Compositions and methods for immunotherapy

Abstract

To provide alternative compositions and methods for administering immunotherapy.SOLUTION: Provided is a method for producing a population of engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising: (a) contacting a population of immune cells obtained from a subject with an activating moiety to obtain a population of contacted immune cells; (b) introducing a polynucleotide encoding a CAR or a TCR into the population of immune cells contacted in (a), thereby producing a population of engineered immune cells expressing a CAR and / or a TCR; and (c) recovering the population of engineered immune cells expressing a CAR or a TCR for infusion into a subject, wherein the total duration of both (a) and (b) is 36 hours or less, and the method includes no ex vivo expansion, except for (a) to (c).SELECTED DRAWING: Figure 24G

Description

[Technical field]

[0001] cross reference

[0001] This application claims priority to Chinese Patent Application No. 201811501797.8, filed on December 07, 2018; PCT Application No. PCT / CN2019 / 073252, filed on January 25, 2019; Chinese Patent Application No. 201910297171.8, filed on April 12, 2019; and PCT Application No. PCT / CN2019 / 097996, filed on July 26, 2019, each of which is incorporated by reference in its entirety into this specification. [Background technology]

[0002]

[0002] Adoptive T cell therapy, as an example of immunotherapy, involves the isolation and ex vivo expansion of tumor-specific T cells to obtain a greater number of T cells than can be obtained by vaccination alone. The tumor-specific T cells are then infused into a patient with cancer to provide the patient's immune system with the ability to defeat remaining tumors via T cells that can attack and kill the cancer. There are many forms of adoptive T cell therapy for cancer, but they generally suffer from various deficiencies. Among them are cell exhaustion, long preparation times, and ineffective composition of engineered cells. Summary of the Invention [Problem to be solved by the invention]

[0003] In view of the foregoing, there exists a significant need for alternative compositions and methods for performing immunotherapy. The compositions and methods of the present disclosure address this need and provide additional advantages as well. In particular, various aspects of the present disclosure provide a system for immune cell modulation. [Means for solving the problem]

[0004] In one aspect, the disclosure provides a method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising injecting a population of immune cells comprising the engineered immune cells into a subject in need thereof, wherein the engineered immune cells have not been subjected to ex vivo expansion for 2 weeks or 1 week or less, and the population is further characterized by central memory T cells (TCM) being enriched over effector memory T cells (TEM) in the population. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 6, 5, 4, 3, 2, or 1 day. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 5 days. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 72, 48, 36, 32, or 24 hours. + CD62L + In some embodiments, the TEM is CD45RO + In some embodiments, the population is further characterized by being less enriched in PD1 and LAG3. In some embodiments, the reduced depletion of cells in the population is observed compared to the depletion of cells in a comparable population that has undergone ex vivo expansion for 2 weeks or 1 week or less, or 10 or 9 days or less.

[0005]

[0005] In some aspects, reduced depletion of the population is characterized by the population comprising fewer cells that express PD1 and LAG3.

[0006] In some embodiments, the engineered immune cell is a T cell, a NK cell, and / or a NKT cell. In some embodiments, the TCR comprises (i) a ligand-binding domain specific to a ligand, and (ii) a transmembrane domain. In some embodiments, the CAR comprises (i) a ligand-binding domain specific to a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some embodiments, the ligand of the TCR or CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.2. In some embodiments, the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1, and / or CD152. In some embodiments, the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10, and / or CD278 (ICOS). In some embodiments, the CAR comprises at least two intracellular signaling domains.In some embodiments, the CAR comprises at least three intracellular signaling domains. In some embodiments, the CAR further comprises a hinge. In some embodiments, the hinge is derived from CD28, IgG1, and / or CD8α. In some embodiments, the CAR further comprises a signal peptide, and the signal peptide is derived from IgG1, GM-CSF, and / or CD8α. In some embodiments, the engineered immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells. In some embodiments, the engineered immune cells are derived from peripheral blood, and the peripheral blood cells are T cells. In some embodiments, greater memory and / or stemness is observed in the population compared to a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In some embodiments, there is 2-fold more TCM compared to the TEM. In some embodiments, there is 4-fold more TCM compared to the TEM. In some embodiments, the infusion is intravenous. In some embodiments, the administration is about 1×10. 4 In some embodiments, the administration comprises injecting about 3×10 5The method further comprises injecting engineered immune cells from 1000 to 10000 / kg body weight. In some embodiments, at least 10% of the immune cells express a CAR and / or a TCR. In some embodiments, at least 20% of the immune cells express a CAR and / or a TCR. In some embodiments, at least 40% of the immune cells express a CAR and / or a TCR. In some embodiments, the method further comprises administering a secondary agent to the subject in need thereof. In some embodiments, the secondary agent is a therapeutically effective amount of an immunostimulant, an immunosuppressant, an antifungal, an antibiotic, an antiangiogenic, a chemotherapeutic, a radioactive, and / or an antiviral agent. In some aspects, the immunostimulant is IL-2. In some aspects, the method further comprises obtaining peripheral blood from the subject in need thereof after administration. In some aspects, the engineered immune cells in the subject are quantified from the peripheral blood. In some embodiments, the level of a growth factor in the subject is quantified. In some embodiments, the growth factor is selected from the group consisting of IL-10, IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-4, IL-8, IL-12, and / or IFN-γ. In aspects, the method includes repeating the infusion. In some embodiments, the population of immune cells is allogeneic to the subject in need thereof. In some aspects, the population of immune cells is autologous to the subject in need thereof. In some aspects, the subject has cancer. In some embodiments, the cancer is a hematological cancer. In some aspects, the hematological cancer is leukemia, myeloma, lymphoma, and / or combinations thereof. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), and / or acute lymphoblastic leukemia (ALL). In some embodiments, the lymphoma is mantle cell lymphoma (MCL), T-cell lymphoma, Hodgkin's lymphoma, and / or non-Hodgkin's lymphoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid cancer is selected from the group including nephroblastoma, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, or bladder cancer. In some embodiments, the subject in need thereof has a BCR-ABL mutation, the mutation being in the BCR-ABL kinase domain. In some embodiments, the subject in need thereof has a T315I and / or V299L mutation in the BCR-ABL kinase domain. In some aspects, the subject exhibits resistance to a tyrosine kinase inhibitor. In some aspects, the subject has or is susceptible to having a tumor after chemotherapy. In some aspects, the subject has been pretreated with chemotherapy prior to administration. In some aspects, the chemotherapy comprises administration of fludarabine, cyclophosphamide and / or cytarabine. In some embodiments, the subject has minimal residual disease (MRD), and the MRD is acute lymphoblastic leukemia.

[0006]

[0007] In some embodiments, the subject population of immune cells is further characterized by observing greater proliferation, cytotoxicity, and / or bone marrow migration in the population compared to a comparable population, e.g., C-CART, undergoing ex vivo expansion for 2 weeks or 1 week or less. In some embodiments, the subject population of cells, such as F-CART, can be evaluated using assays that determine the level of migration, proliferation, cytotoxicity, and effector activity. The level of migration can be determined using a chemotaxis assay. In one aspect, migration can refer to migration to bone marrow. In one aspect, migration can refer to migration from bone marrow. In one aspect, migration can also refer to movement toward a target, e.g., a chemokine or cancer cell. In one aspect, the chemokine system includes more than 40 chemokines and more than 18 chemokine receptors. Chemokine receptors are defined by their ability to induce directional migration of cells, such as engineered immune cells, toward a gradient of chemotactic cytokines (chemotaxis). Chemokine receptors are a family of seven transmembrane domain, G-protein coupled cell surface receptors named CXCR1-CXCR5, CCR1-CCR11, XCR1, and CX3CR1 based on their specific preference for certain chemokines. Chemokines are small secreted proteins that can be divided into two major subfamilies, CXC or CC chemokines, respectively, based on whether the two conserved cysteine ​​residues present in all chemokines are separated by an intervening amino acid. In some embodiments, migration or chemotaxis can be quantified in a population of engineered immune cells. In one aspect, migration or chemotaxis to cancer can be assessed in vitro using the CXCR4 and ligand SDF-1 (CXCL12) axis. In one aspect, a higher percentage of CD3, CD4, and / or CD8 F-CART express CXCR4 compared to CD3, CD4, and / or CD8 C-CART expressing CXCR4.In one embodiment, a greater mean fluorescence intensity (MFI) of CXCR4 is observed in CD3, CD4, and / or CD8 positive F-CARTs compared to CXCR4 in CD3, CD4, and / or CD8 positive C-CARTs. Expression of CXCR4. The present can be an indication of an increased migratory potential of a population of immune cells towards targets expressing CXCR4 ligand CXCL12. In one aspect, the MFI of a receptor such as CXCR4 can be quantified in an engineered immune cell population to determine the density of CXCR4 receptors on cells. Increased MFI or density of CXCR4 on engineered immune cells such as F-CART can indicate an increased migratory potential of the cells. In some cases, migration can be measured in populations of F-CART and C-CART by determining the number of cells migrating towards a target, for example, stromal cell-derived factor 1 (SDF1), also known as C-X-C motif chemokine 12 (CXCL12). In one embodiment, a gradient of SDF-1 (human or mouse) can be established in vitro or in vivo and utilized to determine the migration or chemotaxis of engineered immune cells such as F-CART towards a target. In one embodiment, the percentage of CXCR4 or MFI of CXCR4 in F-CART may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to about 100% higher compared to the percentage or MFI of CXCR4 in C-CART. In one embodiment, cytotoxicity is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1.0 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold greater in a population of cells comprising engineered immune cells compared to a comparable population comprising engineered immune cells that has undergone ex vivo expansion for 2 weeks or 1 week or less when the population comprising engineered immune cells and the comparable population are in contact with a target.

[0007]

[0008] In one embodiment, the proliferation of a population of cells comprising engineered immune cells in vivo is enhanced, and is at least 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, or 10000-fold higher in a population comprising engineered immune cells when the population and the equivalent population are contacted with a target, compared to an equivalent population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, proliferation can be quantified in vitro using a carboxyfluorescein succinimidyl ester (CFSE) assay. In one embodiment, proliferation can be quantified in vitro using a cytometer, for example, using a cytometer. Variables that can be measured by cytometry include, for example, cell size, cell number, cell morphology (shape and structure), cell cycle phase, DNA content, and the presence or absence of certain proteins on the cell surface or in the cytoplasm. The cytometer can evaluate the clumping of cells that can be observed during cell growth. The clumping of cells can be used as a factor to evaluate the enhanced growth, for example, enhanced growth in a population of engineered cells. In another embodiment, the cytometer can be used to count cells. In one embodiment, the cytometer, for example, a flow cytometer, can be used to quantify the number of cells in a sample, for example, blood, cell culture, bone marrow, tumor, and any combination thereof. The flow cytometer can utilize cell surface proteins to quantify cells, for example, engineered immune cells. The cell markers that can be utilized can be CD45, CD2, Beacon, CAR, TCR, CD3, CD4, CD8, CD62, and any combination thereof.

[0008]

[0009] In one embodiment, the proliferation and / or persistence of engineered immune cells can be determined in vivo by quantifying the copy number of engineered immune cells in a subject using quantitative PCR (qPCR). In one embodiment, the copy number of engineered immune cells is calculated as blood cell number / microliter. In one embodiment, the copy number of engineered immune cells is calculated as DNA copy number / microgram. In one embodiment, persistence can also be calculated in vivo.

[0009]

[0010] In one embodiment, bone marrow migration is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold higher in a population comprising engineered immune cells compared to a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less when the population and the comparable population are in contact with a target.

[0010]

[0011] In one embodiment, the target may be a cancer cell or a chemokine. In some cases, the chemokine is stromal cell-derived factor-1 (SDF-1). In some cases, SDF-1 is expressed in the bone marrow of a subject receiving a cell therapy comprising engineered immune cells. In one embodiment, the population comprising engineered immune cells has a greater percentage of CXCR4-positive cells compared to a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the population comprising engineered immune cells has a greater median percentage of CXCR4-positive cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% greater compared to the median percentage of CXCR4-positive cells expressed by a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the population comprising engineered immune cells is cultured for 2 weeks or less than 1 week, ex The population has a median percentage of CXCR4 positive cells that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the median percentage of CXCR4 positive cells expressed by a comparable population undergoing ex vivo expansion. In one aspect, the population comprising engineered immune cells has a greater density of CXCR4 on the cell surface of CXCR4 positive cells compared to the density of CXCR4 on the cell surface of a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one aspect, the density is measured by assessing the mean fluorescence intensity (MFI) of CXCR4 on the cell surface of CXCR4 positive cells. The CXCR4 positive cells can be CD3+, CD4+, CD8+, and any combination thereof. In some embodiments, cytotoxicity can be measured using an in vivo assay. In one embodiment, a reduction in cancer burden is observed in a subject when the subject is administered a population comprising engineered immune cells, as compared to the cancer burden observed in a comparable subject administered a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in a subject treated with a population comprising engineered immune cells, as compared to a comparable subject administered a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less.

[0011]

[0012] In one embodiment, there is more F-CART in the tumor of a mammal expressing the target for which F-CART and the CAR on C-CART show specificity, compared to C-CART. In one embodiment, there is more F-CART in the femur of a mammal expressing the target for which F-CART and the CAR on C-CART show specificity, compared to C-CART. F-CART and C-CART can be quantified in the tumor and / or femur of a mammal via CD45, CD2, and / or CAR expression. In one embodiment, there is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or up to about 20-fold more F-CART in the tumor and / or femur of a mammal compared to C-CART.

[0012]

[0013] In one embodiment, the cytotoxicity of an engineered immune cell, e.g., F-CART, is measured in an in vitro assay and compared to the cytotoxicity of a C-CART. In some embodiments, the cytotoxicity is measured in an in vivo assay. In one embodiment, the cytotoxicity is measured by measuring the cytotoxicity of IFNγ secreted by an engineered immune cell, e.g., a CAR-T+ cell. The level of cytotoxicity can be measured by quantifying the level of tumor suppression. In one embodiment, F-CART can secrete and / or express higher levels of IFNγ and / or IL-2 when contacted with a target for which the CAR shows specificity, compared to C-CART or equivalent cells undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, F-CART can secrete and / or express about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to about 100% more IFNγ and / or IL-2 when contacted with a cell expressing a target for which the CAR shows specificity, compared to C-CART. In one embodiment, greater cytotoxicity is observed in vivo. In one embodiment, cytotoxicity can be measured by quantifying the level of tumor reduction in a mammal with cancer treated with engineered immune cells, such as F-CART with a CAR receptor with specificity for cancer. Cancer reduction in a mammal can be measured by quantifying the level of fluorescence in a mammal with tumor cells containing a fluorescent protein. Lower fluorescence in mammals with tumor cells containing fluorescent proteins can indicate cytotoxicity of engineered immune cells against cancer. In some cases, the reduction of cancer in mammals treated with F-CART can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or up to about 100% more compared to mammals treated with C-CART cells. In one embodiment, the level of cell proliferation can be quantified. Cell proliferation can refer to cell number, cell clumping in culture, and / or cell division. Cell proliferation can be quantified using in vivo or in vitro assays. In some cases, cell proliferation can be measured by quantifying the number of cells using a cytometer and / or via in vitro assays, such as carboxyfluorescein succinimidyl ester (CFSE). In one embodiment, F-CART can proliferate more compared to C-CART when contacted with a target for which the CAR shows specificity.In one aspect, greater proliferation is observed in a population of F-CARTs, which may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to about 100% greater than a comparable population of C-CARTs when contacted with cells expressing a target for which the CAR exhibits specificity. In another aspect, the disclosure provides a method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising injecting a population of immune cells comprising engineered immune cells into a subject in need thereof, wherein the engineered immune cells have not been subjected to ex vivo expansion for 2 weeks or less than 1 week, or for less than 13, 12, 10, 9, 8, 7, or 6 days, and at least 2% of the population are stem memory T cells (TSCM). In some embodiments, at least 5% of the population are TSCM. In some embodiments, at least 10% of the population are TSCM. In some embodiments, at least 15% of the population is a TSCM. In some embodiments, at least 20% of the population is a TSCM. In some embodiments, at least 40% of the population is a TSCM. In some embodiments, at least 50% of the population is a TSCM. In some embodiments, at least 2%, 5%, 10%, 20%, 40%, 50%, or at least 60% of the population is a TSCM. In some embodiments, the TSCM is CD45RO. - CD62L +In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 1 week. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 72, 48, 36, 32, or 24 hours. In some embodiments, the TCR comprises (i) a ligand-binding domain specific for a ligand, and (ii) a transmembrane domain. In some embodiments, the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some embodiments, the ligand of the TCR or CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3 , CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GH R, Flt1, KDR, Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, P D1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.2. In some embodiments, the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1, and / or CD152. In some embodiments, the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS). In some embodiments, the CAR comprises at least two intracellular signaling domains. In some embodiments, the CAR comprises at least three intracellular signaling domains. In some embodiments, the CAR further comprises a hinge. In some embodiments, the hinge is derived from CD28, IgG1 and / or CD8α. In some embodiments, the CAR further comprises a signal peptide, the signal peptide being derived from IgG1, GM-CSF and / or CD8α. In some embodiments, the engineered immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells. In some embodiments, the engineered immune cells are from peripheral blood, and the peripheral blood cells are T cells, NK cells, and / or NKT cells. In some embodiments, the injection is intravenous. In some embodiments, the administration is at a dose of about 1×10 4In some embodiments, the administration comprises injecting about 1×10 5 In some embodiments, the administration comprises injecting about 3×10 5 The method further comprises injecting engineered immune cells from 1000 to 10000 / kg body weight. In some embodiments, at least 20% of the immune cells express a CAR and / or a TCR. In some embodiments, at least 40% of the immune cells express a CAR and / or a TCR. In some aspects, the method further comprises administering a secondary agent to the subject in need thereof. In some embodiments, the secondary agent is a therapeutically effective amount of an immunostimulant, an immunosuppressant, an antifungal, an antibiotic, an antiangiogenic, a chemotherapeutic, a radioactive, and / or an antiviral agent. In some embodiments, the immunostimulant is IL-2. In some aspects, the method further comprises obtaining peripheral blood from the subject in need thereof after the infusion. In some embodiments, the engineered immune cells from the peripheral blood are quantified. In some embodiments, the levels of cytokines are quantified. In some embodiments, the cytokines are IL-10, IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-4, IL-8, IL-12, and / or IFN-γ. In some embodiments, the method includes repeating the injection.

[0013]

[0014] In some embodiments, the populations provided herein are cultured for 2 weeks or 1 week or less, ex It is further characterized in that reduced depletion of cells in the population is observed as compared to depletion of cells in a comparable population undergoing in vivo expansion.

[0014]

[0015] In one aspect, reduced depletion of a population is characterized in that the population contains fewer cells expressing PD1 and LAG3. In some embodiments, the population is further characterized in that more proliferation, cytotoxicity, and / or bone marrow migration is observed in the population compared to the proliferation, cytotoxicity, and / or bone marrow migration of a comparable population that has undergone ex vivo expansion for 2 weeks or 1 week or less. In one aspect, cytotoxicity is measured in an in vitro assay. In one aspect, cytotoxicity is measured in an in vivo assay. In one embodiment, cytotoxicity is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1.0 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold higher in a population of cells comprising engineered immune cells compared to a comparable population comprising engineered immune cells that has undergone ex vivo expansion for 2 weeks or 1 week or less when the population comprising engineered immune cells and the comparable population are contacted with a target.

[0015]

[0016] In one embodiment, in vivo and / or in vitro expansion is at least 0.5 fold, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 50 fold, 100 fold, 500 fold, 1000 fold, 5000 fold, or 10000 fold higher in a population comprising engineered immune cells when the population and the equivalent population are contacted with a target, as compared to an equivalent population undergoing ex vivo expansion for 2 weeks or 1 week or less.

[0016]

[0017] In one embodiment, bone marrow migration is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold higher in a population comprising engineered immune cells compared to a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less when the population and the comparable population are in contact with a target.

[0017]

[0018] In one embodiment, the target may be a cancer cell or a chemokine. In some cases, the chemokine is stromal cell-derived factor-1 (SDF-1). In some cases, SDF-1 is expressed in the bone marrow of a subject receiving a cell therapy comprising engineered immune cells. In one embodiment, the population comprising engineered immune cells has a greater percentage of CXCR4-positive cells compared to a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the population comprising engineered immune cells has a greater median percentage of CXCR4-positive cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% greater compared to the median percentage of CXCR4-positive cells expressed by a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the population containing the engineered immune cells is cultured for 2 weeks or less than 1 week, ex The population has a median percentage of CXCR4 positive cells that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the median percentage of CXCR4 positive cells expressed by a comparable population undergoing ex vivo expansion. In one aspect, the population comprising engineered immune cells has a greater density of CXCR4 on the cell surface of CXCR4 positive cells compared to the density of CXCR4 on the cell surface of a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one aspect, the density is measured by assessing the mean fluorescence intensity (MFI) of CXCR4 on the cell surface of CXCR4 positive cells. The CXCR4 positive cells can be CD3+, CD4+, CD8+, and any combination thereof. In some embodiments, cytotoxicity can be measured using an in vivo assay. In one embodiment, a reduction in cancer burden is observed in a subject when the subject is administered a population comprising engineered immune cells, as compared to the cancer burden observed in a comparable subject administered a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in a subject treated with a population comprising engineered immune cells, as compared to a comparable subject administered a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In yet another embodiment, the present disclosure provides a population of engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). The present invention provides a method of producing a population of engineered immune cells, the method comprising: (a) activating a population of cells, including immune cells, with an activating moiety; and simultaneously (b) introducing a polynucleotide encoding at least a CAR, the CAR comprising (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain, thereby producing a population of engineered immune cells expressing the CAR. In one aspect, the activating moiety binds to the CD3 / T cell receptor complex and / or provides costimulation. In one aspect, the activating moiety is any one of an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the activating moiety is conjugated to a solid phase. In some embodiments, the solid phase is at least one of a bead, a plate, and / or a matrix. In one aspect, the solid phase is a bead. In one aspect, the introducing comprises transducing the population of cells with a viral vector and / or a transposon vector. In one aspect, the viral vector is a retroviral vector, a lentiviral vector, and / or an adeno-associated viral vector. In some embodiments, the transposon-based vector is a sleeping beauty vector and / or a PiggyBac vector. In one aspect, steps (a) and (b) are performed within 48 hours. In one aspect, steps (a) and (b) are performed within 24 hours. In one aspect, steps (a) and (b) are performed within 3 hours. In one aspect, steps (a) and (b) are performed within 1 hour. In some embodiments, steps (a) and (b) are performed within 30 minutes. In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, transduction comprises adding an infectious agent. In some embodiments, the infectious agent is polybrene. In some embodiments, the population of cells is about 10 4 / mL to about 10 8The cells are seeded at a density of 1000 / mL. In some embodiments, the viral vector is plated at a mean of infection (MOI) of about 0.1 to about 10. In some embodiments, the method further comprises stimulating the population of cells with a cytokine. In some embodiments, the cytokine is IL2, IL7, IL15 and / or IL21. In some embodiments, the TCR comprises (i) a ligand-binding domain specific for a ligand, and (ii) a transmembrane domain. In some embodiments, the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some embodiments, the ligand of the TCR or CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.2. In some embodiments, the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1, and / or CD152.In some embodiments, the intracellular signaling domain is from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS). In some embodiments, the CAR comprises at least two intracellular signaling domains. In some embodiments, the CAR comprises at least three intracellular signaling domains. In some embodiments. In some embodiments, the CAR further comprises a hinge. In some embodiments, the hinge is derived from CD28, IgG1 and / or CD8α. In some embodiments, the method further comprises enriching the immune cells prior to manipulation. In some embodiments, the enrichment comprises collecting a monocyte fraction. In some embodiments, the enrichment comprises sorting the immune cells from the monocyte fraction. In some embodiments, the enrichment comprises sorting the immune cells based on expression of one or more markers. In some aspects, the one or more markers comprise CD3, CD28, CD4 and / or CD8. In some aspects, the immune cells are sorted using an anti-CD3 antibody or an antigen-binding fragment thereof, and / or an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the immune cells are sorted using beads conjugated with an anti-CD3 antibody or an antigen-binding fragment thereof, and / or beads conjugated with an anti-CD28 antibody or an antigen-binding fragment thereof. In some embodiments, the engineered immune cell population is characterized by an enrichment of T cell memory cells (TCM) over effector memory T cells (TEM) in the population. In one aspect, at least 2% of the population are stem memory T cells (TSCM). In one aspect, at least 5% of the population are stem memory T cells (TSCM). In one aspect, at least 10% of the population are stem memory T cells (TSCM). In one aspect, at least 15% of the population are stem memory T cells (TSCM). In some embodiments, at least 20% of the population are TSCM. In some embodiments, at least 25% of the population are TSCM. In some embodiments, at least 40% of the population are TSCM. In some embodiments, at least 50% of the population are TSCM. In some embodiments, at least 2%, 5%, 10%, 20%, 40%, 50%, or at least 60% of the population are TSCM. In one aspect, the method further comprises injecting the population of engineered immune cells into a subject in need thereof within 72 hours of completing (a) and (b).In one aspect, the population is further characterized by observing reduced depletion of cells in the population as compared to depletion of cells in a comparable population undergoing a comparable method in which (a) and (b) are not performed simultaneously. In one aspect, reduced depletion of the population is characterized by the population comprising fewer cells that express PD1 and LAG3.

[0018]

[0019] In one embodiment, the population is further characterized by greater proliferation, cytotoxicity, and / or bone marrow migration observed in the population compared to proliferation, cytotoxicity, and / or bone marrow migration of a comparable population undergoing a comparable method in which (a) and (b) are not performed simultaneously. In one aspect, cytotoxicity is measured in an in vitro assay. In one aspect, cytotoxicity is measured in an in vivo assay. In one embodiment, cytotoxicity is quantified and is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1.0 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold higher in the population comprising engineered immune cells compared to a comparable population in which (a) and (b) are performed for greater than 24 hours when the population comprising engineered immune cells and a comparable population are contacted with a target.

[0019]

[0020] In one embodiment, in vivo and / or in vitro proliferation is at least 0.5 fold, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 50 fold, 100 fold, 500 fold, 1000 fold, 5000 fold, or 10000 fold higher in a population comprising engineered immune cells compared to a comparable population in which (a) and (b) are performed for greater than 24 hours when the population and the comparable population are contacted with a target.

[0020]

[0021] In one embodiment, bone marrow migration is at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 1.1-fold, 1.2-fold, 1.3 ...4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 1.9-fold, 1.1-fold, 4x, 1.5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x or 10x higher.

[0021]

[0022] In one aspect, the target may be a cancer cell or a chemokine. The chemokine is stromal cell-derived factor-1 (SDF-1), which may be expressed in the bone marrow of a subject receiving a population comprising engineered immune cells. In some embodiments, the population comprising engineered immune cells has a greater percentage of CXCR4-positive cells compared to a comparable population in which (a) and (b) are performed for more than 24 hours. In one aspect, the population comprising engineered immune cells has a greater median percentage of CXCR4-positive cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% greater compared to the median percentage of CXCR4-positive cells expressed by a comparable population in which (a) and (b) are performed for more than 24 hours. In one embodiment, the population comprising engineered immune cells has a greater median percentage of CXCR4 positive cells that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the median percentage of CXCR4 positive cells expressed by a comparable population in which (a) and (b) are performed for more than 24 hours. In one embodiment, the population comprising engineered immune cells has a greater density of CXCR4 on the cell surface of CXCR4 positive cells compared to the density of CXCR4 on the cell surface of a comparable population in which (a) and (b) are performed for more than 24 hours. The density of receptors on cell surfaces such as CXCR4 can be measured by assessing the mean fluorescence intensity (MFI) of CXCR4 on the cell surface of CXCR4 positive cells. In one embodiment, cytotoxicity is measured in an in vivo assay. In some cases, a reduction in cancer burden is observed in a subject when the subject is administered a population comprising engineered immune cells, compared to the cancer burden observed in a comparable subject administered a comparable population in which (a) and (b) are performed for more than 24 hours. In one embodiment, the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in a subject treated with a population comprising engineered immune cells, compared to a comparable subject administered a comparable population in which (a) and (b) are performed for more than 24 hours.

[0022]

[0023] In one aspect, provided herein is a point-of-care facility comprising a cell infusion device configured to inject a population of immune cells, including engineered immune cells that have not been subjected to ex vivo expansion for 2 weeks or more, or less than 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 day, wherein the population of immune cells is further characterized in that the cell memory T cells (TCM) in the population are enriched over the effector memory T cells (TEM); or at least 2% of the population are stem memory T cells (TSCM). In some embodiments, at least 5% of the population are stem memory T cells (TSCM). In some embodiments, at least 10% of the population are stem memory T cells (TSCM). In some embodiments, at least 15% of the population are stem memory T cells (TSCM). In some embodiments, at least 20% of the population are TSCM. In some embodiments, at least 40% of the population are TSCM. In some embodiments, at least 50% of the population are TSCM. In some embodiments, at least 2%, 5%, 10%, 20%, 40%, 50%, or at least 60% of the population are TSCM. In some embodiments, there are 2-fold more TCM compared to TEM. In some embodiments, there are 4-fold more TCM compared to TEM. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 5 days. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 3 days. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 2 days. In some embodiments, the engineered immune cells have been subjected to ex vivo expansion for less than 1 day. In some embodiments, the immune cells are T cells, NK cells, and / or NKT cells. In some aspects, the population is observed to have reduced depletion of cells in said population. and said reduced depletion is characterized in that said population comprises fewer cells that express PD1 and LAG3.

[0023]

[0024] Provided herein is a point-of-care facility comprising a cell processing device configured to perform the steps of: (a) receiving a population of cells, including immune cells, from a subject; and (b) activating the population of immune cells with an activating moiety and simultaneously introducing into the immune cells a polynucleotide encoding at least a chimeric antigen receptor (CAR), wherein the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain; and (c) injecting the population of immune cells of (b) into the subject within 2 weeks or less prior to performing (b). In some embodiments, step (c) is performed within 1 week or less prior to performing (b). In some embodiments, the immune cells are T cells, NK cells, and / or NKT cells. In some embodiments, the ligand of the CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V In some embodiments, the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1, and / or CD152.In some embodiments, the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10, and / or CD278 (ICOS). In some embodiments, the CAR comprises at least two intracellular signaling domains. In some embodiments, the CAR comprises at least three intracellular signaling domains. In some embodiments, the CAR further comprises a hinge. In some embodiments, the hinge is derived from CD28, IgG1, and / or CD8α. In some embodiments, the CAR further comprises a signal peptide, the signal peptide being derived from IgG1, GM-CSF, and / or CD8α. In some embodiments, the immune cell is a T cell, a NK cell, and / or a NKT cell. In some embodiments, the activating moiety binds to the CD3 / T cell receptor complex and / or provides costimulation. In some embodiments, the activating moiety is one of an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the viral vector and / or transposon vector comprises the polynucleotide. In one aspect, the viral vector is a retroviral vector, a lentiviral vector and / or an adeno-associated viral vector. In some embodiments, steps (a) and (b) are performed within 24 hours. In some embodiments, steps (a) and (b) are performed within 3 hours. In some embodiments, steps (a) and (b) are performed within 1 hour. In some embodiments, steps (a) and (b) are performed within 30 minutes. In some aspects, the population is further characterized by observing a reduced depletion of cells in said population, the reduced depletion being characterized by the population comprising fewer cells expressing PD1 and LAG3.

[0024]

[0025] In one aspect, provided herein is a population of cells comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), the population further characterized in that (i) central memory T cells (TCM) are enriched over effector memory T cells (TEM) in the population; and / or (ii) at least 2% of the population of cells are stem memory T cells (TSCM), the CAR comprising (i) a ligand binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In one aspect, at least 5% of the population is TSCM. In one aspect, at least 10% of the population is TSCM. In some embodiments, there are 2-fold more TCMs compared to TEMs. In some embodiments, there are 4-fold more TCMs compared to TEMs. In some embodiments, the immune cells are T cells, NK cells, and / or NKT cells. In some embodiments, the ligand of the CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V 6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or claudin 18.2. In one embodiment, the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1, and / or CD152.In one aspect, the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS). In some embodiments, the CAR comprises at least two intracellular signaling domains. In some embodiments, the CAR comprises at least three intracellular signaling domains. In some embodiments, the CAR further comprises a hinge. In some embodiments, the hinge is derived from CD28, IgG1 and / or CD8α. In some embodiments, the CAR further comprises a signal peptide, the signal peptide being derived from IgG1, GM-CSF and / or CD8α. In one aspect, the immune cells are T cells, NK cells and / or NKT cells. In one aspect, the population is cryopreserved. In one aspect, the population is not cryopreserved. In one aspect, the population is freshly sourced or comprises freshly sourced cells. In one aspect, the population is further characterized in that a reduced depletion of cells in the population is observed, the reduced depletion being characterized in that the population comprises fewer cells that express PD1 and LAG3.

[0025]

[0026] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering about 1×10 6Provided herein is a method comprising injecting a population of engineered immune cells expressing no more than 10 chimeric antigen receptors (CARs) and / or engineered T cell receptors (TCRs), wherein the engineered immune cells have not been subjected to ex vivo expansion for no more than 2 weeks or 1 week. In one aspect, the population of engineered immune cells exhibits a comparable level of anti-tumor activity in vivo compared to a population of 10 times more engineered immune cells expressing the same chimeric antigen receptors (CARs) and / or engineered T cell receptors (TCRs), but which have been subjected to ex vivo expansion for no more than 2 weeks or 1 week. In one aspect, the population of engineered immune cells is simultaneously activated and transduced with constructs expressing CARs and / or TCRs. In one aspect, the population of engineered immune cells is In one embodiment, the population of engineered immune cells has not been subjected to ex vivo expansion for 72 hours. In one embodiment, the population of engineered immune cells has not been subjected to ex vivo expansion for 72 hours. In one embodiment, the population of engineered immune cells has not been subjected to ex vivo expansion for 72 hours. 6A population of no more than 100 engineered immune cells has been prepared from peripheral blood mononuclear cells (PMBCs) through a process of simultaneous activation and transduction with a construct expressing a CAR and / or a TCR. In some embodiments, the infusion is performed within 1 week of simultaneous activation and transduction with a construct expressing a CAR and / or a TCR. In some embodiments, the simultaneous activation comprises performing activation and transduction within 48 hours. In some embodiments, the simultaneous activation comprises performing activation and transduction within 24 hours. In some embodiments, the simultaneous activation comprises performing activation and transduction within 3 hours. In some embodiments, the simultaneous activation comprises performing activation and transduction within 1 hour. In some embodiments, the simultaneous activation comprises performing activation and transduction within 30 minutes. In some embodiments, the simultaneous activation comprises performing activation and transduction simultaneously. In some embodiments, at least 2% of the population are stem memory T cells (TSCM). In some embodiments, at least 5% of the population are stem memory T cells (TSCM). In some embodiments, at least 10% of the population are stem memory T cells (TSCM). In some embodiments, at least 15% of the population are stem memory T cells (TSCM). In some embodiments, at least 20% of the population are TSCM. In some embodiments, at least 40% of the population are TSCM. In some embodiments, at least 50% of the population are TSCM. In some embodiments, at least 2%, 5%, 10%, 20%, 40%, 50%, or at least 60% of the population are TSCM. In some embodiments, the infusion is about 10 5 In some embodiments, the injection is about 10 4 In some embodiments, the injection is about 10 3In some embodiments, the engineered immune cell is a T cell, a NK cell, and / or a NKT cell. In some embodiments, the TCR comprises (i) a ligand-binding domain specific to a ligand, and (ii) a transmembrane domain. In some embodiments, the CAR comprises (i) a ligand-binding domain specific to a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some embodiments, the ligand of the TCR or CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.2. In some embodiments, the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1, and / or CD152. In some embodiments, the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10, and / or CD278 (ICOS). In some embodiments, the CAR comprises at least two intracellular signaling domains.In some embodiments, the CAR comprises at least three intracellular signaling domains. In some embodiments, the CAR further comprises a hinge. In some embodiments, the hinge is derived from CD28, IgG1 and / or CD8α. In embodiments, the CAR further comprises a signal peptide, the signal peptide being derived from IgG1, GM-CSF and / or CD8α. In some embodiments, the engineered immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells. In some embodiments, the engineered immune cells are derived from peripheral blood, and the peripheral blood cells are T cells. In one aspect, the method further comprises, after administration, obtaining peripheral blood from the subject in need thereof. In one aspect, the engineered immune cells in the subject are quantified from the peripheral blood. In one aspect, the level of a growth factor in the subject is quantified. In some cases, the growth factor is selected from the group consisting of IL-10, IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-4, IL-8, IL-12, and / or IFN-γ. In some embodiments, the method comprises repeating the infusion. In some embodiments, the population of immune cells is allogeneic to the subject in need thereof. In some embodiments, the population of immune cells is autologous to a subject in need thereof. In some embodiments, the subject has cancer. In one aspect, the cancer may be the target. In one aspect, the cancer is a hematological cancer. In one aspect, the hematological cancer is leukemia, myeloma, lymphoma, and / or a combination thereof. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), and / or acute lymphoblastic leukemia (ALL). In some embodiments, the lymphoma is mantle cell lymphoma (MCL), T-cell lymphoma, Hodgkin's lymphoma, and / or non-Hodgkin's lymphoma. In some embodiments, the cancer is the target and is a solid cancer. In some embodiments, the target solid cancer is selected from the group including nephroblastoma, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, or bladder cancer. In some embodiments, the subject has been pretreated with chemotherapy prior to administration.In some embodiments, chemotherapy comprises administration of fludarabine, cyclophosphamide and / or cytarabine. In one aspect, the population is further characterized by observing more proliferation, cytotoxicity and / or bone marrow migration in the population compared to the proliferation, cytotoxicity and / or bone marrow migration of a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one aspect, the cytotoxicity is measured in an in vitro assay. In one aspect, the cytotoxicity is measured in an in vivo assay.

[0026]

[0027] In one aspect, provided herein is a method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising injecting a population of immune cells comprising the engineered immune cells into a subject in need thereof, wherein the engineered immune cells have not been subjected to ex vivo expansion for 2 weeks or 1 week or less, and wherein the population is further characterized in that more proliferation is observed in the population compared to proliferation in a comparable population that has undergone ex vivo expansion for 2 weeks or 1 week or less. In one aspect, proliferation is at least 1-fold higher in the population compared to a comparable population that has undergone ex vivo expansion for 2 weeks or 1 week or less when the population and the comparable population are in contact with a target.

[0027]

[0028] Provided herein is a method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising injecting a population of immune cells comprising the engineered immune cells into a subject in need thereof, wherein the engineered immune cells have not been subjected to ex vivo expansion for no more than 2 weeks or 1 week, and the population is further characterized in that greater cytotoxicity is observed in the population compared to the cytotoxicity of a comparable population that has undergone ex vivo expansion for no more than 2 weeks or 1 week. In one aspect, the cytotoxicity is greater in the population than in the comparable population that has undergone ex vivo expansion for no more than 2 weeks or 1 week, when the population and the comparable population contact a target. At least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1.0 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold higher as compared to a comparable population undergoing expansion.

[0028]

[0029] Provided herein is a method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising injecting a population of immune cells comprising engineered immune cells into a subject in need thereof, wherein the engineered immune cells have not been subjected to ex vivo expansion for 2 weeks or 1 week or less, and wherein the population is further characterized in that greater bone marrow migration is observed in the population compared to a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In some embodiments, bone marrow migration is at least 1-fold higher in the population compared to a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less when the population and the comparable population are in contact with a target. In one aspect, the population is further characterized in that central memory T cells (TCM) are more abundant than effector memory T cells (TEM) in the population. In one aspect, the TCM are CD45RO+CD62L+. In one aspect, the TEM are CD45RO+CD62L-. In one aspect, the engineered immune cells have been subjected to ex vivo expansion for less than 5 days. In one aspect, the engineered immune cells have been subjected to ex vivo expansion for less than 4 days. In one aspect, the engineered immune cells have been subjected to ex vivo expansion for less than 72 hours. In one aspect, the engineered immune cells have been subjected to ex vivo expansion for less than 48 hours. In one aspect, the engineered immune cells have been subjected to ex vivo expansion for less than 24 hours. In one aspect, the target is a cancer cell, a TCR or CAR ligand, or a chemokine. In some embodiments, the chemokine is stromal cell-derived factor-1 (SDF-1), and SDF-1 is expressed in the bone marrow of the subject. In some cases, the population has a greater percentage of CXCR4 positive cells compared to a comparable population that has been subjected to ex vivo expansion for 2 weeks or 1 week or less. In one aspect, the population has a median percentage of CXCR4 positive cells that is at least 10% greater than the median percentage of CXCR4 positive cells expressed by a comparable population that has undergone ex vivo expansion for 2 weeks or 1 week or less.In one embodiment, the population has a greater density of CXCR4 on the cell surface of CXCR4-positive cells compared to the density of CXCR4 on the cell surface of a comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the density is measured by assessing the mean fluorescence intensity (MFI) of CXCR4 on the cell surface of CXCR4-positive cells. In one embodiment, the cytotoxicity is measured in an in vivo assay. In some cases, a reduction in cancer burden is observed in a subject administered the population compared to the cancer burden observed in a comparable subject administered the comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one embodiment, the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in a subject treated with the population compared to the cancer burden observed in a comparable subject administered the comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one aspect, the cancer burden is reduced by at least 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold in subjects treated with the population compared to a comparable subject administered the comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In one aspect, a complete response (CR) is observed in subjects administered the population compared to the cancer burden observed in a comparable subject administered the comparable population undergoing ex vivo expansion for 2 weeks or 1 week or less. In some embodiments, a complete response (CR) is observed in subjects administered the population for 2 weeks or 1 week or less ex. A partial response (PR) is observed compared to the cancer burden observed in a comparable subject receiving a comparable population undergoing in vivo expansion. In one embodiment, the population is up to 1×10 cells / kg / body weight. 4 In one embodiment, the population comprises 1 kg of engineered immune cells. Approximately 1 x 10 cells per body weight 4 engineered immune cells up to approximately 1 x 10 cells per kg body weight 5The CAR comprises an engineered immune cell. In some cases, the engineered immune cell is a T cell, a NK cell, and / or a NKT cell. In some cases, the TCR comprises (i) a ligand-binding domain specific to a ligand, and (ii) a transmembrane domain. In one embodiment, the CAR comprises (i) a ligand-binding domain specific to a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In one embodiment, the ligand of the TCR or CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44 V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or claudin 18.2. In one embodiment, the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1, and / or CD152. In one embodiment, the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10, and / or CD278 (ICOS). In some cases, the CAR comprises at least two intracellular signaling domains. In some cases, the CAR comprises at least three intracellular signaling domains.In some cases, the CAR further comprises a hinge. The hinge may be derived from CD28, IgG1 and / or CD8α. In one embodiment, the engineered immune cell is derived from peripheral blood, umbilical cord blood, bone marrow and / or induced pluripotent stem cells. In some cases, the engineered immune cell is derived from peripheral blood, and the peripheral blood cell is a T cell.

[0029] Incorporating references

[0030] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0030]

[0031] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0031] [Figure 1]

[0032] FIG. 1 shows the multiplicity of infection (MOI) versus percent CAR positivity in F-CART cells. [Diagram 2]

[0033] FIG. 2 shows a comparison of the percent CAR-positive expression between conventional CART (C-CART) and fast-CART (F-CART) generated cells. [Diagram 3]

[0034] FIG. 3 shows phenotypic analysis of control (starting material) and F-CART cells using flow cytometry. [Figure 4A]

[0035] FIG. 4A shows a linear graphical representation of cell proliferation of anti-CD19F-CART cells versus anti-CD19C-CART cells. [Figure 4B] Figure 4B shows the fold expansion of anti-CD19F-CART cells versus anti-CD19C-CART cells. [Diagram 5]

[0036] FIG. 5 shows the in vitro killing efficacy of anti-CD19F-CART cells versus anti-CD19C-CART cells over a 50 hour period. [Figure 6A]

[0037] FIG. 6A shows the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in control cells (non-transduced), F-CART, and C-CART when cocultured with Molt4 (CD19-) or Raji (CD19+) tumor cells at a 1:1 ratio. [Figure 6B] FIG. 6B shows the expression of TNF-α in control cells (non-transduced), F-CART, and C-CART when co-cultured with Molt4 (CD19−) or Raji (CD19+) tumor cells at a 1:1 ratio. [Figure 6C] FIG. 6C shows the expression of IL-2 in control cells, F-CART, and C-CART when cocultured with Molt4 (CD19−) or Raji (CD19+) tumor cells at a 1:1 ratio. [Figure 6D] FIG. 6D shows expression of IFN-γ in control cells (non-transduced), F-CART, and C-CART when co-cultured with Molt4 (CD19−) or Raji (CD19+) tumor cells at a 1:1 ratio. [Figure 7A]

[0038] Figure 7A shows bioluminescence imaging of mice engrafted with Molt4 or Raji tumor cells and treated with control T cells (non-transduced), C-CART cells, or F-CART cells at a total dose of 2e6, 5e5, or 5e4 cells / mouse. [Figure 7B] FIG. 7B shows a graphical summary of bioluminescence imaging days after injection of control (non-transduced) T cells, C-CART cells, or F-CART cells at 0.5×10 6 cells / mouse. [Figure 8]

[0039] FIG. 8 shows the change in body weight of mice engrafted with Raji tumor cells followed by treatment with control (non-transduced) T cells, C-CART cells, or F-CART cells at 0.5×10 6 cells / mouse. [Figure 9]

[0040] Figure 9 shows tumor volumes in mice engrafted with Raji tumor cells and subsequently treated with control, T cells, C-CART cells, or F-CART cells. [Figure 10]

[0041] FIG. 10 shows quantification of cells in peripheral blood of mice engrafted with Raji tumor cells followed by treatment with control (non-transduced) or high dose (2×10 cells / mouse), medium dose (5×10 cells / mouse), or low dose (5×10 cells / mouse) of F-CART cells. [Figure 11A]

[0042] Figure 11A shows phenotypic analysis of Lag3 vs. PD-1 in F-CART and C-CART cells performed on day 6 of three individual donors upon stimulation with K562-CD19+ cells. [Figure 11B] Figure 11B shows phenotypic analysis of Lag3 vs. PD-1 in F-CART and C-CART cells performed on day 10 of three individual donors upon stimulation with K562-CD19+ cells. [Figure 11C] FIG. 11C shows the average expression of PD1+Lag3+ cells on day 6 versus day 10 for three individual donors upon stimulation with K562-CD19+ cells. [Figure 12]

[0043] FIG. 12 shows flow cytometry plots showing the numbers of TSCM, TCM, TEFF and TEM cells on F-CART cells from three individual donors upon stimulation with K562-CD19+ cells. [Figure 13A]

[0044] Figure 13A shows expansion, persistence, and copy number of FAST-CAR+ cells in subject XF001 up to 56 days post-infusion. [Figure 13B] Figure 13B shows the body temperature of subject XF001 after infusion of FAST-CAR+ cells. [Figure 13C] Figure 13C shows the concentration of IL-6 in the blood of subject XF001 after infusion of FAST-CAR+ cells. [Figure 13D]Figure 13D shows the concentration of C-reactive protein (CRP) in the blood of subject XF001 after infusion of FAST-CAR+ cells. [Figure 14A]

[0045] Figure 14A shows the body temperature of subject F01 after infusion of FAST-CAR+ cells. [Figure 14B] Figure 14B shows the FAST-CAR+ cell copy number, FAST-CAR+ copy number in peripheral blood, and FAST-CAR+ copy number in bone marrow for subject F01. [Figure 14C] Figure 14C shows the levels of growth factors (INF-γ, IL-10, sCD25, IL-6, and CRP) in the peripheral blood and body temperature of subject F01 after infusion of FAST-CAR+ cells. [Figure 15]

[0046] Figure 15 shows the treatment outcomes and efficacy of F-CART in 9 different subjects: Cytokine Release Syndrome (CRS), Neurotoxicity (NT), Complete Response (CR), Mean Residual Disease (MRD), Allogeneic Stem Cell Transplant (Allo-SCT). [Figure 16A]

[0047] FIG. 16A shows flow cytometry plots showing the numbers of TSCM, TCM, TEFF, and TEM cells in F-CART versus C-CART cells from individual donors. [Figure 16B] FIG. 16B shows a summary of the flow cytometry results. [Figure 16C] FIG. 16C shows a graphical summary of the percentage of TSCM, TCM, TEM, and TEFF in F-CART vs. C-CART of three individual donors upon stimulation with K562-CD19+ cells. [Figure 17A]

[0048] Figure 17A shows the fold expansion proliferation of F-CART cells versus C-CART cells at days 8, 12, and 18 after manipulation. [Figure 17B] Figure 17B shows the percentage of PD-1 and LAG3 on days 6 and 10 after manipulation of F-CART and C-CART cells. [Figure 17C]Figure 17C shows flow cytometry results of C-CART and F-CART cells from individual donors stained for PD-1 and LAG3. [Figure 17D] FIG. 17D shows the maintenance of in vitro cytotoxicity in co-culture assays of C-CART and F-CART prepared from healthy donors: C-CART cultured with CD19+ tumor cells, F-CART cultured with CD19+ tumor cells, non-transduced cells cultured with CD19+ tumor cells, and tumor only cells (Hela-CD19). [Figure 18] FIG. 18 shows IL-2 and IFNγ secretion of C-CART and F-CART prepared from healthy donors: C-CART cultured with CD19+ tumor cells, F-CART cultured with CD19+ tumor cells, non-transduced cells cultured with CD19+ tumor cells, and a medium only control. [Figure 19A]

[0049] FIG. 19A shows expansion of F-CART and C-CART cells from human sample GC007F. [Figure 19B] FIG. 19B shows the cell phenotypes of F-CART and C-CART in sample GC007F. [Figure 19C] FIG. 19C shows a pie chart of the cell phenotype of sample GC007F. [Figure 19D] FIG. 19D shows a graphical summary of the cell phenotype data via percentages of T cell subsets. [Figure 19E] Figure 19E shows the % of PD1+LAG3+ CAR-T cells in C-CART cells and F-CART cells on days 6 and 9, respectively. [Figure 20A]

[0050] FIG. 20A shows the maintenance of in vitro cytotoxicity in real-time cell analysis (RTCA) assays of C-CART and F-CART prepared from patients: C-CART cultured with CD19+ tumor cells, F-CART cultured with CD19+ tumor cells, non-transduced cells cultured with CD19+ tumor cells, and tumor only cells (Hela-CD19). [Figure 20B]FIG. 20B shows the retention of cytokine profiles in ELISA of the supernatants of co-cultured cells. [Figure 20C] Figure 20C shows the maintenance of cytotoxicity of patient-prepared F-CAR versus C-CART as determined in a luciferase assay. [Figure 21A]

[0051] Figure 21A shows bioluminescence imaging of NOG mice engrafted with Raji tumor cells and treated with control (media only), T cells, C-CART cells or F-CART cells at doses of 2e6 cells / mouse (high dose) or 5e4 cells / mouse (low dose). [Figure 22A]

[0052] FIG. 22A shows a tumor engraftment and treatment profile for a leukemia mouse model. [Figure 22B] Figure 22B shows phenotypic analysis of bone marrow from mice treated with F-CART cells or C-CART cells 10 days after treatment. [Figure 22C] FIG. 22C shows the number of CD45+CD2+CART+ cells in the femurs of F-CART- and C-CART-treated mice. [Figure 22D] FIG. 22D shows the expression of CXCR4 in the CD4 versus CD8 fraction of F-CART- and C-CART-treated mice. [Figure 22E] FIG. 22E shows the percentage of CXCR4 in the CD4 versus CD8 fraction of F-CART- and C-CART-treated mice. [Figure 22F] FIG. 22F shows the MFI of the CXCR4 fraction in the CD4 versus CD8 fraction of F-CART- and C-CART-treated mice. [Figure 22G] FIG. 22G shows a graphical representation of the results of a transwell migration assay of F-CART cells versus C-CART cells and mouse SDF-1α. [Fig. 22H] FIG. 22H shows a graphical representation of the results of a transwell migration assay of F-CART cells versus C-CART cells and human SDF-1α. [Figure 23A]

[0053] FIG. 23A shows a schematic of presentation of NY-ESO-1 fragments by HLA-A*02 on cancer cells and recognition of the NY-ESO-1 fragments by T cells expressing engineered TCRs. [Figure 23B] FIG. 23B shows a comparison of the proliferation capacity of FTCRT and CTRCT cells, both engineered to bind fragments of NY-ESO-1. [Figure 23C] FIG. 23C shows a comparison of lymphocyte subpopulations in FTCRT and CTRCT cells, both of which were engineered to bind fragments of NY-ESO-1. [Figure 23D] FIG. 23D shows a comparison of lymphocyte depletion in FTCRT and CTRCT cells, both engineered to bind fragments of NY-ESO-1. [Figure 23E] FIG. 23E shows a comparison of target cytotoxicity of FTCRT and CTRCT cells, both engineered to bind fragments of NY-ESO-1. [Figure 23F] FIG. 23F shows a differential comparison of target cytotoxicity of FTCRT and CTRCT cells, both engineered to bind fragments of NY-ESO-1. [Figure 24A]

[0054] Figure 24A shows CAR transduction efficiency in GC022 cells via conventional CART and FCART methods. [Figure 24B] Figure 24B shows the cytotoxicity of CAR-expressing GC022 cells produced via traditional CART and FCART methods against target cells. [Figure 24C] Figure 24C shows the cell expansion growth potential of CAR-expressing GC022 cells produced via conventional CART and FCART methods. [Figure 24D] Figure 24D shows the cytotoxicity of CAR-expressing GC022 cells produced via conventional CART and FCART methods against target cells, where the CAR-expressing GC022 cells are expanded via antigen stimulation. [Figure 24E]Figure 24E shows a comparison of lymphocyte subpopulations in CAR-expressing GC022 cells produced via traditional CART and FCART methods. [Figure 24F] Figure 24F shows a comparison of depletion in CAR-expressing GC022 cells produced via traditional CART and FCART methods. [Figure 24G] Figure 24G shows bioluminescence imaging of mice engrafted with tumor cells and treated with control T cells (non-transduced) or CAR-expressing GC022 cells produced via conventional CART and FCART methods. [Fig. 24H] Figure 24H shows a graphical summary of bioluminescence imaging days after injection with control (non-transduced) T cells or CAR-expressing GC022 cells produced via conventional CART and FCART methods. [Figure 24I] Figure 24I shows the change in body weight of mice engrafted with tumor cells and subsequently treated with control (non-transduced) T cells or CAR-expressing GC022 cells produced via conventional CART and FCART methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032]

[0055] The implementation of some methods disclosed herein, unless otherwise specified, employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art.See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); Current Protocols in Molecular Biology series (eds. FMAusubel et al.); Methods In Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (eds. MJMacPherson, BDHames and GRTaylor (1995)); Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (ed. RIFreshney (2010)).

[0033]

[0056] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a chimeric transmembrane receptor polypeptide" includes a plurality of chimeric transmembrane receptor polypeptides.

[0034]

[0057] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" may mean within 20%, 10%, 5%, or 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" is intended to mean within an acceptable error range for the particular value.

[0035]

[0058] "Cell" as used herein may generally refer to a biological cell. A cell may be a structural, functional, and / or biological unit of an organism. A cell may be from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, unicellular eukaryotic cells, protozoan cells, cells from plants, animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), and the like. Sometimes the cells are not from a natural organism (e.g., cells may be synthetically produced, sometimes referred to as artificial cells). Of particular importance are immune cells, e.g., cells from mammals, such as test animals and humans. These are immune cells derived from the body.

[0036]

[0059] The term "antigen" as used herein refers to a molecule or a fragment thereof that can bind to a selective binding agent. As an example, an antigen may be a ligand that can bind to a selective binding agent, such as a receptor. As another example, an antigen may be an antigenic molecule that can bind to a selective binding agent, such as an immunological protein (e.g., an antibody). An antigen may also refer to a molecule or a fragment thereof that can be used in an animal to generate an antibody that can bind to the antigen. In some cases, an antigen may be bound to a substrate (e.g., a cell membrane). Alternatively, an antigen may not be bound to a substrate (e.g., a secreted molecule, such as a secreted polypeptide).

[0037]

[0060] The term "antibody" as used herein refers to a protein-like binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies), as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, different classes of immunoglobulins (Ig) (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Derivatives, variants, or fragments thereof may refer to functional derivatives or fragments that retain the binding specificity of the corresponding antibody (e.g., complete and / or partial). Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable fragments (scFv), minibodies, diabodies, and single domain antibodies ("sdAb" or "nanobody" or "camelid"). The term antibody includes optimized, engineered, or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (e.g., antibodies optimized in fragment crystallizable regions) and multispecific antibodies (e.g., bispecific antibodies). In some cases, an antibody may exhibit binding specificity to at least 1, 2, 3, 4, 5 or more different antigens. In some cases, an antibody may exhibit binding specificity to up to 5, 4, 3, 2, or 1 antigen.

[0038]

[0061] The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance in nucleic acid molecules containing them. The term nucleotide as used herein may also refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be performed using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxaldehyde (C6H ... Examples include carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides include, but are not limited to, [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, available from Molecular Probes, Eugene, Oreg. Nucleotide), BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, Fluorescein-12-UTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP.Nucleotide can also be labeled or marked by chemical modification. Chemically modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTP can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0039]

[0062] The terms "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs, in either single-stranded, double-stranded, or multiple-stranded form. A polynucleotide may be exogenous or endogenous to a cell. A polynucleotide may be present in a cell-free environment. A polynucleotide may be a gene or a fragment thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. A polynucleotide may contain one or more analogs, such as altered backbones, sugars, or nucleobases. Modifications to the nucleotide structure, if present, can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acids, xenonucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), Include thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queosine, and wyosine.Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, one or more loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, such as cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.The sequence of nucleotides may be interrupted by non-nucleotide components.

[0040]

[0063] The term "gene" as used herein refers to nucleic acids (e.g., DNA, e.g., genomic DNA and cDNA) and their corresponding nucleotide sequences involved in encoding an RNA transcript. The term for genomic DNA as used herein includes regulatory regions in addition to intervening non-coding regions, and may include 5' and 3' ends. In some uses, the term encompasses the transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region is expected to contain an "open reading frame" that encodes a polypeptide. In some uses of the term, a "gene" includes only the coding sequences (e.g., "open reading frame" or "coding region") necessary to encode a polypeptide. In some cases, a gene does not encode a polypeptide, e.g., ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only the transcribed sequence, but also non-transcribed regions, such as upstream and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or native gene in its natural location in the genome of an organism. A gene may refer to an "exogenous gene" or non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism, but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) that includes mutations, insertions and / or deletions.

[0041]

[0064] The terms "target polynucleotide" and "target nucleic acid" as used herein refer to a nucleic acid or polynucleotide targeted by an actuator moiety of the present disclosure. A target polynucleotide may be DNA (e.g., endogenous or exogenous). DNA may refer to a template for generating an mRNA transcript and / or various regulatory regions that regulate the transcription of an mRNA from a DNA template. A target polynucleotide may be a portion of a larger polynucleotide, such as a chromosome or a region of a chromosome. A target polynucleotide may refer to an extrachromosomal sequence (e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) or a region of an extrachromosomal sequence. A target polynucleotide may be RNA. RNA may be, for example, an mRNA that may serve as a template for encoding a protein. A target polynucleotide comprising an RNA may contain various regulatory regions that regulate the translation of a protein from an mRNA template. A target polynucleotide may code for a gene product (e.g., DNA encoding an RNA transcript or RNA encoding a protein product) or may contain regulatory sequences that regulate the expression of a gene product. Generally, the term "target sequence" refers to a nucleic acid sequence in a single strand of a target nucleic acid. A target sequence may be a gene, a regulatory sequence, a genomic DNA sequence, or a nucleic acid sequence. The target polynucleotide may be part of a cell-free nucleic acid, such as NA, cfDNA and / or cfRNA, cDNA, fusion genes, and RNA, such as mRNA, miRNA, rRNA, etc. When targeted by an actuator moiety, the target polynucleotide may result in altered gene expression and / or activity. When targeted by an actuator moiety, the target polynucleotide may result in an edited nucleic acid sequence. The target nucleic acid may include a nucleic acid sequence that may not be related to any other sequence in the nucleic acid sample by a single nucleotide substitution. The target nucleic acid may include a nucleic acid sequence that may not be related to any other sequence in the nucleic acid sample by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. In some embodiments, the substitution may not be within 5, 10, 15, 20, 25, 30, or 35 nucleotides from the 5' end of the target nucleic acid. In some embodiments, the substitution may not be within 5, 10, 15, 20, 25, 30, or 35 nucleotides from the 3' end of the target nucleic acid.

[0042]

[0065] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., into an mRNA or other RNA transcript) and / or the transcribed mRNA is then translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in eukaryotic cells. With respect to expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., an RNA, such as an mRNA) and / or a polypeptide sequence compared to its expression level in a wild-type context, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., an RNA, such as an mRNA) and / or a polypeptide sequence compared to its expression level in a wild-type context.

[0043]

[0066] The terms "complement", "complements", "complementary" and "complementarity" as used herein generally refer to a sequence that is sufficiently complementary to a given sequence and can hybridize. In some cases, a sequence hybridized with a given nucleic acid is referred to as the "complement" or "reverse complement" of a given molecule if its base sequence over a given region is capable of complementary binding to the base sequence of its binding partner, for example, so that AT, AU, GC and GU base pairs are formed. Generally, a first sequence that can hybridize to a second sequence can specifically or selectively hybridize to a second sequence, such that hybridization to a second sequence or a set of second sequences is preferred (e.g., thermodynamically more stable under a given set of conditions, e.g., stringent conditions commonly used in the art) over hybridization to non-target sequences during a hybridization reaction. Typically, hybridizable sequences have some degree of sequence complementarity over all or part of their respective lengths, for example, 25% to 100% complementarity, for example, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.Sequence identity, e.g. for purposes of assessing percent complementarity, can be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, e.g., www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html). (See EMBOSS Water aligner, available at http: / / www.emboss.com / .) Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including the default parameters.

[0044]

[0067] Complementarity may be complete or substantial / sufficient. Complete complementarity between two nucleic acids may mean that the two nucleic acids can form a duplex in which every individual base in the duplex is bound to a complementary base by Watson-Crick pairing. Substantial or sufficient complementarity may mean that the sequence of one strand is not completely and / or completely complementary to the sequence of the opposite strand, but sufficient binding occurs between the bases of the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using sequences and standard mathematical calculations to predict the Tm of hybridized strands, or by experimental determination of Tm by using routine methods.

[0045]

[0068] The term "modulate," as used herein in reference to expression or activity, refers to altering the level of expression or activity. Modulation may occur at the transcriptional and / or translational level.

[0046]

[0069] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues linked by peptide bonds. The term does not include a specific length of the polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant technology, chemical synthesis or enzymatic synthesis, or naturally occurring. The term also applies to amino acid polymers that contain at least one modified amino acid in addition to naturally occurring amino acid polymers. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length and includes full-length proteins, as well as proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers that have been modified by any other manipulation, such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with a labeling moiety. The terms "amino acid" and "amino acids" as used herein generally refer to natural and unnatural amino acids, including, but not limited to, modified amino acids and amino acid analogs. Modified amino acids can include natural and unnatural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur in the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D- and L-amino acids.

[0047]

[0070] The terms "derivative," "variant," and "fragment," as used herein with respect to a polypeptide, refer to a polypeptide that is related to a wild-type polypeptide, for example, by either amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide may contain one or more amino acid changes (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, as compared to the wild-type polypeptide.

[0048]

[0071] The term "percent (%) identity" as used herein refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to amino acid (or nucleic acid) residues of a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be disregarded for comparison purposes). Alignment refers to a percentage of identity. Alignment can be achieved in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, ALIGN, or Megalign (DNASTAR) software, for the purpose of determining percent identity. The percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acids or nucleotides at the same position of the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0049]

[0072] The term "peripheral blood lymphocytes" (PBLs) and grammatical equivalents, as used herein, may refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes may refer to lymphocytes that are not localized to an organ. Peripheral blood lymphocytes may include T cells, NK cells, B cells, or any combination thereof.

[0050]

[0073] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0051]

[0074] The terms "treatment" and "treating" as used herein refer to an approach to obtain beneficial or desired results, including, but not limited to, therapeutic benefit and / or preventive benefit. For example, treatment may include administering a system or cell population disclosed herein. Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions or symptoms under treatment. For preventive benefit, the composition can be administered to a subject at risk of developing a particular disease, condition or symptom, or to a subject who has reported one or more physiological symptoms of the disease, even if the disease, condition or symptom has not yet manifested.

[0052]

[0075] The term "TIL" or tumor infiltrating lymphocytes and grammatical equivalents, as used herein, may refer to cells isolated from tumors. For example, TILs may be cells that have migrated into tumors. TILs may also be cells that have infiltrated into tumors. TILs may be any cell found in tumors. For example, TILs may be T cells, B cells, monocytes, natural killer (NK) cells, or any combination thereof. TILs may be a mixed population of cells. A population of TILs may include cells of different phenotypes, cells of different degrees of differentiation, cells of different lineages, or any combination thereof.

[0053]

[0076] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition comprising a system of the present disclosure, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells), sufficient to result in a desired activity when administered to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition sufficient to delay the onset of, halt the progression of, alleviate or relieve at least one symptom of a disorder treated by the method of the present disclosure.

[0054]

[0077] In one aspect, the disclosure provides a method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). In one aspect, the method comprises injecting a population of immune cells comprising the engineered immune cells into a subject in need thereof. In one aspect, the engineered immune cells have not been subjected to ex vivo expansion for more than two weeks. In one aspect, the population comprises a central memory in the population. In one aspect, the present disclosure provides a population of cells comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). In one aspect, the population of cells is further characterized in that (i) the central memory T cells (TCM) in the population are enriched over the effector memory T cells (TEM), and / or (ii) at least 2% of the population of cells are stem memory T cells (TSCM). In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to a subject about 1×10 6 immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). 6 In one embodiment, the method comprises injecting a population of engineered immune cells of about 1×10 6 No more than one population of engineered immune cells have been subjected to ex vivo expansion for more than two weeks.

[0055]

[0078] In some embodiments, the engineered immune cells are subjected to ex vivo expansion for less than 1 week. In one aspect, the engineered immune cells are subjected to ex vivo expansion for less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, less than 1 day, less than 12 hours, less than 6 hours, less than 3 hours, or not subjected to expansion. In one aspect, the engineered immune cells are subjected to ex vivo expansion for less than 1 week, less than 72 hours, less than 48 hours, or less than 24 hours.

[0056]

[0079] In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to a subject about 1×10 6 human T-cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). 6 In one embodiment, the method comprises injecting a population of engineered immune cells of about 1×10 6A population of cells of engineered immune cells or less have not been subjected to ex vivo expansion for more than 2 weeks. In some embodiments, the population of engineered immune cells exhibits a comparable level of anti-tumor activity in vivo compared to a population of 10-fold more engineered immune cells expressing the same chimeric antigen receptor (CAR) and / or engineered T cell receptor (TCR) but subjected to ex vivo expansion for more than 2 weeks. In some embodiments, the population of engineered immune cells exhibits a comparable level of anti-tumor activity in vivo compared to a population of 18-fold, 15-fold, 12-fold, 10-fold, 8-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1-fold more engineered immune cells expressing the same chimeric antigen receptor (CAR) and / or engineered T cell receptor (TCR) but subjected to ex vivo expansion for more than 2 weeks.

[0057]

[0080] In some embodiments, the engineered immune cells are phenotyped to comprise central memory T cells (TCM). In some embodiments, the TCM cells are CD45RO+CD62L+. In some embodiments, the engineered immune cells are phenotyped to comprise effector memory T cells (TEM). In some embodiments, the TEM are CD45RO+CD62L-. In some embodiments, the engineered immune cells are phenotyped to comprise effector T cells (TEFF). In some embodiments, the TEFF cells are CD45RO - CD62L - In some embodiments, the engineered immune cells are phenotyped to comprise stem central memory T cells (TSCM). In some embodiments, the TSCM cells are CD45RO - CD62L +In some embodiments, the cells that can be utilized in the methods provided herein can be positive or negative for a given factor. In some embodiments, the cells utilized in the methods provided herein can be CD3+ cells, CD3- cells, CD5+ cells, CD5- cells, CD7+ cells, CD7- cells, CD14+ cells, CD14- cells, CD8+ cells, CD8- cells, CD103+ cells, CD103- cells, CD11b+ cells, CD11b- cells, BDCA1+ cells, BDCA1- cells, L-selectin+ cells, L-selectin- cells, CD25+, CD25- cells, CD27+, CD27- cells, CD28+ cells, CD28- cells, CD44+ cells, CD44- cells, CD56+ cells, CD56- cells, CD57+ cells, CD57- cells, CD62L+ cells, CD The cell may be 62L-cells, CD69+cells, CD69-cells, CD45RO+cells, CD45RO-cells, CD127+cells, CD127-cells, CD132+cells, CD132-cells, IL-7+cells, IL-7-cells, IL-15+cells, IL-15-cells, lectin-like receptor G1 positive cells, lectin-like receptor G1 negative cells, or differentiated or dedifferentiated cells thereof. The examples of factors expressed by the cells are not intended to be limiting, and one of skill in the art would understand that the cells may be positive or negative for any factor known in the art. In some embodiments, the cells may be positive for two or more factors. For example, the cells may be CD4+ and CD8+. In some embodiments, the cells may be negative for two or more factors. For example, the cells may be CD25-, CD44-, and CD69-. In some embodiments, the cells may be positive for one or more factors or negative for one or more factors. For example, the cells can be CD4+ and CD8-. In some aspects, the cell markers provided herein can be utilized to select, enrich, or deplete a population of cells. In some aspects, enrichment includes selecting a monocyte fraction. In some aspects, enrichment includes sorting a population of immune cells from a monocyte fraction. In some embodiments, the cells may be selected for having or not having one or more given factors (e.g., the cells may be separated based on the presence or absence of one or more factors). In some embodiments, the selected cells may also be transduced and / or expanded in vitro. The selected cells may be expanded in vitro prior to infusion. In some embodiments, the selected cells may be transduced with a vector provided herein. It is to be understood that the cells used in any of the methods disclosed herein may be a mixture of any of the cells disclosed herein (e.g., two or more different cells). For example, the methods of the present disclosure may include cells, where the cells are a mixture of CD4+ cells and CD8+ cells.In another example, the method of the present disclosure may include cells, the cells being a mixture of CD4+ cells and naive cells. In some cases, the cells may be stem memory TSCM cells composed of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, the stem memory cells may also express CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibit a number of functional properties characteristic of stem memory cells. The cells provided herein may also be central memory TCM cells comprising L-selectin and CCR7, where the central memory cells can, for example, secrete IL-2 but cannot secrete IFNγ or IL-4. The cells may also be effector memory TEM cells comprising L-selectin or CCR7, and can produce effector cytokines such as, for example, IFNγ and IL-4. In some cases, the population of cells may be introduced into a subject. For example, the population of cells may be a combination of T cells and NK cells. In other cases, the population may be a combination of naive cells and effector cells. The population of cells may be TILs.

[0058]

[0081] In some embodiments, the methods provided herein may include activating a population of cells. Activation, as used herein, may refer to the process by which cells transition from a resting state to an activated state. This process may include responding to antigens, migration, and / or phenotypic or genetic changes to a functionally active state. In some aspects, activation may refer to the stepwise process of T cell activation. In some cases, T cells may require one or more signals to become activated. For example, T cells may require at least two signals to become fully activated. The first signal may occur after engagement of the TCR by the antigen-MHC complex, and the second signal may occur by binding of a costimulatory molecule. In vitro, an anti-CD3 antibody (or a functional variant thereof) can mimic the first signal, and an anti-CD28 antibody (or a functional variant thereof) can mimic the first signal. The second signal can be simulated.

[0059]

[0082] In some embodiments, the methods provided herein may include activating a population of cells. Activation may be performed by contacting the population of cells with an agent capable of stimulating a signal associated with the CD3 TCR complex and a surface to which a ligand capable of stimulating a costimulatory molecule on the surface of the cells is attached. In particular, the T cell population may be stimulated in vitro, for example, by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contacting with a protein kinase C activator (e.g., bryostatin), sometimes with a calcium ionophore. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds to the accessory molecule may be used. For example, the population of cells may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions that may stimulate proliferation of the T cells. In some cases, 4-1BB may be used to stimulate the cells. For example, the cells may be stimulated with 4-1BB and IL-21 or another cytokine. For activation of either CD4 T cells or CD8 T cells, an anti-CD3 antibody and an anti-CD28 antibody may be used. For example, the agent providing the signal may be in the form of a solution or may be conjugated to a solid surface. The ratio of particles to cells may depend on the particle size relative to the target cells. In a further embodiment, cells, such as T cells, may be combined with beads coated with an agent, in which case the beads and cells may then be separated and optionally cultured. Each bead may be coated with either anti-CD3 or anti-CD28 antibodies, or in some cases a combination of the two. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. Cell surface proteins may be conjugated by contacting the T cells with paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 antibodies can be attached.In one embodiment, cells and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads in a 1:1 ratio) are combined in a buffer, e.g., phosphate buffered saline (PBS) (e.g., without divalent cations such as calcium and magnesium). Any cell concentration can be used. The mixture can be cultured for a few hours or about a few hours (e.g., about 3 hours) to at or about 14 days, or any hourly integer value therebetween. In another embodiment, the mixture can be cultured for at or about 21 days, or up to at or about 21 days. Suitable conditions for T cell culture may include an appropriate medium (e.g., Minimum Essential Medium or RPMI medium 1640, or X-vivo5 (Lonza)) that may contain factors necessary for proliferation and growth, such as serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-21, IL-15, TGF beta, and TNF alpha, or any other additives for cell growth. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media can include RPMI1640, A1M-V, DMEM, MEM, α-MEM, F-12, X-Vivo1, and X-Vivo20, Optimizer, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines sufficient for T cell growth and expansion, with addition of amino acids, sodium pyruvate, and vitamins. Antibiotics, such as penicillin and streptomycin, may only be included in experimental cultures, and are preferably not included in the culture of cells that will be infused into subjects. Target cells can be maintained under conditions necessary to support growth; for example, at an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). In some cases, T cells exposed to various stimulation times may exhibit different characteristics.In some cases, human CD3, CD28, CD2, or any of these. Soluble monospecific tetrameric antibodies for any combination can be used. In some embodiments, activation can utilize an activating moiety, a costimulatory agent, and any combination thereof. In some aspects, the activating moiety binds to the CD3 / T cell receptor complex and / or provides costimulation. In some aspects, the activating moiety is any one of an anti-CD3 antibody and / or an anti-CD28 antibody. In some aspects, the solid phase is at least one of a bead, a plate, and / or a matrix. In some aspects, the solid phase is a bead. Alternatively or in addition, the activating moiety may not be conjugated to a substrate, for example, the activating moiety may be suspended in the medium.

[0060]

[0083] In some cases, the population of cells can be activated or expanded by co-culturing tissues or cells. The cells can be antigen-presenting cells. Artificial antigen-presenting cells (aAPCs) can express ligands for T cell receptors and costimulatory molecules, and can activate and expand T cells for migration, while in some cases improving their efficacy and function. The aAPCs can be engineered to express any gene for T cell activation. The aAPCs can be engineered to express any gene for T cell expansion. The aAPCs can be beads, cells, proteins, antibodies, cytokines, or any combination. The aAPCs can deliver signals to the cell population that may undergo genome transfer. For example, the aAPCs can deliver signal 1, signal 2, signal 3, or any combination. Signal 1 can be an antigen recognition signal. For example, signal 1 can be ligation of the TCR by peptide-MHC complexes, or binding of an agonist antibody to CD3, which can result in activation of the CD3 signal transduction complex. Signal 2 can be a costimulatory signal. For example, the costimulatory signal may be anti-CD28, inducible costimulatory factor (ICOS), CD27, and 4-1BB (CD137), which bind to ICOS-L, CD70, and 4-1BBL, respectively. Signal 3 may be a cytokine signal. The cytokine may be any cytokine. The cytokine may be IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof. In some cases, artificial antigen presenting cells (aAPCs) can be used to activate and / or expand the cell population. In some cases, the artificial may not induce allospecificity. The aAPCs may not express HLA in some cases. The aAPCs may be genetically modified to stably express genes that can be used for activation and / or stimulation. In some cases, K562 cells can be used for activation. The K562 cells can also be used for expansion. The K562 cells may be a human erythroleukemia cell line.K562 cells may be engineered to express a gene of interest. K562 cells may not endogenously express HLA class I, II, or CD1d molecules, but may express ICAM-1 (CD54) and LFA-3 (CD58). K562 may be engineered to deliver signal 1 to T cells. For example, K562 cells may be engineered to express HLA class I. In some cases, K562 cells may be engineered to express additional molecules, such as B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, anti-CD3, anti-CD3mAb, anti-CD28, anti-CD28mAb, CD1d, anti-CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, truncated CD19, or any combination. In some cases, engineered K562 cells can express, in addition to CD80 and CD83, a membrane form of an anti-CD3 mAb, clone OKT3. In some cases, engineered K562 cells can express, in addition to CD80 and CD83, a membrane form of an anti-CD3 mAb, clone OKT3, a membrane form of an anti-CD28 mAb.

[0061]

[0084] The aAPCs may be beads. Spherical polystyrene beads may be coated with antibodies against CD3 and CD28 and used for T cell activation. The beads may have any size. In some cases, the beads may be 3 and 6 micrometers or about 3 and 6 micrometers. The beads may be 4.5 micrometers or about 4.5 micrometers in size. The beads may be utilized in any cell to bead ratio. For example, at 1 million cells per milliliter, a 3 to 1 bead to cell ratio may be used. The aAPCs may also be rigid spherical particles, polystyrene latex microbeads, magnetic nano- or microparticles, nano-sized quantum dots, 4, poly(lactic-co-glycolic acid) (PLGA) microspheres, non-spherical particles, 5, bundles of carbon nanotubes, 6, ellipsoidal PLGA microparticles, 7, nanoworms, fluid lipid bilayer-containing systems, 8, 2D supported lipid bilayers (2D-SLBs), 9, liposomes, 10, RAFTsomes / microdomain liposomes, 11, SLB particles, or any combination thereof. In some cases, aAPCs can expand CD4 T cells. For example, aAPCs can be engineered to mimic the antigen processing and presentation pathway of HLA class II restricted CD4 T cells. K562 can be engineered to express HLA-D, DPα, DPβ chain, Ii, DMα, DMβ, CD80, CD83, or any combination thereof. For example, engineered K562 cells can be pulsed with HLA restricted peptides to expand HLA restricted antigen specific CD4 T cells. In some cases, the use of aAPCs can be combined with exogenously introduced cytokines for T cell activation, expansion, or any combination. Cells can also be expanded in vivo, for example in the blood of a subject, after administering genomically transplanted cells to a subject.

[0062]

[0085] In some embodiments, the methods provided herein may include transducing a population of cells. In some embodiments, the methods include introducing a polynucleotide encoding a cellular receptor, such as a chimeric antigen receptor and / or a T cell receptor. In some cases, transfection of the cells may be performed.

[0063]

[0086] In some embodiments, a viral supernatant is produced that contains polynucleotides encoding cellular receptors such as CAR and / or TCR. In some embodiments, the viral vector may be a retroviral vector, a lentiviral vector, and / or an adeno-associated viral vector. Packaging cells can be used to form viral particles capable of infecting host cells. Such cells can include 293 cells (e.g., for packaging adenovirus), and Psi2 cells or PA317 cells (e.g., for packaging retrovirus). Viral vectors can be produced by producing cell lines that package nucleic acid vectors into viral particles. The vectors may contain minimal viral sequences required for packaging and subsequent integration into the host. The vectors may contain other viral sequences that are replaced with an expression cassette for the polynucleotide to be expressed. Missing viral functions may be automatically supplied by the packaging cell line. For example, AAV vectors may contain ITR sequences from the AAV genome required for packaging and integration into the host genome. Viral DNA may be packaged in cell lines, and such cell lines may contain helper plasmids that code for other AAV genes, namely rep and cap, but lack ITR sequences. Cell lines may also be infected with adenovirus as a helper. Helper viruses can promote the replication of AAV vectors and the expression of AAV genes from the helper plasmid. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus has higher sensitivity than AAV. Additional methods for the delivery of nucleic acid to cells may be used, such as those described in US20030087817, which is incorporated herein by reference.

[0064]

[0087] In some embodiments, the host cell may be transiently or non-transiently transfected with one or more vectors described herein. The cell may be transfected as it naturally occurs in the subject. The cell may be taken from the subject or obtained from the subject and transfected. The cell may be obtained from a cell taken from the subject, such as a cell line. In some embodiments, the cell transfected with one or more vectors described herein is used to establish a new cell line that contains sequences from one or more vectors. Non-limiting examples of vectors for eukaryotic host cells include, but are not limited to: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR54O, pRIT5 (Pharmacia). Eukaryotic: pWL-neo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmiacia). Any other plasmids and vectors can also be used as long as they are replicable and viable in the selected host. Any vector and commercially available ones (and their variants or derivatives) can be engineered to contain one or more recombination sites for use in the method. Such vectors can be obtained, for example, from Vector Laboratories Inc., Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, and Research Genetics.Other vectors of interest include eukaryotic cell expression vectors, such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), as well as pYES2, pAC360, pBlueBa-cHis A, B, and C, pVL1392, pBlueBac111, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen), and variants or derivatives thereof. Other vectors include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), P1 (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSYSPORT1 (Invitrogen) and variants or derivatives thereof.Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBa-cHis2, pcDNA3.1 / His, pcDNA3.1(-) / Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pA081S, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlue-Bac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, and pcDNA from Invitrogen. 2.1, pYES2, pZEr01.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, p VL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1 / Amp, pcDNA3.1, pcDNA3.1 / Zeo, pSe, SV2, pRc / CMV2, pRc / RSV, pREP4, pREP7, pREP8, pREP9, pREP10, p CEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, pCRBac;Pharmacia,X ExCell, X gt11, pTrc99A, pKK223-3, pGEX-1X T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX -5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, pUC4K;Novagen includes pSCREEN-lb(+), pT7Blue(R), pT7Blue-2, pCITE-4-abc(+), pOCUS-2, pTAg, pET-32L1C, pET-30LIC, pBAC-2 cp LIC, pBACgus-2 cp LIC、pT7Blue-2 LIC、pT7Blue-2、X SCREEN-1、X B1ueSTAR、pET-3abcd、pET-7abc、pET9abcd、pET11 abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b, pET-17xb, pET-1 b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd (+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), p ET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3 cp、pBACgus-2 cp、pBACsurf-1、plg、Signal plg、pYX、Select Vecta-Neo、Select Vecta-HygおよびSelect Vecta-Gpt;Clontech's pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFPN, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contra l、pSEAP2-Promoter、pSEAP2-Enhancer、pI3gal-Basic、pl3gal-Control、pI3gal-Promoter、pI3gal-Enhancer、pCMV、pTet-Off pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LU C, pPUR, pSV2neo, pYEX4T-1 / 2 / 3, pYEX-S1, pBacPAK-His, pBacP AK8 / 9, pAcUW31, BacPAK6, pTriplEx, 2Xgt10, Xgt11, pWE15,およびX TriplEx;StratageneかムダZAP II, pBK-CMV, pBK-RSV, pBluescript II KS+ / -, pBluescript II SK+ / -, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, FIX II, DASH, EMBL3, EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct、pBS+ / -、pBC KS+ / -、pBC SK+ / -, Phag-escript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-llabcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI / MCS, pOPI3; CAT, pXT1, pSG5, pPbac, pMbac, pMClneo, pMClneoポリA, pOG44, p0G45, pF RTI3GAL, pNE0I3GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414 The vectors may include pRS415, and pRS416, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp, and variants or derivatives thereof. In some embodiments, the vector may be a minicircle vector. The vectors provided herein can be used to deliver polypeptides encoding CAR and / or TCR.

[0065]

[0088] The transduction and / or transfection can be performed by any one of non-viral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the provided method comprises viral transduction, and the viral transduction comprises lentivirus. The viral particles can be used to deliver the viral vector comprising the polypeptide sequence encoding the cellular receptor to cells ex vivo or in vivo. In some cases, the viral vectors disclosed herein can be measured in pfu (plaque forming units). In some cases, the pfu of the recombinant virus or viral vector of the disclosed compositions and methods is about 10 8 From about 5 × 10 10 In some cases, the recombinant virus of the disclosure can be at least about 1 x 10 pfu. 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×109 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , and 5 × 10 10 In some cases, the recombinant viruses of the disclosure are at most about 1 x 10 pfu. 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , and 5 × 10 10 In some embodiments, the viral vectors of this disclosure can be measured as vector genomes. In some cases, the recombinant viruses of this disclosure can be measured as 1×10 10 From 3×10 12 Vector genome, or 1 × 10 9 From 3×10 13 Vector genome, or 1 × 10 8 From 3×10 14 Vector genome, or at least, approximately 1 × 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×105 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 vector genome, or 1 × 10 8 From 3×10 14 vector genome, or at most, about 1 × 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the viral vectors provided herein can be measured using multiplicity of infection (MOI). In some cases, MOI can refer to the ratio or multiplicity of vector or viral genome to cells to which the nucleic acid can be delivered. In some cases, MOI is 1×10 6 In some cases, the MOI may be 1×10 5 From 1×10 7 In some cases, the MOI may be 1×10 4 From 1×10 8 In some cases, the recombinant virus of the present disclosure may be at least about 1 x 10 1 , 1×10 2 , 1×10 3, 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the recombinant viruses of this disclosure are administered at an MOI of 1 x 10 8 from 3×10 14 MOI is approximately 1 x 10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1 × 10 18 In some cases, the viral vector is administered at an MOI of about 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×106 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 , 3×10 7 at a multiplicity of infection (MOI) of up to approximately 9 × 10 per cell. 9 Transduced via genome copy / viral particle.

[0066]

[0089] The transfection efficiency of cells with any of the nucleic acid delivery platforms described herein, e.g., transduction, is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 ... %, 99.9%, or greater than 99.9%, or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or greater than 99.9%. In some embodiments, the method may include adding an infectious agent to the composition comprising the population of cells. The infectious agent may include polybrene. In some aspects, the infectious agent can enhance the efficiency of viral infection. The infectious agent can enhance the infectivity of the virus by about 100-1,000 fold. Polybrene may be added to the composition at a concentration of about 5ug to 10ug per ml.

[0067]

[0090] In some embodiments, the methods provided herein may include non-viral approaches to introduce cell receptors into cells. Non-viral approaches may include, but are not limited to, CRISPR-associated proteins (Cas proteins, e.g., Cas9), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Argonaute nucleases, and meganucleases. Nucleases may be naturally occurring nucleases, genetically modified, and / or recombinant. Non-viral approaches may also be implemented using transposon-based systems (e.g., PiggyBac, Sleeping Beauty).

[0068]

[0091] In some embodiments, the methods provided herein can utilize the PiggyBac system to introduce exogenous polypeptides into cells. The PiggyBac system includes two components, a transposon and a transposase. The PiggyBac transposase specifically facilitates transposon integration at "TTAA" sites randomly distributed in the genome. The predicted frequency of "TTAA" in the genome is approximately 1 per 256 base pairs of DNA sequence. Unlike other transposons, the PB transposase also allows for the excision of the transposon in a completely seamless manner, leaving no sequence or mutation behind. Furthermore, PiggyBac offers a large cargo carrying capacity (a carrying capacity of over 200 kb has been demonstrated) with no known upper limit. The PB performance level can be increased by codon optimization strategies, mutations, deletions, additions, substitutions, and any combination thereof. In some cases, PBs may have larger cargo (approximately 9.1-14.3 kb), higher transposition activity, and their footprint-free characteristics make them attractive as gene editing tools. In some embodiments, PBs have several features: high efficiency of transposition; large cargo; stable long-term expression; transgene integration as a single copy; tracking of target genes in vivo by non-invasive marks instead of traditional methods such as PCR; and determination of integration sites. The present invention may include simple configurations and combinations thereof.

[0069]

[0092] In some aspects, the methods provided herein can utilize the Sleeping Beauty (SB) system to introduce a polypeptide encoding a cellular receptor into a cell. SB was engineered from a fossil of an ancient Tc1 / Mariner transposon found in the genome of salmonids by in vitro evolution. The SB ITR (230 bp) contains a 32 bp long imperfect direct repeat (DR) that may serve as a recognition signal for the transposase. The binding affinity and spacing between the DR elements in the ITR were associated with transposition activity. The SB transposase may be a 39 kDa protein with a catalytic domain characterized by a DNA binding polypeptide, a nuclear localization signal (NLS) and a conserved amino acid motif (DDE). Various screens mutagenizing the primary amino acid sequence of the SB transposase result in a hyperactivated version of the transposase. In some cases, modified SBs may be utilized. The modified SBs may contain mutations, deletions and additions within the ITRs of the original SB transposon. The modified SB may include pT2, pT3, pT2B, pT4, SB100X, and combinations thereof. Non-limiting examples of modified SBs can be selected from SB10, SB11 (3-fold higher than SB10), SB12 (4-fold higher than SB10), HSB1-HSB5 (up to 10-fold higher than SB10), HSB13-HSB17 (HSB17 is 17-fold higher than SB10), SB100X (100-fold higher than SB10), SB150X (130-fold higher than SB10), and any combinations thereof. In some cases, SB100X is 100-fold overactivated compared to the original restored transposase (SB10). In some embodiments, the excision of SB transposition leaves a footprint (3bp) at the cargo site. Integration into the genomic TA dinucleotide occurs, resulting in duplication of the target site generated by the host repair mechanism. In some cases, SB appears to have a nearly unbiased, near-random integration profile.Although transposon integration may be artificially targeted to a predetermined genomic locus in wild-type systems (approximately 10%), in the chimeric systems provided herein, transposon integration of SB can be directed to a predetermined locus with an efficiency of greater than 10%.

[0070]

[0093] In some embodiments, non-viral approaches can be employed to introduce exogenous polynucleic acids into a population of cells. In some embodiments, non-viral vectors or nucleic acids can be delivered without the use of viruses and can be measured by the amount of nucleic acid. Generally, any suitable amount of nucleic acid can be used with the compositions and methods of this disclosure. In some cases, the nucleic acid can be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μ ... μg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 1 μg, 1 μg, 10 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 1 μg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 50 The amount of the compound may be 100 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g. In some cases, the nucleic acid may be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 90 The amount may be 0 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.

[0071]

[0094] In some embodiments, non-viral approaches to introduce CAR and / or TCR sequences into cells can include electroporation. Electroporation can be performed, for example, using the Neon® Transfection System (ThermoFisher Scientific) or the AMAXA® Nucleofector (AMAXA® Biosystems). Electroporation parameters can be adjusted to optimize transfection efficiency and / or cell viability. Electroporation devices can have multiple wave-form pulse settings, such as exponential decay, time constant, and square wave. Each cell type has a unique optimal field strength (E), which depends on the applied pulse parameters (e.g., voltage, capacitance, and resistance). Application of the optimal field strength results in electropermeabilization through the induction of transmembrane voltage, which allows the nucleic acid to pass through the cell membrane. In some cases, the electroporation pulse voltage, electroporation pulse width, number of pulses, cell density, and tip type can be adjusted to optimize transfection efficiency and / or cell viability.

[0072]

[0095] In some embodiments, the pulse voltage of the electroporation can be altered to optimize transfection efficiency and / or cell viability. In some cases, the electroporation voltage can be less than about 500 volts. In some cases, the electroporation voltage may be at least about 500 volts, at least about 600 volts, at least about 700 volts, at least about 800 volts, at least about 900 volts, at least about 1000 volts, at least about 1100 volts, at least about 1200 volts, at least about 1300 volts, at least about 1400 volts, at least about 1500 volts, at least about 1600 volts, at least about 1700 volts, at least about 1800 volts, at least about 1900 volts, at least about 2000 volts, at least about 2100 volts, at least about 2200 volts, at least about 2300 volts, at least about 2400 volts, at least about 2500 volts, at least about 2600 volts, at least about 2700 volts, at least about 2800 volts, at least about 2900 volts, or at least about 3000 volts. In some cases, the electroporation pulse voltage required for optimal transfection efficiency and / or cell viability may be cell type specific. For example, an electroporation voltage of 1900 volts may be optimal for macrophage cells (e.g., resulting in maximum viability and / or transfection efficiency). In another example, an electroporation voltage of about 1350 volts may be optimal for Jurkat cells or primary human cells, such as T cells (e.g., resulting in maximum viability and / or transfection efficiency). In some cases, there may be a range of optimal electroporation voltages for a given cell type. For example, an electroporation voltage between about 1000 volts and about 1300 volts may be optimal for human 578T cells (e.g., resulting in maximum viability and / or transfection efficiency). In some cases, the primary cells may be primary lymphocytes. In some cases, the population of primary cells may be a population of lymphocytes.

[0073]

[0096] In some embodiments, the pulse width of the electroporation can be altered to optimize transfection efficiency and / or cell viability. In some cases, the pulse width of the electroporation can be less than about 5 milliseconds. In some cases, the electroporation width can be at least about 5 milliseconds, at least about 6 milliseconds, at least about 7 milliseconds, at least about 8 milliseconds, at least about 9 milliseconds, at least about 10 milliseconds, at least about 11 milliseconds, at least about 12 milliseconds, at least about 13 milliseconds, at least about 14 milliseconds, at least about 15 milliseconds, at least about 16 milliseconds, at least about 17 milliseconds, at least about 18 milliseconds, at least about 19 milliseconds, at least about 20 milliseconds, at least about 21 milliseconds, at least about 22 milliseconds, at least about 23 milliseconds, at least about 24 milliseconds, at least about 25 milliseconds, at least about 26 milliseconds, at least about 27 milliseconds, at least about 28 milliseconds, at least about 29 milliseconds, at least about 30 milliseconds, at least about 31 milliseconds, at least about 32 milliseconds, at least about 33 milliseconds, at least about 34 milliseconds, at least about 35 milliseconds, at least about 36 milliseconds, at least about 37 milliseconds, at least about 38 milliseconds, at least about 39 milliseconds, at least about 40 milliseconds, at least about 41 milliseconds, at least about 42 milliseconds, at least about 43 milliseconds, at least about 44 milliseconds, at least about 45 milliseconds, at least about 46 milliseconds, at least about 47 milliseconds, at least about 48 milliseconds, at least about 49 milliseconds, at least about The electroporation pulse width may be at least about 32 ms, at least about 33 ms, at least about 34 ms, at least about 35 ms, at least about 36 ms, at least about 37 ms, at least about 38 ms, at least about 39 ms, at least about 40 ms, at least about 41 ms, at least about 42 ms, at least about 43 ms, at least about 44 ms, at least about 45 ms, at least about 46 ms, at least about 47 ms, at least about 48 ms, at least about 49 ms, or at least about 50 ms. In some cases, the electroporation pulse width required for optimal transfection efficiency and / or cell viability may be cell type specific. For example, an electroporation pulse width of 30 ms may be optimal for macrophage cells (e.g., resulting in the highest viability and / or transfection efficiency). In another example, an electroporation width of about 10 ms may be optimal for Jurkat cells (e.g., resulting in the highest viability and / or transfection efficiency). In some cases, there may be a range of electroporation widths that are optimal for a given cell type. For example, an electroporation width of between about 20 milliseconds and about 30 milliseconds may be optimal for human 578T cells (eg, resulting in maximum viability and / or transfection efficiency).

[0074]

[0097] In some embodiments, the number of electroporation pulses can be varied to optimize transfection efficiency and / or cell viability. In some cases, electroporation may include a single pulse. In some cases, electroporation may include more than one pulse. In some cases, electroporation may include two pulses, three pulses, four pulses, five pulses, six pulses, seven pulses, eight pulses, nine pulses, or ten or more pulses. In some cases, the number of electroporation pulses required for optimal transfection efficiency and / or cell viability may be cell type specific. For example, electroporation with a single pulse may be optimal for macrophage cells (e.g., resulting in the highest viability and / or transfection efficiency). In another example, electroporation with three pulses may be optimal for primary cells (e.g., resulting in the highest viability and / or transfection efficiency). In some cases, there may be a range of electroporation widths that are optimal for a given cell type. For example, electroporation with between about 1 and about 3 pulses may be optimal for human cells (eg, resulting in maximum viability and / or transfection efficiency).

[0075]

[0098] In some cases, the initial cell density for electroporation can be varied to optimize transfection efficiency and / or cell viability. In some cases, the initial cell density for electroporation can be about 1×10 cells. 5 In some cases, the initial cell density for electroporation is at least about 1 x 10 5 At least about 2 x 10 cells 5 cells, at least about 3 x 10 5 cells, at least about 4 x 10 5 cells, at least about 5 x 10 5 cells, at least about 6 x 10 5 cells, at least about 7 x 10 5 cells, at least about 8 x 10 5 cells, at least about 9 x 10 5cells, at least about 1 x 10 6 cells, at least about 1.5 x 10 6 At least about 2 x 10 cells 6 cells, at least about 2.5 x 10 6 cells, at least about 3 x 10 6 cells, at least about 3.5 x 10 6 cells, at least about 4 x 10 6 cells, at least about 4.5 x 10 6 cells, at least about 5 x 10 6 cells, at least about 5.5 x 10 6 cells, at least about 6 x 10 6 cells, at least about 6.5 x 10 6 cells, at least about 7 x 10 6 cells, at least about 7.5 x 10 6 cells, at least about 8 x 10 6 cells, at least about 8.5 x 10 6 cells, at least about 9 x 10 6 cells, at least about 9.5 x 10 6 cells, at least about 1 x 10 7 At least about 1.2 x 10 cells 7 cells, at least about 1.4 x 10 7 cells, at least about 1.6 x 10 7 cells, at least about 1.8 x 10 7 At least about 2 x 10 cells 7 At least about 2.2 x 10 cells 7 At least about 2.4 x 10 cells 7 At least about 2.6 x 10 cells 7 cells, at least about 2. 8×10 7 cells, at least about 3 x 10 7 At least about 3.2 x 10 cells 7 At least about 3.4 x 10 cells 7 At least about 3.6 x 10 cells 7 cells, at least about 3.8 x 10 7 cells, at least about 4 x 107 At least about 4.2 x 10 cells 7 cells, at least about 4.4 x 10 7 cells, at least about 4.6 x 10 7 cells, at least about 4.8 x 10 7 cells, or at least about 5 x 10 7 In some cases, the initial cell density for electroporation required for optimal transfection efficiency and / or cell viability may be cell type specific. For example, 1.5×10 6 An initial cell density for electroporation of 5×10 cells may be optimal for macrophage cells (e.g., resulting in maximum viability and / or transfection efficiency). 6 An initial cell density for electroporation of 5.6×10 cells may be optimal for human cells (e.g., resulting in maximum viability and / or transfection efficiency). In some cases, there may be a range of cell densities for optimal initial electroporation for a given cell type. For example, 5.6×10 cells 6 From 5×10 7 The initial cell density for electroporation between individuals may be optimal for human cells, e.g., T cells (e.g., resulting in maximum viability and / or transfection efficiency).

[0076]

[0099] For example, the efficiency of integration of a nucleic acid sequence encoding a CAR and / or a TCR into the genome of a cell using the CRISPR, Piggy Bac, and / or Sleeping Beauty systems can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. , 99.5%, 99.9%, or greater than 99.9%, or may be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or greater than 99.9%.

[0077]

[0100] In some embodiments, the methods provided herein for producing a population of engineered immune cells expressing a chimeric antigen receptor (CAR) can include the steps of (a) activating a population of cells comprising immune cells with an activating moiety; and (b) simultaneously introducing a polynucleotide encoding at least the CAR. In some embodiments, the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some embodiments, steps (a) and (b) are performed within 48 hours. In some embodiments, steps (a) and (b) are performed within 24 hours. In some embodiments, steps (a) and (b) are performed within 3 hours. In some embodiments, steps (a) and (b) are performed within 1 hour. In some embodiments, steps (a) and (b) are performed within 30 minutes. In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, steps (a) and (b) can be performed within about 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 20 hours, 15 hours, 13 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 3 minutes, 1 minute, and / or simultaneously. In some aspects, the methods provided herein can further include injecting the population of engineered immune cells into the subject within about 1 week of completing (a) and (b). In some aspects, the methods provided herein can further include injecting the population of engineered immune cells into the subject within about 5 days of completing (a) and (b). In some aspects, the methods provided herein can further include injecting the population of engineered immune cells into the subject within about 72 hours of completing (a) and (b). The methods provided herein can further include injecting the population of engineered immune cells into the subject within about 24 hours of completing (a) and (b). In some aspects, the methods provided herein can further include injecting the population of engineered immune cells into the subject within about 12 hours of completing (a) and (b). In some aspects, the methods provided herein can further include injecting the population of engineered immune cells into the subject within about 6 hours of completing (a) and (b). In some aspects, the methods provided herein can further include injecting the population of engineered immune cells into the subject within about 3 hours of completing (a) and (b).

[0078]

[0101] In some embodiments, the method provided herein for producing a population of engineered immune cells expressing a chimeric antigen receptor (CAR) can include (a) activating a population of cells comprising immune cells with an activating moiety; and simultaneously (b) introducing at least a polynucleotide encoding the CAR. In some aspects, the method can further include cryopreserving the population comprising engineered immune cells expressing a CAR and / or a TCR. Cryopreservation can be performed at any time after cell manipulation. Cryopreservation can be performed more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 1.5 weeks, 2 hours, or 2 weeks after (a) and (b). In one aspect, the population comprising engineered immune cells can be freshly sourced. For example, the freshly sourced population can be obtained from a subject and subjected to the method provided herein without cryopreservation.

[0079]

[0102] In some embodiments, the method provided herein for producing a population of engineered immune cells expressing a chimeric antigen receptor (CAR) can include (a) activating a population of cells including immune cells with an activating moiety; and simultaneously (b) introducing at least a polynucleotide encoding a CAR, where (a) and (b) are performed in about 48 hours or less. In some cases, (a) and (b) can be performed in up to 48 hours or less, 36 hours or less, 24 hours or less, 22 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less, 12 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, or less. In some cases, if the processes (a) and (b) do not completely overlap with other processes, the total time it takes to perform both (a) and (b) can be 48 hours or less. In some cases, when processes (a) and (b) do not completely overlap with other processes, the total time taken to perform both (a) and (b) may be up to 48 hours or less, 36 hours or less, 24 hours or less, 22 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less, 12 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, or less.

[0080]

[0103] In one example, a method provided herein for producing a population of engineered immune cells expressing a chimeric antigen receptor (CAR) can include the steps of (a) activating a population of cells comprising immune cells with an activation moiety; and simultaneously (b) introducing at least a polynucleotide encoding the CAR, where (a) and (b) are performed in about 24 hours or less. In another example, a method provided herein for producing a population of engineered immune cells expressing a chimeric antigen receptor (CAR) can include the steps of (a) activating a population of cells comprising immune cells with an activation moiety; and simultaneously (b) introducing at least a polynucleotide encoding the CAR, where the total time taken to perform both (a) and (b) can be 24 hours or less.

[0081]

[0104] In some embodiments, the methods provided herein provide for the production of more centrioles compared to effector memory T cells compared to comparable methods that do not simultaneously activate and transduce. In some embodiments, the generation of cells using the FAST-CAR method provided herein can result in at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, or more increase in central memory T cells (TCM) compared to effector memory T cells (TEM). In some embodiments, the generation of cells using the FAST-CAR method provided herein can result in up to 50-fold, 45-fold, 40-fold, 35-fold, 30-fold, 25-fold, 20-fold, 15-fold, 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, or less increase in TCM compared to TEM. In some embodiments, the methods provided herein can result in more TSCM compared to comparable methods that do not simultaneously activate and transduce. In some embodiments, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or up to about 100% of the cell population are TSCM. In some embodiments, up to 100%, 95%, 90%, 80%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or less of the cell population are TSCM. TSCM are CD45RO - CD62L + In some embodiments, the methods provided herein can include administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). In some aspects, the methods can include injecting a population of immune cells comprising the engineered immune cells into a subject in need thereof. In some aspects, the engineered immune cells have not been subjected to ex vivo expansion for more than two weeks. In some aspects, the engineered immune cells comprise at least 2% stem memory T cells (TSCM).

[0082]

[0105] In some cases, the subject cells (e.g., T cells) may not be pre-activated (e.g., by CD3 / CD28 beads) prior to simultaneous activation and transduction. In such cases, after simultaneous activation and transduction, the duration for activation and transduction of the subject cells may be substantially the same.

[0083]

[0106] In some embodiments, the populations generated by the methods provided herein may be further characterized in that they are not enriched for PD1 and LAG3. In some aspects, the populations generated by the methods provided herein may include lower expression of cell markers associated with depletion. Markers associated with cell depletion include PD-1, LAG3, CTLA-4, TIM-3, 2B4 / CD244 / SLAMF4, CD160, TIGIT, CXCR5, ICOS, to name a few. In some aspects, markers of cell depletion may include loss of IL-2 production, loss of proliferative capacity, loss of ex vivo cytolytic activity, impaired production of TNF-alpha, IFN-gamma, and cc(beta) chemokines, degranulation, expression of high levels of granzyme B, low responsiveness to IL-7 and IL-15, altered expression of GATA-3, Bcl-6, and Helios. In the case of CD4+, depletion can include a bias towards the T follicular helper (Tfh) cell phenotype, secretion of IL-4, IL-6 and / or IL-21, expression of transcription factors Bcl-6, IRF4, STAT4, and any combination thereof.

[0084]

[0107] In some aspects, the immune cells utilized in the methods provided herein are T cells, NK cells, NKT cells, stem cells, induced pluripotent stem cells, B cells, to name a few. In some embodiments, the cells utilized in the methods provided herein are obtained from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells. The cells can be obtained from several non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Additionally, any T cell line can be used. Alternatively, the cells can be obtained from healthy donors, from patients diagnosed with cancer, and from other sources. The cells may be obtained from a patient diagnosed with an infectious disease. In another case, the cells may be part of a mixed population of cells exhibiting different phenotypic characteristics. The cells may be obtained from a cell therapy bank. In one embodiment, the cell population may be selected prior to manipulation. The selection may include at least one of magnetic separation, flow cytometry selection, and antibiotic selection. In one embodiment, the population of cells may include blood cells, such as peripheral blood mononuclear cells (PBMCs), lymphocytes, monocytes, or macrophages. In one embodiment, the immune cells may be lymphocytes, B cells, or T cells. The cells may be obtained from natural foods, apheresis, or a tumor sample of the subject. The cells may be tumor infiltrating lymphocytes (TILs). In some cases, the apheresis may be leukapheresis. Leukapheresis may be a procedure in which blood cells are isolated from blood. During leukapheresis, blood can be drawn from a needle inserted into the subject's arm and circulated through a machine that separates the whole blood into red blood cells, plasma and lymphocytes, and then the plasma and red blood cells are returned to the subject through a needle inserted into the other arm. In some cases, cells are isolated after administration of a treatment regimen and cell therapy. For example, apheresis can be performed consecutively or simultaneously with cell administration. In some cases, apheresis is performed before administration of a cell product and up to about 6 weeks after administration. In some cases, apheresis is performed -3 weeks, -2 weeks, -1 week, 0, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or up to about 10 years after administration of a cell product. In some cases, cells obtained by apheresis may be subjected to testing for specific lysis (e.g., cytotoxicity testing), cytokine release, metabolomic studies, bioenergetics studies, intracellular FAC of cytokine production, ELISA spot assays, and lymphocyte subset analysis. In some cases, samples of the cell product or apheresis product may be cryopreserved for retrospective analysis of the phenotype and function of the infused cells.

[0085]

[0108] The methods provided herein can include activating T cells and simultaneously introducing (e.g., transducing or transfecting) a vector into the T cells. The vector can be a viral vector (e.g., a lentiviral vector). The T cells can be resting (e.g., resting) T cells or non-resting (e.g., activated) T cells. The T cells can be exhausted T cells. In some cases, the T cells introduced with the vector can be a population of T cells including resting T cells, non-resting T cells, and / or exhausted T cells. The population of T cells can be a mixture of resting T cells, non-resting T cells, and exhausted T cells.

[0086]

[0109] The efficiency of transducing cells with viral vectors while simultaneously activating T cells can be higher than the efficiency of transducing resting T cells with viral vectors without simultaneous activation of T cells. The efficiency of transducing cells with simultaneous activation of T cells can be at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or higher than the efficiency of transducing resting T cells without simultaneous activation of T cells. Since the efficiency of simultaneous transduction and activation can be higher, the amount of viral vectors used in the methods provided herein can be lower. The amount of viral vectors used for simultaneous transduction and activation can be at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or lower than the amount used to transduce resting T cells without simultaneous activation of T cells.

[0087]

[0110] The T cells used in the methods described herein may be recovered from frozen cells (e.g., cryopreserved cells). Resting T cells may have a lower recovery efficiency (e.g., percentage of live cells recovered in a population of cells) than activated T cells. For example, the recovery efficiency of resting T cells 24 hours after cryopreservation is up to about 80%, 70%, 60%, 50%, 40%, 30%. , 20%, or lower. Recovery efficiency of activated T cells 24 hours after cryopreservation may be at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or higher. Recovery efficiency of activated T cells may be maintained after 24 hours.

[0088]

[0111] The engineered cells prepared using the simultaneous transduction and activation method described herein can effectively control or inhibit tumor growth. For example, the engineered cells prepared herein can have higher efficiency in controlling tumor growth compared to engineered cells prepared using a method comprising transducing resting T cells with a viral vector without simultaneous activation under the same or substantially the same conditions (e.g., animal models, dosing and experimental conditions). The engineered cells prepared using the simultaneous transduction and activation method described herein can effectively control side effects associated with administration of engineered T cells (e.g., CAR-T cells). Side effects include, but are not limited to, cytokine release syndrome (CRS) and hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, transient hypotension, hypoxemia, and the like. The engineered cells prepared using the simultaneous transduction and activation method described herein may have less CRS than the engineered cells prepared using a method comprising transducing resting T cells with a viral vector without simultaneous activation. The engineered cells prepared using the simultaneous transduction and activation method described herein may produce less proinflammatory cytokines compared to the engineered cells prepared using a method comprising transducing resting T cells with a viral vector without simultaneous activation. The proinflammatory cytokines may be IFN-γ, TNFα, GM-CSF, IL-2 and / or IL-6.

[0089]

[0112] In some embodiments, the methods provided herein include introducing a T cell receptor (TCR) into a cell. In some embodiments, the TCR includes (i) a ligand-binding domain specific for a ligand, and (ii) a transmembrane domain.

[0090]

[0113] In some embodiments, the TCR can be a disulfide-linked membrane-anchored heterodimeric protein. The TCRs provided herein can include variable alpha (α) and / or beta (β) chains. In some aspects, the alpha and / or beta chains can be expressed as part of a complex with an invariant CD3 chain molecule. In some aspects, the TCR can include variable gamma (γ) and / or delta (δ) chains, referred to as γδ T cells. In some aspects, the TCR chains can include an extracellular domain, i.e., a variable (V) region, a constant (C) region, and an immunoglobulin superfamily (IgSF) domain that forms an antiparallel β-sheet. In some embodiments, the constant region is proximal to the cell membrane and is followed by a transmembrane domain and a short cytoplasmic tail. Meanwhile, the variable region, such as a ligand binding domain, binds to the peptide / MHC complex. In some embodiments, the peptide can be a ligand. In some aspects, the variable domains of the TCR α and β chains can each have a hypervariable region or a complementarity determining region (CDR).

[0091]

[0114] In some embodiments, a chimeric antigen receptor is provided herein. The CAR comprises (i) a ligand-binding domain specific to a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some embodiments, the ligand-binding domain of the CAR of the present method can be linked to the intracellular signaling domain via the transmembrane domain. The transmembrane domain can be a transmembrane segment. The transmembrane domain of the present CAR can anchor the CAR to the plasma membrane of a cell, for example, an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide that links the ligand-binding domain and the intracellular signaling domain of the CAR can be any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least one (e.g., at least two, three, four, five, six, seven, eight, nine, ten or more) amino acid substitution, deletion, and insertion compared to the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as a polypeptide linker sequence. The polypeptide linker may be flexible or rigid. The polypeptide linker may be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the extracellular region of the cell to the intracellular region via the ligand binding domain. The natural transmembrane portion of CD28 may be used in the CAR. In other cases, the natural transmembrane portion of CD8 alpha may be used in the CAR. In some embodiments, the transmembrane domain of the CAR is derived from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

[0092]

[0115] In some embodiments, the intracellular signaling domain of the CAR of the present method can include a signaling domain involved in immune cell signaling or any derivative, variant, or fragment thereof. The intracellular signaling domain of the CAR can induce the activity of the immune cell comprising the CAR. The intracellular signaling domain can transmit effector function signals and instruct the cell to perform a specialized function. The signaling domain can include a signaling domain of another molecule. Usually, the signaling domain of another molecule can be used in the CAR, but in many cases, it is not necessary to use the entire chain. In some cases, a truncated portion of the signaling domain is used in the CAR. In some embodiments, the intracellular signaling domain includes multiple signaling domains involved in immune cell signaling or any derivative, variant, or fragment thereof. For example, the intracellular signaling domain can include at least two immune cell signaling domains, for example, at least 2, 3, 4, 5, 7, 8, 9, or 10 signaling domains. In some aspects, the CAR includes at least two intracellular signaling domains. In some aspects, the CAR includes at least three intracellular signaling domains.

[0093]

[0116] The immune cell signaling domain may be involved in regulating the primary activation of the TCR complex in either a stimulatory or inhibitory manner. The intracellular signaling domain may be the intracellular signaling domain of the T cell receptor (TCR) complex. The intracellular signaling domain of the CAR may comprise Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), fetal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the signaling domain comprises an immunoreceptor tyrosine-based activation motif, or ITAM. An ITAM-containing signaling domain may contain two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, resulting in the conserved motif YxxL / Ix(6-8)YxxL / I, where each x is, independently, any amino acid. An ITAM-containing signaling domain may be modified by phosphorylation, for example, when the ligand-binding domain binds to an epitope. The phosphorylated ITAM may serve as a docking site for other proteins, for example, proteins involved in various signaling pathways. In some embodiments, the primary signaling domain is a modified ITAM domain that has altered (e.g., increased or decreased) activity compared to the native ITAM domain. The CAR may comprise an AM domain, e.g., a mutated, truncated, and / or optimized ITAM domain. In some embodiments, the intracellular signaling domain of the CAR comprises an FcγR signaling domain (e.g., an ITAM). The FcγR signaling domain may be selected from FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the intracellular signaling domain comprises an FcεR signaling domain (e.g., an ITAM). The FcεR signaling domain may be selected from FcεRI and FcεRII (CD23). In some embodiments, the intracellular signaling domain comprises an FcαR signaling domain (e.g., an ITAM). The FcαR signaling domain may be selected from FcαRI (CD89) and Fcα / μR. In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the primary signaling domain comprises an ITAM of CD3zeta. In some embodiments, the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10, and / or CD278 (ICOS).

[0094]

[0117] In some embodiments, the intracellular signaling domain of the CAR comprises an immunoreceptor tyrosine-based inhibitory motif, or ITIM. Signaling domains that contain ITIMs may comprise a conserved amino acid sequence (S / I / V / LxYxxI / V / L) found in the cytoplasmic tails of some inhibitory receptors of the immune system. Primary signaling domains that contain ITIMs may be modified and may be phosphorylated, for example, by enzymes such as Src kinase family members (e.g., Lck). After phosphorylation, other proteins, including enzymes, may be recruited to the ITIM. These other proteins include, but are not limited to, enzymes such as phosphotyrosine phosphatases SHP-1 and SHP-2, inositol phosphatases called SHIPs, and proteins with one or more SH2 domains (e.g., ZAP70).The intracellular signaling domains are BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), Fc receptor-like protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin 4 receptor (IL4R), immunoglobulin superfamily receptor translocation-related 1 (IRT A1), Immunoglobulin Superfamily Receptor Translocation Associated 2 (IRTA2), Killer Cell Immunoglobulin-Like Receptor 2DL1 (KIR2DL1), Killer Cell Immunoglobulin-Like Receptor 2DL2 (KIR2DL2), Killer Cell Immunoglobulin-Like Receptor 2DL3 (KIR2DL3), Killer Cell Immunoglobulin-Like Receptor 2DL4 (KIR2DL4), Killer Cell Immunoglobulin-Like Receptor 2DL5 (KIR2DL5), Killer Cell Immunoglobulin-Like Receptor 3DL1 (KIR3DL1), Killer Cell Immunoglobulin-Like Receptor 3D L2 (KIR3DL2), leukocyte immunoglobulin-like receptor subfamily B member 1 (LIR1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LIR2), leukocyte immunoglobulin-like receptor subfamily B member 3 (LIR3), leukocyte immunoglobulin-like receptor subfamily B member 5 (LIR5), leukocyte immunoglobulin-like receptor subfamily B member 8 (LIR8), leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), mast cell function-associated antigen (MAFA), NKG2A, natural killer cell Cytotoxicity triggering receptor 2 (NKp44), NTB-A, programmed cell death protein 1 (PD-1), PILR, SIGLECL1, sialic acid-binding Ig-like lectin 2 (SIGLEC2 or CD22), sialic acid-binding Ig-like lectin 3 (SIGLEC3 or CD33), sialic acid-binding Ig-like lectin 5 (SIGLEC5 or CD170), sialic acid-binding Ig-like lectin 6 (SIGLEC6), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 10 (SIGLEC10), and sialic acid-binding Ig-like lectin 5 (SIGLEC5 or CD170). The intracellular signaling domain may comprise a signaling domain (e.g., an ITIM) of synthetic Ig-like lectin 11 (SIGLEC11), sialic acid-binding Ig-like lectin 4 (SIGLEC4), sialic acid-binding Ig-like lectin 8 (SIGLEC8), sialic acid-binding Ig-like lectin 9 (SIGLEC9), platelet and endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein (SIRP2), and signaling threshold regulating transmembrane adaptor 1 (SIT). In some embodiments, the intracellular signaling domain comprises a modified ITIM domain, e.g., a mutated, truncated, and / or optimized ITIM domain, having altered (e.g., increased or decreased) activity compared to the native ITIM domain. In some embodiments, the intracellular signaling domain comprises at least two ITAM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains). In some embodiments, the intracellular signaling domain comprises at least two ITIM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITIM domains) (e.g., at least two primary signaling domains). In some embodiments, the intracellular signaling domain comprises both an ITAM domain and an ITIM domain.

[0095]

[0118] In some cases, the intracellular signaling domain of the CAR can include a costimulatory domain. In some embodiments, the costimulatory domain, e.g., a costimulatory domain derived from a costimulatory molecule, can provide a costimulatory signal for immune cell signaling, e.g., signaling derived from an ITAM and / or ITIM domain for activation and / or inactivation of immune cell activity. In some embodiments, the costimulatory domain is operable to regulate proliferation and / or survival signals in immune cells. In some embodiments, the costimulatory signaling domain comprises a signaling domain of an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, or a Toll ligand receptor.In some embodiments, the costimulatory domain is selected from the group consisting of 2B4 / CD244 / SLAMF4, 4-1BB / TNFSF9 / CD137, B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R / TNFRSF13C, BAFF / BLyS / TNFSF13B, BLAME / SLAMF8, BTLA / CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229 / SLAMF3, CD27 ligand / TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-1 0 / SLAMF9, CD30 ligand / TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40 ligand / TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58 / LFA-3, CD69, CD7, CD8α, CD8β, CD82 / Kai-1, CD84 / SLAMF5, CD90 / Thy1, CD96, CDS, CEACAM1, CRACC / SLAMF7, CRTAM, CTLA-4, DAP12, Dectin1-1 / CLEC7A, DNAM1(CD226), DPPIV / CD26, DR3 / TNFRSF25, EphB6 , GADS, Gi24 / VISTA / B7-H5, GITR ligand / TNFSF18, GITR / TNFRSF18, HLA class I, HLA-DR, HVEM / TNFRSF14, IA4, ICAM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT / TNFSF14, LTBR, ​​Ly108, Ly9(C. D229), lymphocyte function-associated antigen-1 (LFA-1), lymphotoxin-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), NTB-A / SLAMF6, OX40 ligand / TNFSF4, OX40 / TNFRSF4, PAG / Cbp, PD-1, PDCD6, PD-L2 / B7-DC, PSGL1 , RELT / TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM / CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI / TNFRSF13B, TCL1A, TCL1B, TIM-1 / KIM-1 / HAVCR, TIM-4, TL1A / TNFSF15, TNF RII / TNFRSF1B, TNF-α, TRANCE / RANKL, TSLP, TSLP R, VLA1, and VLA-6. In some embodiments, the intracellular signaling domain comprises a plurality of costimulatory domains, e.g., at least two, e.g., at least three, four, or five costimulatory domains. The costimulatory signaling region may provide a signal synergistically with a primary effector activation signal to meet the requirements for activation of the T cell. In some embodiments, adding a costimulatory domain to a CAR can enhance the potency and persistence of the immune cells provided herein.

[0096]

[0119] Examples of costimulatory signaling domains are provided in Table 1.

[0097] [Table 1-1]

[0098] [Table 1-2]

[0099]

[0120] As an example, a CAR can include a CD3 zeta chain (sometimes referred to as a first generation CAR). As another example, a CAR can include a CD-3 zeta chain and a single costimulatory domain (e.g., CD28 or 4-1BB) (sometimes referred to as a second generation CAR). As another example, a CAR can include a CD-3 zeta chain and two costimulatory domains (CD28 / OX40 or CD28 / 4-1BB) (sometimes referred to as a third generation CAR). These signaling moieties, together with co-receptors such as CD8, can result in downstream activation of kinase pathways that support gene transcription and functional cellular responses.

[0100]

[0121] In some embodiments, the CAR can include a hinge or spacer. A hinge or spacer can refer to a segment between the ligand binding domain and the transmembrane domain. In some embodiments, a hinge can be used to provide flexibility to the ligand binding domain, e.g., scFv. In some embodiments, a hinge can be used to detect the expression of a CAR on the surface of a cell, for example, when an antibody to detect the scFv is not functional or available. In some cases, the hinge is derived from an immunoglobulin molecule and may require optimization depending on the location of the first or second epitope on the target. In some cases, the hinge may not belong to an immunoglobulin molecule, but instead belong to another molecule, such as the natural hinge of the CD8 alpha molecule. The CD8 alpha hinge is a cysteine ​​and proline that may play a role in the interaction of the CD8 co-receptor and the MHC molecule. In some embodiments, cysteine ​​and proline residues can affect the performance of a CAR and therefore can be engineered to affect the performance of a CAR.

[0101]

[0122] The hinge can be of any suitable length. In some embodiments, the hinge of the CAR can be size adjustable, allowing some compensation in standardizing the orthogonal synaptic distance between the CAR-expressing cell and the target cell. This topography of the immunological synapse between the CAR-expressing cell and the target cell can also define distances that cannot be functionally bridged by the CAR due to membrane-distal epitopes on cell surface target molecules, where the synaptic distance cannot be approximated for signaling even with short hinge CARs. Similarly, membrane-proximal CAR target antigen epitopes have been described where signaling output is only observed with long hinge CARs. The hinges disclosed herein can be adjusted depending on the single chain variable fragment region that can be used. In some aspects, the hinge can be derived from CD28, IgG1 and / or CD8α.

[0102]

[0123] As an example, a CAR can include an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain as shown in FIG. 3. A CAR can include a ligand binding domain, generally derived from a single chain antibody, a hinge domain (H) or spacer, a transmembrane domain (TM) that provides an anchorage to the plasma membrane, and a signaling domain responsible for T cell activation. A CAR can include an immune cell signaling domain, such as CD3ζ chain. A CAR can include an immune cell signaling domain and a first costimulatory domain, such as CD3ζ chain and 4-1BB. A CAR can include an immune cell signaling domain and at least two costimulatory domains, such as CD3ζ chain, 4-1BB, and OX40. In some embodiments, a universal CAR can also be utilized in the methods provided herein. A universal CAR can include an intracellular signaling domain fused to a protein domain that binds to a tag (e.g., fluorescein isothiocyanate or biotin) on a monoclonal antibody. Various combinations of immune cell signaling domains and costimulatory domains can be utilized in the present CAR. In some embodiments, the immune cell signaling domain may be derived from CD3, CD4, and / or CD8. The costimulatory domain may be derived from 4-1BB, OX40, CD28, etc.

[0103]

[0124] In some embodiments, the ligand of the TCR or CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA 125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.2. In some embodiments, the ligand of the TCR or CAR may be any one or a portion of a cancer cell, an endogenous cell, a cell of the vasculature, a cell of the tumor microenvironment, and any combination thereof.

[0104]

[0125] In some embodiments, the CAR further comprises a signal peptide. In some aspects, the CAR of the present disclosure may comprise a signal peptide so that when the CAR is expressed in a cell, such as an immune cell, the nascent protein can be directed to the endoplasmic reticulum and then to the cell surface where it can be expressed. The core of the signal peptide may comprise a long stretch of hydrophobic amino acids that have a tendency to form a single alpha helix. The signal peptide may begin with a short stretch of positively charged amino acids, which may help enforce the proper topology of the polypeptide during translocation. At the end of the signal peptide, there may be a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave either during or after translocation is completed to generate a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. The signal peptide may be at the amino terminus of the molecule. In some embodiments, the CAR may have the general formula: signal peptide-ligand binding domain-spacer-domain-transmembrane domain / intracellular T cell signaling domain. The signal peptide may be or may be derived from IgG1, GM-CSF and / or CD8α.

[0105]

[0126] In some embodiments, the method can include administration comprising injection of the engineered cells provided herein. In some aspects, the injection can be intravenous. In some embodiments, the administration can be about 1×10 2 In some embodiments, the administration comprises an infusion of about 1×10 engineered immune cells / kg body weight. 3 In some embodiments, administration comprises injection of about 1×10 4 In some embodiments, administration includes injection of about 1×10 5 In some embodiments, administration includes injection of about 3×10 5 In some embodiments, administration includes injection of about 1×10 5 pieces / kg weight ~ approx. 3×10 5In some embodiments, administration includes injection of about 0.5×10 5 pcs / kg weight ~ approx. 1×10 5 In some embodiments, administration includes injection of about 1×10 4 pieces / kg weight ~ approx. 4×10 5 In some embodiments, administration includes injection of about 0.5×10 5 pcs / kg weight ~ approx. 1×10 5 In some embodiments, administration comprises injection of about 0.5×10 5 kg body weight ~ approx. 1.5 x 10 5 In some embodiments, administration comprises injection of about 1×10 3 Includes injections from 1 / kg body weight.

[0106]

[0127] In some embodiments, a total of about 5×10 10 In some cases, about 5×10 cells are administered to the subject. 10 These cells represent the median amount of cells administered to a subject. In some embodiments, about 5×10 10 In some embodiments, a subject receives at least about 1×10 cells to affect a therapeutic response in a subject. 6 At least about 2 x 10 cells 6 cells, at least about 3 x 10 6 cells, at least about 4 x 10 6 cells, at least about 5 x 10 6 cells, at least about 6 x 10 6 cells, at least about 6 x 10 6 cells, at least about 8 x 10 6 cells, at least about 9 x 10 6 cells, 1 x 10 7 At least about 2 x 10 cells 7 cells, at least about 3 x 10 7 cells, at least about 4 x 10 7 cells, at least about 5 x 10 7 cells, at least about 6 x 10 7 cells, at least about 6 x 10 7cells, at least about 8 x 10 7 cells, at least about 9 x 10 7 cells, at least about 1 x 10 8 At least about 2 x 10 cells 8 cells, at least about 3 x 10 8 cells, at least about 4 x 10 8 cells, at least about 5 x 10 8 cells, at least about 6 x 10 8 cells, at least about 6 x 10 8 cells, at least about 8 x 10 8 cells, at least about 9 x 10 8 cells, at least about 1 x 10 9 At least about 2 x 10 cells 9 cells, at least about 3 x 10 9 cells, at least about 4 x 10 9 cells, at least about 5 x 10 9 cells, at least about 6 x 10 9 cells, at least about 6 x 10 9 cells, at least about 8 x 10 9 cells, at least about 9 x 10 9 cells, at least about 1 x 10 10 At least about 2 x 10 cells 10 cells, at least about 3 x 10 10 pcs Cells, at least about 4 x 10 10 cells, at least about 5 x 10 10 cells, at least about 6 x 10 10 cells, at least about 6 x 10 10 cells, at least about 8 x 10 10 cells, at least about 9 x 10 10 cells, at least about 1 x 10 11 At least about 2 x 10 cells 11 cells, at least about 3 x 10 11 cells, at least about 4 x 10 11 cells, at least about 5 x 10 11 cells, at least about 6 x 10 11cells, at least about 6 x 10 11 cells, at least about 8 x 10 11 cells, at least about 9 x 10 11 cells or at least about 1 x 10 12 A total concentration or dose of cells (cells / kg body weight) may be administered to a subject, or the cells may be administered or dosed on a body weight basis (cells / kg body weight). For example, about 5×10 10 In another example, the cells may be administered to a subject in a dose of 3×10 6 Starting with cells, approximately 5 x 10 10 The cells may be expanded to a number sufficient for treatment, for example, 5×10 cells, and administered to the subject. In some cases, the cells are expanded to a number sufficient for treatment, for example, 5×10 cells. 7 The cells can undergo rapid expansion to generate sufficient numbers for therapeutic use. In some embodiments, a total of about 1×10 6 Fewer than about 1 x 10 cells are administered to a subject. 6 cells represent the median amount of cells administered to a subject. In some cases, up to about 9×10 5 cells, up to about 8 × 10 5 cells, up to about 7 × 10 5 cells, up to about 6 × 10 5 cells, up to about 5 × 10 5 cells, up to about 4 × 10 5 cells, up to about 3 × 10 5 cells, up to about 2 × 10 5 cells, up to about 1 x 10 5 cells, up to about 9 × 10 4 cells, up to about 8 × 10 4 cells, up to about 7 × 10 4 cells, up to about 6 × 10 4 cells, up to about 5 × 10 4 cells, up to about 4 × 10 4 cells, up to about 3 × 10 4 cells, up to about 2 × 10 4 cells, up to about 1 x 10 4 cells, up to about 9 × 10 3 cells, up to about 8 × 103 cells, up to about 7 × 10 3 cells, up to about 6 × 10 3 cells, up to about 5 × 10 3 cells, up to about 4 × 10 3 cells, up to about 3 × 10 3 cells, up to about 2 × 10 3 cells, or up to approximately 1 x 10 3 The cells are administered or dosed to the subject on a body weight basis (cells / kg body weight).

[0107]

[0128] In some embodiments, the methods provided herein are free of cell expansion. In some aspects, the engineered cells, e.g., immune cells, have been subjected to ex vivo expansion for less than 3 weeks. In some aspects, the engineered cells, e.g., immune cells, have been subjected to ex vivo expansion for less than 2 weeks. In some aspects, the engineered cells, e.g., immune cells, have been subjected to ex vivo expansion for less than 1 week. In some aspects, the engineered cells, e.g., immune cells, have been subjected to ex vivo expansion for less than 5 days. In some aspects, the engineered cells, e.g., immune cells, have been subjected to ex vivo expansion for less than 3 days. In some aspects, the engineered cells, e.g., immune cells, have been subjected to ex vivo expansion for less than 2 days. In some aspects, the engineered cells, e.g., immune cells, have been subjected to ex vivo expansion for less than 1 day. The cells are subjected to in vivo expansion. In some cases, the total number of cells (e.g., F-CART cells) can be administered to the subject via a single administration. Alternatively, the total number of cells (e.g., F-CART cells) can be administered to the subject multiple times, for example, via two separate administrations separated by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 20 hours, 24 hours or more.

[0108]

[0129] In some cases, a sufficient number for therapeutic use is about 5×10 4Any number of cells may be injected for therapeutic use, and the cells may be included in a pharmaceutical composition. For example, a patient may be injected with 1×10 cells per kg of body weight. 4 ~5×10 12 A patient may be infused with as many cells as can be generated for sufficient numbers. In some embodiments, the generation of cells is without expansion. In some cases, not all of the cells infused into the patient are engineered. For example, at least 90% of the cells infused into the patient may be engineered. In other examples, at least 40% of the cells infused into the patient may be engineered. The amount of cells required to be therapeutically effective may vary depending on the viability of the cells and the efficiency of modification of the cells. In some cases, the product (e.g., multiplication) of the viability of the cells after genetic modification may correspond to the therapeutic aliquot of cells available for administration to the subject. In some cases, an increase in the viability of the cells after modification may correspond to a decrease in the amount of cells required for administration to be therapeutically effective in the patient. In some embodiments, at least about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of the immune cells express the CAR and / or the TCR. In some embodiments, engineered cells may be selected for administration. In some embodiments, at least 20% of the immune cells express the CAR and / or the TCR. In some embodiments, at least 25% of the immune cells express a CAR and / or a TCR. In some embodiments, at least 30% of the immune cells express a CAR and / or a TCR. In some embodiments, at least 40% of the immune cells express a CAR and / or a TCR.

[0109]

[0130] In some embodiments, the method may further include administering a secondary agent to a subject in need thereof. The secondary agent may be a therapeutically effective amount of an immunostimulant, an immunosuppressant, an antifungal agent, an antibiotic, an antiangiogenic agent, a chemotherapeutic agent, a radioactive agent, and / or an antiviral agent. The secondary agent may be a pharmaceutical composition.

[0110]

[0131] In some embodiments, an immune stimulant may be introduced into the cell or subject. The immune stimulant may be specific or non-specific. The specific immune stimulant may exhibit antigen specificity, such as a vaccine or an antigen. The non-specific immune stimulant may enhance or stimulate an immune response. The non-specific immune stimulant may be an adjuvant. The immune stimulant may be any one of a vaccine, a colony stimulant, an interferon, an interleukin, a virus, an antigen, a co-stimulant, an immunogenic agent, an immunomodulatory agent, or an immunotherapeutic agent. The immune stimulant may be a cytokine, such as an interleukin. One or more cytokines may be introduced with the cells of the provided method. The cytokine may be utilized to boost cytotoxic T lymphocytes (including adoptively transferred tumor-specific cytotoxic T lymphocytes) and expand within the tumor microenvironment. In some cases, IL-2 may be used to promote the expansion of the cells described herein. Cytokines such as IL-15 may also be used. Other related cytokines in the field of immunotherapy, such as IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof, can also be utilized. In some cases, IL-2, IL-7, and IL-15 are used to culture the cells of the present invention. The interleukin can be IL-2, or aldeskeukin.

[0111]

[0132] In some embodiments, an immune stimulant can be administered to the subject. Aldesleukin can be administered in low or high doses. A high dose aldesleukin regimen can include intravenously administering aldesleukin at about 0.037 mg / kg (600,000 IU / kg) every 8 hours for up to about 14 doses, as tolerated. The immune stimulant (e.g., aldesleukin) can be administered within 24 hours after cell administration. The immune stimulant (e.g., aldesleukin) can be administered as an infusion about every 8 hours for about 15 minutes for up to about 4 days after cell infusion. The immune stimulant (e.g., aldesleukin) can be administered at a dose from about 100,000 IU / kg, 200,000 IU / kg, 300,000 IU / kg, 400,000 IU / kg, 500,000 IU / kg, 600,000 IU / kg, 700,000 IU / kg, 800,000 IU / kg, 900,000 IU / kg, or up to about 1,000,000 IU / kg. In some cases, aldesleukin is administered at a dose of about 100,000 IU / kg to 300,000 IU / kg, 300,000 IU / kg to 500,000 IU / kg, 500,000 IU / kg to 700,000 IU / kg, or 700,000 IU / kg to about 1,000,000 IU / kg. The immune stimulant (e.g., aldesleukin) may be administered from 1 dose to about 14 doses. The immune stimulant (e.g., aldesleukin) may be administered from at least about 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses, 13 doses, 14 doses, 15 doses, 16 doses, 17 doses, 18 doses, 19 doses, or up to about 20 doses. In some cases, the immune stimulant, such as aldesleukin, may be administered from about 1 dose to 3 doses, 3 doses to 5 doses, 5 doses to 8 doses, 8 doses to 10 doses, 10 doses to 14 doses, 14 doses to 20 doses. In some cases, the immune stimulant, such as aldesleukin, may be administered in more than 20 doses. In some cases, the immune stimulant, such as aldesleukin, may be administered sequentially or simultaneously with the administration of the cells. For example, the immune stimulant can be administered from about day -14, day -13, day -12, day -11, day -10, day -9, day -8, day -7, day -6, day -5, day -4, day -3, day -2, day -1, day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or up to about day 14. In some cases, the immune stimulant, such as aldesleukin, is administered from day 0 to day 4 after administration of the cell population. In some cases, the immune stimulant (e.g., aldesleukin) is administered for about 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, or up to about 3 hours. In some cases, the immune stimulant (e.g., aldesleukin) can be administered from about 24 hours prior to administration of the engineered cells to about 4 days after administration of the engineered cells. The immune stimulant (e.g., aldesleukin) can be administered from day -7, day -6, day -5, day -4, day -3, day -2, day -1, day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, or up to about day 20 after administration of the engineered cells.

[0112]

[0133] In some embodiments, the immune stimulant is a colony stimulating factor. The colony stimulating factor can be G-CSF (filgrastim). Filgrastim can be stored at 300mcg / ml and 480ug / 1.6ml vial. Filgrastim can be administered daily as a subcutaneous injection. The administration of filgrastim can be from about 5mcg / kg / day. The administration of filgrastim can be from about 1mcg / kg / day. The administration of filgrastim can be from about 2mcg / kg / day. , the administration of filgrastim may be from about 3mcg / kg / day, the administration of filgrastim may be from about 4mcg / kg / day, the administration of filgrastim may be from about 5mcg / kg / day, the administration of filgrastim may be from about 6mcg / kg / day, the administration of filgrastim may be from about 7mcg / kg / day, the administration of filgrastim may be from about 8mcg / kg / day, the administration of filgrastim may be from about 9mcg / kg / day, and the administration of filgrastim may be from about 10mcg / kg / day. In some cases, filgrastim may be administered at a dose ranging from about 0.5 mcg / kg / day to about 1.0 mcg / kg / day, from about 1.0 mcg / kg / day to 1.5 mcg / kg / day, from about 1.5 mcg / kg / day to about 2.0 mcg / kg / day, from about 2.0 mcg / kg / day to about 3.0 mcg / kg / day, from about 2.5 mcg / kg / day to about 3.5 mcg / kg / day, from about 3.5 mcg / kg / day to about 4.0 mcg / kg / day, or from about 4.0 mcg / kg / day to about 4.5 mcg / kg / day. Administration of filgrastim is recommended when the neutrophil count is at least about 1.0 x 10 9 cells / L for 3 days, or at least about 5.0 × 10 9 The immunostimulant, such as filgrastim, may be administered starting on day -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about day 20 after administration of the engineered cells.

[0113]

[0134] In some embodiments, the method further comprises administering an immunosuppressant to the subject. In some cases, the subject may receive an immunosuppressant as part of the treatment regimen. The immunosuppressant may refer to a radiotherapeutic agent, a biological agent, or a chemical agent. In some cases, the immunosuppressant may include a chemical agent. For example, the chemical agent may include at least one member from the group consisting of cyclophosphamide, mechlorethamine, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, fludarabine, nitrosoureas, platinum, methotrexate, azathioprine, mercaptopurine, procarbazine, dacarbazine, temozolomide, carmustine, lomustine, streptozocin, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, and mithramycin. The chemical agent may be cyclophosphamide or fludarabine.

[0114]

[0135] Furthermore, the immunosuppressant may include glucocorticoids, cytostatics, antibodies, anti-immunophilins, or any derivatives thereof. Glucocorticoids can suppress allergic reactions, inflammation, and autoimmune conditions. Glucocorticoids can be prednisone, dexamethasone, and hydrocortisone. Immunosuppressive therapy can include any treatment that suppresses the immune system. Immunosuppressive therapy can help reduce, minimize, or eliminate transplant rejection in the recipient. For example, immunosuppressive therapy can include immunosuppressants. Immunosuppressants that may be used before, during and / or after transplant include MMF (mycophenolate mofetil (Cellcept)), ATG (antithymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10, ASKP1240, CCFZ533X2201), alemtuzumab (Campath), anti-CD20 (rituximab), anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies (sarilumab, olokizumab), CTLA4-Ig (abatacept / Orencia), belatacept (LEA29Y), sirolimus (Rapi mune), everolimus, tacrolimus (Prograf), daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade), cyclosporine, deoxyspergualin, soluble complement receptor 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / Soliris), methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody. In addition, one or more immunosuppressants / immunosuppressants may be used together or sequentially. One or more immunosuppressants / immunosuppressants may be used for induction therapy or maintenance therapy. The same or different drugs may be used in the induction and maintenance phases. In some cases, daclizumab (Zenapax) may be used for induction therapy, and tacrolimus (Prograf) and sirolimus (Rapimune) for maintenance therapy.Daclizumab (Zenapax) may also be used for induction therapy, while low-dose tacrolimus (Prograf) and low-dose sirolimus (Rapimune) may be used for maintenance therapy. Immunosuppression may be achieved using non-drug regimens, including but not limited to total body irradiation, thymic irradiation, and total and / or partial splenectomy.

[0115]

[0136] In some cases, a cytostatic agent may be administered for immunosuppression. The cytostatic agent may inhibit cell division. The cytostatic agent may be a purine analog. The cytostatic agent may be an alkylating agent or an antimetabolite such as methotrexate, azathioprine, or mercaptopurine. The cytostatic agent may be at least one of cyclophosphamide, mechlorethamine, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, fludarabine, nitrosoureas, platinum, methotrexate, azathioprine, mercaptopurine, procarbazine, dacarbazine, temozolomide, carmustine, lomustine, streptozocin, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, and mithramycin.

[0116]

[0137] In some cases, an immunosuppressant such as fludarabine may be administered as part of the treatment regimen. Fludarabine phosphate may be a synthetic purine nucleoside that differs from physiological nucleosides in that the sugar moiety may be arabinose instead of ribose or deoxyribose. Fludarabine may be a purine antagonist antimetabolite. Fludarabine may be supplied in 50 mg vials in the form of a white lyophilized solid cake and as fludarabine phosphate powder. The solution may have a pH of 7.7 after reconstitution with 2 mL of sterile water for injection to a concentration of 25 mg / mL. Fludarabine powder may be stable for at least 18 months at 2-8°C, and when reconstituted, fludarabine is stable at room temperature for at least 16 days. Because it contains no preservatives, reconstituted fludarabine is typically administered within 8 hours. Specialist references should be consulted for specific suitability information. Fludarabine can be dephosphorylated in serum, transported intracellularly, and converted to the nucleotide fludarabine triphosphate. This 2-fluoro-ara-ATP molecule is thought to be necessary for the cytotoxic effect of the drug. Fludarabine can inhibit DNA polymerase, ribonucleotide reductase, and DNA primase, preventing chain elongation, and RNA and protein synthesis. Fludarabine can be administered as an IV infusion over 15-30 minutes in 100 ml of 0.9% sodium chloride, USP. Dosing is based on body surface area (BSA). If the patient is obese (BMI>35), practical body weight is used to calculate drug dosing. In some cases, immunosuppressants such as fludarabine are administered at approximately 20 mg / m2 of the subject's body surface area. 2 ~about 30mg / m 2 In some cases, the immunosuppressant, such as fludarabine, may be administered at a dose of about 5 mg / m2 of the subject's body surface area. 2 ~about 10mg / m 2 , approximately 10 mg / m of the subject's body surface area 2 ~about 15mg / m 2 , approximately 15 mg / m of the subject's body surface area 2 ~about 20mg / m 2 , approximately 20 mg / m of the subject's body surface area 2 ~about 25mg / m2 , approximately 25 mg / m of the subject's body surface area 2 ~about 30mg / m 2 , approximately 30 mg / m of the subject's body surface area 2 ~about 40mg / m 2 In some cases, the immunosuppressant, such as fludarabine, may be administered at a dose of about 1 mg / m2 of the subject's body surface area. 2 , 2 mg / m 2 , 3 mg / m 2 , 4 mg / m 2 , 5 mg / m 2 , 6 mg / m 2 , 7 mg / m 2 , 8 mg / m 2 , 9 mg / m 2 , 10 mg / m 2 , 11 mg / m 2 , 12 mg / m 2 , 13 mg / m 2 , 14 mg / m 2 , 15 mg / m 2 , 16 mg / m 2 , 17 mg / m 2 , 18 mg / m 2 , 19 mg / m 2 , 20 mg / m 2 , 21 mg / m 2 , 22 mg / m 2 , 23 mg / m 2 , 24 mg / m 2 , 25 mg / m 2 , 26 mg / m 2 , 27 mg / m 2 , 28 mg / m 2 , 29 mg / m 2 , 30 mg / m 2 , 31 mg / m 2 , 32 mg / m 2 , 33 mg / m 2 , 34 mg / m 2 , 35 mg / m 2 , 36 mg / m 2 , 37 mg / m 2 , 38 mg / m 2 , 39 mg / m 2 , 40 mg / m 2 , 41 mg / m 2 , 42 mg / m 2 , 43 mg / m 2、44mg / m 2 、45mg / m 2 、46mg / m 2 、47mg / m 2 、48mg / m 2 、49mg / m 2 、50mg / m 2 、51mg / m 2 、52mg / m 2 、53mg / m 2 、54mg / m 2 、55mg / m 2 、56mg / m 2 、57mg / m 2 、58mg / m 2 、59mg / m 2 、60mg / m 2 、61mg / m 2 、62mg / m 2 、63mg / m 2 、64mg / m 2 、65mg / m 2 、66mg / m 2 、67mg / m 2 、68mg / m 2 、69mg / m 2 、70mg / m 2 、71mg / m 2 、72mg / m 2 、73mg / m 2 、74mg / m 2 、75mg / m 2 、76mg / m 2 、77mg / m 2 、78mg / m 2 、79mg / m 2 、80mg / m 2 、81mg / m 2 、82mg / m 2 、83mg / m 2 、84mg / m 2 、85mg / m 2 、86mg / m 2 、87mg / m 2 、88mg / m 2 、89mg / m 2 、90mg / m 2 、91mg / m 2 、92mg / m 2 、93mg / m 2, 94 mg / m 2 , 95 mg / m 2 , 96 mg / m 2 , 97 mg / m 2 , 98 mg / m 2 , 99 mg / m 2 , up to about 100mg / m 2 In some cases, immunosuppressants such as fludarabine can be administered at 25 mg / m in 100 mL of 0.9% sodium chloride, USP. 2 is infused over a period of about 15 to about 30 minutes.

[0117]

[0138] In some cases, immunosuppressive drugs such as cyclophosphamide are part of the treatment regimen. Cyclophosphamide may be administered as a cyclophosphamide infusion. Cyclophosphamide may be a nitrogen mustard derivative alkylating agent. Cyclophosphamide functions as an alkylating agent after being converted to active metabolites in the liver: the drug also has potent immunosuppressant activity. The serum half-life after IV administration is 3-12 hours; the drug and / or its metabolites may be detected in serum for up to 72 hours after administration. After reconstitution as indicated with sterile water for injection, cyclophosphamide may be stable for 24 hours at room temperature, or for 6 days when maintained at 2-8°C. Cyclophosphamide may be diluted in 250 mL of D5W and infused over 1 hour. Dosage is based on the subject's weight. If the subject is obese (BMI>35), drug dosage is calculated using practical weight as described. In some cases, the immunosuppressant, such as cyclophosphamide, may be administered at about 1 mg / kg to about 3 mg / kg, about 3 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, 20 mg / kg to about 30 mg / kg, about 30 mg / kg to about 40 mg / kg, about 40 mg / kg to about 50 mg / kg, about 50 mg / kg to about 60 mg / kg, about 60 mg / kg to about 70 mg / kg, about 70 mg / kg to about 80 mg / kg, about 80 mg / kg to about 90 mg / kg, or about 90 mg / kg to about 100 mg / kg. In some cases, the immunosuppressant, such as cyclophosphamide, may be administered at more than 50 mg / kg of the subject. In some cases, the immunosuppressant, such as cyclophosphamide, is administered at a concentration of about 1 mg / kg of the subject, 2 mg / kg of the subject, 3 mg / kg of the subject, 4 mg / kg of the subject, 5 mg / kg of the subject, 6 mg / kg of the subject, 7 mg / kg of the subject, 8 mg / kg of the subject, 9 mg / kg of the subject, 10 mg / kg of the subject, 11 mg / kg of the subject, 12 mg / kg of the subject, 13 mg / kg of the subject, 14 mg / kg of the subject, 15 mg / kg of the subject, 16 mg / kg of the subject, 17 mg / kg of the subject, 18 mg / kg of the subject, 19 mg / kg of the subject, 20 mg / kg of the subject, 21 mg / kg of the subject, 22 mg / kg of the subject, 23 mg / kg of the subject, 24 mg / kg of the subject, 25 mg / kg of the subject, 26 mg / kg of the subject, 27 mg / kg of the subject, 28 mg / kg of the subject, 29 mg / kg of the subject,30mg / kg subjects, 31mg / kg subjects, 32mg / kg subjects, 33mg / kg subjects, 34mg / kg subjects, 35mg / kg subjects, 36mg / kg subjects, 37mg / kg subjects, 38mg / kg subjects, 39mg / kg subjects, 40mg / kg subjects, 41mg / kg subjects, 42mg / kg subjects, 43mg / kg subjects, 44mg / kg subjects, 45mg / kg subjects, 46mg / kg subjects, 47mg / kg subjects, 48mg / kg subjects, 49mg / kg subjects, 50mg / kg subjects, 51mg / kg subjects, 52mg / kg subjects, 53mg / kg subjects, 54mg / kg subjects, 55mg / kg subjects, 56mg / kg subjects, 57mg / kg subjects, 58mg / kg subjects, 59mg / kg subjects, 60mg / kg subjects, 61mg / kg subjects, 62mg / kg subjects, 63mg / kg subjects, 64mg / kg subjects, 65mg / kg subjects, 66mg / kg subjects, 67mg / kg subjects, 68mg / kg subjects, 69mg / kg subjects, 70mg / kg subjects, 71mg / kg subjects, 72mg / kg subjects, 73mg / kg subjects, 74mg / kg subjects, 75mg / kg subjects, 76mg / kg subjects, 77mg / kg subjects, 78mg / kg subjects, 79mg / kg subjects, 80mg / kg subjects, 81mg / kg subjects, 82mg / kg subjects, 83mg / kg subjects, 84mg / kg of subject, 85mg / kg of subject, 86mg / kg of subject, 87mg / kg of subject, 88mg / kg of subject, 89mg / kg of subject, 90mg / kg of subject, 91mg / kg of subject, 92mg / kg of subject, 93mg / kg of subject, 94mg / kg of subject, 95mg / kg of subject, 96mg / kg of subject, 97mg / kg of subject, 98mg / kg of subject, 99mg / kg of subject, up to about 100mg / kg of subject. In some cases, the immunosuppressant such as cyclophosphamide may be administered for at least about 1 day to about 3 days, 3 days to 5 days, 5 days to 7 days, 7 days to about 10 days, 10 days to 14 days, 14 days to about 20 days. In some cases, the cyclophosphamide may be at a dose of about 60mg / kg, diluted in 250mL of 5% dextrose in water and infused over 1 hour.

[0118]

[0139] The immunosuppressant may be, for example, a regimen of cyclophosphamide and fludarabine. For example, a cyclophosphamide-fludarabine regimen may be administered to a subject receiving engineered cell therapy. The cyclophosphamide-fludarabine regimen is 60 mg / kg qd for 2 days and 25 mg / m 2 The chemotherapy regimen may be administered qd for 5 days. The chemotherapy regimen, e.g., cyclophosphamide-fludarabine, may be administered from 1 hour to 14 days prior to administration of the engineered cells of the invention. The chemotherapy regimen may be administered in different doses. For example, the subject may receive a high dose initially, followed by a low dose. The subject may receive a low dose initially, followed by a high dose.

[0119]

[0140] In some cases, the immunosuppressant may be an antibody. The antibody may be administered in a therapeutically effective amount. The antibody may be a polyclonal or monoclonal antibody. The polyclonal antibody may be administered may be an anti-lymphocyte or anti-thymocyte antigen. The monoclonal antibody may be an anti-IL-2 receptor antibody, an anti-CD25 antibody, or an anti-CD3 antibody. An anti-CD20 antibody may also be used. An agent that reacts with CD20, such as B cell depletion therapy, such as Rituxan, may be used as an immunosuppressant.

[0120]

[0141] The immunosuppressant may be an anti-immunophilin. The anti-immunophilin may be cyclosporine, tacrolimus, everolimus, or sirolimus. The additional immunosuppressant may be an interferon, such as IFN-beta, an opioid, an anti-TNF binding agent, mycophenolate, or fingolimod.

[0121]

[0142] In some embodiments, the method may further include administering radiation therapy to the subject. The radiation therapy may include irradiation. Total body irradiation may be administered at 12 Gy. The irradiation dose may include a cumulative dose of 12 Gy to the whole body, including healthy tissue. The irradiation dose may include 5 Gy to 20 Gy. The irradiation dose may be 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy, 12, Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, or up to 20 Gy. The irradiation may be total body irradiation, or irradiation of a portion of the body. If the irradiation is total body irradiation, it may be uniform or non-uniform. For example, if the irradiation is non-uniform, a small area of ​​the body, such as the neck, may receive a higher dose than a larger area, such as the lower back.

[0122]

[0143] In some embodiments, the methods provided herein can further include administering a chemotherapeutic agent. The chemotherapeutic agent or compound can be a chemical compound useful in the treatment of cancer. Exemplary chemotherapeutic agents that can be used in combination with the methods of the present disclosure include, but are not limited to, mitotic inhibitors (vinca alkaloids). Mitotic inhibitors include vincristine, vinblastine, vindesine, and Navelbine™ (vinorelbine, 5'-noranhydroblastine). In still other cases, the cancer chemotherapeutic agent includes topoisomerase I inhibitors, such as camptothecin compounds. As used herein, "camptothecin compounds" includes Camptosar™ (irinotecan HCL), Hycamtin™ (topotecan HCL), and other compounds derived from camptothecin and its analogs. Another cancer category of chemotherapy agents that can be used in the methods and compositions disclosed herein are podophyllotoxin derivatives, such as etoposide, teniposide, and mitopodozide. The present disclosure further encompasses other cancer chemotherapy agents known as alkylating agents, which alkylate genetic material in tumor cells. Alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, verstine, uracil mustard, chromafazine, and dacarbazine. The present disclosure encompasses antimetabolites as chemotherapy agents. Examples of agents in this class include cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, Azathioprim, and procarbazine. Additional categories of cancer chemotherapeutic agents that can be used in the methods and compositions disclosed herein include antibiotics. Examples include, but are not limited to, doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C, and daunomycin. Many liposomal formulations of these compounds are commercially available. The present disclosure further encompasses other cancer chemotherapeutic agents, such as, but not limited to, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, ifosfamide, and mitoxantrone.

[0123]

[0144] In some embodiments, the method can further comprise administering an antiviral agent to the subject. In some cases, the antiviral agent can be administered as part of the treatment regimen. In some cases, a herpes virus prophylactic agent can be administered to the subject as part of the treatment regimen. The herpes virus prophylactic agent can be valacyclovir (Valtrex). Valtrex can be used orally to prevent the development of herpes virus infection in subjects with positive HSV serology. Additional antiviral agents that can be administered include, but are not limited to, anti-hepatitis B virus (HBV) agents, anti-hepatitis C virus (HCV) agents, anti-human papillomavirus (HPV) agents, and anti-Epstein-Barr virus (EBV) agents.

[0124]

[0145] In some embodiments, the method may further include administering an antibiotic to the subject. The antibiotic may be administered in a therapeutically effective amount. The antibiotic may kill or inhibit the growth of bacteria. The antibiotic may be a broad-spectrum antibiotic capable of targeting a wide range of bacteria. The broad-spectrum antibiotic, either third or fourth generation, may be a cephalosporin or a quinolone. The antibiotic may be a narrow-spectrum antibiotic capable of targeting a specific type of bacteria. The antibiotic may be one that targets the bacterial cell wall, such as penicillins and cephalosporins. The antibiotic may be one that targets the cell membrane, such as polymyxins. The antibiotic may be one that interferes with essential bacterial enzymes, such as rifamycins, lipiarmycins, quinolones, and sulfonamides. The antibiotic may be a protein synthesis inhibitor, such as macrolides, lincosamides, and tetracyclines. The antibiotic may be a cyclic lipopeptide such as daptomycin, a glycylcycline such as tigecycline, an oxazolidinone such as linezolid, and a lipialmycin such as fidaxomicin. In some cases, the antibiotic may be a first generation, second generation, third generation, fourth generation, or fifth generation. The first generation antibiotic may have a narrow spectrum. The first generation antibiotic may be, for example, a penicillin (penicillin G or penicillin V), a cephalosporin (cefazolin, cephalothin, cephapirin, cepharetin, cepharadine, or cephadroxine). In some cases, the antibiotic may be a second generation. The second generation antibiotic may be a penicillin (amoxicillin or ampicillin), a cephalosporin (cefuroxime, cefamandole, cefoxitin, cefaclor, cefrodil, loracarbef). In some cases, the antibiotic may be a third generation. The third generation antibiotic may be a penicillin (carbenicillin and ticarcillin) or a cephalosporin (cefixime, ceftriaxone, cefotaxime, ceftizoxime, and ceftazidime). The antibiotic may be a fourth generation antibiotic. The fourth generation antibiotic may be cefipime. The antibiotic may be a fifth generation. The fifth generation antibiotic may be ceftaroline or ceftobiprole.In some cases, the antibiotic may be a bacterial wall targeting agent, a cell membrane targeting agent, a bacterial enzyme blocking agent, a bactericidal agent, a protein synthesis inhibitor, or a bacteriostatic agent. The bacterial wall targeting agent may be a penicillin derivative (penam), a cephalosporin (cephem), a monobactam, and a carbapenem. β-lactam antibiotics are bactericidal or bacteriostatic and act by inhibiting the synthesis of the peptidoglycan layer of the bacterial cell wall. In some cases, the antibiotic may be a protein synthesis inhibitor. Protein synthesis inhibitors inhibit the synthesis of the peptidoglycan layer that bacteria need to make their cell walls. The bactericidal agent may be ampicillin, which acts as an irreversible inhibitor of the required enzyme transpeptidase. Protein synthesis inhibitors inhibit the third and final step of bacterial cell wall synthesis in binary fission, ultimately leading to cell lysis. Thus, ampicillin is usually bacteriolytic. In some cases, the bactericidal agent may be a cephalosporin or a quinolone. In other cases, the bacteriostatic agent is trimethoprim, sulfamethoxazole, or pentamidine.

[0125]

[0146] In some cases, drugs for the prevention of PCP pneumonia may be administered. For example, trimethoprim and sulfamethoxazole may be administered to prevent pneumonia. The dose of trimethoprim and sulfamethoxazole (TMP / SMX; an exemplary sulfa drug) may be one tablet PO per day, three times per week on non-consecutive days, upon or after the first dose of chemotherapy, continuing for at least about 6 months and until the CD4 count exceeds 200 in at least two laboratory studies. In some cases, trimethoprim may be administered at 160 mg. Trimethoprim may be administered at about 100 to about 300 mg. Trimethoprim may be administered from about 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, or up to about 300 mg. In some cases, sulfamethoxazole is administered at 800 mg. Sulfamethoxazole can be administered at about 500mg to about 1000mg. Sulfamethoxazole can be administered at about 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg, or up to about 1000mg. In some cases, the TMP / SMX regimen can be administered in a therapeutically effective amount. TMP / SMX can be administered at about 1X to about 10X daily. TMP / SMX can be administered 1X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 11X, 12X, 13X, 14X, 15X, 16X, 17X, 18X, 19X, or up to about 20X daily. In some cases, TMP / SMX can be administered on a weekly basis. For example, TMP / SMX can be administered from 1X, 2X, 3X, 4X, 5X, 6X, or up to about 7X per week.The TMP / SMX regimen can be administered from about day -14, day -13, day -12, day -11, day -10, day -9, day -8, day -7, day -6, day -5, day -4, day -3, day -2, day -1, day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or up to about day 14 after administration of a cell therapy such as FAST-CART.

[0126]

[0147] In some embodiments, the methods provided herein may be used in combination with antiangiogenic agents. Antiangiogenic agents suitable for use in the disclosed methods and compositions include anti-VEGF antibodies, such as humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other angiogenesis inhibitors that may be utilized with the provided methods and compositions include angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta), interleukin 12, retinoic acid, and tissue inhibitor of metalloprotease-1 and metalloprotease-2 (TIMP-1 and 2). Small molecules that contain topoisomerase, such as razoxane, a topoisomerase II inhibitor with antiangiogenic activity, may also be used.

[0127]

[0148] In some embodiments, the method can include administration of an additional therapy, such as an antifungal therapy. In some cases, an antifungal agent is administered to a subject receiving a composition comprising engineered cells. The antifungal agent can be a drug that can kill or prevent fungal growth. Targets of the antifungal agent can include sterol biosynthesis, DNA biosynthesis, and β-glucan biosynthesis. The antifungal agent can be a folate synthesis inhibitor or a nucleic acid crosslinker. The folate synthesis inhibitor can be a sulfa drug. For example, the folate synthesis inhibitor can be a folate synthesis inhibitor. The antifungal agent may be a drug or competitive inhibitor that inhibits the fungal synthesis of . Sulfa drugs, i.e., folic acid synthesis inhibitors, may be methotrexate or sulfamethoxazole. In some cases, the antifungal agent may be a nucleic acid crosslinker. The crosslinker may inhibit DNA or RNA processes in fungi. For example, the crosslinker may be 5-fluorocytosine, which may be a fluorinated analog of cytosine. 5-fluorocytosine may inhibit both DNA synthesis and RNA synthesis via cytoplasmic conversion to 5-fluorouracil. Another antifungal agent may be griseofulvin. Griseofulvin is an antifungal antibiotic produced by Penicillium griseofulvum. Griseofulvin inhibits mitosis in fungi and may be considered a crosslinker. Further crosslinkers may be allylamines (naftifine and terbinafine) that inhibit ergosterol synthesis at the level of squalene epoxidase. One morpholene derivative (amorolfine) inhibits a subsequent step in the ergosterol pathway.

[0128]

[0149] In some embodiments, the antifungal agent may be an antifungal agent of the polyene, azole, allylamine or echinocandin class. In some embodiments, the polyene antifungal agent is amphotericin B, candicidin, filipin, hamycin, natamycin, nystatin, or rimocidin. In some cases, the antifungal agent may be an antifungal agent from the azole family. The azole antifungal agent may inhibit lanosterol 14α-demethylase. The azole antifungal agent may be an imidazole, such as bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulcoazole, or tioconazole. The azole antifungal agent can be a triazole, for example, albaconazole, efinaconazole, epoxiconazole, fluconazole, isavonazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, or voriconazole. In some cases, the azole can be a thiazole, for example, abafungin. The antifungal agent can be an allylamine, for example, amorolfine, butenafine, naftifine, or terbinafine. The antifungal agent can be an echinocandin, for example, anidulafungin, caspofungin, or micafungin. Additional agents that can be antifungal agents can be aurone, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, tolnaftate, undecylenic acid, cystal violet, or balsam of Peru. Those skilled in the art can appropriately determine which known antifungal drug to apply based on the fungus that infects the individual. In some cases, the subject receives fluconazole in combination with the engineered cells. Antifungal therapy can be administered prophylactically.

[0129]

[0150] In some aspects, the treated subject may be observed following administration with a composition produced by the methods provided herein. In some embodiments, peripheral blood may be obtained from the subject following administration of the engineered cells. In some embodiments, serum may be isolated from the subject's peripheral blood following administration of the engineered cells. In some embodiments, a spinal tap sample may be taken from the subject following administration of the engineered cells. In some embodiments, the engineered immune cells from a treated subject's sample may be quantified from the sample. In some aspects, the sample from the subject receiving the engineered cells may be peripheral blood. In some embodiments, the engineered cells may be observed by quantitative PCR (qPCR). A qPCR assay of the adoptively transferred cells may indicate the level of engineered cells present in the subject following administration. In some cases, the adoptively transferred cells may be observed using flow cytometry. For example, a flow cytometry assay may determine the level of 4-1BB vs. TCR. In some cases, single cell TCR PCR may be performed. The level of adoptively transferred cells may be identified 7 days after injection. The level of adoptively transferred cells, e.g., modified cells, may be determined using flow cytometry. The le may be identified on any of the following days: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or up to 200 days post-infusion.

[0130]

[0151] In some embodiments, the level of a growth factor is quantified in a subject administered the engineered cells. Determining the level of a growth factor in a subject can indicate the subject's response to the administered engineered cells. In some aspects, quantifying the level of a growth factor is performed to observe the subject's tolerance to the adoptively transferred cells. In some aspects, quantifying the level of a growth factor can indicate that an intervention is required to prevent, stabilize, or top toxicity. In some aspects, the toxicity can be cytokine release syndrome. In some embodiments, the growth factor that can be quantified and / or observed from a sample of a subject is selected from the group consisting of IL-10, IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-4, IL-8, IL-12, and / or IFN-γ.

[0131]

[0152] In some embodiments, the population of cells comprising engineered cells is administered repeatedly.In some embodiments, the subject can receive about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 injections of the population of cells comprising engineered cells.In some embodiments, the engineered cells are allogeneic to the subject to which they are administered.In some embodiments, the engineered cells are autologous to the subject to which they are administered.

[0132]

[0153] In some embodiments, the methods provided herein may be utilized to treat disease. In some aspects, the methods provided herein may be utilized to treat cancer by targeting the cancer with a population of engineered immune cells. In some embodiments, the subject to which the engineered cells are administered has cancer. In some aspects, the cancer is the target and is a hematological cancer. In some embodiments, the hematological cancer includes leukemia, myeloma, lymphoma, and / or combinations thereof. In some aspects, the leukemia can be chronic lymphocytic leukemia (CLL), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), and / or acute lymphoblastic leukemia (ALL). In some embodiments, the lymphoma can be mantle cell lymphoma (MCL), T-cell lymphoma, Hodgkin's lymphoma, and / or non-Hodgkin's lymphoma. In some aspects, the cancer is the target and is a solid cancer. In some embodiments, the solid or liquid cancer target is selected from the group including nephroblastoma, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, or bladder cancer. Non-limiting examples of cancer include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK cell leukemia, AIDS-related Related cancers, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchialAlveolar carcinoma, Brown tumor, Burkitt's lymphoma, Carcinoma of unknown primary site, Carcinoid tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of unknown primary site, Carcinosarcoma, Castleman's disease, Central nervous system embryonal tumor, Cerebellar astrocytoma, Cerebral astrocytoma, Cervical cancer, Bile duct carcinoma, Chondroma, Chondrosarcoma, Spinal cord chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic lymphocytic leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic myeloproliferative disorder, Chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, uterine cancer, endometrial tumor, intestinal injury-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelial sarcoma, erythrocytic leukemia, esophageal cancer , esthesioneuroblastoma, Ewing family of tumors, Ewing family of sarcoma, Ewing sarcoma, extracranial germ cell tumors, extranasal germ cell tumors, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetal in situ, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, biopsy Germ cell tumors, germ cell tumors, gestational choriocarcinoma, gestational trophoblastic tumors, giant cell tumors of bone, glioblastoma multiforme, gliomas, brain glial tumors, glomerular tumors, glucagonomas, gonadoblastomas, granulosa cell tumors, hairy cell leukemia, hairy cell leukemia, head and neck cancer, cardiac cancer, hemangioblastomas, angiohypersensitive cell tumors, angiosarcomas, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer syndromes, Hodgkin lymphoma Lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, renal cancer, Kratzkin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma,Malignant fibrous histiocytoma of bone, Malignant glioma, Malignant mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant Triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid carcinoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesothelioma, Mesothelioma, Metastatic squamous cell neck cancer of unknown primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Oral cancer, Mucinous tumor, Multiple endocrine neoplasia syndrome, Multiple myeloma, Mycosis fungoides, Mycosis fungoides, Myelodysplasia, Myelodysplasia fibroblastoma, myeloid leukemia, myeloid sarcoma, myeloproliferative disorders, myxoma, nasal cavity cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasm, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumor, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic Cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelial cell tumor, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polygerminoma, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary invasive lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primary neuroectodermal tumor, prostate cancer, pseudoperitoneal myxoma, rectal cancer, renal cell carcinoma, airway cancer associated with the NUT gene on chromosome 15, retinoblastoma, rhabdomyoblastoma Myoma, rhabdomyosarcoma, Richter transformation, sacral caudate teratoma, salivary gland cancer, sarcoma, Schwannoma, sebaceous gland carcinoma, secondary neoplasm, seminoma, seroma, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sezary syndrome, Signet ring cell carcinoma, skin cancer, small round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin-producing tumor, sooty wart, spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial stromal tumor, synovial sarcoma,T cell acute lymphoblastic leukemia, T cells, The cancer cells include cells of cancers including large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, end-stage lymphoid cancer, testicular cancer, theca cell tumor, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, genitourinary tumors, uterine sarcoma, urethral melanoma, vaginal cancer, Berner-Morrison syndrome, Verruca carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the target cancer cells represent a subpopulation within a cancer cell population, e.g., cancer stem cells. In some embodiments, the cancer is a cancer of the hematopoietic lineage, such as lymphoma. The antigen can be a tumor-associated antigen. In some aspects, the subject can have minimal residual disease (MRD) following a therapy or administration. The MRD can include any of the aforementioned cancers or cancer cells. In some embodiments, the MRD is acute lymphoblastic leukemia. In one aspect, the cancers provided herein can express chemokines, such as SDF-1. In one aspect, cells in the tumor microenvironment of the cancers provided herein express chemokines. The chemokines can attract immune cells, such as the engineered immune cells provided herein. In one aspect, engineered immune cells, such as F-CART, can migrate toward a cancer or tumor microenvironment with high expression of chemokines, such as SDF-1. In one aspect, cancers that express chemokines, such as SDF-1, can be treated with the engineered immune cells provided herein.

[0133]

[0154] In some embodiments, the subject has BCR-ABL mutation.In some embodiments, the BCR-ABL mutation is in BCR-ABL kinase domain or a part thereof.In some embodiments, the subject has T315I and / or V299L mutation in BCR-ABL kinase domain or a part thereof.In some cases, the subject shows resistance to tyrosine kinase inhibitors.

[0134]

[0155] In some embodiments, the subject is pretreated. For example, the subject may be undergoing first line treatment for a disease such as cancer. In some aspects, the subject may be resistant to the first line treatment and / or prone to having a tumor after the first line treatment such as chemotherapy. In some aspects, the subject is pretreated with chemotherapy prior to administration of the engineered cells.

[0135]

[0156] Methods for administering therapeutic regimens to a subject having a condition, such as cancer, may be provided herein. In some cases, the cell composition (e.g., including a pharmaceutical composition including engineered immune cells) may be provided in unit dosage form. The cell composition may be resuspended in a solution or administered as an infusion. Treatment regimens including immunostimulants, immunosuppressants, antibiotics, antifungals, antiemetics, chemotherapeutics, radiation therapy, and any combination thereof may also be provided herein. Treatment regimens including any of the above may be lyophilized and reconstituted in an aqueous solution (e.g., saline). In some cases, the treatment is administered by a route selected from subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intravenous ("iv") administration, intranasal administration, intralymphatic injection, and oral administration. In some cases, the subject is infused with a cell composition including engineered cells via an intralymphatic microcatheter.

[0136]

[0157] Drugs used in conjunction with the cell therapy of the present disclosure may be administered orally as a liquid, capsule, tablet, or chewable tablet. The oral route is the most frequently used route because it is the most convenient and is usually the safest and least expensive route. However, the oral route is limited because of the way drugs typically pass through the digestive tract. For orally administered drugs, absorption may begin in the mouth or stomach, but most drugs are typically absorbed from the small intestine. The drug passes through the intestinal wall and travels to the liver before being transported to its target site via the bloodstream. The intestinal wall and liver chemically alter (metabolize) many drugs, reducing the amount of drug that reaches the bloodstream. Therefore, these drugs are often given in smaller doses when injected intravenously to produce the same effect.

[0137]

[0158] For subcutaneous administration of drugs used in conjunction with the cell therapy of the present disclosure, a needle is inserted into the fatty tissue just below the skin. After the drug is injected, it travels to small blood vessels (capillaries) and is carried away by the bloodstream. Alternatively, the drug reaches the bloodstream through lymphatic vessels. The intramuscular route is preferred over the subcutaneous route when larger amounts of drug are needed. A longer needle is used because the muscle is under the skin and fatty tissue. Drugs are usually injected into the muscles of the upper arm, thigh, or buttocks. How quickly the drug is absorbed into the bloodstream depends, in part, on the blood supply to the muscle. The less blood supply, the longer it takes for the drug to be absorbed. For the intravenous route, the needle is inserted directly into a vein. The solution containing the drug may be administered by a single dose or continuous infusion. For infusion, the solution is transferred by gravity (from a crushable plastic bag) or, more commonly, by an infusion pump via a thin flexible tube into a tube (catheter) inserted into a vein, usually a vein in the forearm. In some cases, the cells or therapeutic regimen are administered as an infusion. The infusion can be performed for a period of time. For example, the infusion can be administration of the cells or therapeutic regimen for about 5 minutes to about 5 hours. The infusion can be performed for about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or up to about 5 hours.

[0138]

[0159] In some embodiments, intravenous administration is used to deliver precise doses quickly and in a well-controlled manner to the entire body. Intravenous administration is also used for irritating solutions that cause pain and tissue damage when given by subcutaneous or intramuscular injection. Intravenous injections can be more difficult to administer than subcutaneous or intramuscular injections, because it can be difficult to insert a needle or catheter into a vein, especially if the subject is obese. When administered intravenously, the drug is delivered to the bloodstream immediately and is likely to produce an effect faster than when administered by any other route. Therefore, medical personnel closely observe people who receive intravenous injections for signs that the agent is working or causing undesirable side effects. Also, the effects of drugs administered by this route tend to last for a shorter period of time. Therefore, some drugs must be administered by continuous infusion to keep their effects constant. For the intrathecal route, a needle is inserted into the space between two vertebrae at the bottom of the spine and around the spinal cord. The drug is then injected into the spinal canal. A small amount of local anesthetic is often used to numb the injection site. This route is used when drugs need to act rapidly or locally on the brain, spinal cord, or the layers of tissue that cover them (meninges), for example, to treat infections of these structures.

[0139]

[0160] Inhalable drugs used in conjunction with the cell therapy of the present disclosure may be administered via the mouth, sprayed in smaller droplets than those administered by the nasal route. In this way, the drug can enter the lungs through the trachea. Smaller droplets can get to the back of the throat, increasing the amount of drug absorbed. Inside the lungs, the drug is absorbed into the bloodstream. Drugs applied to the skin are usually used for their local effect, and thus are most commonly used for the treatment of superficial skin disorders such as psoriasis, eczema, skin infections (viral, bacterial, and fungal), itching, and dry skin. The drug is mixed with an inert substance. Depending on the robustness of the inert substance, the formulation may be an ointment, cream, lotion, solution, powder, or gel.

[0140]

[0161] In some cases, the treatment regimen may be administered according to the subject's weight. In subjects who are deemed obese (BMI>35), practical weight may need to be utilized. BMI is calculated as follows: BMI=weight (kg) / [height (m)] 2 The ideal weight for men is 50kg + 2.3 * (number of inches over 60), and 45 for women. 5kg + 2.3 (inches over 60 inches). For subjects over 20% of their ideal body weight, an adjusted body weight can be calculated. The adjusted body weight can be the sum of the ideal body weight + (0.4 x (actual body weight - ideal body weight)). In some cases, body surface area can be used to calculate dosage. Body surface area (BSA) can be calculated by: BSA (m2) = √ height (cm) * weight (kg) / 3600.

[0141]

[0162] In some cases, the pharmaceutical composition comprising the cell therapy may be administered by any route, alone or with a pharma- ceutically acceptable carrier or excipient, and such administration may be performed in both single and multiple doses. More specifically, the pharmaceutical composition may be combined with various pharma- ceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hand candies, powders, sprays, aqueous suspensions, injectable solutions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media, and various non-toxic organic solvents, and the like. Moreover, such oral pharmaceutical preparations may be suitably sweetened and / or flavored with various types of agents commonly used for such purposes.

[0142]

[0163] In some cases, the therapeutic regimen can be administered with a carrier or excipient.Exemplary carriers and excipients can include dextrose, sodium chloride, sucrose, lactose, cellulose, xylitol, sorbitol, malitol, gelatin, PEG, PVP, and any combination thereof.In some cases, the excipient such as dextrose or sodium chloride can be about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or up to about 15% percent.

[0143]

[0164] Herein, a method for treating disease (e.g., cancer) in a recipient is described, comprising transplanting one or more cells (including organs and / or tissues) including engineered immune cells into the recipient. The cells prepared by the provided method can be used to treat cancer. In some cases, the level of disease can be determined consecutively or simultaneously with the treatment regimen or cell administration. The disease level on target lesions can be measured as follows: Complete response (CR): disappearance of all target lesions; Partial response (PR): reduction of the sum of the longest diameter (LD) of target lesions by at least 30% based on the total LD ​​of baseline; Progression (PD): increase of the sum of the LD of target lesions by at least 20% based on the minimum total LD ​​recorded since the start of treatment, or appearance of one or more new lesions; Stable disease (SD): neither sufficient shrinkage to qualify as PR nor sufficient increase to qualify as PD based on the minimum total LD. In other cases, non-target lesions can be measured. The disease level of non-target lesions may be: Complete Response (CR): disappearance of all non-target lesions and normalization of tumor marker levels; Non-Complete Response: persistence of one or more non-target lesions; Progressive Disease (PD): appearance of one or more new lesions, definite progression of existing non-target lesions. In some cases, subjects receiving treatment regimens and cell administration may be evaluated for best overall response. Best overall response may be the best response recorded from the start of treatment to disease progression / recurrence (the minimum measurement recorded since the start of treatment is taken as the criterion for progressive disease). Subject best response assignment may depend on the achievement of both measurement criteria and confirmation criteria. Time to progression may be measured from the date of randomization. In one embodiment, response may refer to monitoring of cancer burden or tumor burden in a subject. In one embodiment, a reduced cancer burden is observed when a subject is administered a population containing engineered immune cells, such as F-CART, compared to the cancer burden observed in a comparable subject administered a comparable population undergoing ex vivo expansion for 2 weeks or more, such as C-CART. In one embodiment, the cancer burden is determined by CC in subjects treated with a population comprising engineered immune cells such as F-CART. The amount of HIV-1 induced by the antibody is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to a comparable subject administered a comparable population undergoing ex vivo expansion, such as ART, for 2 weeks or more. In one embodiment, the subject described herein can be a mammal. The mammal can be a human, a dog, a horse, a pig, a mouse, a rat, or a monkey.

[0144]

[0165] To assign PR or CR status, changes in tumor measurements must be confirmed by repeat studies, which should be performed at least approximately 4 weeks after response criteria are first met. For SD, follow-up measurements must meet SD criteria at least once after study entry, at intervals of a minimum of 6-8 weeks. In some cases, the overall response duration may be measured from the time that the measurement criteria for CR / PR are met (whichever is first recorded) to the first date that recurrence or progressive disease is objectively recorded (the criterion for progressive disease is the smallest measurement recorded since treatment was started). The overall response duration of complete response may be measured from the time that the measurement criteria for CR are first met to the first date that recurrent disease is objectively recorded. Stable disease may be measured from the start of treatment until the criteria for progression are met, based on the smallest measurement recorded since treatment initiation. In some cases, measurable disease may be measured and recorded in metric units using a ruler or calipers. All baseline evaluations may be performed as close to the start of treatment as possible. A lesion can be considered measurable if it is superficial (e.g., skin nodules and palpable lymph nodes) and exceeds at least about 10 mm using a caliper. In some cases, color photography can be performed.

[0145]

[0166] In other cases, a computed tomography scan (CT) or magnetic resonance imaging (MRI) may be performed. CT may be performed with a slice thickness of 5 mm or less. If the slice thickness of the CT scan is greater than 5 mm, the minimum size of a measurable lesion must be twice the slice thickness. In some cases, an FDG-PET scan may be used. FDG-PET may be used to evaluate new lesions. A negative FDG-PET at baseline and a positive FDG-PET at follow-up is an indication of progressive disease (PD) based on new lesions. A positive FDG-PET at follow-up with no FDG-PET at baseline and a positive FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, i.e., PD. A positive PDG-PET at follow-up may not be PD if it corresponds to a pre-existing site of disease on CT that may not progress based on anatomical speculation. In some cases, FDG-PET may be used to upgrade the response to CR in a manner similar to a biopsy in cases where residual radiological abnormalities are thought to represent fibrosis or scarring. A positive FDG-PET scan lesion refers to a lesion with high FDG uptake, with uptake greater than twice that of the surrounding tissue on attenuation-corrected images.

[0146]

[0167] In some cases, lesion evaluation may be performed. Complete response (CR) may be the disappearance of all target lesions. Any pathological lymph nodes (target or non-target) may decrease in short axis to less than 10 mm. Partial response (PR) may be at least a 30% decrease in the sum of the diameters of the target lesions based on the sum of the diameters at baseline. Progressive disease (PD) may be at least a 20% increase in the sum of the diameters of the target lesions based on the minimum sum. The sum must also show an absolute increase of at least 5 mm in addition to a relative increase of 20%. Stable disease (SD) may be neither sufficient shrinkage to qualify as PR nor sufficient increase to qualify as PD based on the minimum sum of diameters.

[0147]

[0168] In some cases, non-target lesions may be evaluated. A complete response for non-target lesions may be the disappearance and normalization of tumor marker levels. All lymph nodes must be of non-pathological size (short axis less than 10 mm). If tumor markers initially exceed the upper limit of normal, they must be normalized for the patient to be considered in clinical complete response. Non-CR / Non-P D is the persistence of one or more non-target lesions and / or maintenance of tumor marker levels above the normal range. Progressive disease may be the appearance of one or more new lesions and / or definite progression of existing non-target lesions. Definite progression should not usually be truncated to the status of target lesions. In some cases, the best overall response may be the best response recorded from the start of treatment to disease progression / recurrence.

[0148]

[0169] In some cases, subjects treated with the treatment regimen or cell product described herein may experience adverse events related to the regimen or cell product. An adverse event may be any reaction, side effect, or unintended event occurring during the course of treatment related to the use of a drug in humans, regardless of whether the event is considered treatment-related or clinically significant. In some cases, adverse events may include events reported by subjects, as well as clinically significant abnormal findings in physical examination or clinical laboratory evaluation. Any new disease, symptom, sign, or clinically significant laboratory abnormality, or aggravation of an existing condition or abnormality, may be considered an adverse event. All adverse events, including clinically significant abnormal findings in clinical laboratory evaluation, regardless of severity, will be followed until grade 2 or less, except for lymphopenia and alopecia. If the adverse event is not expected to resolve to grade 2 or less, the subject may cease treatment. In some cases, the treatment regimen may be administered with a toxicity-reducing agent. The toxicity-reducing agent may be a fever or vomiting reducing agent. For example, mesna may be administered to reduce toxicities such as nausea, vomiting, and diarrhea.

[0149]

[0170] In one embodiment, the population of cells may undergo pre-infusion testing prior to or concurrently with administration. Pre-infusion or pre-administration testing may be performed to ensure that the engineered cell product is functional, sterile, and capable of functioning after infusion. Pre-infusion testing may include determining phenotype, cytotoxicity, memory / stemness, depletion, bone marrow migration, ELISA, and any combination thereof. In one embodiment, pre-administration testing may include performing in vitro or in vivo assays. In one embodiment, the level of cytotoxicity may be determined in the population of engineered cells. For example, the population of cells may be assessed by FAC for expression of any one of the following: CD3, CD4, CD8, CD45RO, CCR7, CD45RA, CD62L (L-selectin), CD27, CD28, and IL-7Rα, CD95, IL-2Rβ, CXCR3, and LFA-1. In one embodiment, the functional test may also include co-culture assays, cytotoxicity assays, ELISA (e.g., to quantify interleukin-2 (IL-2) and / or IFN-γ fragments), or ELISPOT assays. In one embodiment, the populations provided herein are further characterized in that greater proliferation, cytotoxicity, and / or bone marrow migration is observed in the population compared to a comparable population undergoing ex vivo expansion for more than 2 weeks, or (a) without simultaneous activation of the population of cells with an activating moiety, and (b) introduction of a polynucleotide encoding a CAR. In one embodiment, the populations provided herein are further characterized in that they contain greater memory and / or stemness compared to a comparable population undergoing ex vivo expansion for more than 2 weeks, or (a) without simultaneous activation of the population of cells with an activating moiety, and (b) introduction of a polynucleotide encoding a CAR.In one embodiment, the levels of proliferation, memory / stemness, cytotoxicity (killing ability), and / or BM migration may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100% greater than a comparable population of cells undergoing ex vivo expansion for 2 weeks, or a comparable population where (a) there is no concomitant activation of the population of cells with an activating moiety, and (b) the introduction of a polynucleotide encoding a CAR, or may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to about 100% greater.

[0150]

[0171] In some embodiments, a point-of-care facility is provided herein. The point-of-care facility may be a hospital, a laboratory, a clinic, a vehicle, a medical center, a recreational vehicle, a home, to name a few exemplary facilities. The point-of-care facility may include a cell infusion device. The cell infusion device may include any one of a bag, a pump, a syringe, a tube, a lumen, a bioreactor, an incubator, a hemocytometer, a centrifuge, a thermometer, a needle, an aspirator, an oxygen tank, a VAD lumen, and any combination thereof. In some embodiments, the point-of-care facility includes a cell infusion device. The cell infusion device may be configured to inject a population of cells including engineered immune cells that have not undergone ex vivo expansion for more than two weeks. In one aspect, the population of immune cells is a pharmaceutical composition. In some aspects, the cell infusion device includes a population of immune cells, which may be further characterized by the enrichment of cell memory T cells (TCM) in the population over effector memory T cells (TEM). In some aspects, the cell infusion device includes a population of immune cells, where at least 2% of the population are stem memory T cells (TSCM). In some embodiments, the point-of-care facility includes a cell processing device configured to (a) receive a population of cells including immune cells from a subject, (b) activate the population of immune cells with an activating moiety, and simultaneously introduce a polynucleotide encoding at least a chimeric antigen receptor (CAR) into said immune cells. In some aspects, the CAR includes (i) a ligand binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. In some aspects, the cell processing device is further configured to (c) inject the population of immune cells of (b) into the subject within 2 weeks of performing (b), and in some aspects, step (c) is performed within 1 week of performing (b). In some embodiments, the cell processing device of the point-of-care facility is configured to perform steps (a) and (b) within 24 hours. In some embodiments, the cell processing device of the point-of-care facility is configured to perform steps (a) and (b) within 3 hours. In some embodiments, the cell processing device of the point-of-care facility is configured to perform steps (a) and (b) within 1 hour.In some embodiments, the cell processing device at the point-of-care facility is configured to perform steps (a) and (b) in 30 minutes or less. EXAMPLES

[0151]

[0172] Various aspects of the present disclosure are further illustrated by the following non-limiting examples. Example 1

[0173] Construction of lentiviral vectors

[0174] A nucleotide sequence including the EF1a promoter (shown in sequence number 1), anti-CD19scFv (shown in sequence number 3), CD28 hinge region, CD28 transmembrane region, and CD28 costimulatory signal molecule (shown in sequence number 5), CD3ζ intracellular domain (shown in sequence number 7), and CAR (shown in sequence number 9) was artificially synthesized.

[0152]

[0175] SEQ ID NO:1

[0176] gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt tctgttctgc gccgttacag atc

[0177] Sequence number 3

[0178] atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc ccatcaaggt tcagtggcag tgggtctgga acagattatt ctct caccat tagcaacctg gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc agcggccgca gactacaaag acgatgacga caag

[0179] Sequence number 5

[0180] attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg gggagtcctg gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc c

[0181] Sequence number 7

[0182] agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgc

[0183] Sequence number 9

[0184] atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac tccaagagcc aagtttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga acctcagtca ccgtctctc agcggccgca gactacaaag acgatgacga caagattgaa gttatgtatc ctctcctta cctagacaat gagagagca atggaaccat tatccatgtg aaagggaaac acctttgtcc aagtccccta tttcccggac cttctaagcc cttttgggtg ctggtggtgg ttgggggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt atttctggg tgaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc gcagcctatc gctccagagt gaagttcagc aggagcgcag acgccccgc gtaccagcag ggccagaacc agctcatataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctaa

[0185] The nucleic acid sequence encoding CAR was cloned into the lentiviral vector FUGW, a plasmid named GCP042.293. T cells were co-transfected with the plasmid GCP042 and other packaging plasmids (helper plasmids). Briefly, 2 × 10 6 293T cells were cultured at 150 cm 2 In a culture dish, 1.3 × 10 4 cells / cm 2 The cells were then cultured at 37°C, 5% CO2 and saturated humidity for 3 days before being transfected.

[0153]

[0186] 18 μg of helper plasmid and 24 μg of GCP042 were added to a centrifuge tube containing 3 mL of DMEM medium, and then 126 mg of PEI was added to the tube to obtain a mixture. The mixture was left at room temperature for 30 minutes, and then supplemented with 12 mL of DMEM containing 2% FBS to obtain a transfection medium. For transfection, after removing the culture medium, 293T cells were incubated with the transfection medium for 4 hours, and then the transfection medium was replaced with 20 mL of DMEM medium containing 2% FBS. After 72 hours, the medium was collected and centrifuged at 3000 g for 10 minutes at 4°C. The supernatant was then filtered through a 0.45 μm filter for further purification. The filtrate was further centrifuged at 27000 g for 2 hours at 4°C. The pellet was collected and resuspended in 100 μL of pre-cooled PBS to obtain a GCL042 lentivirus suspension, which was kept at 4°C overnight. The next day, the virus suspension was aliquoted for further use.

[0154] Example 2

[0187] 1. Preparation of F-CART cells

[0188] To prepare F-CART cells, 100 mL of peripheral blood was collected from healthy donors. PBMCs were isolated by density gradient centrifugation at 500–600 g for 20–30 min. T cells were sorted and enriched using magnetic beads conjugated with CD28 and CD3 antibodies (CD3 / CD28 Dynabeads, purchased from ThermoFisher). T cells were then cultured at 37°C in X-vivo15 medium at 0.1 × 10 6 cells / mL~10×10 6 The cells were transfected with GCL042 prepared in Example 1 at a cell density of 10 ...

[0155]

[0189] Similarly, approximately 100 mL of peripheral blood was collected from healthy donors and PBMCs were isolated by density gradient centrifugation at 500–600 g for 20–30 min. T cells were sorted and enriched using magnetic beads conjugated with CD28 and CD3 antibodies (CD3 / CD28 Dynabeads, purchased from ThermoFisher). T cells were further activated by incubating them with CD3 / CD28 Dynabeads at a ratio of 0.1:1–5:1 (CD3 / CD28 Dynabeads:T cells) and 300 IU / mL IL2. Meanwhile or thereafter, T cells were cultured at 0.1–10 × 10 6 The cells were transfected with GCL042 at a cell density of 10000000 cells / mL at 37° C. for 4 hours, and then washed with buffered saline. The F-CART cells of the present disclosure were obtained without further expansion. This procedure activated the cells. These cells are also referred to as F-CART-FV2 cells in the present disclosure.

[0156]

[0190] Table 1 summarizes the preparation process of F-CART-FV1 cells versus F-CART-FV2 cells.

[0191]

[0157] [Table 2]

[0158] Example 3

[0192] Lentivirus MOI and ratio of CAR-positive cells

[0193] We then compared the percentage of CAR-positive cells using the FV1 and FV2 preparation processes. Briefly, approximately 100 mL of peripheral blood was collected from healthy donors, and PBMCs were isolated by density gradient centrifugation at 500-600 g for 20-30 min. T cells were then sorted and enriched using CD3 / CD28 Dynabeads.

[0159]

[0194] 1×10 7The sorted T cells were divided into two groups, F-CART-FV1 cells and F-CART-FV2 cells, respectively, prepared as described in Example 2. GCL042 was used to obtain 2×10 6 T cells were transfected at a cell density of 1000 cells / mL and an MOI of 3-4. In particular, the amount of virus used was calculated as virus number = cell number × MOI / virus titer. The calculated virus was then incubated with T cells for 16-24 hours to infect the T cells. After transfection, the virus was removed by centrifugation, and the cells were then harvested and cultured for another 3-5 days. 0.5 × 10 6 ~1.0×10 6 Cells were harvested and subjected to flow cytometry for analysis of CAR-positive ratio. The results are shown in Table 2.

[0160]

[0195]

[0161] [Table 3]

[0162]

[0196] From Table 2, it can be seen that the CAR positive ratio in the F-CART-FV2 preparation is higher than that in the F-CART-FV1 preparation. The correspondence between the MOI and the CAR positive ratio was then tested using F-CART-FV2 cells. The results are shown in Figure 1. As can be seen from Figure 1, with MOIs of 0.8 and 1.6, the CAR positive ratios were 31.0% and 39.8%, respectively, indicating that at this stage, the positive ratio was also influenced by additional factors other than MOI. MOIs of 1.6 and 3.2 With MOIs of 3.2, 6.4 and 12.8, the CAR positive ratios were 39.8% and 57.2%, respectively, and the positive ratios changed by nearly 1.5-fold with increasing amounts of virus, indicating that at this stage, the positive ratios were basically correlated with the amount of virus used. With MOIs of 3.2, 6.4 and 12.8, the CAR positive ratios were 57.2%, 64.6% and 69.3%, respectively, and at this stage, the positive ratios did not increase significantly with the amount of virus, indicating that virus infection had entered the platform. This result suggests that the preferred MOI for preparing the F-CART of the present disclosure, particularly F-CART-FV2, is between 1.6 and 6.4, which allows the positive ratio of F-CART to be more efficiently and stably controlled.

[0163] Example 4

[0197] 1. Preparation of c-CART cells

[0198] Control cells (also referred to as second reference cells, C-CART or C-CAR-T cells) were prepared according to the method described in Kochenderfer, JN et al. (2013), "Donor-derived CD19-targeted T cells cause regression of malignancy persisting after allogeneic hematopoietic stem cell transplantation"), Blood 122(25):4129-4139.

[0164]

[0199] Briefly, approximately 100 mL of peripheral blood was collected from healthy donors and PBMCs were isolated by density gradient centrifugation at 500–600 g for 20–30 min. CD3 / CD28 Dynabeads were used to select and enrich T cells. T cells were then activated by incubating them with CD3 / CD28 Dynabeads at a ratio of 0.1:1–5:1 (CD3 / CD28 Dynabeads:T cells) and 300 IU / mL IL2. Meanwhile or subsequently, T cells were incubated with 0.1–10 × 10 6The cells were transfected with GCL042 at a cell density of 10000000 cells / mL for 4 hours at 37° C. and then washed with buffered saline. After activation and transfection, the modified cells were expanded in culture for 8 days to obtain control cells (also referred to as second reference cells, C-CART cells or C-CAR-T cells).

[0165] Example 5

[0200] Positive ratio of F-CART cells to C-CART cells

[0201] Changes in the proportion of CAR-positive cells (CAR+) during in vitro culture of F-CART cells (e.g., F-CART-FV2 cells) and C-CART cells after recovery from cryopreservation were analyzed using a CD19 + The results were compared with and without tumor cell stimulation. The results are shown in Figure 2. As can be seen from Figure 2, for the recovered F-CART cells, the CAR+ ratio decreased with the culture time. After tumor antigen was added, the CAR+ ratio increased again (the solid line in Figure 2 represents the group without tumor antigen addition, and the dashed line represents the group with tumor antigen), indicating that CAR+ cells can be enriched by tumor antigen stimulation, and that the polynucleotide encoding CAR was stably integrated into the genome of T cells. Figure 2 also shows that before tumor antigen stimulation, at the starting point, the CAR+ ratio in F-CART cells was much higher than that in C-CART cells, and after day 16, the CAR+ ratio in C-CART cells decreased significantly, but the CAR+ ratio in F-CART still increased slightly. After stimulation with tumor antigen, both F-CART cells and C-CART cells showed a significantly increased CAR+ ratio.

[0166] Example 6

[0202] Analysis of lymphocyte subpopulations

[0203] Lymphocyte subpopulations were analyzed by conventional flow cytometry in the starting material (corresponding T cells without viral transfection) and in F-CART cells (such as F-CART-FV2 cells). (Garcia RL et al., Analysis of lymphocyte subpopulations in vitro and in vivo). f proliferative grade using anti-PCNA / cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis, The American journal of pathology, 1989, 134(4):733).

[0167]

[0204] Expression of markers including CD3, CD4, CD8, CD45RO and CD62L was assessed using 2-3 × 10 starting T cells and F-cART cells, respectively. 6 The results were analyzed by flow cytometry using the results shown in Table 3 and FIG.

[0168]

[0205]

[0169] [Table 4]

[0170]

[0206] In Table 3, TEM is CD45RO + / CD62L - TCM represents effector T cells with CD45RO + / CD62L + represents central memory T cells having N CD45RO - / CD62L + They represent early (or naive) T cells with great differentiation potential that can differentiate into subpopulations of cells such as TEMs and TCMs.

[0171]

[0207] The results showed that CD3 + The percentage of T cells in the starting material is CD3 + The percentage of CD4+ T cells in F-CART was significantly higher than that in CD4+ T cells, indicating effective sorting and enrichment of T cells. + T cells and CD8 +The percentage of T cells in the starting material was determined by + T cells and CD8 + The proportion of lymphocyte subpopulations was altered in F-CART compared to the starting material, with the proportions of TCM and TEM increased in F-CART, and T N The proportion of N This may be due to the differentiation of the population after T cell activation. These results indicate that the F-CART preparation process of the present application can be used to prepare activated T cells with differentiation potential.

[0172] Example 7

[0208] In vitro expansion of CAR-T cells

[0209] To compare the in vitro proliferation of F-CART (e.g., F-CART-FV2) and C-CART preparations, cell viability and viable cell counts were detected on days 0, 2, 5, 8, 11, and 13 in F-CART cells (e.g., F-CART-FV2 cells) and C-CART cells.

[0173]

[0210] Briefly, X-vivo15 medium with 300 IU / mL IL-2 was pre-warmed in a 37 °C water bath. F-CART cells (such as F-CART-FV2 cells) and C-CART cells were thawed in a 37 °C water bath for 2-3 min, then transferred to the pre-warmed medium, mixed well, and the total volume of the cell suspension was measured. After the cell viability and cell density in 300 μL of F-CART cell suspension and C-CART cell suspension were calculated by NC-200 counter, the cell number was calculated based on the volume. The cells in the two groups were then separated into 100 μL of IL-2 and 100 μL of IL-2. The cells were then centrifuged at 250-300 g for 8-10 minutes. The supernatant was then removed, and the cells were diluted to 0.5 × 10 6 cells / mL~1.0×10 6After resuspension to a density of 1000 cells / mL, they were seeded on plates and cultured at 37 °C and 5% CO2. To compare the proliferation of F-CART cells versus C-CART cells, cell viability and viable cell counts were calculated on days 0, 2, 5, 8, 11, and 13. The cell density was maintained at 0.5 × 10 6 cells / mL~1.0×10 6 Maintained in cells / mL.

[0174]

[0211] Furthermore, the proliferation of the two groups was compared under the stimulation of tumor antigens. Briefly, primary tumor cells expressing CD19 (derived from a patient with B-ALL tumor) were cocultured with F-CART cells and C-CART cells from day 0. Cell viability and viable cell counts were calculated on days 2, 5, 8, 11, and 13. The cell density was maintained at 0.5 × 10 6 cells / mL~1.0×10 6 The results are shown in Table 4, Figure 4A and Figure 4B.

[0175]

[0212]

[0176] [Table 5]

[0177]

[0213] The results show that F-CART cells had a significantly stronger proliferation ability than that of C-CART cells, regardless of the presence or absence of tumor cell stimulation, indicating a superior expansion proliferation ability of F-CART cells.

[0178] Example 8

[0214] In vitro tumor-killing efficacy of CAR-T

[0215] To compare the in vitro tumor-killing efficacy of F-CART (such as F-CART-FV2) and C-CART preparations, F-CART cells (such as F-CART-FV2 cells) and C-CART cells were thawed and cultured in X-vivo15 medium containing 10% (v / v) AB serum and 300 IU / mL IL-2, and tumor-killing efficacy was detected on day 2.

[0179]

[0216] Briefly, X-vivo15 medium containing 10% (v / v) AB serum and 300 IU / mL IL-2 was pre-warmed in a 37 °C water bath. The prepared F-CART cells (such as F-CART-FV2 cells) and C-CART cells were thawed in a 37 °C water bath for 2–3 min, then transferred to the pre-warmed medium, mixed well, and the total volume of the cell suspension was measured. After the cell viability and cell density in 300 μL of F-CART cell suspension and C-CART cell suspension were calculated by NC-200 counter, the cell number was calculated based on the volume. The cells in the two groups were centrifuged at 250–300 g for 8–10 min. The supernatant was removed, and the cells were diluted to 0.5 × 10 with an appropriate amount of X-vivo15 medium containing 10% (v / v) AB serum and 300 IU / mL IL-2. 6 cells / mL~1.0×10 6 After resuspending to a density of 100 cells / mL, the cells were plated and cultured at 37°C and 5% CO2.

[0180]

[0217] On day 2, 0.5 × 10 6 ~1.0×10 6 10 cells were subjected to analysis of CAR+ ratio. As target cells, HELA-CD19 cells, HELA cells and HEK293T cells under P2-P10 were washed with 10 mL of DPBS, digested with 5-10 mL of 0.25% trypsin at 37 °C for 1-3 min, then neutralized with 5-10 mL of complete medium (containing 10% FBS) and pipetted repeatedly to form a single cell suspension. Then, the three groups of target cells were centrifuged at 300 g for 5 min. The supernatant was removed, and the cells were resuspended in RPMI1640 complete medium containing 10% FBS. The cell number was counted, and the cell viability was ≥ 85% in all three groups. The cell density was then adjusted to 1 × 10 5 Adjusted to cells / mL.

[0181]

[0218] The detection procedure was established according to the xCELLLigence instruction manual. In particular, each group of target cells was added to an E-PlateView 96-well plate, and after standing in an incubator for 30 min, the attachment of cells was examined. If the Cell-Index of HELA-CD19 cells detected by xCELLLIgence was ≥2, the required number of CAR+ cells were collected based on the CAR+ ratio analyzed above and centrifuged at 300g for 5 min. The supernatant was removed, and the cells were resuspended to an appropriate density in RPMI1640 complete medium. The E-PlateView 96 plate was removed from the xCELLLigence detection, and the supernatant was discarded. Based on the required ratio of CAR-T cells to target cells, CAR-T cells were added to each well of the plate. The E-PlateView 96 plate was then subjected to cell death detection for 24-48 h.

[0182]

[0219] As shown in Table 5 and Figure 5, the data was collected and plotted, and the time to death of half of the target cells was calculated as the time point with the maximum Cell-Index (CImax) minus the time point with half the maximum Cell-Index (CImax / 2).

[0183]

[0220] In FIG. 5, 1 is HELA-CD19, 2 is NT+HELA-CD19 (5:1), 3 is C-CART+HELA-CD19 (1:1), 4 is F-CART+HELA-CD19 (1:1), 5 is C-CART+HELA-CD19 (5:1), and 6 is F-CART+HELA-CD19 (5:1). A is the starting point, time is 7.90 hours, and Cell-Index is 1.02. B is F-CART+HELA-CD19 (5:1), time is 8.91 hours, and Cell-Index is 0.51. C is C-CART+HELA-CD19 (5:1), time is 15.23 hours, and Cell-Index is 0.51. D is F-CART+HELA-CD19 (1:1), time is 16.77 hours, and Cell-Index is 0.51. E is C-CART+HELA-CD19 (1:1), time is 51.23 hours, and Cell-Index is 0.52.

[0184]

[0221]

[0185] [Table 6]

[0186]

[0222] The results showed that the median time to death of F-CART was shorter than that of C-CART in two models with effector cell:target cell ratios of 5:1 and 1:1, suggesting that the killing efficacy of F-CART against tumor cells is significantly stronger than that of C-CART. Similar results were also observed using HELA cells and HEK293T cells.

[0187] Example 9

[0223] CAR-T response to tumor antigen stimulation

[0224] Cytokines released by F-CART (such as F-CART-FV2) and C-CART preparations were compared to assess the response to tumor antigen stimulation. In this comparative assessment, F-CART (such as F-CART-FV2) and C-CART cells were subjected to cytokine detection on day 2, and non-transfected T cells (starting material) as described in Example 2 were used as a control.

[0188]

[0225] Briefly, culture medium was pre-warmed in a 37 °C water bath. F-CART cells (such as F-CART-FV2 cells) and C-CART cells were thawed in a 37 °C water bath for 2-3 min, then transferred to the pre-warmed medium, mixed well, and the total volume of the cell suspension was measured. After the cell viability and cell density in 300 μL of F-CART cell suspension and C-CART cell suspension were calculated by NC-200 counter, the cell number was calculated based on the volume. Then, the cells were centrifuged at 250-300 g for 8-10 min. The supernatant was removed, and the cells were diluted to 0.5 × 10 with an appropriate amount of medium. 6 cells / mL~1.0×10 6After resuspending at a density of 1000 cells / mL, the cells were plated and cultured at 37°C and 5% CO2. On the second day, the cells were mixed well by pipetting, and 0.3 × 10 cells from each group were removed. 6 ~1.0×10 6 The cells were subjected to analysis of the CAR+ ratio. Based on the CAR+ ratio, an appropriate amount of cells was collected and centrifuged at 300 g for 5 minutes, after which the supernatant was removed and the cells were diluted to 1 × 10 6 The cells were resuspended to a density of 1000 cells / mL.

[0189]

[0226] Molt4 cells (without tumor antigen stimulation) and Raji cells (with tumor antigen stimulation) were used as negative and positive targets, respectively. 0.3-0.5 mL of the target cell suspension was first subjected to calculation of cell number and cell viability. Then, the two groups of target cells were further centrifuged at 300 g for 5 min. The supernatant was removed, and the cells were diluted to 1 × 10 in RPMI1640 complete medium. 6 The cells were resuspended to a density of 1000 cells / mL.

[0190]

[0227] Subsequently, 100 μL of both effector cells (control T cells, F-CART cells, and C-CART cells) and target cells (Molt4 cells or Raji cells) were added to each well of a 96-well plate in a 1:1 ratio and incubated together for 18-24 hours at 37°C and 5% CO2. After incubation, the cells were centrifuged at 300g for 5 minutes. 100-150 μL of supernatant was collected and transferred to a new 96-well plate to detect the release of cytokines, including GM-CSF, IL-2, TNF-α, and IFN-γ, by ELISA, as shown in Figure 6.

[0191]

[0228] From Figure 6, it can be seen that there is no significant difference in the level of GM-CSF released between F-CART and C-CART. IL-2 is a factor released by activated T cells, and the level of IL-2 released by F-CART is significantly higher than that released by C-CART, indicating that F-CART has a strong activation by tumor antigen stimulation. The levels of TNF-α and IFN-γ both indicate the direct killing efficacy of T cells against target cells, and the levels of these two factors are both significantly higher in F-CART than that in C-CART, indicating that the killing activity of F-CART is superior to that of C-CART.

[0192] Example 10

[0229] In vivo tumor killing efficacy

[0230] The CDX model (allogeneic tumor model) of immunodeficient mice (NDG mice) was developed by expressing CD19 + The Raji B malignant cell line was established using Raji-Luc cells. Briefly, Raji-Luc cells were cultured at 5 × 10 5 The cells were suspended at a density of 10 cells / 0.2 mL, and 0.2 mL of cells were injected into each B-NDG (B-NSG) mouse through the tail vein. From day 0 of injection onwards, the mice were subjected to imaging to observe the growth of the tumor. The average signal of the mouse images was approximately 5 × 10 6 When P / S was reached, mice with moderate signals were selected and then randomly divided into different groups, 3 per group. On the same day, mice were tail vein injected with F-CART cells (such as F-CART-FV2 cells), C-CART cells, non-transfected T cells (starting material), and blanks containing only cell cryopreservation solution. After injection, tumor growth in each animal was observed by imaging twice a week. The results are shown in Figures 7A and 7B. The results show that F-CART exhibited significantly stronger inhibition against tumors in mice and eventually eliminated tumors. As shown in Figure 7B, 5×10 5Using a dose of (5E5), C-CART showed some tumor-inhibiting effect but was unable to eliminate the tumor, whereas F-CART completely eliminated the tumor, demonstrating stronger antitumor efficacy.

[0193] Example 11

[0231] In vivo toxicity

[0232] As in Example 10, 2×10 F-CART cells (such as F-CART-FV2 cells), C-CART cells, non-transfected T cells (starting material), and blanks were 6 The body weight of the mice was measured before and after administration of the dose of (2E6). The changes in body weight of the animals before and after administration were compared, as shown in Figure 8. As can be seen from Figure 8, the change in body weight (%) is expressed as a percentage of the body weight after administration to the original body weight, calculated as: change in body weight (%)=body weight after administration / original body weight×100%.

[0194]

[0233] The results show that compared with the blank group and the starting material group, the body weight of the animals in the F-CART group and C-CART group did not show any significant changes, and the body weight of the animals in the F-CART group seemed to be more stable, indicating the absence of obvious toxicity of the preparation.

[0195] Example 12

[0234] Dose-dependent tumor suppression in vivo

[0235] High dose (2×10 6 cells / 0.2 mL), medium dose (5 × 10 5 cells / 0.2 mL), low dose (5 × 10 4 F-CART cells (e.g., F-CART-FV2 cells) at 100 μg / 0.2 mL as well as a blank were administered to the mice established in Example 10 to observe changes in tumor size. The results are shown in Figure 9. As can be seen in Figure 9, the inhibition of F-CART cells against tumors was dose-dependent, and the tumor size decreased as the dosage increased.

[0196] Example 13

[0236] In vivo expansion of CAR-T cells

[0237] High dose (2×10 6 cells / 0.2 mL), medium dose (5 × 10 5 cells / 0.2 mL), low dose (5 × 10 4 F-CART cells (e.g., F-CART-FV2 cells) at 100 μg / 0.2 mL of F-CART cells and blanks were administered to the mice established in Example 10, and then peripheral blood was collected from the mice to analyze the expansion and proliferation of CAR-T cells by flow cytometry (Garcia RL et al., Analysis of proliferative grade using anti-PCNA / cyclin monoclonal antibodies in fixed, embedded tissue). es. Comparison with flow cytometric analysis, The American journal of pathology, 1989, 134(4):733. The results are shown in FIG.

[0197]

[0238] As can be seen from Figure 10, F-CART cells showed rapid expansion and proliferation up to 28 days after administration, particularly between days 14 and 21. The proliferation ability of F-CART cells was dose-dependent and increased with the dose.

[0198] Example 14

[0239] Subpopulation of F-CART cells versus C-CART cells

[0240] T cell status and depletion were assessed to compare subpopulations of F-CART (e.g., F-CART-FV2) and C-CART preparations. Subpopulations of F-CART (e.g., F-CART-FV2) and C-CART cells were analyzed by flow cytometry (see Garcia RL et al., Analysis of proliferative grade using anti-PCNA / cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis, The American journal of pathology, 1989, 134(4):733).

[0199]

[0241] Briefly, cells were harvested from three healthy donors, and F-CART cells and C-CART cells were prepared as described in Examples 2 and 4. Then, 1 × 10 cells from each group were 6 The prepared cells were co-cultured with irradiated K562-CD19 cells at a ratio of 1:1 for 10 days. During the culture, the irradiated K562-CD19 cells were replenished every 3 days to maintain the ratio of 1:1. On the 6th and 10th days, the expression of PD1 and LAG3 on the surface of F-CART cells and C-CART cells was analyzed by flow cytometry. The results are shown in Figure 11A and Figure 11B. From Figure 11A and Figure 11B, the expression of PD1 in the F-CART group was significantly higher than that in the C-CART group. + LAG3 + It was found that the percentage of cells was significantly lower than that of C-CART on both days 6 and 10, indicating that fewer cells were inhibited or depleted in the F-CART group.

[0200]

[0242] Similarly, cells were harvested from three healthy donors and F-CART cells and C-CART cells were prepared as described in Examples 2 and 4. Then, 1 × 10 cells from each group were cultured in a 30-well plate. 6The prepared cells were co-cultured with irradiated K562-CD19 cells at a ratio of 1:1 for 10 days. During the culture, the irradiated K562-CD19 cells were replenished every 3 days to maintain a ratio of 1:1. Then, the expression of CD62L and CD45RO on the surface of F-CART cells and C-CART cells was analyzed by flow cytometry to evaluate the differentiation state of the cells. The results are shown in Table 6 and Figure 12.

[0201]

[0243]

[0202] [Table 7]

[0203]

[0244]

[0245] The results showed that the proportion of TSCM cells and TCM cells in the F-CART group was higher than that of TSCM cells and TCM cells in the C-CART group, and the proportion of TEM cells was lower, indicating that F-CART cells were less differentiated, in a younger state, and had stronger proliferation and differentiation ability.

[0204] Example 15

[0246] Safety and clinical efficacy of F-CART cell preparations

[0247] F-CART cells were administered to human patient XF001 to test the safety and efficacy of the preparation. Patient XF001 was a woman, 39 years old, 150 cm tall, 70 kg in weight, and had been diagnosed with chronic myeloid leukemia (CML) for 15 years. Four months prior to F-CART treatment, B-cell acute lymphoblastic leukemia (B-ALL) was found and diagnosed in the patient along with central nervous system leukemia. The patient also had a drug resistance mutation T315I / V299L in BCR-ABL. Various chemotherapy treatments were poorly effective. The patient further showed resistance to tyrosine kinase inhibitors and did not respond to conventional chemotherapy.

[0205]

[0248] White blood cells were collected from patients, and patient-derived F-CART cells containing CD19-CAR were prepared according to the method described in Example 2. The interval between apheresis and cell infusion was only 10 days, and the preparation process of F-CART cells took only 24 hours. First, the patient was pretreated with 3 days of FC chemotherapy (Fludarabine 50 mg and cyclophosphamide 300 mg administered daily on days 1-3), and then the F-CART preparation was infused into the patient at a dose of 4.2 × 10 6 The number of cells (approximately 6 × 10 4 cells / kg body weight) and injected into patients.

[0206]

[0249] As shown in Figure 13B, on day 8, the patient's temperature was normal, and symptoms such as fever and infection occurred from day 10 to day 13 after infusion of F-CART cells. The symptoms were determined to be grade 1 cytokine release syndrome (grade 1 CRS). The patient then received antipyretic therapy and was treated with meropenem for infection. On day 16, the patient's temperature returned to normal (as shown in Figure 13B).

[0207]

[0250] As shown in Figure 13A, on days 1 to 7 after F-CART infusion, CD19 + Flow cytometry results showed that few B cells appeared in peripheral blood, and copies of the nucleic acid molecule encoding CAR (CAR copy number) were not detected by PCR. + No B cells were detected in peripheral blood. At day 13 after infusion of F-CART, the CAR copy number peaked (4670.2 copies / μg DNA), and at day 28, the copy number returned to 15.4 copies / μg DNA. At day 56, the copy number decreased to 0. The results of CAR-T analyzed by flow cytometry (change in the number of CAR+ cells) were consistent with the results of CAR copy number.

[0208]

[0251] On the 18th day after infusion of F-CART, analysis of a sample obtained from a bone puncture showed that protozoan lymphocytes with normal morphology accounted for 0.5%, granulocytic proliferation was active, and minimal residual disease (MRD) was evaluated as CML, indicating the clearance of B-ALL MRD in the patient. The patient was therefore diagnosed with chronic phase CML (CP-CML). The patient received an HLA7 / 12 half-matched hematopoietic stem cell transplant (HSCT) from his son on the 40th day after infusion of F-CART, showed leukocyte reconstitution, and was discharged on the 20th day after transplantation. The changes in the patient's factors related to immune response (such as IL-6 and C-reactive protein CRP) were as shown in Figure 13C and Figure 13D.

[0209]

[0252] The above results demonstrated that after administering low doses of F-CART cells to patients, the MRD of B-ALL was successfully eradicated without causing severe CRS and neurotoxicity.

[0210]

[0253] Based on the rapid preparation, the infusion of our F-CART was accelerated by at least 7-10 days compared to conventional CD19CAR-T (C-CART), suggesting an advantage of F-CART in the timing of treatment. In addition, the peak in CAR+ ratio and CAR copy number appeared in patients at a later time point with the F-CART preparation compared to conventional CD19CAR-T (C-CART).

[0211] Example 16

[0254] Safety and clinical efficacy of F-CART cell preparations

[0255] F-CART cells were administered to human patient F01 to test the safety and efficacy of the preparation. After enrollment, PBMCs were isolated from the patient, and patient-derived F-CART cells containing CD19-CAR were prepared according to the method described in Example 2. First, the patient was pretreated with 3 days of chemotherapy (Fludarabine 50 mg x 3 days + Cyclophosphamide 0.4 g x 3 days + Cytarabine 0.5 g x 4 days), and then approximately 1.07 x 10 5 Patients were infused at a dose of cells / kg body weight.

[0212]

[0256] As shown in FIG. 14A, on the 10th day after infusion of F-CART, the patient developed a fever, and the temperature rose to 39.4°C. The fever was alleviated after the procedure. No symptoms of hypoxia or hypotension, symptoms of CRS, or neurotoxicity were found. The patient did not receive tocilizumab or other hormonal drugs. CRS was determined to be grade 1. In addition, as shown in FIG. 14C, changes in cytokines including CRP, IL-6, IL-10, INF-γ, and sCD25 in the patient's peripheral blood were assayed. Compared with baseline, the secretion of IL-6 increased on the 21st day after infusion of F-CART. Other cytokines showed no significant changes.

[0213]

[0257] As shown in FIG. 14B, significant proliferation of F-CART cells was observed in peripheral blood (PB) from day 7 to day 14 after infusion. At day 7 after infusion, the CAR copy number (qPCR) and F-CART cell number (FACS) in peripheral blood were 195,297 copies / μg DNA and 27.5 cells / μl, respectively. At day 10 after infusion, the CAR copy number (qPCR) and F-CART cell number (FACS) in peripheral blood were 106822 copies / μg DNA and 20 cells / μl, respectively. At day 14 after infusion, the CAR copy number (qPCR) and F-CART cell number (FACS) in peripheral blood were 162464 copies / μg DNA and 26.5 cells / μl, respectively. At day 21 after infusion, proliferation was significantly reduced, and at day 28 after infusion, F-CART cells could still be detected. At days 14 and 28 post-infusion, bone marrow (BM) CAR copy numbers were detected as 26429 copies / μg DNA and 68135.6 copies / μg DNA, respectively, and CAR expansion was not detected by qPCR. Fourteen and 28 days after infusion, no abnormal B cells were detected in the patient's peripheral blood by flow cytometry, and no abnormal cells or tumors were found in bone marrow samples.

[0214]

[0258] Administration of low doses of F-CART cells to patients proves safe and effective in killing tumor cells in vivo. Example 17

[0259] Clinical efficacy of F-CART cell preparations

[0260] F-CART cell preparations were administered to human patients DF06, GF001, XF002, TF003, TF002, DF04, DF01, XF001 and TF001, diagnosed with relapsed or refractory B-ALL, respectively.

[0215]

[0261] White blood cells were collected from the patients, and F-CART cells containing CD19-CAR from each patient were prepared according to the method described in Example 2. The interval between apheresis and cell infusion was only 10 days, and the preparation process of F-CART cells took only 24 hours. During ...

Claims

1. 1. A method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising: Injecting the population of immune cells comprising the engineered immune cells into a subject in need thereof. wherein said engineered immune cells have not been subjected to ex vivo expansion for one week or more, and said population is further characterized in that central memory T cells (TCM) are enriched over effector memory T cells (TEM) in said population.

2. 10. The method of claim 1, wherein the engineered immune cells have been subjected to ex vivo expansion for less than 6 days.

3. The method of any one of claims 1 to 2, wherein the engineered immune cells are subjected to ex vivo expansion for less than 5 days.

4. The method of any one of claims 1 to 3, wherein the engineered immune cells are subjected to ex vivo expansion for less than 72 hours.

5. The method of any one of claims 1 to 4, wherein the engineered immune cells are subjected to ex vivo expansion for less than 48 hours.

6. The method of any one of claims 1 to 5, wherein the engineered immune cells are subjected to ex vivo expansion for less than 24 hours.

7. The method of any one of claims 1 to 6, wherein the TCM is CD45RO+CD62L+.

8. The method of any one of claims 1 to 7, wherein the TEM is CD45RO+CD62L-.

9. 9. The method of any one of claims 1 to 8, further characterized in that reduced depletion of cells in said population is observed compared to cell depletion in an equivalent population that has undergone ex vivo expansion for one week or more.

10. 10. The method of claim 9, wherein said reduced depletion of said population is characterized in that said population comprises fewer cells that express PD1 and LAG3.

11. The method of any one of claims 1 to 10, wherein the engineered immune cells are T cells, NK cells, and / or NKT cells.

12. The method of any one of claims 1 to 11, wherein the TCR comprises (i) a ligand-binding domain specific for a ligand, and (ii) a transmembrane domain.

13. The method of any one of claims 1 to 12, wherein the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

14. The ligand of the TCR or CAR is selected from the group consisting of VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, C D80, CD86, CD81, CD123, CD171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER- 4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL 6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.

2.

15. The method of any one of claims 13 to 14, wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

16. The method of any one of claims 13 to 15, wherein the intracellular signaling domain is derived from CD3ζ, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS).

17. The method of any one of claims 13 to 16, wherein the CAR comprises at least two intracellular signaling domains.

18. The method of any one of claims 13 to 17, wherein the CAR comprises at least three intracellular signaling domains.

19. The method of any one of claims 13 to 18, wherein the CAR further comprises a hinge.

20. The method of claim 19, wherein the hinge is derived from CD28, IgG1 and / or CD8α.

21. The method of any one of claims 13 to 20, wherein the CAR further comprises a signal peptide, and the signal peptide is derived from IgG1, GM-CSF and / or CD8α.

22. The method of any one of claims 1 to 21, wherein the engineered immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells.

23. 23. The method of claim 22, wherein the engineered immune cells are derived from peripheral blood and the peripheral blood cells are T cells.

24. The method of any one of claims 1 to 23, wherein greater memory and / or stemness is observed in said population compared to a comparable population that has undergone ex vivo expansion for one week or more.

25. 25. The method of claim 24, wherein there is twice as much TCM as TEM.

26. 25. The method of claim 24, wherein there is four times more TCM compared to TEM.

27. The method of any one of claims 1 to 26, wherein the injection is intravenous.

28. The administration is about 1×10 ４ 28. The method of any one of claims 1 to 27, comprising injecting engineered immune cells from 100 / kg body weight.

29. The administration is about 3×10 ５ 29. The method of any one of claims 1 to 28, comprising injecting engineered immune cells from 100 / kg body weight.

30. 30. The method of any one of claims 1 to 29, wherein at least 10% of the immune cells express the CAR and / or the TCR.

31. The method of any one of claims 1 to 29, wherein at least 40% of the immune cells express the CAR and / or the TCR.

32. The method of any one of claims 1 to 31, further comprising administering a secondary agent to the subject in need thereof.

33. 33. The method of claim 32, wherein the secondary agent is a therapeutically effective amount of an immunostimulant, an immunosuppressant, an antifungal agent, an antibiotic, an antiangiogenic agent, a chemotherapeutic agent, a radioactive agent, and / or an antiviral agent.

34. The method of claim 33, wherein the immune stimulant is IL-2.

35. 35. The method of any one of claims 1 to 34, further comprising the step of obtaining peripheral blood from the subject in need thereof after said administration.

36. 36. The method of claim 35, wherein the engineered immune cells in the subject are quantified from the peripheral blood.

37. The method of claim 35, wherein the level of the growth factor in the subject is quantified.

38. 38. The method of claim 37, wherein the growth factor is selected from the group consisting of IL-10, IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-4, IL-8, IL-12, and / or IFN-γ.

39. 39. The method of any one of claims 1 to 38, comprising repeating the injection.

40. 40. The method of any one of claims 1 to 39, wherein the population of immune cells is allogeneic to the subject in need thereof.

41. 40. The method of any one of claims 1 to 39, wherein the population of immune cells is autologous to the subject in need thereof.

42. The method of any one of claims 1 to 41, wherein the subject has cancer.

43. 43. The method of claim 42, wherein the cancer is a hematological cancer.

44. 44. The method of claim 43, wherein the hematological cancer is leukemia, myeloma, lymphoma, and / or a combination thereof.

45. 45. The method of any one of claims 42 to 44, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), and / or acute lymphoblastic leukemia (ALL).

46. The method of any one of claims 43 to 44, wherein the lymphoma is mantle cell lymphoma (MCL), Hodgkin's lymphoma, T-cell lymphoma, and / or non-Hodgkin's lymphoma.

47. 43. The method of claim 42, wherein the cancer is a solid cancer.

48. 48. The method of claim 47, wherein the solid cancer is selected from the group including nephroblastoma, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, or bladder cancer.

49. The method of any one of claims 1 to 48, wherein the subject in need thereof has a BCR-ABL mutation, and the mutation is in the BCR-ABL kinase domain.

50. 50. The method of claim 49, wherein the subject in need thereof has a T315I and / or V299L mutation in the BCR-ABL kinase domain.

51. The method of any one of claims 1 to 50, wherein the subject exhibits resistance to a tyrosine kinase inhibitor.

52. 52. The method of any one of claims 1 to 51, wherein the subject has or is prone to have a tumor following chemotherapy.

53. The method of any one of claims 1 to 52, wherein the subject has been pretreated with chemotherapy prior to said administering.

54. 54. The method of claim 53, wherein the chemotherapy comprises administration of fludarabine, cyclophosphamide and / or cytarabine.

55. 55. The method of any one of claims 1-54, wherein the subject has minimal residual disease (MRD), and the MRD is acute lymphoblastic leukemia.

56. 56. The method of any one of claims 1-55, further characterized in that greater proliferation, cytotoxicity, and / or bone marrow migration is observed in said population compared to proliferation, cytotoxicity, and / or bone marrow migration of a comparable population that has undergone ex vivo expansion for one week or more.

57. 57. The method of claim 56, wherein the cytotoxicity is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, or 3.0 fold greater in the population when the population and a comparable population are in contact with a target, as compared to a comparable population that has undergone ex vivo expansion for one week or more.

58. The proliferation occurs when the population and a comparable population contact a target, 57. The method of claim 56, wherein the cell proliferation rate is at least 1-fold higher as compared to a comparable population undergoing ex vivo expansion for more than a week.

59. 57. The method of claim 56, wherein said bone marrow migration is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold greater in said population when said population and a comparable population are in contact with a target compared to a comparable population that has undergone ex vivo expansion for one or more weeks.

60. 60. The method of claim 59, wherein the target is a cancer cell, a ligand of the TCR or the CAR, or a chemokine.

61. 61. The method of claim 60, wherein said chemokine is stromal cell-derived factor-1 (SDF-1), and said SDF-1 is expressed in the bone marrow of said subject.

62. 62. The method of any one of claims 56-61, wherein the population has a greater percentage of CXCR4 positive cells compared to an equivalent population that has undergone ex vivo expansion for one week or more.

63. The method of claim 62, wherein the population has a median percentage of CXCR4 positive cells that is at least 10% greater than the median percentage of CXCR4 positive cells expressed by a comparable population that has undergone ex vivo expansion for one week or more.

64. 64. The method of claim 63, wherein the population has a greater density of CXCR4 on the cell surface of the CXCR4 positive cells compared to the density of CXCR4 on the cell surface of a comparable population that has undergone ex vivo expansion for one week or more.

65. 65. The method of claim 64, wherein the density is measured by assessing the mean fluorescent intensity (MFI) of CXCR4 on the cell surface of the CXCR4 positive cells.

66. 57. The method of claim 56, wherein the cytotoxicity is measured in an in vivo assay.

67. 67. The method of claim 66, wherein when the subject is administered the population, a reduction in cancer burden is observed in the subject as compared to the cancer burden observed in a comparable subject administered a comparable population that has undergone ex vivo expansion for one or more weeks.

68. 68. The method of claim 67, wherein the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the subject treated with the population compared to the comparable subject administered a comparable population undergoing ex vivo expansion for one or more weeks.

69. 1. A method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising: Injecting the population of immune cells comprising the engineered immune cells into a subject in need thereof. wherein the engineered immune cells have not been subjected to ex vivo expansion for one week or more, and at least 15% of the population are stem memory T cells (TSCM).

70. 70. The method of claim 69, wherein at least 20% of the population is TSCM.

71. 71. The method of any one of claims 69-70, wherein at least 25% of the population is TSCM.

72. 72. The method of any one of claims 69 to 71, wherein at least 30% of the population is TSCM.

73. 73. The method of any one of claims 69 to 72, wherein at least 50% of the population is TSCM.

74. The method of any one of claims 69 to 73, wherein the TSCM is CD45RO-CD62L+.

75. 75. The method of any one of claims 69-74, wherein the engineered immune cells are subjected to ex vivo expansion for less than 5 days.

76. 76. The method of any one of claims 69-75, wherein the engineered immune cells are subjected to ex vivo expansion for less than 72 hours.

77. 77. The method of any one of claims 69-76, wherein the engineered immune cells are subjected to ex vivo expansion for less than 48 hours.

78. 78. The method of any one of claims 69-77, wherein the engineered immune cells are subjected to ex vivo expansion for less than 24 hours.

79. 79. The method of any one of claims 69-78, wherein the TCR comprises (i) a ligand-binding domain specific for a ligand, and (ii) a transmembrane domain.

80. The method of any one of claims 69 to 79, wherein the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

81. The ligand of the TCR or CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125 , CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.

2.

82. The method of any one of claims 80 to 81, wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

83. 83. The method of any one of claims 80 to 82, wherein the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS).

84. The method of any one of claims 80-83, wherein the CAR comprises at least two intracellular signaling domains.

85. The method of any one of claims 80-84, wherein the CAR comprises at least three intracellular signaling domains.

86. The method of any one of claims 80 to 85, wherein the CAR further comprises a hinge.

87. The method of claim 19, wherein the hinge is derived from CD28, IgG1 and / or CD8α.

88. The method of any one of claims 80 to 87, wherein the CAR further comprises a signal peptide, the signal peptide being derived from IgG1, GM-CSF and / or CD8α.

89. 89. The method of any one of claims 69 to 88, wherein the engineered immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells.

90. 90. The method of claim 89, wherein the engineered immune cells are derived from peripheral blood, and the peripheral blood cells are T cells, NK cells, and / or NKT cells.

91. 91. The method of any one of claims 69 to 90, wherein the injection is intravenous.

92. The administration is about 1×10 ４ 92. The method of any one of claims 69 to 91, comprising injecting engineered immune cells from 100 / kg body weight.

93. The administration is about 3×10 ５ 93. The method of any one of claims 69 to 92, comprising injecting engineered immune cells from 100 / kg body weight.

94. 94. The method of any one of claims 69 to 93, wherein at least 10% of said immune cells express said CAR and / or said TCR.

95. 95. The method of any one of claims 69 to 94, wherein at least 40% of said immune cells express said CAR and / or said TCR.

96. 96. The method of any one of claims 69-95, further comprising administering a secondary agent to the subject in need thereof.

97. 97. The method of claim 96, wherein the secondary agent is a therapeutically effective amount of an immunostimulant, an immunosuppressant, an antifungal agent, an antibiotic, an antiangiogenic agent, a chemotherapeutic agent, a radioactive agent, and / or an antiviral agent.

98. 98. The method of claim 97, wherein the immune stimulant is IL-2.

99. 99. The method of any one of claims 69-98, further comprising the step of obtaining peripheral blood from the subject in need thereof after said infusion.

100. 100. The method of claim 99, wherein the engineered immune cells from the peripheral blood are quantified.

101. 101. The method of claim 100, wherein the level of the cytokine is quantified.

102. 102. The method of claim 101, wherein the cytokine is IL-10, IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-4, IL-8, IL-12, and / or IFN-γ.

103. 103. The method of any one of claims 69 to 102, comprising repeating the injection.

104. 104. The method of any one of claims 69-103, further characterized in that reduced depletion of cells in said population is observed compared to cell depletion in an equivalent population that has undergone ex vivo expansion for one week or more.

105. 105. The method of claim 104, wherein the reduced depletion of the population is characterized in that the population comprises fewer cells that express PD1 and LAG3.

106. 106. The method of any one of claims 69-105, further characterized in that greater proliferation, cytotoxicity, and / or bone marrow migration is observed in said population compared to proliferation, cytotoxicity, and / or bone marrow migration of a comparable population that has undergone ex vivo expansion for one week or more.

107. 107. The method of claim 106, wherein the cytotoxicity is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, or 3.0 fold greater in the population when the population and a comparable population are in contact with a target, as compared to a comparable population that has undergone ex vivo expansion for one week or more.

108. 107. The method of claim 106, wherein said expansion is at least 0.5 fold, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 50 fold, 100 fold, 500 fold, 1000 fold, 5000 fold, or 10000 fold greater in said population when said population and a comparable population are contacted with a target, as compared to a comparable population that has undergone ex vivo expansion for one week or more.

109. 107. The method of claim 106, wherein said bone marrow migration is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold greater in said population when said population and a comparable population are in contact with a target compared to a comparable population that has undergone ex vivo expansion for one or more weeks.

110. 110. The method of claim 109, wherein the target is a cancer cell or a chemokine.

111. The method of claim 110, wherein said chemokine is stromal cell-derived factor-1 (SDF-1), and said SDF-1 is expressed in bone marrow of said subject.

112. 112. The method of any one of claims 109-111, wherein the population has a greater percentage of CXCR4 positive cells compared to an equivalent population that has undergone ex vivo expansion for one week or more.

113. The method of claim 112, wherein the population has a median percentage of CXCR4 positive cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% greater than the median percentage of CXCR4 positive cells expressed by a comparable population that has undergone ex vivo expansion for one week or more.

114. The method of claim 113, wherein the population has a greater density of CXCR4 on the cell surface of the CXCR4 positive cells compared to the density of CXCR4 on the cell surface of a comparable population that has undergone ex vivo expansion for one week or more.

115. The method of claim 114, wherein the density is measured by assessing the mean fluorescent intensity (MFI) of CXCR4 on the cell surface of the CXCR4 positive cells.

116. The method of claim 106, wherein the cytotoxicity is measured in an in vivo assay.

117. The method of claim 116, wherein when the subject is administered the population, a reduction in cancer burden is observed in the subject as compared to the cancer burden observed in a comparable subject administered a comparable population that has undergone ex vivo expansion for one or more weeks.

118. 117. The method of claim 117, wherein the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the subject treated with the population compared to the comparable subject administered a comparable population undergoing ex vivo expansion for one or more weeks.

119. 1. A method for producing a population of engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising: (a) activating a population of cells comprising immune cells with an activating moiety; and (b) introducing a polynucleotide encoding at least said CAR and / or said TCR, thereby producing a population of engineered immune cells expressing said CAR and / or said TCR. wherein the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain; A method wherein (a) and (b) are carried out in 24 hours or less.

120. The method of claim 119, wherein the activating moiety binds to the CD3 / T cell receptor complex and / or provides costimulation.

121. 121. The method of claim 120, wherein the activating moiety is any one of anti-CD3 and / or anti-CD28.

122. The method of any one of claims 119 to 121, wherein the activating moiety is conjugated to a solid phase.

123. The solid phase is at least one of a bead, a plate, and / or a matrix. The method of claim 122.

124. 124. The method of claim 123, wherein the solid phase is a bead.

125. 125. The method of any one of claims 119 to 124, wherein said introducing comprises transducing said population of cells with a viral vector and / or a transposon vector.

126. The method of claim 125, wherein the viral vector is a retroviral vector, a lentiviral vector and / or an adeno-associated viral vector.

127. 126. The method of claim 125, wherein the transposon-based vector is a Sleeping Beauty vector and / or a PiggyBac vector.

128. 128. The method of any one of claims 119 to 127, wherein steps (a) and (b) are carried out within 72 hours.

129. 128. The method of any one of claims 119 to 127, wherein steps (a) and (b) are carried out within 48 hours.

130. 128. The method of any one of claims 119 to 127, wherein steps (a) and (b) are carried out within 24 hours.

131. 128. The method of any one of claims 119 to 127, wherein steps (a) and (b) are carried out within 3 hours.

132. 128. The method of any one of claims 119 to 127, wherein steps (a) and (b) are carried out within one hour.

133. 128. The method of any one of claims 119 to 127, wherein steps (a) and (b) are carried out simultaneously.

134. 134. The method of any one of claims 125 to 133, wherein said transduction comprises adding an infectious agent.

135. 136. The method of claim 135, wherein the infectious agent is polybrene.

136. The population of cells is about 10 ４ / mL to about 10 ８ The method of any one of claims 119 to 135, wherein the cells are seeded at a density of 100 / mL.

137. 137. The method of any one of claims 125-136, wherein the viral vectors are plated at a mean of infectivity (MOI) of about 0.1 to about 10.

138. 138. The method of any one of claims 119 to 137, further comprising stimulating the population of cells with a cytokine.

139. 139. The method of claim 138, wherein the cytokine is IL2, IL7, IL15 and / or IL21.

140. The method of any one of claims 119 to 139, wherein the TCR comprises (i) a ligand binding domain specific for a ligand, and (ii) a transmembrane domain.

141. The method of any one of claims 119 to 140, wherein the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

142. The ligand of the TCR or CAR is selected from the group consisting of VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, 142. The method of any one of claims 140-141, wherein the target polypeptide is CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.

2.

143. The method of any one of claims 140 to 142, wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

144. The method of any one of claims 140 to 143, wherein the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS).

145. The method of any one of claims 141-144, wherein the CAR comprises at least two intracellular signaling domains.

146. The method of any one of claims 141-145, wherein the CAR comprises at least three intracellular signaling domains.

147. The method of any one of claims 141 to 146, wherein the CAR further comprises a hinge.

148. The method of claim 147, wherein the hinge is derived from CD28, IgG1 and / or CD8α.

149. 149. The method of any one of claims 119 to 148, further comprising enriching said immune cells prior to said manipulation.

150. 150. The method of claim 149, wherein said enrichment comprises collecting a monocyte fraction.

151. 150. The method of claim 149, wherein said enrichment comprises sorting said immune cells from a monocyte fraction.

152. The enrichment comprises sorting the immune cells based on expression of one or more markers.

150. The method of claim 149, comprising:

153. 153. The method of claim 152, wherein the one or more markers include CD3, CD28, CD4, and / or CD8.

154. The method of any one of claims 151 to 153, wherein the immune cells are selected using an anti-CD3 antibody or an antigen-binding fragment thereof, and / or an anti-CD28 antibody or an antigen-binding fragment thereof.

155. 154. The method of any one of claims 151 to 153, wherein the immune cells are selected using beads conjugated with the anti-CD3 or an antigen-binding fragment thereof, and / or beads conjugated with the anti-CD28 antibody or an antigen-binding fragment thereof.

156. 156. The method of any one of claims 119 to 155, wherein the engineered population of immune cells is characterized in that cellular memory T cells (TCM) are enriched over effector memory T cells (TEM) in the population.

157. 157. The method of any one of claims 119-156, wherein at least 15% of the population are stem memory T cells (TSCM).

158. 157. The method of any one of claims 119-156, wherein at least 20% of the population are stem memory T cells (TSCM).

159. 157. The method of any one of claims 119-156, wherein at least 25% of the population are stem memory T cells (TSCM).

160. 157. The method of any one of claims 119-156, wherein at least 30% of the population are stem memory T cells (TSCM).

161. 157. The method of any one of claims 119-156, wherein at least 50% of the population are stem memory T cells (TSCM).

162. 162. The method of any one of claims 119-161, further comprising the step of injecting said population of engineered immune cells into a subject in need thereof within 72 hours of completing (a) and (b).

163. 163. The method of any one of claims 119-162, further characterized in that reduced depletion of cells in the population is observed compared to depletion of cells in a comparable population undergoing a comparable method in which (a) and (b) are not performed simultaneously.

164. 164. The method of claim 163, wherein the reduced depletion of the population is characterized in that the population comprises fewer cells that express PD1 and LAG3.

165. The method of any one of claims 119-164, further characterized in that greater proliferation, cytotoxicity, and / or bone marrow migration is observed in said population compared to proliferation, cytotoxicity, and / or bone marrow migration in a comparable population undergoing a comparable method in which (a) and (b) are not performed simultaneously.

166. said cytotoxicity is at least 166. The method of claim 165, wherein the concentration of the β-amino acid is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, or 3.0 times higher.

167. 166. The method of claim 165, wherein said proliferation is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, or 10000 times greater in said population when said population and a comparable population are contacted with a target, compared to a comparable population in which (a) and (b) are performed for greater than 24 hours.

168. 166. The method of claim 165, wherein said bone marrow migration is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold higher in said population when said population and a comparable population are in contact with a target compared to a comparable population in which (a) and (b) are performed for greater than 24 hours.

169. The method of claim 168, wherein the target is a cancer cell or a chemokine.

170. 170. The method of claim 169, wherein said chemokine is stromal cell-derived factor-1 (SDF-1), and said SDF-1 is expressed in bone marrow of said subject.

171. 171. The method of any one of claims 168-170, wherein the population has a greater percentage of CXCR4 positive cells compared to an equivalent population in which (a) and (b) are performed for greater than 24 hours.

172. The method of claim 171, wherein the population has a median percentage of CXCR4 positive cells that is at least 10% greater than the median percentage of CXCR4 positive cells expressed by a comparable population in which (a) and (b) are performed for greater than 24 hours.

173. The method of claim 172, wherein the population has a greater density of CXCR4 on the cell surface of the CXCR4-positive cells compared to the density of CXCR4 on the cell surface of an equivalent population in which (a) and (b) are performed for greater than 24 hours.

174. The method of claim 173, wherein the density is measured by assessing the mean fluorescent intensity (MFI) of CXCR4 on the cell surface of the CXCR4 positive cells.

175. 166. The method of claim 165, wherein the cytotoxicity is measured in an in vivo assay.

176. The method of claim 175, wherein when the subject is administered the population, a reduction in cancer burden is observed in the subject compared to the cancer burden observed in a comparable subject administered a comparable population in which (a) and (b) are performed for greater than 24 hours.

177. 177. The method of claim 176, wherein the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in said subjects treated with said population compared to said comparable subjects administered a comparable population in which (a) and (b) are performed for greater than 24 hours.

178. Injecting a population of immune cells, including engineered immune cells, that have not been subjected to ex vivo expansion for more than one week. wherein the population of immune cells is further characterized in that cellular memory T cells (TCM) are enriched over effector memory T cells (TEM) in the population; or at least 15% of the population are stem memory T cells (TSCM).

179. The point-of-care facility of claim 178, wherein at least 20% of said population are stem memory T cells (TSCM).

180. The point-of-care facility of claim 178, wherein at least 25% of said population are stem memory T cells (TSCM).

181. The point-of-care facility of claim 178, wherein at least 30% of said population are stem memory T cells (TSCM).

182. The point-of-care facility of any one of claims 178 to 181, wherein at least 50% of said population are stem memory T cells (TSCM).

183. 182. The point-of-care facility of any one of claims 178 to 181, wherein there is twice as much TCM as TEM.

184. 182. The point-of-care facility of any one of claims 178 to 181, wherein there is four times more TCM compared to TEM.

185. The point-of-care facility of any one of claims 178 to 184, wherein the engineered immune cells have been subjected to ex vivo expansion for less than 5 days.

186. The point-of-care facility of any one of claims 178 to 185, wherein the engineered immune cells have been subjected to ex vivo expansion for less than 72 hours.

187. The point-of-care facility of any one of claims 145 to 186, wherein the engineered immune cells have been subjected to ex vivo expansion for less than 48 hours.

188. The point-of-care facility of any one of claims 178 to 187, wherein the engineered immune cells have been subjected to ex vivo expansion for less than 24 hours.

189. The point-of-care facility of any one of claims 178 to 188, wherein the immune cells are T cells, NK cells, and / or NKT cells.

190. (a) receiving a population of cells from a subject, the population comprising immune cells; (b) activating the population of immune cells with an activating moiety and simultaneously introducing into the immune cells a polynucleotide encoding at least a chimeric antigen receptor (CAR), the CAR comprising (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain; and (c) within two weeks or less prior to performing (b), injecting the population of immune cells of (b) into the subject. A point-of-care facility including a cell processing device configured to perform the steps of:

191. Step (c) is performed within one week or prior to performing (b).

191. The point-of-care equipment of claim 190.

192. The point-of-care facility of any one of claims 190 to 191, wherein the immune cells are T cells, NK cells, and / or NKT cells.

193. The ligand of the CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, CD151, C 193. The point of care device of any one of claims 190 to 192, wherein the antigen is TLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or Claudin 18.

2.

194. The point-of-care facility of any one of claims 190 to 193, wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

195. 195. The point of care device of any one of claims 190 to 194, wherein the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS).

196. The point of care device of any one of claims 190 to 195, wherein the CAR comprises at least two intracellular signaling domains.

197. The point of care device of any one of claims 190 to 196, wherein the CAR comprises at least three intracellular signaling domains.

198. The point-of-care equipment of any one of claims 190 to 197, wherein the CAR further comprises a hinge.

199. The point-of-care facility of claim 198, wherein the hinge is derived from CD28, IgG1 and / or CD8α.

200. The point-of-care facility of any one of claims 190 to 199, wherein the CAR further comprises a signal peptide, the signal peptide being derived from IgG1, GM-CSF and / or CD8α.

201. The point-of-care facility of any one of claims 190 to 200, wherein the immune cells are T cells, NK cells, and / or NKT cells.

202. The activating moiety binds to the CD3 / T cell receptor complex and / or induces costimulation. A point-of-care facility according to any one of claims 190 to 201, providing

203. The point-of-care facility of claim 202, wherein the activating moiety is one of anti-CD3 and / or anti-CD28.

204. A point-of-care facility according to any one of claims 190 to 203, wherein a viral vector and / or a transposon vector comprises the polynucleotide.

205. The point-of-care device of claim 204, wherein the viral vector is a retroviral vector, a lentiviral vector and / or an adeno-associated viral vector.

206. A point-of-care facility according to any one of claims 190 to 205, wherein steps (a) and (b) are carried out within 72 hours.

207. A point-of-care facility according to any one of claims 190 to 205, wherein steps (a) and (b) are carried out within 48 hours.

208. A point-of-care facility according to any one of claims 190 to 205, wherein steps (a) and (b) are carried out within 24 hours.

209. A point-of-care facility according to any one of claims 190 to 205, wherein steps (a) and (b) are carried out within one hour.

210. 210. The point of care facility of any one of claims 190 to 209, wherein the population is further characterized by observing a reduced depletion of cells in the population, wherein the reduced depletion is characterized by the population comprising fewer cells that express PD1 and LAG3.

211. A population of cells comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), The population, (i) central memory T cells (TCM) are more abundant than effector memory T cells (TEM) in said population; and / or (ii) at least 15% of the population of cells are stem memory T cells (TSCM), and the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain. A group further characterized by:

212. The population of claim 211, wherein at least 20% of the population is TSCM.

213. The population of any one of claims 211-212, wherein at least 25% of the population is TSCM.

214. The population of any one of claims 211-213, wherein at least 30% of the population is TSCM.

215. The population of any one of claims 211-214, wherein at least 50% of the population is TSCM.

216. Any one of claims 211 to 215, wherein there is twice as much TCM as TEM. The population described in.

217. The population of any one of claims 211-216, wherein there is four times more TCM compared to TEM.

218. The population of any one of claims 211 to 217, wherein the immune cells are T cells, NK cells, and / or NKT cells.

219. The ligand of the CAR is VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, CD1 51, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or claudin 18.

2.

220. The population of any one of claims 211-219, wherein the transmembrane domain is from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

221. 221. The population of any one of claims 211-220, wherein the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS).

222. The population of any one of claims 211-221, wherein the CAR comprises at least two intracellular signaling domains.

223. The population of any one of claims 211-221, wherein the CAR comprises at least three intracellular signaling domains.

224. The population of any one of claims 211-223, wherein the CAR further comprises a hinge.

225. The population of claim 224, wherein the hinge is derived from CD28, IgG1 and / or CD8α.

226. The population of any one of claims 211-225, wherein the CAR further comprises a signal peptide, wherein the signal peptide is derived from IgG1, GM-CSF and / or CD8α.

227. The population of any one of claims 211 to 226, wherein the immune cells are T cells, NK cells, and / or NKT cells.

228. 228. The population of any one of claims 211-227, wherein the population is cryopreserved or the population is supplied fresh.

229. 229. The population of any one of claims 211-228, wherein said population is further characterized by observing a reduced depletion of cells in said population, said reduced depletion being characterized in that said population comprises fewer cells that express PD1 and LAG3.

230. 1. A method of treating cancer in a subject in need thereof, comprising administering to a subject an amount of about 1×10 6 human leukocytes expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), the method ... ６ 20. A method comprising injecting a population of engineered immune cells in a culture medium comprising administering to the patient ...

231. The method of claim 230, wherein the population of engineered immune cells exhibits a comparable level of anti-tumor activity in vivo compared to a population of 10-fold more engineered immune cells expressing the same chimeric antigen receptor (CAR) and / or engineered T cell receptor (TCR) but that have been subjected to ex vivo expansion for one or more weeks.

232. 232. The method of any one of claims 230-231, wherein said engineered population of immune cells are simultaneously activated and transduced with constructs expressing said CAR and / or TCR.

233. 233. The method of any one of claims 230-232, wherein the population of engineered immune cells has not been subjected to ex vivo expansion for one week.

234. 233. The method of any one of claims 230-232, wherein the population of engineered immune cells has not been subjected to ex vivo expansion for 72 hours.

235. Approximately 1×10 ６ The method of any one of claims 230-234, wherein a population of not more than 10 engineered immune cells is prepared from peripheral blood mononuclear cells (PMBCs) via a process of simultaneous activation and transduction with constructs expressing said CAR and / or TCR.

236. The method of any one of claims 230-235, wherein said injection is performed within one week of concomitant activation and transduction with constructs expressing said CAR and / or TCR.

237. 237. The method of claim 236, wherein said simultaneous activation comprises performing activation and transduction within 72 hours.

238. 237. The method of claim 236, wherein said simultaneous activation comprises performing activation and transduction within 48 hours.

239. 237. The method of claim 236, wherein said simultaneous activation comprises performing activation and transduction within a 24 hour period.

240. 237. The method of claim 236, wherein said simultaneous activation comprises performing activation and transduction within 3 hours.

241. The simultaneous activation comprises performing activation and transduction within one hour. The method of claim 236.

242. 237. The method of claim 236, wherein said simultaneous activation comprises performing activation and transduction simultaneously.

243. 243. The method of any one of claims 230-242, wherein at least 15% of the population are stem memory T cells (TSCM).

244. 244. The method of any one of claims 230-243, wherein at least 20% of the population are stem memory T cells (TSCM).

245. The method of any one of claims 230-244, wherein at least 25% of the population are stem memory T cells (TSCM).

246. The method of any one of claims 230-245, wherein at least 30% of the population are stem memory T cells (TSCM).

247. The method of any one of claims 230-246, wherein at least 50% of the population are stem memory T cells (TSCM).

248. 248. The method of any one of claims 230-247, further characterized in that reduced depletion of cells in said population is observed compared to depletion of cells in an equivalent population undergoing ex vivo expansion for one week or more.

249. The method of claim 248, wherein the reduced depletion of the population is characterized in that the population comprises fewer cells that express PD1 and LAG3.

250. The injection is performed for about 10 ５ 250. The method of any one of claims 230-249, wherein the engineered immune cell is no more than 100 cells / mL.

251. The injection is performed for about 10 ４ 250. The method of any one of claims 230-249, wherein the engineered immune cell is no more than 100 cells / mL.

252. The injection is performed for about 10 ３ 250. The method of any one of claims 230-249, wherein the engineered immune cell is no more than 100 cells / mL.

253. The method of any one of claims 230 to 252, wherein the engineered immune cells are T cells, NK cells, and / or NKT cells.

254. The method of any one of claims 230-253, wherein the TCR comprises (i) a ligand binding domain specific for a ligand, and (ii) a transmembrane domain.

255. The method of any one of claims 230-254, wherein the CAR comprises (i) a ligand-binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

256. The ligand of the TCR or CAR is selected from the group consisting of VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met , EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4 , CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, 256. The method of any one of claims 254-255, wherein the target polypeptide is HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or claudin 18.

2.

257. The method of any one of claims 254 to 255, wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

258. The method of any one of claims 255 to 257, wherein the intracellular signaling domain is derived from CD3zeta, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or CD278 (ICOS).

259. The method of any one of claims 255-258, wherein the CAR comprises at least two intracellular signaling domains.

260. The method of any one of claims 255-258, wherein the CAR comprises at least three intracellular signaling domains.

261. The method of any one of claims 255-260, wherein the CAR further comprises a hinge.

262. The method of claim 261, wherein the hinge is derived from CD28, IgG1 and / or CD8α.

263. The method of any one of claims 255-262, wherein the CAR further comprises a signal peptide, the signal peptide being derived from IgG1, GM-CSF and / or CD8α.

264. 264. The method of any one of claims 230-263, wherein the engineered immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells.

265. 265. The method of claim 264, wherein the engineered immune cells are derived from peripheral blood and the peripheral blood cells are T cells.

266. 266. The method of any one of claims 230-265, further comprising the step of obtaining peripheral blood from the subject in need thereof after said administering.

267. The method of claim 266, wherein the engineered immune cells in the subject are quantified from the peripheral blood.

268. The method of claim 267, wherein the level of the growth factor in the subject is quantified.

269. 269. The method of claim 268, wherein the growth factor is selected from the group consisting of IL-10, IL-6, tumor necrosis factor alpha (TNF-α), IL-1β, IL-2, IL-4, IL-8, IL-12, and / or IFN-γ.

270. 270. The method of any one of claims 230 to 269, comprising repeating the injection.

271. 271. The method of any one of claims 230-270, wherein the population of immune cells is allogeneic to the subject in need thereof.

272. 271. The method of any one of claims 230-270, wherein the population of immune cells is autologous to the subject in need thereof.

273. The method of any one of claims 230 to 272, wherein the subject has cancer.

274. 274. The method of claim 273, wherein the cancer is a blood cancer.

275. 275. The method of claim 274, wherein the hematological cancer is leukemia, myeloma, lymphoma, and / or a combination thereof.

276. 276. The method of claim 275, wherein the leukemia is chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), and / or acute lymphoblastic leukemia (ALL).

277. 276. The method of claim 275, wherein the lymphoma is mantle cell lymphoma (MCL), Hodgkin's lymphoma, T-cell lymphoma, and / or non-Hodgkin's lymphoma.

278. The method of claim 273, wherein the cancer is a solid cancer.

279. The method of claim 278, wherein the solid cancer is selected from the group including nephroblastoma, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, or bladder cancer.

280. 280. The method of any one of claims 230-279, wherein the subject has been pretreated with chemotherapy prior to said administering.

281. 281. The method of claim 280, wherein the chemotherapy comprises administration of fludarabine, cyclophosphamide and / or cytarabine.

282. 282. The method of any one of claims 230-281, further characterized in that greater proliferation, cytotoxicity, and / or bone marrow migration is observed in said population compared to proliferation, cytotoxicity, and / or bone marrow migration of a comparable population that has undergone ex vivo expansion for one or more weeks.

283. the cytotoxicity is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 283. The method of claim 282, wherein the antibody is 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, or 3.0 times higher.

284. The method of claim 282, wherein said expansion is at least 0.5 fold, 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 50 fold, 100 fold, 500 fold, 1000 fold, 5000 fold, or 10000 fold greater in said population when said population and a comparable population are contacted with a target, as compared to a comparable population that has undergone ex vivo expansion for one week or more.

285. The method of claim 282, wherein said bone marrow migration is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold greater in said population when said population and a comparable population are in contact with a target compared to a comparable population that has undergone ex vivo expansion for one or more weeks.

286. The method of claim 285, wherein the target is a cancer cell or a chemokine.

287. 287. The method of claim 286, wherein said chemokine is stromal cell-derived factor-1 (SDF-1), and said SDF-1 is expressed in bone marrow of said subject.

288. 288. The method of any one of claims 285-287, wherein the population has a greater percentage of CXCR4 positive cells compared to an equivalent population that has undergone ex vivo expansion for one week or more.

289. The method of claim 288, wherein the population has a median percentage of CXCR4 positive cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% greater than the median percentage of CXCR4 positive cells expressed by a comparable population that has undergone ex vivo expansion for one week or more.

290. The method of claim 289, wherein the population has a greater density of CXCR4 on the cell surface of the CXCR4 positive cells compared to the density of CXCR4 on the cell surface of a comparable population that has undergone ex vivo expansion for one week or more.

291. The method of claim 290, wherein the density is measured by assessing the mean fluorescent intensity (MFI) of CXCR4 on the cell surface of the CXCR4 positive cells.

292. The method of claim 282, wherein the cytotoxicity is measured in an in vivo assay.

293. The method of claim 292, wherein when the subject is administered the population, a reduction in cancer burden is observed in the subject compared to the cancer burden observed in a comparable subject administered a comparable population that has undergone ex vivo expansion for one or more weeks.

294. The method of claim 293, wherein the cancer burden is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% in the subject treated with the population compared to the comparable subject administered a comparable population undergoing ex vivo expansion for one or more weeks.

295. 1. A method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising: Injecting the population of immune cells comprising the engineered immune cells into a subject in need thereof. wherein said engineered immune cells have not been subjected to ex vivo expansion for one week or more, and said population is further characterized in that greater proliferation is observed in said population as compared to proliferation in a comparable population that has undergone ex vivo expansion for one week or more.

296. 300. The method of claim 295, wherein said expansion is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000, or 10,000 times greater in said population when said population and said equivalent population are contacted with a target, as compared to an equivalent population that has undergone ex vivo expansion for said one or more weeks.

297. 1. A method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising: Injecting the population of immune cells comprising the engineered immune cells into a subject in need thereof. wherein said engineered immune cells have not been subjected to ex vivo expansion for one week or more, and said population is further characterized in that greater cytotoxicity is observed in said population compared to the cytotoxicity of a comparable population that has undergone ex vivo expansion for one week or more.

298. The method of claim 296, wherein the cytotoxicity is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0, 2.5, or 3.0 times greater in the population when the population and the equivalent population contact a target, compared to an equivalent population that has undergone ex vivo expansion for one or more weeks.

299. 1. A method of administering a cell therapy comprising engineered immune cells expressing a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR), comprising: Injecting the population of immune cells comprising the engineered immune cells into a subject in need thereof. wherein said engineered immune cells have not been subjected to ex vivo expansion for one week or more, and said population is further characterized in that greater bone marrow migration is observed in said population as compared to bone marrow migration of a comparable population that has undergone ex vivo expansion for one week or more.

300. 300. The method of claim 299, wherein said bone marrow migration is at least 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold, 0.7 fold, 0.8 fold, 0.9 fold, 1 fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold or 10 fold greater in said population when said population and said comparable population are in contact with a target compared to said comparable population undergoing ex vivo expansion for one week or more.

301. 301. The method of any one of claims 295-300, wherein said population is further characterized by central memory T cells (TCM) being enriched over effector memory T cells (TEM) in said population.

302. The method of claim 301, wherein the TCM is CD45RO+CD62L+.

303. The method of any one of claims 301 to 302, wherein the TEM is CD45RO+CD62L-.

304. The method of any one of claims 295-303, wherein the engineered immune cells are subjected to ex vivo expansion for less than 5 days.

305. The method of any one of claims 295-304, wherein the engineered immune cells are subjected to ex vivo expansion for less than 5 days.

306. The method of any one of claims 295-305, wherein the engineered immune cells are subjected to ex vivo expansion for less than 72 hours.

307. The method of any one of claims 295-306, wherein the engineered immune cells are subjected to ex vivo expansion for less than 48 hours.

308. The method of any one of claims 295-307, wherein the engineered immune cells are subjected to ex vivo expansion for less than 24 hours.

309. The method of any one of claims 296-300, wherein the target is a cancer cell, a ligand of the TCR or CAR, or a chemokine.

310. The method of claim 309, wherein said chemokine is stromal cell-derived factor-1 (SDF-1), and said SDF-1 is expressed in bone marrow of said subject.

311. 311. The method of any one of claims 299-310, wherein said population has a greater percentage of CXCR4 positive cells compared to said comparable population that has been undergoing said ex vivo expansion for one week or more.

312. The method of claim 311, wherein the population has a median percentage of CXCR4 positive cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% greater than the median percentage of CXCR4 positive cells expressed by the comparable population that has undergone ex vivo expansion for one week or more.

313. The method of claim 312, wherein the population has a greater density of CXCR4 on the cell surface of the CXCR4 positive cells compared to the density of CXCR4 on the cell surface of the equivalent population that has undergone ex vivo expansion for one week or more.

314. The method of claim 313, wherein the density is measured by assessing the mean fluorescent intensity (MFI) of CXCR4 on the cell surface of the CXCR4 positive cells.

315. The method of any one of claims 297 to 314, wherein the cytotoxicity is measured in an in vivo assay.

316. The method of claim 315, wherein a reduction in cancer burden is observed in the subject administered the population compared to the cancer burden observed in a comparable subject administered the population that has undergone ex vivo expansion for one week or more.

317. The cancer burden is increased ex vivo for one week or more in the subjects treated with the cohort. o The method of claim 316, wherein the level of IL-16 expression is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% compared to a comparable subject administered a comparable population undergoing expansion.

318. The method of claim 316, wherein a complete response (CR) is observed in the subject administered the population compared to the cancer burden observed in a comparable subject administered the population undergoing ex vivo expansion for one week or more.

319. The method of claim 316, wherein a partial response (PR) is observed in the subject administered the population compared to the cancer burden observed in a comparable subject administered the population undergoing ex vivo expansion for one week or more.

320. The population has a maximum of 1×10 cells / kg body weight ４ 320. The method of any one of claims 295-319, comprising an engineered immune cell.

321. The population is about 1 x 10 cells per kg body weight ４ From 1 to 1 kg / body weight, up to about 1 x 10 cells ５ 320. The method of any one of claims 295-319, comprising an engineered immune cell.

322. The method of any one of claims 295 to 321, wherein the engineered immune cells are T cells, NK cells, and / or NKT cells.

323. The method of any one of claims 295-322, wherein the TCR comprises (i) a ligand binding domain specific for a ligand, and (ii) a transmembrane domain.

324. The method of any one of claims 295-323, wherein the CAR comprises (i) a ligand binding domain specific for a ligand, (ii) a transmembrane domain, and (iii) an intracellular signaling domain.

325. The ligand of the TCR or CAR is selected from the group consisting of VEGFR-2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, BCMA, CD133, EGFR, GPC3, PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, CD44V6, CEA, CA125, 325. The method of any one of claims 295-324, wherein the target polypeptide is CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, mesothelin, NY-ESO-1, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79, Notch-1-4, and / or claudin 18.

2.

326. The method of any one of claims 324 to 325, wherein the transmembrane domain is derived from CD8α, CD4, CD28, CD45, PD-1 and / or CD152.

327. The intracellular signaling domain is selected from the group consisting of CD3ζ, CD28, CD54 (ICAM), CD134 (OX40), CD137 (4-1BB), GITR, CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), DAP10 and / or The method of any one of claims 324 to 326, which is derived from CD278 (ICOS).

328. The method of any one of claims 324-327, wherein the CAR comprises at least two intracellular signaling domains.

329. The method of any one of claims 324-327, wherein the CAR comprises at least three intracellular signaling domains.

330. The method of any one of claims 324-329, wherein the CAR further comprises a hinge.

331. The method of claim 330, wherein the hinge is derived from CD28, IgG1 and / or CD8α.

332. The method of any one of claims 324-331, wherein the engineered immune cells are derived from peripheral blood, umbilical cord blood, bone marrow, and / or induced pluripotent stem cells.

333. The method of claim 332, wherein the engineered immune cells are derived from peripheral blood and the peripheral blood cells are T cells.

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