Methods of improving cell therapy using organelle complexes

Abstract

The contents disclosed herein include methods, compositions, and kits suitable for use in enhanced adoptive T cell therapy. In some embodiments, the methods include contacting an isolated organelle complex with a population of T cells to produce a population of T cells comprising the organelle complex. The organelle complex can include mitochondria and one or more of endoplasmic reticulum, peroxisome, lysosome, and Golgi. The population of T cells exhibits one or more of the following: enhanced expansion capability, enhanced cytotoxicity against a target cell, enhanced resistance to depletion, and enhanced persistence compared to a population of T cells not comprising an exogenous organelle complex.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 394,546, filed on August 2, 2022; U.S. Provisional Application No. 63 / 403,268, filed on September 1, 2022; and U.S. Provisional Application No. 63 / 406,022, filed on September 13, 2022. The entire contents of these applications are hereby expressly incorporated by reference in their entirety. Background Art Technical Field

[0003] The present disclosure generally relates to methods of enhancing the proliferation, migration, persistence, and / or activity of T cells for adoptive T cell therapy.

[0004] Description of Related Technology

[0005] Mitochondria are intracellular organelles responsible for many metabolic conversions and regulatory functions. Mitochondria are also highly dynamic organelles, which move throughout the cell and undergo structural changes, changing length, morphology, shape and size. In addition, mitochondria are constantly eliminated and regenerated in a process called mitochondrial biogenesis. Although most mitochondrial genes have been transferred to the nuclear genome, the mitochondrial genome still encodes 13 subunits of rRNA, tRNA and the electron transport chain (ETC). Therefore, functional communication between the nuclear genome and the mitochondrial genome is crucial for mitochondrial biogenesis, effective oxidative phosphorylation and normal health. Mitochondria are also the main source of free radicals and reactive oxygen species (ROS) that cause oxidative stress. In addition, mitochondria play a key role in the control of intracellular signal transduction and cell death (including apoptosis and necrosis).

[0006] Chimeric antigen receptor (CAR) T-cell therapy is a potentially curative treatment for patients with relapsed or refractory (r / r) hematologic malignancies. However, approximately half of patients with r / r B-cell lymphomas experience treatment failure following CAR-T-cell therapy, which may be due to poor expansion, limited persistence, and exhaustion of CAR-T cells. Given that metabolic adaptation plays a central role in regulating T-cell function, manipulating mitochondrial function is a promising approach to enhance the anti-tumor effects 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

[0007] Provided herein are methods and compositions for solving the above-mentioned problems in the art. For example, as described herein, the organelle complex of separation is transplanted into T cells to perform metabolic reprogramming on T cells, and thus improve the performance of the T cells in terms of multiple parameters (including cytokine production, proliferation, persistence and anti-tumor activity) in vitro and in vivo. Disclosed herein include methods for producing T cell colonies for adoptive T cell therapy. In certain embodiments, the method includes: contacting the organelle complex of separation with a T cell colony to produce a T cell colony comprising these organelle complexes, wherein these organelle complexes comprise mitochondria and one or more of endoplasmic reticulum, peroxisomes, lysosomes and Golgi bodies. In certain embodiments, the method includes stimulating the T cell colony. In certain embodiments, the stimulation step amplifies the T cell colony.

[0008] Disclosed herein include methods for enhancing the proliferation, migration, persistence, and / or activity of a T cell population for adoptive T cell therapy. In some embodiments, the method comprises contacting an isolated organelle complex with a T cell population to produce a T cell population comprising the organelle complexes, wherein the organelle complexes comprise mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus. The method may comprise stimulating the T cell population to expand the T cell population.

[0009] Disclosed herein are methods for generating a population of T cells that are resistant to exhaustion. In some embodiments, the methods include contacting isolated organelle complexes with a population of T cells to generate a population of T cells comprising the organelle complexes, wherein the organelle complexes comprise mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus. The methods may include stimulating the population of T cells to expand the population of T cells.

[0010] In some embodiments, the stimulation is carried out before, after and / or during the contact. In some embodiments, the stimulation is included in the presence of one or more stimulating agents to cultivate the T cell colony. In some embodiments, the one or more stimulating agents include agents that stimulate CD3 / TCR complex-related signals and agents that stimulate costimulatory molecules on the T cell surface. In some embodiments, the one or more stimulating agents include: molecules (e.g., one or more of anti-CD28 antibodies, CD80, and CD86) in conjunction with CD28; and / or molecules (e.g., anti-CD3 antibodies) in conjunction with CD3. In some embodiments, the stimulation and / or contact are carried out 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, when these separated organelle complexes are in contact with T cell colonies, these organelle complexes can be incorporated into these T cells. In some embodiments, the contacting step is repeated at least 2, 3, or 4 times. In some embodiments, the contact is carried out during one or both of the stimulation step and the introduction step. In some embodiments, the T cell population is contacted with an effective amount of an isolated organelle complex sufficient to enhance the proliferation, migration, persistence, and / or activity of the T cells. In some embodiments, the effective amount comprises about 20 μg of an isolated organelle complex. In some embodiments, the T cell population is contacted with an effective amount of an isolated organelle complex sufficient to resist exhaustion of the T cells. In some embodiments, the T cell population is derived from lymph node cells (e.g., purified by a magnetic particle-based enrichment procedure selected from the group consisting of: manual and In some embodiments, the T cell population comprises one or more of the following: CD4 + cells, CD8 + T cells, cytotoxic T cells, terminal 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).

[0011] In some embodiments, the organelle complexes include a first organelle complex, a second organelle complex, or a combination of a first organelle complex and a second organelle complex, wherein the first organelle complex and the second organelle complex are depleted of cytoplasmic macromolecules, wherein the first organelle complex is derived from (i) frozen cells; (ii) floating cells; and / or (iii) cells contacted with a surfactant at a concentration equal to or higher than the critical micelle concentration (CMC) of the surfactant, and wherein the second organelle complex is derived from (i) adherent cells; and / or (ii) cells contacted with a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. In some embodiments, the cytoplasmic macromolecule comprises a cytoplasmic protein (e.g., the cytoplasmic protein is p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), wherein the abundance of one or more cytoplasmic proteins is depleted by at least about 90% compared to the cells from which the organelle complex populations were derived. In some embodiments, the organelle complexes comprise: 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., lysosomal associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR) and / or lysosomal associated membrane protein 1 (LAMP1)); one or more peroxy In some embodiments, the organelle complexes are derived from cells treated with a mitochondrial activator (e.g., resveratrol). In some embodiments, the organelle complexes are derived from cells of a subject different from the subject from which the T cells were derived. In some embodiments, the organelle complexes are derived from cells of the same subject from which the T cells were derived.

[0012] In some embodiments, the method includes introducing a heterologous nucleic acid (e.g., a vector) encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR) into a T cell. In some embodiments, the introduction step is performed before, after, and / or during the contact. 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 comprises 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 CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10 and DAP12, or functional variants thereof.In some embodiments, the costimulatory domain comprises a functional domain of one or more proteins 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, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, C D11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2 , TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD22 9), 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. 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, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single domain antibody (SDAB), a VH or VL domain, a camelid VHH domain, Fab, Fab', F(ab')2, Fv, scFv, dsFv, a diabody, a triabody, a tetrabody, a multispecific antibody formed by an antibody fragment, a single domain antibody (sdAb), a single chain comprising an anti-complement scFv (tandem scFv) or a bispecific tandem scFv, an Fv construct, a disulfide-linked Fv, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody, an aptamer, an affibody, an affilin, an affitin, an affimer, an alpha body, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain peptide, a monomer or any combination thereof. In some embodiments, the antigen binding domain is connected to the transmembrane domain via a hinge region.In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: the alpha, beta, or zeta chain of a 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 (KLR F1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f ,ITGAD,CD11d,ITGAE,CD103,ITGAL,CD11a,LFA-1,ITGAM,CD11b,ITGAX,CD11c,ITGB1,CD29,ITGB 2. 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 functional variants 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.

[0013] In some embodiments, the T cell recovery efficiency is at least about 5% greater than that of a method in which the organelle complex of separation is contacted with the T cell colony. In some embodiments, the T cell recovery efficiency is the ratio of the number of T cells recovered after stimulation (e.g., after about 48 hours of stimulation) to the number of T cells immediately before the start of stimulation. In some embodiments, the T cell colony, compared with a T cell colony not comprising an exogenous organelle complex, exhibits one or more of the following: enhanced amplification capacity (in vivo and / or in vitro), enhanced cytotoxicity for target cells (in vivo and / or in vitro), enhanced anti-exhaustion (in vivo and / or in vitro) and enhanced persistence (in vivo and / or in vitro). In some embodiments, the T cell colony, compared with a T cell colony not comprising an exogenous organelle complex, exhibits an increase of at least about 1.1 times of basal oxygen consumption rate and / or maximum oxygen consumption rate (in vivo and / or in vitro). In some embodiments, the T cell population exhibits an increase of at least about 1.1 times in basal glycolytic rate and / or maximum glycolytic rate (in vivo and / or in vitro) compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the T cell population exhibits an increase of at least about 1.1 times in glycolytic capacity and / or respiratory capacity (in vivo and / or in vitro) compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the T cell population exhibits an increase of at least about 1.1 times in mitochondrial mass (in vivo and / or in vitro) compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the T cell population exhibits an increase of at least about 1.1 times in cytotoxic activity (in vivo and / or in vitro) against target cells compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the T cell population exhibits a decrease of at least about 1.1 times in the level of one or more exhaustion markers (in vivo and / or in vitro) compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the exhaustion marker is selected from the group comprising PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD 160, CD39, VISTA, TIGIT, or any combination thereof. In some embodiments, the T cell population exhibits at least about a 1.1-fold reduction in the level 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 T cell population that does not comprise an exogenous organelle complex. In some embodiments, the reactive oxygen species include superoxide (O2. - ), hydroperoxide (HO.2), hydrogen peroxide (H2O2), peroxynitrite (ONOO -), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO.), peroxyl radical (ROO.), alkoxyl radical (RO.), singlet oxygen ( 1 O2), lipid peroxides, lipid peroxyl radicals or lipid alkoxyl radicals or any combination thereof. In some embodiments, the T cell population shows an increase of at least about 1.1 times in the production (in vivo and / or in vitro) of one or more of the cytokines compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, these cytokines include interleukin-2 (IL-2), interferon-γ (IFNγ), interleukin-4 (IL-4), TNF-α (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF), CD107a and / or TGF-β (TGFβ). In some embodiments, the T cell population shows an increase of at least about 1.1 times in cell proliferation (in vivo and / or in vitro) compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the T cell population exhibits at least about a 1.1-fold increase in cell viability following chronic TCR stimulation (in vivo and / or in vitro) compared to a T cell population that does not comprise the exogenous organelle complex.

[0014] In some embodiments, a T cell population generated by the methods disclosed herein is provided. In some embodiments, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises: a T cell population generated by the methods disclosed herein; and one or more pharmaceutically acceptable carriers.

[0015] In some embodiments, a T cell population for adoptive T cell therapy is provided, wherein the T cells comprise exogenous organelle complexes. In some embodiments, the organelle complexes comprise mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. In some embodiments, the T cell populations exhibit one or more of the following compared to T cell populations that do not comprise exogenous organelle complexes: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

[0016] Disclosed herein include methods for treating or preventing a disease or condition in a subject. In some embodiments, the methods include administering to the subject an effective amount of a T cell population generated according to the methods disclosed herein, thereby treating or preventing the disease or condition in the subject. In some embodiments, the T cell populations exhibit one or more of the following compared to T cell populations that do not contain exogenous organelle complexes: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

[0017] Disclosed herein include methods for enhancing adoptive T cell therapy in a subject. In some embodiments, the method comprises: generating an effective amount of T cells comprising an exogenous organelle complex according to the methods disclosed herein; and adoptively transferring the T cells to the subject. In some embodiments, the T cell population exhibits one or more of the following compared to a T cell population that does not comprise an exogenous organelle complex: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

[0018] Disclosed herein include methods for treating or preventing a disease or illness in a subject. In some embodiments, the method includes: administering an effective amount of a T cell population (e.g., a T cell comprising a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR)) to the subject; and administering an effective amount of an isolated organelle complex to the subject, thereby treating or preventing the subject's disease or illness. In some embodiments, these isolated organelle complexes are able to contact these T cell populations in vivo to produce a T cell population comprising these organelle complexes. In some embodiments, these T cell populations exhibit one or more of the following compared to T cell populations that do not comprise exogenous organelle complexes: enhanced amplification capacity, enhanced cytotoxicity for target cells, enhanced resistance to exhaustion, and enhanced persistence. In some embodiments, an effective amount of these T cell populations are administered to the subject before an effective amount of an isolated organelle complex is administered to the subject. In some embodiments, an effective amount of the isolated organelle complex is administered to the subject before an effective amount of these T cell populations are administered to the subject. In some embodiments, an effective amount of a T cell population and an effective amount of these isolated organelle complexes are administered to the subject simultaneously.

[0019] Disclosed herein include methods for treating or preventing a disease or illness of a subject. In some embodiments, the method includes: administering to the subject an effective amount of heterologous nucleic acid (e.g., vector, 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 subject's disease or illness. In some embodiments, the heterologous nucleic acid and isolated organelle complex can contact the subject's T cells in vivo to produce T cells comprising these organelle complexes and CAR and / or engineered TCR. In some embodiments, the T cells exhibit one or more of the following compared to T cells that do not comprise exogenous organelle complexes: enhanced amplification capacity, enhanced cytotoxicity for target cells, enhanced anti-exhaustion and enhanced persistence.

[0020] In some embodiments, the in vivo persistence of the T cell colony includes a time period of about 15 days, about 30 days, about 60 days, about 90 days, or about one year. In some embodiments, the T cell colony reduces the tumor volume, tumor growth, and / or tumor burden of the subject. In some embodiments, the T cell colony reduces the tumor volume of the subject by at least about 1.1 times compared to the tumor volume of an untreated subject or a subject to which a T cell colony not comprising an exogenous organelle complex is administered. In some embodiments, the T cell colony increases total survival or progression-free survival. In some embodiments, the T cell colony increases total survival or progression-free survival by at least about 1.1 times compared to an untreated subject or a subject to which a T cell colony not comprising an exogenous organelle complex is administered. In some embodiments, these T cells are autologous for the subject. In some embodiments, these T cells are allogeneic for the subject. In some embodiments, the adoptive T cell therapy is a CAR-T cell therapy. In some embodiments, the adoptive T cell therapy is an engineered TCR-T cell therapy. In some embodiments, the adoptive T cell therapy is a tumor infiltrating lymphocyte (TIL) therapy. In some embodiments, the administering comprises administering (e.g., intravenously) at least about 1×10 6 In some embodiments, the method comprises repeatedly using the population of T cells.

[0021] In some embodiments, the subject is a mammal. In some embodiments, the disease or disorder is associated with expression of a tumor antigen, and wherein the disease associated with expression of a tumor antigen is selected from the group consisting of proliferative diseases, precancerous conditions, cancers, and non-cancer related indications 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 lung cancer, mesothelioma, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endometrial cancer, uterine cancer, ovarian cancer, rectal cancer, anal 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, endometrial cancer, uterine ... Endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers. In some embodiments, the cancer is a hematological cancer selected from one or more of the following: chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid 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 disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or preleukemia.

[0022] In some embodiments, administration comprises systemic administration (e.g., intravenous, intramuscular, intraperitoneal, or intraarticular), intrathecal administration, intracranial injection, aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, oral delivery, ocular delivery, topical delivery, topical delivery, intracisternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous injection, subcutaneous injection, intranodal injection, intratumoral injection, intraperitoneal injection, intradermal injection, or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Depicted are data related to enhanced OXPHOS in Q-treated CAR-T cells compared to vehicle-treated CAR-T cells.

[0024] Figure 2 Depicted are data relating to tumor growth inhibition in mice injected with Q-treated CAR-T cells.

[0025] Figure 3 Depicted are data relating to T cell recovery efficiency following stimulation following transplantation with the second organelle complex (2nd OC) or vehicle.

[0026] Figures 4A to 4D Depicts data related to mitochondrial mass assessment using MitoTracker Deep Red dye. CD4 T cells ( Figures 4A to 4B ) and CD8 T cells ( Figures 4C to 4D ) were transplanted with the second organelle complex (2nd OC) or vehicle and assessed on day 4.

[0027] Figures 5A to 5D Depicted are data related to T cell proliferation capacity assessed by dye dilution assay using Cell Trace Violet dye. CD4 T cells ( Figures 5A to 5B ) and CD8 T cells ( Figures 5C to 5D ) were transplanted with the second organelle complex (2nd OC) or vehicle and assessed on day 4.

[0028] Figures 6A to 6D Depicts data related to the measurement of mitochondrial selective oxidative stress using MitoSOX Red dye. CD4 T cells ( Figures 6A to 6B ) and CD8 T cells ( Figures 6C to 6D ) were transplanted with the second organelle complex (2nd OC) or vehicle and assessed on day 4.

[0029] Figures 7A to 7D Depicted are data related to the level of cellular oxidative stress measured using Cell ROX Green dye. Figures 7A to 7B ) and CD8 T cells ( Figures 7C to 7D ) were transplanted with the second organelle complex (2nd OC) or vehicle and assessed on day 4.

[0030] Figure 8 Depicted are data related to T cell oxygen consumption rate measured with a Seahorse extracellular flux analyzer. T cells were transplanted with the second organelle complex (2nd OC) or vehicle and assessed on day 4.

[0031] Figures 9A to 9BData related to cytokine production capacity assessed using PMA and ionomycin are depicted. T cells were transplanted with the second organelle complex (2nd OC) or vehicle and assessed on day 4.

[0032] Figures 10A to 10B Data related to the number of surviving cells after chronic stimulation of TCR in vitro are depicted. T cells were transplanted with the second organelle complex (2nd OC) or vehicle, and viable cell counts were assessed on day 9 using a dead cell staining dye.

[0033] Figure 11 Depicted are data related to CAR-T cell glycolytic rates measured using a Seahorse extracellular flux analyzer.

[0034] Figures 12A to 12D Depicted are data related to the proliferation capacity of CAR-T cells assessed by dye dilution assay during co-culture with A20. CD4 CAR-T cells ( Figures 12A to 12B ) and CD8 CAR-T cells ( Figures 12C to 12D ) were transplanted with a second organelle complex (2nd OC) or vehicle.

[0035] Figures 13A to 13B Depicted are data related to cytokine production capacity assessed using PMA and ionomycin.

[0036] Figure 14 Depicted are data relating to cytotoxicity assays performed by co-culture with A20 cells.

[0037] Figure 15 Depicted are data related to overall survival of mice injected with CAR-T cells treated with the second organelle complex compared to vehicle-treated CAR-T cells or naive T cells.

[0038] Figures 16A to 16B Flow cytometric analysis data related to TCF-1 precursor cells are depicted. T cells were transplanted with the second organelle complex (2nd OC) or vehicle and assessed 72 hours after TCR stimulation.

[0039] Figures 17A to 17B Flow cytometric analysis data related to TCF-1 precursor CAR-T cells are depicted. CAR-T cells were transplanted with the second organelle complex (2nd OC) or vehicle and assessed 96 hours after the start of TCR stimulation.

[0040] Figures 18A to 18B Figure 3. CD4 T cells transplanted with the second organelle complex (Q) or vehicle after 72 hours of stimulation with anti-CD3 / CD28 antibodies. + T cells ( Figure 18A ) and CD8 + T cells ( Figure 18B ) related data on GPX4 expression.

[0041] Figures 19A to 19B Figure 3. CD4 T cells transplanted with the second organelle complex (Q) or vehicle after 72 hours of stimulation with anti-CD3 / CD28 antibodies. + T cells ( Figure 19A ) and CD8 + T cells ( Figure 19B ) in relation to lipid peroxidation levels.

[0042] Figures 20A to 20B Figure 3 shows apoptotic CD4 T 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 + T cells ( Figure 20B ) percentage-related data.

[0043] Figures 21A to 21B Depicts the relationship between CD4 + ( Figure 21A ) and CD8 + ( Figure 21B Data related to ROS accumulation in vehicle CAR-T cells and Q-treated CAR-T cells assessed with CellROX Green dye in a ) environment.

[0044] Figures 22A to 22B Depicts the CD4 + CAR-T cells ( Figure 22A ) and CD8 + CAR-T cells ( Figure 22B ) in relation to lipid peroxidation levels.

[0045] Figures 23A to 23B Figure 3 shows the expression of CD4 T cells transplanted with the second organelle complex (Q) or vehicle in tumor infiltrating lymphocytes (TILs) at 17 days after tumor cell inoculation. + CAR-T cells ( Figure 23A ) and CD8 + CAR-T cells ( Figure 23B ) percentage-related data.

[0046] Figures 24A to 24D Figure 3. CD4 T cells transplanted with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibodies. + T cells ( Figure 24A、 Figure 24C ) and CD8 + T cells ( Figure 24B 、 Figure 24D ) in cells ROS( Figures 24A to 24B ) and mitochondrial ROS ( Figures 24C to 24D ) accumulate relevant data.

[0047] Figures 25A to 25B Depicts CD4 T cells transplanted with the second organelle complex (Q) or vehicle after chronic stimulation. + T cells ( Figure 25A ) and CD8 + T cells ( Figure 25B ) related to the proliferation capacity.

[0048] Figures 26A to 26B Depicted are the percentage of viable cells ( Figure 26A ) and the number of viable cells ( Figure 26B ) related data. DETAILED DESCRIPTION

[0049] In the following detailed description, reference is made to the accompanying drawings, which form a part of this document. Unless the context indicates otherwise, similar symbols typically identify similar components in the accompanying drawings. The illustrative embodiments described in the detailed description, drawings, and claims are not intended 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 various aspects of the present disclosure, as generally described herein and shown in the accompanying drawings, may be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and form a part of the disclosure herein.

[0050] All patents, published patent applications, other publications concerning the related art, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety.

[0051] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. 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 the purposes of this disclosure, the following terms are defined as follows.

[0052] As used herein, "isolated" shall be given its ordinary meaning and shall also refer to a substance or entity that has been (1) separated from at least some of the components with which it was originally produced (whether in nature or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. In some embodiments, isolated mitochondria or isolated populations of organelle complexes are processed to be obtained from a cellular environment via the methods provided herein.

[0053] As used herein, the term "mitochondria" should be given its ordinary meaning, and should also refer to an organelle present in a eukaryotic cell having a double lipid membrane, an inner membrane and an outer membrane, and a matrix surrounded by cristae and the inner membrane. Mitochondria (more than one mitochondria) have enzymes involved in oxidative phosphorylation, such as respiratory chain complexes, on their inner membranes. The inner and outer proton gradients formed by the effects of respiratory chain complexes, etc., and the inner membrane has a membrane potential. It is believed that when the inner membrane is damaged, the mitochondria cannot maintain this membrane potential.

[0054] As used herein, the term "organelle complex" should be given its common meaning and should also refer to a complex of mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. The organelle complex can be depleted of cytoplasmic macromolecules (e.g., cytoplasmic proteins). In some embodiments, the organelle complex does not include cytoplasmic macromolecules. In some embodiments, the organelle complex population includes homogenized mitochondria. As used herein, the term "population" should have its common meaning and should also refer to a collection of multiple identical or different substances. For example, a "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 a physical, chemical, and / or physiological distribution. Physical distribution includes, for example, particle size and polydispersity index. Chemical distribution includes, for example, zeta potential distribution and lipid composition distribution. Physiological distribution includes, 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 also refer to mitochondria isolated via a method comprising one or more homogenization steps.

[0055] As used herein, the term "surfactant" shall be given its ordinary meaning and shall also refer to a molecule having a hydrophilic portion and a hydrophobic portion in one molecule. Surfactants have the function of reducing the surface tension of an interface or mixing 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, while ionic surfactants are those in which the hydrophilic portion contains cations or anions or both cations and anions.

[0056] As used herein, the term "critical micelle concentration" (CMC) shall be given its ordinary meaning and shall also refer to the concentration (particularly the bulk concentration) at which the surfactant forms micelles and further addition of surfactant to the system facilitates micelle formation. When the concentration is above the critical micelle concentration, adding surfactant to the system may desirably increase the amount, and particularly the number, of micelles.

[0057] As used herein, the term "regulation" should give its ordinary meaning, and should also refer to a change or change in biological activity. Regulation includes but is not limited to stimulation or inhibition activity. Regulation can be an increase or decrease in the activity of a biological property, functional property or immunological property related to the activity of a cell, cell colony, protein, approach, system or other biological target of interest, a change in binding characteristics or any other variation, and can be at least about 1.1 times (for example, 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or a numeral or scope between any two of these values).

[0058] As used herein, "subject" refers to an animal that is the subject of treatment, observation, or experiment. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates, such as fish, shellfish, reptiles, and in particular mammals. As used herein, "mammal" refers to an individual belonging to the class mammals, and includes, but is not limited to, humans, domestic and farm animals, zoo animals, sports animals, and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cattle; horses; primates, such as monkeys, chimpanzees, and apes, and in particular humans. In some embodiments, the mammal is a human. However, in some embodiments, the mammal is not a human. As used herein, the term "host cell" should be given its ordinary meaning and should also refer to cells in vivo, in vitro, and / or isolated cells into which exogenous mitochondria and / or organelle complexes are incorporated.

[0059] As used herein, the term "treatment" refers to an intervention that reacts to a disease, illness or physiological condition that a patient exhibits. The purpose of treatment may include, but is not limited to, one or more of the following: alleviating or preventing symptoms, slowing down or stopping the progression or worsening of a disease, illness or condition, and alleviating a disease, illness or condition. The terms "treat" and "treatment" include, for example, therapeutic treatments, preventative treatments, and applications that reduce the risk of a subject suffering from an illness or other risk factors. Treatment does not require complete cure of the illness and encompasses embodiments that reduce symptoms or potential risk factors. In certain embodiments, "treatment" refers to both therapeutic treatments and preventive or preventive measures. Those that require treatment include those that have been affected by a disease or illness or an undesirable physiological condition and those that are intended to prevent a disease or illness or an undesirable physiological condition. As used herein, the term "prevention" refers to any activity that reduces the burden on the individual that subsequently exhibits these symptoms. This can occur at the primary, secondary, and / or tertiary prevention levels, where: a) primary prevention avoids the progression of a symptom / disorder / condition; b) secondary prevention activities target the early stages of treatment of a symptom / disorder / symptom, thereby increasing the chances of intervention to prevent the progression of the symptom / disorder / symptom and the onset of symptoms; and c) tertiary prevention reduces the negative impact of an established symptom / disorder / symptom by, for example, restoring function and / or reducing any symptom / disorder / symptom or associated complications. The term "prevention" does not require 100% elimination of the likelihood of an event. Rather, it indicates that the likelihood of an event occurring has been reduced in the presence of a compound or method.

[0060] As used herein, the term "anti-tumor effect" shall be given its ordinary meaning and shall also refer to the inhibition of the formation, growth and / or viability of tumors and / or tumor cells (in vivo and / or in vitro), and includes 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 a number or range between any two of these values) in one or more of tumor cell death, tumor volume, tumor number, tumor viability, metastasis, and survival.

[0061] As used herein, the term "oxidative stress" shall be given its ordinary meaning and shall also 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.

[0062] As used herein, the term "effective amount" refers to an amount sufficient to achieve beneficial or desired biological and / or clinical results.

[0063] As used herein, the term "contact" should be given its ordinary meaning, and should also refer to placing two or more entities so close that they actually come into physical contact with each other, for example, by combining two or more entities (e.g., separated organelle complexes and T cells) to come into physical contact. Contact can include co-incubation. Contact 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 in physical contact. Contacting the separated organelle complex with a T cell colony can include contacting the organelle complex colony with a T cell colony. Contacting the separated organelle complex with a T cell colony can produce a T cell colony comprising an exogenous organelle complex. When the organelle complex is contacted with the T cell colony, the organelle complex provided herein can be incorporated into the T cell. In some embodiments, incorporating (e.g., transplanting) the separated organelle complex into the T cell includes co-localizing and / or fusing endogenous mitochondria into the T cell. The T cell can be in vivo, in vitro or ex vivo. In some embodiments, the organelle complex that has been incorporated (e.g., transplanted) into a T cell can be detected (e.g., distinguished from the endogenous organelles of the T cell) within 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 organelle complex transplantation provided herein can last until the organelle complex is no longer detected in the T cell colony. The T cell colony comprising the organelle complex provided herein includes a T cell colony that has undergone one or more cell divisions and / or continuous culture after initially contacting the organelle complex separated. In some embodiments, the offspring of the T cell comprising the organelle complex also comprises the organelle complex (e.g., via the random distribution of the organelle complex during division). As used herein, the term "exogenous" should be given its ordinary meaning, and should also refer to cellular material (e.g., organelle complex) that has been removed from one cell and incorporated into another cell.

[0064] The term "autologous" shall be given its ordinary meaning and shall also refer to any material derived from the same individual that is subsequently reintroduced into that individual.

[0065] The term "allogeneic" shall be given its ordinary meaning and shall also refer 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 each other when the genes at one or more loci differ. In some aspects, allogeneic material from individuals of the same species may be sufficiently genetically different to produce antigenic interactions.

[0066] In some embodiments, the methods, compositions, systems and kits provided herein can be used in conjunction 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 entirety).

[0067] In some embodiments, a method for producing a T cell population for adoptive T cell therapy is provided. In some embodiments, the method comprises contacting an isolated organelle complex with a T cell population to produce a T cell population comprising the organelle complexes, wherein the organelle complexes comprise mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus. The method may include stimulating the T cell population. The stimulation step may amplify the T cell population.

[0068] In some embodiments, a method for enhancing the proliferation, migration, persistence, and / or activity of a T cell population for adoptive T cell therapy is provided. In some embodiments, the method comprises contacting an isolated organelle complex with a T cell population to produce a T cell population comprising the organelle complexes, wherein the organelle complexes comprise mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus. The method may comprise stimulating the T cell population to expand the T cell population.

[0069] In some embodiments, a method for generating a T cell population that is resistant to exhaustion is provided. In some embodiments, the method comprises: contacting isolated organelle complexes with a T cell population to generate a T cell population comprising the organelle complexes, wherein the organelle complexes comprise mitochondria and one or more of an endoplasmic reticulum, a peroxisome, a lysosome, and a Golgi apparatus. The method may comprise: stimulating the T cell population to expand the T cell population.

[0070] In certain embodiments, T cells are obtained from a donor subject. In some embodiments, the donor subject is a human patient suffering from a cancer or tumor. In other embodiments, the donor subject is a human patient not suffering from a cancer or tumor.

[0071] The T cells provided herein can be obtained from any source known in the art. For example, T cells can be differentiated from hematopoietic stem cell colonies in vitro, or T cells can be obtained from a subject. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In addition, T cells can be derived from one or more T cell lines available in the art. Various techniques known to those skilled in the art (such as FICOLL) can also be used. TM In some embodiments, the cells are washed with PBS. As will be appreciated, a washing step may be used, such as by using a semi-automatic flow-through centrifuge, such as a Cobe TM 2991 Cell Processor, Baxter CytoMate TM Etc. In some embodiments, the washed cells are resuspended in one or more biocompatible buffers, or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample are removed. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0072] In certain embodiments, the monocytes are depleted by lysing the red blood cells and depleting the monocytes (e.g., by using a PERCOLL TM Gradient centrifugation) to separate T cells from PBMCs. In some embodiments, specific T cell subsets (such as CD4 + 、CD8 + 、CD28 + 、CD45RA + and CD45RO + T cells). For example, enrichment of T cell populations by negative selection can be achieved using 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 using a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells can be used. For example, to enrich CD4 + Cells, monoclonal antibody cocktails typically include 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.

[0073] In some embodiments, T cells are genetically modified after being separated using known methods, or T cells are activated and amplified (or differentiated in the case of progenitor cells) in vitro before being genetically modified. In another embodiment, T cells are genetically modified with chimeric antigen receptors as described herein (for example, transduced with a viral vector comprising one or more nucleotide sequences encoding CAR), and then activated and / or amplified in vitro. Methods for activating and amplifying T cells are known in the art and are described in, for example, U.S. Patent No. 6,905,874; 6,867,041; and 6,797,514; and PCT application No. WO 2012 / 079000, the contents of which are hereby incorporated by reference in their entirety. In general, such methods include contacting PBMC or separated T cells with stimulators and costimulators (such as anti-CD3 antibodies and anti-CD28 antibodies) that are typically attached to beads or other surfaces in a culture medium with appropriate cytokines (such as IL-2). Anti-CD3 antibodies and anti-CD28 antibodies attached to the same beads act as "alternative" antigen presenting cells (APCs). An example is System, which is a CD3 / CD25 activator / stimulator system for physiological activation of human "T" cells. In other embodiments, T cells are activated and T cell proliferation is stimulated 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 Application No. WO 2012 / 129514 (the contents of which are hereby incorporated by reference in their entirety).

[0074] Stimulation can be carried out before contact, after contact and / or during contact.The term "stimulation" should give its common meaning, and should also refer to such a primary response, which is combined with its cognate ligand (or tumor antigen in the case of CAR) by a stimulating molecule (e.g., TCR / CD3 complex or CAR) to thereby mediate a signal transduction event (such as, but not limited to, signal transduction via TCR / CD3 complex or signal transduction via the signal transduction domain of CAR) induction. Stimulation can mediate the expression of changes in certain molecules. Stimulation can include culturing T cell colonies in the presence of one or more stimulating agents. One or more stimulating agents can include agents that stimulate CD3 / TCR complex-related signals and agents that stimulate costimulatory molecules on the T cell surface. One or more stimulating agents can include: molecules (e.g., one or more of anti-CD28 antibodies, CD80, and CD86) in conjunction with CD28; and / or molecules (e.g., anti-CD3 antibodies) in conjunction with CD3. The stimulation and / or contacting can be 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, 7 days, or a number or range between any two of these values.

[0075] In some embodiments, the T cell recovery efficiency can be at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or a number or range between any two of these values) compared to a method that does not include contacting the isolated organelle complex with the T cell population. The T cell recovery efficiency is the ratio of the number of T cells recovered after stimulation (e.g., after about 48 hours of stimulation) to the number of T cells immediately before the start of stimulation. In some embodiments, the T cell population exhibits one or more of the following compared to a T cell population that does not include the exogenous organelle complex: 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). In some embodiments, the T cell population exhibits an increase in basal and / or maximal oxygen consumption rate (in vivo and / or in vitro) 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 a number or range between any two of these values) compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the T cell population exhibits an increase in basal glycolytic rate and / or maximum glycolytic rate (in vivo and / or in vitro) of at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or a number or range between any two of these values) compared to a T cell population that does not comprise the exogenous organelle complex. In some embodiments, the T cell population 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 a number or range between any two of these values) increase in glycolytic capacity and / or respiratory capacity (in vivo and / or in vitro) compared to a T cell population that does not comprise the exogenous organelle complex. In some embodiments, the T cell population exhibits at least about a 1.1-fold increase (e.g., a 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 a number or range between any two of these values) in mitochondrial mass (in vivo and / or in vitro) compared to a T cell population that does not comprise the exogenous organelle complex.In some embodiments, the T cell population exhibits at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or a number or range between any two of these values) of cytotoxic activity (in vivo and / or in vitro) against target cells compared to a T cell population that does not comprise an exogenous organelle complex. The exhaustion marker can be selected from the group comprising PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD 160, CD39, VISTA, TIGIT, or any combination thereof. In some embodiments, the T cell population exhibits at least about a 1.1-fold (e.g., a 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 a number or range between any two of these values) reduction in the level 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), as compared to a T cell population that does not comprise the exogenous organelle complex. Reactive oxygen species can include superoxide (O2). - ), hydroperoxide (HO.2), hydrogen peroxide (H2O2), peroxynitrite (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO.), peroxyl radical (ROO.), alkoxyl radical (RO.), singlet oxygen ( 1O 2), lipid peroxides, lipid peroxyl radicals or lipid alkoxyl radicals or any combination thereof. In some embodiments, the T cell population exhibits an increase in the production (in vivo and / or in vitro) of one or more of the cytokines compared to a T cell population that does not comprise the exogenous organelle complex 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 a number or range between any two of these values). Cytokines can include interleukin-2 (IL-2), interferon-γ (IFNγ), interleukin-4 (IL-4), TNF-α (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF), CD107a and / or TGF-β (TGFβ). In some embodiments, the T cell population exhibits an increase in cell proliferation (in vivo and / or in vitro) of at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or a number or range between any two of these values) compared to a T cell population that does not comprise an exogenous organelle complex. In some embodiments, the T cell population exhibits an increase in cell viability (in vivo and / or in vitro) after chronic TCR stimulation compared to a T cell population that does not comprise an exogenous organelle complex. For example, 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 a number or range between any two of these values. In some embodiments, the introduction of the organelle complex metabolically reprograms the CAR-T cells via redox regulation. In some embodiments, the T cell population 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 a number or range between any two of these values) decrease and / or increase (in vivo and / or in vitro) in one or more of: NAD + / NADH, ATP level, sirtuin (Sirt1,3) level, TP-53 level, NF-κB level, Bax level, memory T cell (Tcm, Tscm) percentage, telomere activity and SA-βGal activity.

[0076] "Exhaustion" or "unresponsiveness" refers to a cellular state in which a cell does not perform its usual function or activity in response to normal input signals, and includes the refractoriness of an immune cell to stimulation (such as stimulation via an activating receptor or cytokine). Such functions or activities include, but are not limited to, proliferation or cell division, entry into the cell cycle, cytokine production, cytotoxicity, trafficking, phagocytic activity, or any combination thereof. Normal input signals may include, but are not limited to, stimulation via receptors (e.g., T cell receptors, B cell receptors, co-stimulatory receptors, etc.).

[0077] The cytotoxic activity, cytokine production, proliferation, trafficking, phagocytic activity, or any combination thereof of the depleted immune cells can be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more relative to corresponding control immune cells of the same type. In one embodiment, the depleted cells are CD8 + T cells (e.g., antigen-specific effector CD8 + T cells). CD8 cells typically proliferate (e.g., clonally expand) in response to T cell receptor and / or co-stimulatory receptor stimulation and in response to cytokines (such as IL-2). Therefore, exhausted CD8 T cells are cells that do not proliferate and / or produce cytokines in response to normal input signals. It is well known that the exhaustion of effector function can be described according to several stages, which 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 the "partial exhaustion I" stage, which is characterized by the loss of partial effector function, including the loss of IL-2 production, reduced TNFa production, and reduced proliferation capacity and / or ex vivo lysis capacity. In the second stage, when both IL-2 and TNFa production stop and IFNy production decreases after antigen stimulation, partially exhausted T cells enter the "partial exhaustion II" stage. When CD8 + "Complete exhaustion" or "terminal exhaustion" occurs when T cells lose all effector functions, including the lack of production of IL-2, TNFa, and IFNy upon antigen stimulation, as well as loss of ex vivo lytic capacity and proliferative potential. +T cells are cells that respond to normal input signals and do not proliferate, do not lyse target cells (cytotoxicity) and / or do not produce appropriate cytokines (such as IL-2, TNFa or IFNy). When antigen load is high and / or CD4 is low, the lack of this effector function may occur. This graded loss of function is also related to the expression of co-inhibitor immune receptors (such as PD-1, 'HM-3, LAG-3, etc.) (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 the hierarchical stages of immune cell exhaustion, such as high eomesodermin (EOMES) and low TBET expression as markers of terminally exhausted T cells (Paley et al. (2012) Science 338:1220-1225). Additional markers of exhausted T cells, such as a decrease in Bcl-b and increased production of BLIMP-1 (Pdrml). T cell exhaustion can include the expression of one or more T cell exhaustion biomarkers selected from the group consisting of checkpoint inhibitors, PD-1 (Pdcdl), 'IM-3 (Havcr2), LAG-3 (Lag3), CTLA-4 (Ctla4), 2B4 (CD244), CD39 (Entpdl), CD 160, 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 T cell population 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 a number or range between any two of these values) reduction in the level of one or more exhaustion markers (in vivo and / or in vitro) compared to a T cell population that does not comprise the exogenous organelle complex.The in vivo persistence of a T cell population can include about 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, days, 19 days, 20 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.

[0078] In some embodiments, a T cell population produced by the methods disclosed herein is provided. In some embodiments, a T cell population for adoptive T cell therapy is provided, wherein the T cell comprises an exogenous organelle complex. The organelle complex may comprise mitochondria and one or more of the endoplasmic reticulum, peroxisomes, lysosomes, and Golgi bodies. In some embodiments, these T cell populations exhibit one or more of the following compared to T cell populations that do not comprise an exogenous organelle complex: enhanced amplification capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

[0079] In some embodiments, a method for treating or preventing a disease or condition in a subject is provided. In some embodiments, the method comprises administering to the subject an effective amount of a T cell population generated according to the methods disclosed herein, thereby treating or preventing the disease or condition in the subject. In some embodiments, the T cell population exhibits one or more of the following compared to a T cell population that does not contain an exogenous organelle complex: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

[0080] In some embodiments, a method for enhancing adoptive T cell therapy in a subject is provided. In some embodiments, the method comprises: generating an effective amount of T cells comprising an exogenous organelle complex according to the methods disclosed herein; and adoptively transferring the T cells to the subject. In some embodiments, the T cell population exhibits one or more of the following compared to a T cell population that does not comprise an exogenous organelle complex: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

[0081] In some embodiments, a method for treating or preventing a disease or illness of a subject is provided. In some embodiments, the method includes: administering an effective amount of a T cell colony (e.g., a T cell comprising a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR)) to the subject; and administering an effective amount of an isolated organelle complex to the subject, thereby treating or preventing the subject's disease or illness. The isolated organelle complex can contact a T cell colony in vivo to produce a T cell colony comprising an organelle complex. In some embodiments, these T cell colonies, compared to T cell colonies that do not comprise exogenous organelle complexes, exhibit one or more of the following: enhanced amplification capacity, enhanced cytotoxicity for target cells, enhanced anti-exhaustion and enhanced persistence. Before administering an effective amount of an isolated organelle complex to the subject, an effective amount of a T cell colony can be administered to the subject. Before administering an effective amount of an isolated organelle complex to the subject, an effective amount of an isolated organelle complex can be administered to the subject. An effective amount of a T cell colony and an effective amount of an isolated organelle complex can be administered to the subject simultaneously.

[0082] Disclosed herein include methods for treating or preventing a disease or illness of a subject. In some embodiments, the method includes: administering to the subject an effective amount of heterologous nucleic acid (e.g., vector, 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 subject's disease or illness. Heterologous nucleic acid and isolated organelle complex can contact the subject's T cells in vivo to produce T cells comprising organelle complexes and CAR and / or engineered TCR. In some embodiments, the T cells exhibit one or more of the following compared to T cells that do not comprise exogenous organelle complexes: enhanced amplification capacity, enhanced cytotoxicity for target cells, enhanced anti-exhaustion and enhanced persistence.

[0083] In certain embodiments, T cell colonies reduce the tumor volume, tumor growth and / or tumor load of a subject. In certain embodiments, T cell colonies reduce the tumor volume of a subject by at least about 1.1 times (e.g., 1.1 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times or any two of these values) compared to the tumor volume of an untreated subject or a subject using a T cell colony not comprising an exogenous organelle complex. In certain embodiments, the T cell colony increases total survival or progression-free survival. In some embodiments, the T cell population 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 a number or range between any two of these values) compared to an untreated subject or a subject administered a T cell population that does not comprise an exogenous organelle complex.

[0084] 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 can include administering (e.g., intravenously) at least about 1×10 6 The method may include repeated administration of the T cell population. The subject may be a mammal.

[0085] The disease or condition may be associated with the expression of a tumor antigen. The disease associated with the expression of a tumor antigen may be selected from the group consisting of proliferative diseases, precancerous conditions, cancers and non-cancer related indications associated with the expression of a tumor antigen.

[0086] The cancer may be selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell lung cancer, mesothelioma, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, 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. cancer of the kidney or ureter, renal pelvis, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of these cancers, and metastatic lesions of these cancers.

[0087] The cancer can be a blood cancer selected from one or more of the following: 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 disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or preleukemia.

[0088] Organelle complex

[0089] In some embodiments, when the isolated organelle complex is contacted with the T cell population, the organelle complex can be incorporated into the T cell. The contacting step can be repeated at least 2, 3, or 4 times. The contacting can be performed during one or both of the stimulation step and the introduction step. The T cell population can be contacted with an effective amount of the isolated organelle complex sufficient to enhance the proliferation, migration, persistence, and / or activity of the T cells. The effective amount can include about 20 μg of the isolated organelle complex. An effective amount of an isolated organelle complex can be, can be about, can be at least, or can be at most 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, 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、200 0, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000 or a number or range between any two of these values ​​ug, mg, ug / mL and / or mg / mL. The T cell population can be contacted with an effective amount of the isolated organelle complex sufficient to resist exhaustion of the T cells. The T cell population can be derived from lymph node cells (e.g., by purification based on a magnetic particle enrichment procedure selected from the group consisting of: manual and The T cell population may comprise one or more of the following: CD4 + cells, CD8 + T cells, cytotoxic T cells, terminal 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).

[0090] The organelle complex provided herein can comprise the first organelle complex, the second organelle complex or the combination of the first organelle complex and the second organelle complex. The organelle complex separated can be an organelle complex colony. The organelle complex colony can comprise the first organelle complex or the combination of the first organelle complex and the second organelle complex. The first organelle complex and / or the second organelle complex can be depleted of cytoplasm macromolecules. The cytoplasm macromolecules can comprise cytoplasmic proteins (for example, cytoplasmic proteins are p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). Compared with the cells derived from the organelle complex colony, the abundance of one or more cytoplasmic proteins can be depleted by at least about 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or the numeral or scope between any two of these values). The organelle complex may comprise: 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., lysosomal associated membrane protein 2 (LAMP2), mannose-6-phosphate receptor (M6PR) and / or lysosomal associated membrane protein 1 (LAMP1)); one or more peroxisomes Protein (e.g., catalase and / or ATP-binding cassette transporter 1, subfamily D, type 3 (ABCD3)); one or more Golgi proteins (e.g., Golgi protein-97, syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130) and / or mannosidase alpha 2A class member 1 (MAN2A1)); and / or one or more endoplasmic reticulum proteins (e.g., calreticulin and / or calnexin). The organelle complex can be derived from cells of a subject different from the subject from which the T cell is derived. The organelle complex can be derived from cells of the same subject from which the T cell is derived. The organelle complex provided herein (e.g., a first organelle complex, a second organelle complex) can comprise one, two, three or four of mitochondria and the endoplasmic reticulum, peroxisomes, lysosomes and the Golgi apparatus.The organelle complex (e.g., a first organelle complex, a second organelle complex) may comprise: (i) mitochondria and endoplasmic reticulum; (ii) mitochondria and peroxisomes; (iii) mitochondria and lysosomes; (iv) mitochondria and Golgi apparatus; (v) mitochondria, endoplasmic reticulum, and peroxisomes; (vi) mitochondria, endoplasmic reticulum, and lysosomes; (vii) mitochondria, endoplasmic reticulum, and Golgi apparatus; (viii) mitochondria, endoplasmic reticulum, peroxisomes, and lysosomes The ratio of mitochondria to other organelles (e.g., endoplasmic reticulum, peroxisomes, lysosomes, and Golgi apparatus) may vary within a population of organelle complexes.

[0091] Disclosed herein are methods for producing a first population of organelle complexes. In some embodiments, the method comprises: incubating cells in a first solution comprising a surfactant at a first temperature; removing the surfactant to form a second solution; and recovering the first organelle complexes from the second solution. The first organelle complexes can be derived from: (i) frozen cells; (ii) floating cells; and / or (iii) cells contacted with a surfactant at a concentration equal to or greater than the critical micelle concentration (CMC) of the surfactant. Generating the first organelle complexes can comprise: (Step A) providing adherent, floating, and / or frozen cells, thawing, and placing them in a tube. Generating the first organelle complexes can comprise: (Step A) providing adherent, floating, and / or frozen cells, centrifuging, and collecting the precipitate. Generating the first organelle complexes can comprise: (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. Generation 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 solution containing a surfactant and incubating; (Step E) centrifuging and collecting the precipitate; (Step F) adding Tris buffer, centrifuging, and collecting the precipitate; (Step G) adding Tris buffer and pipetting; (Step H) transferring to another tube and collecting the buffer solution in the original tube and rinsing; (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 collecting the supernatant and / or precipitate. One or more of the above steps can be omitted and one or more additional steps can be included. The time, volume, concentration, and centrifugal force can vary depending on the embodiment. Incubating the cells in the first solution and / or incubating the second solution can include applying a physical stimulus to the first solution and / or the second solution, respectively, such as, for example, pipetting, shaking, and / or stirring. Applying the physical stimulus to the first solution and / or the second solution may include flowing the first solution and / or the second solution through a flow device (e.g., a flow reduction device). The flow device may include a fluid channel comprising two or more segments having different cross-sectional diameters.In some embodiments, the present invention provides a flow reducing device.Described cross-sectional diameter can be, can be about, can be at least or can be 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 2mm, 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, 18mm, 19mm, 20mm, 21mm, 22mm, 23mm, 24mm, 25mm or numeral or scope between any two of these values.Flow reducing device provided herein can have various configurations, for example, as square reducer, tapered reducer, concentric reducer and / or eccentric reducer.Flow device can comprise the pipe of various types and sizes, to produce other flow and shearing force, for the first organelle complex to extract.In certain embodiments, described flowing through flow device produces other flow and / or shearing. Recovering these first organelle complexes from the second solution can include tangential flow filtration (TFF). In some embodiments, TFF can be used to purify and / or buffer exchange. Recovering these first organelle complexes from the second solution can include tangential flow filtration (TFF). TFF can be performed with a low viscosity buffer, and in some embodiments, the low viscosity buffer reduces the shear rate.The viscosity of the TFF buffer may be, may be about, may be at least, or may be at most 1 centipoise (cP), 2cP, 3cP, 4cP, 5cP, 6cP, 7cP, 8cP, 9cP, 10cP, 11cP, 12cP, 13cP, 14cP, 15cP, 16cP, 17cP, 18cP, 19cP, 20cP, 21cP, 22cP, 23cP, 24cP, 25cP, 26cP, 27cP, cP, 28cP, 29cP, 30cP, 31cP, 32cP, 33cP, 34cP, 35cP, 36cP, 37cP, 38cP, 39cP, 40cP, 41cP, 42cP, 43cP, 44cP, 45cP, 46cP, 47cP, 48cP, 49cP, 50cP, 51cP, 52cP, 53cP, 54cP, 55cP, 56cP, 57cP, 58c P, 59cP, 60cP, 61cP, 62cP, 63cP, 64cP, 65cP, 66cP, 67cP, 68cP, 69cP, 70cP, 71cP, 72cP, 73cP, 7 4cP, 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, 300cP, 400cP, 500cP, 600cP, 700cP, 800cP, 900cP, 1000cP, 2500cP, 5000cP, 7500cP, 10000cP or a number or range between any two of these values. The temperature at which TFF is performed can be, can be about, can be at least, or can be 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 of the TFF procedure and / or flow device (eg, flow reduction device) may be, may be about, may be at least, or may be at most 1 sec. -1 , 2sec -1 , 3sec -1 , 4sec -1 5 sec -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 、30sec -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 -1or a number or range between any two of these values. TFF can be performed using a buffer comprising human albumin (HA). Recovering the first organelle complex from the second solution can include performing TFF using a TFF membrane. The TFF membrane has a molecular weight cutoff of, can be about, can be at least, or can be at least 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18 kDa, 19 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 128 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, kDa, 200kDa, 210kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa, 330kDa, 340kDa, 350kDa , 360kDa, 370kDa, 380kDa, 390kDa, 400kDa, 410kDa, 420kDa, 430kDa, 440kDa, 450kDa, 460kDa, 470kDa, 480kDa, 490kDa, 500kDa, 510kDa, 520 kDa, 530kDa, 540kDa, 550kDa, 560kDa, 570kDa, 580kDa, 590kDa, 600kDa, 610kDa, 620kDa, 630kDa, 640kDa, 650kDa, 660kDa, 670kDa, 680kDa , 690kDa, 700kDa, 710kDa, 720kDa, 730kDa, 740kDa, 750kDa, 760kDa, 770kDa, 780kDa, 790kDa, 800kDa, 810kDa, 820kDa, 830kDa, 840kDa, 85 0kDa, 860kDa, 870kDa, 880kDa, 890kDa, 900kDa, 910kDa, 920kDa, 930kDa, 940kDa, 950kDa, 960kDa, 970kDa, 980kDa, 990kDa, 1000kDa, 1100 kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa, 2100kDa, 2200kDa, 2300kDa, 2400kDa, 2500kDa,000 kDa, 2600 kDa, 2700 kDa, 2800 kDa, 2900 kDa, 3000 kDa, 3250 kDa, 3500 kDa, 3750 kDa, 4000 kDa, 4250 kDa, 4500 kDa, 4750 kDa, 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. Methods for obtaining organelle complexes from cells and organelle complexes obtained by such methods are disclosed in PCT patent application number PCT / US23 / 27014, filed on July 6, 2023, entitled “ORGANELLE COMPLEXES,” the contents of which are incorporated herein by reference in their entirety. In some embodiments, a second organelle complex is provided. In some embodiments, the method for separating the second organelle complex from the cell includes treating the cells in the first solution with a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant, removing the surfactant 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 a surfactant at a concentration lower than the critical micelle concentration (CMC) of the surfactant. In some embodiments, Q mitochondria are provided. The second organelle complex can include or can be Q mitochondria. The method for obtaining Q mitochondria from cells and the Q mitochondria obtained by this method are disclosed in PCT Patent Application Publication No. WO / 2021 / 015298, the contents of which are incorporated herein by reference in their entirety. The organelle complex population can be derived from cells treated with a mitochondrial activator (e.g., resveratrol). The organelle complex can be depleted of cytoplasmic macromolecules. Cytoplasmic macromolecules may not be present in the organelle complex population provided herein. The organelle complex (e.g., the first organelle complex, the second organelle complex) population provided herein may contain negligible and / or undetectable amounts of cytoplasmic macromolecules. Cytoplasmic macromolecules may include cytoplasmic proteins (e.g., p70S6K and / or glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). The first organelle complex and the second organelle complex may be derived from cells treated with a mitochondrial activator. Homogenized mitochondria, the first organelle complex, and / or the second organelle complex may be encapsulated in lipid membrane-based vesicles. The method of encapsulation in lipid membrane-based vesicles is disclosed in PCT Patent Application Publication No. WO2021 / 132735, the contents of which are incorporated herein by reference in their entirety.

[0092] Chimeric antigen receptors and engineered T cell receptors

[0093] The T cells provided herein may include a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, CAR includes a T cell receptor (TCR) antigen binding domain. The method may include introducing a heterologous nucleic acid (e.g., a vector) encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR) into a T cell. The introduction step may be performed before contact, after contact, and / or during contact. The term "chimeric antigen receptor" or "CAR" refers to a group of polypeptides, typically two in the simplest embodiment, which, when in immune effector cells, provide cells with specificity for target cells (typically cancer cells) and the generation of intracellular signals. The terms "CAR" and "CAR molecule" are used interchangeably. In some embodiments, CAR includes at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some embodiments, the group of polypeptides is located in the same polypeptide chain (e.g., constituting a chimeric fusion protein). In some embodiments, the group of polypeptides is continuous with each other. In some embodiments, the group of polypeptides is discontinuous with each other, for example, located in different polypeptide chains. In some embodiments, the group of polypeptides includes a dimerization switch, which can couple polypeptides to each other when a dimerization molecule is present, for example, the antigen binding domain can be coupled to the intracellular signaling domain. On the one hand, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. On the one hand, the cytoplasmic signaling domain further includes 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, for example, 4-1BB (i.e., CD137), CD27 and / or CD28. In some embodiments, CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.In some embodiments, CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, CAR comprises an optional leader sequence located at the amino terminus (N terminus) of the CAR fusion protein. In some embodiments, CAR further comprises a leader sequence at the N terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cut from the antigen binding domain (e.g., scFv) during cell processing and localization of CAR by the cell.

[0094] CAR and / or TCR may include one or more of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. CAR or TCR may further include a leader peptide. TCR may further include a constant region and / or CDR4. The term "signaling domain" refers to a functional portion of a protein that plays a role by transmitting information within the cell, to regulate cell activity by generating a second messenger via a determined signaling pathway or by acting as an effector in response to such a messenger. As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain produces a signal that promotes the immune effector function of cells (e.g., CAR-T cells) containing CAR. Examples of immune effector functions (e.g., in CAR-T cells) include cytolytic activity and auxiliary activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent simulation. In an embodiment, the intracellular signaling domain may include a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may include the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may include the cytoplasmic sequence from a co-receptor or a costimulatory molecule. The primary intracellular signaling domain may include a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from: CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0095] The intracellular signaling domain may comprise 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 an intracellular signaling domain. The intracellular signaling domain is generally responsible for activating the normal effector function of at least one immune cell introduced into the CAR. The term "effector function" refers to a special function of a cell. For example, the effector function of a T cell may be a cytolytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and instructs cells to perform specialized functions. Although the entire intracellular signaling domain can generally be used, in many cases it is not necessary to use the entire chain. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used to replace the complete chain as long as it transduces the effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal.

[0096] The term "costimulatory molecules" refers to T cells that specifically bind to costimulatory ligands, thereby mediating the cognate binding partners of the costimulatory responses (such as but not limited to proliferation) of T cells.Costimulatory molecules are cell surface molecules that contribute to effective immune responses in addition to antigen receptors or their ligands.Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1BB (CD137). Other examples of such costimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, 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, CD18, I TGB7, 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 a ligand that specifically binds to CD83. The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can represent 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 derived therefrom, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0097] Examples of intracellular signal transduction domains for CARs provided herein include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors (which work together to start signal transduction after antigen receptor engagement), and any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities. It is known that the signal generated only by TCR is not enough to fully activate T cells, and secondary and / or costimulatory signals are also required. Therefore, T cell activation can be considered to be mediated by two different types of cytoplasmic signal transduction sequences: one class starts antigen-dependent primary activation by TCR (primary intracellular signal transduction domain), and the other class plays a role in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic domains, such as costimulatory domains). The primary signal transduction domain regulates the primary activation of the TCR complex in a stimulating manner or in an inhibitory manner. The primary intracellular signal transduction domain that works in a stimulating manner may contain a signal transduction motif, which is referred to as an activation motif or ITAM based on immunoreceptor tyrosine. The primary signaling domain comprises a functional signaling domain of one or more proteins selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10 and DAP12, or functional variants thereof.

[0098] In some embodiments, the intracellular signaling domain is designed to include two or more (e.g., 2, 3, 4, 5 or more) costimulatory signaling domains. In an embodiment, two or more (e.g., 2, 3, 4, 5 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 comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue. The co-stimulatory domain can comprise a functional domain of one or more proteins selected from the group consisting of 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, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, 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, 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 their functional variants.

[0099] The CAR portion comprising an antibody or its antibody fragment can exist in various forms, wherein the antigen binding domain is expressed as a part of a continuous polypeptide chain, including, for example, single domain antibody fragments (sdAb), single-chain antibodies (scFv), 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 domains of CAR provided herein include antibody fragments. In a further aspect, CAR includes antibody fragments containing scFv.

[0100] In some embodiments, the CARs provided herein comprise a target-specific binding element, also referred to as an antigen binding domain. The choice of part depends on the type and number of ligands that define the surface of the target cell.

[0101] In some embodiments, the T cell reaction of CAR mediation can be directed to the antigen of interest by engineering the antigen binding domains specifically binding to the desired antigen into CAR. In some embodiments, the CAR portion comprising an antigen binding domain includes the antigen binding domains for targeting tumor antigens (e.g., tumor antigens as described herein).Antigen binding domains can be any domain combined with 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 camel-derived antibodies Heavy chain variable domains (VH), light chain variable domains (VL) and variable domains (VHH), and alternative scaffolds serving as antigen binding domains known in the art, such as recombinant fibronectin domains, T cell receptors (TCR) or its fragments, such as single-chain TCRs, etc. In some cases, it is beneficial for the antigen binding domains to be derived from CAR for use in the same species thereof. For example, for use in humans, it may be beneficial for the antigen binding domains of CAR to include human residues or humanized residues of the antigen binding domains of antibodies or antibody fragments. In some embodiments, antigen binding domains include humanized antibodies or antibody fragments. In some aspects, non-human antibodies are humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to naturally occurring antibodies or fragments thereof in humans.In some embodiments, the antigen binding domain is humanized.

[0102] The antigen binding domain may comprise an antibody, an antibody fragment, a scFv, a Fv, a Fab, (Fab')2, a single domain antibody (SDAB), a VH or VL domain, a Camelid VHH domain, a Fab, Fab', F(ab')2, a Fv, a scFv, a dsFv, a diabody, a triabody, a tetrabody, a multispecific antibody formed from antibody fragments, a single domain antibody (sdAb), a single chain comprising an anti-complement scFv (tandem scFv) or a bispecific tandem scFv, an Fv construct, a disulfide-linked Fv, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody, an aptamer, an affibody, an affilin, an affitin, an affimer, an alphabody, anticalin, avimer, DARPin, Fynomer, a Kunitz domain peptide, a monomer or any combination thereof.

[0103] 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 manufacturing 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 references are incorporated herein in their entirety). For example, scTCRs containing Vα and Vβ genes from T cell clones connected by a linker (e.g., a flexible peptide) can be engineered. This approach is very useful for cancer-associated targets that are themselves located within the cell, however, fragments of this antigen (peptide) are presented on the surface of cancer cells by MHC.

[0104] In some embodiments, the antigen binding domain is a multispecific antibody molecule. In some embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. The bispecific antibody molecule is characterized in that a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In an embodiment, the first epitope and the second epitope are located on the same antigen, such as on the same protein (or subunit of a multimeric protein). In an embodiment, the first epitope and the second epitope overlap. In an embodiment, the first epitope and the second epitope do not overlap. In an embodiment, the first epitope and the second epitope are located on different antigens, such as on different proteins (or different subunits of a multimeric protein). In an embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for the first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for the second epitope. In an embodiment, the bispecific antibody molecule comprises a half antibody that has binding specificity for the first epitope and a half antibody that has binding specificity for the second epitope. In an embodiment, the bispecific antibody molecule comprises a half antibody or fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In an embodiment, the bispecific antibody molecule comprises a scFv or fragment thereof having binding specificity for a first epitope and a scFv or fragment thereof having binding specificity for a second epitope.

[0105] Antigen binding domains can be configured to be combined with tumor antigens. The term "cancer associated antigen" or "tumor antigen" refers interchangeably to a molecule (typically a protein, carbohydrate, or lipid) that is expressed completely or as a fragment (e.g., MHC / peptide) on the surface of a cancer cell and is preferentially targeted to cancer cells for pharmacological agents. In some embodiments, tumor antigens are markers expressed by both normal cells and cancer cells, such as lineage markers, such as CD19 on B cells. In some embodiments, tumor antigens are cell surface molecules that are overexpressed (e.g., 1 times overexpressed, 2 times overexpressed, 3 times overexpressed, or more) in cancer cells compared to normal cells. In some embodiments, tumor antigens are cell surface molecules that are inappropriately synthesized in cancer cells, such as molecules that contain deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, tumor antigens are expressed exclusively on the cell surface of cancer cells, either completely or as a fragment (e.g., MHC / peptide), and are not synthesized or expressed on the surface of normal cells. In some embodiments, CAR provided herein includes a CAR comprising an antigen binding domain (e.g., an antibody or antibody fragment) that is combined with an MHC presenting peptide. Typically, peptides derived from endogenous proteins fill the pocket of major histocompatibility complex (MHC) class I molecules and are taken up by CD8+ 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, 2011117(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 201219(2):84-100). For example, TCR-like antibodies can be identified by screening libraries, such as human scFv phage display libraries.

[0106] The tumor antigen may be a solid tumor antigen. The tumor antigen may be selected from the group consisting of: 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 member B cell maturation protein (BCMA); Tn antigen ((Tn Ag) 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 CD213A) 2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); proteinase serine 21 (Testisin 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 kinase ERBB2 (Her2 / neu); mucin 1, cell Mucin-associated cell surface protein (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic enzyme; prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100) Oncogene fusion protein composed of the breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-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); the hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); urokinase 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pan-nexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (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 variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); 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; prostate-specific protein; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis protein (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma 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 (BORIS or brother of imprinted site regulator), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); pre-acrosomal protein-binding protein sp32 (OY-TES1);Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitin 1 (RU1); renal ubiquitin 2 (RU2); legumin; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of the 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 molecule-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).

[0107] The tumor antigen may be selected from the group consisting of 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, CD56, 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-11Rα, IL-13R-α2, IL-2, IL-22R-α, 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 factors, exogenous cognate binding molecules (ExoCBM), oncogene products, antifolate receptor, c-Met, carcinoembryonic antigen (CEA), cyclin (D1) , ephrinB2, epithelial tumor antigen, estrogen receptor, fetal acetylcholine receptor, folate binding protein, gp100, hepatitis B virus surface antigen, kappa chain, kappa light chain, kdr, lambda chain, inhibitor of apoptosis protein (livin), melanoma-associated antigen, mesothelin, mouse double minute 2 homolog (MDM2), mucin 16 (MUC16), mutant p53, mutant ras, cell necrosis antigen, carcinoembryonic antigen, ROR2, progesterone receptor, prostate-specific antigen, tegmented epidermal growth factor receptor (tEGFR), tenascin, beta2-microglobulin, Fc receptor-like 5 (FcRL5), or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0108] Antigen binding domain is connected to membrane spaning domain by hinge region.In some cases, membrane spaning domain can be connected via hinge (for example, hinge from human protein) and CAR extracellular region (for example CAR antigen binding domain).For example, in one embodiment, hinge can be human Ig (immunoglobulin) hinge (for example, IgG4 hinge, IgD hinge), GS joint (for example, GS joint as described herein), KIR2DS2 hinge or CD8a hinge.

[0109] About membrane spaning domain, in various embodiments, CAR can be designed to include the membrane spaning domain attached to the extracellular domain of CAR.The membrane spaning domain may include one or more other amino acids adjacent to the membrane spaning region, such as one or more amino acids related to the extracellular region of the protein derived from it (for example, 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the extracellular region) and / or one or more other amino acids related to the intracellular region of the protein derived from it (for example, 1,2,3,4,5,6,7,8,9,10 to 15 amino acids in the intracellular region). In certain embodiments, membrane spaning domain is a membrane spaning domain related to one of the other domains of CAR, for example, in one embodiment, membrane spaning domain can be from the same protein derived from its signal transduction domain, costimulatory domain or hinge domain. In certain embodiments, membrane spaning domain is not derived from the same protein as any other domain from which CAR is derived. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In some embodiments, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a cell expressing CAR. In different aspects, the amino acid sequence of the transmembrane domain can be modified or replaced to minimize interactions with the binding domain of a natural binding partner present in the same cell expressing CAR.

[0110] The transmembrane domain may comprise a transmembrane domain of a protein selected from the group consisting of: the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, 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 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, L Y9 (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 their functional variants. The transmembrane domain can be derived from a natural source or a recombinant source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. In some embodiments, as long as the CAR binds to the target, the transmembrane domain is able to send a signal to one or more intracellular domains.

[0111] Pharmaceutically acceptable compositions and methods of administration

[0112] In some embodiments, a pharmaceutical composition is provided. In some embodiments, the pharmaceutical composition comprises: a T cell population produced by the methods disclosed herein; and one or more pharmaceutically acceptable carriers.

[0113] The present disclosure also provides the use of a T cell population comprising an organelle complex in the manufacture of a medicament for treating a disease or condition provided herein. In some embodiments, a T cell population comprising an organelle complex is administered to a subject in combination with one or more additional agents and / or additional therapies designed to treat a disease or condition.

[0114] Contacting the cells of the subject can include a route of administration selected from the group consisting of intravenous administration, intraarterial administration, intratracheal administration, subcutaneous administration, intramuscular administration, inhalation, intrapulmonary administration, and intraocular administration. The T cell population comprising the organelle complex can be administered locally or systemically.

[0115] As used herein, the term "local administration" or "topic administration" refers to any route of administration that brings a population of T cells comprising an organelle complex into contact with the body of an individual such that the location of the resulting T cells within the body is local (limited to the specific tissue, organ, or other body part to be imaged). Exemplary routes of local administration include injection into a specific tissue by needle, tube feeding into the gastrointestinal tract, and application of a solution containing a population of T cells comprising an organelle complex onto the surface of the skin.

[0116] As used herein, the phrase "systemic administration" represents any such route of administration, so that the T cell colony comprising the organelle complex is contacted with the body of the individual so that the position of the resulting T cells in vivo is systemic (i.e., not limited to the specific tissues, organs, or other body parts that require imaging). Systemic administration includes enteral administration and parenteral administration. Enteral administration is a systemic route of administration in which a substance is given via the digestive tract, and includes but is not limited to oral administration, administration by a gastric feeding tube, administration by a duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration. Parenteral administration is a systemic route of administration in which a substance is given by a route other than the digestive tract, and includes but is not limited to intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration, and intravesical infusion.

[0117] In another aspect, the present disclosure provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of a T cell population comprising an organelle complex disclosed herein. As described in detail below, the pharmaceutical compositions of the present disclosure can be specifically formulated for administration in solid or liquid form, including those suitable for the following: (1) oral administration, such as an immersion (aqueous or non-aqueous solution or suspension), tablet, pill, powder, granule, paste; (2) parenteral administration, such as by subcutaneous, intramuscular or intravenous injection, such as as a sterile solution or suspension; (3) topical administration, such as as a cream, ointment or spray applied to the skin; (4) intravaginal or intrarectal, such as as a pessary, cream or foam; or (5) an aerosol containing a T cell population comprising an organelle complex, such as as an aqueous aerosol, liposomal formulation or solid particles. The pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers. As used herein, the phrase "therapeutically effective amount" may refer to an amount of a T cell population comprising an organelle complex disclosed herein that is effective to produce some desired therapeutic effect, such as cancer treatment, at a reasonable benefit / risk ratio.

[0118] As used herein, the phrase "pharmaceutically acceptable / pharmaceutically acceptable" refers to those agents, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0119] As used herein, the phrase "pharmaceutically acceptable carrier" means 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 the subject chemical 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 causing harm to the subject. Some examples of materials that can be used 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, cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and tantalum oil. soybean oil; (10) glycols such as propylene glycol; (1) polyols such as glycerol, 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) ethanol; (20) phosphate buffer solution; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0120] Preparations that can be used for the methods of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, spray and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of the active ingredient (e.g., a T cell population comprising 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 specific mode of administration. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the T cell population comprising an organelle complex that produces a therapeutic effect. Overall, in one hundred percent, this amount will be in the range of 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%.

[0121] Suspensions, in addition to the active agents, may contain 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.

[0122] 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 can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0123] Ointments, pastes, creams and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0124] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also contemplated as being within the scope of this disclosure.

[0125] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Suitable fluidity can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0126] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can ensure that prevention of microbial action is achieved by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents (e.g., sugar, sodium chloride, etc.) in the composition. In addition, the absorption of injectable drug forms can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0127] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be determined by the methods of the present disclosure so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without causing toxicity to the subject.

[0128] The T cells comprising the organelle complex can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells comprising the organelle complex can be 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, at least about 10 9 or at least about 10 10 In another embodiment, the therapeutically effective amount of T cells comprising the organelle complex is about 10 4cells, about 10 5 cells, about 10 6 cells, about 10 7 cells or about 10 8 In a specific embodiment, the therapeutically effective amount of T cells comprising the organelle complex is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7 cells / kg, about 8×10 7 cells / kg or approximately 9×10 7 cells / kg.

[0129] Also provided herein are kits comprising one or more compositions described herein (e.g., a preparation comprising a T cell colony containing an organelle complex colony) in suitable packaging, and may further comprise written materials that may include instructions for use, discussions of clinical studies, lists of side effects, and the like. Such kits may also include information such as scientific literature references, package inserts, clinical trial results, and / or summaries of these, which indicate or establish the activity and / or advantages of the compositions, and / or describe administration, application, side effects, drug interactions, or other information useful to healthcare providers. Such information may be based on the results of various studies, such as studies using experimental animals involving in vivo models and studies based on human clinical trials. Kits may comprise one or more unit doses as described herein.

[0130] Other medications

[0131] In some embodiments, the method comprises administering one or more additional agents to the subject. In some embodiments, the one or more additional agents increase the efficacy of T cells containing the exogenous organelle complex. The one or more additional agents may include protein phosphatase inhibitors, kinase inhibitors, cytokines, inhibitors of immunosuppressive molecules, and / or inhibitors that reduce T REG Cell level or active agent.One or more other agents may include immunomodulators, anti-metastatic agents, chemotherapeutic agents, hormones or growth factor antagonists, alkylating agents, TLR agonists, cytokine antagonists, cytokine antagonists or any combination thereof.One or more other agents may include check point inhibitors or check point stimulating molecules (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, TIM-3) with specific target antibodies or antagonistic antibodies.

[0132] The one or more additional agents may be selected from the group consisting of: alkylating agents (nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes); Uracilnitrogen ); bendamustine Chloromethylamine Cyclophosphamide ( Revimmune TM ); ifosfamide Melphalan Chlorambucil Pipobrom triethyleneimine triazine Triethylenethiophosphamide; Temozolomide Thiotepa Busulfan Carmustine Lomustine Streptozotocin Estramustine Fotemustine; ilofofen; mannosulfan; dibromomannitol; nimustine; procarbazine; ranimustine; semustine; triazone; trooxsulfan; and dacarbazine Anti-EGFR antibodies (eg, cetuximab) Panitumumab and gefitinib ); anti-Her-2 antibodies (eg, trastuzumab and other antibodies from Genentech); antimetabolites (including but not limited to folate antagonists (also referred to herein as antifolates), pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors): methotrexate 5-Fluorouracil Floxuridine Carmofur, cytarabine ( Tarabine PFS), 6-mercaptopurine ), 6-thioguanine ), fludarabine phosphate Pentostatin Pemetrexed Raltitrexed Cladribine Clofarabine Mercaptopurine Capecitabine Nilarabine Azacitidine Decitabine Enocitabine Sapacitabine, tegafur-uracil, thiazofurine, thioguanine, trofosfamide, and gemcitabine Vinca alkaloids: vinblastine vincristine Vindesine Vinorelbine Vinflunine Platinum-based agents: Carboplatin Cisplatin Oxaliplatin Nedaplatin, satraplatin, and triplatin; anthracyclines: daunorubicin Doxorubicin Epirubicin Idabit Mitoxantrone Valrubicin Aclarubicin, amrubicin, liposomal doxorubicin, liposomal daunorubicin, pirarubicin, pixentron, and daunorubicin; topoisomerase inhibitors: topotecan Irinotecan Etoposide Teniposide Lamellar sphingomyelin D, SN-38, camptothecins (e.g., IT-101), belotecan, and rubitecan; taxanes: paclitaxel Docetaxel Larotaxel, cabazitaxel, ortataxel, and tecitaxel; antibiotic: actinomycin Bleomycin Hydroxyurea Mitomycin Immunomodulator: Lenalidomide Thalidomide Immune cell antibody: alentuzumab Gemtuzumab Rituximab Tositumomab Interferons (e.g., IFN-α ( -A) or IFN-γ ); interleukins: IL-1, IL-2 IL-24, IL-6 IL-12; HSP90 inhibitors (e.g., geldanamycin or any derivative thereof). In certain embodiments, the HSP90 inhibitor is selected from geldanamycin, 17-alkylamino-17-desmethoxygeldanamycin ("17-AAG"), or 17-(2-dimethylaminoethyl)amino-17-desmethoxygeldanamycin ("17-DMAG"); antiandrogens, including but not limited to nilutamide and bicalutamide Antiestrogens, including but not limited to tamoxifen Toremifene Letrozole Testolactone Anastrozole Bicalutamide Exemestane Flutamide Fulvestrant Raloxifene and raloxifene hydrochloride; antihypercalcemic agents including, but not limited to, gallium (III) nitrate hydrate and pamidronate disodium Apoptosis inducers, including but not limited to ethanol, 2-[[3-(2,3-dichlorophenoxy)propyl]amino]-(9Cl), gambogic acid, ilisimol, anthracene and arsenic trioxide Aurora kinase inhibitors, including but not limited to binucleine 2; Bruton's tyrosine kinase inhibitors, including but not limited to terrenic acid; calcineurin inhibitors, including but not limited to cypermethrin, deltamethrin, fenvalerate, and tyrosine phosphorylation inhibitor 8; CaM kinase II inhibitors, including but not limited to 5-isoquinolinesulfonic acid, 4-[{2S)-2-[(5-isoquinolinesulfonyl)methylamino]-3-oxo-3-{4-phenyl-1-piperazinyl)propyl]phenyl ester, and benzenesulfonamide; CD45 tyrosine phosphatase inhibitors, including but not limited to Phosphonic acid; CDC25 phosphatase inhibitors, including but not limited to 1,4-naphthalenedione, 2,3-bis[(2-hydroxyethyl)thio]-(9Cl); CHK kinase inhibitors, including but not limited to desbromoaldehyde dioxin; 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 Rofecoxib Etoricoxib Lumiracoxib Valdecoxib or 5-alkyl-2-arylaminophenylacetic acid); cRAF kinase inhibitors, including but not limited to 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindolin-2-one and benzamide, 3-(dimethylamino)-N-[3-[(4-hydroxybenzoyl)amino]-4-methylphenyl]-(9Cl); cyclin-dependent kinase inhibitors, including but not limited to olomocin and its derivatives, purvalanol B, roascovitine Indirubin, kemperolone, 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 intercalators, including but not limited to plicamycin and daptomycin DNA strand breakers, including but not limited to bleomycin E3 ligase inhibitors, including but not limited to N-((3,3,3-trifluoro-2-trifluoromethyl)propionyl)sulfonamide; EGF pathway inhibitors, including but not limited to tyrosine phosphorylation inhibitor 46, EKB-569, erlotinib Gefitinib Lapatinib and those compounds disclosed generally and specifically in WO 97 / 02266, EP 0 564409, WO 99 / 03854, EP 0 520722, EP 0 566 226, EP 0 787 722, EP 0 837 063, U.S. Pat. No. 5,747,498, WO 98 / 10767, WO 97 / 30034, WO 97 / 49688, WO 97 / 38983 and WO 96 / 33980; Farnesyl transferase inhibitors including, but not limited to, non-hydroxy farnesylphosphonic acid, butyric 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 and manumitin A; Flk-1 kinase inhibitors, including but not limited to 2-acrylamide, 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; histidine deacetylase (HDAC) inhibitors, including but not limited to suberoylanilide hydroxamic acid (SAHA), [4-(2-amino-phenylcarbamoyl)-benzyl]carbamic acid pyridin-3-ylmethyl ester and its derivatives, butyric acid, pyroxamide, trichostatin A, oxamflatin, apicidin, depsipeptide, depudecin, trapoxin, vorinostat and compounds disclosed in WO 02 / 22577; I-κB-α kinase inhibitors (IKK), including but not limited to 2-acrylonitrile, 3-[(4-methylphenyl)sulfonyl]-(2E)-(9Cl); imidazole tetrazinones, including but not limited to temozolomide ( and its derivatives (e.g., as generally and specifically disclosed in U.S. Patent No. 5,260,291) and mitozolamide; insulin tyrosine kinase inhibitors, including but not limited to hydroxy 1-2-naphthylmethylphosphonic acid; c-Jun-N-terminal kinase (JNK) inhibitors, including but not limited to pyrazolonafurone 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′-boryl-chalcone; MEK inhibitors, including but not limited to succinonitrile, bis[amino[2-aminophenyl)thio]methylene]-(9Cl); MMP inhibitors, including but not limited to actinonin, epigallocatechin gallate, collagen peptide mimetic inhibitors and non-peptide mimetic inhibitors, tetracycline derivatives Marimastat Pumastatin, incyclinide Shark Cartilage Extract AE-941 Tanostat, TAA211, MMI270B or AAJ996; mTor inhibitors, including but not limited to rapamycin and analogs and derivatives thereof, AP23573 (also known as lirolimus, deforolimus or MK-8669), CCI-779 (also known as temsirolimus) and SDZ-RAD; NGFR tyrosine kinase inhibitors, including but not limited to tyrosine phosphorylation inhibitor AG 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 tanazolin and tyrosine phosphorylation inhibitor 46; PDGF pathway inhibitors, including but not limited to tyrosine phosphorylation inhibitor AG 1296, tyrosine phosphorylation inhibitor 9, 1,3-butadiene-1,1,3-tricarboxylic acid, 2-amino-4-(1H-indol-5-yl)-(9Cl), imatinib and gefitinib and those compounds disclosed generally and specifically in European Patent No. 0 564 409 and PCT Publication No. WO 99 / 03854; phosphatidylinositol 3-kinase inhibitors including, but not limited to, wortmannin and quercetin dihydrate; phosphatase inhibitors including, but not limited to, cantharidic acid, cantharidin, and L-leucinamide; protein phosphatase inhibitors including, but not limited to, cantharidic acid, cantharidin, LP-bromotetramidazole oxalate, 2(5H)-furanone, 4-hydroxy-5-(hydroxymethyl)-3-(1-oxohexadecanyl)-(5R)-(9Cl), and benzylphosphonic acid; PKC inhibitors including, but not limited to, 1-H-pyridinium chloride. Pyrrol-2,5-dione, 3-[1-3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-(9Cl), bisindolemaleimide IX, sphingosine, staurosporine and hypericin; PKCδ kinase inhibitors, including but not limited to crude furfural shilomycin; polyamine synthesis inhibitors, including but not limited to DMFO; PTP1B inhibitors, including but not limited to L-leucinamide; protein tyrosine kinase inhibitors, including but not limited to tyrosine phosphorylation inhibitor Ag. 216, tyrosine phosphorylation inhibitor Ag 1288, tyrosine phosphorylation inhibitor Ag 1295, geldanamycin, genistein and 7H-pyrrolo[2,3-d]pyrimidine derivatives, as generally and specifically described in PCT Publication No. WO 03 / 013541 and U.S. Publication No. 2008 / 0139587; SRC family tyrosine kinase inhibitors, including but not limited to PP1 and PP2; Syk tyrosine kinase inhibitors, including but not limited to bisantrol; Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors, including but not limited to tyrosine phosphorylation inhibitor AG 490 and 2-naphthyl vinyl ketone; retinoids, including but not limited to isotretinoin and retinoic acid ( Retin-A ); RNA polymerase H elongation inhibitors, including but not limited to 5,6-dichloro-1-β-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 Sorafenib ZD6474 (also known as vandetanib) (Zactima TM ), SU6668, CP-547632 and AZD2171 (also known as cediranib) (Recent TM).

[0133] Examples

[0134] Some 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 present disclosure in any way.

[0135] Example 1

[0136] Mitochondrial transplantation enhances the metabolic adaptability and anti-tumor effects of CAR-T cells

[0137] introduce

[0138] Chimeric antigen receptor (CAR) T-cell therapy is a potentially curative treatment for patients with relapsed or refractory (r / r) hematological malignancies. However, approximately half of patients with r / r B-cell lymphomas experience treatment failure after CAR-T cell therapy, which may be due to poor expansion, limited persistence, and exhaustion of CAR-T cells. Given that metabolic adaptability plays a central role in regulating T-cell function, manipulating mitochondrial function is a promising approach to enhance the anti-tumor effects of CAR-T cells. Recently, it was demonstrated that transferring viable mitochondria (mitochondrial transfer) prepared by traditional homogenization methods (Mito-conv) into T cells can enhance the expression of inhibitory receptors and reduce T-cell proliferation (Court AC: EMBO Rep2020). In the current study, we tested whether transferring "Q" mitochondria isolated from HeLa cells using the novel iMIT isolation technology (WO / 2021 / 015298A1) into T cells could enhance the metabolic adaptability and anti-tumor effects of CAR-T cells.

[0139] method

[0140] 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 mitochondria. Culture medium and Q were refreshed every 24 or 48 hours. Mitochondrial respiratory function was evaluated using an XFp flux analyzer. For CAR-T cell production, purified T cells were stimulated with anti-CD3 / -CD28 antibodies for 48 hours and then incubated with a retroviral vector encoding anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hours.

[0141] result

[0142] First, it was confirmed that the mitochondrial structure and ATP synthesis of Q mitochondria were better maintained than those of Mito-conv. When mouse purified T cells were incubated with fluorescently labeled Q mitochondria for 24 hours during TCR stimulation, approximately 70% of the T cells endocytosed Q mitochondria, and the mitochondrial mass of each cell increased significantly. As expected, after TCR stimulation, oxidative phosphorylation (OXPHOS) in Q-treated T cells was significantly enhanced compared to vehicle-treated controls. After 72 hours of TCR stimulation, the levels of reactive oxygen species (ROS) increased in vehicle-treated T cells, while Q mitochondria significantly reduced the accumulation of ROS in activated T cells. Q mitochondria inhibited the differentiation of terminally exhausted T cells and increased TCF-1 in memory and exhausted T cells, which was associated with improved survival after chronic TCR stimulation (196 hours) and enhanced production of IFN-g and TNF-a. + Precursor populations (e.g., TCF-1 + PD-1 + Tim3-precursor depleted T cells (pTex)). Based on these findings, we next investigated whether Q mitochondria would affect CAR-T cell function. CAR-T cells were generated using purified T cells from naive BALB / c mice, and Q mitochondria were added during CAR-T cell generation, starting with TCR stimulation. Compared to vehicle-treated CAR-T cells, Q-treated CAR-T cells exhibited enhanced OXPHOS and cytokine production ( Figure 1 When CAR-T cells were incubated with syngeneic B-cell lymphoma cells (A20) for 12 hours, Q-treated CAR-T cells showed significantly enhanced in vitro cytotoxicity against A20 lymphoma cells. Next, the in vivo anti-tumor effect of CAR-T cells was examined. 14 days after tumor inoculation, naive BALB / c mice were injected subcutaneously in the right flank with 15 x 10 6 A20 cells, and 1 x 10 6 Q-treated or control CAR-T cells or naive BALB / c T cells were injected. Although control CAR-T cells significantly inhibited tumor growth compared with naive T cells, all mice died due to tumor growth by day 31 after tumor inoculation. Importantly, tumor growth was further inhibited in mice injected with Q-treated CAR-T cells, resulting in significantly prolonged survival ( Figure 2 ).

[0143] in conclusion

[0144] For the first time, CAR-T cells transplanted with Q mitochondria prepared using the iMIT method were found to have improved metabolic fitness, leading to enhanced proliferation capacity and cytokine production, as well as anti-tumor effects in vitro and in vivo. Mitochondrial transplantation can also be used as an adjunct approach in combination with various other genetic and pharmacological approaches to enhance the effectiveness of CAR-T cell therapy.

[0145] Example 2

[0146] Modulating T cell function using a second organelle complex transplantation

[0147] Immature T cells rely on oxidative phosphorylation (OXPHOS) to produce energy, while activated T cells show an increase in overall metabolic activity (such as glycolysis and glutamate degradation). In the case where T cells receive chronic antigen stimulation (such as in a tumor microenvironment), reactive oxygen species (ROS) accumulate and mitochondrial function is impaired. Mitochondrial dysfunction and mitochondrial mass reduction may cause the production of effector cytotoxic cytokines in T cells to decrease. In addition, the accumulation of ROS in mitochondria inhibits OXHPOS and induces T cell exhaustion. CAR-T cells from patients whose CAR-T therapy fails can show a decrease in mitochondrial nascent potential and a decrease in mitochondrial mass, which is associated with a decrease in proliferation capacity and persistence. In some embodiments, the second organelle complex transplant improves the efficacy of CAR-T therapy when CAR-T is produced. This example shows that the transfer of the second organelle complex improves T cell function by restoring mitochondrial energy production and reducing mitochondrial ROS.

[0148] The second organelle complex was isolated from HeLa cells. After permeabilization of the cell membrane with digitonin and a rinse step, the cells were incubated on ice. Subsequently, pipetting and centrifugation were performed to recover the isolated second organelle complex.

[0149] Administration of the second organelle complex was found to improve the efficiency of T cell recovery after stimulation. Figure 3 Data related to post-stimulation T cell recovery efficiency are depicted. Secondary organelle complexes (or vehicle) were transplanted on day 1, followed by stimulation with anti-CD3 / CD28 antibodies. T cell recovery was measured 48 hours later. T cell recovery efficiency (%) was defined as {(number of T cells recovered 48 hours after stimulation) / (number of T cells before stimulation)} x 100.

[0150] The effect of the second organelle complex transplantation on long-term stimulated T cells was examined. Chronic T cell stimulation was performed to reproduce the tumor microenvironment. T cells were purified from C57BL / 6 mice (MACS negative selection) and then stimulated on day 1 (via plate-bound anti-CD3 / CD28 Ab). The second organelle complex was administered on day 1, day 3, day 5, and day 7 (or control vehicle), followed by FACS analysis on day 4 or day 9.

[0151] It was found that transplantation of the second organelle complex increased the mass of T cell mitochondria ( Figures 4A to 4D Next, the effect of transplantation of the second organelle complex on T cell proliferation was examined. Figures 5A to 5D Depicted are data related to T cell proliferation capacity assessed by dye dilution assay using Cell Trace Violet dye. CD4 T cells ( Figures 5A to 5B ) and CD8 T cells ( Figures 5C to 5D ) were transplanted with the second organelle complex or vehicle and assessed on day 4. It was found that the second organelle complex transplantation improved T cell proliferation. Next, the effect of the second organelle complex transplantation on mitochondrial ROS levels was determined, and it was found that the second organelle complex transplantation reduced the level of ROS accumulated in mitochondria ( Figures 6A to 6D It was also found that transplantation of the second organelle complex reduced the accumulation of ROS levels in the entire T cell ( Figures 7A to 7D Next, we determined the effect of transplantation of the second organelle complex on the mitochondrial respiratory capacity of T cells. Figure 8 Data related to T cell oxygen consumption rate measured with an XFp flux analyzer are depicted. T cells were transplanted with the second organelle complex or vehicle and assessed on day 4. It was found that transplantation of the second organelle complex improved T cell mitochondrial respiratory capacity. It was found that transplantation of the second organelle complex enhanced the cytokine production capacity of CD8 T cells ( Figures 9A to 9B ). Figures 10A to 10B Data related to the number of surviving cells after chronic stimulation of TCR in vitro are depicted. T cells were transplanted with the second organelle complex or vehicle, and the viable cell count was assessed on day 9 using a dead cell staining dye. It was found that transplantation of the second organelle complex increased the number of viable T cells after chronic stimulation. Flow cytometric analysis of TCF-1 precursor cells was performed 72 hours after TCR stimulation to compare T cells transplanted with the second organelle complex (2nd OC) or vehicle ( Figures 16A to 16B ).

[0152] Mouse CD19 CAR-T is produced as follows. T cells are purified from BALB / c mice (MACS negative selection), and then T cell stimulation (plate-bound anti-CD3 / CD28 antibodies) is performed for 48 hours on the 1st day. Retroviral particles are produced (via transfection) using a vector plasmid (CD19 CAR) comprising inactive 1D3 scFv, CD28, and CD3ζ 1st and 3rd ITAM. CAR gene transduction (using retroviral vectors) is performed on the 3rd and 4th days. The second organelle complex is applied three times (1st day, 3rd day, and 4th day). Figure 11 Depicted are data related to CAR-T cell glycolytic rates measured using a Seahorse extracellular flux analyzer. Figure 1 The oxygen consumption rate of CAR-T cells measured using a Seahorse extracellular flux analyzer after T cells were transplanted with a second organelle complex or vehicle was depicted. It was found that the transplantation of the second organelle complex improved the mitochondrial respiratory capacity and glycolytic capacity of T cells. It was found that the transplantation of the second organelle complex improved the proliferation capacity of CAR-T cells ( Figures 12A to 12D ). Next, the cytokine production capacity was evaluated. It was found that the transplantation of the second organelle complex enhanced the cytokine production capacity of CD8 CAR-T cells ( Figures 13A to 13B ). It was also found that transplantation of the second organelle complex enhanced the cytotoxic activity of CAR-T cells in vitro ( Figure 14 Flow cytometric analysis of TCF-1 precursor CAR-T cells was performed 96 hours after the start of TCR stimulation to compare CAR-T cells transplanted with the second organelle complex (2nd OC) or vehicle ( Figures 17A to 17B ).

[0153] Next, we used a B-cell lymphoma model (A20, 15×10 6 The anti-tumor effect of CAR-T cells was evaluated in vivo. 6 The cells were intravenously injected into BALB / c mice bearing A20 tumors, and the 2 nd OC(+)CAR-T cells (n=7), 2 nd OC(-)CAR-T cells (n=7) and naive T cells (n=4). Figure 2 Depicted are data relating to inhibition of tumor growth in mice injected with CAR-T cells transplanted with the second organelle complex, resulting in significantly prolonged survival. Figure 15 Data related to overall survival of mice injected with CAR-T cells transplanted with the second organelle complex compared to vehicle-transplanted CAR-T cells or naive T cells are depicted. It was found that transplantation of the second organelle complex enhanced the anti-tumor effect of CAR-T and improved survival.

[0154] This study demonstrates that transplantation of the second organelle complex improves T cell proliferation and increases the number of surviving cells during chronic TCR stimulation in vitro. Transplantation of the second organelle complex reduced cellular and mitochondrial ROS levels, while improving mitochondrial respiratory capacity. Furthermore, transplantation of the second organelle complex during the production of CD19 CAR-T cells from naive T cells improved T cell recovery efficiency after anti-CD3 / CD28 antibody stimulation. Importantly, transplantation of the second organelle complex improved mitochondrial respiratory capacity and anti-tumor effects in CD19 CAR-T cells in mice.

[0155] Example 3

[0156] Study on the effect of organelle complex transplantation on the metabolic adaptability and anti-tumor effect of CAR-T cells

[0157] introduce

[0158] In this study, we will test whether the transfer of organelle complexes isolated from HeLa cells (e.g., organelle complex 1, organelle complex 2) into T cells can enhance the metabolic adaptability and anti-tumor effects of CAR-T cells.

[0159] method

[0160] In the presence or absence of 50-100 μg / ml organelle complex (e.g., the first organelle complex, the second organelle complex), purified T cells from naive mice or healthy volunteers are stimulated with plate-bound anti-CD3 / -CD28 antibodies. Culture medium and organelle complex (e.g., the first organelle complex, the second organelle complex) will be updated every 24 or 48 hours. Mitochondrial respiratory function will be evaluated using an XFp flux analyzer. For CAR-T cell production, purified T cells are stimulated with anti-CD3 / -CD28 antibodies for 48 hours and then incubated for another 48 hours with a retroviral vector (1D3-28Z.1-3; Addgene) encoding anti-CD19 CAR.

[0161] Expected Results

[0162] First, it will be confirmed that the mitochondrial structure and ATP synthesis of T cells transplanted with organelle complexes (e.g., the first organelle complex, the second organelle complex) are better maintained compared to the mitochondrial structure and ATP synthesis of Mito-conv. During TCR stimulation, mouse purified T cells are incubated with fluorescently labeled organelle complexes (e.g., the first organelle complex, the second organelle complex) for 24 hours, and it is expected that approximately 70% of the T cells will have endocytosed organelle complexes and the mitochondrial mass of each cell will increase significantly. After TCR stimulation, oxidative phosphorylation (OXPHOS) in T cells treated with organelle complexes (OC) will be significantly enhanced compared to vehicle-treated controls. After 72 hours of TCR stimulation, the level of reactive oxygen species (ROS) in vehicle-treated T cells will increase, while organelle complexes (e.g., the first organelle complex, the second organelle complex) will significantly reduce the accumulation of ROS in activated T cells. Organelle complexes (e.g., organelle complex I, organelle complex II) will inhibit the differentiation of terminally exhausted T cells and will increase TCF-1 in memory and exhausted T cells associated with improved survival and enhanced production of IFN-g and TNF-a after chronic TCR stimulation (96 hours) + Precursor populations (e.g., TCF-1 + PD-1 + Tim3-precursor depleted T cells (pTex)). Next, it will be studied whether organelle complexes (e.g., the first organelle complex, the second organelle complex) can affect CAR-T cell function. CAR-T cells will be produced by purified T cells from naive BALB / c mice, and organelle complexes (e.g., the first organelle complex, the second organelle complex) will be added from the beginning of TCR stimulation during CAR-T cell production. OC-treated CAR-T cells will exhibit enhanced OXPHOS and cytokine production compared to vehicle-treated CAR-T cells. CAR-T cells will be incubated with isogenic B cell lymphoma cells (A20) for 12 hours, and OC-treated CAR-T cells will exhibit significantly enhanced in vitro cytotoxicity against A20 lymphoma cells. Next, the in vivo anti-tumor effect of CAR-T cells will be examined. 14 days after tumor inoculation, naive BALB / c mice will be injected subcutaneously in the right flank with 1.5x10 7 A20 cells, and 1 x 10 6The researchers injected mice with OC-treated or control CAR-T cells or naive BALB / c T cells. It was expected that although control CAR-T cells would significantly inhibit tumor growth compared to naive T cells, all mice died due to tumor growth by day 31 after tumor inoculation. Mice injected with OC-treated CAR-T cells would further inhibit tumor growth, resulting in significantly prolonged survival.

[0163] Expected conclusions

[0164] CAR-T cells transplanted with organelle complexes (e.g., organelle complex I, organelle complex II) will exhibit improved metabolic adaptability, which will lead to enhanced proliferation capacity and cytokine production as well as anti-tumor effects in vitro and in vivo. Organelle complex transplantation can also be used as an adjuvant method in combination with various other genetic and pharmacological approaches to enhance the effectiveness of CAR-T cell therapy.

[0165] Example 4

[0166] Organelle complex transplantation inhibits T cell ferroptosis

[0167] introduce

[0168] This example investigates the effect of organelle complex transplantation (e.g., organelle complex 1, organelle complex 2 (Q)) on ferroptosis in T cells. Ferroptosis is a unique form of regulated cell death that can lead to the loss of a significant number of antigen-specific T cells, including virus-specific CD4 T cells. + Helper T cells (see 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 lethal accumulation of iron-dependent membrane lipid peroxidation and can occur under resting 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 the majority of endogenous ROS, such as superoxide anions and hydrogen peroxide, and both can serve as substrates for the Fenton reaction to generate hydroxyl radicals, which in turn can promote the propagation of phospholipid peroxidation and the degradation of membrane lipids. In contrast, GPX4, a glutathione-dependent peroxidase, can convert toxic PL-PUFA-OOH to non-toxic PL-PUFA-OH (promoted by the oxidation of glutathione (GSH) to glutathione disulfide (GSSG)), thereby inhibiting membrane lipid hydroperoxidation. It is hypothesized that transplantation of the second organelle complex (Q) can inhibit ferroptosis and improve T cell survival.

[0169] Q transplantation promotes GPX4 expression in activated T cells

[0170] Figures 18A to 18B Figure 3. CD4 T cells transplanted with the second organelle complex (Q) or vehicle after 72 hours of stimulation with anti-CD3 / CD28 antibodies. + T cells ( Figure 18A ) and CD8 + T cells ( Figure 18B ) and found that Q transplantation promoted the expression of GPX4, a molecule that inhibits ferroptosis.

[0171] Q transplantation inhibits lipid peroxidation in activated T cells

[0172] Figures 19A to 19B Figure 3. CD4 T cells transplanted with the second organelle complex (Q) or vehicle after 72 hours of stimulation with anti-CD3 / CD28 antibodies. + T cells ( Figure 19A ) and CD8 + T cells ( Figure 19B ). Lipid peroxidation was assessed using a lipid peroxidation kit based on the fluorescence shift from PE to FITC. As the PE / FITC ratio increased, lipid peroxidation levels decreased. These results suggest that transplantation of the second organelle complex can inhibit lipid peroxidation.

[0173] Q transplantation inhibits cell death of activated T cells

[0174] Figures 20A to 20BFigure 3 shows apoptotic CD4 T 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 + T cells ( Figure 20B ). It was found that transplantation of the second organelle complex could inhibit cell death of activated T cells, which can be considered as ferroptosis inhibition.

[0175] Q transplantation inhibits ROS accumulation in mouse CAR-T cells

[0176] Figures 21A to 21B Depicts the relationship between CD4 + ( Figure 21A ) and CD8 + ( Figure 21B Data related to ROS accumulation in vehicle CAR-T cells and CAR-T cells treated with Q, assessed with CellROX Green dye in a ) environment. A significant reduction in ROS accumulation was found in CAR-T cells transplanted with the second organelle complex.

[0177] Q transplantation inhibits lipid peroxidation in mouse CAR-T cells

[0178] Figures 22A to 22B Depicts the CD4 + CAR-T cells ( Figure 22A ) and CD8 + CAR-T cells ( Figure 22B ). Lipid peroxidation was assessed using a lipid peroxidation kit based on the fluorescence shift from PE to FITC. As the PE / FITC ratio decreased, lipid peroxidation levels increased. Transplantation of the second organelle complex was found to inhibit lipid peroxidation.

[0179] Q transplantation of mouse CAR-T cells prolongs survival in tumors after infusion

[0180] Figures 23A to 23B Figure 3 shows the expression of CD4 T cells transplanted with the second organelle complex (Q) or vehicle in tumor infiltrating lymphocytes (TILs) at 17 days after tumor cell inoculation. + CAR-T cells ( Figure 23A ) and CD8 + CAR-T cells ( Figure 23B CD4 was found in the tumors of the second organelle complex transplantation group. + and CD8 + The percentages of both CAR-T cells increased.

[0181] Q transplantation inhibits ROS accumulation in human activated T cells

[0182] Figures 24A to 24D Figure 3. CD4 T cells transplanted with the second organelle complex (Q) or vehicle after 192 hours of stimulation with anti-CD3 / CD28 antibodies. + T cells ( Figure 24A 、 Figure 24C ) and CD8 + T cells ( Figure 24B 、 Figure 24D ) in cells ROS( Figures 24A to 24B ) and mitochondrial ROS ( Figures 24C to 24D CellROX Green and MitoSOX Red dyes were used to assess cellular and mitochondrial ROS accumulation, respectively. Transplantation of the second organelle complex in human activated T cells was found to inhibit ROS accumulation.

[0183] Q transplantation promotes the proliferation of human activated T cells

[0184] Figures 25A to 25B Depicts CD4 T cells transplanted with the second organelle complex (Q) or vehicle after chronic stimulation. + T cells ( Figure 25A ) and CD8 + T cells ( Figure 25B T cell proliferation was assessed using the Cell Trace Violet dilution assay. Following 192 hours of stimulation with anti-CD3 / -CD28 antibodies, the proportion and number of viable T cells were assessed using a dead cell stain. Figures 26A to 26B Depicted are the percentage of viable cells ( Figure 26A ) and the number of viable cells ( Figure 26B ) data related to the discovery that transplantation of the second organelle complex enhances the proliferation of human activated T cells.

[0185] in conclusion

[0186] Ferroptosis is an important apoptotic pathway in T cells that is associated with mitochondrial-generated ROS. + T cells and CD8 +T cells), it was demonstrated that the transplantation of the second organelle complex (Q) can promote GPX4 expression, inhibit lipid peroxidation, inhibit cell death, inhibit ROS accumulation, promote proliferation, and prolong tumor survival. The results provided in this example show that the transplantation of the second organelle complex (Q) can inhibit ferroptosis in T cells and further demonstrate its practicality in cell therapy.

[0187] Unless such substitution is technically infeasible, in at least some previously described embodiments, one or more elements used in one embodiment may be interchangeably used in another embodiment. It will be understood by those skilled in the art that various other omissions, additions, and modifications may be made to the above-described methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0188] Regarding the use of substantially any plural and / or singular terms herein, those skilled in the art can translate the plural into the singular and / or translate the singular into the plural according to the context and / or application. For the sake of clarity, various singular / plural arrangements can be explicitly set forth herein. Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references. Unless otherwise indicated, any reference to "or" herein is intended to encompass "and / or".

[0189] Those skilled in the art will understand that, in general, the terms used herein and particularly in the appended claims (e.g., the bodies of the appended claims) are generally intended to be "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 "comprising" should be interpreted as "including but not limited to," etc.). Those skilled in the art will also understand that if a specific number of claim recitations is intended to be introduced, such intent will be explicitly recited in the claim, and in the absence of such recitation, no such intent is present. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that introducing a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article, such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); the same is true for the use of definite articles to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be construed to mean at least the recited number (e.g., reciting "two recitations" alone without mentioning other modifiers means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention similar to “at least one of A, B, and C, etc.” is used, generally speaking, such construction is intended to be in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, systems having: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention similar to “at least one of A, B, or C, etc.” is used, generally speaking, such construction is intended to be in the sense that one skilled in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include, but is not limited to, systems having: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will also understand that virtually any disjunctive word and / or phrase presenting two or more alternative terms (whether in the specification, claims or drawings) should be understood to contemplate the possibility of including one of the terms, either one of the terms, or both of the terms.

[0190] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0191] As will be understood by those skilled in the art, for any and all purposes, such as in providing written specifications, all scopes disclosed herein also encompass any and all possible sub-ranges and combinations thereof.Any listed scope can easily be considered to fully describe and enable the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As non-limiting examples, each scope discussed herein can be described as lower one-third, middle one-third, and upper one-third, etc. Those skilled in the art can also understand that all languages ​​(such as "until," "at least," "greater than," "less than," etc.) include cited numbers and refer to the scopes that can subsequently be decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, scope includes each individual member. Therefore, for example, a group with 1 to 3 articles refers to a group with 1, 2, or 3 articles. Similarly, a group with 1 to 5 articles refers to a group with 1, 2, 3, 4, or 5 articles, etc.

[0192] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A method for producing a T cell population for adoptive T cell therapy, comprising: The isolated organelle complex is contacted with a population of T cells to produce 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.

2. The method of claim 1, wherein the method comprises stimulating the T cell population.

3. The method of claim 2, wherein the stimulation step expands the T cell population.

4. A method for enhancing the proliferation, migration, persistence and / or activity of a T cell population for adoptive T cell therapy, comprising: contacting the isolated organelle complex with a population of T cells to produce 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 The T cell population is stimulated to expand the T cell population.

5. A method for generating a T cell population resistant to exhaustion, comprising: contacting the isolated organelle complex with a population of T cells to produce 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 The T cell population is stimulated to expand the T cell population.

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

7. The method of any one of claims 2 to 6, wherein the stimulation comprises culturing the T cell population 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 include agents that stimulate CD3 / TCR complex-associated signals and agents that stimulate co-stimulatory molecules on the surface of the T cells.

9. The method according to any one of claims 2 to 8, wherein the one or more stimulants include: a molecule that binds to CD28, optionally one or more of an anti-CD28 antibody, CD80, and CD86; and / or A molecule that binds CD3, optionally an anti-CD3 antibody.

10. The method of any one of claims 1 to 9, wherein the stimulation 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. The method according to any one of claims 1 to 10, wherein the method comprises introducing a heterologous nucleic acid encoding a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR) into the T cell, optionally the introduction step is performed before, after and / or during the contacting, and further optionally the heterologous nucleic acid is a vector.

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

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

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

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. The method according to any one of claims 1 to 15, wherein the T cell population is derived from lymph node cells, which are optionally purified by a magnetic particle-based enrichment procedure selected from the group consisting of: manual and 17. The method according to any one of claims 1 to 16, wherein the T cell population comprises one or more of the following: CD4 + Cells, CD8 + T cells, cytotoxic T cells, terminal 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).

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

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

20. The method according to any one of claims 1 to 19, wherein 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 proteins, optionally Golgi protein-97, syntaxin-6, TGOLN2 / trans-Golgi network protein 2 (TGN46), Golgi matrix protein 130 (GM130) and / or mannosidase alpha 2A class member 1 (MAN2A1); and / or One or more endoplasmic reticulum proteins, optionally calreticulin and / or calnexin.

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

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

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

24. The method of any one of claims 1 to 23, wherein the T cell comprises 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. The method of claim 25, wherein the intracellular signaling domain comprises a primary signaling domain, a co-stimulatory domain, or both a primary signaling domain and a co-stimulatory domain.

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 CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10 and DAP12 or functional variants thereof.

28. The method of any one of claims 26 to 27, wherein the co-stimulatory domain comprises a functional domain of one or more proteins 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, L IGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD4 9f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITG B7, 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 their functional variants.

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

30. The method of any one of claims 25 to 29, wherein the antigen binding domain comprises an antibody, an antibody fragment, a scFv, a Fv, a Fab, (Fab')2, a single domain antibody (SDAB), a VH or VL domain, a Camelid VHH domain, a Fab, Fab', F(ab')2, Fv, scFv, dsFv, a diabody, a triabody, a tetrabody, a multispecific antibody formed from antibody fragments, a single domain antibody (sdAb), a single chain comprising an anti-complement scFv (tandem scFv) or a bispecific tandem scFv, an Fv construct, a disulfide-linked Fv, a dual variable domain immunoglobulin (DVD-Ig) binding protein or a nanobody, an aptamer, an affibody, an affilin, an affitin, an affimer, an alpha body, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, a monomer 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 via a hinge region.

32. The method of any one of claims 25 to 31, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: the alpha, beta or zeta chain of a 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), SLAM F7, NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, V LA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD 29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, C RTAM, 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 their functional variants.

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

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

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

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

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

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

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

42. A method according to any one of claims 1 to 41, wherein the T cell population exhibits at least about a 1.1-fold reduction in the level of one or more exhaustion markers, optionally in vivo and / or in vitro, compared to a T cell population that does not contain an exogenous organelle complex, optionally the exhaustion marker is selected from the group comprising: PD-1, CTLA-4, TIM-3, LAG-3, BTLA, 2B4, CD 160, CD39, VISTA, TIGIT or any combination thereof.

43. The method of any one of claims 1 to 42, wherein the T cell population exhibits at least about a 1.1-fold reduction in the level 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 T cell population that does not comprise an exogenous organelle complex, further optionally wherein the reactive oxygen species comprise superoxide (O2. - ), hydroperoxide (HO.2), hydrogen peroxide (H2O2), peroxynitrite (ONOO - ), hypochlorous acid (HOCl), hypobromous acid (HOBr), hydroxyl radical (HO.), peroxyl radical (ROO.), alkoxyl radical (RO.), singlet oxygen ( 1 O2), lipid peroxides, lipid peroxyl radicals or lipid alkoxyl radicals or any combination thereof.

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

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

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

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

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

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

50. A method of treating or preventing a disease or disorder in a subject, comprising: administering to the subject an effective amount of the T cell population 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 T cell population exhibits one or more of the following compared to a T cell population that does not contain the exogenous organelle complex: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

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

52. 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); as well as administering to the subject an effective amount of the isolated organelle complex, thereby treating or preventing the disease or disorder in the subject, wherein the isolated organelle complex is capable of contacting the T cell population in vivo to produce a T cell population comprising the organelle complex, The T cell population exhibits one or more of the following compared to a T cell population that does not contain the exogenous organelle complex: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

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

54. 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; and administering to the subject an effective amount of the isolated organelle complex, thereby treating or preventing the disease or disorder in the subject, wherein the heterologous nucleic acid and the isolated organelle complex are capable of contacting the subject's T cells in vivo to generate T cells comprising the organelle complex and CAR and / or engineered TCR, and The T cells exhibit one or more of the following compared to T cells that do not contain the exogenous organelle complex: enhanced expansion capacity, enhanced cytotoxicity against target cells, enhanced resistance to exhaustion, and enhanced persistence.

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

56. A method according to any one of claims 1 to 46 or 50 to 55, wherein the T cell population reduces the tumor volume, tumor growth and / or tumor burden of the subject, optionally the T cell population reduces the tumor volume of the subject by at least about 1.1 times compared to the tumor volume of an untreated subject or a subject administered with a T cell population that does not contain an exogenous organelle complex.

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

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

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

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 method of any one of claims 50 to 62, wherein the administering comprises administering at least about 1×10 6 T cells, optionally administered intravenously.

64. The method of any one of claims 50 to 63, wherein the method comprises repeated administration of the T cell population.

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

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 wherein the disease associated with expression of a tumor antigen is selected from the group consisting of a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the tumor antigen.

67. The method of claim 66, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, small cell or non-small cell lung cancer, 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, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's disease, Golden lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers and metastatic lesions of said cancers.

68. The method of any one of claims 66 to 67, wherein the cancer is a blood cancer selected from one or more of the following: 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 carcinoma, Dendritic cell neoplasms, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, or preleukemia.

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

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