, which includes by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, the compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used here refer to modes of administration other than enteral and topical, usually by injection and include, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal injection and infusion , intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal. In one embodiment, the compositions contemplated here are administered to an individual by direct injection into a tumor, lymph node, or field of infection.
[0395] In one embodiment, an individual in need of it is administered an effective amount of a composition to increase the cellular immune response to cancer in the individual. The immune response can include cellular immune response mediated by cytotoxic T cells capable of exterminating infected cells, regulatory T cells and helper T cell responses. Humoral immune response, mediated mainly by T helper cells capable of activating B cells thus leading to antibody production, can also be induced. A variety of techniques can be used to analyze the type of immune response induced by the compositions of the present invention, which are well described in the art; for example, Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Etan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.
[0396] In the case of T cell-mediated extermination, binding to the CAR-ligand initiates CAR signaling to the T cell, which results in the activation of a variety of T cell signaling pathways that induce the T cell to produce or release proteins capable of inducing apoptosis of the target cell by various mechanisms. Said T cell-mediated mechanisms include (but are not limited to) the transfer of intracellular cytotoxic granules from the T cell into a target cell, pro-inflammatory T cell cytokine secretion that can directly induce target cell extermination (or indirectly by recruiting other effector killer cells) and regulating more than death receptor ligands (for example FasL) on the surface of the T cell that induces apoptosis of the target cell following binding to its cognate death receptor (for example In the target cell.
[0397] In one embodiment, the present invention provides a method of treating an individual diagnosed with a cancer, which comprises removing the immune effector cells from the individual, genetically modifying said immune effector cells with a vector comprising a nucleic acid that encodes a TCR or CAR modified by genetic engineering as contemplated here, thereby producing a population of modified immune effector cells and administering the population of modified immune effector cells to the same individual. In the preferred embodiment, the immune effector cells comprise T cells.
[0398] In certain embodiments, the present invention also provides methods for stimulating an immune effector cell-mediated immune modulator response to a target cell population in an individual comprising the steps of administering to the individual an immune effector cell population that express a nucleic acid builder that encodes a TCR or CAR modified by genetic engineering molecule.
[0399] The method for administering the cell compositions described here includes any method that is effective in resulting in ex vivo reintroduction of genetically modified immune effector cells that are directly expressed in a genetically engineered TCR or CAR in the individual or in the reintroduction of the genetically modified progenitors of immune effector cells that on introduction into an individual differentiate into mature immune effector cells that express the genetically modified TCR or CAR. One method comprises transducing peripheral blood T cells ex vivo with a nucleic acid builder according to the present invention and returning the transduced cells to the subject.
[0400] All publications, patent applications and issued patents cited in this specification are hereby incorporated by reference as if each individual publication, patent application or issued patent was specifically and individually indicated to be incorporated by reference.
[0401] Although the prior invention has been described in some detail by way of illustration and example for the sake of clarity of understanding, it will be readily apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications can be made without deviate from the spirit or scope of the attached claims. The following examples are offered by way of illustration only and not by way of limitation. Those skilled in the art can easily recognize a variety of non-fundamental parameters that could be altered or modified to produce essentially similar results.
Examples Example 1 Proliferation of T cells cultured with an AKT inhibitor
[0402] The purpose of this experiment was to determine the effect of AKT inhibitors on T cell proliferation. The impact of MK-2206 (Seleckchem) on T cell proliferation was assessed by measuring T cell division.
[0403] Peripheral blood mononuclear cells (PBMC) are the heterogeneous cell population that contains T cells. PBMC were collected from normal donors and marked with a fluorescent ink (CelTrace® Violet, Molecular Probes), whose intensity is progressively diluted by a factor of two with each cell division. The labeled PBMCs were used as the source of cells for T cell expansion. T cells were activated and expanded by culturing the PBMCs labeled with CD3 and CD28 antibodies (Miltenyi Biotec) in medium containing IL-2 (CelGenix).
[0404] The impact of MK-2206 on cell division was tested by assessing CelTrace Violet dilution with flow cytometry after adding 0.025 μΜ, 0.074 μΜ, 0.222 μΜ, 0.67 μΜ, or 2.0 μΜ, MK-2206 to T cultures on day 0. XVIVO-15 fresh-based culture medium containing IL-2 and MK-2206 was added to T cell cultures every two to three days for a total of seven days to allow for development and expansion of T cells, MK-2206 treatment did not substantially decrease T cell division three days after the start of culture (Figure 1A). In addition, T cells cultured in the presence of MK-2206 for seven days did not show statistically significant differences in T cell division compared to the vehicle or untreated controls (Figure 1B). A pairwise t-test was performed on cultures on day seven; (each MK-2206 concentration was compared to 0 μΜ MK-2206: the cultures were not statistically different at the p <0.05 level). EXAMPLE 2 EXPRESSION OF CD62L IN TT CELLS TREATED WITH MK-2206 [0405] The aim of the present experiment was to determine the effect of AKT inhibitors on T cell markers in terms of T cell power. The impact of MK-2206 (Seleckchem ) on T cell potency was assessed by measuring CD62L expression in T cell cultures manufactured in Example 1.
[0406] Mouse models have shown that CD62L expression identifies potent T cells; T cells with greater anti-tumor efficacy after adoptive transfer. However, T cells cultured with IL-2 in vitro have been shown to reduce CD62L expression and therefore correlate with the reduced anti-tumor potency of tumor-specific T cells. Surprisingly, the present inventor found that the AKT MK-2206 inhibitor significantly increased the expression of CD62L at all concentrations of MK-2206 tested after seven days of culture and in the presence of IL-2. Figure 2. A paired T test was performed on all seven days of cultures (each concentration of MK-2206 was compared to 0 μΜ MK-2206: * p <.05, ** p <.01). EXAMPLE 3 EXPRESSION OF CD62L IN T-CAR CELLS TREATED WITH MK-2206 OR ZSTK474 [0407] T-CAR cells were cultured with either MK-2206 or trichiribin phosphate - TCN (AKT inhibitors) or ZSTK474 (PI3K inhibitor). CAR T cells specific for antigen maturing B cell (BCMA) were used in this set of experiments. T CAR cell cultures were performed using a directly scalable system of major clinical manufacturing processes. In short, peripheral blood mononuclear cells (PBMC) were grown in static flasks in medium containing IL-2 (CelGenix) and specific antibodies for CD3 and CD28 (Miltenyi Biotec). 2x108 make the lentivirus transduction units encoding anti-BCMA CARs were added one day after the initiation culture. The anti-BCMA CAR T cells were maintained in log-phase by adding fresh medium containing IL-2 and an optimized dose of an inhibitor for a total of ten days of culture. At the end of the culture, the anti-BCMA CAR T cells were interrogated for the phenotype.
[0408] CD62L expression in anti-BCMA CAR T cells was assessed by flow cytometry at the end of the culture. Anti-BCMA CAR T cells were specifically identified using PE-conjugated recombinant human BCMA-IgG Fc. CD62L expression was determined with a co-stain using an antibody specific for CD62L. CAR anti-BCMA T cells from three normal donors cultured with IL-2 and MK2206 had significantly higher CD62L expression compared to cultures with IL-2 alone. Lentiviral transduction to express anti-BCMA CARs did not reduce the enhanced phenotype that caused MK2206. A similar improvement was seen using ZST474 during culture but not with TCN. (Figure 3). EXAMPLE 4 CAR T cells treated with Mk-2206 or ZSTK474 Show Enhanced Therapeutic Activity [0409] Anti-BCMA CAR T cells treated with MK-2206, TCN, or ZSTK474 as described in Example 3 were tested to determine whether the highest expression CD62L was associated with increased antitumor activity. T cells expressing anti-BCMA CARs were generated after culturing with iL-2 and either MK2206, TCN or ZsTK474. an aliquot of T CAR cells was also cultured in medium supplemented with IL-7 and IL-15 at doses previously demonstrated to improve the therapeutic efficacy of T CAR cells. Animals with 100mm3 subcutaneous multiple myeloma tumors (RpMI-8226) were infused with equivalent doses of T-CAR cell (1x106 CAR-positive cells).
[0410] Anti-BCMA CAR T cells cultured with IL-2 were sufficient to cause complete tumor regression. Anti-BCMA T CAR cells cultured with IL-7 and IL-15, MK-2206, or ZsT747 showed similar levels of anti-tumor activity compared to standard anti-BCMA T cells cultured with standard IL-2 in this animal model of multiple myeloma. Differently, anti-BCMA T cell CAR cultured with TCN completely abolished any anti-tumor response. The results of these experiments are shown in figure 4. EXAMPLE 5 CAR T cells treated with Mk-2206 or ZSTK474 Show Enhanced Therapeutic Activity in an Aggressive Tumor Model [0411] A more aggressive and difficult to treat tumor model was used to check the anti-tumor activity of T CAR cells treated with iL-2, MK-2206, TCN, ZsTK474, iL7 / 15 or vehicle. Daudi tumor cells express a low level of BCMA protein and can be effectively treated using anti-BCMA CAR T cells. Daudi's tumor progression was not affected after treatment with anti-BCMA T-CAR cells cultured with IL-2- or IL7 / 15. Strictly, anti-BCMA T CAR cells cultured with either MK-2206 or ZsT474 caused complete tumor regression. the results of these experiments are shown in figure 5. EXAMPLE 6 [0412] CAR T Cells Treated with Mk-2206 or ZSTK474 Show Improved Persistence [0413] Clinical data in patients treated with CAR T cells suggest that persistence is associated with objective responses of the tumor. The persistence of anti-BCMA T-CAR cells was assessed by again challenging tumors in mice that had a complete regression. Animals treated with anti-BCMA T CAR cells cultured with iL-2-, MK-2206-, or ZsTK474 that completely regressed 100mm3 RPMI-8226 tumors were challenged again 13 days later with RPMI-8226 on the opposite flank. Animals treated with CAR T cells cultured with iL-2 were unable to prevent tumor development. Differently, none of the animals treated with anti-BCMA T-CAR cells cultured with ZSTK474 showed any evidence of tumor grafting. Said data show that therapeutically active anti-BCMA T CAR cells persist in anti-BCMA T CAR cells cultured with ZSTK474. The results of these experiments are shown in figure 6.
[0414] In general, the following claims, the terms used should not be construed in such a way as to limit the claims to the specific modalities described in the specification and the claims, but should be constructed to include all possible modalities along with the total scope of equivalents to which the claims are entitled. Accordingly, the claims are not limited by the description.

权利要求:
Claims (22)
[1]
1. In vitro or ex vivo method for making T cells CHARACTERIZED by the fact that it comprises: (a) activating a population of T cells and stimulating the population of T cells to proliferate; (b) transducing T cells with a viral vector comprising a polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen (CAR) receptor; (c) cultivating the transduced T cells to proliferate; where steps (a) to (c) are performed in the presence of a phosphatidyl-inositol-3 kinase inhibitor (PI3K) selected from the group consisting of: BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX -221, AS25242, CAL-101, IPI-145 and ZSTK474; and wherein the proliferation of T cells is maintained in the transduced T cells manufactured according to steps (a) to (c) performed in the presence of the PI3K inhibitor compared to the proliferation of transduced T cells manufactured according to steps (a) a (c) performed in the absence of the PI3K inhibitor.
[2]
2. Method, according to claim 1, CHARACTERIZED by the fact that: (a) the source of T cells is peripheral blood mononuclear cells; (b) T cell activation comprises contacting T cells with an anti-CD3 antibody or a single anti-CD3 variable chain (scFv) fragment; (c) stimulation of T cells comprises contacting T cells with an anti-CD28 antibody or an anti-CD28 scFv; (d) T cells are transduced with the viral vector before T cell proliferation; or (e) T cells are transduced with the viral vector after T cell proliferation.
[3]
3. Method, according to claim 1 or 2, CHARACTERIZED by the fact that: (a) the viral vector is a retroviral vector; (b) the viral vector is a lentiviral vector; or (c) T cells comprise a CAR.
[4]
4. Method, according to claim 3, CHARACTERIZED by the fact that the CAR comprises: (a) an extracellular domain that binds to an antigen selected from the group consisting of: BCMA, alpha folate receptor, 5T4, integrin OvP6, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, family EGFR including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glipican-3 (GPC3), HLA-A1 + MAGE1, HLA-A2 + MAGE1, HLA-A3 + MAGE1, HLA-A1 + NY-ESO-1, HLA-A2 + NY-ESO-1, HLA-A3 + NY-ESO-1, IL-11Ra, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivina, TAG72, TEMs and VEGFR2 ligands; (b) a transmembrane domain derived from a polypeptide selected from the group consisting of: CD8o; CD4, CD28, CD45, PD-1 and CD152; (c) one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2) , CD274 (PD-L1) and CD278 (ICOS); and (d) a CD3Z signaling domain.
[5]
5. Method according to claim 4, CHARACTERIZED by the fact that: (a) the extracellular domain comprises an antibody or antigen-binding fragment that binds to the antigen, in which the antigen-binding fragment is selected from of the group consisting of: an Ig Camel (camelid antibody (VHH)), an Ig NAR, a Fab fragment, a Fab 'fragment, an F (ab)' 2 fragment, an F (ab) '3 fragment, an Fv fragment , a single-chain Fv antibody (“scFv”), a bis-scFv, (scFv) 2, a minibody, a bicorpt, a tri-body, a tetribody, a disulfide-stabilized Fv protein (“dsFv”) and an single domain (sdAb, Nanocorpo); (b) the transmembrane domain is derived from CD8o or CD28; (c) the one or more co-stimulatory signaling domains are selected from the group consisting of: CD28, CD134 and CD137; (d) the CAR further comprises a hinge region polypeptide; (e) the CAR further comprises a hinge region polypeptide that comprises an IgG1 or CD8o hinge region; (f) the CAR further comprises a signal peptide; (g) the CAR further comprises a signal peptide comprising an IgG1 heavy chain signal polypeptide or a CD8o signal polypeptide.
[6]
6. Method according to claim 5, CHARACTERIZED by the fact that the CAR comprises an antibody or antigen-binding fragment that binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79a, CD79b, CD123 , CD138, CSPG4, EGFR, HER2, EGFRvIII or MUC16, in which the antigen binding fragment is selected from the group consisting of: an Ig Camel (a camelid antibody (VHH)), an Ig NAR, a Fab fragment , a Fab 'fragment, an F (ab)' 2 fragment, an F (ab) '3 fragment, an Fv, a single chain Fv antibody (“scFv”), a bis-scFv, (scFv) 2, an minibody, a bicorpt, a tricorpt, a tetrabody, a disulfide stabilized Fv protein (“dsFv”) and a single domain antibody (sdAb, Nanocorp).
[7]
7. Method, according to claim 5, CHARACTERIZED by the fact that the CAR is an anti-BCMA CAR.
[8]
8. Method, according to claim 7, CHARACTERIZED by the fact that the anti-BCMA CAR comprises a scFv that binds to BCMA, a CD8o joint, a CD8o transmembrane domain, a co-stimulatory domain of 4- 1 BB and a CD3Z primary signaling domain.
[9]
9. Method according to any one of claims 1 to 8, CHARACTERIZED by the fact that the PI3K inhibitor is ZSTK474.
[10]
10. Method according to any one of claims 1 to 9, CHARACTERIZED by the fact that the population of T cells manufactured in the presence of the PI3K inhibitor has an increased number of T cells that express one or more markers selected from the group which consists of: CD62L, CCR7, CD28, CD27, CD122 and CD127 compared to a population of T cells manufactured in the absence of the PI3K inhibitor.
[11]
11. Method according to claim 10, CHARACTERIZED by the fact that the population of T cells manufactured in the presence of the PI3K inhibitor does not express CD57 or KLRG1 or expresses less CD57 or KLRG1 compared to the population of T cells manufactured in the absence of the inhibitor PI3K.
[12]
12. Composition CHARACTERIZED by the fact that it comprises a cryoprotective agent and a population of immune effector cells comprising a vector comprising a polynucleotide encoding a TCR or CAR, in which the immune effector cells were manufactured in the presence of a PI3K inhibitor, and where the population of T cells manufactured in the presence of the PI3K inhibitor has an increased number of T cells expressing one or more markers selected from the group consisting of: CD62L, CCR7, CD28, CD27, CD122 and CD127 compared to a population of T cells manufactured in the absence of the PI3K inhibitor.
[13]
13. Composition according to claim 12, CHARACTERIZED by the fact that immune effector cells comprise T cells.
[14]
14. Composition according to claim 12 or 13, CHARACTERIZED by the fact that immune effector cells comprise a lentiviral vector comprising a polynucleotide encoding a CAR, wherein the CAR comprises: a) an antibody or fragment binding to antigen that binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79a, CD79b, CD123, CD138, CSPG4, EGFR, HER2, EGFRvIII or MUC16, in which the antigen binding fragment is selected from the group consisting of: an Ig Camel (a camelid antibody (VHH)), an Ig NAR, a Fab fragment, a Fab 'fragment, an F (ab)' 2 fragment, an F (ab) '3 fragment, an Fv , a single-chain Fv antibody (“scFv”), a bis-scFv, (scFv) 2, a minibody, a bicorpt, a tri-body, a tetribody, a disulfide-stabilized Fv protein (“dsFv”) and an single domain (sdAb, Nanocorpo); b) a transmembrane domain derived from a polypeptide selected from the group consisting of: CD8o; CD4, CD28, CD45, PD-1 and CD152; c) one or more intracellular co-stimulatory signaling domains selected from the group consisting of: CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1) and CD278 (ICOS); and d) a CD3Z signaling domain.
[15]
15. Composition according to any one of claims 12 to 14, CHARACTERIZED by the fact that the immune effector cells comprise a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR.
[16]
16. Composition according to any one of claims 12 to 15, CHARACTERIZED by the fact that the immune effector cells comprise a lentiviral vector comprising a polynucleotide encoding an anti-BCMA CAR, wherein the anti-BCMA CAR comprises an scFv that binds to BCMA, a CD8o joint, a CD8o transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3Z primary signaling domain.
[17]
17. Composition according to any one of claims 12 to 16, CHARACTERIZED by the fact that the PI3K inhibitor is ZSTK474.
[18]
18. Use of the composition, as defined in any of claims 12 to 17, CHARACTERIZED by the fact that it is for the preparation of a pharmaceutical composition for the treatment of cancer in an individual in need thereof.
[19]
19. Use, according to claim 18, CHARACTERIZED by the fact that: (a) the cancer is selected from the group consisting of myeloma, Wilms' tumor, Ewing's sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma , a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile cancer, cervical cancer, endometrial cancer, esophageal cancer, cancer gastric, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and cancer urinary bladder; (b) the cancer is a B cell malignancy and the binding domain binds to a BCMA epitope; (c) cancer is bladder cancer and the extracellular binding domain binds to a PSCA or MUC1 epitope; (d) the cancer is glioblastoma multiforme and the extracellular binding domain binds to an EPHA2, EGFRvIII or CSPG4 epitope; (e) cancer is lung cancer and the extracellular binding domain binds to a PSCA or GD2 epitope; (f) cancer is breast cancer and the extracellular binding domain binds to an epitope of CSPG4 or HER2; (g) the cancer is melanoma and the extracellular binding domain binds to an epitope of CSPG4 or GD2; or (h) the cancer is pancreatic cancer and the extracellular binding domain binds to a PSCA or MUC1 epitope.
[20]
20. Use of the composition, as defined in any one of claims 12 to 17, CHARACTERIZED by the fact that it is for the preparation of a pharmaceutical composition to treat multiple myeloma (MM), chronic lymphocytic leukemia (CLL), or lymphoma non-Hodgkin (NHL) in an individual in need of it.
[21]
21. Use, according to claim 20, CHARACTERIZED by the fact that MM is selected from the group consisting of: evident multiple myeloma, latent multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, myeloma osteosclerotic, solitary bone plasmacytoma and extramedullary plasmacytoma.
[22]
22. Use, according to claim 20, CHARACTERIZED by the fact that NHL is selected from the group consisting of: Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), large B-cell lymphoma diffuse, follicular lymphoma, immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma and mantle cell lymphoma.

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优先权:

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申请号 | 申请日 | 专利标题

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US201462008957P| true| 2014-06-06|2014-06-06|

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PCT/US2015/034515|WO2015188119A1|2014-06-06|2015-06-05|Improved t cell compositions|

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