Methods for improving cell therapy with organelle complexes

Abstract

Disclosed herein are methods, compositions, and kits suitable for use in enhancing adoptive T cell therapy. In some embodiments, the methods include contacting a population of T cells with an isolated organelle complex to generate a population of T cells containing the organelle complex. The organelle complex can include mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The population of T cells can exhibit one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence compared to a population of T cells that does not contain the exogenous organelle complex.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 394,546, filed August 2, 2022, U.S. Provisional Application No. 63 / 403,268, filed September 1, 2022, and U.S. Provisional Application No. 63 / 406,022, filed September 13, 2022, the entire contents of which are expressly incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to methods for enhancing the proliferation, migration, persistence and / or activity of T cells for adoptive T cell therapy. [Background technology]

[0003] Mitochondria are intracellular organelles responsible for multiple metabolic transformations and regulatory functions. They are also highly dynamic organelles, mitochondria moving throughout the cell, undergoing structural transitions and changing length, morphology, shape, and size. Furthermore, mitochondria are continuously removed and regenerated in a process known as mitochondrial biogenesis. Although most mitochondrial genes are transferred to the nuclear genome, the mitochondrial genome still encodes rRNA, tRNA, and 13 subunits of the electron transport chain (ETC). Therefore, functional communication between the nuclear and mitochondrial genomes is essential for mitochondrial biogenesis, efficient oxidative phosphorylation, and normal health. Mitochondria are also a major source of free radicals and reactive oxygen species (ROS), which cause oxidative stress. Furthermore, mitochondria play an important role in intracellular signaling and the regulation of cell death, including apoptosis and necrosis.

[0004] Chimeric antigen receptor (CAR) T-cell therapy is a potentially curative treatment for patients with relapsed or refractory (r / r) hematopoietic malignancies. However, approximately half of patients with r / r B-cell lymphoma experience treatment failure after CAR-T-cell therapy, likely due to poor proliferation, limited persistence, and exhaustion of CAR-T cells. Given that metabolic fitness plays a central role in regulating T-cell function, manipulating mitochondrial function is a promising approach to enhance the antitumor efficacy of CAR-T cells. Methods to enhance the proliferation, migration, persistence, and / or activity of T cells for adoptive T-cell therapy are needed. Summary of the Invention

[0005] Provided herein are methods and compositions that solve the above-mentioned problems in the art. For example, transplantation of isolated organelle complexes as described herein into T cells can metabolically reprogram T cells, thereby improving their performance across multiple parameters both in vitro and in vivo, including cytokine production, proliferation, persistence, and anti-tumor activity. Disclosed herein are methods for generating a population of T cells for adoptive T cell therapy. In some embodiments, the method includes contacting an isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, wherein the organelle complex comprises mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the method includes stimulating the population of T cells. In some embodiments, the stimulating step expands the population of T cells.

[0006] Disclosed herein are methods for enhancing the proliferation, migration, persistence, and / or activity of a population of T cells for adoptive T cell therapy. In some embodiments, the methods include contacting an isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, the organelle complex comprising mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The methods can also include stimulating the population of T cells to expand the population of T cells.

[0007] Disclosed herein are methods for generating a population of T cells resistant to exhaustion. In some embodiments, the method includes contacting an isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, the organelle complex comprising mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The method can include stimulating the population of T cells to expand the population of T cells.

[0008] In some embodiments, the stimulation occurs before, after, and / or during the contacting. In some embodiments, the stimulation comprises culturing the population of T cells in the presence of one or more stimulatory agents. In some embodiments, the one or more stimulatory agents comprise an agent that stimulates a CD3 / TCR complex-associated signal and an agent that stimulates a costimulatory molecule on the surface of the T cells. In some embodiments, the one or more stimulatory agents comprise a molecule that binds to CD28 (e.g., one or more of an anti-CD28 antibody, CD80, and CD86) and / or a molecule that binds to CD3 (e.g., an anti-CD3 antibody). In some embodiments, the stimulation and / or contacting occurs for a period of at least about 6 hours, 12 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some embodiments, contacting the population of T cells with the isolated organelle complex allows the organelle complex to be internalized into the T cells. In some embodiments, the contacting step is repeated at least 2, 3, or 4 times. In some embodiments, the contacting occurs during one or both of the stimulating step and the introducing step. In some embodiments, the population of T cells is contacted with an effective amount of the isolated organelle complex sufficient to enhance proliferation, migration, persistence, and / or activity of the T cells. In some embodiments, the effective amount comprises about 20 μg of the isolated organelle complex. In some embodiments, the population of T cells is contacted with an effective amount of the isolated organelle complex sufficient to render the T cells resistant to exhaustion. In some embodiments, the population of T cells is derived from lymph node cells (e.g., purified by a magnetic particle-based enrichment method selected from the group consisting of manualMACS®, AutoMACS®, CliniMACS®, EasySep®, and RoboSep®). In some embodiments, the population of T cells is CD4 + T cells, CD8 +The present invention relates to a method for treating inflammatory bowel disease, including the treatment of inflammatory bowel disease, including one or more of T cells, cytotoxic T cells, peripheral effector T cells, effector T cells, memory or central memory T cells, naive T cells, regulatory T cells, natural killer T cells, gamma delta T cells, cytokine-induced killer (CIK) T cells, and tumor-infiltrating lymphocytes (TILs).

[0009] In some embodiments, the organelle complex comprises a first organelle complex, a second organelle complex, or a combination of the first organelle complex and the second organelle complex, wherein the first organelle complex and the second organelle complex are depleted of cytoplasmic macromolecules, and the first organelle complex is derived from (i) frozen cells, (ii) suspension cells, and / or (iii) cells contacted with a detergent at a concentration equal to or greater than the critical micelle concentration (CMC) of the detergent, and the second organelle complex is derived from (i) adherent cells and / or (ii) cells contacted with a detergent at a concentration less than the critical micelle concentration (CMC) of the detergent. In some embodiments, the cytoplasmic macromolecules include cytoplasmic proteins (e.g., the cytoplasmic proteins are p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), and the abundance of one or more cytoplasmic proteins is at least about 90% depleted compared to the cell from which the organelle complex population was derived. In some embodiments, the organelle complexes include one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM) and / or citrate synthase (CS)), one or more mitochondrial outer membrane proteins (e.g., mitochondrial outer membrane complex subunit 20 (TOMM20)), one or more lysosomal proteins (e.g., lysosome-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosome-associated membrane protein 1 (LAMP1)), one or more peroxisomes (e.g., mitochondrial matrix proteins ... and / or one or more Golgi apparatus proteins (e.g., Golgin-97, Sintaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha class 2A member 1 (MAN2A1)), and / or one or more endoplasmic reticulum proteins (e.g., calticarin and / or caldisin).In some embodiments, the organelle complex is derived from cells treated with a mitochondrial activator (e.g., resveratrol). In some embodiments, the organelle complex is derived from cells of a subject different from the subject from which the T cells are derived. In some embodiments, the organelle complex is derived from cells of the same subject from which the T cells are derived.

[0010] In some embodiments, the method comprises introducing a heterologous nucleic acid (e.g., a vector) encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR) into the T cell. In some embodiments, the introducing step occurs before, after, and / or during said contacting. In some embodiments, the T cell comprises a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). In some embodiments, the CAR and / or TCR comprise one or more of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain, a costimulatory domain, or both a primary signaling domain and a costimulatory domain. In some embodiments, the primary signaling domain comprises a functional signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12, or a functional variant thereof.In some embodiments, the costimulatory domain is selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD28-OX40, CD28-4-1BB, CD30, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAMl(CD226), SLAMF 4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), C In some embodiments, the antigen-binding domain comprises a functional domain of one or more proteins selected from the group consisting of D69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D, or a functional variant thereof. In some embodiments, the antigen-binding domain binds to a tumor antigen (e.g., a solid tumor antigen).In some embodiments, the antigen-binding domain comprises an antibody, antibody fragment, scFv, Fv, Fab, a(Fab'), single domain antibody (SDAB), VH or VL domain, camelid VHH domain, Fab, Fab', F(ab'), Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single domain antibody (sdAb), single chain comprising anti-complementary scFv (tandem scFv) or bispecific tandem scFv, Fv construct, disulfide-linked Fv, dual variable domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, affibody, affilin, affitin, affimer, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, or any combination thereof. In some embodiments, the antigen-binding domain is connected to the transmembrane domain by a hinge region.In some embodiments, the transmembrane domain is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), IC OS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLEF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, C D103, ITGAL, CD11a, LFA-1, ITGAM, CD11d, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1( CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, C The CAR or TCR further comprises a transmembrane domain of a protein selected from the group consisting of D100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or a functional variant thereof. In some embodiments, the CAR or TCR further comprises a leader peptide. In some embodiments, the TCR further comprises a constant region and / or CDR4.

[0011] In some embodiments, the T cell recovery efficiency is at least about 5 percent greater than a method that does not include contacting the population of T cells with an isolated organelle complex. In some embodiments, T cell recovery is the ratio of T cells recovered after stimulation (e.g., about 48 hours after stimulation) to the number of T cells immediately before the start of stimulation. In some embodiments, the population of T cells exhibit one or more of enhanced expansion capacity (in vivo and / or in vitro), enhanced cytotoxicity against target cells (in vivo and / or in vitro), enhanced resistance to exhaustion (in vivo and / or in vitro), and enhanced persistence (in vivo and / or in vitro) compared to a population of T cells that does not include an exogenous organelle complex. In some embodiments, the population of T cells exhibits at least about a 1.1-fold increase in basal and / or maximal oxygen consumption rate (in vivo and / or in vitro) compared to a population of T cells that does not include an exogenous organelle complex. In some embodiments, the population of T cells exhibit at least about a 1.1-fold increase in basal and / or maximal glycolytic rates (in vivo and / or in vitro) compared to a population of T cells that do not comprise exogenous organelle complexes. In some embodiments, the population of T cells exhibit at least about a 1.1-fold increase in glycolytic and / or respiratory capacity (in vivo and / or in vitro) compared to a population of T cells that do not comprise exogenous organelle complexes. In some embodiments, the population of T cells exhibit at least about a 1.1-fold increase in mitochondrial mass (in vivo and / or in vitro) compared to a population of T cells that do not comprise exogenous organelle complexes. In some embodiments, the population of T cells exhibit at least about a 1.1-fold increase in cytotoxic activity (in vivo and / or in vitro) against target cells compared to a population of T cells that do not comprise exogenous organelle complexes. In some embodiments, the population of T cells exhibits at least about a 1.1-fold increase in the levels (in vivo and / or in vitro) of one or more exhaustion markers compared to a population of T cells that does not contain the exogenous organelle complex.In some embodiments, the exhaustion marker is selected from the group consisting of PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, TIGIT, or any combination thereof. In some embodiments, the population of T cells exhibits at least about a 1.1-fold decrease in levels of one or more of cellular ROS (in vivo and / or in vitro), mitochondrial ROS (in vivo and / or in vitro), cellular oxidative stress (in vivo and / or in vitro), and mitochondrial oxidative stress (in vivo and / or in vitro) compared to a population of T cells that does not contain the exogenous organelle complex. In some embodiments, the reactive oxygen species is superoxide (O2). - ), hydroperoxy (HO.2), hydrogen peroxide (H2O2), peroxynitrite (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO), peroxy radical (ROO), alkoxy radical (RO), singlet oxygen ( 1In some embodiments, the population of T cells comprises at least about a 1.1-fold increase in production of one or more cytokines (in vivo and / or in vitro) compared to a population of T cells not comprising the exogenous organelle complex. In some embodiments, the cytokines comprise interleukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and / or transforming growth factor-beta (TGFβ). In some embodiments, the population of T cells exhibits at least about a 1.1-fold increase in cell proliferation (in vivo and / or in vitro) compared to a population of T cells not comprising the exogenous organelle complex. In some embodiments, the population of T cells exhibits at least about a 1.1-fold increase in cell viability (in vivo and / or in vitro) following chronic TCR stimulation compared to a population of T cells that does not contain the exogenous organelle complex.

[0012] In some embodiments, populations of T cells generated by the methods disclosed herein are provided. In some embodiments, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise a population of T cells generated by the methods disclosed herein and one or more pharmaceutically acceptable carriers.

[0013] In some embodiments, a population of T cells for adoptive T cell therapy is provided, wherein the T cells comprise an exogenous organelle complex. In some embodiments, the organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, peroxisomes, lysosomes, and a Golgi apparatus. In some embodiments, the population of T cells exhibit one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not comprise the exogenous organelle complex.

[0014] Disclosed herein are methods for treating or preventing a disease or disorder in a subject. In some embodiments, the method comprises administering to the subject an effective amount of the population of T cells produced according to the methods disclosed herein, thereby treating or preventing the disease or disorder in the subject. In some embodiments, the population of T cells exhibit one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not contain an exogenous organelle complex.

[0015] Disclosed herein are methods for enhancing adoptive T cell therapy in a subject. In some embodiments, the method includes generating an effective amount of T cells comprising an exogenous organelle complex according to the methods disclosed herein and adoptively transferring the T cells into the subject. In some embodiments, the population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence compared to a population of T cells that does not comprise the exogenous organelle complex.

[0016] Disclosed herein are methods for treating or preventing a disease or disorder in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a population of T cells (e.g., T cells comprising a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR)) and administering to the subject an effective amount of an isolated organelle complex, thereby treating or preventing the disease or disorder in the subject. In some embodiments, the isolated organelle complex can be contacted with the population of T cells in vivo to generate a population of T cells comprising the organelle complex. In some embodiments, the population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not comprise the exogenous organelle complex. In some embodiments, an effective amount of the population of T cells is administered to the subject before administering an effective amount of the isolated organelle complex to the subject. In some embodiments, an effective amount of the isolated organelle complex is administered to the subject before administering an effective amount of the population of T cells to the subject. In some embodiments, the effective amount of the population of T cells and the effective amount of the isolated organelle complex are administered to the subject simultaneously.

[0017] Disclosed herein are methods for treating or preventing a disease or disorder in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a heterologous nucleic acid (e.g., a vector, a viral vector) encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), and administering to the subject an effective amount of an isolated organelle complex, thereby treating or preventing the disease or disorder in the subject. In some embodiments, the heterologous nucleic acid and the isolated organelle complex can be contacted in vivo with T cells from the subject to generate T cells comprising the organelle complex and the CAR and / or engineered TCR. In some embodiments, the T cells exhibit one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to T cells that do not comprise the exogenous organelle complex.

[0018] In some embodiments, the in vivo persistence of the population of T cells comprises a period of about 15 days, about 30 days, about 60 days, about 90 days, or about 1 year. In some embodiments, the population of T cells reduces tumor volume, tumor growth, and / or tumor burden in the subject. In some embodiments, the population of T cells reduces tumor volume in the subject by at least about 1.1 fold compared to tumor volume in an untreated subject or a subject administered a population of T cells that does not include exogenous organelle complexes. In some embodiments, the population of T cells increases overall survival or progression-free survival. In some embodiments, the population of T cells increases overall survival or progression-free survival by at least about 1.1 fold compared to an untreated subject or a subject administered a population of T cells that does not include exogenous organelle complexes. In some embodiments, the T cells are autologous to the subject. In some embodiments, the T cells are allogeneic to the subject. In some embodiments, the adoptive T cell therapy is CAR-T cell therapy. In some embodiments, the adoptive T cell therapy is engineered TCR-T cell therapy. In some embodiments, the adoptive T cell therapy is tumor infiltrating lymphocyte (TIL) therapy. In some embodiments, the administration is at least about 1 x 10 6 In some embodiments, the method comprises administering a population of T cells. In some embodiments, the method comprises repeat administration of the population of T cells.

[0019] In some embodiments, the subject is a mammal. In some embodiments, the disease or disorder is associated with expression of a tumor antigen, and the disease associated with expression of the tumor antigen is selected from the group consisting of a proliferative disease, a precancerous condition, cancer, and a non-cancer-related indication associated with expression of the tumor antigen. In some embodiments, the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell carcinoma of the lung, mesothelioma, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, or a combination thereof. The cancer is selected from the group consisting of adenocarcinoma, parathyroid carcinoma, adrenal gland carcinoma, soft tissue sarcoma, urethral carcinoma, penile carcinoma, childhood cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of the above cancers, and metastatic lesions of the above cancers. In some embodiments, the cancer is a hematological cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or preleukemia.

[0020] In some embodiments, the administration comprises systemic administration (intravenous, intramuscular, intraperitoneal, or intraarticular), intrathecal administration, intracranial injection, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, topical delivery, local delivery, intraciliary delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows data regarding enhanced OXPHOS of Q-treated CAR-T cells compared to vehicle-treated CAR-T cells. [Figure 2] 1 shows data regarding the suppression of tumor growth in mice injected with Q-treated CAR-T cells. [Figure 3] Data are presented regarding the efficiency of T cell recovery after stimulation following transplantation of a second organelle complex (2ndOC) or vehicle. [Figure 4A] Data are shown for mitochondrial mass assessment using MitoTracker Deep Red dye. CD4 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 4B] Data are shown for mitochondrial mass assessment using MitoTracker Deep Red dye. CD4 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 4C] Data are shown for mitochondrial mass assessment using MitoTracker Deep Red dye. CD8 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 4D] Data are shown for mitochondrial mass assessment using MitoTracker Deep Red dye. CD8 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 5A]Data are shown for T cell proliferation capacity assessed by dye dilution assay using Cell Trace Violet dye. CD4 T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 5B] Data are shown for T cell proliferation capacity assessed by dye dilution assay using Cell Trace Violet dye. CD4 T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 5C] Data are shown for T cell proliferation capacity assessed by dye dilution assay using Cell Trace Violet dye. CD8 T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 5D] Data are shown for T cell proliferation capacity assessed by dye dilution assay using Cell Trace Violet dye. CD8 T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 6A] Data are shown for mitochondrial-selective oxidative stress measurements using MitoSOX Red dye. CD4 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 6B] Data are shown for mitochondrial-selective oxidative stress measurements using MitoSOX Red dye. CD4 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 6C] Data are shown for mitochondrial-selective oxidative stress measurements using MitoSOX Red dye. CD8 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 6D]Data are shown for mitochondrial-selective oxidative stress measurements using MitoSOX Red dye. CD8 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 7A] Data are shown for cellular oxidative stress levels measured using cellular ROX Green dye, and CD4 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 7B] Data are shown for cellular oxidative stress levels measured using cellular ROX Green dye, and CD4 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 7C] Data are shown for cellular oxidative stress levels measured using cellular ROX Green dye, and CD8 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 7D] Data are shown for cellular oxidative stress levels measured using cellular ROX Green dye, and CD8 T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 8] Data are shown for T cell oxygen consumption rate measured with a Seahose Extracellular Flux Analyzer. T cells were transplanted with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 9A] Data are shown for cytokine production capacity assessed using PMA and ionomycin; T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 9B] Data are shown for cytokine production capacity assessed using PMA and ionomycin; T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed on day 4. [Figure 10A]Data are shown for the number of viable cells after chronic in vitro TCR stimulation. T cells were transplanted with a second organelle complex (2nd OC) or vehicle, and viable cell numbers were assessed on day 9 using a dead cell staining dye. [Figure 10B] Data are shown for the number of viable cells after chronic in vitro TCR stimulation. T cells were transplanted with a second organelle complex (2nd OC) or vehicle, and viable cell numbers were assessed on day 9 using a dead cell staining dye. [Figure 11] Data are shown for CAR-T cell glycolytic rates measured with a Seahorse Extracellular Flux Analyzer. [Figure 12A] Data are presented regarding CAR-T cell proliferation capacity assessed by dye dilution assay during co-culture with A20, CD4 CAR-T cells transplanted with a second organelle complex (2ndOC) or vehicle. [Figure 12B] Data are presented regarding CAR-T cell proliferation capacity assessed by dye dilution assay during co-culture with A20, CD4 CAR-T cells transplanted with a second organelle complex (2ndOC) or vehicle. [Figure 12C] Data are presented regarding CAR-T cell proliferation capacity assessed by dye dilution assay during co-culture with A20, CD8 CAR-T cells transplanted with a second organelle complex (2ndOC) or vehicle. [Figure 12D] Data are presented regarding CAR-T cell proliferation capacity assessed by dye dilution assay during co-culture with A20, CD8 CAR-T cells transplanted with a second organelle complex (2ndOC) or vehicle. [Figure 13A] Data on cytokine production capacity assessed using PMA and ionomycin are shown. [Figure 13B] Data on cytokine production capacity assessed using PMA and ionomycin are shown. [Figure 14] Data are shown for a cytotoxicity assay performed by co-culture with A20 cells. [Figure 15] 1 shows data regarding overall survival of mice injected with second organelle complex-treated CAR-T cells compared to vehicle-treated CAR-T cells or naive T cells. [Figure 16A] Flow cytometry analysis data for TCF-1 progenitor cells are shown, and T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed after 72 hours of TCR stimulation. [Figure 16B] Flow cytometry analysis data for TCF-1 progenitor cells are shown, and T cells were transferred with the second organelle complex (2ndOC) or vehicle and assessed after 72 hours of TCR stimulation. [Figure 17A] Flow cytometry analysis data for TCF-1 precursor CAR-T cells are shown; CAR-T cells were transplanted with a second organelle complex (2ndOC) or vehicle and evaluated 96 hours after the initiation of TCR stimulation. [Figure 17B] Flow cytometry analysis data for TCF-1 precursor CAR-T cells are shown; CAR-T cells were transplanted with a second organelle complex (2ndOC) or vehicle and evaluated 96 hours after the initiation of TCR stimulation. [Figure 18A] Data are shown regarding GPX4 expression in CD4+ T cells transferred with the second organelle complex (Q) or vehicle after stimulation with anti-CD3 / CD28 antibody for 72 hours. [Figure 18B] Data are shown regarding GPX4 expression in CD8+ T cells transplanted with the second organelle complex (Q) or vehicle after 72 hours of stimulation with anti-CD3 / CD28 antibodies. [Figure 19A] Data are shown regarding lipid peroxidation levels in CD4+ T cells transferred with the second organelle complex (Q) or vehicle after 72 hours of stimulation with anti-CD3 / CD28 antibody. [Figure 19B] Data are shown regarding lipid peroxidation levels in CD8+ T cells transplanted with the second organelle complex (Q) or vehicle after 72 hours of stimulation with anti-CD3 / CD28 antibodies. [Figure 20A] Data are shown for the percentage of apoptotic CD4+ T cells engrafted with the second organelle complex (Q) or vehicle after 120 hours of stimulation with anti-CD3 / CD28 antibody. [Figure 20B] Data are shown for the percentage of apoptotic CD8+ T cells engrafted with the second organelle complex (Q) or vehicle after 120 hours of stimulation with anti-CD3 / CD28 antibodies. [Figure 21A] 1 shows data on ROS accumulation in vehicle and Q-treated CAR-T cells assessed with CellROX Green dye in a CD4+ context. [Figure 21B] 1 shows data on ROS accumulation in vehicle and Q-treated CAR-T cells assessed with CellROX Green dye in a CD8+ context. [Figure 22A] Data are shown regarding lipid peroxidation levels in CD4+ CAR-T cells engrafted with the second organelle complex (Q) or vehicle. [Figure 22B] Data are shown regarding lipid peroxidation levels in CD8+ CAR-T cells engrafted with the second organelle complex (Q) or vehicle. [Figure 23A] Data are shown for the percentage of CD4+ CAR-T cells engrafted with the second organelle complex (Q) or vehicle to tumor infiltrating lymphocytes (TILs) 17 days after tumor cell inoculation. [Figure 23B] Data are presented regarding the percentage of CD8+ CAR-T cells engrafted with the second organelle complex (Q) or vehicle to tumor infiltrating lymphocytes (TILs) 17 days after tumor cell inoculation. [Figure 24A] Data are shown regarding the accumulation of cellular ROS in CD4+ T cells transferred with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibodies. [Figure 24B]Data are shown regarding the accumulation of cellular ROS in CD8+ T cells transferred with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibodies. [Figure 24C] Data are shown regarding the accumulation of mitochondrial ROS in CD4+ T cells transferred with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibodies. [Figure 24D] Data are shown regarding the accumulation of mitochondrial ROS in CD8+ T cells transferred with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibodies. [Figure 25A] Data are presented regarding the proliferative capacity of CD4+ T cells transferred with the second organelle complex (Q) or vehicle after chronic stimulation. [Figure 25B] Data are presented regarding the proliferative capacity of CD8+ T cells transferred with the second organelle complex (Q) or vehicle after chronic stimulation. [Figure 26A] Data are shown regarding the percentage of viable T cells transferred with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibody. [Figure 26B] Data are shown regarding the number of viable T cells transferred with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0022] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like elements, unless context dictates otherwise. The illustrative embodiments described in the detailed description, the drawings, and the claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described and illustrated in the figures herein, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and form a part of this disclosure.

[0023] All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated by reference in their entirety with respect to the relevant art.

[0024] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0025] As used herein, "isolated" shall be given its ordinary meaning and shall refer to a substance or entity that (1) has been separated from at least some of the components with which it was associated when originally produced (in nature or in an experimental setting) and / or (2) has been produced, prepared, and / or manufactured by the hand of man. In some embodiments, isolated mitochondrial or isolated organelle complex populations have been processed to obtain them from a cellular environment via the methods provided herein.

[0026] As used herein, the term "mitochondria" shall be given its ordinary meaning and shall refer to an organelle present within a eukaryotic cell having a matrix surrounded by a bilayer lipid membrane, inner and outer membranes, and cristae and inner membranes. Mitochondria (plural mitochondria) have enzymes, such as respiratory chain complexes involved in oxidative phosphorylation, on their inner membrane. The inner membrane has a membrane potential, such as a transverse proton gradient formed by the action of the respiratory chain complexes. It is believed that mitochondria cannot maintain their membrane potential when the inner membrane is disrupted.

[0027] As used herein, the term "organelle complex" shall be given its ordinary meaning and shall refer to a complex of mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. An organelle complex can be depleted of cytoplasmic macromolecules (e.g., cytoplasmic proteins). In some embodiments, an organelle complex does not contain cytoplasmic macromolecules. In some embodiments, an organelle complex population comprises homogenized mitochondria. As used herein, the term "population" shall be given its ordinary meaning and shall refer to a group of multiple identical or different substances. For example, an "organelle complex population" is a group of at least multiple identical or different organelle complexes. A population may not always be homogenous and may have physical, chemical, and / or physiological distributions. Physical distributions include, for example, particle size and polydispersity index. Chemical distributions include, for example, zeta potential distribution and lipid composition distribution. Physiological distributions include, for example, differences in physiological function (e.g., respiratory activity). The organelle complex population can include a first organelle complex, a second organelle complex, homogenized mitochondria, or any combination thereof. As used herein, the term "homogenized mitochondria" shall be given its ordinary meaning and shall refer to mitochondria isolated via a method that includes one or more homogenization steps.

[0028] As used herein, the term "surfactant" shall be given its ordinary meaning and shall refer to a molecule having a hydrophilic portion and a hydrophobic portion in one molecule. Surfactants serve to reduce surface tension at interfaces or to mix polar and non-polar substances by forming micelles. Surfactants are broadly classified into nonionic surfactants and ionic surfactants. Nonionic surfactants are those in which the hydrophilic portion is not ionized, and ionic surfactants are those in which the hydrophilic portion contains a cation, an anion, or both a cation and an anion.

[0029] As used herein, the term "critical micelle concentration" (CMC) shall be given its ordinary meaning and shall refer to the concentration, particularly the concentration in the bulk, at which a surfactant forms micelles and at which further surfactant added to the system contributes to micelle formation. At concentrations above the critical micelle concentration, adding surfactant to the system ideally increases the amount of micelles, particularly the number of micelles.

[0030] As used herein, the term "modulation" shall be given its ordinary meaning and shall also refer to an alteration or change in biological activity. Modulation includes, but is not limited to, stimulating or inhibiting activity. Modulation may be an increase or decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a cell, cell population, protein, pathway, system, or other biological target of interest, and may be a change of at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values).

[0031] As used herein, "subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold-blooded vertebrates, warm-blooded vertebrates, and invertebrates, such as fish, shellfish, reptiles, and especially mammals. As used herein, "mammal" refers to an individual belonging to the class Mammalia, including, but not limited to, humans, farm and livestock animals, zoo animals, and sport and pet animals. Mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, and primates, such as monkeys, chimpanzees, and apes, especially humans. In some embodiments, a mammal is a human. However, in some embodiments, a mammal is not a human. As used herein, the term "host cell" shall be given its ordinary meaning and shall refer to in vivo, in vitro, and / or ex vivo cells into which incorporation of exogenous mitochondria and / or organelle complexes is intended.

[0032] As used herein, the term "treatment" refers to an intervention made in response to a disease, disorder, or physiological condition manifested by a patient. The objectives of treatment include, but are not limited to, one or more of: alleviating or preventing symptoms; slowing or halting the progression or worsening of a disease, disorder, or condition; and ameliorating a disease, disorder, or condition. The terms "treat" and "treatment" include, for example, therapeutic treatment, prophylactic treatment, and applications in which a subject reduces the risk of developing a disorder or other risk factors. Treatment does not require a complete cure of the disorder, but encompasses embodiments in which symptoms or underlying risk factors are reduced. In some embodiments, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include those already affected by a disease or disorder or undesirable physiological condition, as well as those in whom a disease or disorder or undesirable physiological condition is to be prevented. As used herein, the term "prevention" refers to any activity that reduces an individual's burden of subsequently developing those symptoms. This can be done at the primary, secondary, and / or tertiary prevention levels, where a) primary prevention avoids the onset of the condition / disorder / condition, b) secondary prevention activity targets the early stages of condition / disorder / symptom treatment, thereby increasing the chances of intervention to prevent the progression of the condition / disorder / symptom and the appearance of symptoms, and c) tertiary prevention reduces the adverse effects of an already established condition / disorder / symptom, for example, by restoring function and / or reducing any condition / disorder / symptom or associated complications. The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it indicates that the likelihood of the event occurring in the presence of the compound or method is reduced.

[0033] As used herein, the term "anti-tumor effect" shall be given its ordinary meaning and refers to the inhibition of tumor and / or tumor cell (in vivo and / or in vitro) formation, growth, and / or viability, including an alteration of at least about 1.1 fold (e.g., 1.1 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, or any number or range between these values) in one or more of tumor cell death, tumor volume, tumor number, tumor viability, metastasis, and viability.

[0034] As used herein, the term "oxidative stress" shall be given its ordinary meaning and shall refer to an imbalance between the production of reactive oxygen species, reactive nitrogen species, and / or free radicals and the antioxidant capacity of a biological system.

[0035] As used herein, the term "effective amount" refers to an amount sufficient to effect beneficial or desired biological and / or clinical results.

[0036] As used herein, the term "contacting" shall be given its ordinary meaning and refers to placing two or more entities (e.g., an isolated organelle complex and T cells) in such proximity that they actually come into physical contact with one another, for example, by combining the two or more entities. Contacting can include co-incubation. Contacting can occur in vitro, in situ, or in vivo. In some embodiments, contacting two entities includes incorporating (e.g., transplanting) one entity into another entity that has been physically contacted. Contacting an isolated organelle complex with a population of T cells can include contacting the organelle complex population with a population of T cells. Contacting an isolated organelle complex with a population of T cells can generate a population of T cells containing the exogenous organelle complex. Upon contacting the organelle complex with a T cell population, the organelle complex provided herein can be taken up by the T cells. In some embodiments, incorporation (e.g., transplantation) of the isolated organelle complex into T cells involves colocalization and / or fusion with endogenous mitochondria within the T cells. The T cells can be in vivo, in vitro, or ex vivo. In some embodiments, the organelle complex incorporated (e.g., transplanted) into the T cells can be detected (e.g., distinguished from endogenous organelles of the T cells) for at least a period of time (e.g., 6 hours, 12 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or a number or range between any two of these values). The beneficial effects of transplantation of the organelle complexes provided herein can persist beyond the time the organelle complex is detectable in the population of T cells. Populations of T cells containing the organelle complexes provided herein include populations of T cells that have undergone one or more cell divisions and / or continuous culture after initial contact with the isolated organelle complex. In some embodiments, the progeny of a T cell that comprises an organelle complex also comprise said organelle complex (eg, through random distribution of the organelle complex between divisions).As used herein, the term "exogenous" shall be given its ordinary meaning and shall refer to cellular material (e.g., an organelle complex) that has been removed from one cell and incorporated into another cell.

[0037] The term "autologous" shall be given its ordinary meaning and shall refer to any material derived from the same individual that is later reintroduced into the same individual.

[0038] The term "allogeneic" shall be given its ordinary meaning and refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another if their genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently different genetically and antigenically to interact.

[0039] The methods, compositions, systems, and kits provided herein can, in some embodiments, be used in combination with the methods, compositions, systems, and kits described in PCT Patent Application Publication Nos. WO2018 / 092839, WO2017 / 090763, WO2020 / 230601, WO2019 / 164003, WO2020 / 054824, WO2020 / 203961, WO2020 / 054829, WO2021 / 015298, and WO2021 / 132735, the contents of which are incorporated herein by reference in their entireties.

[0040] In some embodiments, a method for generating a population of T cells for adoptive T cell therapy is provided. In some embodiments, the method includes contacting an isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, the organelle complex comprising mitochondria and one or more of an endoplasmic reticulum, peroxisomes, lysosomes, and a Golgi apparatus. The method can include stimulating the population of T cells. The stimulating step can expand the population of T cells.

[0041] In some embodiments, methods are provided for enhancing the proliferation, migration, persistence, and / or activity of a population of T cells for adoptive T cell therapy. In some embodiments, the methods include contacting an isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, the organelle complex comprising mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The methods can include stimulating the population of T cells to expand the population of T cells.

[0042] In some embodiments, a method for generating a population of T cells resistant to exhaustion is provided. In some embodiments, the method includes contacting an isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, the organelle complex comprising mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus. The method can include stimulating the population of T cells to expand the population of T cells.

[0043] In certain embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from cancer or a tumor. In other embodiments, the donor subject is a human patient not suffering from cancer or a tumor.

[0044] The T cells provided herein may be obtained through any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. In certain embodiments, cells collected by apheresis are washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing. In some embodiments, the cells are washed with PBS. As will be appreciated, a washing step can be used, such as by using a semi-automated flow-through centrifuge, such as a Cobe™ 2991 cell processor or a Baxter CytoMate™. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other saline solutions, with or without buffer. In certain embodiments, undesirable components of the apheresis sample are removed. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, the entire contents of which are incorporated herein by reference.

[0045] In certain embodiments, T cells are isolated from PBMCs by lysing red blood cells and depleting monocytes, for example, by using centrifugation through a PERCOLL™ gradient. + , CD8 + , CD28 + , CD45RA + , and CD45RO +Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a population of T cells by negative selection can be achieved with a combination of antibodies against surface markers unique to the negatively selected cells. In some embodiments, cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry can be used, using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can be used to enrich a population of T cells by negative selection, such as CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD8, CD11b, CD14, CD16, CD20, and HLA-DR. In certain embodiments, flow cytometry and cell sorting are used to isolate cell populations of interest for use in the methods provided herein.

[0046] In some embodiments, T cells are genetically modified following isolation using known methods, or T cells are activated and expanded (or differentiated, in the case of precursor cells) in vitro before being genetically modified. In another embodiment, T cells are genetically modified with a chimeric antigen receptor described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then activated and / or expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Pat. Nos. 6,905,874, 6,867,041, and 6,797,514, and PCT Publication No. WO 2012 / 079000, the entire contents of which are incorporated herein by reference. Generally, such methods involve contacting PBMCs or isolated T cells with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as "surrogate" antigen-presenting cells (APCs). One example is the Dynaheads® system, a CD3 / CD2S activation / stimulation system for physiologically activating human "I" cells. In other embodiments, T cells are activated and stimulated to proliferate using feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent Nos. 6,040,177 and 5,827,642, and PCT Publication No. WO 2012 / 129514, the contents of which are incorporated herein by reference in their entirety.

[0047] Stimulation can occur before, after, and / or during contact. The term "stimulation" shall be given its ordinary meaning and refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, including, but not limited to, signaling through the TCR / CD3 complex or signaling through the signaling domain of the CAR. Stimulation can mediate altered expression of specific molecules. Stimulation can include culturing a population of T cells in the presence of one or more stimulatory agents. The one or more stimulatory agents can include an agent that stimulates a CD3 / TCR complex-associated signal and an agent that stimulates a costimulatory molecule on the surface of the T cells. The one or more stimulatory agents can include a molecule that binds to CD28 (e.g., one or more of an anti-CD28 antibody, CD80, and CD86) and / or a molecule that binds to CD3 (e.g., an anti-CD3 antibody). The stimulation and / or contacting can be for a period of at least about 6 hours, 12 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or a number or range between any two of these values.

[0048] In some embodiments, T cell recovery can be at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) greater than a method that does not include contacting the population of T cells with an isolated organelle complex. T cell recovery can be the ratio of T cells recovered after stimulation (e.g., about 48 hours after stimulation) to the number of T cells immediately before the start of stimulation. In some embodiments, the population of T cells exhibit one or more of an enhanced expansion capacity (in vivo and / or in vitro), enhanced cytotoxicity against target cells (in vivo and / or in vitro), enhanced resistance to exhaustion (in vivo and / or in vitro), and enhanced persistence (in vivo and / or in vitro) compared to a population of T cells that does not comprise the exogenous organelle complex. In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) increase in basal and / or maximal oxygen consumption rate (in vivo and / or in vitro) compared to a population of T cells that does not comprise the exogenous organelle complex. In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) increase in basal and / or maximal glycolytic rate (in vivo and / or in vitro) compared to a population of T cells that does not comprise the exogenous organelle complex.In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) increase in glycolytic and / or respiratory capacity (in vivo and / or in vitro) compared to a population of T cells that does not comprise the exogenous organelle complex. In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) increase in mitochondrial mass (in vivo and / or in vitro) compared to a population of T cells that does not contain the exogenous organelle complex. In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range therebetween) increase in cytotoxic activity (in vivo and / or in vitro) against target cells compared to a population of T cells that does not contain the exogenous organelle complex. The exhaustion marker can be selected from the group including PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, TIGIT, or any combination thereof. In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) decrease in levels of one or more of cellular ROS (in vivo and / or in vitro), mitochondrial ROS (in vivo and / or in vitro), cellular oxidative stress (in vivo and / or in vitro), and mitochondrial oxidative stress (in vivo and / or in vitro) compared to a population of T cells that does not contain the exogenous organelle complex.Reactive oxygen species include superoxide (O2). - ), hydroperoxy (HO.2), hydrogen peroxide (H2O2), peroxynitrite (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO), peroxy radical (ROO), alkoxy radical (RO), singlet oxygen ( 1In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) increase in production (in vivo and / or in vitro) of one or more cytokines compared to a population of T cells that does not include the exogenous organelle complex. Cytokines can include interleukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), TNF-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and / or TGF-beta (TGFβ). In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) increase in cell proliferation (in vivo and / or in vitro) compared to a population of T cells that does not comprise the exogenous organelle complex. In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) increase in cell viability (in vivo and / or in vitro) following chronic TCR stimulation compared to a population of T cells that does not include the exogenous organelle complex. In some embodiments, the introduction of the organelle complex metabolically reprograms the CAR-T cells via redox modulation. In some embodiments, the population of T cells exhibits an increase in NAD+ / -β-glucan (NAD) levels compared to a population of T cells that does not include the exogenous organelle complex. + / NADH, ATP levels, sirtuin (Sirt1,3) levels, TP-53 levels, NF-κB levels, Bax levels, percentage of memory T cells (Tcm, Tscm), telomere activity, and SA-β Gal activity (in vivo and / or in vitro) are decreased and / or increased by at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values).

[0049] "Exhausted" or "unresponsive" refers to a cellular state in which a cell does not perform its useful function or activity in response to normal input signals, including the refractoriness of immune cells to stimuli, such as stimulation via activating receptors or cytokines. Such functions or activities include, but are not limited to, proliferation or cell division, participation in the cell cycle, cytokine production, cytotoxicity, transport, phagocytic activity, or any combination thereof. Normal input signals can include, but are not limited to, stimulation via receptors (e.g., T cell receptors, B cell receptors, costimulatory receptors, etc.).

[0050] The exhausted immune cells can have at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduced cytotoxic activity, cytokine production, proliferation, trafficking, phagocytic activity, or any combination thereof, relative to corresponding control immune cells of the same type. In one embodiment, the exhausted cells are CD8 + T cells (e.g., antigen-specific effector CD8 +CD8 T cells are typically CD8 T cells. CD8 T cells typically proliferate (e.g., clonally expand) in response to T cell receptor and / or costimulatory receptor stimulation and cytokines such as IL-2. Thus, exhausted CD8 T cells do not proliferate and / or produce cytokines in response to normal input signals. The exhaustion of effector function can be divided into several stages that ultimately lead to terminal or complete exhaustion and ultimately to loss (Yi et al. (2010) Immunol. 129:474-481; Wherry and Ahmed (2004) J. Virol. 78:5535-5545). In the first stage, functional T cells enter a "partial exhaustion I" stage characterized by the loss of a subset of effector functions, including loss of IL-2 production, reduced TNFα production, and reduced capacity for proliferation and / or ex vivo lysis. In the second stage, partially exhausted T cells enter a "partial exhaustion II" stage when both IL-2 and TNFα production cease following antigen stimulation and IFNγ production is reduced. "Complete exhaustion" or "terminal exhaustion" is characterized by the CD8 + This occurs when T cells lose all effector functions following antigen stimulation, including loss of IL-2, TNFα, and IFNγ production, and loss of ex vivo lytic and proliferative capabilities. +In response to normal input signals, T cells fail to proliferate, lyse target cells (cytotoxicity), and / or produce appropriate cytokines such as IL-2, TNFα, or IFNγ. This loss of effector function can occur when antigen load is high and / or CD4 help is low. This hierarchical loss of function is also associated with the expression of co-inhibitory immune receptors such as PD-1, ΜΜ-3, and LAG-3 (Day et al. (2006) Nature 443:350-4; Trauttnann et al. (2006) Nat. Med 12:1198-202; and Urbani et al. (2006) J. Virol. 80:1398-1403). Other molecular markers distinguish hierarchical stages of immune cell exhaustion, such as high eomesodermin (EOMES) expression and low TBET expression as markers of terminally exhausted T cells (Paley et al. (2012) Science 338:1220-1225). Reduced Bcl-b and increased BLIMP-1 (PdrmL) production are further markers of exhausted T cells. T cell exhaustion can include expression of one or more T cell exhaustion biomarkers selected from the group including checkpoint inhibitors, PD-1 (Pdcdl), ΠΜ-3 (Havcr2), LAG-3 (Lag3), CTLA-4 (Ctla4), 2B4 (CD244), CD39 (Entpdl), CD160, eomesodermin (Eomes), T-BET (Tbx21), BATF, BLIMP-1 (Prdml), NFATC1, NR4A2, MAFB, OCT-2 (Pou2f2), Foxpl, retinoic acid receptor alpha (Rara), or any combination thereof. In some embodiments, the population of T cells exhibits at least about a 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) decrease in levels (in vivo and / or in vitro) of one or more exhaustion markers compared to a population of T cells that does not contain the exogenous organelle complex.The in vivo persistence of T cell populations was approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days ...1 hours, 22 hours, 23 hours, 24 hours, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 The period may include 0 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, or any number or range between these values.

[0051] In some embodiments, a population of T cells generated by the methods disclosed herein is provided. In some embodiments, a population of T cells for adoptive T cell therapy is provided, wherein the T cells comprise an exogenous organelle complex. The organelle complex can include mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. In some embodiments, the population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence compared to a population of T cells that does not comprise the exogenous organelle complex.

[0052] In some embodiments, methods of treating or preventing a disease or disorder in a subject are provided. In some embodiments, the methods include administering to the subject an effective amount of a population of T cells generated according to the methods disclosed herein, thereby treating or preventing the disease or disorder in the subject. In some embodiments, the population of T cells exhibit one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not contain an exogenous organelle complex.

[0053] In some embodiments, methods are provided for enhancing adoptive T cell therapy in a subject. In some embodiments, the methods include generating an effective amount of T cells comprising an exogenous organelle complex according to the methods disclosed herein and adoptively transferring the T cells into a subject. In some embodiments, the population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence compared to a population of T cells that does not comprise the exogenous organelle complex.

[0054] In some embodiments, methods for treating or preventing a disease or disorder in a subject are provided. In some embodiments, the methods include administering to the subject an effective amount of a population of T cells (e.g., the T cells comprise a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR)) and administering to the subject an effective amount of an isolated organelle complex, thereby treating or preventing the disease or disorder in the subject. The isolated organelle complex can be contacted in vivo with a population of T cells to generate a population of T cells comprising the organelle complex. In some embodiments, the population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not comprise the exogenous organelle complex. An effective amount of the population of T cells can be administered to the subject before administering the effective amount of the isolated organelle complex to the subject. An effective amount of the population of T cells can be administered to the subject before administering the effective amount of the isolated organelle complex to the subject. An effective amount of the population of T cells and an effective amount of the isolated organelle complex can be administered to the subject simultaneously.

[0055] Disclosed herein are methods for treating or preventing a disease or disorder in a subject. In some embodiments, the methods include administering to the subject an effective amount of a heterologous nucleic acid (e.g., a vector, a viral vector) encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), and administering to the subject an effective amount of an isolated organelle complex, thereby treating or preventing the disease or disorder in the subject. The heterologous nucleic acid and the isolated organelle complex can be contacted with the subject's T cells in vivo to generate T cells comprising the organelle complex and the CAR and / or engineered TCR. In some embodiments, the T cells exhibit one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to T cells that do not contain the exogenous organelle complex.

[0056] In some embodiments, the population of T cells reduces tumor volume, tumor growth, and / or tumor burden in a subject. In some embodiments, the population of T cells reduces tumor volume in a subject by at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) compared to the tumor volume in an untreated subject or a subject administered a population of T cells that does not include the exogenous organelle complex. In some embodiments, the population of T cells increases overall survival or progression-free survival. In some embodiments, the population of T cells increases overall survival or progression-free survival by at least about 1.1-fold (e.g., 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or any number or range between these values) compared to an untreated subject or a subject administered a population of T cells that does not include the exogenous organelle complex.

[0057] The T cells can be autologous to the subject. The T cells can be allogeneic to the subject. The adoptive T cell therapy can be CAR-T cell therapy. The adoptive T cell therapy can be engineered TCR T cell therapy. The adoptive T cell therapy can be tumor infiltrating lymphocyte (TIL) therapy. Administration is at least about 1 x 10 6 The method can include administering (e.g., intravenously) the T cells. The method can include repeated administration of the population of T cells. The subject can be a mammal.

[0058] The disease or disorder can be associated with expression of a tumor antigen. The disease associated with expression of a tumor antigen can be selected from the group consisting of a proliferative disease, a precancerous condition, a cancer, and a non-cancer-related indication associated with expression of a tumor antigen.

[0059] Cancers include colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell carcinoma of the lung, mesothelioma, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, parathyroid cancer, The cancer may be selected from the group consisting of renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of the above cancers, and metastatic lesions of the above cancers.

[0060] The cancer can be a hematological cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or preleukemia. organelle complex

[0061] In some embodiments, contacting a population of T cells with the isolated organelle complex allows the organelle complex to be taken up by the T cells. The contacting step can be repeated at least two, three, or four times. Contacting can occur during one or both of the stimulation and induction steps. The population of T cells can be contacted with an effective amount of the isolated organelle complex sufficient to enhance T cell proliferation, migration, persistence, and / or activity. An effective amount can include about 20 μg of the isolated organelle complex. An effective amount of an isolated organelle complex is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 10, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780 , 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, It can be, about, or up to 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000 ug, mg, ug / mL, and / or mg / mL, or a number or range between any two of these values.A population of T cells can be contacted with an effective amount of the isolated organelle complex sufficient to render the T cells resistant to exhaustion. The population of T cells can be derived from lymph node cells (e.g., purified by a magnetic particle-based enrichment method selected from the group consisting of manual MACS®, AutoMACS®, CliniMACS®, EasySep®, and RoboSep®). The population of T cells can be CD4+. + cells, CD8 + The cells may include one or more of T cells, cytotoxic T cells, peripheral effector T cells, effector T cells, memory or central memory T cells, naive T cells, regulatory T cells, natural killer T cells, gamma delta T cells, cytokine-induced killer (CIK) T cells, and tumor infiltrating lymphocytes (TILs).

[0062] The organelle complexes provided herein can include a first organelle complex, a second organelle complex, or a combination of the first and second organelle complexes. The isolated organelle complex can be an organelle complex population. The organelle complex population can include the first organelle complex, or a combination of the first and second organelle complexes. The first organelle complex and / or the second organelle complex can be depleted of cytoplasmic macromolecules. The cytoplasmic macromolecules can include cytoplasmic proteins (e.g., the cytoplasmic proteins are p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). The abundance of one or more cytoplasmic proteins can be depleted by at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values) compared to the cells from which the organelle complex population was derived. The organelle complex can include one or more mitochondrial matrix proteins (e.g., mitochondrial transcription factor A (TFAM) and / or citrate synthase (CS)), one or more mitochondrial outer membrane proteins (e.g., mitochondrial outer membrane complex subunit 20 (TOMM20)), one or more lysosomal proteins (e.g., lysosome-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosome-associated membrane protein 1 (LAMP1)), one or more peroxisomal proteins (e.g., , catalase and / or ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)), one or more Golgi apparatus proteins (e.g., Golgin-97, Sintaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or mannosidase alpha class 2A member 1 (MAN2A1)), and / or one or more endoplasmic reticulum proteins (e.g., calticarin and / or caldisin).The organelle complex can be derived from cells of a subject different from the subject from which the T cells are derived. The organelle complex is derived from cells of the same subject from which the T cells are derived. The organelle complexes provided herein (e.g., a first organelle complex, a second organelle complex) can include mitochondria and one, two, three, or four of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus. The organelle complexes (e.g., a first organelle complex, a second organelle complex) can include (i) mitochondria and an endoplasmic reticulum, (ii) mitochondria and a peroxisome, (iii) mitochondria and a lysosome, (iv) mitochondria and a Golgi apparatus, (v) mitochondria, an endoplasmic reticulum, and a peroxisome, (vi) mitochondria, an endoplasmic reticulum, and a lysosome, (vii) mitochondria, an endoplasmic reticulum, and a Golgi apparatus, (viii) mitochondria, an endoplasmic reticulum, a peroxisome, and a lysosome, (ix) mitochondria. The organelle complexes can include (x) mitochondria, endoplasmic reticulum, peroxisomes, and Golgi apparatus, (x) mitochondria, endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus, (xi) mitochondria, endoplasmic reticulum, lysosomes, and Golgi apparatus, (xii) mitochondria, peroxisomes, and lysosomes, (xiii) mitochondria, peroxisomes, and Golgi apparatus, (xiv) mitochondria, peroxisomes, lysosomes, and Golgi apparatus, and / or (xv) mitochondria, lysosomes, and Golgi apparatus. The ratio of mitochondria to additional organelles (e.g., endoplasmic reticulum, peroxisomes, lysosomes, and / or Golgi apparatus) within the organelle complex population can vary.

[0063] Disclosed herein are methods for generating a first organelle complex population. In some embodiments, the method includes incubating cells in a first solution containing a detergent at a first temperature, removing the detergent to form a second solution, and recovering the first organelle complex from the second solution. The first organelle complex can be derived from (i) frozen cells, (ii) suspension cells, and / or (iii) cells contacted with a detergent at a concentration equal to or greater than the critical micelle concentration (CMC) of the detergent. The generation of the first organelle complex can include (Step A) providing adherent cells, suspension cells, and / or frozen cells, thawing them, and placing them in a tube. The generation of the first organelle complex can include (Step A) providing adherent cells, suspension cells, and / or frozen cells, centrifuging, and collecting the precipitate. The production of the first organelle complex can include (Step A) providing adherent cells, aspirating, adding a solution (e.g., PBS(-)), aspirating, adding TrypLE, incubating, adding a solution (e.g., PBS(-)), placing the cell suspension in a tube, centrifuging, and collecting the precipitate. The production of the first organelle complex can include one or more of the following steps: (Step B) adding Tris buffer, centrifuging, and collecting the precipitate; (Step C) adding Tris buffer and vortexing; (Step D) adding a surfactant-containing solution and incubating; (Step E) centrifuging and collecting the precipitate; (Step F) adding Tris buffer and centrifuging and collecting the precipitate; (Step G) adding Tris buffer and pipetting; (Step H) transferring to another tube and collecting and washing out the buffer solution in the original tube; (Step I) centrifuging and collecting the supernatant; (Step J) centrifuging and collecting the precipitate; and (Step K) pipetting. One or more of the above steps can include an incubation period. One or more of the above steps can include a centrifugation step followed by collection of the supernatant and / or precipitate. One or more of the above steps can be omitted, and one or more additional steps can be included.Depending on the embodiment, the time, volume, concentration, and centrifugal force can be varied. Incubating the cells in the first solution and / or incubating the second solution can include applying a physical stimulus, such as pipetting, shaking, and / or stirring, to the first solution and / or the second solution, respectively. Applying a physical stimulus to the first solution and / or the second solution can include flowing the first solution and / or the second solution through a flow device (e.g., a reducing agent flow device). The flow device can include a channel including two or more portions with different cross-sectional diameters. The cross-sectional diameter can be about, at least, or at most about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, or 25 mm, or any number or range thereof. The reducing agent flow devices provided herein can have a variety of configurations, including, for example, angular reducers, tapered reducers, concentric reducers, and / or eccentric reducers. The flow device can include various types and sizes of tubes to generate additional flow and shear for extraction of the first organelle complex. In some embodiments, flow through the flow device generates additional rouleaux and / or shear. Recovering the first organelle complex from the second solution can include tangential flow filtration (TFF). In some embodiments, TFF can be used for purification and / or buffer exchange. Recovering the first organelle complex from the second solution can include tangential flow filtration (TFF). TFF can be performed using a low-viscosity buffer, which in some embodiments reduces the shear rate.The viscosity of TFF buffers is 1 centipoise (cP), 2 cP, 3 cP, 4 cP, 5 cP, 6 cP, 7 cP, 8 cP, 9 cP, 10 cP, 11 cP, 12 cP, 13 cP, 14 cP, 15 cP, 16 cP, 17 cP, 18 cP, 19 cP, 20 cP, 21 cP, 22 cP, 23 cP, 24 cP, 25 cP, 26 cP, 27 cP, 28 cP, 29 cP, 30 cP, 31 cP, 32 cP, 33 cP, 34cP, 35cP, 36cP, 37cP, 38cP, 39cP, 40cP, 41cP, 42cP, 43cP, 44cP, 45cP, 46cP, 47cP, 48cP, 49cP, 50c P, 51cP, 52cP, 53cP, 54cP, 55cP, 56cP, 57cP, 58cP, 59cP, 60cP, 61cP, 62cP, 63cP, 64cP, 65cP, 66cP, 67cP , 68cP, 69cP, 70cP, 71cP, 72cP, 73cP, 74cP, 75cP, 76cP, 77cP, 78cP, 79cP, 80cP, 81cP, 82cP, 83cP, 84cP, 85cP, 86cP, 87cP, 88cP, 89cP, 90cP, 91cP, 92cP, 93cP, 94cP, 95cP, 96cP, 97cP, 98cP, 99cP, 100cP, 200cP , 300 cP, 400 cP, 500 cP, 600 cP, 700 cP, 800 cP, 900 cP, 1000 cP, 2500 cP, 5000 cP, 7500 cP, 10000 cP, or a number or range between any two of these values, can be about these numbers, can be at least these numbers, or can be up to these numbers. The temperature at which TFF is performed can be, about, at least, or at most 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or a number or range between any two of these values.The shear rate for the TFF procedure and / or flow device (e.g., reducing agent flow device) is 1 sec. -1 , 2sec -1 , 3sec -1 , 4sec -1 , 5sec -1 , 6sec -1 , 7sec -1 , 8sec -1 , 9sec -1 , 10sec -1 , 11sec -1 , 12sec -1 , 13sec -1 , 14sec -1 , 15sec -1 , 16sec -1 , 17sec -1 , 18sec -1 , 19sec -1 , 20sec -1 , 25sec -1 , 30 seconds -1 , 35sec -1 , 40sec -1 , 45sec -1 , 50sec -1 , 60sec -1 , 70sec -1 , 80sec -1 , 90sec -1 , 100sec -1 , 110sec -1 , 120sec -1 , 128sec -1 , 130sec -1 , 140sec -1 , 150sec -1 , 160sec -1 , 170sec -1 , 180sec -1 , 190sec -1 , 200sec -1 , 210sec -1 , 220sec -1 , 230sec -1 , 240sec -1 , 250sec -1 , 260sec -1 , 270sec -1 , 280sec -1、290sec -1 、300sec -1 、310sec -1 、320sec -1 、330sec -1 、340sec -1 、350sec -1 、360sec -1 、370sec -1 、380sec -1 、390sec -1 、400sec -1 、410sec -1 、420sec -1 、430sec -1 、440sec -1 、450sec -1 、460sec -1 、470sec -1 、480sec -1 、490sec -1 、500sec -1 、510sec -1 、520sec -1 、530sec -1 、540sec -1 、550sec -1 、560sec -1 、570sec -1 、580sec -1 、590sec -1 、600sec -1 、610sec -1 、620sec -1 、630sec -1 、640sec -1 、650sec -1 、660sec -1 、670sec -1 、680sec -1 、690sec -1 、700sec -1 、710sec -1 、720sec -1 、730sec -1 、740sec -1 、750sec -1 、760sec -1 、770sec -1 、780sec -1、790sec -1 、800sec -1 、810sec -1 、820sec -1 、830sec -1 、840sec -1 、850sec -1 、860sec -1 、870sec -1 、880sec -1 、890sec -1 、900sec -1 、910sec -1 、 920sec -1 、930sec -1 、940sec -1 、950sec -1 、960sec -1 、970sec -1 、980sec -1 、990sec -1 、1000sec -1 、1100sec -1 、1200sec -1 、1300sec -1 、1400sec -1 、1500sec -1 、1600sec -1 、1700sec -1 、1800sec -1 、1900sec -1 、2000sec -1 、2100sec -1 、2200sec -1 、2300sec -1 、2400sec -1 、2500sec -1 、2600sec -1 、2700sec -1 、2800sec -1 、2900sec -1 、3000sec -1 、3250sec -1 、3500sec -1 、3750sec -1 、4000sec -1 、4250sec -1 、4500sec -1、4750sec -1 、5000sec -1 、5500sec -1 、6000sec -1 、6500sec -1 、7000sec -1 、7500sec -1 、8000sec -1 、8500sec -1 、9000sec -1 、9500sec -1 、10000sec -1The first organelle complex can be a number or range between any two of these values, can be about, at least, or at most these numbers. TFF can be performed in a buffer containing human albumin (HA). Recovering the first organelle complex from the second solution can include TFF performed using a TFF membrane. The molecular weight cutoffs of the TFF membranes are 10kDa, 11kDa, 12kDa, 13kDa, 14kDa, 15kDa, 16kDa, 17kDa, 18kDa, 19kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 128kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320k Da, 330kDa, 340kDa, 350kDa, 360kDa, 370kDa, 380kDa, 390kDa, 400kDa, 410kDa, 420kDa, 430kDa, 440kDa, 450kDa, 460kDa, 47 0kDa, 480kDa, 490kDa, 500kDa, 510kDa, 520kDa, 530kDa, 540kDa, 550kDa, 560kDa, 570kDa, 580kDa, 590kDa, 600kDa, 610kDa , 620kDa, 630kDa, 640kDa, 650kDa, 660kDa, 670kDa, 680kDa, 690kDa, 700kDa, 710kDa, 720kDa, 730kDa, 740kDa, 750kDa, 760 kDa, 770kDa, 780kDa, 790kDa, 800kDa, 810kDa, 820kDa, 830kDa, 840kDa, 850kDa, 860kDa, 870kDa, 880kDa, 890kDa, 900kDa, 910kDa, 920kDa, 930kDa, 940kDa, 950kDa, 960kDa, 970kDa, 980kDa, 990kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa,1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa, 260 0kDa, 2700kDa, 2800kDa, 2900kDa, 3000kDa, 3250kDa, 3500kDa, 3750kDa, 4000kDa, 4250kDa, 4500kDa, 4750kDa , 5000 kDa, 5500 kDa, 6000 kDa, 6500 kDa, 7000 kDa, 7500 kDa, 8000 kDa, 8500 kDa, 9000 kDa, 9500 kDa, 10000 kDa, or a number or range between any two of these values, can be about those numbers, can be at least those numbers, or can be up to those numbers. Methods for obtaining organelle complexes from cells and organelle complexes obtained by such methods are disclosed in PCT Patent Application No. PCT / US23 / 27014, filed July 6, 2023, and entitled "ORGANELLE COMPLEXES," the entire contents of which are incorporated herein by reference. In some embodiments, a second organelle complex is provided. In some embodiments, a method for isolating a second organelle complex from cells includes treating cells in a first solution with a detergent at a concentration below the critical micelle concentration (CMC) of the detergent, removing the detergent to form a second solution, incubating the cells in the second solution, and recovering the second organelle complex from the second solution. The second organelle complex can be derived from (i) adherent cells and / or (ii) cells contacted with the detergent at a concentration below the critical micelle concentration (CMC) of the detergent. In some embodiments, Q mitochondria are provided. The second organelle complex can comprise or be Q mitochondria. Methods for obtaining Q mitochondria from cells and Q mitochondria obtained by such methods are disclosed in PCT Patent Application Publication No. WO / 2021 / 015298, the entire contents of which are incorporated herein by reference. The organelle complex population can be isolated by treatment with a mitochondrial activator (e.g.,The organelle complexes can be derived from cells treated with a mitochondrial activator (e.g., resveratrol). The organelle complexes can be depleted of cytoplasmic macromolecules. Cytoplasmic macromolecules can be absent from the organelle complex populations provided herein. The organelle complex (e.g., first organelle complex, second organelle complex) populations provided herein can contain negligible and / or undetectable amounts of cytoplasmic macromolecules. The cytoplasmic macromolecules can include cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). The first organelle complex and the second organelle complex can be derived from cells treated with a mitochondrial activator. The homogenized mitochondria, the first organelle complex, and / or the second organelle complex can be encapsulated in lipid membrane-based vesicles. Methods for encapsulation into lipid membrane-based vesicles are disclosed in PCT Patent Application Publication No. WO2021 / 132735, the entire contents of which are incorporated herein by reference.

[0064] Chimeric antigen receptors and engineered T cell receptors The T cells provided herein can comprise a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the CAR comprises a T cell receptor (TCR) antigen-binding domain. The method can include introducing a heterologous nucleic acid (e.g., a vector) encoding the chimeric antigen receptor (CAR) and / or engineered T cell receptor (TCR) into the T cell. The introducing step can occur before, after, and / or during contact. The term "chimeric antigen receptor" or alternatively "CAR," in its simplest embodiment, refers to a set of polypeptides, typically two polypeptides, which, when present in an immune effector cell, provide the cell with specificity for a target cell, typically a cancer cell, and intracellular signaling. The terms "CAR" and "CAR molecule" are used interchangeably. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), with functional signaling domains derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. In some embodiments, the set of polypeptides is in the same polypeptide chain (e.g., comprising a chimeric fusion protein). In some embodiments, the set of polypeptides are contiguous with one another. In some embodiments, the set of polypeptides are not contiguous with one another, e.g., on different polypeptide chains. In some embodiments, the set of polypeptides comprises a dimerization switch that, in the presence of a dimerization molecule, can link the polypeptides to one another, e.g., link an antigen binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28.In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, with a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, with a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, with two or more functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, with at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0065] A CAR and / or TCR can comprise one or more of an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. A CAR or TCR can further comprise a leader peptide. A TCR can further comprise a constant region and / or CDR4. The term "signaling domain" refers to a functional portion of a protein that acts by transmitting intracellular information to regulate cellular activity through a defined signaling pathway by responding to such messengers, generating second messengers, or functioning as an effector. As used herein, an "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates signals that promote immune effector functions of CAR-containing cells, such as CAR-T cells. For example, immune effector functions in CAR-T cells include cytolytic activity and helper activity, including cytokine secretion. In one embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Primary intracellular signaling domains include domains derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Costimulatory intracellular signaling domains include domains derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain can include the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can include the cytoplasmic sequence from a coreceptor or costimulatory molecule. The primary intracellular signaling domain can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0066] The intracellular signaling domain can include a primary signaling domain, a costimulatory domain, or both a primary signaling domain and a costimulatory domain. The cytoplasmic domain or region of the CAR includes the intracellular signaling domain. The intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire chain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used instead of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0067] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell that is not limited to proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11 a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83. The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors.The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived or a functional fragment or derivative thereof, or the entire naturally occurring intracellular signaling domain.

[0068] Intracellular signaling domains for use in the CARs provided herein include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities. It is known that signals generated via the TCR alone are insufficient for full activation of T cells; secondary and / or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, e.g., costimulatory domains). Primary signaling domains regulate primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. The primary signaling domain can comprise a functional signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12, or functional variants thereof.

[0069] In some embodiments, the intracellular signaling domain is designed to include two or more, e.g., two, three, four, five, or more, costimulatory signaling domains. In one embodiment, the two or more, e.g., two, three, four, five, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain includes two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue. Costimulatory domains include CD27, CD28, 4-1BB (CD137), OX40, CD28-OX40, CD28-4-1BB, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11 b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D, or functional variants thereof.

[0070] Portions of CARs comprising antibodies or antibody fragments thereof may exist in various forms in which the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), humanized antibodies, or bispecific antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CARs provided herein comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment comprising an scFv.

[0071] In some embodiments, the CARs provided herein comprise a target-specific binding element, also referred to as an antigen-binding domain. The choice of moiety depends on the type and number of ligands that define the surface of the target cell.

[0072] In some embodiments, CAR-mediated T cell responses can be directed to an antigen of interest by engineering an antigen-binding domain that specifically binds the desired antigen into the CAR. In some embodiments, the portion of the CAR that includes the antigen-binding domain includes an antigen-binding domain that targets a tumor antigen, such as a tumor antigen described herein. The antigen-binding domain can be any domain that binds to an antigen, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including, but not limited to, single-domain antibodies such as the heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH) of camelid-derived nanobodies, and alternative scaffolds known in the art to function as antigen-binding domains, such as recombinant fibronectin domains, T cell receptors (TCRs), or fragments thereof, e.g., single-chain TCRs. In some instances, it is beneficial to have an antigen-binding domain derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to include human or humanized residues for the antigen-binding domain of an antibody or antibody fragment. In some embodiments, the antigen-binding domain comprises a humanized antibody or antibody fragment. In some aspects, a non-human antibody is humanized, where certain sequences or regions of the antibody are modified to increase similarity to antibodies or fragments thereof that are naturally produced in humans. In some embodiments, the antigen-binding domain is humanized.

[0073] The antigen binding domain can comprise an antibody, antibody fragment, scFv, Fv, Fab, a(Fab')2, single domain antibody (SDAB), VH or VL domain, camelid VHH domain, Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single domain antibody (sdAb), single chain including anti-complementary scFv (tandem scFv) or bispecific tandem scFv, Fv construct, disulfide-linked Fv, dual variable domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, affibody, affilin, affitin, affimer, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, or any combination thereof.

[0074] In some embodiments, the antigen-binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single-chain TCR (scTCR). Methods for generating such TCRs are known in the art. See, for example, Willemsen RA et al., Gene Therapy 7:1369-1377 (2000); Zhang T et al., Cancer Gene Ther 11:487-496 (2004); Aggen et al., Gene Ther. 19(4):365-74 (2012), the contents of which are incorporated herein by reference in their entirety. For example, a scTCR can be engineered that includes Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is highly useful for cancer-associated targets that are themselves intracellular, while fragments of such antigens (peptides) are presented on the surface of cancer cells by MHC.

[0075] In some embodiments, the antigen-binding domain is a multispecific antibody molecule. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable region sequence that has binding specificity for a first epitope and a second immunoglobulin variable region sequence that has binding specificity for a second epitope. In embodiments, the first epitope and the second epitope are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In embodiments, the first epitope and the second epitope overlap. In embodiments, the first epitope and the second epitope do not overlap. In embodiments, the first epitope and the second epitope are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, a bispecific antibody molecule comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope, and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In embodiments, a bispecific antibody molecule comprises a half antibody that has binding specificity for a first epitope and a half antibody that has binding specificity for a second epitope. In embodiments, a bispecific antibody molecule comprises a half antibody, or fragment thereof, that has binding specificity for a first epitope and a half antibody, or fragment thereof, that has binding specificity for a second epitope. In embodiments, a bispecific antibody molecule comprises an scFv, or fragment thereof, that has binding specificity for a first epitope and an scFv, or fragment thereof, that has binding specificity for a second epitope.

[0076] The antigen-binding domain can be configured to bind to a tumor antigen. The terms "cancer-associated antigen" or "tumor antigen" refer interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either entirely or as fragments (e.g., MHC / peptides), and that is useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or more overexpression compared to normal cells. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, a tumor antigen is overexpressed on the cell surface of cancer cells, either entirely or as fragments (e.g., MHC / peptides), and is not synthesized or expressed on the surface of normal cells. In some embodiments, the CARs provided herein include CARs with an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the pocket of a major histocompatibility complex (MHC) class I molecule and are expressed by CD8 +They are recognized by T cell receptors (TCRs) on T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified from the screening of a library such as a human scFv phage display library.

[0077] The tumor antigen can be a solid tumor antigen, such as CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family members, B cell maturation (B CMA), Tn antigen ((TnAg) or (GalNAcα-Ser / Thr)), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), mesothelin, interleukin IL-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21 (testosin or PRSS21), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis (Y) antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-β), stage-specific embryonic antigen 4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine protein kinase ERBB2 (Her2 / neu), mucin 1, cell surface associated (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid Protein phosphatase (PAP), mutant elongation factor 2 (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunit, beta type 9 (LMP2), glycoprotein 100 (gp100), oncogene fusion protein (bcr-abl) consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), tyrosinase,Ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR) ), G protein-coupled receptor class C group 5, member D (GPRC5D), chromosome X open reading frame 61 (CXORF61), CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), hexasaccharide moiety of globoH glycoceramide (GloboH), mammary differentiation antigen (NY-BR-1), uroplakin 2 (UPK2), hepatitis A virus cellular receptor 1 (HAVCR1), adrenergic receptor beta 3 (ADRB3), pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K9 (LY6K), olfactory receptor 51E2 (OR51E2), TCR gamma alternative reading frame protein (TARP), Wilms tumor protein (WT1), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), melanoma-associated antigen 1 (MAGE-A1), ETS translocation mutated gene 6, located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie2), melanoma cancer testis antigen 1 (MAD-CT-1), melanoma cancer testis antigen 2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutants, prostein, survivin, telomerase, prostate cancer tumor antigen 1 (PCTA-1 or galectin 8), melanoma antigen 1 recognized by T cells (MelanA or MART1), rat sarcoma (Ras) mutants, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease,Serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), paired-box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (sibling of BORIS or regulator of imprinted sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), paired box protein Pax-5 (PAX5), proacrosin-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), kinase anchor protein 4 (AKAP-4), synovial sarcoma, X-breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), legumain, human papillomavirus E6 (HPVE6), human papillomavirus E7 (HPVE7), intestinal carboxylesterase, heat shock protein Protein 70-2 variant (muthsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican 3 (GPC3), Fc receptor-like 5 (FCRL5), and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0078] The tumor antigens are CD150, 5T4, ActRIIA, B7, BMCA, CA-125, CCNA1, CD123, CD126, CD138, CD14, CD148, CD15, CD19, CD20, CD200, CD21, CD22, CD23, CD24, CD25, CD26, CD261, CD262, CD30, CD33, CD362, CD37, CD38, CD4, CD40, CD40L, CD44, CD46, CD5, CD52, CD53, CD54, CD5 6, CD66a-d, CD74, CD8, CD80, CD92, CE7, CS-1, CSPG4, ED-B fibronectin, EGFR, EGFRvIII, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, GD2, GD3, HER1-HER2 combination, HER2-HER3 combination, HERV-K, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, HLA-DR, HM1.24, HMW-MAA, Her2, Her2 / neu, IGF-1R, IL-11Ralpha, IL-13R-alpha2, IL-2, IL-22R-alpha, IL-6, IL-6R, Ia, Ii, L1-CAM, L1-cell adhesion molecule, Lewis Y, L1-CAM, MAGE A3, MAGE-A1, MART-1, MUC1, NKG2C ligand, NKG2D ligand, NY-ESO-1, OEPHa2, PIGF, PSCA, PSMA, ROR1, T101, TAC, TAG72, TIM-3, TRAIL-R1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), VEGF, VEGFR2, WT-1, G protein-coupled receptor, alpha-fetoprotein (AFP), angiogenic factor, exogenous cognate binding molecule (ExoCBM), oncogene product, anti-folate receptor, c-Met, carcinoembryonic antigen (CEA), cyclin (D1), ephrin B2, epithelial tumor anti The antigen can be selected from the group including: ribonuclease, estrogen receptor, fetal acetate choline receptor, folate binding protein, gp100, hepatitis B surface antigen, kappa chain, kappa light chain, kdr, lambda chain, livin, melanoma-associated antigen, mesothelin, mouse double minute 2 homolog (MDM2), mucin 16 (MUC16), mutated p53, mutated ras, necrosis antigen, oncofetal antigen, ROR2, progesterone receptor, prostate-specific antigen, tEGFR, tenascin, β2-microglobulin, Fc receptor-like 5 (FcRL5), or a molecule expressed by HIV, HCV, HBV, or other pathogens.

[0079] The antigen-binding domain can be connected to the transmembrane domain by a hinge region. In some examples, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge, or a CD8a hinge.

[0080] With respect to the transmembrane domain, in various embodiments, a CAR can be designed to include a transmembrane domain attached to the extracellular domain of the CAR. The transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the intracellular region). In some embodiments, the transmembrane domain is a domain associated with one of the other domains of the CAR; for example, in one embodiment, the transmembrane domain may be derived from the same protein from which the signaling domain, costimulatory domain, or hinge domain is derived. In some embodiments, the transmembrane domain is not derived from the same protein from which any other domain of the CAR is derived. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to prevent binding of such domain to the transmembrane domain of the same or a different surface membrane protein, for example, to minimize interaction with other members of the receptor complex. In some embodiments, the transmembrane domain allows homodimerization with another CAR on the cell surface of the CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of a natural binding partner present in the same CAR-expressing cell.

[0081] Transmembrane domains include the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4- 1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLEF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL , CD11a, LFA-1, ITGAM, CD11d, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or a functional variant thereof. The transmembrane domain can be derived from either natural or recombinant sources.When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.In some embodiments, the transmembrane domain can transmit signals to the intracellular domain(s) whenever CAR binds to the target. Pharmaceutically acceptable compositions and methods of administration

[0082] In some embodiments, a pharmaceutical composition is provided, which comprises a population of T cells generated by the methods disclosed herein and one or more pharmaceutically acceptable carriers.

[0083] The present disclosure also provides for the use of a population of T cells comprising an organelle complex in the manufacture of a medicament for treating the diseases and disorders provided herein. In some embodiments, the population of T cells comprising an organelle complex is administered to a subject in combination with one or more additional agents and / or therapies designed to treat the disease or disorder.

[0084] Contacting the subject's cells can include a route of administration selected from the group consisting of intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation administration, intrapulmonary administration, and intraocular administration. The population of T cells containing the organelle complex can be administered locally or systemically.

[0085] As used herein, the phrases "local administration" or "topic administration" refer to any route of administration in which a population of T cells containing organelle complexes contacts an individual's body such that the location of the resulting T cells within the body is topical (limited to a particular tissue, organ, or other body part for which imaging is desired). Exemplary local administration routes include injection into a specific tissue using a needle, gavage into the gastrointestinal tract, and spreading a solution containing a population of T cells containing organelle complexes onto the surface of the skin.

[0086] As used herein, the phrase "systemic administration" refers to any route of administration in which a population of T cells, including organelle complexes, contacts an individual's body such that the location of the resulting T cells in the body is systemic (i.e., not limited to a particular tissue, organ, or other body part for which imaging is desired). Systemic administration includes enteral and parenteral administration. Enteral administration is a systemic administration route in which a substance is given via the digestive tract, including, but not limited to, oral administration, administration via a gastric feeding tube, administration via a duodenal feeding tube, gastrostomy, enteral feeding, and rectal administration. Parenteral administration is a systemic administration route in which a substance is given via a route other than the digestive tract, including, but not limited to, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical instillation.

[0087] In another aspect, this disclosure provides a pharmaceutically acceptable composition comprising a population of T cells comprising a therapeutically effective amount of the organelle complexes disclosed herein. As described in detail below, the pharmaceutical compositions of this disclosure may be specially formulated for administration in solid or liquid form, including dosage forms suitable for: (1) oral administration, e.g., drops (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, or pastes; (2) parenteral administration, e.g., by subcutaneous, intramuscular, or intravenous injection, e.g., as a sterile solution or suspension; (3) topical application, e.g., as a cream, ointment, or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; or (5) aerosol, e.g., as an aqueous aerosol, liposomal preparation, or solid particles containing a population of T cells comprising the organelle complexes. The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers. As used herein, the phrase "therapeutically effective amount" can refer to an amount of a population of T cells containing an organelle complex disclosed herein that is effective in producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit / risk ratio.

[0088] The phrase "pharmaceutically acceptable" is used herein to refer to those agents, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0089] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, vehicle, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a target chemical substance from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can act as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, and corn oil. and soybean oil, (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer solutions, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0090] Formulations useful in the methods of this disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient (e.g., a population of T cells containing an organelle complex) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of a population of T cells containing an organelle complex that achieves a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0091] Suspensions may contain, in addition to the active agent, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0092] Dosage forms for topical or transdermal administration of T cells containing organelle complexes include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that may be required.

[0093] The ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0094] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this disclosure.

[0095] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0096] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0097] The actual dosage level of the active ingredient in the pharmaceutical compositions of this disclosure can be determined by the methods of the present disclosure to obtain an amount of the active ingredient that is non-toxic to the subject and effective to achieve the desired therapeutic response for the particular subject, composition, and mode of administration.

[0098] The T cells containing the organelle complex can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells containing the organelle complex can be administered in a dose of at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, or at least about 10 9 cells, at least about 10 10 In another embodiment, the T cells containing the therapeutically effective amount of organelle complexes can be about 10 4 cells, approximately 10 5cells, approximately 10 6 cells, approximately 10 7 cells, or approximately 10 8 In one particular embodiment, the therapeutically effective amount of T cells containing the organelle complex is about 2 x 10 6 cells / kg, approximately 3 x 10 6 cells / kg, approximately 4 x 10 6 cells / kg, approximately 5 x 10 6 cells / kg, approximately 6 x 10 6 cells / kg, approximately 7 x 10 6 cells / kg, approximately 8 x 10 6 cells / kg, approximately 9 x 10 6 cells / kg, approximately 1 x 10 7 cells / kg, approximately 2 x 10 7 cells / kg, approximately 3 x 10 7 cells / kg, approximately 4 x 10 7 cells / kg, approximately 5 x 10 7 cells / kg, approximately 6 x 10 7 cells / kg, approximately 7 x 10 7 cells / kg, approximately 8 x 10 7 cells / kg, or approximately 9 x 10 7 cells / kg.

[0099] The present specification also provides kits containing one or more compositions described herein (e.g., formulations comprising a population of T cells containing organelle complexes) in suitable packaging and may further include written materials that may include instructions for use, conclusions of clinical studies, lists of side effects, etc. Such kits may also include information such as scientific literature references, package inserts, clinical trial results, and / or summaries thereof, demonstrating or establishing the activity and / or benefits of the compositions and / or describing dosage, administration, side effects, drug interactions, or other information useful to healthcare providers. Such information may be based on the results of various studies, for example, studies using experimental animals including in vivo models and studies based on human clinical trials. The kits may also include one or more unit doses described herein. Additional medications

[0100] In some embodiments, the method includes administering one or more additional agents to the subject. In some embodiments, the one or more additional agents increase the efficacy of T cells comprising the exogenous organelle complex. The one or more additional agents include a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor of an immunosuppressive molecule, and / or a T REG The one or more additional agents can include an agent that reduces the level or activity of a cell. The one or more additional agents can include an immunomodulator, an anti-metastatic agent, a chemotherapeutic agent, a hormone or growth factor antagonist, an alkylating agent, a TLR agonist, a cytokine antagonist, a cytokine antagonist, or any combination thereof. The one or more additional agents can include an agonist or antagonist antibody specific for a checkpoint inhibitor or checkpoint stimulatory molecule, such as PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, or TIM-3.

[0101] The one or more additional agents may be alkylating agents (nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes); uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard); Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®; bendamustine (Treakisym®, Ribomustin®, Treanda®); chlormethine (Mustartgen®); cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™); ifosfamide (Mitoxana®); melphalan (Alkeran®); chlorambucil (Leukeran®); pipebroman (Amedel®, Vercyte®) (registered trademark); triethylenemelamine (Hemel®, Hexylen®, Hexastat®); triethylenethiophosphoramine; temozolomide (Temodar®); thiotepa (Thioplex®); busulfan (Busilvex®, Myleran®); carmustine (BiCNU®); lomustine (CeeNU®); streptozocin (Zanosar®); estramustine (Emcyt®, Estracit®); fotemustine; irofulven; mannosulfan; mitobronitol; nimustine; procarbazine; ranimustine; semustine; triaziconazole; treosulfan; and dacarbazine (DTIC-Dome®);anti-EGFR antibodies (e.g., cetuximab (Erbitux®), panitumumab (Vectibix®), and gefitinib (Iressa®)); anti-Her-2 antibodies (e.g., trastuzumab (Herceptin®) and other antibodies from Genentech); antimetabolites (including, but not limited to, antifolates (also referred to herein as antifolates), pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate (Rheumatrex®, Trexall®), 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), floxuridine (FUDF®), carmofur, cytarabine (Cytosar-U®, Tarabine®), PFS), 6-mercaptopurine (Puri-Nethol®), 6-thioguanine (Thioguanine Tabloid®), fludarabine phosphate (Fludara®), pentostatin (Nipent®), pemetrexed (Alimta®), raltitrexed (Tomudex®), cladribine (Leustatin®), clofarabine (Clofarex®, Clolar®), mercaptopurine (Puri-Nethol®), capecitabine (Xeloda®), nelarabine (Arranon®), azacitidine (Vidaza®) ), decitabine (Dacogen®), enocitabine (Sunrabin®), sapacitabine, tegafur-uracil, tiazofurin, thioguanine, trofosfamide, and gemcitabine (Gemzar®); Vinca alkaloids: vinblastine (Velban®, Velsar®), vincristine (Vincasar®, Oncovin®), vindesine (Eldisine®), vinorelbine (Navelbine®), vinflunine (Javlor®);Platinum-based agents: carboplatin (Paraplat®, Paraplatin®), cisplatin (Platinol®), oxaliplatin (Eloxatin®), nedaplatin, satraplatin, and triplatin; anthracyclines: daunorubicin (Cerubidine®, Rubidomycin®), doxorubicin (Adriamycin®), epirubicin (Ellence®), idarubicin (Idamycin®) ), mitoxantrone (Novantrone®), valrubicin (Valstar®), aclarubicin, amrubicin, liposomal doxorubicin, liposomal daunorubicin, pirarubicin, pixantrone, and zorubicin; topoisomerase inhibitors: topotecan (Hycamtin®), irinotecan (Camptosar®), etoposide (Toposar®, VePesid®), teniposide (Vumon®), lamellarin D, SN-38, Campto Tecins (e.g., IT-101), belotecan, and rubitecan; taxanes: paclitaxel (Taxol®), docetaxel (Taxotere®), larotaxel, cabazitaxel, ortataxel, and tesetaxel; antibiotics: actinomycin (Cosmegen®), bleomycin (Blenoxane®), hydroxyurea (Droxia®, Hydrea®), mitomycin (Mitozytrex®, Mutamycin®); Immunomodulators: lenalidomide (Revlimid®), thalidomide (Thalomid®); immune cell antibodies: alemtuzamab (Campath®), gemtuzumab (Myelotarg®), rituximab (Rituxan®), tositumomab (Bexxar®); interferons (e.g., IFN-alpha (Alferon®, Roferon-A®, Intron®-A) or IFN-gamma (Actimmune®));Interleukins: may be selected from the group consisting of IL-1, IL-2 (Proleukin®), IL-24, IL-6 (Sigosix®), IL-12; HSP90 inhibitors (e.g., geldanamycin or any of its derivatives). In certain embodiments, the HSP90 inhibitor is geldanamycin, 17-alkylamino-17-desmethoxygeldanamycin ("17-AAG") or 17-(2-dimethylaminoethyl)amino-17-desmethoxygeldanamycin ("17-DMAG"), antiandrogens (nilutamide (Nilandron®) and bicalutamide (Caxodex®); tamoxifen (Nolvadex®), toremifene (Fareston®), letrozole (Femara®), testolactone (Teslac®), anastrozole (Arimidex®), bicalutamide (Casodex®), exemestane (Aromasin®), flutamide (Eulexin®), fulvestrant (Faslodex®), raloxifene anti-estrogen drugs (including but not limited to Evista®, Keoxifene®, and raloxifene hydrochloride); anti-hypercalcemic drugs, including but not limited to gallium(III) nitrate hydrate (Ganite®) and pamidronate disodium (Aredia®); apoptosis inducers, including but not limited to ethanol, 2-[[3-(2,3-dichlorophenoxy)propyl]amino]-(9Cl), gambogic acid, elesclomol, embelin, and arsenic trioxide (Trisenox®); Aurora kinase inhibitors, including but not limited to binuclein 2; Bruton's tyrosine kinase inhibitors, including but not limited to tereic acid; calcineurin inhibitors, including but not limited to cypermethrin, deltamethrin, fenvalerate, and tyrphostin 8;CaM kinase II inhibitors, including but not limited to, 5-isoquinolinesulfonic acid, 4-[{2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-{4-phenyl-1-piperazinyl)propyl]phenyl ester and benzenesulfonamide; CD45 tyrosine phosphatase inhibitors, including but not limited to, phosphonic acids; CDC25 phosphatase inhibitors, including but not limited to, 1,4-naphthalenedione, 2,3-bis[(2-hydroxyethyl)thio]-(9Cl); Debromohymenia CHK kinase inhibitors, including but not limited to rudisin; cyclooxygenase inhibitors, including but not limited to 1H-indole-3-acetamide, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-N-(2-phenylethyl)-(9Cl), 5-alkyl-substituted 2-arylaminophenylacetic acid and its derivatives (e.g., celecoxib (Celebrex®), rofecoxib (Vioxx®), etoricoxib (Arcoxia®), lumiracoxib (Prexige®)). , valdecoxib (Bextra®) or 5-alkyl-2-arylaminophenylacetic acid; cRAF kinase inhibitors including, but not limited to, 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindol-2-one and benzamide, 3-(dimethylamino)-N-[3-[(4-hydroxybenzoyl)amino]-4-methylphenyl]-(9Cl); olomoucine and its derivatives, purvalanol B, roascovitine (Seliciclib®), indirubin, kempa Cyclin-dependent kinase inhibitors, including but not limited to, uron, purvalanol A, and indirubin-3'-monoxime; cysteine protease inhibitors, including but not limited to, 4-morpholinecarboxamide, N-[(1S)-3-fluoro-2-oxo-1-(2-phenylethyl)propyl]amino]-2-oxo-1-(phenylmethyl)ethyl]-(9Cl); DNA inhibitors, including but not limited to, plicamycin (Mithracin®) and daptomycin (Cubicin®);DNA strand breakers, including but not limited to bleomycin (Blenoxane®); E3 ligase inhibitors, including but not limited to N-((3,3,3-trifluoro-2-trifluoromethyl)propionyl)sulfanilamide; tyrphostin 46, EKB-569, erlotinib (Tarceva®), gefitinib (Iressa®), lapatinib (Tyke rb®), as well as compounds disclosed generically and specifically in WO 97 / 02266, EP 0564409, WO 99 / 03854, EP 0520722, EP 0566226, EP 0787722, EP 0837063, U.S. Pat. No. 5,747,498, WO 98 / 10767, WO 97 / 30034, WO 97 / 49688, WO 97 / 38983, and WO 96 / 33980; EGF pathway inhibitors; farnesyltransferase inhibitors, including but not limited to, ahydroxyfarnesylphosphonic acid, butanoic acid, 2-[(2S)-2-[[(2S,3S)-2-[[(2R)-2-amino-3-mercaptopropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]amino]-4-(methylsulfonyl)-1-methylethyl ester (2S)-(9Cl), tipifarnib (Zarnestra®), and manumycin A; Flk-1 kinase inhibitors, including but not limited to, 2-propenamide, 2-cyano-3-[4-hydroxy-3,5-bis(1-methylethyl)phenyl]-N-(3-phenylpropyl)-(2E-)-(9Cl); glycogen synthase kinase-3 (GSK3) inhibitors, including but not limited to, indirubin-3'-monoxime; suberoylanilide hydroxamic acid (SAHA), [4-(2-aminophenylcarbamoyl)benzyl]carbamic acid pyridin-3-ylmethyl ester and its derivatives. histone deacetylase (HDAC) inhibitors, including but not limited to, tetracycline, butyric acid, pyroxamide, trichostatin A, oxamflatin, apicidin, depsipeptide, depudecin, trapoxin, vorinostat (Zolinza®), and compounds disclosed in WO 02 / 22577; I-kappa B-alpha kinase inhibitors (IKK), including but not limited to, 2-propenenitrile, 3-[(4-methylphenyl)sulfonyl]-(2E)-(9Cl); temozolomide (M imidazotetrazinones, including but not limited to ethazolastone®, Temodar®) and their derivatives (e.g., as generally or specifically disclosed in U.S. Pat. No. 5,260,291), and mitozolomide; insulin tyrosine kinase inhibitors, including but not limited to hydroxyl-2-naphthalenylmethylphosphonic acid; c-Jun-N-terminal kinase (JNK) inhibitors, including but not limited to pyrazolianthrone and epigallocatechin gallate;Mitogen-activated protein kinase (MAP) inhibitors, including but not limited to, benzenesulfonamide, N-[2-[[[3-(4-chlorophenyl)-2-propenyl]methyl]amino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxy-(9Cl); MDM2 inhibitors, including but not limited to, trans-4-iodo, 4'-boranyl-chalcone; MEK inhibitors, including but not limited to, butanedinitrile, bis[amino[2-aminophenyl)thio]methylene]-(9Cl); actinonin, epigallocatechin gallate, collagen peptidomimetic and non-peptidomimetic inhibitors, tetracycline derivatives marimastat (Marimastat®), prinomastat, incyclinide (Metastat®), shark cartilage extract MMP inhibitors, including but not limited to AE-941 (Neovastat®), tanomastat, TAA211, MMI270B, or AAJ996; mTor inhibitors, including but not limited to rapamycin (Rapamune®) and its analogs and derivatives, AP23573 (also known as ridaforolimus, deforolimus, or MK-8669), CCI-779 (also known as temsirolimus) (Torisel®), and SDZ-RAD; Tyrphostin AG NGFR tyrosine kinase inhibitors, including but not limited to 879; p38 MAP kinase inhibitors, including but not limited to phenol, 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-(9Cl), and benzamide, 3-(dimethylamino)-N-[3-[(4-hydroxybenzoyl)amino]-4-methylphenyl]-(9Cl); p56 tyrosine kinase inhibitors, including but not limited to damnacanthal and tyrphostin 46;PDGF pathway inhibitors, including but not limited to tyrphostin AG 1296, tyrphostin 9, 1,3-butadiene-1,1,3-tricarbonitrile, 2-amino-4-(1H-indol-5-yl)-(9Cl), imatinib (Gleevec®) and gefitinib (Iressa®), and compounds disclosed generically and specifically in European Patent No. 0564409 and PCT Publication No. WO 99 / 03854; phosphatidylinositol 3-kinase inhibitors, including but not limited to wortmannin and quercetin dihydrate; cantharidic acid, cantharidin and L-leucinamide; protein phosphatase inhibitors including, but not limited to, cantharidic acid, cantharidin, LP-bromotetramisole oxalate, 2(5H)-furanone, 4-hydroxy-5-(hydroxymethyl)-3-(1-oxohexadecyl)-(5R)-(9Cl) and benzylphosphonic acid; 1-H-pyrrolo-2,5-dione, 3-[1-3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-(9Cl), bisindolylmaleimide PKC inhibitors, including but not limited to IX, sfinogosine, staurosporine, and hypericin; PKC delta kinase inhibitors, including but not limited to rottlerin; polyamine synthesis inhibitors, including but not limited to DMFO; PTP1B inhibitors, including but not limited to L-leucinamide; protein tyrosine kinase inhibitors, such as those generally and specifically described in PCT Publication No. WO 03 / 013541 and US Publication No. 2008 / 0139587, including but not limited to tyrphostin Ag 216, tyrphostin Ag 1288, tyrphostin Ag 1295, geldanamycin, genistein, and 7H-pyrrolo[2,3-d]pyrimidine derivatives; SRC family tyrosine kinase inhibitors, including but not limited to PP1 and PP2; Syk tyrosine kinase inhibitors, including but not limited to piceatannol; tyrphostin Ag Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors, including but not limited to 490 and 2-naphthyl vinyl ketone;Isotretinoin (Accutane®, Amnesteem®, Cistane®, Claravis®, Sotret®) and tretinoin (Aberel®, Aknoten®, Avita®, Renova®, Retin-A®, Retin-A retinoids, including but not limited to MICRO®, Vesanoid®; RNA polymerase H elongation inhibitors, including but not limited to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole; serine / threonine kinase inhibitors, including but not limited to 2-aminopurine; sterol biosynthesis inhibitors, including but not limited to squalene epoxidase and CYP2D6; VEGF pathway inhibitors, including but not limited to anti-VEGF antibodies, such as bevacizumab, and small molecules, such as sunitinib (Sutent®), sorafinib (Nexavar®), ZD6474 (also known as vandetanib) (Zactima™), SU6668, CP-547632, and AZD2171 (also known as cediranib) (Recentin™);

[0102] Example Certain aspects of the above-described embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0103] Example 1 Mitochondrial transfer enhances the metabolic fitness and antitumor efficacy of CAR-T cells.

[0104] Introduction Chimeric antigen receptor (CAR) T-cell therapy is a potentially curative treatment for patients with relapsed or refractory (r / r) hematopoietic malignancies. However, approximately half of patients with r / r B-cell lymphoma experience treatment failure after CAR-T-cell therapy, likely due to poor proliferation, limited persistence, and exhaustion of CAR-T cells. Given that metabolic fitness plays a central role in regulating T-cell function, manipulating mitochondrial function is a promising approach to enhance the antitumor efficacy of CAR-T cells. Recently, it has been shown that the transfer of viable mitochondria prepared by conventional homogenization (Mito-conv) into T cells (mitochondrial transplantation) enhanced the expression of inhibitory receptors and reduced T-cell proliferation (Court AC:EMBO Rep 2020). In this study, we tested whether the transfer of "Q" mitochondria isolated from HeLa cells using the novel iMIT isolation technology (WO / 2021 / 015298 A1) into T cells could enhance the metabolic fitness and antitumor efficacy of CAR-T cells.

[0105] method Purified T cells from naive mice or healthy volunteers were stimulated with plate-bound anti-CD3 / CD28 antibodies in the presence or absence of 50–100 μg / ml Q-mitochondrial. Culture medium and Q-mitochondrial were refreshed every 24 or 48 hours. Mitochondrial respiratory function was assessed using an XFp Flux Analyzer. For CAR-T cell generation, purified T cells were stimulated with anti-CD3 / CD28 antibodies for 48 hours and then incubated with a retroviral vector encoding an anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hours.

[0106] result First, we confirmed that mitochondrial structure and ATP synthesis in Q mitochondria were much better maintained than in mito-converted T cells. When purified murine T cells were incubated with fluorescently labeled Q mitochondria for 24 hours during TCR stimulation, approximately 70% of T cells contained Q mitochondria, and the mitochondrial mass per cell significantly increased. As expected, oxidative phosphorylation (OXPHOS) was significantly enhanced in Q-treated T cells compared with vehicle-treated controls after TCR stimulation. Although reactive oxygen species (ROS) levels in vehicle-treated T cells increased after 72 hours of TCR stimulation, Q mitochondria significantly reduced ROS accumulation in activated T cells. Q mitochondria suppressed differentiation of terminally discharged T cells and enhanced TCF-1 production in memory and discharged T cells, associated with improved survival and enhanced production of IFN-g and TNF-α after chronic TCR stimulation (196 hours). + Progenitor populations (e.g., TCF-1 + PD-1 + The number of Tim3-excreting T cells (pTex) was increased. Based on these findings, we next investigated whether Q mitochondria could affect CAR-T cell function. CAR-T cells were generated using purified T cells from naive BALB / c mice, and Q mitochondria were added from the start of TCR stimulation during CAR-T cell generation. Q-treated CAR-T cells exhibited enhanced OXPHOS and cytokine production compared with vehicle-treated CAR-T cells (Figure 1). When CAR-T cells were incubated with syngeneic B-cell lymphoma cells (A20) for 12 hours, Q-treated CAR-T cells demonstrated significantly enhanced in vitro cytotoxicity against A20 lymphoma cells. Next, we investigated the in vivo antitumor effects of CAR-T cells. Naive BALB / c mice were inoculated with 15 × 10 mitochondria into the right flank. 6 A20 cells were injected subcutaneously, and 1 x 10 cells were injected 14 days after tumor inoculation. 6Q-treated or control CAR-T cells or naive BALB / c T cells were intravenously injected into mice. Although control CAR-T cells significantly suppressed tumor growth compared to naive T cells, all mice died of tumor growth 31 days after tumor inoculation. Importantly, tumor growth was further suppressed in mice injected with Q-treated CAR-T cells, resulting in significantly prolonged survival (Figure 2).

[0107] conclusion In the first study, we found that CAR-T cells transplanted with Q mitochondria prepared by the iMIT method had improved metabolic fitness, resulting in enhanced proliferation capacity, cytokine production, and antitumor effects in vitro and in vivo. Mitochondrial transplantation can also be useful as an adjunctive method in combination with various other genetic and pharmacological approaches to enhance the efficacy of CAR-T cell therapy.

[0108] Example 2 Regulation of T cell function by transplantation of a second organelle complex

[0109] Naive T cells rely on oxidative phosphorylation (OXPHOS) for energy generation, whereas activated T cells exhibit increased overall metabolic activity, including glycolysis and glutamate degradation. In situations where T cells are chronically stimulated by antigens, such as in the tumor microenvironment, reactive oxygen species (ROS) accumulate and mitochondrial function is impaired. Mitochondrial dysfunction and reduced mitochondrial mass can lead to decreased production of effector cytotoxic cytokines in T cells. Furthermore, mitochondrial ROS accumulation inhibits OXPHOS and induces T cell exhaustion. CAR-T cells from patients who have failed CAR-T therapy can exhibit reduced mitochondrial biogenesis and reduced mitochondrial mass, which are associated with reduced proliferation and persistence. In some embodiments, transplantation of a second organelle complex at the time of CAR-T generation improves the efficacy of CAR-T therapy. This example demonstrates that transfer of a second organelle complex improves T cell function by restoring mitochondrial energy generation and reducing mitochondrial ROS.

[0110] Secondary organelle complexes were isolated from HeLa cells. After permeabilization of the cell membrane with digitonin and a washout step, the cells were incubated on ice. Subsequently, the isolated secondary organelle complexes were collected by pipetting and centrifugation.

[0111] Administration of the second organelle complex was found to improve the efficiency of T cell recovery after stimulation. Figure 3 shows data on the efficiency of T cell recovery after stimulation. The second organelle complex (or vehicle) was transplanted on day 1, followed by stimulation with anti-CD3 / CD28 antibodies. T cell recovery was measured 48 hours later. The T cell recovery rate (%) is defined as {(number of recovered T cells after 48 hours of stimulation) / (number of T cells immediately before the start of stimulation)} × 100.

[0112] The effect of transplantation of a second organelle complex on chronically stimulated T cells was examined. Chronic T cell stimulation was performed to mimic the tumor microenvironment. T cell purification (negative selection by MACS) from C57BL / 6 mice was followed by T cell stimulation (via plate-bound anti-CD3 / CD28 Ab) on day 1. Administration of the second organelle complex was performed on days 1, 3, 5, and 7 (or vehicle control), followed by FACS analysis on days 4 or 9.

[0113] Transplantation of the second organelle complex was found to increase T cell mitochondrial mass (Figures 4A-4D). The effect of transplantation of the second organelle complex on T cell proliferation capacity was examined. Figures 5A-5D show data on T cell proliferation capacity assessed by a dye dilution assay using Cell Trace Violet dye. CD4 T cells (Figures 5A-5B) and CD8 T cells (Figures 5C-5D) were transplanted with the second organelle complex or vehicle and assessed on day 4. Transplantation of the second organelle complex was found to improve T cell proliferation capacity. Next, the effect of transplantation of the second organelle complex on mitochondrial ROS levels was assayed, and transplantation of the second organelle complex was found to reduce ROS levels accumulated in mitochondria (Figures 6A-6D). Transplantation of the second organelle complex was also found to reduce ROS accumulation throughout the T cell (Figures 7A-7D). Next, we assayed the effect of transplantation of a second organelle complex on T cell mitochondrial respiratory capacity. Figure 8 shows data on T cell oxygen consumption rate measured with an XFp Flux Analyzer. T cells were transplanted with the second organelle complex or vehicle and evaluated on day 4. Transplantation of the second organelle complex was found to improve T cell mitochondrial respiratory capacity. Transplantation of the second organelle complex was found to enhance the cytokine production capacity of CD8 T cells (Figures 9A-9B). Figures 10A-10B show data on the number of viable cells after chronic in vitro TCR stimulation. T cells were transplanted with the second organelle complex or vehicle, and viable cell counts were assessed using a dead cell staining dye on day 9. Transplantation of the second organelle complex was found to increase the number of viable T cells after chronic stimulation. Flow cytometry analysis of TCF-1 progenitor cells was performed after 72 hours of TCR stimulation to determine whether the second organelle complex (2 nd T cells transferred with OC or vehicle were compared (Figures 16A-16B).

[0114] Murine CD19 CAR-T generation was performed as follows: T cell purification from BALB / c mice (negative selection by MACS) was followed by 48 hours of T cell stimulation (plate-bound anti-CD3 / CD28 antibody) on day 1. Retroviral particles (via transfection) were generated using a vector plasmid (CD19 CAR) containing 1D3scFv, CD28, and the first and third inactive ITAMs of CD3ζ. CAR gene transduction (using a retroviral vector) was performed on days 3 and 4. The second organelle complex was administered three times (on days 1, 3, and 4). Figure 11 shows data on CAR-T cell glycolysis rate measured with a Seahorse Extracellular Flux Analyzer. Figure 1 shows CAR-T cell oxygen consumption rate measured with a Seahorse Extracellular Flux Analyzer following T cell transplantation with the second organelle complex or vehicle. Transplantation of the second organelle complex was found to improve T cell mitochondrial respiratory and glycolytic capacities. Transplantation of the second organelle complex was found to improve CAR-T cell proliferation capacity (Figures 12A-12D). Next, cytokine production capacity was evaluated. Transplantation of the second organelle complex was found to enhance the cytokine production capacity of CD8 CAR-T cells (Figures 13A-13B). Transplantation of the second organelle complex was also found to enhance the cytotoxic activity of CAR-T cells in vitro (Figure 14). Flow cytometry analysis of TCF-1 precursor CAR-T cells was performed 96 hours after the start of TCR stimulation to determine the effect of the second organelle complex (2 nd CAR-T cells implanted with OC or vehicle were compared (Figures 17A-17B).

[0115] Next, the antitumor effect of CAR-T was evaluated in vivo using a B-cell lymphoma model (A20, 15 × 10 6 On day 14, 1 × 10 cells were injected into A20 tumor-bearing BALB / c mice. 6 cells were injected intravenously, and 2 nd OC(+) CAR-T cells (n=7), 2 ndOC(-) CAR-T cells (n=7) and naive T cells (n=4) were compared. Figure 2 shows data regarding the suppression of tumor growth in mice injected with CAR-T cells implanted with a second organelle complex, resulting in significantly extended survival. Figure 15 shows data regarding the overall survival of mice injected with CAR-T cells implanted with a second organelle complex compared to vehicle-implanted CAR-T cells or naive T cells. Implantation of a second organelle complex was found to enhance the antitumor effect of CAR-T and improve survival.

[0116] This example demonstrates that transplantation of a second organelle complex during chronic in vitro TCR stimulation improved T cell proliferation and increased the number of viable cells. Transplantation of the second organelle complex reduced cellular and mitochondrial ROS levels while improving mitochondrial respiratory capacity. Thus, transplantation of the second organelle complex during CD19 CAR-T cell generation from naive T cells improved T cell recovery after anti-CD3 / CD28 antibody stimulation. Importantly, transplantation of the second organelle complex improved the mitochondrial respiratory capacity of murine CD19 CAR-T cells and improved their antitumor efficacy.

[0117] Example 3 Investigating the effects of organelle complex transplantation on the metabolic fitness and antitumor efficacy of CAR-T cells

[0118] Introduction In this study, we examine whether the transfer of isolated organelle complexes (e.g., first organelle complex, second organelle complex) from HeLa cells to T cells enhances the metabolic fitness and antitumor efficacy of CAR-T cells. method

[0119] Purified T cells from naive mice or healthy volunteers were stimulated with plate-bound anti-CD3 / CD28 antibodies in the presence or absence of 50–100 μg / ml of organelle complexes (e.g., first organelle complex, second organelle complex). The culture medium and organelle complexes (e.g., first organelle complex, second organelle complex) were renewed every 24 or 48 hours. Mitochondrial respiratory function was assessed using an XFp Flux Analyzer. For CAR-T cell generation, purified T cells were stimulated with anti-CD3 / CD28 antibodies for 48 hours and then incubated with a retroviral vector encoding an anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hours.

[0120] Predicted results First, we confirmed that the mitochondrial structure and ATP synthesis of T cells transplanted with organelle complexes (e.g., organelle complex 1, organelle complex 2) were significantly better maintained than those of mito-conjugated T cells. Purified murine T cells were incubated with fluorescently labeled organelle complexes (e.g., organelle complex 1, organelle complex 2) for 24 hours during TCR stimulation. Approximately 70% of T cells were expected to have endocytosed organelle complexes, resulting in a significant increase in mitochondrial mass per cell. Oxidative phosphorylation (OXPHOS) was significantly enhanced in organelle complex (OC)-treated T cells compared with vehicle-treated controls after TCR stimulation. Although reactive oxygen species (ROS) levels in vehicle-treated T cells increased after 72 hours of TCR stimulation, organelle complexes (e.g., organelle complex 1, organelle complex 2) significantly reduced ROS accumulation in activated T cells. Organelle complexes (e.g., first organelle complex, second organelle complex) suppress differentiation of terminally expelled T cells and increase TCF-1 expression in memory and expelled T cells, associated with improved survival and enhanced production of IFN-g and TNF-a after chronic TCR stimulation (96 hours). + Progenitor populations (e.g., TCF-1 + PD-1 +Tim3-excreting T cells (pTex) are increased. Next, we examine whether organelle complexes (e.g., first organelle complex, second organelle complex) affect CAR-T cell function. CAR-T cells are generated from purified T cells from naive BALB / c mice, and organelle complexes (e.g., first organelle complex, second organelle complex) are added from the start of TCR stimulation during CAR-T cell generation. OC-treated CAR-T cells exhibit enhanced OXPHOS and cytokine production compared with vehicle-treated CAR-T cells. When CAR-T cells were incubated with syngeneic B-cell lymphoma cells (A20) for 12 hours, OC-treated CAR-T cells demonstrated significantly enhanced in vitro cytotoxicity against A20 lymphoma cells. Next, we examine the in vivo antitumor effect of CAR-T cells. Naive BALB / c mice were inoculated with 1.5 × 10 cells into the right flank. 7 A20 cells were injected subcutaneously, and 1 x 10 cells were injected 14 days after tumor inoculation. 6 OC-treated or control CAR-T cells or naive BALB / c T cells are intravenously injected. Although control CAR-T cells significantly suppress tumor growth compared to naive T cells, all mice are expected to die from tumor growth 31 days after tumor inoculation. Importantly, tumor growth is further suppressed in mice injected with OC-treated CAR-T cells, resulting in significantly prolonged survival.

[0121] Predicted conclusion CAR-T cells transplanted with organelle complexes (e.g., a first organelle complex, a second organelle complex) exhibit improved metabolic fitness, leading to improved proliferation capacity and cytokine production, as well as in vitro and in vivo antitumor effects. Organelle complex transplantation can also be useful as an adjunctive method in combination with various other genetic and pharmacological approaches to enhance the efficacy of CAR-T cell therapy.

[0122] Example 4 Transplantation of organelle complexes suppresses ferroptosis in T cells Introduction

[0123] In this example, we examine the effect of transplantation of organelle complexes (e.g., a first organelle complex, a second organelle complex (Q)) on ferroptosis in T cells. Ferroptosis is induced by virus-specific CD4 + It is a unique form of regulated cell death that can cause a significant loss of antigen-specific T cells, including helper T cells (Wang, Yifei, et al. "The kinase complex mTORC2 promotes the longevity of virus-specific memory CD4 + T cells by preventing ferroptosis.”Nature immunology 23.2 (2022):303-317). Ferroptosis can be driven by the iron-dependent lethal accumulation of membrane lipid peroxidation and can occur under quiescent conditions when the polyunsaturated fatty acid (PUFA) tails of membrane phospholipids are excessively peroxidized to toxic lipid peroxides (PL-PUFA-OOH). Mitochondria are responsible for the generation of most endogenous ROS, such as superoxide anion and hydrogen peroxide. Both of these species can act as substrates for the Fenton reaction to generate hydroxyl radicals, which can promote the progression of phospholipid peroxidation and membrane lipid degradation. In contrast, the glutathione-dependent peroxidase GPX4 can convert toxic PL-PUFA-OOH to non-toxic PL-PUFA-OH, driven by the oxidation of glutathione (GSH) to glutathione disulfide (GSSG), thereby suppressing membrane lipid hydroperoxides. The transplantation of a second organelle complex (Q) has been hypothesized to suppress ferroptosis and improve T cell survival.

[0124] Q-transplantation promotes the expression of GPX4 in activated T cells 18A-18B show the results of 72-hour stimulation with anti-CD3 / CD28 antibodies in CD4+ cells transplanted with the second organelle complex (Q) or vehicle. + T cells (Figure 18A) and CD8 +Data on GPX4 expression in T cells (FIG. 18B) are shown. Q transplantation was found to promote the expression of GPX4, a molecule that suppresses ferroptosis.

[0125] Q transplantation suppresses lipid peroxidation in activated T cells 19A-19B show the results of 72-hour stimulation with anti-CD3 / CD28 antibodies in CD4+ cells transplanted with the second organelle complex (Q) or vehicle. + T (Figure 19A) and CD8 + Data on lipid peroxidation levels in T cells (Figure 19B) are shown. Lipid peroxidation was assessed as the fluorescence change from PE to FITC using a lipid peroxidation kit. As the PE / FITC ratio increased, the lipid peroxidation level decreased. These results indicate that transplantation of a second organelle complex can suppress lipid peroxidation.

[0126] Q transplantation suppresses cell death of activated T cells Figures 20A-20B show apoptotic CD4+ cells transplanted with the second organelle complex (Q) or vehicle after 120 hours of stimulation with anti-CD3 / CD28 antibodies. + T cells (Figure 20A) and CD8 + Data on the proportion of T cells (Figure 20B) are shown. It was found that transplantation of the second organelle complex can suppress cell death of activated T cells, which can be considered as suppression of ferroptosis.

[0127] Q-implantation suppresses ROS accumulation in mouse CAR-T cells Figures 21A-21B show CD4 + (Figure 21A) and CD8 + (Figure 21B) Data on ROS accumulation in vehicle and Q-treated CAR-T cells assessed with CellROX Green dye in context. A significant decrease in ROS accumulation was found in CAR-T cells implanted with the second organelle complex.

[0128] Q-transplantation suppresses lipid peroxidation in mouse CAR-T cells Figures 22A-22B show the results of CD4 + CAR-T cells (Figure 22A) and CD8 + Figure 22B shows data on lipid peroxidation levels in CAR-T cells. Lipid peroxidation was assessed as the fluorescence change from PE to FITC using a lipid peroxidation kit. As the PE / FITC ratio decreased, the level of lipid peroxidation increased. Transplantation of the second organelle complex was found to suppress lipid peroxidation.

[0129] Q-transplantation of mouse CAR-T cells extends tumor survival after infusion Figures 23A-23B show the effect of the second organelle complex (Q) or vehicle on tumor-infiltrating lymphocytes (TILs) transferred to CD4+ T cells 17 days after tumor cell inoculation. + CAR-T cells (Figure 23A) and CD8 + Data on the percentage of CAR-T cells (Figure 23B) are shown. + and CD8 + Increased proportions of both CAR-T cells were found in tumors in the second organelle complex transplant group.

[0130] Q-transplantation suppresses ROS accumulation in human activated T cells Figures 24A-24D show the results of 192 hours of stimulation with anti-CD3 / CD28 antibodies in CD4 cells transplanted with the second organelle complex (Q) or vehicle. + T cells (Fig. 24A, Fig. 24C) and CD8 + Data are shown for the accumulation of cellular ROS (Figures 24A-24B) and mitochondrial ROS (Figures 24C-24D) in T cells (Figures 24B, 24D). Cellular ROS accumulation and mitochondrial ROS accumulation were assessed using CellROX Green and MitoSOX Red dyes, respectively. Transplantation of the second organelle complex in human activated T cells was found to have an inhibitory effect on ROS accumulation.

[0131] Q-transplantation promotes the expansion of human activated T cells Figures 25A-25B show the results of CD4+ cells transplanted with the second organelle complex (Q) or vehicle after chronic stimulation. + T cells (Figure 25A) and CD8 + Figure 25B shows data on the proliferation capacity of T cells. T cell proliferation capacity was assessed using a Cell Trace Violet dilution assay. Evaluation of the percentage and number of viable T cells using a dead cell staining dye was also performed after 192 hours of stimulation with anti-CD3 / CD28 antibodies. Figures 26A-26B show data on the percentage (Figure 26A) and number (Figure 26B) of viable T cells transplanted with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibodies. Transplantation of the second organelle complex was found to enhance the proliferation of human activated T cells.

[0132] conclusion Ferroptosis is a key apoptotic pathway in T cells coupled with mitochondrially generated ROS. In various models tested (human, mouse, and CAR-T contexts), CD4 + T cells and CD8 + In tumor-associated cells (including T cells), transplantation of the second organelle complex (Q) has been demonstrated to promote GPX4 expression, inhibit lipid peroxidation, inhibit cell death, inhibit ROS accumulation, promote proliferation, and extend tumor survival. The results provided in this example demonstrate that transplantation of the second organelle complex (Q) inhibits ferroptosis in T cells, further demonstrating its usefulness in cell therapy.

[0133] In at least some of the above-described embodiments, one or more elements used in an embodiment may be used interchangeably with another embodiment, unless such substitution is technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be within the scope of the subject matter defined by the appended claims.

[0134] With respect to the use of virtually any plural and / or singular term herein, those of ordinary skill in the art can convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference herein to "or" is intended to include "and / or" unless expressly stated otherwise.

[0135] Those skilled in the art will understand that the terms used herein generally, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including, but not limited to," etc.). Those skilled in the art will further understand that where a specific number is intended in the introduced claim recitation, such intention will be expressly set forth in the claim; in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitation. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" limits a particular claim that includes such an introduced claim recitation to embodiments that include only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). Also, even if a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the mere recitation "two recitations," without any other modifier, means at least two recitations, or more than two recitations). Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such interpretation is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together).When a convention similar to "at least one of A, B, or C, etc." is used, such interpretation is generally intended in the sense that one of ordinary skill in the art would understand that convention (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together). Those of ordinary skill in the art will further understand that whether in the specification, claims, or drawings, virtually any disjunctive word and / or phrase presenting two or more alternative terms should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms.

[0136] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, one skilled in the art will recognize that the disclosure also is described in terms of any individual member or subgroup of members of the Markush group.

[0137] As will be understood by those skilled in the art, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations thereof. Any recited range is readily recognizable to fully describe and enable that same range to be divided into at least one half, one third, one quarter, one fifth, one tenth, etc. As a non-limiting example, each range described herein can be easily subdivided into a lower third, middle third, upper third, etc. As will also be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc., are inclusive of the recited numbers and refer to ranges that can be subsequently subdivided into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0138] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are intended to be illustrative only and not limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for generating a population of T cells for adoptive T cell therapy, comprising: and contacting an isolated organelle complex with a population of T cells to generate a population of T cells comprising said organelle complex, wherein said organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus.

2. 2. The method of claim 1, comprising stimulating the population of T cells.

3. 3. The method of claim 2, wherein the stimulating step expands the population of T cells.

4. 1. A method of enhancing the proliferation, migration, persistence and / or activity of a population of T cells for adoptive T cell therapy, comprising: contacting the isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, wherein the organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus; and stimulating said population of T cells to expand said population of T cells.

5. 1. A method for generating a population of T cells resistant to depletion, comprising: contacting the isolated organelle complex with a population of T cells to generate a population of T cells comprising the organelle complex, wherein the organelle complex comprises mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus; and stimulating said population of T cells to expand said population of T cells.

6. The method according to any one of claims 2 to 5, wherein the stimulation is performed before, after, and / or during the contact.

7. The method of any one of claims 2 to 6, wherein said stimulating comprises culturing said population of T cells in the presence of one or more stimulating agents.

8. The method of any one of claims 2 to 7, wherein the one or more stimulatory agents comprise an agent that stimulates a CD3 / TCR complex-associated signal and an agent that stimulates a costimulatory molecule on the surface of the T cell.

9. The one or more stimulants are a molecule that binds to CD28, optionally one or more of an anti-CD28 antibody, CD80, and CD86; and / or The method of any one of claims 2 to 8, comprising a molecule that binds to CD3, optionally an anti-CD3 antibody.

10. 10. The method of any one of claims 1 to 9, wherein the stimulating and / or contacting is performed for a period of at least about 6 hours, 12 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

11. 11. The method of any one of claims 1 to 10, comprising introducing a heterologous nucleic acid encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR) into the T cell, optionally wherein said introducing step occurs before, after, and / or during said contacting, and further optionally wherein said heterologous nucleic acid is a vector.

12. The method according to any one of claims 1 to 11, wherein the isolated organelle complex is capable of being taken up into a population of T cells when the isolated organelle complex is contacted with the population of T cells.

13. 12. The method of any one of claims 1 to 11, wherein the contacting step is repeated at least 2, 3, or 4 times, and optionally, the contacting occurs during one or both of the stimulating step and the introducing step.

14. 14. The method of any one of claims 1-13, wherein the population of T cells is contacted with an effective amount of the isolated organelle complex sufficient to enhance proliferation, migration, persistence and / or activity of the T cells, optionally wherein the effective amount comprises about 20ug of the isolated organelle complex.

15. 15. The method of any one of claims 1 to 14, wherein the population of T cells is contacted with an effective amount of the isolated organelle complex sufficient to render the T cells resistant to exhaustion.

16. 16. The method of any one of claims 1 to 15, wherein the population of T cells is derived from lymph node cells and is optionally purified by a magnetic particle-based enrichment method selected from the group consisting of manualMACS®, AutoMACS®, CliniMACS®, EasySep®, and RoboSep®.

17. The population of T cells is CD4 ＋ T cells, CD8 ＋ 17. The method of any one of claims 1 to 16, comprising one or more of T cells, cytotoxic T cells, peripheral effector T cells, effector T cells, memory or central memory T cells, naive T cells, regulatory T cells, natural killer T cells, gamma delta T cells, cytokine-induced killer (CIK) T cells and tumor infiltrating lymphocytes (TIL).

18. the organelle complex comprises a first organelle complex, a second organelle complex, or a combination of a first organelle complex and a second organelle complex; the first organelle complex and the second organelle complex are depleted of cytoplasmic macromolecules; the first organelle complex is derived from (i) frozen cells, (ii) suspension cells, and / or (iii) cells contacted with a detergent at a concentration equal to or greater than the critical micelle concentration (CMC) of the detergent; The method of any one of claims 1 to 17, wherein the second organelle complex is derived from (i) adherent cells and / or (ii) cells contacted with a detergent at a concentration below the critical micelle concentration (CMC) of the detergent.

19. 19. The method of claim 18, wherein the cytoplasmic macromolecules comprise cytoplasmic proteins, and the abundance of one or more cytoplasmic proteins is at least about 90% depleted compared to the cell from which the organelle complex population is derived, and optionally, the cytoplasmic proteins are p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

20. The organelle complex comprises: one or more mitochondrial matrix proteins, optionally mitochondrial transcription factor A (TFAM) and / or citrate synthase (CS); one or more mitochondrial outer membrane proteins, optionally mitochondrial outer membrane complex subunit 20 (TOMM20); one or more lysosomal proteins, optionally lysosomal-associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR), and / or lysosomal-associated membrane protein 1 (LAMP1); one or more peroxisomal proteins, optionally catalase and / or ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3), one or more Golgi apparatus proteins, optionally Golgin-97, Sintaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130), and / or Mannosidase alpha class 2A member 1 (MAN2A1), and / or 20. The method of any one of claims 1 to 19, comprising one or more endoplasmic reticulum proteins, optionally calticalin and / or caldisin.

21. 21. The method of any one of claims 1 to 20, wherein the organelle complexes are derived from cells treated with a mitochondrial activator, optionally resveratrol.

22. The method of any one of claims 1 to 21, wherein the organelle complex is derived from cells of a subject different from the subject from which the T cells are derived.

23. The method of any one of claims 1 to 22, wherein the organelle complex is derived from cells of the same subject from which the T cells are derived.

24. 24. The method of any one of claims 1 to 23, wherein the T cells comprise a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR).

25. The method of any one of claims 1 to 24, wherein the CAR and / or TCR comprises one or more of an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

26. 26. The method of claim 25, wherein the intracellular signaling domain comprises a primary signaling domain, a costimulatory domain, or both a primary signaling domain and a costimulatory domain.

27. 27. The method of claim 26, wherein the primary signaling domain comprises a functional signaling domain of one or more proteins selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12, or functional variants thereof.

28. The costimulatory domain may be any of the following: CD27, CD28, 4-1BB (CD137), OX40, CD28-OX40, CD28-4-1BB, CD30, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHT TR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGA X, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAMl (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly 108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D, or a functional domain thereof.

29. 29. The method of any one of claims 25 to 28, wherein the antigen-binding domain binds to a tumor antigen, and optionally the tumor antigen is a solid tumor antigen.

30. The antigen-binding domain may be an antibody, antibody fragment, scFv, Fv, Fab, a(Fab'), single domain antibody (SDAB), VH or VL domain, camelid VHH domain, Fab, Fab', F(ab') ２ 30. The method of any one of claims 25 to 29, comprising an Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single domain antibody (sdAb), single chain comprising anti-complementary scFv (tandem scFv) or bispecific tandem scFv, Fv construct, disulfide-linked Fv, dual variable domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, affibody, affilin, affitin, affimer, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, or any combination thereof.

31. The method of any one of claims 25 to 30, wherein the antigen-binding domain is connected to the transmembrane domain by a hinge region.

32. The transmembrane domain may be selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1B B (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLEF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11d, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD24 4, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or a functional variant thereof.

33. The method of any one of claims 24 to 32, wherein the CAR or TCR further comprises a leader peptide.

34. The method of any one of claims 24 to 33, wherein the TCR further comprises a constant region and / or CDR4.

35. the T cell recovery efficiency is at least about 5 percent greater than a method that does not include contacting the population of T cells with an isolated organelle complex; 35. The method of any one of claims 1 to 34, wherein T cell recovery is the ratio of T cells recovered after stimulation, optionally about 48 hours after stimulation, to the number of T cells immediately before the start of stimulation.

36. 36. The method of any one of claims 1 to 35, wherein the population of T cells optionally exhibits one or more of an enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence in vivo and / or in vitro compared to a population of T cells that does not comprise the exogenous organelle complex.

37. 37. The method of any one of claims 1 to 36, wherein the population of T cells optionally exhibits an increase in basal and / or maximal oxygen consumption rate of at least about 1.1 fold in vivo and / or in vitro compared to a population of T cells that does not comprise the exogenous organelle complex.

38. 38. The method of any one of claims 1 to 37, wherein the population of T cells optionally exhibits at least about a 1.1-fold increase in basal and / or maximal glycolytic rate in vivo and / or in vitro compared to a population of T cells that does not comprise the exogenous organelle complex.

39. 39. The method of any one of claims 1 to 38, wherein the population of T cells optionally exhibits at least about a 1.1-fold increase in glycolytic and / or respiratory capacity in vivo and / or in vitro compared to a population of T cells that does not comprise the exogenous organelle complex.

40. 40. The method of any one of claims 1 to 39, wherein the population of T cells optionally exhibits at least about a 1.1-fold increase in mitochondrial mass in vivo and / or in vitro compared to a population of T cells that does not comprise the exogenous organelle complex.

41. 41. The method of any one of claims 1 to 40, wherein the population of T cells optionally exhibits at least about a 1.1-fold increase in cytotoxic activity against target cells in vivo and / or in vitro compared to a population of T cells that does not contain the exogenous organelle complex.

42. 42. The method of any one of claims 1-41, wherein the population of T cells optionally exhibits at least about a 1.1-fold decrease in the level of one or more exhaustion markers in vivo and / or in vitro compared to a population of T cells that does not comprise the exogenous organelle complex, and optionally the exhaustion markers are selected from the group comprising PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD160, CD39, VISTA, TIGIT, or any combination thereof.

43. The population of T cells optionally exhibits at least about a 1.1-fold reduction in levels of one or more of cellular ROS, mitochondrial ROS, cellular oxidative stress, and mitochondrial oxidative stress in vivo and / or in vitro compared to a population of T cells that does not contain the exogenous organelle complex, and further optionally, the reactive oxygen species is selected from the group consisting of superoxide (O ２ . － ), hydroperoxy (HO. ２ ), hydrogen peroxide (H ２ O ２ ), peroxynitrite (ONOO － ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO), peroxy radical (ROO), alkoxy radical (RO), singlet oxygen ( １ O ２ ), lipid peroxides, lipid peroxy radicals or lipid alkoxyl radicals, or any combination thereof.

44. 44. The method of any one of claims 1 to 43, wherein the population of T cells optionally exhibits at least about a 1.1-fold increase in production of one or more of cytokines in vivo and / or in vitro compared to a population of T cells that does not comprise the exogenous organelle complex, and further optionally, the cytokines include interleukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), TNF-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and / or TGF-beta (TGFβ).

45. 45. The method of any one of claims 1 to 44, wherein the population of T cells exhibits, optionally in vivo and / or in vitro, at least about a 1.1-fold increase in cell proliferation compared to a population of T cells that does not comprise the exogenous organelle complex.

46. 46. The method of any one of claims 1 to 45, wherein the population of T cells exhibits at least about a 1.1-fold increase in cell viability following chronic TCR stimulation, optionally in vivo and / or in vitro, compared to a population of T cells that does not contain the exogenous organelle complex.

47. A population of T cells produced by the method of any one of claims 1 to 46.

48. 1. A pharmaceutical composition comprising: A population of T cells generated by the method of any one of claims 1 to 46; and one or more pharmaceutically acceptable carriers.

49. 1. A population of T cells for adoptive T cell therapy, comprising: the T cell comprises an exogenous organelle complex; the organelle complex includes a mitochondrion and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus; The population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not contain the exogenous organelle complex.

50. 1. A method of treating or preventing a disease or disorder in a subject, comprising: administering to the subject an effective amount of the population of T cells produced according to the method of any one of claims 1 to 46, thereby treating or preventing the disease or disorder in the subject; The population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not contain the exogenous organelle complex.

51. 1. A method for enhancing adoptive T cell therapy in a subject, comprising: generating an effective amount of T cells comprising an exogenous organelle complex according to the method of any one of claims 1 to 46; adoptively transferring the T cells into the subject; The population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not contain the exogenous organelle complex.

52. 1. A method of treating or preventing a disease or disorder in a subject, comprising: administering to the subject an effective amount of a population of T cells, optionally wherein the T cells comprise a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR); administering to said subject an effective amount of the isolated organelle complex, thereby treating or preventing said disease or disorder in said subject; the isolated organelle complex can be contacted in vivo with the population of T cells to generate a population of T cells comprising the organelle complex; The population of T cells exhibits one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to a population of T cells that does not contain the exogenous organelle complex.

53. administering to the subject an effective amount of the population of T cells prior to administering to the subject an effective amount of the isolated organelle complex; administering to the subject an effective amount of an isolated organelle complex prior to administering to the subject an effective amount of the population of T cells; or 53. The method of claim 52, wherein an effective amount of the population of T cells and an effective amount of the isolated organelle complex are administered to the subject simultaneously.

54. 1. A method of treating or preventing a disease or disorder in a subject, comprising: administering to the subject an effective amount of a heterologous nucleic acid encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), optionally wherein the heterologous nucleic acid is a vector, further optionally a viral vector; administering to said subject an effective amount of the isolated organelle complex, thereby treating or preventing a disease or disorder in said subject; the heterologous nucleic acid and isolated organelle complex can be contacted in vivo with T cells of the subject to generate T cells comprising the organelle complex and a CAR and / or an engineered TCR; The method, wherein the T cells exhibit one or more of enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to attrition, and enhanced persistence compared to T cells that do not contain the exogenous organelle complex.

55. 55. The method of any one of claims 1-46 or 50-54, wherein the in vivo persistence of the population of T cells comprises a period of about 15 days, about 30 days, about 60 days, about 30 days, or about 1 year.

56. 56. The method of any one of claims 1-46 or 50-55, wherein the population of T cells reduces tumor volume, tumor growth, and / or tumor burden in the subject, and optionally, the population of T cells reduces tumor volume in the subject by at least about 1.1 fold compared to tumor volume in an untreated subject or a subject administered a population of T cells that does not include the exogenous organelle complex.

57. 57. The method of any one of claims 1-46 or 50-56, wherein the population of T cells enhances overall survival or progression-free survival, and optionally, the population of T cells enhances overall survival or progression-free survival by at least about 1.1 fold compared to an untreated subject or a subject administered a population of T cells that does not comprise the exogenous organelle complex.

58. 58. The method of any one of claims 1-46 or 50-57, wherein the T cells are autologous to the subject.

59. 59. The method of any one of claims 1-46 or 50-58, wherein the T cells are allogeneic to the subject.

60. The method of any one of claims 1 to 46 or 50 to 59, wherein the adoptive T cell therapy is CAR-T cell therapy.

61. 61. The method of any one of claims 1-46 or 50-60, wherein the adoptive T cell therapy is an engineered TCR-T cell therapy.

62. 62. The method of any one of claims 1 to 46 or 50 to 61, wherein the adoptive T cell therapy is tumor infiltrating lymphocyte (TIL) therapy.

63. The administration is at least about 1 x 10 ６ 63. The method of any one of claims 50 to 62, comprising administering the T cells, optionally intravenously.

64. 64. The method of any one of claims 50 to 63, comprising repeated administration of said population of T cells.

65. 65. The method of any one of claims 50 to 64, wherein the subject is a mammal.

66. 66. The method of any one of claims 50 to 65, wherein the disease or disorder is associated with expression of a tumor antigen, and the disease associated with expression of a tumor antigen is selected from the group consisting of a proliferative disease, a precancerous condition, cancer, and a non-cancer-related indication associated with expression of the tumor antigen.

67. The cancers include colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell carcinoma of the lung, mesothelioma, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, 67. The method of claim 66, wherein the cancer is selected from the group consisting of carcinoma, soft tissue sarcoma, urethral cancer, penile cancer, childhood cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, tumor of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of said cancers, and metastatic lesions of said cancers.

68. The cancer may be chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma 68. The method of any one of claims 66-67, wherein the cancer is a hematological cancer selected from one or more of: lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or preleukemia.

69. 69. The method of any one of claims 50-68, wherein the administration comprises systemic administration, intrathecal administration, intracranial injection, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, topical delivery, local delivery, intraciliary delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof, and optionally the systemic administration is intravenous, intramuscular, intraperitoneal, or intraarticular.

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