Process for generating therapeutic compositions from manipulated cells

Abstract

To provide a process for generating compositions of engineered cells.SOLUTION: A process comprises: (a) incubating, under stimulating conditions, an input composition comprising T cells enriched for CD4+ primary human T cells, the stimulating conditions comprising the presence of (i) a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules and (ii) one or more cytokines, wherein at least one cytokine is or comprises recombinant human IL-2, thereby generating a stimulated composition; and (b) introducing a recombinant receptor into the stimulated composition, thereby generating an engineered composition comprising engineered T cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Patent Application No. 62 / 580,409, filed on November 1, 2017, entitled "PROCESS FOR GENERATING THERAPEUTIC COMPOSITIONS OF ENGINEERED CELLS," U.S. Provisional Patent Application No. 62 / 596,771, filed on December 8, 2017, entitled "PROCESS FOR GENERATING THERAPEUTIC COMPOSITIONS OF ENGINEERED CELLS," and U.S. Provisional Patent Application No. 62 / 721,603, filed on August 22, 2018, entitled "PROCESS FOR GENERATING THERAPEUTIC COMPOSITIONS OF ENGINEERED CELLS," the entire contents of which are incorporated by reference for all purposes.

[0002] Inclusion by referencing sequence listings This application is filed together with an electronic sequence listing. The sequence listing is provided as a file named 735042013240SeqList.txt, created on October 31, 2018, and is 35,994 bytes in size. The electronic information of the sequence listing is incorporated in its entirety by reference.

[0003] field This disclosure provides a method for genetically engineering T cells, such as CD4+ T cells and / or CD8+ T cells, for use in cell therapy. In some aspects, the method provided comprises one or more steps for incubating cells under stimulating conditions, introducing recombinant polypeptides into cells via transduction or transfection, and culturing cells under conditions that promote proliferation and / or expansion. In some aspects, incubation and / or culture are carried out in the presence of recombinant IL-2. In some aspects, the method provided is an efficient and reliable means for producing genetically engineered T cells with a high success rate. [Background technology]

[0004] background Various cell therapies are available to treat diseases and conditions. These cell therapies include methods involving immune cells, such as T cells, that have been genetically modified using recombinant receptors, such as chimeric antigen receptors. Improved methods for manufacturing and / or manipulating such cell therapies are needed, including providing more efficient processes and / or improved cell composition products. Methods, kits, and products that meet such needs are provided. [Overview of the project]

[0005] overview In several aspects, a method is provided for preparing a composition of engineered cells, the method comprising (a) (i) incubating an input composition containing CD4+ primary human T cells enriched T cells under stimulating conditions comprising the presence of a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, and (ii) one or more cytokines in which at least one cytokine is recombinant human IL-2 or contains recombinant human IL-2, thereby producing a stimulated composition; and (b) introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition containing engineered T cells.

[0006] In some embodiments of the methods provided herein, the input composition comprises more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% CD4+ primary human T cells, and / or the input composition consists essentially of CD4+ primary human T cells. In some embodiments, the concentration of recombinant IL-2 is 10 IU / mL to 200 IU / mL or about 10 to about 200 IU / mL. In some embodiments of the methods provided herein, one or more cytokines further comprise IL-7 and / or IL-15, and optionally, the concentration of IL-7 is 100 IU / mL to 1000 IU / mL or about 100 IU / mL to about 1000 IU / mL, and / or the concentration of IL-15 is 1 IU / mL to 50 IU / mL or about 1 IU / mL to about 50 IU / mL. In some embodiments of the methods provided herein, incubation is carried out in the presence of one or more antioxidants.

[0007] In several aspects, a method is provided for preparing a composition of engineered cells, the method comprising (a) incubating an input composition containing T cells enriched with one or both CD4+ and CD8+ primary human T cells to produce a stimulated composition, the incubation being carried out under one or more stimulating conditions, (1) (i) a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules and (ii) one or more cytokines; and / or (2) one or more antioxidants; and (b) introducing recombinant receptors into the stimulated composition to produce an engineered composition containing engineered T cells.

[0008] In some embodiments of the methods provided herein, the input composition comprises more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ and / or CD8+ primary human T cells, and / or the input composition consists essentially of CD4+ and / or CD8+ primary human T cells. In some embodiments of the methods provided herein, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In some embodiments, the concentration of recombinant IL-2 is 10–200 IU / mL or about 10–about 200 IU / mL, the concentration of recombinant IL-7 is 100 IU / mL–1000 IU / mL or about 100 IU / mL–about 1000 IU / mL, and / or the concentration of recombinant IL-15 is 1 IU / mL–25 IU / mL or about 1 IU / mL–about 25 IU / mL.

[0009] In some embodiments of the methods provided herein, the input composition comprises more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98%, and / or the input composition consists essentially of CD4+ primary human T cells. In some embodiments, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15. In some embodiments, the input composition comprises more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98%, and / or the input composition consists essentially of CD8+ primary human T cells. In some embodiments of the methods provided herein, one or more cytokines are selected from recombinant IL-2 and recombinant IL-15.

[0010] In some embodiments of the methods provided herein, the stimulating agent comprises a primary agent that specifically binds to a member of the TCR complex, and optionally specifically binds to CD3. In some embodiments, the stimulating agent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, and optionally the costimulatory molecule is selected from CD28, CD137(4-1-BB), OX40, or ICOS. In some embodiments of the methods provided herein, the primary and / or secondary agents comprise antibodies, and optionally the stimulating agent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody, or their antigen-binding fragments. In some embodiments, the primary and / or secondary agents are present on the surface of a solid support.

[0011] In some embodiments of the methods provided herein, the solid support is or comprises beads. In some embodiments, the beads have a diameter greater than or about 3.5 μm but less than or equal to about 9 μm, or less than or equal to about 8 μm, or less than or equal to about 7 μm, or less than or equal to about 6 μm, or less than or equal to about 5 μm. In some embodiments, the beads have a diameter of 4.5 μm or about 4.5 μm. In some embodiments, the beads are inert. In some embodiments, the beads are a polystyrene surface or comprises a polystyrene surface. In some embodiments, the beads are magnetic or superparamagnetic. In some embodiments, the bead-to-cell ratio is less than 3:1 or less than about 3:1. In some embodiments, the bead-to-cell ratio is 2:1 to 0.5:1 or about 2:1 to about 0.5:1. In some embodiments, the bead-to-cell ratio is 1:1 or about 1:1.

[0012] In some embodiments of the methods provided herein, one or more antioxidants include sulfur-containing antioxidants. In some embodiments, one or more antioxidants include glutathione precursors. In some embodiments, one or more antioxidants include N-acetylcysteine ​​(NAC), optionally, the NAC is at a concentration of 0.2 mg / mL to 2.0 mg / mL or about 0.2 mg / mL to about 2.0 mg / mL.

[0013] In some embodiments of the methods provided herein, the introduction involves transducing cells of a stimulated composition with a viral vector containing a polynucleotide encoding a recombinant receptor. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector or a gamma retroviral vector. In some embodiments of the methods provided herein, the introduction is carried out in the presence of a transduction adjuvant. In some embodiments, the transduction adjuvant is or comprises protamine sulfate, optionally 1 μg / ml to 50 μg / ml or about 1 μg / ml to about 50 μg / ml of protamine sulfate, a transduction adjuvant derived from fibronectin, and / or RetroNectin. In other embodiments of the methods provided herein, the introduction involves transfecting cells of a stimulated composition with a vector containing a polynucleotide encoding a recombinant receptor. In some embodiments, the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a Piggybac transposon.

[0014] In some embodiments of the methods provided herein, the method further comprises culturing an engineered composition under conditions that promote the proliferation or expansion of engineered cells, thereby producing an output composition containing engineered T cells. In some embodiments, the culturing is carried out in the presence of one or more cytokines, at least one of which is recombinant human IL-2 or comprises recombinant human IL-2. In some embodiments, the stimulating reagent is removed from the engineered composition before culturing.

[0015] In some embodiments, the stimulant is removed within or less than 7 days after the start of incubation. In some embodiments, the stimulant is removed 3 to 6 days or approximately 3 to approximately 6 days after the start of incubation. In some embodiments, the stimulant is removed 4 days or approximately 4 days after the start of incubation. In some embodiments, removing the beads involves exposing the cells of the manipulated composition to a magnetic field.

[0016] In other aspects, a method is provided for preparing a composition of engineered cells, the method comprising culturing an engineered cell composition containing CD4+ primary human T cells, which include recombinant receptor engineered cells, in the presence of one or more cytokines, wherein the at least one cytokine is recombinant human IL-2 or comprises recombinant human IL-2, the method resulting in proliferation or expansion of the cells in the composition to prepare an output composition containing engineered CD4+ cells. In some embodiments, the proliferation or expansion results in a 2x or about 2x or at least 2x increase in the number of recombinant receptor engineered CD4+ T cells, a 3x or about 3x or at least 3x increase, a 4x or about 4x or at least 4x increase, a 5x or about 5x or at least 5x increase, or more than a 5x increase.

[0017] In some aspects of any of the methods provided herein, the manipulated cell composition comprises more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98%, or more than 98%, and / or the manipulated cell composition consists essentially of CD4+ primary human T cells. In some aspects of the methods provided herein, the concentration of recombinant IL-2 is 50 IU / mL to 500 IU / mL or about 50 IU / mL to about 500 IU / mL. In some embodiment of any of the methods provided herein, one or more cytokines further comprise IL-7 and / or IL-15, optionally, the concentration of IL-7 being 500 IU / mL to 2000 IU / mL or about 500 IU / mL to about 2000 IU / mL, and / or the concentration of IL-15 being 5 IU / mL to 50 IU / mL or about 5 IU / mL to about 50 IU / mL.

[0018] In some embodiment of any of the methods provided herein, the engineered cell composition is prepared by a method comprising: (i) incubating an input composition containing CD4+ primary human T cells enriched under stimulating conditions including the presence of a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules; and (ii) in the presence of at least one cytokine being recombinant human IL-2 or one or more cytokines containing recombinant human IL-2, thereby generating a stimulated composition; and (b) introducing a recombinant receptor into the stimulated composition, thereby generating an engineered composition containing engineered T cells.

[0019] In some embodiments of any of the methods provided herein, the input composition comprises more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ primary human T cells, and / or the input composition consists essentially of CD4+ primary human T cells. In some embodiments of any of the methods provided herein, one or more cytokines further comprise IL-7 and / or IL-15. In some embodiments of any of the methods provided herein, the culture is carried out in the presence of a surfactant. In some embodiments, at least a portion of the culture is carried out using continuous mixing and / or perfusion.

[0020] In another aspect, a method is provided for producing an engineered cell composition comprising culturing an engineered cell composition containing one or both CD4+ and CD8+ primary human T cells, which include recombinant receptor-engineered cells, in the presence of one or more cytokines, wherein the culture is carried out in the presence of a surfactant and / or using continuous mixing and / or perfusion, the method resulting in proliferation or expansion of cells in the composition to produce an output composition containing engineered CD4+ and / or CD8+ T cells.

[0021] In some embodiment of any of these methods described above, the manipulated cell composition comprises more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ and / or CD8+ recombinant receptor-expressing primary T cells, and / or the manipulated cell composition consists essentially of CD4+ and / or CD8+ primary human T cells.

[0022] In some embodiments of any of the methods provided herein, proliferation or expansion results in a doubling or approximately doubling or at least doubling increase in the number of recombinant receptor-engineered CD4+ and / or CD8+ T cells, a tripling or approximately tripling or at least tripling increase, a quadrupling or approximately quadrupling or at least quadrupling increase, a quintupling or approximately quintupling or at least quintupling increase, or more than a quintupling increase.

[0023] In some embodiments of any of the methods provided herein, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15. In some embodiments, the concentration of recombinant IL-2 is 50 IU / mL to 500 IU / mL or about 50 IU / mL to about 500 IU / mL, the concentration of recombinant IL-7 is 500 IU / mL to 2000 IU / mL or about 500 IU / mL to about 2000 IU / mL, and / or the concentration of recombinant IL-15 is 5 IU / mL to 50 IU / mL or about 5 IU / mL to about 50 IU / mL.

[0024] In some embodiment of any of the methods provided herein, the manipulated cell composition comprises more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of CD4+ primary human T cells or CD4+ and recombinant receptor-expressing primary human T cells, and / or the manipulated cell composition consists essentially of CD4+ primary human T cells.

[0025] In some embodiments of any of the methods provided herein, proliferation or expansion results in a doubling or approximately doubling or at least doubling increase in the number of recombinant receptor-engineered CD8+ T cells, a tripling or approximately tripling or at least tripling increase, a quadrupling or approximately quadrupling or at least quadrupling increase, a quintupling or approximately quintupling or at least quintupling increase, or more than a quintupling increase. In some embodiments, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15.

[0026] In some embodiment of any of the methods provided herein, the manipulated cell composition comprises more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of CD8+ primary human T cells or CD8+ and recombinant receptor-expressing primary human T cells, and / or the manipulated cell composition consists essentially of CD8+ primary human T cells.

[0027] In some embodiments of any of the methods provided herein, one or more cytokines are selected from recombinant IL-2 and recombinant IL-15. In some embodiments of any of the methods provided herein, the surfactant comprises a poloxamer, which is optionally present at a concentration of 0.5 μL / mL to 5 μL / mL or about 0.5 μL / mL to about 5 μL / mL. In some embodiments, the poloxamer is poloxamer 188.

[0028] In some aspect of any of the methods provided herein, the engineered cell composition is prepared by a method comprising: (i) incubating an input composition containing primary T cells enriched with one or both CD4+ and CD8+ primary human T cells under stimulating conditions including the presence of a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, and (ii) in the presence of one or more cytokines, thereby producing a stimulated composition; and (b) introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition containing engineered T cells.

[0029] In some embodiments of any of the methods provided herein, the input composition comprises more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ and / or CD8+ primary human T cells, and / or the input composition consists essentially of CD4+ and / or CD8+ primary human T cells. In some embodiments, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15.

[0030] In some embodiments of any of the methods provided herein, the input composition comprises more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98%, and / or the input composition consists essentially of CD4+ primary human T cells. In some embodiments, one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15.

[0031] In some embodiments of any of the methods provided herein, the input composition comprises more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98%, and / or the input composition consists essentially of CD8+ primary human T cells. In some embodiments, one or more cytokines are selected from recombinant IL-2 and recombinant IL-15.

[0032] In some embodiments of any of the methods provided herein, the stimulating agent comprises a primary agent that specifically binds to a member of the TCR complex, and optionally specifically binds to CD3. In some embodiments, the stimulating agent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, and optionally the costimulatory molecule is selected from CD28, CD137(4-1-BB), OX40, or ICOS.

[0033] In some embodiments, the primary and / or secondary agents comprise antibodies, and optionally, the stimulating reagent comprises incubation with anti-CD3 antibodies and anti-CD28 antibodies, or their antigen-binding fragments. In some embodiments, the primary and / or secondary agents are present on the surface of a solid support. In some embodiments, the solid support is or comprises beads. In some embodiments, the beads have a diameter greater than or about 3.5 μm but less than or equal to about 9 μm, or less than or equal to about 8 μm, or less than or equal to about 7 μm, or less than or equal to about 6 μm, or less than or equal to about 5 μm. In some embodiments, the beads comprise a diameter of 4.5 μm or about 4.5 μm. In some embodiments, the beads are inert. In some embodiments, the beads are on or comprise a polystyrene surface. In some embodiments, the beads are magnetic or superparamagnetic. In some embodiments, the bead-to-cell ratio is less than or about 3:1. In some embodiments, the bead-to-cell ratio is 2:1 to 0.5:1 or about 2:1 to about 0.5:1. In some embodiments, the bead-to-cell ratio is 1:1 or approximately 1:1.

[0034] In some embodiments of any of the methods provided herein, incubation is carried out in the presence of one or more antioxidants. In some embodiments, one or more antioxidants include sulfur-containing antioxidants. In some embodiments, one or more antioxidants include glutathione precursors. In some embodiments, one or more antioxidants include N-acetylcysteine ​​(NAC), and optionally, the NAC is at a concentration of 0.2 mg / mL to 2.0 mg / mL or about 0.2 mg / mL to about 2.0 mg / mL.

[0035] In some aspects of any of the methods provided herein, the introduction involves transducing cells of a stimulated composition with a viral vector containing a polynucleotide encoding a recombinant receptor. In some aspects, the viral vector is a retroviral vector. In some aspects, the viral vector is a lentiviral vector or a gamma retroviral vector. In some aspects of any of the methods provided herein, the introduction is carried out in the presence of a transduction adjuvant. In some aspects, the transduction adjuvant is or comprises protamine sulfate, optionally 1 μg / ml to 50 μg / ml or about 1 μg / ml to about 50 μg / ml of protamine sulfate, a transduction adjuvant derived from fibronectin, and / or RetroNectin. In some aspects of any of the methods provided herein, the introduction involves transfecting cells of a stimulated composition with a vector containing a polynucleotide encoding a recombinant receptor. In some aspects, the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a Piggybac transposon.

[0036] In some embodiment of any of the methods provided herein, the manipulated cell composition does not contain a stimulating reagent, and / or the stimulating reagent is substantially removed from the composition before culture, wherein the stimulating reagent comprises a reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules.

[0037] In some aspects of any of the methods provided herein, the culture is carried out until the output composition contains at least a threshold number of T cells. In some aspects, the culture is continued for at least one day after the threshold number of T cells has been reached. In some aspects, the threshold number is at least twice, at least three times, at least four times, at least five times, or more than the number of manipulated cell compositions before culture. In some aspects of any of the methods provided herein, the culture is carried out for 2 to 10 days, including both end values, and / or the culture is carried out for at least 10 days. In some aspects of any of the methods provided herein, after the culture, the cells of the output composition are collected. In some aspects, the time between the start of incubation of the output composition and the collection of cells is 7 to 15 days or about 7 to about 15 days. In some aspects, the time between the start of incubation of the output composition and the collection of cells is 9 to 13 days or about 9 to about 13 days. In some aspects, the time between the start of incubation of the output composition and the collection of cells is 8 to 13 days or about 8 to about 13 days.

[0038] In some embodiments of any of the methods provided herein, the method further comprises formulating cells of the output composition for cryopreservation and / or administration to a subject, in the presence of optionally pharmaceutically acceptable excipients. In some embodiments, the cells of the output composition are formulated in the presence of a cryoprotective agent. In some embodiments, the cryoprotective agent comprises DMSO. In some embodiments, the cells of the output composition are formulated in a container, optionally in a vial or bag.

[0039] In some aspects of any of the methods provided herein, the method further comprises isolating CD4+ and / or CD8+ T cells from a biological sample prior to incubation. In some aspects, isolation comprises selecting cells based on the surface expression of CD4 and / or CD8, optionally by positive selection or negative selection. In some aspects, isolation comprises performing selection based on immunoaffinity. In some aspects, the biological sample comprises primary T cells obtained from a subject. In some aspects, the subject is a human subject. In some aspects, the biological sample is or comprises whole blood sample, buffy coat sample, peripheral blood mononuclear cell (PBMC) sample, unfractionated T cell sample, lymphocyte sample, leukocyte sample, apheresis product, or leukocyte apheresis product.

[0040] In some embodiments of any of the methods provided herein, a recombinant receptor can bind to a target antigen that is specific to, and / or expressed on, a cell or tissue associated with, a disease, disorder, or pathological condition. In some embodiments, the disease, disorder, or pathological condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the target antigens are 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, cyclin, cyclin A2, c-Met, biantigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrin B2, erb-B2, erb-B3, erb-B4, erbB dimer, EGFR vIII, estrogen receptor, fetal AchR, folate receptor α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Ra2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAG) The antigens selected are from among E)-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), fetal neoplastic antigen, melanoma preferential expression antigen (PRAME), PSCA, progesterone receptor, survivorbin, ROR1, TAG72, tEGFR, VEGF receptor, VEGF-R2, Wilms tumor 1 (WT-1), pathogen-specific antigens, and antigens associated with the universal tag.

[0041] In some aspects of any of the methods provided herein, the recombinant receptor is a functional non-TCR antigen receptor or a TCR or its antigen-binding fragment, or comprises the same. In some aspects of any of the methods provided herein, the recombinant receptor is a chimeric antigen receptor (CAR). In some aspects of any of the methods provided herein, the recombinant receptor is an anti-CD19 CAR. In some aspects, the chimeric antigen receptor comprises an extracellular domain including an antigen-binding domain. In some aspects, the antigen-binding domain is an antibody or an antibody fragment, which is optionally a single-chain fragment, or comprises the same. In some aspects, the fragment comprises an antibody variable region linked by a flexible linker. In some aspects, the fragment comprises an scFv.

[0042] In some embodiments of any of the methods provided herein, the chimeric antigen receptor further comprises a spacer and / or hinge region. In some embodiments, the chimeric antigen receptor comprises an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain of a T cell receptor (TCR) component, and / or an immunoreceptor tyrosine activation motif (ITAM). In some embodiments, the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3 zeta (CD3ζ) chain, or a signaling portion thereof.

[0043] In some embodiments of any of the methods provided herein, the chimeric antigen receptor further comprises a transmembrane domain located between an extracellular domain and an intracellular signaling domain. In some embodiments, the intracellular signaling domain further comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain comprises the intracellular signaling domain or signaling moiety of a T cell co-stimulatory molecule. In some embodiments, the co-stimulatory signaling domain comprises the signaling domain or signaling moiety of CD28, 4-1BB, or ICOS. In some embodiments, the co-stimulatory signaling domain is located between the transmembrane domain and the intracellular signaling domain.

[0044] In some embodiments of any of the methods provided herein, an output composition containing a threshold number or greater number of cells is prepared by repeating the method more than 85% or about 85%, more than 90% or about 90%, or more than 95% or about 95%. In some embodiments of any of the methods described herein, the method is carried out for less than 21 days, including 21 days.

[0045] In other aspects, compositions comprising manipulated cells prepared by a method described in any aspect of this specification are provided. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a cryoprotectant, optionally DMSO.

[0046] In another aspect, a product is provided comprising any of the compositions described herein and instructions for administering the output composition to a subject. In some embodiments of the product, the subject has a disease or pathological condition, and optionally, the recombinant receptor specifically recognizes or specifically binds to an antigen that is associated with the disease or pathological condition, or that is expressed on or present on cells of the disease or pathological condition. In some embodiments, the output composition is a composition of engineered CD4+ T cells. In some embodiments, the output composition is an engineered composition of CD8+ T cells.

[0047] In some aspects, a product is provided comprising a composition of engineered CD4+ T cells prepared by any of the methods described herein, a composition of engineered CD8+ T cells prepared by any of the methods described herein, and instructions for administering the engineered CD4+ T cells and engineered CD8+ T cells to a subject. In some aspects, the instructions specify that the CD4+ T cells and CD8+ T cells be administered separately to the subject. In other aspects, the instructions specify that the CD4+ T cells and CD8+ T cells be administered to the subject in a desired ratio.

[0048] In some embodiments of any of the methods described herein, the method is carried out in less than 21 days, including 21 days. [Invention 1001] A method for preparing a composition of manipulated cells, comprising the following steps: (a) (i) a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, and (ii) an input composition comprising CD4+ primary human T cells enriched under stimulating conditions comprising the presence of one or more cytokines, at least one of which is recombinant human IL-2 or comprises recombinant human IL-2, thereby generating a stimulated composition; and (b) A step of introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition comprising engineered T cells. [Invention 1002] The input composition contains more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ primary human T cells, and / or The input composition essentially consists of CD4+ primary human T cells. The method of the present invention 1001. [Invention 1003] The method of Invention 1001 or Invention 1002, wherein the concentration of recombinant IL-2 is 10 IU / mL to 200 IU / mL, or about 10 IU / mL to about 200 IU / mL. [Invention 1004] One or more cytokines further include IL-7 and / or IL-15, Any method according to Invention 1001 to 1003, wherein the concentration of IL-7 is optionally 100 IU / mL to 1000 IU / mL or approximately 100 IU / mL to approximately 1000 IU / mL, and / or the concentration of IL-15 is 1 IU / mL to 50 IU / mL or approximately 1 IU / mL to approximately 50 IU / mL. [Invention 1005] A method according to any one of the present invention 1001 to 1004, wherein the incubation step is carried out in the presence of one or more antioxidants. [Invention 1006] A method for preparing a composition of manipulated cells, comprising the following steps: (a) A step of incubating an input composition containing T cells enriched with one or both CD4+ and CD8+ primary human T cells, thereby producing a stimulated composition, wherein the incubation step is: (1)(i) a stimulating agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, and (ii) one or more stimulating conditions including the presence of one or more cytokines; and / or (2) In the presence of one or more antioxidants It will be done; and (b) A step of introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition comprising engineered T cells. [Invention 1007] The input composition contains more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ and / or CD8+ primary human T cells; and / or The input composition essentially consists of CD4+ and / or CD8+ primary human T cells. The method of the present invention 1006. [Invention 1008] The method of the present invention 1006 or 1007, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. [Invention 1009] The recombinant IL-2 concentration is 10 IU / mL to 200 IU / mL or approximately 10 IU / mL to approximately 200 IU / mL; The recombinant IL-7 concentration is 100 IU / mL to 1000 IU / mL or approximately 100 IU / mL to approximately 1000 IU / mL; and / or The concentration of recombinant IL-15 is 1 IU / mL to 25 IU / mL or approximately 1 IU / mL to approximately 25 IU / mL. The method of the present invention 1008. [Invention 1010] The input composition contains more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 95%, or more than 98% or more than 98% of CD4+ primary human T cells, and / or The input composition essentially consists of CD4+ primary human T cells. The method of the present invention 1006. [Invention 1011] The method of the present invention 1006 or 1010, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15. [Invention 1012] The input composition contains more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 95%, or more than 98% or more than 98% of CD8+ primary human T cells, and / or The input composition essentially consists of CD8+ primary human T cells. The method of the present invention 1006. [Invention 1013] The method of the present invention 1006 or 1012, wherein one or more cytokines are selected from recombinant IL-2 and recombinant IL-15. [Invention 1014] A method according to any one of the present invention 1001 to 1013, wherein the stimulating reagent comprises a primary agent that specifically binds to a member of the TCR complex, and optionally a primary agent that specifically binds to CD3. [Invention 1015] The method of the present invention 1014, wherein the stimulating agent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, the costimulatory molecule optionally selected from CD28, CD137(4-1-BB), OX40, or ICOS. [Invention 1016] The method of Invention 1014 or Invention 1015, wherein the primary and / or secondary agent comprises an antibody, and optionally the stimulating agent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody, or an antigen-binding fragment thereof. [Invention 1017] A method according to any one of the present invention 1014 to 1016, wherein the primary agent and / or secondary agent are present on the surface of a solid support. [Invention 1018] The method of the present invention 1017, wherein the solid support is a bead or includes a bead. [Invention 1019] The method of the present invention 1018, wherein the beads include a diameter greater than 3.5 μm or greater than approximately 3.5 μm but less than or equal to approximately 9 μm, or less than or equal to approximately 8 μm, or less than or equal to approximately 7 μm, or less than or equal to approximately 6 μm, or less than or equal to approximately 5 μm. [Invention 1020] The method of Invention 1018 or Invention 1019, wherein the beads include a diameter of 4.5 μm or about 4.5 μm. [Invention 1021] Any method of the present invention 1018 to 1020, wherein the beads are inert. [Invention 1022] A method according to any of items 1018 to 1021 of the present invention, wherein the beads are polystyrene surfaces or include polystyrene surfaces. [Invention 1023] A method according to any of items 1018 to 1022 of the present invention, wherein the beads are magnetic or superparamagnetic. [Invention 1024] Any method of the present invention 1018 to 1023, wherein the bead-to-cell ratio is less than 3:1 or approximately less than 3:1. [Invention 1025] A method according to any of the present invention 1018 to 1024, wherein the bead-to-cell ratio is 2:1 to 0.5:1, or approximately 2:1 to approximately 0.5:1. [Invention 1026] A method according to any of the present invention 1018 to 1025, wherein the bead-to-cell ratio is 1:1 or approximately 1:1. [Invention 1027] A method according to any one of the present invention 1005 to 1026, wherein one or more antioxidants include a sulfur-containing antioxidant. [Invention 1028] A method according to any one of the present invention 1005 to 1027, wherein one or more antioxidants include a glutathione precursor. [Invention 1029] A method according to any one of the present invention 1005 to 1028, wherein one or more antioxidants comprises N-acetylcysteine ​​(NAC), and optionally, the NAC is at a concentration of 0.2 mg / mL to 2.0 mg / mL, or about 0.2 mg / mL to about 2.0 mg / mL. [Invention 1030] A method according to any one of the present invention 1001 to 1029, wherein the introduction step includes transduction of cells of a stimulated composition with a viral vector containing a polynucleotide encoding a recombinant receptor. [Invention 1031] The method of the present invention 1030, wherein the viral vector is a retroviral vector. [Invention 1032] The method of the present invention 1030 or 1031, wherein the viral vector is a lentiviral vector or a gamma retroviral vector. [Invention 1033] Any method 1030 to 1032 of the present invention, wherein the introduction step is carried out in the presence of a transduction adjuvant. [Invention 1034] The method of the present invention 1033, wherein the transduction adjuvant is protamine sulfate, optionally 1 μg / ml to 50 μg / ml or about 1 μg / ml to about 50 μg / ml of protamine sulfate, a transduction adjuvant derived from fibronectin, and / or RetroNectin, or comprising the same. [Invention 1035] A method according to any one of the present invention 1001 to 1034, wherein the introduction step includes transfecting cells of the stimulated composition with a vector containing a polynucleotide encoding a recombinant receptor. [Invention 1036] The method of the present invention 1035, wherein the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a Piggybac transposon. [Invention 1037] A step of culturing an engineered composition under conditions that promote the proliferation or expansion of engineered cells, thereby producing an output composition containing engineered T cells. Any method of the present invention 1001 to 1036, further comprising the above. [Invention 1038] The method of the present invention 1037, wherein the culturing step is carried out in the presence of one or more cytokines, and at least one cytokine is recombinant human IL-2 or contains recombinant human IL-2. [Invention 1039] The method of Invention 1037 or Invention 1038, wherein the irritant is removed from the manipulated composition before the culturing step. [Invention 1040] The method of the present invention 1039, wherein the irritant is removed within or less than 7 days after the start of the incubation step. [Invention 1041] The method of Invention 1039 or Invention 1040, wherein the irritating reagent is removed 3 to 6 days after the start of the incubation step or approximately 3 to approximately 6 days later. [Invention 1042] A method according to any one of the invention 1037 to 1041, wherein the irritating reagent is removed 4 days or approximately 4 days after the start of the incubation step. [Invention 1043] Any method of the present invention 1039-1042, wherein removing the beads involves exposing the cells of the manipulated composition to a magnetic field. [Invention 1044] A method for preparing a composition of manipulated cells, A step of culturing an engineered cell composition containing CD4+ primary human T cells, including cells engineered with recombinant receptors, in the presence of one or more cytokines, wherein at least one cytokine is recombinant human IL-2 or contains recombinant human IL-2. Includes, A method for producing an output composition containing manipulated CD4+ cells by causing proliferation or expansion of cells in the composition. [Invention 1045] The method of the present invention 1044, wherein proliferation or expansion results in a twofold or approximately twofold or at least twofold increase in the number of recombinant receptor-engineered CD4+ T cells, a threefold or approximately threefold or at least threefold increase, a fourfold or approximately fourfold or at least fourfold increase, a fivefold or approximately fivefold or at least fivefold increase, or more than a fivefold increase. [Invention 1046] The manipulated cell composition contains more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of CD4+ primary human T cells or CD4+ recombinant receptor-expressing cells, and / or The manipulated cell composition consists essentially of CD4+ primary human T cells. The method of the present invention 1044 or 1045. [Invention 1047] A method according to any of the invention 1044 to 1046, wherein the concentration of recombinant IL-2 is 50 IU / mL to 500 IU / mL or approximately 50 IU / mL to approximately 500 IU / mL. [Invention 1048] One or more cytokines further include IL-7 and / or IL-15, The method according to any of Invention 1038 to 1047, optionally wherein the concentration of IL-7 is 500 IU / mL to 2000 IU / mL or approximately 500 IU / mL to approximately 2000 IU / mL, and / or the concentration of IL-15 is 5 IU / mL to 50 IU / mL or approximately 5 IU / mL to approximately 50 IU / mL. [Invention 1049] The manipulated cell composition (a) (i) a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, and (ii) an input composition comprising CD4+ primary human T cells enriched with primary T cells under stimulating conditions comprising the presence of one or more cytokines, at least one of which is recombinant human IL-2 or comprises recombinant human IL-2, thereby producing a stimulated composition; and (b) A step of introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition containing engineered T cells. A method of any of the present invention 1044 to 1048, which is produced by a method including the above. [Invention 1050] The input composition contains more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ primary human T cells, and / or The input composition essentially consists of CD4+ primary human T cells. The method of the present invention 1049. [Invention 1051] A method according to Invention 1049 or Invention 1050, wherein one or more cytokines further comprise IL-7 and / or IL-15. [Invention 1052] A method according to any of the present invention 1037 to 1051, wherein the culturing step is carried out in the presence of a surfactant. [Invention 1053] A method according to any one of the present invention 1037 to 1052, wherein at least part of the culturing step is performed using continuous mixing and / or perfusion. [Invention 1054] A method for preparing a composition of manipulated cells, A step of culturing an engineered cell composition containing one or both CD4+ and CD8+ primary human T cells, including cells engineered with recombinant receptors, in the presence of one or more cytokines, wherein the culturing is carried out in the presence of a surfactant and / or at least a portion of the culturing is carried out using continuous mixing and / or perfusion. Includes, A method for producing an output composition containing manipulated CD4+ and / or CD8+ T cells by causing proliferation or expansion of cells in the composition. [Invention 1055] The manipulated cell composition contains more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ and / or CD8+ primary human T cells or CD4+ and / or CD8+ recombinant receptor-expressing primary T cells, and / or The manipulated cell composition essentially consists of CD4+ and / or CD8+ primary human T cells. The method of the present invention 1054. [Invention 1056] A method of the Invention 1054 or Invention 1055, wherein proliferation or expansion results in a twofold or approximately twofold or at least twofold increase in the number of recombinant receptor-engineered CD4+ and / or CD8+ T cells, a threefold or approximately threefold or at least threefold increase, a fourfold or approximately fourfold or at least fourfold increase, a fivefold or approximately fivefold or at least fivefold increase, or more than a fivefold increase. [Invention 1057] The method of the present invention 1055, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. [Invention 1058] The recombinant IL-2 concentration is 50–500 IU / mL or approximately 50–500 IU / mL; The recombinant IL-7 concentration is 500 IU / mL to 2000 IU / mL or approximately 500 IU / mL to approximately 2000 IU / mL; and / or The concentration of recombinant IL-15 is 5 IU / mL to 50 IU / mL or approximately 5 IU / mL to approximately 50 IU / mL. The method of Invention 1056 or Invention 1057. [Invention 1059] The manipulated cell composition contains more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of CD4+ primary human T cells or CD4+ and recombinant receptor-expressing primary human T cells, and / or The manipulated cell composition consists essentially of CD4+ primary human T cells. The method of the present invention 1054. [Invention 1060] The method of the present invention 1059, wherein proliferation or expansion results in a twofold or approximately twofold or at least twofold increase in the number of recombinant receptor-engineered CD8+ T cells, a threefold or approximately threefold or at least threefold increase, a fourfold or approximately fourfold or at least fourfold increase, a fivefold or approximately fivefold or at least fivefold increase, or more than a fivefold increase. [Invention 1061] The method of the present invention 1054 or 1055, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15. [Invention 1062] The manipulated cell composition contains more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of CD8+ primary human T cells or CD8+ and recombinant receptor-expressing primary human T cells, and / or The manipulated cell composition consists essentially of CD8+ primary human T cells. The method of the present invention 1054. [Invention 1063] The method of the present invention 1054 or 1062, wherein one or more cytokines are selected from recombinant IL-2 and recombinant IL-15. [Invention 1064] A method according to any one of the present invention 1052 to 1063, wherein the surfactant contains poloxamer, and optionally the poloxamer is present at a concentration of 0.5 μL / mL to 5 μL / mL or about 0.5 μL / mL to about 5 μL / mL. [Invention 1065] The method of the present invention 1064, wherein the poloxamer is poloxamer 188. [Invention 1066] The manipulated cell composition (a) a stimulating reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules, and (ii) an input composition comprising primary T cells enriched with one or both CD4+ and CD8+ primary human T cells, wherein the composition is stimulated; and (b) A step of introducing a recombinant receptor into the stimulated composition, thereby producing an engineered composition containing engineered T cells. A method of any of the present invention 1054 to 1065, which is produced by a method including the above. [Invention 1067] The input composition contains more than 70% or about 70%, more than 75% or about 75%, more than 80% or about 80%, more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, or more than 98% or about 98% of CD4+ and / or CD8+ primary human T cells, and / or The input composition essentially consists of CD4+ and / or CD8+ primary human T cells. The method of the present invention 1066. [Invention 1068] The method of the present invention 1066 or 1067, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. [Invention 1069] The input composition contains more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 95%, or more than 98% or more than 98% of CD4+ primary human T cells, and / or The input composition essentially consists of CD4+ primary human T cells. The method of the present invention 1066. [Invention 1070] The method of the present invention 1066 or 1069, wherein one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15. [Invention 1071] The input composition contains more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 95%, or more than 98% or more than 98% of CD8+ primary human T cells, and / or The input composition essentially consists of CD8+ primary human T cells. The method of the present invention 1066. [Invention 1072] The method of the present invention 1066 or 1071, wherein one or more cytokines are selected from recombinant IL-2 and recombinant IL-15. [Invention 1073] A method of any of the present invention 1049-1053 and 1066-1072, wherein the stimulating agent comprises a primary agent that specifically binds to a member of the TCR complex, and optionally a primary agent that specifically binds to CD3. [Invention 1074] The method of the present invention 1073, wherein the stimulating agent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, the costimulatory molecule optionally selected from CD28, CD137(4-1-BB), OX40, or ICOS. [Invention 1075] A method of the present invention 1073 or 1074, wherein the primary and / or secondary agent comprises an antibody, and optionally the stimulating agent comprises incubation with an anti-CD3 antibody and an anti-CD28 antibody, or an antigen-binding fragment thereof. [Invention 1076] A method according to any one of the present invention 1073 to 1075, wherein the primary agent and / or secondary agent are present on the surface of a solid support. [Invention 1077] The method of the present invention 1076, wherein the solid support is a bead or includes a bead. [Invention 1078] The method of the present invention 1077, wherein the beads include a diameter greater than 3.5 μm or greater than approximately 3.5 μm but less than or equal to approximately 9 μm, or less than or equal to approximately 8 μm, or less than or equal to approximately 7 μm, or less than or equal to approximately 6 μm, or less than or equal to approximately 5 μm. [Invention 1079] The method of Invention 1077 or Invention 1078, wherein the beads include a diameter of 4.5 μm or approximately 4.5 μm. [Invention 1080] Any method 1077 to 1079 of the present invention, wherein the beads are inert. [Invention 1081] A method according to any of the present invention 1077 to 1080, wherein the beads are polystyrene surfaces or include polystyrene surfaces. [Invention 1082] A method according to any of the present invention 1077 to 1081, wherein the beads are magnetic or superparamagnetic. [Invention 1083] Any method of the present invention 1077 to 1082, wherein the bead-to-cell ratio is less than 3:1 or approximately less than 3:1. [Invention 1084] A method according to any of the present invention 1077 to 1083, wherein the bead-to-cell ratio is 2:1 to 0.5:1 or approximately 2:1 to approximately 0.5:1. [Invention 1085] Any method of the present invention 1077 to 1084, wherein the bead-to-cell ratio is 1:1 or approximately 1:1. [Invention 1086] The method according to any one of the present invention 1049-1053 and 1066-1085, wherein the incubation step is carried out in the presence of one or more antioxidants. [Invention 1087] The method of the present invention 1086, wherein one or more antioxidants include a sulfur-containing antioxidant. [Invention 1088] A method of the present invention 1086 or 1087, wherein one or more antioxidants comprise a glutathione precursor. [Invention 1089] A method according to any one of the present invention 1086 to 1088, wherein one or more antioxidants comprises N-cetylcysteine ​​(NAC), and optionally, the NAC is at a concentration of 0.2 mg / mL to 2.0 mg / mL or about 0.2 mg / mL to about 2.0 mg / mL. [Invention 1090] A method of the present invention, any one of items 1049-1053 and 1066-1089, wherein the introduction step includes transduction of cells of a stimulated composition with a viral vector containing a polynucleotide encoding a recombinant receptor. [Invention 1091] The method of the present invention 1090, wherein the viral vector is a retroviral vector. [Invention 1092] The method of the present invention 1090 or 1091, wherein the viral vector is a lentiviral vector or a gamma retroviral vector. [Invention 1093] A method of the present invention, any of items 1049-1053 and 1066-1092, wherein the introduction step is carried out in the presence of a transduction adjuvant. [Invention 1094] The method of the present invention 1093, wherein the transduction adjuvant is protamine sulfate, optionally 1 μg / ml to 50 μg / ml or about 1 μg / ml to about 50 μg / ml of protamine sulfate, a transduction adjuvant derived from fibronectin, and / or RetroNectin, or comprising the same. [Invention 1095] A method according to any one of the present invention 1049-1053 and 1066-1089, wherein the introduction step includes transfecting cells of the stimulated composition with a vector containing a polynucleotide encoding a recombinant receptor. [Invention 1096] The method of the present invention 1095, wherein the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a Piggybac transposon. [Invention 1097] Any method of the present invention 1044 to 1069, wherein the manipulated cell composition does not contain a stimulating reagent, and / or the stimulating reagent is substantially removed from the composition before the culturing step, and the stimulating reagent comprises a reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules. [Invention 1098] A method according to any one of the present invention 1037 to 1097, wherein the culturing step is carried out until the output composition contains at least a threshold number of T cells, a threshold number of viable T cells, T cells at a threshold concentration, or viable T cells at a threshold concentration. [Invention 1099] The method of the present invention 1098, wherein the culturing step is continued for at least one day after reaching a threshold number of T cells, a threshold number of viable T cells, a threshold concentration of T cells, or a threshold concentration of viable T cells. [Invention 1100] The method of Invention 1098 or Invention 1099, wherein the threshold number of T cells, the threshold number of viable T cells, the threshold concentration of T cells, or the threshold concentration of viable T cells is at least 2 times, at least 3 times, at least 4 times, at least 5 times or more than the number or concentration of the manipulated cell composition before culture, or the viable number or concentration. [Invention 1101] A method according to any one of the present invention 1037 to 1100, wherein the culturing step is performed for 2 to 10 days, including both end values, and / or the culturing step is performed for at least 10 days. [Invention 1102] Any method of the present invention 1037 to 1100, wherein the culturing step is performed for 2 to 10 days including both end values, and / or the culturing step is performed until at least 9 days after the start of the incubation step. [Invention 1103] A method according to any of the present invention 1037 to 1100, wherein the culturing step is performed for at least 4 days. [Invention 1104] A method according to any of the present invention 1037 to 1101, comprising collecting cells of the output composition after the culture step. [Invention 1105] The method of the present invention 1104, wherein the time between the start of the incubation step of the output composition and the collection of cells is 7 to 15 days or about 7 to about 15 days. [Invention 1106] The method of Invention 1104 or Invention 1105, wherein the time between the start of the incubation step of the output composition and the collection of cells is 9 to 13 days or about 9 to about 13 days. [Invention 1107] Any method 1104 to 1106 of the present invention, wherein the time between the start of the incubation step of the output composition and the collection of cells is 8 to 13 days or about 8 to about 13 days. [Invention 1108] Any method of the present invention 1037 to 1107, further comprising the step of formulating cells of an output composition for cryopreservation and / or administration to a subject in the presence of optionally pharmaceutically acceptable excipients. [Invention 1109] The method of the present invention 1108, wherein the cells of the output composition are formulated in the presence of a cryoprotective agent. [Invention 1110] The method of invention 1109, wherein the cryoprotectant contains DMSO. [Invention 1111] Any method 1108 to 1110 of the present invention, wherein the cells of the output composition are formulated in a container, optionally in a vial or bag. [Invention 1112] Any method of the present invention 1001-1043, 1049-1053, and 1066-1089, further comprising the step of isolating CD4+ and / or CD8+ T cells from a biological sample before the incubation step. [Invention 1113] The method of the present invention 1112, wherein the isolation step optionally includes selecting cells based on the surface expression of CD4 and / or CD8 by positive selection or negative selection. [Invention 1114] A method according to Invention 1112 or Invention 1113, wherein the isolation step includes selection based on immunoaffinity. [Invention 1115] A method according to any of the present invention 1112 to 1114, wherein the biological sample contains primary T cells obtained from the subject. [Invention 1116] The method of the present invention 1115, wherein the subject is a human subject. [Invention 1117] A method according to any one of the present invention 1112 to 1114, wherein the biological sample is a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukocyte apheresis product, or includes such a sample. [Invention 1118] Any method of the present invention 1001 to 1117, wherein the recombinant receptor is associated with, specific to, cells or tissues of a disease, disorder, or pathological condition, and / or can bind to a target antigen expressed on cells or tissues of a disease, disorder, or pathological condition. [Invention 1119] The method of the present invention 1118, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. [Invention 1120] The method of the present invention 1118 or 1119, wherein the target antigen is a tumor antigen. [Invention 1121] The target antigens are 5T4, 8H9, avb6 integrin, B7-H6, B cell maturation antigen (BCMA), CA9, cancer testis antigen, carbonic anhydrase 9 (CAIX), CCL-1, CD19, CD20, CD22, CEA, hepatitis B surface antigen, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD138, CD171, carcinoembryonic antigen (CEA), CE7, cyclin, cyclin A2, c-Met, biantigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, ephrin B2, erb-B2, erb-B3, erb-B4, erbB dimer, and EGFR. vIII, estrogen receptor, fetal AchR, folate receptor α, folate-binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, G250 / CAIX, GD2, GD3, gp100, G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erbB2), HMW-MAA, IL-22R-α, IL-13 receptor α2 (IL-13Rα2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE- Any method according to items 1118 to 1120 of the present invention, selected from A3, MAGE-A6, MART-1, mesothelin, mouse CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), fetal neoplastic antigen, melanoma preferential expression antigen (PRAME), PSCA, progesterone receptor, survivorbin, ROR1, TAG72, tEGFR, VEGF receptor, VEGF-R2, Wilms tumor 1 (WT-1), pathogen-specific antigens, and antigens associated with the universal tag. [Invention 1122] A method according to any one of the present invention 1001 to 1121, wherein the recombinant receptor is a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof, or comprises a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. [Invention 1123] A method according to any of the present invention 1001 to 1122, wherein the recombinant receptor is a chimeric antigen receptor (CAR). [Invention 1124] A method according to any of the present invention 1001 to 1123, wherein the recombinant receptor is an anti-CD19 CAR. [Invention 1125] The method of the present invention 1123, wherein the chimeric antigen receptor includes an extracellular domain containing an antigen-binding domain. [Invention 1126] The method of the present invention 1125, wherein the antigen-binding domain is an antibody or optionally a single-chain fragment thereof, or comprises an antibody or optionally a single-chain fragment thereof. [Invention 1127] The method of the present invention 1126, wherein the fragment comprises an antibody variable region linked by a flexible linker. [Invention 1128] A method of the present invention 1126 or 1127, wherein the fragment comprises scFv. [Invention 1129] Any method of the present invention 1125 to 1128, wherein the chimeric antigen receptor further comprises a spacer and / or hinge region. [Invention 1130] A method according to any one of the present invention 1125 to 1129, wherein the chimeric antigen receptor includes an intracellular signaling region. [Invention 1131] The method of the present invention 1130, wherein the intracellular signaling region includes an intracellular signaling domain. [Invention 1132] The method of the present invention 1131, wherein the intracellular signaling domain is a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain for T cell receptor (TCR) components, and / or a signaling domain comprising an immunoreceptor tyrosine activation motif (ITAM), or comprising the same. [Invention 1133] The method of the present invention 1132, wherein the intracellular signaling domain is the intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof, or includes the same. [Invention 1134] Any method of the present invention 1130 to 1133, wherein the chimeric antigen receptor further comprises a transmembrane domain positioned between an extracellular domain and an intracellular signaling region. [Invention 1135] Any method of the present invention 1130 to 1134, wherein the intracellular signaling region further comprises a co-stimulatory signaling region. [Invention 1136] The method of the present invention 1135, wherein the co-stimulatory signaling region includes the intracellular signaling domain or signaling portion of a T cell co-stimulatory molecule. [Invention 1137] The method of Invention 1135 or Invention 1136, wherein the co-stimulatory signaling region includes an intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. [Invention 1138] A method according to any one of the present invention 1135 to 1137, wherein the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region. [Invention 1139] A method according to any of the 1098-1101 of the present invention, wherein an output composition containing a threshold number or greater number of cells is produced by repeating the method more than 85% or about 85%, more than 90% or about 90%, or more than 95% or about 95%. [Invention 1140] A composition comprising manipulated cells prepared by any of the methods described in invention 1001 to 1137. [Invention 1141] The composition of the present invention 1140, further comprising a pharmaceutically acceptable carrier. [Invention 1142] A composition according to Invention 1140 or Invention 1141, comprising a cryoprotectant and optionally DMSO. [Invention 1143] A product comprising any of the compositions of Invention 1138 to 1140 and instructions for administering the output composition to a target. [Invention 1144] A product of the present invention 1143, wherein the subject has a disease or pathological condition, and optionally, the recombinant receptor specifically recognizes or specifically binds to an antigen that is associated with the disease or pathological condition, or that is expressed or present on cells of the disease or pathological condition. [Invention 1145] A product of the present invention 1143 or 1144, wherein the output composition is a composition of manipulated CD4+ T cells. [Invention 1146] A product of the present invention 1143 or 1144, wherein the output composition is a composition of manipulated CD8+ T cells. [Invention 1147] A product comprising a composition of engineered CD4+ T cells prepared by any of the methods 1001-1011, 1013-1060, 1063-1070, or 1072-1139 of the present invention, a composition of engineered CD8+ T cells prepared by any of the methods 1006-1008, 1011-1043, 1054-1058, 1060-1068, or 1070-1139 of the present invention, and instructions for administering the engineered CD4+ T cells and the engineered CD8+ T cells to a target. [Invention 1148] The product of Invention 1147, wherein the instructions specify that the CD4+ T cells and CD8+ T cells be administered separately. [Invention 1149] A product of the present invention 1147 or 1148, wherein the instructions specify that the CD4+ T cells and CD8+ T cells be administered to the subject in a desired ratio. [Invention 1150] Any method 1001 to 1139 of the present invention, performed in less than 21 days, including the 21st day. [Invention 1151] A method of any of the Invention 1001 to 1139 for producing an output composition that is administered to a subject and / or ready to be administered to a subject within a 95% confidence interval of less than 21 days, including 21 days. [Invention 1152] A method according to any of the Invention 1037-1137, wherein the cells are monitored for cell viability, concentration, density, number, or a combination thereof during at least part of the culturing process. [Invention 1153] The method of the present invention 1152, wherein monitoring is performed by optical methods, optionally by microscopy. [Invention 1154] The method of the present invention 1152 or 1153, wherein monitoring is performed by bright-field microscopy, fluorescence microscopy, differential interference contrast microscopy, phase-contrast microscopy, digital holography (DHM), differential digital holography (DDHM), or a combination thereof. [Invention 1155] Any method according to 1152 to 1154 of the present invention, wherein monitoring is performed by differential digital holography microscopy (DDHM). [Invention 1156] The method according to any one of the invention 1152 to 1155, wherein monitoring is performed intermittently or continuously for at least a portion of the culture, and optionally every hour, every 6 hours, every 12 hours, every 18 hours, every 24 hours, or every 26 hours during the culture. [Invention 1157] Any method 1152 to 1156 of the present invention, wherein monitoring is performed until the cells reach a threshold number of T cells, a threshold number of viable T cells, a threshold concentration of T cells, or a threshold concentration of viable T cells. [Invention 1158] Any method 1152 to 1157 of the present invention, wherein monitoring and culturing are performed in a closed system. [Brief explanation of the drawing]

[0049] [Figure 1]The graph shows the total number of cells in CD4+ (black symbols) and CD8+ (white symbols) cell compositions obtained from the same leukocyte apheresis sample, measured at different time points during cell stimulation, transduction, and expansion in the alternative (triangle) and exemplary (circle) processes for generating anti-CD19 chimeric antigen receptor (CAR) expressing cells described in Example 1. The horizontal dashed line indicates the threshold cell number required to meet the criteria for collection. [Figure 2A] Figure 2A shows the viable cell count (VCC; ×10⁶ cells / mL) and cell viability (%) assessed using sequential monitoring ("sequential," linear) or manual sampling ("manual") with differential DHM for CD4+ cells from donor 1 in Experiment 1. The upper panel shows the respective measurements, and the lower panel shows linear regression analysis, as well as R² and gradient(s) (one or more) to compare sequential monitoring and manual sampling. [Figure 2B] Figure 2B shows the viable cell count (VCC; ×10⁶ cells / mL) and cell viability (%) assessed using sequential monitoring ("sequential," linear) or manual sampling ("manual") with differential DHM for CD4+ cells from donor 2 in Experiment 1. The upper panel shows the respective measurements, and the lower panel shows linear regression analysis, as well as R² and gradient(s) (one or more) to compare sequential monitoring and manual sampling. [Figure 2C] Figure 2C shows the viable cell count (VCC; ×10⁶ cells / mL) and cell viability (%) assessed using sequential monitoring ("sequential," linear) or manual sampling ("manual") with differential DHM for CD4+ cells from donor 3 in Experiment 2. The upper panel shows the respective measurements, and the lower panel shows linear regression analysis, as well as R² and gradient(s) (one or more) to compare sequential monitoring and manual sampling. [Figure 2D]Figure 2D shows the viable cell count (VCC; ×10⁶ cells / mL) and cell viability (%) assessed using sequential monitoring ("sequential," linear) or manual sampling ("manual") with differential DHM for CD8+ cells from donor 3 in Experiment 2. The upper panel shows the respective measurements, and the lower panel shows linear regression analysis, as well as R² and gradient(s) (one or more) to compare sequential monitoring and manual sampling. [Figure 3] This shows the number of viable cells (VCC; ×10⁶ cells / mL) and cell viability (%) as assessed using continuous monitoring by differential DHM in the automated expansion process compared to the manual expansion process. [Modes for carrying out the invention]

[0050] Detailed explanation Methods for generating or producing compositions of engineered cells, such as engineered CD4+ and / or CD8+ T cells, that express recombinant receptors are provided herein. In certain embodiments, the methods are used in connection with methods comprising incubating cells under stimulating conditions, genetically engineering cells by introducing polynucleotides encoding recombinant receptors, for example, and culturing engineered cells under conditions that promote cell proliferation and / or expansion. In some embodiments, the cells are a composition of cells enriched with CD4+ T cells (also referred to herein hereafter as a composition of enriched CD4+ T cells). In some embodiments, the cells are a composition of cells enriched with CD8+ T cells (also referred to herein hereafter as a composition of enriched CD8+ T cells). In some embodiments, the methods are carried out to generate or produce two or separate compositions of engineered T cells, each engineered with the same recombinant receptor from cells of the same biological sample (e.g., from the same subject), by separately incubating, engineering, and culturing the separate compositions of CD4+ T cells and CD8+ T cells.

[0051] Various methods are available for generating genetically engineered T cell populations, including generating engineered T cells that express chimeric antigen receptors. However, in some embodiments, some of these processes may require a long or relatively long time to generate the engineered cells. In some embodiments, some of the existing processes may require different amounts of time to generate engineered T cells from samples obtained from different subjects. For example, in some embodiments, the same process may require 5, 6, 7, or more days to generate engineered cells for one subject than for another. In some specific embodiments, some of the processes may have different abilities to successfully generate therapeutically suitable engineered cells from various subjects. In certain embodiments, the variability and / or lack of predictability of some processes may pose problems to clinicians, such as difficulty in determining whether cell therapy can be generated for a given subject, or difficulty in planning or adjusting the administration of cell therapy when the timing of its availability is unknown.

[0052] The embodiments provided address one or more of these problems. In certain embodiments, the methods provided generate engineered T cells suitable for therapy, such as autologous cell therapy, in a short or relatively short time compared to some existing processes. Furthermore, in some embodiments, the methods provided result in a more consistent and less volatile process with respect to the time required to produce engineered cells from samples collected from various subjects. In certain embodiments, the methods provided can successfully generate engineered T cells suitable for cell therapy from a high percentage of subjects. Thus, in some specific embodiments, the methods described herein provide a relatively rapid and efficient means for generating engineered T cells for therapy. These features may enable more potential subjects to be treated with T cell therapies, such as autologous T cell therapy, and may alleviate some of the difficulties associated with planning and tailoring cell therapies for subjects.

[0053] In some embodiments, the provided process can shorten the expansion period and / or produce the product within a narrower time window compared to other products, across a wider range of starting samples (including those that might otherwise not meet the sampling threshold), thereby reducing failures due to insufficient cell expansion in some aspects.

[0054] In some specific embodiments, the methods provided are used in connection with a process to generate engineered CD4+ T cells expressing recombinant receptors, such as chimeric antigen receptors. In some specific embodiments, the CD4+ T cells are incubated and / or cultured in the presence of recombinant IL-2. Generally, alternative processes for generating engineered CD4+ T cells do not involve or require the addition of recombinant IL-2 with respect to CD4+ T cells, since it is generally understood that cultured CD4+ T cells produce and / or secrete IL-2 in some embodiments. However, without wishing to be bound by theory, some embodiments assume that CD4+ T cells derived from certain patients, such as affected subjects and / or subjects whose T cells include one or more characteristics associated with unhealthy cells, do not produce or secrete sufficient amounts of IL-2. In some specific embodiments, such CD4+ T cells cannot grow, proliferate, and / or expand without supplementation with recombinant IL-2. Therefore, in some specific embodiments, by including recombinant IL-2 in the process for culturing and manipulating CD4+ T cells, the provided method expands the pool of subjects that can provide manipulable CD4+ T cells, and thus expands the pool of subjects that can be treated with autologous cell therapies containing manipulable CD4+ T cells.

[0055] In some specific embodiments, cells are incubated under stimulating conditions with stimulating reagents, such as anti-CD3 and anti-CD28 antibody-conjugated beads, before genetically modifying the cells, e.g., transduction or transfecting them. In some embodiments, the stimulating reagents are isolated or removed from the cells before the start of the culture step and within 7 days or earlier after the start of incubation, e.g., on day 4 or around day 4 or day 5 or around day 5. Certain embodiments intend that if the stimulating reagents are removed or separated from the cells at an earlier point in the process, the cells will have improved viability and will undergo more robust growth and / or expansion during the culture step than cells cultured in alternative processes where the stimulating reagents are not isolated or removed, or are isolated or removed at a later point in the process. In such embodiments, the cultured cells will achieve the target or threshold cell number, density, and / or expansion faster than cells cultured in alternative processes. Therefore, in some embodiments, removing or separating the stimulating reagent from the cells within 7 days or earlier from the start or commencement of incubation allows the process of generating or producing the manipulated cells to be completed in a shorter time than alternative processes.

[0056] In some embodiments, the methods provided herein are used in connection with a process for generating genetically engineered T cells over a short period of time. In some specific embodiments, a short process period may increase the rate, instance, and / or probability of generating a composition of engineered T cells that can be administered to a subject for cell therapy. In some embodiments, a manufacturing protocol for a therapeutic cell composition may require that the cell composition be verified and / or determined to be suitable for administration to a subject, e.g., to be prepared and released for injection within a certain time. In some aspects, a short process period provided can be expected to reduce or eliminate process failures that would occur from cell compositions that cannot be expanded within the required time.

[0057] In certain embodiments, the methods provided herein are used in connection with culturing engineered cells under conditions that promote proliferation and / or expansion. In some embodiments, the culture is carried out in a setting that allows constant mixing and / or perfusion of the cells to be cultured, for example, in or in connection with a bioreactor. In some embodiments, at least a portion of the culture is carried out with constant mixing and a slow, constant perfusion that replaces used medium with fresh medium. In some specific embodiments, the cells are initially cultured under static conditions, for example, without perfusion or mixing, and then cultured with constant mixing and perfusion when the cultured cells reach a predetermined number or density and / or when the cells have been cultured for approximately a predetermined time for the first time. In some such embodiments, the cells to be cultured achieve a target or threshold number, density, and / or expansion faster than cells cultured by alternative processes. Thus, in some embodiments, culture with constant mixing and / or perfusion allows the process of generating or producing engineered cells to be completed in a shorter time than alternative processes in which cells are cultured under static conditions.

[0058] In certain embodiments, the methods provided are used in connection with a process for efficiently producing or generating manipulated cells suitable for use in cell therapy. In some specific embodiments, the timing, conditions, and reagents used in each step of the process improve the efficiency of each subsequent step and / or the process as a whole. For example, in some embodiments, cells may be incubated with reagents, such as stimulating reagents or transduction adjuvants, at a concentration high enough to achieve the desired effect, such as cell stimulation or improved transduction efficiency, but low enough to avoid delayed growth or reduced viability in subsequent processing steps. Furthermore, in some embodiments, the steps of the process are timed to start or end at specific points in time to improve the efficiency of subsequent process steps and / or the process as a whole. For example, in some embodiments, the incubation and manipulation steps (e.g., cell transduction or transfestation) are completed earlier in the process than in alternative methods, which, in some specific embodiments, improves the viability and / or health of cells during subsequent culture steps, as well as the rate of proliferation and expansion. Therefore, in one aspect, the specific timing, conditions, and reagents of each step affect the cells beyond the individual steps and, in some specific embodiments, affect the overall performance of the process.

[0059] In some embodiments, the methods are used in connection with the process of generating or producing genetically engineered cells suitable for cell therapy in a manner that may be faster and more efficient than alternative processes. In some specific embodiments, the methods provided herein have a high success rate for generating or producing compositions of engineered cells from a broader target population than may be possible from alternative processes. In some specific embodiments, engineered cells produced or generated by the methods provided may have higher health, viability, and activation than cells produced by alternative methods, and may have higher recombinant receptor expression. Thus, in some aspects, the speed and efficiency of the methods provided for generating engineered cells for cell therapy makes it easier to plan and adjust cell therapy treatments, such as autotherapy, for a broader target population than may be possible by some alternative methods.

[0060] Unless otherwise defined, all technical terms, notations, and other technical and scientific or specialized terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in which the claimed subject matter pertains. In some cases, terms having a generally understood meaning are defined herein for the sake of clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from the generally understood meaning in the art.

[0061] All publications, including patent documents, scientific articles, and databases, referenced herein are incorporated by reference in their entirety for the same degree as each individual publication is incorporated by reference individually. If any definition provided herein conflicts with or otherwise contradicts any definition provided in a patent, application, published application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

[0062] The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subjects discussed.

[0063] I. Process for generating manipulated cells Methods for producing output compositions of engineered cells, such as engineered CD4+ T cells and / or engineered CD8+ T cells, that express recombinant proteins, such as recombinant receptors such as T cell receptors (TCRs) or chimeric antigen receptors (CARs), are provided herein. In some embodiments, the methods provided herein may be used in connection with the manufacture, generation, or preparation of cell therapies, as well as in connection with additional processing steps such as cell isolation, separation, selection, activation or stimulation, transduction, washing, suspension, dilution, concentration, and / or formulation. In some embodiments, a method for generating or preparing engineered cells, such as engineered CD4+ T cells and / or engineered CD8+ T cells, comprises one or more of the following: isolation of cells from a subject, preparation of cells, processing, incubation under stimulating conditions, and / or manipulation (e.g., transduction). In some embodiments, the method includes processing steps carried out in the following order: first isolating input cells, e.g., primary cells, from a biological sample, e.g., by selection or separation; incubating the input cells under stimulating conditions and manipulating them with vector particles, e.g., viral vector particles, to introduce recombinant polynucleotides into the cells, e.g., by transfection or induction; culturing the manipulated cells, e.g., transfected cells, for purposes such as cell expansion; and collecting, harvesting, and / or filling containers all or part of the cells to formulate the cells into an output composition. In some embodiments, the cells of the resulting output composition are reintroduced into the same subject before or after cryopreservation. In some embodiments, the output composition of the manipulated cells is suitable for use in therapies, e.g., autologous cell therapy.

[0064] In certain embodiments, the methods provided are used in connection with generating an output composition of cells expressing recombinant receptors from an initial or input composition of cells. In some embodiments, the cell composition is a composition of enriched T cells, enriched CD4+ T cells, and / or enriched CD8+ T cells (hereinafter also referred to herein as a composition of enriched T cells, a composition of enriched CD4+ T cells, and a composition of enriched CD8+ T cells, respectively). In some embodiments, the methods provided are used in connection with one or more of the following: activating or stimulating a composition of enriched cells with T cells; genetically engineering a composition of enriched T cells to introduce polynucleotides encoding recombinant proteins, for example by transduction or transfection; and / or culturing an engineered composition of enriched T cells under conditions that promote proliferation and / or expansion, for example. In some specific embodiments, the methods may also be used in connection with isolating or selecting cells from a biological sample, such as a biological sample collected, gathered, and / or obtained from a subject, to generate an input composition of enriched T cells. In certain embodiments, the methods provided may be used in connection with harvesting, collecting, and / or formulating enriched T cell compositions after cells have been incubated, activated, stimulated, manipulated, transfected, and / or cultured.

[0065] In some embodiments, the methods provided are used in connection with the isolation, separation, selection, activation or stimulation, transduction, washing, suspension, dilution, concentration, and / or formulation of a single composition of enriched T cells. In some embodiments, the enriched T cell composition is a cell composition comprising enriched CD4+ T cells. In some specific embodiments, the enriched CD4+ T cell composition comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.9% CD4+ T cells. In certain embodiments, the enriched CD4+ T cell composition comprises 100% CD4+ T cells or approximately 100% CD4+ T cells. In some specific embodiments, the enriched T cell composition contains or includes less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or does not contain CD8+ T cells, and / or is CD8+ T cell-free or substantially CD8+ T cell-free. In some embodiments, the cell population consists essentially of CD4+ T cells.

[0066] In some embodiments, the method provided is used in connection with generating two or more distinct output compositions of enriched T cells. In some embodiments, the method provided is carried out separately for two or more distinct compositions of enriched T cells, e.g., one or more distinct compositions of enriched CD4+ T cells and one or more distinct compositions of enriched CD8+ T cells. In some specific embodiments, the method may be used in connection with separately activating and / or stimulating two or more compositions of enriched T cells; separately manipulating two or more compositions of enriched T cells; and / or separately culturing two or more compositions of enriched T cells. In some specific embodiments, the method may also be used in connection with isolating or selecting different cells from a biological sample to generate distinct input compositions of enriched T cells, such as distinct compositions of enriched CD4+ T cells and enriched CD8+ T cells. In certain embodiments, the method provided may be used in connection with separately harvesting, collecting, and / or formulating distinct compositions of enriched T cells after the T cells have been incubated, activated, stimulated, manipulated, transfected, and / or cultured.

[0067] In some specific embodiments, the method involves separately incubating at least one distinct composition of enriched CD4+ T cells and at least one distinct composition of enriched CD8+ T cells; separately activating and / or stimulating at least one distinct composition of enriched CD4+ T cells and at least one distinct composition of enriched CD8+ T cells after incubation; separately manipulating, transfecting, and / or transfecting at least one distinct composition of enriched CD4+ T cells and at least one distinct composition of enriched CD8+ T cells after activation and / or stimulation; and also This may be used in connection with separately culturing at least one distinct composition of enriched CD4+ T cells and at least one distinct composition of enriched CD8+ T cells after transfection; separately harvesting and / or collecting at least one distinct composition of enriched CD4+ T cells and at least one distinct composition of enriched CD8+ T cells after culturing; separately formulating at least one distinct composition of enriched CD4+ T cells and at least one distinct composition of enriched CD8+ T cells after harvesting and / or collection; and / or separately administering the formulated compositions to subjects requiring them.

[0068] In some embodiments, two or more distinct compositions of enriched T cells include a composition of enriched CD4+ T cells. In some specific embodiments, two or more distinct compositions include CD8+ T cells. In some embodiments, two or more distinct compositions include a composition of enriched CD4+ T cells and a composition of enriched CD8+ T cells. In certain embodiments, the created distinct compositions of enriched CD4+ T cells and distinct compositions of enriched CD8+ T cells were first isolated, selected, and / or enriched from the same biological sample, such as the same biological sample obtained, collected, and / or extracted from a single subject. In some embodiments, the same biological sample is first subjected to the selection of CD4+ T cells, where both negative and positive fractions are retained, and the negative fraction is further subjected to the selection of CD8+ T cells. In other embodiments, the same biological sample is first subjected to the selection of CD8+ T cells, where both negative and positive fractions are retained, and the negative fraction is further subjected to the selection of CD4+ T cells.

[0069] In some embodiments, the enriched T cell composition is a enriched CD8+ T cell composition. In some specific embodiments, the enriched CD8+ T cell composition contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.9% CD8+ T cells, or 100% or about 100% CD8+ T cells. In some specific embodiments, the enriched CD8+ T cell composition contains or includes less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and / or does not contain CD4+ T cells, and / or is CD4+ T cell-free or substantially CD4+ T cell-free. In some embodiments, the cell population consists essentially of CD8+ T cells.

[0070] In some embodiments, enriched CD4+ T cell compositions and / or engineered enriched CD4+ T cell compositions are incubated, activated, stimulated, engineered, transfected, and / or cultured with and / or in the presence of recombinant IL-2. In certain embodiments, the method is used in connection with incubating an enriched CD4+ T cell input composition under stimulating conditions with and / or in the presence of recombinant IL-2. In some embodiments, the method is used in connection with culturing an engineered enriched CD4+ T cell composition under conditions that promote proliferation and / or expansion with and / or in the presence of recombinant IL-2.

[0071] In some embodiments, incubating an enriched T cell input composition under stimulating conditions involves or includes incubating cells with a stimulating agent, e.g., a stimulating agent described in Section IB-1. In some specific embodiments, the stimulating agent is removed or separated from the cells before the culture step. In some specific embodiments, the stimulating agent is removed or separated from the cells after genetic manipulation, e.g., after transfection or transduction. In some embodiments, the stimulating agent is removed from the cells within a set time from the start or commencement of incubation with the stimulating agent, e.g., within 7 days or less from the start or commencement of incubation. In certain embodiments, incubation under stimulating conditions is carried out in the presence of one or more antioxidants, e.g., sulfur-containing antioxidants and / or glutathione precursors.

[0072] In certain embodiments, a composition of enriched T cells, such as a stimulated composition of enriched T cells, is manipulated in the presence of polycations to improve the efficiency of transfection or transduction, for example. In some specific embodiments, the polycations are present in low and / or relatively low amounts and / or concentrations.

[0073] In some specific embodiments, at least part of the culture process is carried out with constant mixing and / or perfusion, for example, using a closed-system bioreactor. In some specific embodiments, the mixing and / or perfusion incorporates a steady and / or stepwise exchange of used or old cell medium or solution with fresh medium or solution. In certain embodiments, cells are cultured in the presence of surfactants and / or agents that reduce or prevent cell shear, such as shear during constant mixing and / or perfusion.

[0074] In some embodiments, the provided method is carried out such that one, more, or all steps in the preparation of cells for clinical use, such as adoptive cell therapy, are performed without exposing the cells to non-sterile conditions. In some embodiments of such a process, the cells are isolated, separated or selected, transduced, washed, optionally activated or stimulated, and formulated, all within a closed system. In some embodiments, one or more steps are carried out separately from the closed system or device. In some such embodiments, the enriched cell composition is transferred separately from the closed system or device under sterile conditions, such as by sterile transfer to another closed system.

[0075] In certain embodiments, enriched T cell compositions may be collected before, during, or after any step or process of the process for producing an enriched T cell output composition expressing recombinant receptors, formulated for cryoprotection, cryo-frozen, and / or stored below 0°C, below -20°C, or below -70°C or below -70°C or below -80°C or below -80°C. In some embodiments, cells may be stored for a period of less than 1 day, less than 2 days, less than 3 days, less than 4 days, less than 5 days, less than 6 days, less than 7 days, less than 8 days, or less than 10 days, or for a period of less than 1 week, less than 2 weeks, less than 3 weeks, less than 4 weeks, less than 5 weeks, less than 6 weeks, less than 7 weeks, or less than 8 weeks, or for a period of at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, or at least 8 weeks, or for a period of more than 8 weeks. After storage, enriched T cell compositions may be thawed and processing may be resumed from the same point in time of the process. In some embodiments, the enriched T cell input composition is cryopreserved and stored before further processing, such as incubation under stimulating conditions. In certain embodiments, the enriched T cell culture and / or formulated composition is cryopreserved and stored before being administered to a subject, for example, as autologous cell therapy.

[0076] In some specific embodiments, separate cell compositions of enriched T cells are combined into a single composition. For example, in some embodiments, a composition of enriched CD4+ T cells is combined with a composition of enriched CD8+ T cells to form a single composition of enriched CD4+ and CD8+ T cells. In some specific embodiments, the created separate compositions are initially isolated, selected and / or enriched from the same biological sample, such as, for example, obtained, collected and / or harvested from a single subject. In some embodiments, the separate compositions are processed separately for one or more steps or stages of a process for producing an output composition, such as a process related to the method provided. In some embodiments, the separate compositions may be combined into a single composition before, during, or after any step or stage of a process for producing an output composition. Thus, in some embodiments, separate input, stimulated, manipulated, cultured, formulated, and / or harvested compositions of enriched T cells from the same biological sample are combined into a single composition, and in some specific embodiments, further processed as a single composition. In some specific embodiments, the separate output compositions of enriched cells are combined into a single output composition before the cells are administered to a subject.

[0077] In some specific embodiments, a portion of the cells may be sampled or collected at any stage or step of the process, for example, while the composition remains in a closed system during isolation, incubation, manipulation, culture, and / or formulation, cells may be taken from the enriched T cell composition. In some specific embodiments, such cells may be analyzed for markers, features, or properties including, but not limited to, viability, apoptosis, activation, stimulation, growth, and / or depletion. In some embodiments, the cells are sampled or collected by an automated process while the enriched T cell composition remains in a closed system. In some embodiments, the analysis of the sampled or collected cells is automated. In certain embodiments, the analysis is performed in a closed system under sterile conditions.

[0078] In some embodiments, cells or cell compositions prepared and / or processed by the provided method may be compared to cells or cell compositions processed or prepared by exemplary and / or alternative processes. In some embodiments, alternative and / or exemplary processes may differ in one or more specific aspects, but otherwise include similar or identical features, aspects, steps, stages, reagents, and / or conditions to the aspects or aspects of the provided method being compared. For example, if the provided method is used in connection with incubating cells in the presence of a reagent, such cells may be compared to cells that are not incubated with the reagent in an exemplary and / or alternative process. In some embodiments, unless otherwise specified, the provided method and exemplary and / or alternative processes are otherwise similar and / or identical, such as using similar or identical steps for isolation, selection, concentration, activation, stimulation, manipulation, transfection, transduction, culture, and / or formulation. In some embodiments, unless otherwise specified, the methods and alternative processes provided isolate, select, and / or concentrate cells from the same or similar types of biological samples, and / or process cells of the same cell type and / or input cells.

[0079] Cells and compositions prepared by methods comprising pharmaceutical compositions and formulations, as well as kits, systems, and devices for carrying out the methods, are also provided. Furthermore, methods for the use of cells and compositions prepared by such methods, including therapeutic methods such as adoptive cell therapy, are also provided, as well as pharmaceutical compositions for administration to subjects.

[0080] A. Sample and cell preparation In certain embodiments, the methods provided are used in connection with isolating, selecting, and / or concentrating cells from a biological sample to produce one or more input compositions of concentrated cells, such as T cells. In some embodiments, the methods provided include isolating cells or compositions thereof from a biological sample, such as those obtained from or derived from a subject having a particular disease or condition, or a subject requiring or being administered cell therapy. In some aspects, the subject is a human, such as a patient requiring a particular therapeutic intervention, such as adoptive cell therapy, in which the cells are isolated, processed, and / or manipulated. Thus, the cells in some embodiments are primary cells, such as primary human cells. Samples include tissues, bodily fluids, and other samples taken directly from a subject. Biological samples may be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, and tissue and organ samples, including processed samples derived from tissue and organ samples.

[0081] In some contexts, the sample is either blood or a blood-derived sample, or apheresis or leukocyte apheresis product or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived therefrom. In the context of cell therapy, such as adoptive cell therapy, the sample may include autologous and allogeneic source-derived samples.

[0082] In some cases, cells derived from the circulating blood of interest are obtained, for example, by apheresis or leukocyte apheresis. The sample, in some aspects, includes lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and / or platelets, and in some aspects, includes cells other than erythrocytes and platelets.

[0083] In some embodiments, blood cells collected from the subject are washed to remove, for example, the plasma fraction and to place the cells into a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution does not contain calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is achieved by a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is achieved by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are treated with, for example, Ca ++ / Mg ++ The cells are resuspended in various biocompatible buffers, such as PBS, which do not contain [specific component]. In some specific embodiments, components of the blood cell sample are removed, and the cells are directly resuspended in culture medium.

[0084] In some embodiments, the preparation method includes a step of freezing the cells, for example, for cryopreservation, before or after isolation, selection and / or concentration and / or incubation for transduction and manipulation, and / or after culture and / or harvesting of the manipulated cells. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution after a washing step to remove, for example, plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. In some embodiments, the cells are suspended in a freezing solution, for example, with a final concentration of 12.5% ​​or about 12.5%, 12.0% or about 12.0%, 11.5% or about 11.5%, 11.0% or about 11.0%, 10.5% or about 10.5%, 10.0% or about 10.0%, 9.5% or about 9.5%, 9.0% or about 9.0%, 8.5% or about 8.5%, 8.0% or about Freeze in a medium and / or solution containing 8.0%, 7.5% or approximately 7.5%, 7.0% or approximately 7.0%, 6.5% or approximately 6.5%, 6.0% or approximately 6.0%, 5.5% or approximately 5.5%, or 5.0% or approximately 5.0% DMSO, or 1%-15%, 6%-12%, 5%-10%, or 6%-8% DMSO, e.g., cryofreeze or cryopreserve. In certain embodiments, cells are frozen, for example, cryopreserved or cryopreserved in a medium and / or solution having a final concentration of 5.0% or about 5.0%, 4.5% or about 4.5%, 4.0% or about 4.0%, 3.5% or about 3.5%, 3.0% or about 3.0%, 2.5% or about 2.5%, 2.0% or about 2.0%, 1.5% or about 1.5%, 1.25% or about 1.25%, 1.0% or about 1.0%, 0.75% or about 0.75%, 0.5% or about 0.5%, or 0.25% or about 0.25% HSA, or 0.1% to -5%, 0.25% to 4%, 0.5% to 2%, or 1% to 2% HSA. One example involves using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HSA).Next, dilute this 1:1 with culture medium to achieve final concentrations of 10% and 4% DMSO and HSA, respectively. Then freeze the cells to -80°C or approximately -80°C at a rate of generally 1° or approximately 1° per minute and store them in the vapor phase of a liquid nitrogen storage tank.

[0085] In some embodiments, the isolation of cells or populations comprises one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents to remove unwanted components, concentrate desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, cells are separated based on one or more properties such as density, adhesion characteristics, size, sensitivity, and / or resistance to a particular component. In some embodiments, the method comprises density-based cell separation methods, such as the preparation of leukocytes from peripheral blood by lysing erythrocytes, and centrifugation by Percoll or Ficoll gradient.

[0086] In some embodiments, at least part of the selection step includes incubation of cells with a selection reagent. For example, incubation with one or more selection reagents as part of a selection method that may be performed using one or more selection reagents to select one or more different cell types based on the intracellular or cellular expression or presence of one or more specific molecules such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method using one or more selection reagents for separation based on such markers may be used. In some embodiments, one or more selection reagents result in separation which is separation based on affinity or immunoaffinity. For example, selection in some aspects includes incubation with one or more reagents for separation of cells and cell populations based on the expression level of one or more markers, typically cell surface markers, e.g., incubation with antibodies or binding partners that specifically bind to such markers, followed generally by a washing step and separation of cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner.

[0087] In some aspects of such a process, a certain volume of cells is mixed with a certain amount of a selection reagent based on a desired affinity. Immunoaffinity-based selection can be carried out using any system or method that results in a favorable energy interaction between the cells to be separated and molecules that specifically bind to markers on the cells, such as antibodies or other binding partners on a solid surface, such as particles. In some embodiments, the method is carried out using particles such as beads, e.g., magnetic beads, coated with a selection agent (e.g., antibody) specific to the cell marker. The particles (e.g., beads) may be incubated or mixed with cells in a container such as a tube or bag while shaking or mixing at a constant cell density to particle (e.g., beads) ratio that helps to promote an energetically favorable interaction. In other cases, the method involves the selection of cells, in whole or in part, being carried out in the internal cavity of a centrifugal chamber, e.g., under centrifugal rotation. In some embodiments, the incubation of cells with a selection reagent, such as an immunoaffinity-based selection reagent, is carried out in a centrifugal chamber. In certain specific embodiments, isolation or separation is carried out using a system, device, or apparatus described in International Publication No. 2009 / 072003 or U.S. Patent Application No. 20110003380A1. For example, the system is the system described in International Publication No. 2016 / 073602.

[0088] In some embodiments, performing such a selection process or part thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in the cavity of a centrifugal chamber allows the user to control certain parameters such as the volume of various solutions, the addition of solutions during processing, and the timing of these additions, which may offer advantages compared to other available methods. For example, the ability to reduce the liquid volume in the cavity during incubation can increase the concentration of particles used for selection (e.g., bead reagents), and thus increase the chemical potential of the solution without affecting the total number of cells in the cavity. This, in turn, can enhance the pair interaction between the cells being processed and the particles used for selection. In some embodiments, as relating to the systems, circuits, and controls described herein, performing the incubation process in the chamber allows the user to perform agitation of the solution at desired times (one or more) during incubation, which can also improve the interaction.

[0089] In some embodiments, at least part of the selection step, which includes incubating cells with a selection reagent, is performed in a centrifuge chamber. In some aspects of such a process, a certain volume of cells is mixed with a selection reagent based on a desired affinity in a much smaller amount than would normally be used when performing a similar selection in a tube or container for the selection of the same number and / or volume of cells according to the manufacturer's instructions. In some embodiments, one or more amounts of one or more selection reagents are used that are 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 50% or less, 60% or less, 70% or less, or 80% or less of the amount of the same selection reagent(s) used for cell selection in a tube or container-based incubation of the same number and / or volume of cells according to the manufacturer's instructions.

[0090] In some embodiments, for cell selection, e.g., selection based on immunoaffinity, cells are incubated in a chamber cavity in a composition that also includes a selection buffer along with a selection reagent, such as an antibody, optionally a scaffold such as a polymer or surface, e.g., beads, e.g., magnetic beads, e.g., magnetic beads conjugated with monoclonal antibodies specific to CD4 and CD8, and molecules that specifically bind to surface markers on cells to be concentrated and / or depleted, but not to other surface markers on cells in the composition. In some embodiments, as described, the selection reagent is added to the cells in the chamber cavity in substantially less amount (e.g., less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80%) compared to the amount of selection reagent typically used or required to achieve approximately the same or similar efficiency for the selection of the same number of cells or the same volume of cells when performing selection in a tube with shaking or rotation. In some embodiments, incubation is performed by adding a selection buffer to the cells and the selection reagent, and the target volume is achieved by incubation of, for example, 10 mL to 200 mL of reagent, for example, at least 10 mL or at least about 10 mL or about 10 mL, at least 20 mL or at least about 20 mL or about 20 mL, at least 30 mL or at least about 30 mL or about 30 mL, at least 40 mL or at least about 40 mL or about 40 mL, at least 50 mL or at least about 50 mL or about 50 mL, at least 60 mL or at least about 60 mL or about 60 mL, at least 70 mL or at least about 70 mL or about 70 mL, at least 80 mL or at least about 80 mL or about 80 mL, at least 90 mL or at least about 90 mL or about 90 mL, at least 100 mL or at least about 100 mL or about 100 mL, at least 150 mL or at least about 150 mL or about 150 mL, or at least 200 mL or at least about 200 mL or about 200 mL.In some embodiments, the selection buffer and selection reagent are pre-mixed before being added to the cells. In some embodiments, the selection buffer and selection reagent are added to the cells separately. In some embodiments, the selection incubation is carried out under periodic, gentle mixing conditions, which helps to promote energetically favorable interactions, thereby allowing for the use of less overall selection reagent while achieving high selection efficiency.

[0091] In some embodiments, the total incubation period with the selected reagent is 5 minutes to 6 hours or about 5 minutes to about 6 hours, for example 30 minutes to 3 hours, for example at least 30 minutes or at least about 30 minutes, at least 60 minutes or at least about 60 minutes, at least 120 minutes or at least about 120 minutes, or at least 180 minutes or at least about 180 minutes.

[0092] In some embodiments, incubation is generally carried out under mixed conditions, for example, at a relatively low force or speed, for example, a speed lower than the speed used to pelletize the cells, for example, 600 rpm to 1700 rpm or about 600 rpm to about 1700 rpm (for example, 600 rpm or about 600 rpm or at least 600 rpm, 1000 rpm or about 1000 rpm or at least 1000 rpm, or 1500 rpm or about 1500 rpm or at least 1500 rpm, or The spinning is performed in the presence of spins at RCF on the sample or wall of a chamber or other container of 1700 rpm or approximately 1700 rpm or at least 1700 rpm, for example, 80g to 100g or approximately 80g to approximately 100g (for example, 80g or approximately 80g or at least 80g, 85g or approximately 85g or at least 85g, 90g or approximately 90g or at least 90g, 95g or approximately 95g or at least 95g, or 100g or approximately 100g or at least 100g). In some embodiments, the spinning is performed using repeated intervals of spinning at such low speeds followed by pauses, for example, spinning and / or pausing for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, for example, spinning for about 1 or 2 seconds followed by pauses for about 5, about 6, about 7, or about 8 seconds.

[0093] In some embodiments, such a process is carried out within a fully enclosed system in which the chambers are integrated. In some embodiments, this process (and in some aspects, one or more additional steps, such as a preceding washing step for washing cell-containing samples, such as apheresis samples) is carried out in an automated manner, using an automated program to complete the washing and binding steps within a single closed system, such that cells, reagents, and other components are drawn into and pushed out of the chambers at appropriate times and centrifugation is performed.

[0094] In some embodiments, after incubation and / or mixing of cells with one and / or more selection reagents, the incubated cells are subjected to separation to select cells based on the presence or absence of a particular one or more reagents. In some embodiments, the separation is performed within the same closed system in which the incubation of cells with the selection reagents was performed. In some embodiments, after incubation with the selection reagents, the incubated cells, including cells bound to the selection reagents, are transferred to a system for separation of cells based on immunoaffinity. In some embodiments, the system for separation based on immunoaffinity is or includes a magnetic separation column.

[0095] Such separation steps may be based on positive selection, in which cells bound to a reagent, such as an antibody or binding partner, are retained for further use, and / or negative selection, in which cells not bound to a reagent, such as an antibody or binding partner, are retained. In some cases, both fractions are retained for further use. In some situations, negative selection may be particularly useful when antibodies that specifically identify cell types are not available in heterogeneous populations, and therefore separation is best performed based on markers expressed by cells other than the desired population.

[0096] In some embodiments, the process steps further include negative and / or positive selection of incubated cells, such as using a system or apparatus capable of performing affinity-based selection. In some embodiments, isolation is performed by enriching a particular cell population by positive selection or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is performed by expressing (marker+) or expressing (marker) at relatively high levels on the positively or negatively selected cells, respectively. 高This is achieved by incubating cells with one or more antibodies or other conjugates that specifically bind to one or more surface markers. The same selection step, for example, a positive or negative selection step, can be performed multiple times. In some specific embodiments, positive or negative selected fractions are subjected to the selection process, such as by repeating the positive or negative selection step. In some embodiments, the selection is repeated two, three, four, five, six, seven, eight, nine, or more times. In some specific embodiments, the same selection is performed up to five times. In some specific embodiments, the same selection step is performed three times.

[0097] Isolation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0098] In some examples, multiple separation steps are performed if the positively or negatively selected fractions from a single step are subjected to further separation steps, such as subsequent positive or negative selection. In some examples, cells expressing multiple markers simultaneously can be depleted in a single separation step, for example, by incubating cells with multiple antibodies or binding partners specific to markers that are targets for negative selection, each of which is a target marker. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types. In some specific embodiments, one or more separation steps are repeated and / or performed more than once. In some embodiments, the positively or negatively selected fractions obtained from a separation step are subjected to the same separation step, for example, by repeating the positive or negative selection step. In some embodiments, a single separation step is repeated and / or performed more than once, for example, to increase the yield of positively selected cells, increase the purity of negatively selected cells, and / or further remove positively selected cells from the negatively selected fraction. In some specific embodiments, one or more separation steps are performed and / or repeated two, three, four, five, six, seven, eight, nine, ten, or more than ten times. In some specific embodiments, one or more selection steps are performed and / or repeated one to ten times, one to five times, or three to five times. In some specific embodiments, one or more selection steps are repeated three times.

[0099] For example, in some aspects, specific subpopulations of T cells, such as cells that are positive for or express at high levels one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibodies or binding partners that specifically bind to such markers. In some embodiments, the antibodies or binding partners can be conjugated, for example, directly or indirectly, to a solid support or matrix for carrying out the selection, e.g., magnetic beads or paramagnetic beads. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 binding magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander, and / or ExpACT® beads).

[0100] In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells, such as CD14, B cells, monocytes, or other leukocytes. In some aspects, CD4+ or CD8+ selection steps are used to isolate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further subdivided into subpopulations by positive or negative selection for markers expressed or expressed to relatively high degrees on one or more naive T cell, memory T cell, and / or effector T cell subpopulations.

[0101] In some embodiments, CD8+ T cells are further enriched or depleted, such as by positive or negative selection based on surface antigens associated with each subpopulation, resulting in naive, central memory, effector memory, and / or central memory stem cells. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, for example, to improve long-term survival, expansion, and / or engraftment after administration, which is particularly robust in some aspects in such subpopulations. See Terakura et al., (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells with CD4+ T cells further enhances efficacy.

[0102] In this embodiment, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted of the CD62L-CD8+ and / or CD62L+CD8+ fractions using anti-CD8 antibodies and anti-CD62L antibodies, for example.

[0103] In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of the CD8+ population enriched with TCM cells is performed by depletion of cells expressing CD4, CD14, and CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting from a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on the expression of CD14 and CD45RA, and positive selection based on CD62L.

[0104] In some aspects, such selection is performed simultaneously, and in others, it is performed sequentially in any order. In some aspects, the same CD4 expression-based selection step used to prepare a CD8+ T cell population or subpopulation is also used to generate a CD4+ T cell population or subpopulation, so that both positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally followed by one or more further positive or negative selection steps. In some embodiments, the selection of the CD4+ T cell population and the selection of the CD8+ T cell population are performed simultaneously. In some embodiments, the selection of the CD4+ T cell population and the CD8+ T cell population are performed sequentially in any order. In some embodiments, the method for selecting cells may include that described in U.S. Patent Application Publication No. 20170037369. In some embodiments, after selection, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined. In some aspects, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined in a bioreactor bag as described herein. In some embodiments, selected CD4+ T cell populations and selected CD8+ T cell populations are processed separately, thereby the selected CD4+ T cell population is enriched, incubated with a stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR), and cultured under conditions for T cell expansion, and the selected CD8+ T cell population is enriched, incubated with a stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads), transduced with a viral vector encoding a recombinant protein (e.g., CAR), such as the same recombinant protein used in the manipulation of CD4+ T cells from the same donor, and cultured under conditions for T cell expansion, such as according to the provided method.

[0105] In certain embodiments, a biological sample, such as PBMCs or other leukocyte samples, is subjected to the selection of CD4+ T cells that retain both negative and positive fractions. In some specific embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to the selection of CD8+ T cells that retain both negative and positive fractions. In some specific embodiments, CD4+ T cells are selected from the negative fraction.

[0106] In certain cases, PBMC samples or other leukocyte samples are subjected to selection of CD4+ T cells that retain both negative and positive fractions. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or CD19, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, where positive and negative selection are performed in either order.

[0107] CD4+ T helper cells can be classified into naive cells, central memory cells, and effector cells by identifying cell populations possessing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, or CD4+ T cells. In some embodiments, central memory CD4+ T cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ T cells are CD62L- and CD45RO-.

[0108] In one example, to enrich CD4+ T cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as magnetic or paramagnetic beads, which allows for the separation of cells for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, pp. 17-25 Edited by: SABrooks and U.Schumacher (copyright) Humana Press Inc., Totowa, NJ).

[0109] In some cases, incubated samples or compositions of cells to be separated are incubated with a selection reagent containing small magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynalbeads or MACS® beads). The magnetically responsive material, such as particles, is generally bound directly or indirectly to one or more cells that are to be separated, for example, negative or positive selection, or to molecules present on a cell population, such as surface markers, to binding partners, such as antibodies, that specifically bind to these molecules.

[0110] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials for use in magnetic separation methods are known, such as those described in Molday's U.S. Patent No. 4,452,773 and European Patent No. 452342B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in Owen's U.S. Patent No. 4,795,698 and Liberti et al.'s U.S. Patent No. 5,200,084, may also be used.

[0111] Incubation is generally carried out under conditions in which an antibody or binding partner, or a molecule attached to magnetic particles or beads that specifically binds to such antibody or binding partner, such as a secondary antibody or other reagent, specifically binds to cell surface molecules if cells are present in the sample.

[0112] In some specific embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In some specific embodiments, magnetic particles are attached to cells via a coating of a primary antibody specific to one or more markers. In some specific embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell-type specific secondary antibody or other binding partner (e.g., streptavidin) are added. In some specific embodiments, streptavidin-coated magnetic particles are used in combination with a biotinylated primary or secondary antibody.

[0113] In some cases, separation is achieved by placing the sample in a magnetic field, attracting cells with magnetically responsive or magnetizable particles to the magnet, and separating them from unlabeled cells. In the case of positive selection, cells attracted to the magnet are retained, and in the case of negative selection, cells that are not attracted (unlabeled cells) are retained. In some cases, a combination of positive and negative selection occurs during the same selection process, in which case the positive and negative fractions are retained and subjected to further processing or further separation steps.

[0114] In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetically activated cell sorting (MACS), such as the CliniMACS system, allows for the selection of highly pure cells to which magnetized particles are attached. In some specific embodiments, MACS operates in a mode in which non-target and target species are successively eluted after the application of an external magnetic field. That is, cells attached to magnetized particles are retained in place while non-attached species are eluted. Then, after this initial elution step is complete, species that were trapped by the magnetic field and whose elution was prevented are released in some way so that they can be eluted and recovered. In some specific embodiments, non-target cells are labeled and removed from the heterogeneous population of cells.

[0115] In some embodiments, the magnetically responsive particles remain attached to cells that will subsequently be incubated, cultured, and / or manipulated, and in some aspects, the particles remain attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, antibodies conjugated to magnetizable particles or cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0116] In some embodiments, isolation and / or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more distinct input compositions are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, separate input compositions are isolated, selected, enriched, and / or obtained from separate biological samples collected, harvested, and / or obtained from the same subject.

[0117] In some specific embodiments, one or more input compositions are enriched T cell compositions comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD3+ T cells, or comprising the same. In some specific embodiments, the enriched T cell input composition consists essentially of CD3+ T cells.

[0118] In some specific embodiments, one or more input compositions are enriched CD4+ T cell compositions containing at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In some specific embodiments, the CD4+ T cell input composition contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells and / or does not contain CD8+ T cells and / or is CD8+ T cell-free or substantially CD8+ T cell-free. In some embodiments, the enriched T cell composition consists essentially of CD4+ T cells.

[0119] In some specific embodiments, one or more compositions are CD8+ T cell compositions or compositions comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells. In some specific embodiments, the CD8+ T cell compositions contain less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells and / or do not contain CD4+ T cells and / or are CD4+ T cell-free or substantially CD4+ T cell-free. In some embodiments, the enriched T cell composition consists essentially of CD8+ T cells.

[0120] In some embodiments, one or more input compositions of enriched T cells are frozen, e.g., cryopreserved and / or cryo-freezed, after isolation, selection, and / or enrichment. In some embodiments, one or more input compositions are frozen, e.g., cryopreserved and / or cryo-freezed, before any of the steps of incubation, activation, stimulation, manipulation, transduction, transfection, culture, expansion, harvesting, and / or formulation of the cell composition. In certain embodiments, one or more cryo-frozen input compositions are stored, e.g., at -80°C or about -80°C for 12 to 7 hours, 24 to 120 hours, or 2 to 5 days. In certain embodiments, one or more cryo-frozen input compositions are stored at -80°C or about -80°C for a period of less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, or less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments, one or more cryogenically frozen input compositions are stored at -80°C or about -80°C for 1 day or about 1 day, 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, or 6 days or about 6 days.

[0121] B. Cell activation and stimulation In some embodiments, the methods provided are used in connection with incubating cells under stimulating conditions. In some embodiments, the stimulating conditions include conditions that activate or stimulate, and / or can activate or stimulate, signals in cells, e.g., CD4+ T cells or CD8+ T cells, such as signals produced from TCRs and / or co-receptors. In some embodiments, the stimulating conditions include one or more steps of culturing, cultivating, incubating, activating, and growing cells with and / or in the presence of a stimulating reagent, e.g., a reagent that activates or stimulates, and / or can activate or stimulate, signals in cells. In some embodiments, the stimulating reagent stimulates and / or activates TCRs and / or co-receptors. In certain embodiments, the stimulating reagent is a reagent described in Section IB-1.

[0122] In certain embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions before genetically engineering the cells, for example, before transfecting and / or transducing the cells by the techniques provided in Section IC. In certain embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions after one or more compositions have been isolated, selected, enriched, or obtained from a biological sample. In certain embodiments, one or more compositions are input compositions. In certain embodiments, one or more input compositions are pre-cryogenically frozen and stored, and thawed before incubation.

[0123] In some specific embodiments, one or more compositions of enriched T cells are or comprise two distinct compositions of enriched T cells, e.g., distinct input compositions. In some specific embodiments, two distinct compositions of enriched T cells, e.g., two distinct compositions of enriched T cells selected, isolated, and / or enriched from the same biological sample, are incubated separately under stimulating conditions. In some specific embodiments, the two distinct compositions comprise a composition of enriched CD4+ T cells. In some specific embodiments, the two distinct compositions comprise a composition of enriched CD8+ T cells. In some embodiments, the two distinct compositions of enriched CD4+ T cells and enriched CD8+ T cells are incubated separately under stimulating conditions.

[0124] In some embodiments, a single composition of enriched T cells is incubated under stimulating conditions. In some specific embodiments, the single composition is a composition of enriched CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells combined from separate compositions prior to incubation.

[0125] In some embodiments, a composition of enriched CD4+ T cells incubated under stimulating conditions contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In some specific embodiments, a composition of enriched CD4+ T cells incubated under stimulating conditions contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells and / or does not contain CD8+ T cells and / or is CD8+ T cell-free or substantially CD8+ T cell-free.

[0126] In some embodiments, a composition of enriched CD8+ T cells incubated under stimulating conditions contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells. In some specific embodiments, a composition of enriched CD8+ T cells incubated under stimulating conditions contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells and / or does not contain CD4+ T cells and / or is CD4+ T cell-free or substantially CD4+ T cell-free.

[0127] In some embodiments, separate compositions of enriched CD4+ and CD8+ T cells are combined into a single composition and incubated under stimulating conditions. In some specific embodiments, separate stimulated compositions of enriched CD4+ and enriched CD8+ T cells are combined into a single composition after incubation has been performed and / or completed. In some embodiments, separate stimulated compositions of stimulated CD4+ and stimulated CD8+ T cells are processed separately after incubation has been performed and / or completed, thereby stimulating the CD4+ T cell population (e.g., incubated with a stimulating anti-CD3 / anti-CD28 magnetic bead stimulating reagent) into a viral vector encoding a recombinant protein (e.g., CAR) and culturing under conditions to expand the T cells, and stimulating the CD8+ T cell population (e.g., incubated with a stimulating anti-CD3 / anti-CD28 magnetic bead stimulating reagent) into a viral vector encoding a recombinant protein (e.g., CAR), such as the same recombinant protein used in the manipulation of CD4+ T cells from the same donor, and culturing under conditions to expand the T cells, such as according to the provided method.

[0128] In some embodiments, incubation under stimulating conditions may include culture, cultivation, stimulation, activation, and proliferation, including incubation in the presence of stimulating conditions designed to induce cell proliferation, expansion, activation, and / or survival in a population, mimic antigen exposure, and / or prime cells for genetic manipulation such as the introduction of recombinant antigen receptors. In certain embodiments, stimulating conditions may include one or more of specific media, temperature, oxygen content, carbon dioxide content, time, active agents such as nutrients, amino acids, antibiotics, ions, and / or stimulating factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0129] In some cases, stimulation and / or incubation under stimulating conditions are carried out according to techniques such as those described in Riddell et al. U.S. Patent No. 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0130] In some embodiments, cells, such as T cells, a composition of cells, and / or CD4 + and CD8 +Cell populations, such as T cells or their composition, population, or subpopulation, are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture initiation composition (for example, so that the resulting cell population contains at least about 5, at least about 10, at least about 20, or at least about 40 or more PBMC feeder cells per T lymphocyte in the initial population being expanded), and incubating the culture (for example, for a time sufficient to expand the number of T cells). In some aspects, non-dividing feeder cells may include gamma-irradiated PBMC feeder cells. In some aspects, PBMCs are irradiated with gamma rays in the range of about 3000–3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium before the addition of the T cell population.

[0131] In some embodiments, the stimulation conditions include a temperature suitable for the proliferation of human T lymphocytes, e.g., at least about 25°C, generally at least about 30°C, generally 37°C or about 37°C. In some embodiments, a temperature shift, e.g., a temperature shift from 37°C to 35°C, is performed during culture. Optionally, incubation may further include the addition of non-dividing EBV-transformed lymphoblast-like cells (LCLs) as feeder cells. The LCLs can be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some aspects, the LCL feeder cells are provided in any suitable amount, such as an LCL feeder cell to initial T lymphocyte ratio of at least about 10:1.

[0132] In this embodiment, the antigen-specific CD4 + and CD8 + Populations can be obtained by stimulating naive or antigen-specific T lymphocytes with antigens. For example, antigen-specific T cell lines or clones against cytomegalovirus antigens can be generated by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen. Naive T cells can also be used.

[0133] In certain embodiments, stimulating conditions include incubating, culturing, and / or cultivating cells with a stimulating reagent. In certain embodiments, the stimulating reagent is the reagent described in Section IB-1. In some specific embodiments, the stimulating reagent includes or contains beads. An exemplary stimulating reagent is an anti-CD3 / anti-CD28 magnetic bead or includes an anti-CD3 / anti-CD28 magnetic bead. In some specific embodiments, the start and / or commencement of incubation, culturing, and / or cultivating of cells under stimulating conditions occurs when the cells come into contact with the stimulating reagent and / or are incubated with the stimulating reagent. In certain embodiments, cells are incubated before, during, and / or after genetically engineering cells, such as introducing recombinant polynucleotides into cells by transduction or transfection.

[0134] In some embodiments, the enriched T cell composition is incubated with stimulating reagents and / or beads, such as anti-CD3 / anti-CD28 magnetic beads against cells in a ratio of 3:1 or approximately 3:1, 2.5:1 or approximately 2.5:1, 2:1 or approximately 2:1, 1.5:1 or approximately 1.5:1, 1.25:1 or approximately 1.25:1, 1.2:1 or approximately 1.2:1, 1.1:1 or approximately 1.1:1, 1:1 or approximately 1:1, 0.9:1 or approximately 0.9:1, 0.8:1 or approximately 0.8:1, 0.75:1 or approximately 0.75:1, 0.67:1 or approximately 0.67:1, 0.5:1 or approximately 0.5:1, 0.3:1 or approximately 0.3:1, or 0.2:1 or approximately 0.2:1. In certain embodiments, the stimulating reagent and / or bead-to-cell ratio is 2.5:1 to 0.2:1, 2:1 to 0.5:1, 1.5:1 to 0.75:1, 1.25:1 to 0.8:1, or 1.1:1 to 0.9:1. In certain embodiments, the stimulating reagent-to-cell ratio is approximately 1:1 or 1:1.

[0135] In certain embodiments, incubating cells with a stimulating agent at a ratio of less than 3:1 or less than 3 per cell, such as anti-CD3 / anti-CD28 magnetic beads, at a ratio of, for example, 1:1, reduces the amount of cell death that occurs during incubation, such as activation-induced cell death. In some embodiments, cells are incubated with a stimulating agent, such as anti-CD3 / anti-CD28 magnetic beads, at a bead-to-cell ratio of less than 3 (or 3:1 or less than 3 beads per cell). In certain embodiments, incubating cells at a ratio of less than 3:1 or less than 3 per cell, such as 1:1, reduces the amount of cell death that occurs during incubation, such as activation-induced cell death.

[0136] In a particular embodiment, a composition of enriched T cells is incubated with a stimulating agent, such as anti-CD3 / anti-CD28 magnetic beads, at a ratio of less than 3:1 of stimulating agent and / or beads per cell, such as 1:1, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the T cells survive for 1 day or at least 1 day, 2 days or at least 2 days, 3 days or at least 3 days, 4 days or at least 4 days, 5 days or at least 5 days, 6 days or at least 6 days, 7 days or at least 7 days, or more than 7 days after the incubation is complete, e.g., viable and / or not undergoing necrosis, programmed cell death, or apoptosis. In a particular embodiment, a composition of enriched T cells is incubated with a stimulating agent and / or beads in a ratio of less than 3:1 per cell, e.g., 1:1, and less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of cells undergo activation-induced cell death during incubation.

[0137] In some particular embodiments, the composition of enriched T cells is incubated with a stimulating reagent, such as anti-CD3 / anti-CD28 magnetic beads, at a ratio of beads less than 3:1 per cell, such as a ratio of 1:1, and the cells of the composition have a survival rate that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater compared to cells that undergo an exemplary and / or alternative process where the composition of enriched T cells is incubated with the stimulating reagent at a ratio of 3:1 or greater.

[0138] In some embodiments, the composition of enriched T cells incubated with a stimulating reagent is at 1.0×10 5 cells / mL to 1.0×10 8 cells / mL or about 1.0×10 5 cells / mL to about 1.0×10 8 cells / mL, such as at least 1.0×10 5 cells / mL or at least about 1.0×10 5 cells / mL or about 1.0×10 5 cells / mL, at least 5×10 5 cells / mL or at least about 5×10 5 cells / mL or about 5×10 5 cells / mL, at least 1×10 6 cells / mL or at least about 1×10 6 cells / mL or about 1×10 6 cells / mL, at least 5×10 6 cells / mL or at least about 5×10 6 cells / mL or about 5×10 6 cells / mL, at least 1×10 7 cells / mL or at least about 1×10 7 cells / mL or about 1×10 7Cells / mL, at least 5 × 10⁶ 7 cells / mL or at least approximately 5 × 10⁶ 7 cells / mL or approximately 5 × 10⁴ 7 cells / mL, or at least 1 × 10⁶ 8 cells / mL or at least about 1 × 10⁶ 8 cells / mL or approximately 1 × 10⁶ 8 Contains cells / mL. In some embodiments, the enriched T cell composition incubated with the stimulating reagent contains approximately 0.5 × 10⁶ cells. 6 cells / mL, approximately 1×10 6 cells / mL, approximately 1.5×10 6 cells / mL, approximately 2×10 6 cells / mL, approximately 2.5×10 6 cells / mL, approximately 3×10 6 cells / mL, approximately 3.5×10 6 cells / mL, approximately 4×10 6 cells / mL, approximately 4.5×10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 5.5×10 6 cells / mL, approximately 6×10 6 cells / mL, approximately 6.5×10 6 cells / mL, approximately 7×10 6 cells / mL, approximately 7.5×10 6 cells / mL, approximately 8×10 6 cells / mL, approximately 8.5×10 6 cells / mL, approximately 9×10 6 cells / mL, approximately 9.5×10 6 cells / mL, or approximately 10 × 10 6 Cells / mL, e.g., approximately 2.4 × 10⁶ 6 Contains cells / mL.

[0139] In some embodiments, the enriched T cell composition is incubated with a stimulating reagent at a temperature of approximately 25 to approximately 38°C, for example, approximately 30 to approximately 37°C, for example, 37°C ± 2°C or approximately 37°C ± 2°C. In some embodiments, the enriched T cell composition is incubated with a stimulating reagent at a CO2 level of approximately 2.5% to approximately 7.5%, for example, approximately 4% to approximately 6%, for example, 5% ± 0.5% or approximately 5% ± 0.5%. In some embodiments, the enriched T cell composition is incubated with a stimulating reagent at a temperature of 37°C or approximately 37°C and / or at a CO2 level of 5% or approximately 5%.

[0140] In certain embodiments, the stimulation conditions include incubating, culuring, and / or cultivating a composition of enriched T cells with and / or in the presence of one or more cytokines. In certain embodiments, one or more cytokines are recombinant cytokines. In some embodiments, one or more cytokines are human recombinant cytokines. In some specific embodiments, one or more cytokines bind to and / or can bind to receptors expressed by and / or endogenous to T cells. In certain embodiments, one or more cytokines are members of the 4-α-helix bundle family of cytokines, or include them. In some embodiments, members of the 4-α-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin-12 (IL-12), interleukin-15 (IL-15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, one or more cytokines are IL-15 or comprise IL-15. In certain embodiments, one or more cytokines are IL-7 or comprise IL-17. In certain embodiments, one or more cytokines are IL-2 or comprise IL-2. In some embodiments, the stimulation conditions include incubating a composition of enriched T cells, such as enriched CD4+ T cells or enriched CD8+ T cells, in the presence of a stimulation reagent, e.g., anti-CD3 / anti-CD28 magnetic beads, and in the presence of one or more recombinant cytokines, as described.

[0141] In certain embodiments, enriched CD4+ T cell compositions are incubated with IL-2, for example, recombinant IL-2. While not wishing to be bound by theory, certain embodiments intend that CD4+ T cells obtained from certain subjects will not produce, or will not produce, in sufficient quantities of IL-2 to enable growth, division, and expansion throughout the entire process of generating output cells, e.g., a composition of engineered cells suitable for use in cell therapy. In some embodiments, incubating enriched CD4+ T cell compositions under stimulating conditions in the presence of recombinant IL-2 increases the probability or likelihood that the CD4+ T cells of the composition will remain viable, grow, expand, and / or remain active during the incubation step and throughout the entire process. In some embodiments, incubating a composition of enriched CD4+ T cells in the presence of recombinant IL-2 reduces the probability and / or possibility that an output composition of enriched CD4+ T cells, e.g., engineered CD4+ T cells suitable for cell therapy, will be produced from the enriched CD4+ T cell composition by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, compared to alternative and / or exemplary methods that do not involve incubating the composition of enriched CD4+ T cells in the presence of recombinant IL-2, by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, and less. Increase by at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 25x, at least 50x, or at least 100x.

[0142] In certain specific embodiments, the amount or concentration of one or more cytokines is measured and / or quantified in International Units (IU). International Units can be used to quantify vitamins, hormones, cytokines, vaccines, blood products, and similar bioactive substances. In some embodiments, IU is or includes the unit of measurement for the potency of a biological product by comparison with an international reference standard of a specific weight and strength, e.g., the WHO 1st International Standard for Human IL-2, 86 / 504. International Units are the only recognized and standardized method for reporting bioactive units published and derived from the results of international collaborative research. In certain embodiments, the IU of a cytokine composition, sample, or source may be obtained by product comparison testing using similar WHO standards. For example, in some embodiments, the IU / mg of a composition, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared to WHO standard IL-2 product (NIBSC code: 86 / 500), WHO standard IL-17 product (NIBSC code: 90 / 530), and WHO standard IL-15 product (NIBSC code: 95 / 554), respectively.

[0143] In some aspects, the biological activity at IU / mg is (ED in ng / ml units). 50 ) -1 ×10 6 Equivalent to. In certain embodiments, recombinant human IL-2 or IL-15 ED 50 This is equal to the concentration required for semi-maximal stimulation of cell proliferation (XTT cleavage) by CTLL-2 cells. In some specific embodiments, recombinant human IL-7 ED 50This is equal to the concentration required for semi-maximal stimulation for proliferation of PHA-activated human peripheral blood lymphocytes. Details regarding the assay and calculation of IU for IL-2 are discussed in Wadhwa et al., Journal of Immunological Methods (2013), 379(1-2):1-7; and Gearing and Thorpe, Journal of Immunological Methods (1988), 114(1-2):3-9, which are incorporated herein by reference in their entirety. Details regarding the assay and calculation of IU for IL-15 are discussed in Soman et al., Journal of Immunological Methods (2009), 348(1-2):83-94, which are incorporated herein by reference in their entirety.

[0144] In certain embodiments, the enriched CD8+ T cell composition is incubated under stimulating conditions in the presence of IL-2 and / or IL-15. In some specific embodiments, the enriched CD4+ T cell composition is incubated under stimulating conditions in the presence of IL-2, IL-7, and / or IL-15. In some embodiments, IL-2, IL-7, and / or IL-15 are recombinant. In some specific embodiments, IL-2, IL-7, and / or IL-15 are human. In certain embodiments, one or more cytokines are or comprise human recombinant IL-2, IL-7, and / or IL-15. In some aspects, incubation of the enriched T cell composition also comprises the presence of stimulating reagents, such as anti-CD3 / anti-CD28 magnetic beads.

[0145] In some ways, cells are incubated with cytokines, such as recombinant human cytokines, at concentrations of 1 IU / ml to 1,000 IU / ml, 10 IU / ml to 50 IU / ml, 50 IU / ml to 100 IU / ml, 100 IU / ml to 200 IU / ml, 100 IU / ml to 500 IU / ml, 250 IU / ml to 500 IU / ml, or 500 IU / ml to 1,000 IU / ml.

[0146] In some embodiments, the enriched T cell composition is incubated with IL-2 at concentrations of 1 IU / ml to 200 IU / ml, 10 IU / ml to 200 IU / ml, 10 IU / ml to 100 IU / ml, 50 IU / ml to 150 IU / ml, 80 IU / ml to 120 IU / ml, 60 IU / ml to 90 IU / ml, or 70 IU / ml to 90 IU / ml, such as human recombinant IL-2. In a particular embodiment, the enriched T cell composition may be 50 IU / ml or approximately 50 IU / ml, 55 IU / ml or approximately 55 IU / ml, 60 IU / ml or approximately 60 IU / ml, 65 IU / ml or approximately 65 IU / ml, 70 IU / ml or approximately 70 IU / ml, 75 IU / ml or approximately 75 IU / ml, 80 IU / ml or approximately 80 IU / ml, 85 IU / ml or approximately 85 IU / ml, 90 IU / ml, or The T cells are incubated with recombinant IL-2 at concentrations of approximately 90 IU / ml, 95 IU / ml, 100 IU / ml, 110 IU / ml, 120 IU / ml, 130 IU / ml, 140 IU / ml, or 150 IU / ml. In some embodiments, the enriched T cell composition is incubated in the presence of recombinant IL-2 at 85 IU / ml or approximately 85 IU / ml. In some embodiments, the composition incubated with recombinant IL-2 is enriched with a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the population of T cells is a population of CD4+ T cells. In some embodiments, the enriched T cell composition is a composition of enriched CD8+ T cells. In certain embodiments, the enriched T cell composition is enriched with CD8+ T cells, but not with CD4+ T cells, and / or CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched T cell composition is a composition of enriched CD4+ T cells.In certain embodiments, the enriched T cell composition is enriched with CD4+ T cells, but not with CD8+ T cells, and / or CD8+ T cells are negatively selected from the composition or depleted from the composition. In some embodiments, the enriched CD4+ T cell composition incubated with recombinant IL-2 may also be incubated with recombinant IL-7 and / or recombinant IL-15, such as in the amounts described. In some embodiments, the enriched CD8+ T cell composition incubated with recombinant IL-2 may also be incubated with recombinant IL-15, such as in the amounts described.

[0147] In some embodiments, the enriched T cell composition is incubated with recombinant IL-7, such as human recombinant IL-7, at concentrations of 100 IU / ml to 2,000 IU / ml, 500 IU / ml to 1,000 IU / ml, 100 IU / ml to 500 IU / ml, 500 IU / ml to 750 IU / ml, 750 IU / ml to 1,000 IU / ml, or 550 IU / ml to 650 IU / ml. In a particular embodiment, the enriched T cell composition is 50 IU / ml or approximately 50 IU / ml, 100 IU / ml or approximately 100 IU / ml, 150 IU / ml or approximately 150 IU / ml, 200 IU / ml or approximately 200 IU / ml, 250 IU / ml or approximately 250 IU / ml, 300 IU / ml or approximately 300 IU / ml, 350 IU / ml or approximately 350 IU / ml, 400 IU / ml or approximately 400 IU / ml, 450 IU / ml or approximately 450 IU / ml, 500 IU / ml or approximately 500 IU / ml, 550 IU / The T cell enrichment composition is incubated with recombinant IL-7 at concentrations of approximately 550 IU / ml, 600 IU / ml, 650 IU / ml, 700 IU / ml, 750 IU / ml, 800 IU / ml, 750 IU / ml, 750 IU / ml, 750 IU / ml, 750 IU / ml, or 1,000 IU / ml. In certain embodiments, the enriched T cell composition is incubated in the presence of recombinant IL-7 at 600 IU / ml or approximately 600 IU / ml. In some embodiments, the composition incubated with recombinant IL-7 enriches a population of T cells, such as CD4+ T cells. In some embodiments, enriched CD4+ T cell compositions incubated with recombinant IL-7 may also be incubated with recombinant IL-2 and / or recombinant IL-15, such as in the amounts described. In certain embodiments, the enriched T cell composition is enriched with CD4+ T cells, but not with CD8+ T cells, and / or CD8+ T cells are negatively selected from the composition or depleted from the composition.In some embodiments, the enriched CD8+ T cell composition is not incubated with recombinant IL-7.

[0148] In some embodiments, the enriched T cell composition is incubated with recombinant IL-15, such as human recombinant IL-15, at concentrations of 0.1 IU / ml to 100 IU / ml, 1 IU / ml to 100 IU / ml, 1 IU / ml to 50 IU / ml, 5 IU / ml to 25 IU / ml, 25 IU / ml to 50 IU / ml, 5 IU / ml to 15 IU / ml, or 10 IU / ml to 100 IU / ml. In a particular embodiment, the enriched T cell composition is 1 IU / ml or about 1 IU / ml, 2 IU / ml or about 2 IU / ml, 3 IU / ml or about 3 IU / ml, 4 IU / ml or about 4 IU / ml, 5 IU / ml or about 5 IU / ml, 6 IU / ml or about 6 IU / ml, 7 IU / ml or about 7 IU / ml, 8 IU / ml or about 8 IU / ml, 9 IU / ml or about 9 IU / ml, 10 IU / ml or about 10 IU / ml, 11 IU / ml or about 1 The T cell enrichment composition is incubated with recombinant IL-15 at concentrations of 1 IU / ml, 12 IU / ml or approximately 12 IU / ml, 13 IU / ml or approximately 13 IU / ml, 14 IU / ml or approximately 14 IU / ml, 15 IU / ml or approximately 15 IU / ml, 20 IU / ml or approximately 20 IU / ml, 25 IU / ml or approximately 25 IU / ml, 30 IU / ml or approximately 30 IU / ml, 40 IU / ml or approximately 40 IU / ml, or 50 IU / ml or approximately 50 IU / ml. In some embodiments, the enriched T cell composition is incubated in recombinant IL-15 at 10 IU / ml or approximately 10 IU / ml. In some embodiments, the composition incubated with recombinant IL-15 is enriched with a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the T cell population is a population of CD4+ T cells. In some embodiments, the enriched T cell composition is a composition of enriched CD8+ T cells. In certain embodiments, the enriched T cell composition is enriched with CD8+ T cells, but not with CD4+ T cells, and / or CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched T cell composition is a composition of enriched CD4+ T cells.In certain embodiments, the enriched T cell composition is enriched with CD4+ T cells, but not with CD8+ T cells, and / or CD8+ T cells are negatively selected from the composition or depleted from the composition. In some embodiments, the enriched CD4+ T cell composition incubated with recombinant IL-15 may also be incubated with recombinant IL-7 and / or recombinant IL-2, such as in the amounts described. In some embodiments, the enriched CD8+ T cell composition incubated with recombinant IL-15 may also be incubated with recombinant IL-2, such as in the amounts described.

[0149] In certain embodiments, cells such as enriched CD4+ T cells and / or enriched CD8+ T cells are incubated with a stimulating reagent in the presence of one or more antioxidants. In some embodiments, the one or more antioxidants include tocopherol, tocotrienol, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, α-tocopherolquinone, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, Examples of antioxidants include, but are not limited to, isoflavones, lycopene, β-carotene, selenium, ubiquinone, ruetin, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine ​​(NAC), citrate, L-carnitine, BHT, monothioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine and cystathionine, and / or glycine-glycine-histidine. In some aspects, incubation of enriched T cell compositions, such as enriched CD4+ T cells and / or enriched CD8+ T cells, with antioxidants also includes the presence of stimulating reagents, such as anti-CD3 / anti-CD28 magnetic beads, and one or more recombinant cytokines as described.

[0150] In some embodiments, one or more antioxidants are sulfur-containing oxidizing agents or comprise a sulfur-containing oxidizing agent. In some specific embodiments, the sulfur-containing antioxidant may comprise a thiol-containing antioxidant and / or an antioxidant exhibiting one or more sulfur moieties in a ring structure, for example. In some embodiments, the sulfur-containing antioxidant may comprise, for example, N-acetylcysteine ​​(NAC) and 2,3-dimercaptopropanol (DMP), L-2-oxo-4-thiazolidine carboxylate (OTC), and lipoic acid. In certain embodiments, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified in one or more intracellular processes to yield glutathione. In certain embodiments, the glutathione precursor may comprise, but is not limited to, N-acetylcysteine ​​(NAC), L-2-oxothiazolidine-4-carboxylic acid (procysteine), lipoic acid, S-allylcysteine, or methylmethionine sulfonium chloride.

[0151] In some embodiments, incubating cells such as enriched CD4+ T cells and / or enriched CD8+ T cells under stimulating conditions involves incubating the cells in the presence of one or more antioxidants. In certain embodiments, the cells are stimulated with a stimulating reagent in the presence of one or more antioxidants. In some embodiments, the cells are incubated in the presence of one or more antioxidants in concentrations of 1 ng / ml to 100 ng / ml, 10 ng / ml to 1 μg / ml, 100 ng / ml to 10 μg / ml, 1 μg / ml to 100 μg / ml, 10 μg / ml to 1 mg / ml, 100 μg / ml to 1 mg / ml, 1500 μg / ml to 2 mg / ml, 500 μg / ml to 5 mg / ml, 1 mg / ml to 10 mg / ml, or 1 mg / ml to 100 mg / ml. In some embodiments, cells are found to be 1 ng / ml or approximately 1 ng / ml, 10 ng / ml or approximately 10 ng / ml, 100 ng / ml or approximately 100 ng / ml, 1 μg / ml or approximately 1 μg / ml, 10 μg / ml or approximately 10 μg / ml, 100 μg / ml or approximately 100 μg / ml, 0.2 mg / ml or approximately 0.2 mg / ml, 0.4 mg / ml or approximately 0.4 mg / ml, 0.6 mg / ml or approximately 0.6 mg / ml, 0.8 mg / ml or approximately 0.8 mg / ml, 1 mg / ml or approximately 1 mg / ml, 2 mg / ml or approximately 2 mg / ml, 3 mg / ml Alternatively, the mixture is incubated in the presence of one or more antioxidants in concentrations of approximately 3 mg / ml, 4 mg / ml or approximately 4 mg / ml, 5 mg / ml or approximately 5 mg / ml, 10 mg / ml or approximately 10 mg / ml, 20 mg / ml or approximately 20 mg / ml, 25 mg / ml or approximately 25 mg / ml, 50 mg / ml or approximately 50 mg / ml, 100 mg / ml or approximately 100 mg / ml, 200 mg / ml or approximately 200 mg / ml, 300 mg / ml or approximately 300 mg / ml, 400 mg / ml or approximately 400 mg / ml, 500 mg / ml or approximately 500 mg / ml. In some embodiments, one or more antioxidants are sulfur-containing antioxidants or comprise sulfur-containing antioxidants. In certain embodiments, one or more antioxidants are glutathione precursors or comprise glutathione precursors.

[0152] In some embodiments, one or more antioxidants are or comprise N-acetylcysteine ​​(NAC). In some embodiments, incubating cells such as enriched CD4+ T cells and / or enriched CD8+ T cells under stimulating conditions comprises incubating cells in the presence of NAC. In certain embodiments, cells are stimulated with a stimulating reagent in the presence of NAC. In some embodiments, cells are incubated in the presence of NAC at concentrations of 1 ng / ml to 100 ng / ml, 10 ng / ml to 1 μg / ml, 100 ng / ml to 10 μg / ml, 1 μg / ml to 100 μg / ml, 10 μg / ml to 1 mg / ml, 100 μg / ml to 1 mg / ml, 1-500 μg / ml to 2 mg / ml, 500 μg / ml to 5 mg / ml, 1 mg / ml to 10 mg / ml, or 1 mg / ml to 100 mg / ml. In some embodiments, the cells are 1 ng / ml or approximately 1 ng / ml, 10 ng / ml or approximately 10 ng / ml, 100 ng / ml or approximately 100 ng / ml, 1 μg / ml or approximately 1 μg / ml, 10 μg / ml or approximately 10 μg / ml, 100 μg / ml or approximately 100 μg / ml, 0.2 mg / ml or approximately 0.2 mg / ml, 0.4 mg / ml or approximately 0.4 mg / ml, 0.6 mg / ml or approximately 0.6 mg / ml, 0.8 mg / ml or approximately 0.8 mg / ml, 1 mg / ml or approximately 1 mg / ml, 2 mg / ml or approximately 2 mg / ml, 3 The cells are incubated in the presence of NAC at a concentration of mg / ml or approximately 3mg / ml, 4mg / ml or approximately 4mg / ml, 5mg / ml or approximately 5mg / ml, 10mg / ml or approximately 10mg / ml, 20mg / ml or approximately 20mg / ml, 25mg / ml or approximately 25mg / ml, 50mg / ml or approximately 50mg / ml, 100mg / ml or approximately 100mg / ml, 200mg / ml or approximately 200mg / ml, 300mg / ml or approximately 300mg / ml, 400mg / ml or approximately 400mg / ml, 500mg / ml or approximately 500mg / ml. In some embodiments, the cells are incubated with 0.8mg / ml or approximately 0.8mg / ml.

[0153] In certain embodiments, incubating a composition of enriched T cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, in the presence of one or more antioxidants, e.g., NAC, reduces cell activation compared to cells incubated in alternative and / or exemplary processes in the absence of antioxidants. In some specific embodiments, the reduction in activation is measured by the expression of one or more activation markers in the cells. In some specific embodiments, markers of activation include, but are not limited to, increased intracellular complexity (determined, e.g., by measuring lateral scattering (SSC)), increased cell size (determined, e.g., by measuring cell diameter and / or forward scattering (FSC)), increased CD27 expression, and / or decreased CD25 expression. In some embodiments, cells of a composition, when examined during or after incubation, manipulation, transduction, transfection, expansion, or formulation, or at or after any stage of the process performed after incubation, have negative, reduced, or low expression and / or levels of markers of activation. In some embodiments, the cells of the composition have negative, reduced, or low expression and / or levels of the marker of activation after the process is complete. In certain embodiments, the cells of the output composition have negative, reduced, or low expression and / or levels of the marker of activation.

[0154] In some embodiments, flow cytometry is used to determine the relative size of cells. In certain embodiments, FSC and SSC parameters are used to analyze cells and distinguish them from one another based on size and internal complexity. In certain embodiments, the actual size of cells can be determined by measuring using particles or beads of known size as a standard. In some embodiments, flow cytometry is used in combination with staining, e.g., labeled antibodies, to measure or quantify the expression of surface proteins such as activation markers, e.g., CD25 or CD27.

[0155] In some embodiments, a composition of enriched T cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, is incubated in the presence of one or more antioxidants, e.g., NAC, and the cell diameter is reduced by at least 0.25 μm, at least 0.5 μm, at least 0.75 μm, at least 1.0 μm, at least 1.5 μm, at least 2 μm, at least 2.5 μm, at least 3 μm, at least 3.5 μm, at least 4 μm, at least 4.5 μm, at least 5 μm, or more than 5 μm compared to cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of antioxidants. In a particular embodiment, a composition of enriched T cells is incubated in the presence of one or more antioxidants, e.g., NAC, and the cell size, as measured by FSC, is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of antioxidants.

[0156] In some embodiments, enriched T cell compositions, such as enriched CD4+ T cells and / or enriched CD8+ T cells, are incubated in the presence of one or more antioxidants, e.g., NAC, and the intracellular complexity, as measured by SSC, is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of antioxidants.

[0157] In certain embodiments, enriched T cell compositions, such as enriched CD4+ T cells and / or enriched CD8+ T cells, are incubated in the presence of one or more antioxidants, e.g., NAC, and the expression of CD27, as measured by flow cytometry, is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of antioxidants.

[0158] In some specific embodiments, enriched T cell compositions, such as enriched CD4+ T cells and / or enriched CD8+ T cells, are incubated in the presence of one or more antioxidants, e.g., NAC, and the expression of CD25, as measured by flow cytometry, is increased by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 150%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 25x, at least 50x, or at least 100x compared to cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of antioxidants.

[0159] In certain embodiments, incubating a composition of enriched T cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, in the presence of one or more antioxidants, such as NAC, increases expansion during the incubation or culture process or stage described, for example, in Section ID. In some embodiments, the enriched cell composition achieves a 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, or more than 10x expansion within 14 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, or 3 days from the start of culture. In some embodiments, a composition of enriched T cells is incubated in the presence of one or more antioxidants, and the cells of the composition expand during culture at a rate at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 100%, at least 150%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, and at least 10x faster than cultured cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of antioxidants.

[0160] In certain embodiments, incubating a composition of enriched cells, such as enriched CD4+ T cells and / or enriched CD8+ T cells, in the presence of one or more antioxidants, such as NAC, reduces the amount of cell death, for example, by apoptosis. In some embodiments, a composition of enriched T cells is incubated in the presence of one or more antioxidants, such as NAC, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the cells survive for one day or at least one day, two days or at least two days, three days or at least three days, four days or at least four days, five days or at least five days, six days or at least six days, seven days or at least seven days, or more than seven days after the incubation is complete, without undergoing apoptosis. In some embodiments, the composition is incubated in the presence of one or more antioxidants, e.g., NAC, and the cells of the composition have a viability at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 25x, at least 50x, or at least 100x greater than cells undergoing alternative and / or exemplary processes in which the cells are not incubated in the presence of one or more antioxidants.

[0161] In certain embodiments, enriched T cell compositions, such as enriched CD4+ T cells and / or enriched CD8+ T cells, are incubated in the presence of one or more antioxidants, e.g., NAC, and caspase expression, e.g., caspase 3 expression, is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of antioxidants.

[0162] In some embodiments, compositions or cells such as enriched CD4+ T cells and / or enriched CD8+ T cells are incubated under stimulating conditions or in the presence of stimulants as described. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime cells for genetic manipulation such as the introduction of recombinant antigen receptors. Exemplary stimulating reagents, such as anti-CD3 / anti-CD28 magnetic beads, are described below. Incubation with stimulating reagents may also be carried out in the presence of one or more stimulating cytokines, for example, one or more recombinant IL-2, recombinant IL-7, and / or recombinant IL-15, and / or in the presence of at least one antioxidant, such as NAC as described above. In some embodiments, compositions of enriched CD4+ T cells are incubated under stimulating conditions with stimulants, such as described amounts, recombinant IL-2, recombinant IL-7, recombinant IL-15, and NAC. In some embodiments, compositions of enriched CD8+ T cells are incubated under stimulating conditions with stimulants, such as described amounts, recombinant IL-2, recombinant IL-15, and NAC.

[0163] In some embodiments, the conditions for stimulation and / or activation may include one or more of a specific culture medium, temperature, oxygen content, carbon dioxide content, time, active agents such as nutrients, amino acids, antibiotics, ions, and / or stimulating factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0164] In some cases, incubation is carried out according to techniques such as those described in Riddell et al. U.S. Patent No. 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0165] In some embodiments, at least a portion of the incubation in the presence of one or more stimulating conditions or stimulants is carried out in the internal cavity of a centrifugal chamber under centrifugal rotation, for example, as described in International Publication No. 2016 / 073602. In some embodiments, at least a portion of the incubation carried out in a centrifugal chamber includes mixing with one or more reagents for inducing stimulation and / or activation. In some embodiments, cells, such as selected cells, are mixed with the stimulating conditions or stimulants in a centrifugal chamber. In some aspects of such a process, a certain volume of cells is mixed with one or more stimulating conditions or stimulants in amounts far less than those typically used when performing similar stimulation in a cell culture plate or other system.

[0166] In some embodiments, the stimulant is added to the cells in the chamber cavity in substantially less amount (e.g., less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, or less than 80%) compared to the amount of stimulant typically used or required to achieve nearly the same or similar efficiency for the selection of the same number of cells or the same volume of cells when the selection is performed without mixing in the centrifugation chamber, e.g., in a tube or bag, while periodically shaking or rotating.In some embodiments, incubation is performed by adding incubation buffer to the cells and stimulant, for example, about 10 mL to about 200 mL, or about 20 mL to about 125 mL, for example, at least 10 mL or at least about 10 mL or about 10 mL, at least 20 mL or at least about 20 mL or about 20 mL, at least 30 mL or at least about 30 mL or about 30 mL, at least 40 mL or at least about 40 mL or about 40 mL, at least 50 mL or at least about 50 mL. Or approximately 50 mL, at least 60 mL or at least about 60 mL or about 60 mL, at least 70 mL or at least about 70 mL or about 70 mL, at least 80 mL or at least about 80 mL or about 80 mL, at least 90 mL or at least about 90 mL or about 90 mL, at least 100 mL or at least about 100 mL or about 100 mL, at least 105 mL or at least about 105 mL or about 105 mL, at least 110 mL or at least about 110 mL or about 110 mL , at least 115 mL or at least approximately 115 mL or approximately 115 mL, at least 120 mL or at least approximately 120 mL or approximately 120 mL, at least 125 mL or at least approximately 125 mL or approximately 125 mL, at least 130 mL or at least approximately 130 mL or approximately 130 mL, at least 135 mL or at least approximately 135 mL or approximately 135 mL, at least 140 mL or at least approximately 140 mL or approximately 140 mL, at least 145 mL or at least approximately 145 mL or approximately The target volume is achieved by incubation of 145 mL, at least 150 mL or at least approximately 150 mL or approximately 150 mL, at least 160 mL or at least approximately 160 mL or approximately 160 mL, at least 170 mL or at least approximately 170 mL or approximately 170 mL, at least 180 mL or at least approximately 180 mL or approximately 180 mL, at least 190 mL or at least approximately 190 mL or approximately 190 mL, or at least 200 mL or at least approximately 200 mL or approximately 200 mL of reagent.In some embodiments, the incubation buffer and stimulant are pre-mixed before being added to the cells. In some embodiments, the incubation buffer and stimulant are added to the cells separately. In some embodiments, the stimulating incubation is carried out under periodic, gentle mixing conditions, which helps to promote energetically favorable interactions, thereby allowing for the use of less overall stimulant while achieving cell stimulation and activation.

[0167] In some embodiments, incubation is generally carried out under mixed conditions, for example, at a relatively low force or speed, for example, a speed lower than the speed used to pelletize the cells, for example, 600 rpm to 1700 rpm or about 600 rpm to about 1700 rpm (for example, 600 rpm or about 600 rpm or at least 600 rpm, 1000 rpm or about 1000 rpm or at least 1000 rpm, or 1500 rpm or about 1500 rpm or at least 1500 rpm, or The spinning is performed in the presence of spins at RCF on the sample or wall of a chamber or other container of 1700 rpm or approximately 1700 rpm or at least 1700 rpm, for example, 80g to 100g or approximately 80g to approximately 100g (for example, 80g or approximately 80g or at least 80g, 85g or approximately 85g or at least 85g, 90g or approximately 90g or at least 90g, 95g or approximately 95g or at least 95g, or 100g or approximately 100g or at least 100g). In some embodiments, the spinning is performed using repeated intervals of spinning at such low speeds followed by pauses, for example, spinning and / or pausing for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, for example, spinning for about 1 or 2 seconds followed by pauses for about 5, about 6, about 7, or about 8 seconds.

[0168] In some embodiments, for example, the total duration of incubation with the stimulant is 1 to 96 hours or about 1 to 96 hours, 1 to 72 hours or about 1 to 72 hours, 1 to 48 hours or about 1 to 48 hours, 4 to 36 hours or about 4 to 36 hours, 8 to 30 hours or about 8 to 30 hours, 18 to 30 hours or about 18 to 30 hours, or 12 to 24 hours or about 12 to 24 hours, for example, at least 6 hours or at least about 6 hours or about 6 hours, at least 12 hours or at least about 12 hours or about 12 hours, at least 18 hours or at least about 18 hours or about 18 hours, at least 24 hours or at least about 24 hours or about 24 hours, at least 36 hours or at least about 36 hours or about 36 hours, or at least 72 hours or at least about 72 hours or about 72 hours. In some embodiments, further incubation includes values ​​at both ends, such as 1 hour to 48 hours or approximately 1 hour to approximately 48 hours, 4 hours to 36 hours or approximately 4 hours to approximately 36 hours, 8 hours to 30 hours or approximately 8 hours to approximately 30 hours, or 12 hours to 24 hours or approximately 12 hours to approximately 24 hours.

[0169] In some embodiments, cells are cultured, cultivated, and / or incubated under stimulating conditions before and / or during the process of introducing polynucleotides, e.g., polynucleotides encoding recombinant receptors, by transduction and / or transfection, as described in Section IC. In some specific embodiments, cells are cultured, cultivated, and / or incubated under stimulating conditions for periods of 30 minutes to 2 hours, 1 hour to 8 hours, 1 hour to 6 hours, 6 hours to 12 hours, 12 hours to 18 hours, 16 hours to 24 hours, 12 hours to 36 hours, 24 hours to 48 hours, 24 hours to 72 hours, 42 hours to 54 hours, 60 hours to 120 hours, 96 hours to 120 hours, 90 hours, 1 day to 7 days, 3 days to 8 days, 1 day to 3 days, 4 days to 6 days, or 4 days to 5 days prior to the genetic manipulation. In some embodiments, the cells are incubated for 2 days or approximately 2 days before the procedure.

[0170] In some specific embodiments, cells are incubated with and / or in the presence of a stimulating agent before and / or during genetic engineering. In some specific embodiments, cells are incubated with and / or in the presence of a stimulating agent for periods of 12–36 hours, 24–48 hours, 24–72 hours, 42–54 hours, 60–120 hours, 96–120 hours, 90 hours, 2–7 days, 3–8 days, 1–8 days, 4–6 days, or 4–5 days. In certain embodiments, cells are cultured, cultivated, and / or incubated under stimulating conditions for a period of less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, or less than 168 hours, less than 162 hours, less than 156 hours, less than 144 hours, less than 138 hours, less than 132 hours, less than 120 hours, less than 114 hours, less than 108 hours, less than 102 hours, or less than 96 hours before and / or during genetic engineering of the cells. In certain embodiments, cells are incubated with and / or in the presence of a stimulating agent for 4 days or about 4 days, 5 days or about 5 days, 6 days or about 6 days, or 7 days or about 7 days. In some embodiments, cells are incubated with and / or in the presence of a stimulating agent for 4 days or about 4 days. In certain embodiments, cells are incubated with and / or in the presence of a stimulating agent for 5 days or about 5 days. In some specific embodiments, cells are incubated with and / or in the presence of a stimulating agent for less than 7 days.

[0171] In some embodiments, incubating cells under stimulating conditions involves incubating cells with a stimulating reagent described in Section IB-1. In some embodiments, the stimulating reagent contains or includes beads, such as paramagnetic beads, and the cells are incubated with the stimulating reagent in a ratio of less than 3:1 (beads:cells), for example, 1:1. In certain embodiments, the cells are incubated with the stimulating reagent in the presence of one or more cytokines and / or one or more antioxidants. In some embodiments, enriched CD4+ T cell compositions are incubated with a stimulating reagent in a 1:1 (beads:cells) ratio in the presence of recombinant IL-2, IL-7, IL-15, and NAC. In some specific embodiments, enriched CD8+ T cell compositions are incubated with a stimulating reagent in a 1:1 (beads:cells) ratio in the presence of recombinant IL-2, IL-15, and NAC. In some embodiments, the stimulating agent is removed and / or separated from the cells on or within 6 days or approximately 6 days from the start or commencement of incubation, for example, on or within 5 days or approximately 5 days from the time the stimulating agent was added to or came into contact with the cells, or on or within 4 days or approximately 4 days.

[0172] 1. Stimulating reagent In some embodiments, incubating a composition of enriched cells under stimulating conditions involves incubating and / or contacting the composition of enriched cells with a stimulating reagent that can activate and / or enlarge T cells. In some embodiments, the stimulating reagent can stimulate and / or activate one or more intracellular signals. In some embodiments, one or more signals are mediated by receptors. In certain embodiments, one or more signals are or are related to signaling and / or secondary messengers, e.g., cAMP and / or changes in the level or amount of intracellular calcium, changes in the amount, cellular localization, identification, phosphorylation, ubiquitination, and / or truncation of one or more cellular proteins, and / or changes in cellular activity, e.g., transcription, translation, proteolysis, cell morphology, activation state, and / or changes in cell division. In certain embodiments, the stimulating reagent can activate and / or activate one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more co-stimulatory molecules.

[0173] In some specific embodiments, the stimulating reagent includes one or more agents, such as particles conjugated or linked to biomolecules, such as beads, which can activate and / or enlarge cells, such as T cells. In some embodiments, one or more agents are bound to beads. In some embodiments, the beads are biocompatible, i.e., made of a material suitable for biological use. In some embodiments, the beads are nontoxic to cultured cells, such as cultured T cells. In some embodiments, the beads can be any particles to which the agents can be attached in a manner that allows for interaction between the agents and cells.

[0174] In some embodiments, the stimulating agent comprises one or more agents that can activate and / or enlarge beads, for example, cells bound to or otherwise attached to the surface of the beads, such as T cells. In some specific embodiments, the beads are non-cellular particles. In certain embodiments, the beads may include colloidal particles, microspheres, nanoparticles, magnetic beads, etc. In some embodiments, the beads are agarose beads. In some specific embodiments, the beads are cephalos beads.

[0175] In certain embodiments, the irritant comprises monodisperse beads. In some specific embodiments, the monodisperse beads comprise a size dispersion with a diameter standard deviation of less than 5% from one another.

[0176] In some embodiments, the beads include one or more agents, such as conjugated, conjugated, or linked agents, on the surface of the beads (directly or indirectly). In some embodiments, the agents contemplated herein may include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipids, lectins, or any other biomolecules having affinity for a desired target. In some embodiments, the desired target is a T cell receptor and / or a component of a T cell receptor. In some specific embodiments, the desired target is CD3. In some specific embodiments, the desired target is a T cell costimulatory molecule, e.g., CD28, CD137(4-1-BB), OX40, or ICOS. One or more agents may be attached directly or indirectly to the beads by a variety of methods known and available in the art. Attachment may be covalent, non-covalent, electrostatic, or hydrophobic, and may be achieved by a variety of attachment means, including, for example, chemical, mechanical, or enzymatic means. In some embodiments, a biomolecule (e.g., a biotinylated anti-CD3 antibody) can be indirectly attached to the beads via another biomolecule (e.g., an anti-biotin antibody) that is directly attached to the beads.

[0177] In some embodiments, the stimulating reagent comprises one or more agents that directly interact with the beads and polymers on the cell surface. In some specific embodiments, the beads (e.g., paramagnetic beads) interact with the cell via one or more agents (e.g., antibodies) that are specific to one or more polymers (e.g., one or more cell surface proteins) on the cell. In some specific embodiments, the beads (e.g., paramagnetic beads) are labeled with a first agent described herein, such as a primary antibody (e.g., an anti-biotin antibody) or other biomolecule, and then a second agent, such as a secondary antibody (e.g., a biotinylated anti-CD3 antibody) or other secondary biomolecule (e.g., streptavidin), is added so that the secondary antibody or other secondary biomolecule specifically binds to such primary antibody or other biomolecule on the particle.

[0178] In some embodiments, the stimulating agent adheres to beads (e.g., paramagnetic beads) and on cells (e.g., T cells) the following macromolecules: CD2, CD3, CD4, CD5, CD8, CD25, CD27, CD28, CD29, CD31, CD44, CD45RA, CD45RO, CD54 (ICAM-1), CD127, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BB (CD137), 4-1BBL, CD30L, LIGHT, IL-2R, IL-12R, IL-1R, IL-15R; IFN-γR, TNF-αR, IL-4R, IL-10R, CD18 / CD1 The present invention comprises one or more agents (e.g., antibodies) that specifically bind to one or more of the following polymers on a cell (e.g., CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., delta-like 1 / 4, Jagged 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3, or ligands corresponding to these polymers or fragments thereof. In some embodiments, the agent (e.g., antibody) attached to the beads specifically binds to one or more of the following polymers on a cell (e.g., T cell): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO.

[0179] In some embodiments, one or more of the agents attached to the beads are antibodies. Antibodies may include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). In some embodiments, the stimulating agent is an antibody fragment (including an antigen-binding fragment), e.g., Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. Any isotype constant region, including IgG, IgM, IgA, IgD, and IgE constant regions, can be used in the antibodies intended herein, and it will be understood that such constant regions are available from any human or animal species (e.g., mouse species). In some embodiments, the agent is an antibody that binds to and / or recognizes one or more components of a T cell receptor. In certain embodiments, the agent is an anti-CD3 antibody. In some specific embodiments, the agent is an antibody that binds to and / or recognizes a co-receptor. In some embodiments, the stimulating agent includes an anti-CD28 antibody. In some embodiments, the beads have diameters greater than approximately 0.001 μm, greater than approximately 0.01 μm, greater than approximately 0.1 μm, greater than approximately 1.0 μm, greater than approximately 10 μm, greater than approximately 50 μm, greater than approximately 100 μm, or greater than approximately 1000 μm, and less than or equal to approximately 1500 μm. In some embodiments, the beads have diameters of approximately 1.0 μm to approximately 500 μm, approximately 1.0 μm to approximately 150 μm, approximately 1.0 μm to approximately 30 μm, approximately 1.0 μm to approximately 10 μm, approximately 1.0 μm to approximately 5.0 μm, approximately 2.0 μm to approximately 5.0 μm, or approximately 3.0 μm to approximately 5.0 μm. In some embodiments In this configuration, the beads have a diameter of approximately 3 μm to approximately 5 μm. In some embodiments, the beads are at least 0.001 μm or at least about 0.001 μm or about 0.001 μm, at least 0.01 μm or at least about 0.01 μm or about 0.01 μm, at least 0.1 μm or at least about 0.1 μm or about 0.1 μm, at least 0.5 μm or at least about 0.5 μm or about 0.5 μm, at least 1.0 μm or at least about 1.0 μm or about 1.0 μm, at least 1.5 μm or at least approximately 1.5 μm or approximately 1.5 μm, at least 2.0 μm or at least approximately 2.0 μm or approximately 2.0 μm, at least 2.5 μm or at least approximately 2.5 μm or approximately 2.5 μm, at least 3.0 μm or at least approximately 3.0 μm or approximately 3.0 μm, at least 3.5 μm or at least approximately 3.5 μm or approximately 3.5 μm, at least 4.0 μm or at least approximately 4.0 μm or approximately 4.0 μm, at least 4.5 μm or at least approximately 4.5 μm or approximately 4.5 μm, at least 5.0 μm or at least approximately 5.0 μm or approximately 5.0 μm, at least 5.5 μm or at least approximately 5.5 μm or approximately 5.5 μm, at least 6.0 μm or at least approximately 6.0 μm or approximately 6.0 μm, at least 6.5 μm or at least approximately 6.5 μm or approximately 6.5 μm, at least 7.0 μm or less Each has a diameter of approximately 7.0 μm or approximately 7.0 μm, at least 7.5 μm or at least approximately 7.5 μm or approximately 7.5 μm, at least 8.0 μm or at least approximately 8.0 μm or approximately 8.0 μm, at least 8.5 μm or at least approximately 8.5 μm or approximately 8.5 μm, at least 9.0 μm or at least approximately 9.0 μm or approximately 9.0 μm, at least 9.5 μm or at least approximately 9.5 μm or approximately 9.5 μm, at least 10 μm or at least approximately 10 μm or approximately 10 μm, at least 12 μm or at least approximately 12 μm or approximately 12 μm, at least 14 μm or at least approximately 14 μm or approximately 14 μm, at least 16 μm or at least approximately 16 μm or approximately 16 μm, at least 18 μm or at least approximately 18 μm or approximately 18 μm or at least 20 μm or at least approximately 20 μm or approximately 20 μm. In some specific embodiments, the beads have a diameter of 4.5 μm or approximately 4.5 μm. In some specific embodiments, the beads have a diameter of 2.8 μm or approximately 2.8 μm.

[0180] In some embodiments, the beads are 0.001 g / cm³ 3 Super, 0.01g / cm 3Over, 0.05 g / cm 3 Over, 0.1 g / cm 3 Over, 0.5 g / cm 3 Over, 0.6 g / cm 3 Over, 0.7 g / cm 3 Over, 0.8 g / cm 3 Over, 0.9 g / cm 3 Over, 1 g / cm 3 Over, 1.1 g / cm 3 Over, 1.2 g / cm 3 Over, 1.3 g / cm 3 Over, 1.4 g / cm 3 Over, 1.5 g / cm 3 Over, 2 g / cm 3 Over, 3 g / cm 3 Over, 4 g / cm 3 Over, or 5 g / cm 3 Having a density of over. In some embodiments, the beads are about 0.001 g / cm 3 ~ about 100 g / cm 3 ~ about 0.01 g / cm 3 ~ about 50 g / cm 3 ~ about 0.1 g / cm 3 ~ about 10 g / cm 3 ~ about 0.1 g / cm 3 ~ about 0.5 g / cm 3 ~ about 0.5 g / cm 3 ~ about 1 g / cm 3 [[ID=5Y]]~ about 0.5 g / cm 3 ~ about 1.5 g / cm 3 ~ about 1 g / cm 3 ~ about 1.5 g / cm 3 ~ about 1 g / cm 3 ~ about 2 g / cm 3 ~ about 1 g / cm 3 ~ about 5 g / cm 3 Having a density of. In some embodiments, the beads are about 0.5 g / cm 3 ~ about 0.5 g / cm 3 ~ about 0.6 g / cm 3 ~ about 0.7 g / cm [[ID=Y7]] 3 ~ about 0.8 g / cm 3 ~ about 0.9 g / cm 3 ~ about 1.0 g / cm 3 ~ about 1.1 g / cm 3 [[ID=8Y]]~ about 1.2 g / cm 3, about 1.3g / cm 3 Approximately 1.4 g / cm³ 3 , about 1.5g / cm 3 , about 1.6g / cm 3 , about 1.7g / cm 3 , about 1.8g / cm 3 , about 1.9g / cm 3 , or approximately 2.0 g / cm³ 3 It has a density of approximately 1.6 g / cm³. In some specific embodiments, the beads have a density of approximately 1.6 g / cm³. 3 It has a density of about 1.5 g / cm³. In a particular embodiment, the beads or particles have a density of about 1.5 g / cm³. 3 It has a density of approximately 1.3 g / cm³. In some specific embodiments, the particles are approximately 1.3 g / cm³. 3 It has a density of .

[0181] In some specific embodiments, the beads have a uniform density. In some specific embodiments, the uniform density includes a density standard deviation of less than 10%, less than 5%, or less than 1% of the average bead density.

[0182] In some embodiments, the beads are (m³) per gram of each particle. 2 / g) approx. 0.001m 2 ~about 1,000m 2 / g, approx. .010m 2 / g~about 100m 2 / g, approx. 0.1m 2 / g~about 10m 2 / g, approx. 0.1m 2 / g ~ approx. 1m 2 / g, approx. 1m 2 / g~about 10m 2 / g, approx. 10m 2 / g~about 100m 2 / g, approx. 0.5m 2 / g~about 20m 2 / g, approx. 0.5m 2 / g~about 5m 2 / g, or approximately 1m 2 / g~about 4m 2 It has a surface area of ​​about 1 m² / g. In some embodiments, the particles or beads are about 1 m². 2 / g~about 4m 2 It has a surface area of ​​ / g.

[0183] In some embodiments, the beads include at least one material on or near the bead surface that can be conjugated, linked, or conjugated to an agent. In some embodiments, the beads are surface-functionalized, i.e., they include functional groups that can form covalent bonds with binding molecules, such as polynucleotides or polypeptides. In certain embodiments, the beads include carboxyl, amino, hydroxyl, tosyl, epoxy, and / or chloromethyl groups exposed on the surface. In certain embodiments, the beads include agarose and / or Sepharose exposed on the surface. In some specific embodiments, the bead surface includes an adhesion stimulating agent that can bind to or adhere to a binding molecule. In certain embodiments, the biomolecule is a polypeptide. In some embodiments, the beads include protein A, protein G, or biotin exposed on the surface.

[0184] In some embodiments, the beads react in a magnetic field. In some embodiments, the beads are magnetic beads. In some embodiments, magnetic beads are paramagnetic. In certain embodiments, magnetic beads are superparamagnetic. In some specific embodiments, the beads exhibit no magnetic properties unless exposed to a magnetic field.

[0185] In certain embodiments, the beads include a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core includes a metal. In some embodiments, the metal may be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In some specific embodiments, the magnetic core includes metal oxides (e.g., iron oxide), ferrites (e.g., manganese ferrite, cobalt ferrite, nickel ferrite, etc.), hematite, and metal alloys (e.g., CoTaZn). In some embodiments, the magnetic core includes one or more of ferrite, metal, metal alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core includes elemental iron or a compound thereof. In some embodiments, the magnetic core includes one or more of magnetite (Fe3O4), maghemite (γFe2O3), or greigite (Fe3S4). In some embodiments, the inner core includes iron oxide (e.g., Fe3O4).

[0186] In some specific embodiments, the beads comprise a magnetic, paramagnetic, and / or superparamagnetic core covered with a surface-functionalized coating or coating. In some embodiments, the coating may include, but is not limited to, polymers, polysaccharides, silica, fatty acids, proteins, carbon, agarose, Sepharose, or combinations thereof. In some embodiments, the polymer may be polyethylene glycol, poly(lactic acid-coglycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In some specific embodiments, the outer coating or coating comprises polystyrene. In certain embodiments, the outer coating is surface-functionalized.

[0187] In some embodiments, the stimulating reagent comprises beads containing a metal oxide core (e.g., an iron oxide core) and a coat, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and the coat comprises at least one polysaccharide (e.g., aminodextran), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In some specific embodiments, one or more agents comprise an antibody or its antigen-binding fragment. In certain embodiments, one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulating reagent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulating reagent comprises an anti-biotin antibody. In some embodiments, the beads have a diameter of about 3 μm to about 10 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In some specific embodiments, the beads have a diameter of about 3.5 μm.

[0188] In some embodiments, the stimulating reagent comprises one or more agents that adhere to beads containing a metal oxide core (e.g., an iron oxide internal core) and a coating (e.g., a protective coating), the coating comprising polystyrene. In some specific embodiments, the beads are monodisperse paramagnetic (e.g., superparamagnetic) beads comprising a core containing a paramagnetic (e.g., superparamagnetic) iron core, e.g., magnetite (Fe3O4) and / or maghemite (γFe2O3)c and a polystyrene coating or coating. In some embodiments, the beads are non-porous. In some embodiments, the beads include a functionalized surface to which one or more agents adhere. In some specific embodiments, one or more agents are covalently bonded to the beads at the surface. In some embodiments, one or more agents comprise an antibody or its antigen-binding fragment. In some embodiments, one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulating reagent is an anti-CD3 / anti-CD28 magnetic bead or comprises an anti-CD3 / anti-CD28 magnetic bead. In some embodiments, one or more agents comprise an anti-CD3 antibody and / or an anti-CD28 antibody, as well as an antibody or antigen fragment that can bind to a labeled antibody (e.g., a biotinylated antibody), such as a labeled anti-CD3 or anti-CD28 antibody. In some specific embodiments, the beads are approximately 1.5 g / cm³. 3 The density and approximately 1 m 2 / g~about 4m 2 It has a surface area of ​​approximately 1.5 g / cm². In a particular embodiment, the beads have a diameter of approximately 4.5 μm and a surface area of ​​approximately 1.5 g / cm². 3 These are monodisperse superparamagnetic beads having a density of . In some embodiments, the beads have an average diameter of about 2.8 μm and a density of about 1.3 g / cm². 3 These are monodisperse superparamagnetic beads having a density of [density].

[0189] In some embodiments, enriched T cell compositions are incubated with stimulating reagents in bead-to-cell ratios of 3:1 or approximately 3:1, 2.5:1 or approximately 2.5:1, 2:1 or approximately 2:1, 1.5:1 or approximately 1.5:1, 1.25:1 or approximately 1.25:1, 1.2:1 or approximately 1.2:1, 1.1:1 or approximately 1.1:1, 1:1 or approximately 1:1, 0.9:1 or approximately 0.9:1, 0.8:1 or approximately 0.8:1, 0.75:1 or approximately 0.75:1, 0.67:1 or approximately 0.67:1, 0.5:1 or approximately 0.5:1, 0.3:1 or approximately 0.3:1, or 0.2:1 or approximately 0.2:1. In certain embodiments, the bead-to-cell ratio is 2.5:1–0.2:1, 2:1–0.5:1, 1.5:1–0.75:1, 1.25:1–0.8:1, or 1.1:1–0.9:1. In certain embodiments, the stimulating agent-to-cell ratio is approximately 1:1 or 1:1.

[0190] 2. Removal of stimulating reagents from cells In some specific embodiments, stimulating agents, such as anti-CD3 / anti-CD28 magnetic beads, are removed and / or separated from the cells. While not wishing to be bound by theory, certain embodiments intend that the binding and / or association between the stimulating agent and the cells may, in some circumstances, decrease over time during incubation. In some specific embodiments, the addition of one or more agents may reduce the binding and / or association between the stimulating agent and the cells. In certain embodiments, changes in cell culture conditions, such as the temperature or pH of the culture medium, may reduce the binding and / or association between the stimulating agent and the cells. Therefore, in some embodiments, the stimulating agent may be removed from the incubation, cell culture system, and / or solution separately from the cells, for example, without removing the cells from the incubation, cell culture system, and / or solution.

[0191] Methods for removing stimulating reagents (which are particles, such as bead particles or magnetizable particles, or stimulating reagents containing such particles) from cells are known. In some embodiments, a competing antibody, such as an unlabeled antibody, can be used, which binds to the primary antibody of the stimulating reagent, altering its affinity for its antigen on the cell, thereby enabling gentle separation. In some cases, after separation, the competing antibody may remain bound to the particles (e.g., bead particles), but the unreacted antibody may be washed away or flushed out, and the cells do not isolate, select, enrich, and / or activate the antibody. An example of such a reagent is DETACaBEAD (Friedl et al. 1995; Entschladen et al. 1997). In some embodiments, the particles (e.g., bead particles) may be removed in the presence of a cleavable linker (e.g., a DNA linker), thereby conjugating the antibody bound to the particles to the linker (e.g., CELLection, Dynal). In some cases, the linker region provides a cleavable site for removing the particles (e.g., bead particles) from the cells after isolation, for example, by adding DNase or other release buffer. In some embodiments, other enzymatic methods can also be used for the release of particles (e.g., bead particles) from cells. In some embodiments, the particles (e.g., bead particles or magnetizable particles) are biodegradable.

[0192] In some embodiments, the stimulating agent is magnetic, paramagnetic, and / or superparamagnetic, and / or includes beads that are magnetic, paramagnetic, and / or superparamagnetic, and the stimulating agent can be removed from the cells by exposing them to a magnetic field. Examples of suitable devices including magnets for generating a magnetic field include DynaMag CTS (Thermo Fisher), Magnetic Separator (Takara), and EasySep Magnet (Stem Cell Technologies).

[0193] In certain embodiments, the stimulating agent is removed or separated from the cells before completion of the provided method, for example, before harvesting, collecting, and / or formulating the manipulated cells produced by the method provided herein. In some embodiments, the stimulating agent is removed and / or separated from the cells before manipulating the cells, for example, transfecting or transfecting them. In certain embodiments, the stimulating agent is removed and / or separated from the cells after the cell manipulation step. In some specific embodiments, the stimulating agent is removed before culturing the cells, for example, before culturing cells that have been manipulated under conditions that promote proliferation and / or expansion, for example, transfected or transfected cells.

[0194] In certain embodiments, the stimulating agent is separated and / or removed from the cells after a certain period of time. In certain embodiments, the time is the time from the start and / or commencement of incubation under stimulating conditions. In certain embodiments, the start of incubation is considered to be the time when the cells come into contact with the stimulating agent and / or the culture medium or solution containing the stimulating agent, or approximately the time when the cells come into contact with the stimulating agent. In certain embodiments, the stimulating agent is removed or separated from the cells within 10 days or about 10 days, within 9 days or about 9 days, within 8 days or about 8 days, within 7 days or about 7 days, within 6 days or about 6 days, within 5 days or about 5 days, within 4 days or about 4 days, within 3 days or about 3 days, or within 2 days or about 2 days after the start or commencement of incubation. In certain embodiments, the stimulating agent is removed and / or separated from the cells on day 9 or approximately day 9, day 8 or approximately day 8, day 7 or approximately day 7, day 6 or approximately day 6, day 5 or approximately day 5, day 4 or approximately day 4, day 3 or approximately day 3, or day 2 or approximately day 2 after the start or commencement of incubation. In some specific embodiments, the stimulating agent is removed and / or separated from the cells at 168 hours or approximately 168 hours, 162 hours or approximately 162 hours, 156 hours or approximately 156 hours, 144 hours or approximately 144 hours, 138 hours or approximately 138 hours, 132 hours or approximately 132 hours, 120 hours or approximately 120 hours, 114 hours or approximately 114 hours, 108 hours or approximately 108 hours, 102 hours or approximately 102 hours, or 96 hours or approximately 96 hours after the start or commencement of incubation. In certain embodiments, the stimulating agent is removed and / or separated from the cells on day 5 or approximately day 5 after the start and / or commencement of incubation. In some embodiments, the stimulating agent is removed and / or separated from the cells on day 4 or approximately day 4 after the start and / or commencement of incubation.

[0195] C. Cell manipulation In some embodiments, the methods provided include administering cells expressing recombinant antigen receptors to subjects having a disease or pathological condition. Various methods for introducing genetically modified components, such as antigen receptors, such as CARs or TCRs, are well known and can be used in conjunction with the methods and compositions provided. Exemplary methods include methods for transferring nucleic acids encoding receptors, including via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.

[0196] Among the cells that express receptors and are administered by the provided method, there are engineered cells. Genetic engineering generally involves introducing nucleic acids encoding recombinant or engineered components into cell-containing compositions, such as by retroviral transduction, transfection, or transformation.

[0197] In some embodiments, the methods provided herein are used in connection with manipulating one or more compositions of enriched T cells. In some specific embodiments, the manipulation is or includes the introduction of polynucleotides, for example, recombinant polynucleotides encoding recombinant proteins. In certain embodiments, the recombinant protein is a recombinant receptor, such as those described in Section II. The introduction of nucleic acid molecules encoding recombinant proteins, such as recombinant receptors, into cells can be carried out using any of several known vectors. Such vectors include viral and non-viral systems, including lentiviruses and gamma-retrovirus systems, as well as transposon-based systems, such as PiggyBac or Sleeping Beauty-based gene transfer systems. Exemplary methods include methods for the transfer of receptor-encoding nucleic acids, including via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation. In some embodiments, the manipulation produces one or more manipulated compositions of enriched T cells.

[0198] In certain embodiments, one or more compositions of enriched T cells are manipulated, e.g., transduced or transfected, before culturing the cells under conditions that promote proliferation and / or expansion, for example, by a method provided in Section ID. In certain embodiments, one or more compositions of enriched T cells are manipulated after the one or more compositions have been stimulated, activated, and / or incubated under the stimulating conditions described in a method provided in Section IB. In certain embodiments, one or more compositions are stimulated compositions. In certain embodiments, one or more stimulated compositions are cryopreserved and stored beforehand and thawed before manipulation.

[0199] In some specific embodiments, one or more compositions of stimulated T cells are or comprise two distinct stimulated compositions of enriched T cells. In some specific embodiments, two distinct compositions of enriched T cells, e.g., selected, isolated, and / or enriched from the same biological sample, are operated separately. In some specific embodiments, the two distinct compositions comprise a composition of enriched CD4+ T cells. In some specific embodiments, the two distinct compositions comprise a composition of enriched CD8+ T cells. In some embodiments, two distinct compositions of enriched CD4+ T cells and enriched CD8+ T cells, such as after incubation under the stimulation conditions described above, are genetically engineered separately. In some embodiments, a single composition of enriched T cells is genetically engineered. In some specific embodiments, the single composition is a composition of enriched CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells combined from distinct compositions before the operation.

[0200] In some embodiments, a composition of enriched CD4+ T cells, such as engineered, for example transfected or transfected, stimulated CD4+ T cells, contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In some specific embodiments, a composition of enriched CD4+ T cells, such as engineered, stimulated CD4+ T cells, contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells and / or does not contain CD8+ T cells and / or is CD8+ T cell-free or substantially CD8+ T cell-free.

[0201] In some embodiments, a composition of enriched CD8+ T cells, such as engineered, for example transfected or transfected, stimulated CD8+ T cells, contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells. In some specific embodiments, a composition of enriched CD8+ T cells, such as engineered, stimulated CD8+ T cells, contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells and / or does not contain CD4+ T cells and / or is CD4+ T cell-free or substantially CD4+ T cell-free.

[0202] In some embodiments, separate compositions of enriched CD4+ and CD8+ T cells are combined into a single composition and genetically modified, e.g., transfected or transfected. In some specific embodiments, separate modified compositions of enriched CD4+ and enriched CD8+ T cells are combined into a single composition after genetic modification has been performed and / or completed. In certain embodiments, separate compositions of enriched CD4+ and CD8+ T cells, such as separate compositions of stimulated CD4+ and CD8+ T cells, are modified separately and processed separately for T cell culture and / or expansion after genetic modification has been performed and / or completed.

[0203] In some embodiments, the introduction of polynucleotides, such as recombinant polynucleotides encoding recombinant proteins, is carried out by contacting enriched CD4+ or CD8+ T cells, such as stimulated CD4+ or CD8+ T cells, with viral particles containing polynucleotides. In some embodiments, contact can be carried out using centrifugation, such as spinoculation (e.g., centrifugation inoculation). In some embodiments, the composition comprising cells, viral particles, and reagents can generally be rotated at a relatively low force or speed, for example, a speed lower than the speed used to pelletize cells, such as 600 rpm to 1700 rpm or about 600 rpm to about 1700 rpm (e.g., 600 rpm or about 600 rpm or at least 600 rpm, 1000 rpm or about 1000 rpm or at least 1000 rpm, or 1500 rpm or about 1500 rpm or at least 1500 rpm, or 1700 rpm or about 1700 rpm or at least 1700 rpm).In some embodiments, rotation is measured, for example, at the inner or outer wall of the chamber or cavity, in the range of 100g to 3200g or approximately 100g to approximately 3200g (e.g., 100g or approximately 100g or at least 100g or at least approximately 100g, 200g or approximately 200g or at least 200g or at least approximately 200g, 300g or approximately 300g or at least 300g or at least approximately 300g, 400g or approximately 400g or at least 400g or at least approximately 400g, 500g or approximately 500g or at least 500g or at least approximately 500g, 1000g or approximately 10 The force is such that 00g or at least 1000g or at least about 1000g, 1500g or about 1500g or at least 1500g or at least about 1500g, 2000g or about 2000g or at least 2000g or at least about 2000g, 2500g or about 2500g or at least 2500g or at least about 2500g, 3000g or about 3000g or at least 3000g or at least about 3000g, or 3200g or about 3200g or at least 3200g or at least about 3200g), for example 693g or about 693g, for example, relative centrifugal force. The term “relative centrifugal force” or RCF is generally understood to be the effective force exerted on an object or substance (such as a cell, sample, or pellet, and / or a point in a rotating chamber or other container) relative to Earth’s gravity at a particular point in space with respect to the axis of rotation. This value can be determined using a well-known formula, taking into account gravity, rotational speed, and radius of rotation (the distance from the axis of rotation to the object, substance, or particle on which the RCF is measured). In some embodiments, at least part of the contact with, incubation with, and / or manipulation with the virus of cells, e.g., cells derived from a stimulated composition of enriched CD4+ T cells or enriched CD8+ T cells, is carried out at rotations of about 100g to 3200g, about 1000g to 2000g, about 1000g to 3200g, about 500g to 1000g, about 400g to 1200g, about 600g to 800g, about 600g to 700g, or about 500g to 700g.In some embodiments, the rotation is 600g to 700g, for example, 693g or approximately 693g.

[0204] In some specific embodiments, at least part of the operation, transduction, and / or transfection is carried out using rotation, e.g., spinoculation and / or centrifugation. In some embodiments, the rotation is performed for 5 minutes or about 5 minutes or at least 5 minutes or at least about 5 minutes, 10 minutes or about 10 minutes or at least 10 minutes or at least about 10 minutes, 15 minutes or about 15 minutes or at least 15 minutes or at least about 15 minutes, 30 minutes or about 30 minutes or at least 30 minutes or about 30 minutes, 60 minutes or about 60 minutes or at least 60 minutes or at least about 60 minutes, 90 minutes or about 90 minutes or at least 90 minutes or at least about 90 minutes, 1 hour or about 1 hour or at least 1 hour or at least about 1 hour, 2 hours or about 2 hours or at least 2 hours or at least about 2 hours, 3 hours or about 3 hours or at least 3 hours or at least about 3 hours, 4 hours or about 4 hours or at least 4 hours or at least about 4 hours, 6 hours or about 6 hours or less The rotation may last for 6 hours or at least about 6 hours, 8 hours or at least about 8 hours or at least about 8 hours, 12 hours or at least about 12 hours or at least about 12 hours, 24 hours or at least about 24 hours or at least about 24 hours, 48 ​​hours or at least about 48 hours or at least about 48 hours, 72 hours or at least about 72 hours or at least about 72 hours, 2 days or at about 2 days or at least about 2 days, 3 days or at about 3 days or at least about 3 days, 4 days or at about 4 days or at least about 4 days, 5 days or at about 5 days or at least about 5 days, 6 days or at about 6 days or at least about 6 days, or at least about 7 days. In some embodiments, the rotation may last for 60 minutes or about 60 minutes. In some specific embodiments, the rotation may last for about 30 minutes.In some embodiments, the rotation is performed at 600g to 700g, for example, 693g or approximately 693g, for about 30 minutes.

[0205] In some specific embodiments, the number of living cells manipulated, transfected, and / or transfected is approximately 5 × 10 6 Cells ~ approx. 100×10 7 Cells, for example, about 10 x 10 6 Cells ~ approx. 100×10 6 cells, approximately 100 x 10 6 Cells ~ approx. 200×10 6 cells, approximately 200 x 10 6 Cells ~ approx. 300×10 6 Cells, approximately 300 x 10 6 Cells ~ approx. 400×10 6 Cells, approximately 400 x 10 6 Cells ~ approx. 500×10 6 Cells, or approximately 500 x 10 6 Cells ~ approx. 100×10 7 The range of cells is extensive. In a particular example, the number of living cells manipulated, transduced, and / or transfected is approximately 300 × 10⁶. 6 Cells or approximately 300 x 10 6 It is smaller than a cell.

[0206] In some specific embodiments, at least part of the manipulation, transduction, and / or transfection is performed in a volume of approximately 5 mL to approximately 100 mL, e.g., approximately 10 mL to approximately 50 mL, approximately 15 mL to approximately 45 mL, approximately 20 mL to approximately 40 mL, approximately 25 mL to approximately 35 mL, or 30 mL or approximately 30 mL (e.g., spinocuration volume). In some specific embodiments, the post-spinocuration cell pellet volume ranges from approximately 1 mL to approximately 25 mL, e.g., approximately 5 mL to approximately 20 mL, approximately 5 mL to approximately 15 mL, approximately 5 mL to approximately 10 mL, or 10 mL or approximately 10 mL.

[0207] In some embodiments, gene transfer is achieved by first stimulating cells, such as by combining them with stimuli that induce responses such as proliferation, survival, and / or activation, as measured by the expression of cytokines or activation markers, followed by transduction of the activated cells and expansion to a number sufficient for clinical application under culture. In some specific embodiments, gene transfer is achieved by first incubating the cells under stimulating conditions, such as by one of the methods described in Section IB.

[0208] In some embodiments, methods for genetic manipulation are carried out by contacting one or more cells of a composition with a recombinant protein, such as a nucleic acid molecule encoding a recombinant receptor. In some embodiments, contact can be carried out using centrifugation, such as spinoculation (e.g., centrifugation inoculation). Such methods include any of the methods described in International Publication No. 2016 / 073602. Exemplary centrifugation chambers include those for use in Sepax® and Sepax® 2 systems, including the A-200 / F and A-200 centrifugation chambers, and various kits for use in such systems, manufactured and sold by Biosafe SA. Exemplary chambers, systems, and processing equipment and cabinets are described, for example, in U.S. Patent No. 6,123,655, U.S. Patent No. 6,733,433 and U.S. Patent Application Publication No. 2008 / 0171951 and International Publication No. 00 / 38762, the contents of which are incorporated herein by reference in their entirety. Examples of kits for use in such systems include, but are not limited to, the single-use kits sold by BioSafe SA under the product names CS-430.1, CS-490.1, CS-600.1, or CS-900.2.

[0209] In some embodiments, the system is arranged with and / or in conjunction with other equipment, including equipment for operating, automating, controlling and / or monitoring aspects of one or more different processing steps carried out in the system, such as those of the centrifugal chamber system described herein or in connection with International Publication No. 2016 / 073602. In some embodiments, this equipment is contained within a cabinet. In some embodiments, the equipment includes a cabinet containing a housing with a control circuit, a centrifuge, a cover, a motor, a pump, sensors, a display, and a user interface. Exemplary devices are described in U.S. Patent No. 6,123,655, U.S. Patent No. 6,733,433 and U.S. Patent Application No. 2008 / 0171951.

[0210] In some embodiments, the system includes a series of containers, such as bags, tubes, stopcocks, clamps, connectors, and a centrifuge chamber. In some embodiments, the containers, such as bags, include one or more containers, such as bags, that contain transduced cells and viral vector particles in the same container or separate containers, for example, in the same bag or separate bags. In some embodiments, the system further includes one or more containers, such as a medium, such as a diluent and / or washing solution, drawn into the chamber, and / or bags containing other components for diluting, resuspending, and / or washing components and / or compositions during the process. The containers can be connected to one or more locations in the system, for example, locations corresponding to input lines, diluent lines, washing lines, waste lines, and / or output lines.

[0211] In some embodiments, the chamber is coupled with a centrifuge that can cause the chamber to rotate, such as around its axis of rotation. The rotation may occur before, during, and / or after incubation related to cell transduction, and / or in one or more other processing steps. Thus, in some embodiments, one or more of the various processing steps are carried out under rotation, for example, with a specific force. The chamber is typically capable of vertical or approximately vertical rotation, such that the chamber is vertically positioned during centrifugation, with the side walls and axis perpendicular or approximately perpendicular, and the end walls (one or more) horizontal or approximately horizontal.

[0212] In some embodiments, a composition containing cells and a composition containing viral vector particles, and optionally air, may be combined or mixed before the compositions are provided to the cavity. In some embodiments, a composition containing cells and a composition containing viral vector particles, and optionally air, may be provided separately and combined and mixed within the cavity. In some embodiments, a composition containing cells, a composition containing viral vector particles, and optionally air may be provided to the internal cavity in any order. In any of these embodiments, a composition containing cells and viral vector particles is an input composition when combined or mixed together, regardless of whether it is combined or mixed inside or outside the centrifugation chamber, and / or whether the cells and viral vector particles are provided to the centrifugation chamber together or separately, for example, simultaneously or sequentially.

[0213] In some embodiments, the incorporation of a certain volume of gas, such as air, is performed before the incubation of cells and viral vector particles, such as rotation in transduction methods. In some embodiments, the incorporation of a certain volume of gas, such as air, is performed during the incubation of cells and viral vector particles, such as rotation in transduction methods.

[0214] In some embodiments, the liquid volume of the cell or viral vector particles constituting the transduction composition, and optionally the volume of air, may be predetermined volumes. These volumes may be programmed into and / or controlled by the system.

[0215] In some embodiments, the intake of a transdermal composition and, optionally, a gas such as air, is controlled manually, semi-automatically, and / or automatically until a desired or predetermined volume is taken into the internal cavity of the chamber. In some embodiments, sensors associated with the system can detect liquids and / or gases entering and leaving the centrifugal chamber by their color, flow rate, and / or density, etc., and communicate with associated circuits to stop or continue the intake as needed until such desired or predetermined volume is achieved. In some aspects, sensors programmed to detect only liquids in the system and not gases (e.g., air), or capable of detecting gases in this way, may allow gases such as air to pass through the system without stopping the intake. In some such embodiments, opaque portions of tubing can be placed in the line near the sensors while the intake of gases such as air is desired. In some embodiments, the intake of gases such as air can be controlled manually.

[0216] In aspects of the provided method, the internal cavity of the centrifugal chamber is subjected to high-speed rotation. In some embodiments, the rotation is performed before, simultaneously with, after, or intermittently with, the intake of the liquid input composition and optionally air. In some embodiments, the rotation is performed after the intake of the liquid input composition and optionally air. In some embodiments, the rotation is 800g or approximately 800g or at least 800g or at least approximately 800g, 1000g or approximately 1000g or at least 1000g or at least approximately 1000g, 1100g or approximately 1100g or at least 1100g or at least approximately 1100g, 1500 or approximately 1500 or at least 1500 or at least approximately 1500, 1600g or approximately 1600g or at least 1600g or at least approximately 1600g, 1800g or approximately 1800g or at least 1800g or at least approximately 1800g, 2000g or approximately 2000g or at least 200 This is achieved by centrifugation of a centrifugal chamber using relative centrifugal force on the inner surface of the side walls of the internal cavity and / or the surface layer of the cells, resulting in a volume of 0g or at least approximately 2000g, 2200g or approximately 2200g or at least approximately 2200g, 2500g or approximately 2500g or at least approximately 2500g, 3000g or approximately 3000g or at least approximately 3000g, 3500g or approximately 3500g or at least approximately 3500g, or 4000g or approximately 4000g or at least 4000g or at least approximately 4000g. In some embodiments, the rotation is due to centrifugal separation with a force greater than or about 1100g, for example, greater than or about 1200g, greater than or about 1400g, greater than or about 1600g, greater than or about 1800g, greater than or about 2000g, greater than or about 2400g, greater than or about 2800g, greater than or about 3000g, or greater than or about 3200g.In some embodiments, the rotation is achieved by centrifugal separation with a force of 1600g or approximately 1600g.

[0217] In some embodiments, the transduction method includes rotating or centrifugating the transduction composition and optionally air in a centrifugal chamber for more than 5 minutes or about 5 minutes, for example, more than 10 minutes or about 10 minutes, more than 15 minutes or about 15 minutes, more than 20 minutes or about 20 minutes, more than 30 minutes or about 30 minutes, more than 45 minutes or about 45 minutes, more than 60 minutes or about 60 minutes, more than 90 minutes or about 90 minutes, or more than 120 minutes or about 120 minutes. In some embodiments, the transduction composition and optionally air are rotated or centrifuged in a centrifugal chamber for longer than 5 minutes but 60 minutes or less, 45 minutes or less, 30 minutes or less, or 15 minutes or less. In certain embodiments, transduction includes rotating or centrifugating for 60 minutes or about 60 minutes.

[0218] In some embodiments, the transduction method includes values ​​at both ends, ranging from 10 to 60 minutes or approximately 10 to 60 minutes, 15 to 60 minutes or approximately 15 to 60 minutes, 15 to 45 minutes or approximately 15 to 45 minutes, 30 to 60 minutes or approximately 30 to 60 minutes, or 45 to 60 minutes or approximately 45 to 60 minutes, with a minimum of 1000g or more than 1000g or approximately 1000g, at least 1100g or more than 1100g or approximately 1100g, at least 1200g or more than 1200g or approximately 1200g, at least 1400g or more than 1400g or approximately 1400g, at least 1500g or more than 1500g or approximately 1500g, and less A transduction composition in a centrifugal chamber, applied by force on the inner surface of the side walls of the internal cavity and / or the surface layer of the cells, at least 1600g or more than 1600g or about 1600g, at least 1800g or more than 1800g or about 1800g, at least 2000g or more than 2000g or about 2000g, at least 2200g or more than 2200g or about 2200g, at least 2400g or more than 2400g or about 2400g, at least 2800g or more than 2800g or about 2800g, at least 3200g or more than 3200g or about 3200g, or at least 3600g or more than 3600g or about 3600g, and / or the surface layer of the cells, and optionally includes rotation of air or centrifugation. In a particular embodiment, the transduction method comprises rotating or centrifugating a transduction composition, such as cells and viral vector particles, at 1600 g or about 1600 g for 60 minutes or about 60 minutes.

[0219] In some embodiments, a gas, such as air, in the chamber cavity is released from the chamber. In some embodiments, the gas, such as air, is released into a container functionally connected to the centrifugal chamber as part of a closed system. In some embodiments, the container is empty or contains nothing. In some embodiments, the gas, such as air, in the chamber cavity is released through a filter functionally connected to the internal cavity of the chamber via a sterile tubular line. In some embodiments, the air is released using a manual, semi-automated, or automated process. In some embodiments, air is released from the chamber cavity intermittently or after extrusion, simultaneously with extrusion, before extruding an output composition containing incubated cells, such as cells transduced or cells transduced with a viral vector, and viral vector particles.

[0220] In some embodiments, transduction and / or other incubations are carried out as a continuous or semi-continuous process, or as part thereof. In some embodiments, the continuous process includes the continuous incorporation of cells and viral vector particles, e.g., transduction compositions (either as a single existing composition or by continuously drawing them into the same container, e.g., cavity, and thereby mixing them) for at least part of the incubation, e.g., during centrifugation, and / or the continuous extrusion or discharge of liquid from the container, and optionally the release of gas (e.g., air). In some embodiments, the continuous incorporation and continuous extrusion are carried out at least partially simultaneously. In some embodiments, the continuous incorporation is carried out during part of the incubation, e.g., during part of centrifugation, and the continuous extrusion is carried out during another part of the incubation. These two may be carried out alternately. Thus, continuous incorporation and extrusion allow for processing, e.g., transduction, of a larger overall volume of sample while carrying out the incubation.

[0221] In some embodiments, incubation is part of a continuous process, and the method includes, at least for part of the incubation, carrying out continuous intake of the transdermal composition into the cavity while the chamber is rotating and during part of the incubation, continuous extrusion of the liquid from the cavity through at least one opening while the chamber is rotating, and optionally releasing a gas (e.g., air).

[0222] In some embodiments, semi-continuous incubation is carried out by alternating between taking up a composition into a cavity, incubation, purging the liquid from the cavity into an output container, and optionally releasing a gas (e.g., air) from the cavity, and then taking up subsequent compositions (e.g., second, third, etc.) containing more cells and other reagents for processing, such as viral vector particles, and repeating the process. For example, in some embodiments, incubation is part of a semi-continuous process, and the method includes taking up a transduction composition into a cavity through the at least one opening before incubation, and purging the liquid from the cavity after incubation; taking up another transduction composition containing cells and viral vector particles into the internal cavity; and incubating the other transduction composition in the internal cavity under conditions in which cells in the other transduction composition are transduced with the vector. This process may be continued iteratively over several further rounds. In this respect, semi-continuous or continuous methods may allow for the production of even larger volumes and / or numbers of cells.

[0223] In some embodiments, part of the transduction incubation is carried out in a centrifugal chamber under conditions including rotation or centrifugation.

[0224] In some embodiments, the method includes incubation in which a further portion of the incubation of cells and viral vector particles is carried out without rotation or centrifugation, which generally follows at least a portion of incubation that includes rotation or centrifugation of the chamber. In some specific embodiments, the incubation of cells and viral vector particles is carried out without rotation or centrifugation for at least 1 hour, at least 6 hours, at least 12 hours, at least 24 hours, at least 32 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 90 hours, at least 96 hours, at least 3 days, at least 4 days, at least 5 days, or more than 5 days. In some specific embodiments, the incubation is carried out for 72 hours or about 72 hours.

[0225] In some such embodiments, further incubation is carried out under conditions that result in the integration of the viral vector into the host genome of one or more cells. Evaluating or determining whether incubation has resulted in the integration of viral vector particles into the host genome, and thus empirically determining the conditions for further incubation, is within the scope of the art. In some embodiments, the integration of the viral vector into the host genome can be evaluated by measuring the expression level of recombinant proteins, such as heterologous proteins, encoded by nucleic acids contained in the genome of the viral vector particles after incubation. Many well-known methods for evaluating the expression level of recombinant molecules can be used, for example, in relation to cell surface proteins, such as affinity-based detection, such as immunoaffinity detection, and such as flow cytometry detection. In some examples, expression is measured by the detection of transduction markers and / or reporter constructs. In some embodiments, nucleic acids encoding truncated surface proteins are included in the vector and used as markers for expression and / or enhancement thereof.

[0226] In some embodiments, compositions comprising cells, vectors, such as viral particles, and reagents can generally be rotated with relatively low force or speed, at speeds lower than, for example, the speed used to pelletize cells, such as 600 rpm to 1700 rpm or about 600 rpm to about 1700 rpm (e.g., 600 rpm or about 600 rpm or at least 600 rpm, 1000 rpm or about 1000 rpm or at least 1000 rpm, or 1500 rpm or about 1500 rpm or at least 1500 rpm, or 1700 rpm or about 1700 rpm or at least 1700 rpm). In some embodiments, rotation is measured, for example, at the inner or outer wall of the chamber or cavity, from 100g to 3200g or approximately 100g to approximately 3200g (e.g., 100g or approximately 100g or at least 100g or at least approximately 100g, 200g or approximately 200g or at least 200g or at least approximately 200g, 300g or approximately 300g or at least 300g or at least approximately 300g, 400g or approximately 400g or at least 400g or at least approximately 400g, 500g or approximately 500g or at least 500g or at least approximately 500g, 1000 The force is exerted by a force of g or 1000g or at least 1000g or at least about 1000g, 1500g or about 1500g or at least 1500g or at least about 1500g, 2000g or about 2000g or at least 2000g or at least about 2000g, 2500g or about 2500g or at least 2500g or at least about 2500g, 3000g or about 3000g or at least 3000g or at least about 3000g, or 3200g or about 3200g or at least 3200g or at least about 3200g, for example, relative centrifugal force. The term “relative centrifugal force” or RCF is generally understood to be the effective force exerted on an object or substance (such as a cell, sample, or pellet, and / or a point in a rotating chamber or other container) against Earth’s gravity at a particular point in space relative to the axis of rotation.This value can be determined using a well-known formula, taking into account gravity, rotational speed, and radius of rotation (the distance from the axis of rotation to the object, substance, or particle on which the RCF is measured).

[0227] In some embodiments, at least part of the genetic manipulation, for example during transduction and / or following the genetic manipulation, the cells are transferred to a bioreactor bag assembly for culturing the genetically modified cells, such as cell culture or expansion as described above.

[0228] In some specific embodiments, the enriched T cell composition is operated in the presence of a transduction adjuvant, for example, transduced or transfected. In some embodiments, the enriched T cell composition is operated in the presence of one or more polycations. In some embodiments, the enriched T cell composition is transduced in the presence of one or more transduction adjuvants and incubated, for example, with viral vector particles. In certain embodiments, the enriched T cell composition is transfected in the presence of one or more transduction adjuvants and incubated, for example, with a non-viral vector. In some specific embodiments, the presence of one or more transduction adjuvants enhances the efficiency of gene delivery, such as by increasing the amount, portion, and / or percentage of cells in the composition being operated (e.g., transduced or transfected). In some specific embodiments, the presence of one or more transduction adjuvants enhances the efficiency of transfection. In some specific embodiments, the presence of one or more transduction adjuvants enhances the efficiency of transduction. In certain embodiments, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of cells manipulated in the presence of polycations contain or express recombinant polynucleotides. In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 25x, at least 50x, or at least 100x more cells of the composition are manipulated to contain or express recombinant transduction adjuvants in the presence of polycations compared to alternative and / or exemplary methods of manipulating cells without the presence of transduction adjuvants.

[0229] In some embodiments, the enriched cell composition is manipulated in the presence of a transduction adjuvant in the presence of less than 100 μg / ml, less than 90 μg / ml, less than 80 μg / ml, less than 75 μg / ml, less than 70 μg / ml, less than 60 μg / ml, less than 50 μg / ml, less than 40 μg / ml, less than 30 μg / ml, less than 25 μg / ml, less than 20 μg / ml, or less than μg / ml or less than 10 μg / ml. In some specific embodiments, transduction adjuvants suitable for use in the provided method include, but are not limited to, polycations, fibronectin or fibronectin-derived fragments or variants, RetroNectin, and combinations thereof.

[0230] In some aspects, cells are manipulated in the presence of cytokines, such as recombinant human cytokines, at concentrations of 1 IU / ml to 1,000 IU / ml, 10 IU / ml to 50 IU / ml, 50 IU / ml to 100 IU / ml, 100 IU / ml to 200 IU / ml, 100 IU / ml to 500 IU / ml, 250 IU / ml to 500 IU / ml, or 500 IU / ml to 1,000 IU / ml.

[0231] In some embodiments, enriched T cell compositions are manipulated in the presence of IL-2 at concentrations of 1 IU / ml to 200 IU / ml, 10 IU / ml to 100 IU / ml, 50 IU / ml to 150 IU / ml, 80 IU / ml to 120 IU / ml, 60 IU / ml to 90 IU / ml, or 70 IU / ml to 90 IU / ml, such as human recombinant IL-2. In a particular embodiment, the enriched T cell composition is 50 IU / ml or approximately 50 IU / ml, 55 IU / ml or approximately 55 IU / ml, 60 IU / ml or approximately 60 IU / ml, 65 IU / ml or approximately 65 IU / ml, 70 IU / ml or approximately 70 IU / ml, 75 IU / ml or approximately 75 IU / ml, 80 IU / ml or approximately 80 IU / ml, 85 IU / ml or approximately 85 IU / ml, or 90 IU / ml Alternatively, the composition is manipulated in the presence of recombinant IL-2 at concentrations of approximately 90 IU / ml, 95 IU / ml, 100 IU / ml, 110 IU / ml, 120 IU / ml, 130 IU / ml, 140 IU / ml, or 150 IU / ml. In some embodiments, the enriched T cell composition is manipulated in the presence of 85 IU / ml or approximately 85 IU / ml. In some embodiments, the T cell population is a population of CD4+ T cells. In certain embodiments, the enriched T cell composition is enriched with CD4+ T cells, but not with CD8+ T cells, and / or CD8+ T cells are negatively selected from the composition or depleted from the composition. In a particular embodiment, the enriched T cell composition is a enriched CD8+ T cell composition. In a particular embodiment, the enriched T cell composition is enriched with CD8+ T cells, but not with CD4+ T cells, and / or CD4+ T cells are negatively selected or depleted from the composition.

[0232] In some embodiments, enriched T cell compositions are manipulated in the presence of recombinant IL-7, such as human recombinant IL-7, at concentrations of 100 IU / ml to 2,000 IU / ml, 500 IU / ml to 1,000 IU / ml, 100 IU / ml to 500 IU / ml, 500 IU / ml to 750 IU / ml, 750 IU / ml to 1,000 IU / ml, or 550 IU / ml to 650 IU / ml. In a particular embodiment, the enriched T cell composition is 50 IU / ml or approximately 50 IU / ml, 100 IU / ml or approximately 100 IU / ml, 150 IU / ml or approximately 150 IU / ml, 200 IU / ml or approximately 200 IU / ml, 250 IU / ml or approximately 250 IU / ml, 300 IU / ml or approximately 300 IU / ml, 350 IU / ml or approximately 350 IU / ml, 400 IU / ml or approximately 400 IU / ml, 450 IU / ml or approximately 450 IU / ml, 500 IU / ml or approximately 500 IU / ml, 550 The compositions are manipulated in the presence of IL-7 at concentrations of IU / ml or approximately 550 IU / ml, 600 IU / ml or approximately 600 IU / ml, 650 IU / ml or approximately 650 IU / ml, 700 IU / ml or approximately 700 IU / ml, 750 IU / ml or approximately 750 IU / ml, 800 IU / ml or approximately 800 IU / ml, 750 IU / ml or approximately 750 IU / ml, 750 IU / ml or approximately 750 IU / ml, 750 IU / ml or approximately 750 IU / ml, or 1,000 IU / ml or approximately 1,000 IU / ml. In certain embodiments, the enriched T cell composition is manipulated in the presence of 600 IU / ml or approximately 600 IU / ml of IL-7. In some embodiments, the composition manipulated in the presence of recombinant IL-7 is enriched with a population of T cells, such as CD4+ T cells. In a particular embodiment, the enriched T cell composition is enriched with CD4+ T cells, but not with CD8+ T cells, and / or CD8+ T cells are negatively selected from the composition or depleted from the composition.

[0233] In some embodiments, enriched T cell compositions are manipulated in the presence of recombinant IL-15, such as human recombinant IL-15, at concentrations of 0.1 IU / ml to 100 IU / ml, 1 IU / ml to 50 IU / ml, 5 IU / ml to 25 IU / ml, 25 IU / ml to 50 IU / ml, 5 IU / ml to 15 IU / ml, or 10 IU / ml to 100 IU / ml. In a particular embodiment, the enriched T cell composition may be 1 IU / ml or approximately 1 IU / ml, 2 IU / ml or approximately 2 IU / ml, 3 IU / ml or approximately 3 IU / ml, 4 IU / ml or approximately 4 IU / ml, 5 IU / ml or approximately 5 IU / ml, 6 IU / ml or approximately 6 IU / ml, 7 IU / ml or approximately 7 IU / ml, 8 IU / ml or approximately 8 IU / ml, 9 IU / ml or approximately 9 IU / ml, 10 IU / ml or approximately 10 IU / ml, 11 IU / ml, or The enriched T cell composition is operated in the presence of IL-15 at concentrations of approximately 11 IU / ml, 12 IU / ml, 13 IU / ml, 14 IU / ml, 15 IU / ml, 20 IU / ml, 25 IU / ml, 30 IU / ml, 40 IU / ml, or 50 IU / ml. In some embodiments, the enriched T cell composition is operated in 10 IU / ml or approximately 10 IU / ml of IL-15. In some embodiments, the enriched T cell composition is incubated in 10 IU / ml or approximately 10 IU / ml of recombinant IL-15. In some embodiments, a composition manipulated in the presence of recombinant IL-15 is enriched with a population of T cells, e.g., CD4+ T cells and / or CD8+ T cells. In some embodiments, the enriched T cell composition is a composition enriched with CD8+ T cells. In certain embodiments, the enriched T cell composition is enriched with CD8+ T cells, but not with CD4+ T cells, and / or CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the enriched T cell composition is a composition enriched with CD4+ T cells.In a particular embodiment, the enriched T cell composition is enriched with CD4+ T cells, but not with CD8+ T cells, and / or CD8+ T cells are negatively selected from the composition or depleted from the composition.

[0234] In certain embodiments, the enriched CD8+ T cell composition is manipulated in the presence of IL-2 and / or IL-15. In some specific embodiments, the enriched CD4+ T cell composition is manipulated in the presence of IL-2, IL-7, and / or IL-15. In some embodiments, IL-2, IL-7, and / or IL-15 are recombinant. In some specific embodiments, IL-2, IL-7, and / or IL-15 are human. In certain embodiments, one or more cytokines are or contain human recombinant IL-2, IL-7, and / or IL-15.

[0235] In certain embodiments, cells are manipulated in the presence of one or more antioxidants. In some embodiments, one or more antioxidants include tocopherol, tocotrienol, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, α-tocopherolquinone, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, Examples of such substances include, but are not limited to, isoflavones, lycopene, beta-carotene, selenium, ubiquinone, ruetin, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine ​​(NAC), citric acid, L-carnitine, BHT, monothioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine and cystathionine, and / or glycine-glycine-histidine.

[0236] In some embodiments, one or more antioxidants are sulfur-containing oxidizing agents or comprise a sulfur-containing oxidizing agent. In some specific embodiments, the sulfur-containing antioxidant may comprise a thiol-containing antioxidant and / or an antioxidant exhibiting one or more sulfur moieties in a ring structure, for example. In some embodiments, the sulfur-containing antioxidant may comprise, for example, N-acetylcysteine ​​(NAC) and 2,3-dimercaptopropanol (DMP), L-2-oxo-4-thiazolidine carboxylate (OTC), and lipoic acid. In certain embodiments, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified in one or more intracellular processes to yield glutathione. In certain embodiments, the glutathione precursor may comprise, but is not limited to, N-acetylcysteine ​​(NAC), L-2-oxothiazolidine-4-carboxylic acid (procysteine), lipoic acid, S-allylcysteine, or methylmethionine sulfonium chloride.

[0237] In some embodiments, cells are manipulated in the presence of one or more antioxidants. In some embodiments, cells are manipulated in the presence of one or more antioxidants in concentrations of 1 ng / ml to 100 ng / ml, 10 ng / ml to 1 μg / ml, 100 ng / ml to 10 μg / ml, 1 μg / ml to 100 μg / ml, 10 μg / ml to 1 mg / ml, 100 μg / ml to 1 mg / ml, 1500 μg / ml to 2 mg / ml, 500 μg / ml to 5 mg / ml, 1 mg / ml to 10 mg / ml, or 1 mg / ml to 100 mg / ml. In some aspects, the cells are 1 ng / ml or approximately 1 ng / ml, 10 ng / ml or approximately 10 ng / ml, 100 ng / ml or approximately 100 ng / ml, 1 μg / ml or approximately 1 μg / ml, 10 μg / ml or approximately 10 μg / ml, 100 μg / ml or approximately 100 μg / ml, 0.2 mg / ml or approximately 0.2 mg / ml, 0.4 mg / ml or approximately 0.4 mg / ml, 0.6 mg / ml or approximately 0.6 mg / ml, 0.8 mg / ml or approximately 0.8 mg / ml, 1 mg / ml or approximately 1 mg / ml, 2 mg / ml or approximately 2 mg / ml, 3 mg The process is carried out in the presence of one or more antioxidants in amounts of / ml or approximately 3 mg / ml, 4 mg / ml or approximately 4 mg / ml, 5 mg / ml or approximately 5 mg / ml, 10 mg / ml or approximately 10 mg / ml, 20 mg / ml or approximately 20 mg / ml, 25 mg / ml or approximately 25 mg / ml, 50 mg / ml or approximately 50 mg / ml, 100 mg / ml or approximately 100 mg / ml, 200 mg / ml or approximately 200 mg / ml, 300 mg / ml or approximately 300 mg / ml, 400 mg / ml or approximately 400 mg / ml, 500 mg / ml or approximately 500 mg / ml. In some embodiments, one or more antioxidants are sulfur-containing antioxidants or comprise sulfur-containing antioxidants. In certain embodiments, one or more antioxidants are glutathione precursors or comprise glutathione precursors.

[0238] In some embodiments, cells are manipulated in the presence of NAC. In some embodiments, cells are manipulated in the presence of NAC in concentrations of 1 ng / ml to 100 ng / ml, 10 ng / ml to 1 μg / ml, 100 ng / ml to 10 μg / ml, 1 μg / ml to 100 μg / ml, 10 μg / ml to 1 mg / ml, 100 μg / ml to 1 mg / ml, 1500 μg / ml to 2 mg / ml, 500 μg / ml to 5 mg / ml, 1 mg / ml to 10 mg / ml, or 1 mg / ml to 100 mg / ml. In some embodiments, the cells are 1 ng / ml or approximately 1 ng / ml, 10 ng / ml or approximately 10 ng / ml, 100 ng / ml or approximately 100 ng / ml, 1 μg / ml or approximately 1 μg / ml, 10 μg / ml or approximately 10 μg / ml, 100 μg / ml or approximately 100 μg / ml, 0.2 mg / ml or approximately 0.2 mg / ml, 0.4 mg / ml or approximately 0.4 mg / ml, 0.6 mg / ml or approximately 0.6 mg / ml, 0.8 mg / ml or approximately 0.8 mg / ml, 1 mg / ml or approximately 1 mg / ml, 2 mg / ml or approximately 2 mg / ml The cells are manipulated in the presence of NAC at concentrations of 3 mg / ml or approximately 3 mg / ml, 4 mg / ml or approximately 4 mg / ml, 5 mg / ml or approximately 5 mg / ml, 10 mg / ml or approximately 10 mg / ml, 20 mg / ml or approximately 20 mg / ml, 25 mg / ml or approximately 25 mg / ml, 50 mg / ml or approximately 50 mg / ml, 100 mg / ml or approximately 100 mg / ml, 200 mg / ml or approximately 200 mg / ml, 300 mg / ml or approximately 300 mg / ml, 400 mg / ml or approximately 400 mg / ml, 500 mg / ml or approximately 500 mg / ml. In some embodied methods, the cells are manipulated at 0.8 mg / ml or approximately 0.8 mg / ml.

[0239] In some embodiments, a composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is manipulated in the presence of one or more polycations. In some embodiments, a composition of enriched T cells, such as stimulated CD4+ T cells or stimulated CD8+ T cells, is transfected in the presence of one or more polycations and incubated, for example, with viral vector particles. In certain embodiments, a composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is transfected in the presence of one or more polycations and incubated, for example, with a non-viral vector. In some specific embodiments, the presence of one or more polycations enhances the efficiency of gene delivery, such as by increasing the amount, portion, and / or percentage of cells in the composition being manipulated (e.g., transfected or transfected). In some specific embodiments, the presence of one or more polycations enhances the efficiency of transfection. In some specific embodiments, the presence of one or more polycations enhances the efficiency of transfection. In certain embodiments, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of cells manipulated in the presence of polycations contain or express recombinant polynucleotides.In some embodiments, cells are manipulated to contain or express recombinant polynucleotides in the presence of polycations at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 25x, at least 50x, or at least 100x more cells of the composition compared to alternative and / or exemplary methods of manipulating cells without the presence of polycations.

[0240] In some specific embodiments, enriched cell compositions, such as enriched CD4+ T cells or enriched CD8+ T cells, or compositions of the stimulated T cells, are operated in the presence of lower concentrations or amounts of polycations compared to exemplary and / or alternative methods for operating cells in the presence of polycations, for example. In some specific embodiments, enriched cell compositions, such as stimulated T cells, such as stimulated CD4+ T cells or stimulated CD8+ T cells, are operated in the presence of amounts and / or concentrations of polycations less than 90%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the amount and / or concentration of polycations compared to exemplary and / or alternative processes for operating cells. In some embodiments, enriched cell compositions such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, are manipulated in the presence of polycations in concentrations of less than 100 μg / ml, less than 90 μg / ml, less than 80 μg / ml, less than 75 μg / ml, less than 70 μg / ml, less than 60 μg / ml, less than 50 μg / ml, less than 40 μg / ml, less than 30 μg / ml, less than 25 μg / ml, less than 20 μg / ml, or less than μg / ml or less than 10 μg / ml. In a particular embodiment, a composition of enriched cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is manipulated in the presence of polycations at a concentration of 1 μg / ml or approximately 1 μg / ml, 5 μg / ml or approximately 5 μg / ml, 10 μg / ml or approximately 10 μg / ml, 15 μg / ml or approximately 15 μg / ml, 20 μg / ml or approximately 20 μg / ml, 25 μg / ml or approximately 25 μg / ml, 30 μg / ml or approximately 30 μg / ml, 35 μg / ml or approximately 35 μg / ml, 40 μg / ml or approximately 40 μg / ml, 45 μg / ml or approximately 45 μg / ml, or 50 μg / ml or approximately 50 μg / ml.

[0241] In certain embodiments, manipulating a composition of enriched cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, in the presence of polycations reduces the amount of cell death, for example, by necrosis, programmed cell death, or apoptosis. In some embodiments, a composition of enriched T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is manipulated in the presence of a small amount of polycation, e.g., less than 100 μg / ml, less than 50 μg / ml, or less than 10 μg / ml, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 99.9% of the cells survive for 1 day or at least 1 day, 2 days or at least 2 days, 3 days or at least 3 days, 4 days or at least 4 days, 5 days or at least 5 days, 6 days or at least 6 days, 7 days or at least 7 days, or more than 7 days after the completion of the manipulation step, without undergoing necrosis, programmed cell death, or apoptosis. In some embodiments, the composition is operated in the presence of a lower concentration or amount of polycation compared to alternative and / or exemplary methods that operate on cells in the presence of a higher amount or concentration of polycation, e.g., greater than 50 μg / ml, greater than 100 μg / ml, greater than 500 μg / ml, or greater than 1,000 μg / ml, and the cells of the composition have a viability at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 1x, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 25x, at least 50x, or at least 100x higher than cells undergoing exemplary and / or alternative processes.

[0242] In some embodiments, polycations are positively charged. In certain embodiments, polycations reduce the repulsive force between cells and vectors, such as viruses or non-viral vectors, and mediate the contact and / or binding of vectors to the cell surface. In some embodiments, polycations are polybrene, DEAE-dextran, protamine sulfate, poly-L-lysine, or cationic liposomes.

[0243] In certain embodiments, the polycation is protamine sulfate. In some embodiments, the composition of enriched T cells such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is less than or about 500 μg / ml, less than or about 400 μg / ml, less than or about 300 μg / ml, less than or about 200 μg / ml, less than or about 150 μg / ml, less than or about 100 μg / ml, less than or about 90 μg / ml, less than or about 80 μg / ml The procedure is performed in the presence of protamine sulfate at concentrations of μg / ml, less than 75 μg / ml or approximately 75 μg / ml, less than 70 μg / ml or approximately 70 μg / ml, less than 60 μg / ml or approximately 60 μg / ml, less than 50 μg / ml or approximately 50 μg / ml, less than 40 μg / ml or approximately 40 μg / ml, less than 30 μg / ml or approximately 30 μg / ml, less than 25 μg / ml or approximately 25 μg / ml, less than 20 μg / ml or approximately 20 μg / ml, less than 15 μg / ml or approximately 15 μg / ml, or less than 10 μg / ml or approximately 10 μg / ml.In a particular embodiment, the enriched cell composition, such as stimulated T cells, e.g., stimulated CD4+ T cells or stimulated CD8+ T cells, is 1 μg / ml or approximately 1 μg / ml, 5 μg / ml or approximately 5 μg / ml, 10 μg / ml or approximately 10 μg / ml, 15 μg / ml or approximately 15 μg / ml, 20 μg / ml or approximately 20 μg / ml, 25 μg / ml or approximately 25 μg / ml, 30 μg / ml or approximately 30 μg / ml, 35 μg / ml or approximately 35 μg / ml, 40 μg / ml or approximately 40 μg / ml, 45 μg / ml or approximately 45 μg / ml, 50 μg / ml or approximately 50 μg / ml, 55 μg / ml or approximately 55 μg / ml, 60 μg / ml or approximately 60 μg / ml, 75 μg / ml or approximately 75 μg / ml, 80 μg / ml or approximately 80 μg / ml, 85 μg / ml or approximately 85 μg / ml, 90 μg / ml or approximately 90 μg / ml, 95 μg / ml or approximately 95 μg / ml, 100 μg / ml or approximately 100 μg / ml, 105 μg / ml or approximately 105 μg / ml, 110 μg / ml or approximately 110 μg / ml, 115 μg / ml or approximately 115 μg / ml, 120 μg / ml The procedure is performed in the presence of protamine sulfate at concentrations of g / ml or approximately 120 μg / ml, 125 μg / ml or approximately 125 μg / ml, 130 μg / ml or approximately 130 μg / ml, 135 μg / ml or approximately 135 μg / ml, 140 μg / ml or approximately 140 μg / ml, 145 μg / ml or approximately 145 μg / ml, or 150 μg / ml or approximately 150 μg / ml.

[0244] In some embodiments, an engineered composition of enriched CD4+ T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells, contains at least 40%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD4+ T cells. In some specific embodiments, an engineered composition of enriched CD4+ T cells, such as stimulated T cells, e.g., stimulated CD4+ T cells, contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and / or does not contain CD8+ T cells, and / or is CD8+ T cell-free or substantially CD8+ T cell-free.

[0245] In some embodiments, a composition of enriched CD8+ T cells, such as engineered, stimulated T cells, e.g., stimulated CD8+ T cells, contains at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% or about 100% CD8+ T cells. In some specific embodiments, a composition of enriched CD8+ T cells, such as engineered, stimulated T cells, e.g., stimulated CD8+ T cells, contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells and / or does not contain CD4+ T cells and / or is CD4+ T cell-free or substantially CD4+ T cell-free.

[0246] In some embodiments, manipulating cells involves culturing, contacting, or incubating them with a vector, such as a viral vector or a non-viral vector. In some specific embodiments, the manipulation involves culturing, contacting, and / or incubating cells with a vector for 4 hours or about 4 hours or at least 4 hours, 6 hours or about 6 hours or at least 6 hours, 8 hours or about 8 hours or at least 8 hours, 12 hours or about 12 hours or at least 12 hours, 16 hours or about 16 hours or at least 16 hours, 18 hours or about 18 hours or at least 18 hours, 24 hours or about 24 hours or at least 24 hours, 30 hours or about 30 hours or at least 30 hours, 36 hours or about 36 hours or at least 36 hours, 40 hours or about 40 hours or at least 40 hours, 4 8 hours or approximately 48 hours or at least 48 hours, 54 hours or approximately 54 hours or at least 54 hours, 60 hours or approximately 60 hours or at least 60 hours, 72 hours or approximately 72 hours or at least 72 hours, 84 hours or approximately 84 hours or at least 84 hours, 1 day or approximately 1 day or at least 1 day, 2 days or approximately 2 days or at least 2 days, 3 days or approximately 3 days or at least 3 days, 4 days or approximately 4 days or at least 4 days, 5 days or approximately 5 days or at least 5 days, 6 days or approximately 6 days or at least 6 days, or 7 days or approximately 7 days or at least 7 days, or more than 7 days. In certain embodiments, the operation involves culturing, contacting, and / or incubating cells with a vector for 24 hours or about 24 hours, 36 hours or about 36 hours, 48 ​​hours or about 48 hours, 60 hours or about 60 hours, 72 hours or about 72 hours, 84 hours or about 84 hours, or 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, or 5 days or about 5 days.In some embodiments, the operation process is carried out for 24 hours or approximately 24 hours, 36 hours or approximately 36 hours, 48 ​​hours or approximately 48 hours, 60 hours or approximately 60 hours, 72 hours or approximately 72 hours, or 84 hours or approximately 84 hours. In some specific embodiments, the operation is carried out for approximately 60 hours or approximately 84 hours, 72 hours or approximately 72 hours, or 2 days or approximately 2 days.

[0247] In some embodiments, the operation is carried out at a temperature of approximately 25 to approximately 38°C, for example, approximately 30 to approximately 37°C, approximately 36 to approximately 38°C, or 37°C ± 2°C or approximately 37°C ± 2°C. In some embodiments, the enriched T cell composition is operated at a CO2 level of approximately 2.5% to approximately 7.5%, for example, approximately 4% to approximately 6%, for example, 5% ± 0.5% or approximately 5% ± 0.5%. In some embodiments, the enriched T cell composition is operated at a temperature of 37°C or approximately 37°C and / or at a CO2 level of 5% or approximately 5%.

[0248] In some embodiments, cells, such as CD4+ and / or CD8+ T cells, are cultured after one or more steps have been performed to transduce or transfect them to include a genetic modification, such as a polynucleotide encoding a recombinant receptor. In some embodiments, the culture may include culture, incubation, stimulation, activation, expansion, and / or proliferation. In some such embodiments, further culture is carried out under conditions that result in the integration of a viral vector into the host genome of one or more cells. Incubation and / or manipulation may be carried out in a culture vessel such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other container for culture or for culturing cells. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to prime cells for genetic modifications such as inducing proliferation, expansion, activation, and / or survival of cells in a population, mimicking antigen exposure, and / or introducing recombinant antigen receptors.

[0249] In some embodiments, further incubation is carried out at a temperature higher than room temperature, for example, higher than 25°C or about 25°C, for example, generally higher than 32°C or about 32°C, higher than 35°C or about 35°C, or higher than 37°C or about 37°C. In some embodiments, further incubation is carried out at a temperature of 37°C ± 2°C or about 37°C ± 2°C, for example, 37°C or about 37°C.

[0250] In some embodiments, further incubation is carried out under conditions for stimulating and / or activating cells, the conditions may include one or more of a specific culture medium, temperature, oxygen content, carbon dioxide content, time, active agents such as nutrients, amino acids, antibiotics, ions, and / or stimulating factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0251] In some embodiments, the stimulating condition or stimulant comprises one or more active agents (e.g., stimulants and / or auxiliary agents), e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, active agents suitable for initiating the activation of ITAM-inducible signals, such as those suitable for delivering primary signals, e.g., those specific to TCR components, and / or active agents that promote costimulatory signals, such as those specific to T cell costimulatory receptors, e.g., anti-CD3, anti-CD28, or anti-41-BB, optionally bound to a solid support such as beads, and / or one or more cytokines, activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Among the stimulants are anti-CD3 / anti-CD28 beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander and / or ExpACT® beads). Optionally, the expansion method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium. In some embodiments, the stimulant comprises IL-2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL.

[0252] In some embodiments, the stimulating condition or stimulant comprises one or more activators, e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the activators activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such activators may comprise antibodies specific to TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, and / or one or more cytokines, conjugated to a solid support, e.g., beads. Optionally, the expansion method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulant comprises IL-2 and / or IL-15, e.g., IL-2 concentrations of at least about 10 units / mL, at least about 50 units / mL, at least about 100 units / mL, or at least about 200 units / mL.

[0253] The conditions may include one or more of the following: a specific culture medium, temperature, oxygen content, carbon dioxide content, time, active agents such as nutrients, amino acids, antibiotics, ions, and / or stimulants such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0254] In some cases, incubation is carried out according to techniques such as those described in Riddell et al. U.S. Patent No. 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0255] In some embodiments, further incubation is carried out in the same container or apparatus in which contact occurred. In some embodiments, further incubation is carried out without rotation or centrifugation, which generally follows at least a portion of incubation carried out under rotation, for example in connection with centrifugation or spinoculation. In some embodiments, further incubation is carried out outside of a stationary phase, such as outside a chromatographic matrix, for example in solution.

[0256] In some embodiments, further incubation is carried out in a different container or device from the one in which contact occurred, such as by transferring the cell composition to a different container or device after contact with the virus particles and reagents, for example, by an automated transfer.

[0257] In some embodiments, for example, further culture or incubation to promote ex vivo expansion is performed for more than 24 hours or approximately more than 24 hours, more than 2 days or approximately more than 2 days, more than 3 days or approximately more than 3 days, more than 4 days or approximately more than 4 days, more than 5 days or approximately more than 5 days, more than 6 days or approximately more than 6 days, more than 7 days or approximately more than 7 days, more than 8 days or approximately more than 8 days, more than 9 days or approximately more than 9 days, more than 10 days or approximately more than 10 days, more than 11 days or approximately more than 11 days, more than 12 days or approximately more than 12 days, more than 13 days or approximately more than 13 days, or more than 14 days or approximately more than 14 days. In some embodiments, further culture or incubation is performed for 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 24 hours or less.

[0258] In some embodiments, for example, the total duration of incubation with the stimulant is 1 to 96 hours or about 1 to about 96 hours, 1 to 72 hours or about 1 to about 72 hours, 1 to 48 hours or about 1 to about 48 hours, 4 to 36 hours or about 4 to about 36 hours, 8 to 30 hours or about 8 to about 30 hours, or 12 to 24 hours or about 12 to about 24 hours, for example, at least 6 hours or at least about 6 hours or about 6 hours, at least 12 hours or at least about 12 hours or about 12 hours, at least 18 hours or at least about 18 hours or about 18 hours, at least 24 hours or at least about 24 hours or about 24 hours, at least 36 hours or at least about 36 hours or about 36 hours, or at least 72 hours or at least about 72 hours or about 72 hours. In some embodiments, further incubation includes values ​​at both ends, such as 1 hour to 48 hours or approximately 1 hour to approximately 48 hours, 4 hours to 36 hours or approximately 4 hours to approximately 36 hours, 8 hours to 30 hours or approximately 8 hours to approximately 30 hours, or 12 hours to 24 hours or approximately 12 hours to approximately 24 hours.

[0259] In some embodiments, the methods provided herein do not involve further culture or incubation, for example, they do not include an ex vivo expansion step, or they include a substantially shorter ex vivo expansion step.

[0260] In some embodiments, the stimulating agent is removed and / or separated from the cells before the operation. In certain embodiments, the stimulating agent is removed and / or separated from the cells after the operation. In some specific embodiments, the stimulating agent is removed and / or separated from the cells following the operation, and before culturing the operated cells under conditions that promote proliferation and / or expansion, for example. In some specific embodiments, the stimulating agent is the stimulating agent described in Section IB-1. In certain embodiments, the stimulating agent is removed and / or separated from the cells as described in Section IB-2.

[0261] 1. Vectors and Methods Provided are one or more polynucleotides (e.g., nucleic acid molecules) encoding recombinant receptors, and vectors for genetically engineering cells to express such receptors according to a method provided, in order to produce genetically engineered cells. In some embodiments, the vector comprises nucleic acids encoding recombinant receptors. In certain embodiments, the vector is a viral vector or a non-viral vector. In some cases, the vector is a viral vector, such as a retroviral vector, e.g., a lentiviral vector or a gamma retroviral vector.

[0262] In some cases, nucleic acid sequences encoding recombinant receptors, such as chimeric antigen receptors (CARs), include signal sequences encoding signal peptides. Non-exclusive exemplary examples of signal peptides include, for example, the GMCSFR alpha-chain signal peptide shown in SEQ ID NO: 61 and encoded by the nucleotide sequence shown in SEQ ID NO: 60, the CD8 alpha-signal peptide shown in SEQ ID NO: 59, or the CD33 signal peptide shown in SEQ ID NO: 58.

[0263] In some embodiments, the vectors include viral vectors, such as retroviral or lentiviral vectors; nonviral vectors or transposons, such as the Sleeping Beauty transposon system; vectors derived from Simian virus 40 (SV40); adenoviruses; adeno-associated viruses (AAV); lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors; Moloney's mouse leukemia virus (MoMLV); myeloproliferative sarcoma virus (MPSV); mouse embryonic stem cell virus (MESV); mouse stem cell virus (MSCV); spleen fociforming virus (SFFV); or retroviral vectors derived from adeno-associated viruses (AAV).

[0264] In some embodiments, the viral vector or nonviral DNA comprises nucleic acids encoding heterorecombinant proteins. In some embodiments, the heterorecombinant molecule is or comprises homologous double-stranded nucleic acids for recombinant receptors, e.g., antigen receptors, e.g., SB transposons for gene silencing, capsid-encapsulated transposons, e.g., genomic recombination or reporter genes (e.g., GFP) or fluorescent proteins such as luciferase.

[0265] a. Viral vector particles In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as vectors derived from Simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3.doi:10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2,e93; Park et al., Trends Biotechnol. 2011 November 29(11):550-557).

[0266] In some embodiments, retroviral vectors derived from retroviral vectors, such as Moloney's mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV), have long terminal repeat sequences (LTRs). Most retroviral vectors are derived from mouse retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphitropic, meaning they can infect host cells of several species, including humans. In one embodiment, the expressed gene replaces the gag, pol, and / or env sequences of the retrovirus. Many exemplary retroviruses have been described (see, for example, U.S. Patent Nos. 5,219,740, 6,207,453, and 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0267] Methods for lentiviral transduction are well known. Exemplary methods are described, for example, in Wang et al. (2012) J.Immunother. 35(9):689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2):497-505.

[0268] In some embodiments, the viral vector particles include a genome derived from a retroviral genome-based vector, for example, a lentiviral genome-based vector. In some aspects of the provided viral vector, heterologous nucleic acids encoding recombinant receptors, such as antigen receptors like CARs, are contained and / or located between the 5'LTR and 3'LTR sequences of the vector genome.

[0269] In some embodiments, viral vector genomes are lentiviral genomes, such as the HIV-1 genome or SIV genome. For example, lentiviral vectors are produced by multiple attenuation of virulence genes, for instance, by deleting genes such as env, vif, vpu, and nef, making the vector safer for therapeutic purposes. Lentiviral vectors are well known (see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry sequences essential for the incorporation, selection, and transfer of foreign nucleic acids into host cells. Known lentiviruses are readily available from depositaries or collections such as the American Type Culture Collection ("ATCC"; 10801 University Blvd., Manassas, Va. 20110-2209), or can be isolated from known sources using generally available techniques.

[0270] Non-exclusive examples of lentiviral vectors include those derived from lentiviruses such as human immunodeficiency virus 1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), human T-lymphotropic virus 1 (HTLV-1), HTLV-2, or equine infectious anemia virus (E1AV). For example, lentiviral vectors are produced by multiple attenuation of HIV virulence genes, for instance, by deleting genes such as env, vif, vpr, vpu, and nef, making the vector safer for therapeutic purposes. Lentiviral vectors are well known in the art (see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry sequences essential for the incorporation, selection, and transfer of foreign nucleic acids into host cells. Known lentiviruses are readily available from depositaries or collections such as the American Type Culture Collection ("ATCC"; 10801 University Blvd., Manassas, Va. 20110-2209) or can be isolated from known sources using generally available techniques.

[0271] In some embodiments, a viral genome vector may contain the 5'LTR and 3'LTR sequences of a retrovirus, such as a lentivirus. In some aspects, a viral genome construct may contain sequences from the 5'LTR and 3'LTR of a lentivirus, and in particular, the R and U5 sequences from the 5'LTR of a lentivirus, as well as the inactivated or self-inactivated 3'LTR from the lentivirus. The LTR sequences can be LTR sequences from any lentivirus of any species. For example, they can be LTR sequences from HIV, SIV, FIV, or BIV. Typically, the LTR sequence is an HIV LTR sequence.

[0272] In some embodiments, the nucleic acids of viral vectors, such as HIV virus vectors, lack additional transcription units. The vector genome may contain an inactivated or self-inactivated 3'LTR (Zufferey et al., J Virol 72:9873, 1998; Miyoshi et al., J Virol 72:8150, 1998). For example, a deletion in the U3 region of the 3'LTR of a nucleic acid used to construct viral vector RNA can be used to generate a self-inactivated (SIN) vector. This deletion can then be transferred to the 5'LTR of the proviral DNA during reverse transcription. Self-inactivated vectors generally have deletions of enhancer and promoter sequences from the 3' long terminal repeat (LTR), which are copied to the 5'LTR during vector incorporation. In some embodiments, sufficient sequences, including the removal of a TATA box, can be eliminated to inactivate the transcriptional activity of the LTR. This can prevent the production of full-length vector RNA in transduced cells. In some aspects, the U3 element of the 3'LTR contains deletions of its enhancer sequence, TATA box, Sp1, and NF-κ B site. As a result of the self-inactivating 3'LTR, the provirus generated after entry and reverse transcription contains an inactivated 5'LTR. This can improve safety by reducing the risk of vector genome recruitment and the impact of the LTR on nearby cell promoters. The self-inactivating 3'LTR can be constructed by any method known in the art. In some embodiments, this does not affect the vector titer or the in vitro or in vivo properties of the vector.

[0273] Optionally, the U3 sequence from the lentivirus 5'LTR can be replaced with a promoter sequence from a viral construct, such as a heterologous promoter sequence. This can increase the titer of the virus recovered from the packaging cell line. Enhancer sequences may also be included. Any enhancer / promoter combination can be used to increase the expression of the viral RNA genome in the packaging cell line. For example, the CMV enhancer / promoter sequence is used (US Patent Nos. 5,385,839 and 5,168,062).

[0274] In some specific embodiments, the risk of insertional mutagenesis can be minimized by constructing retroviral vector genomes, such as lentiviral vector genomes, to be inclusion-delete. Various approaches can be pursued to construct non-inclusion vector genomes. In some embodiments, one or more mutations can be manipulated into the integrase enzyme component of the pol gene so that it encodes a protein having an inactive integrase. In some embodiments, inclusion can be prevented by modifying the vector genome itself, for example by mutating or deleting one or both attachment sites, or by rendering the 3'LTR proximal polyprint lact (PPT) nonfunctional through deletion or modification. In some embodiments, non-genetic approaches are available, which include pharmacological agents that inhibit one or more functions of integrase. These approaches are not mutually exclusive, i.e., multiple of the...

Claims

1. A method for preparing a composition of manipulated cells, comprising the following steps: (a) A step of incubating an input composition containing one or both CD4+ and CD8+ primary human T cells under stimulating conditions, thereby producing a stimulated composition; wherein the incubation step is (i) an anti-CD3 antibody or its CD3-binding fragment, and an anti-CD28 antibody or its CD28-binding fragment, (ii) Recombinant IL-2 and recombinant IL-15, and (iii) N-acetylcysteine It is carried out in the presence of; (b) a step of introducing a recombinant receptor, which is a chimeric antigen receptor (CAR) or a T cell receptor (TCR), into cells of the stimulated composition, thereby producing an engineered composition comprising engineered T cells; wherein the recombinant receptor can bind to a target antigen expressed on cells of disease or pathological condition; and (c) A step of culturing the manipulated composition under conditions that promote the expansion of the manipulated T cells, thereby producing an output composition containing the manipulated T cells; where, (i) The culturing step is carried out in the presence of recombinant IL-2 and recombinant IL-15; (ii) At least part of the culturing process is carried out using mixing and perfusion, wherein the culturing process is started under conditions without perfusion, and then the concentration of the cells is increased to at least 0.2 × 10 6 When a predetermined concentration of live cells / mL is reached, the perfusion is initiated; and (iii) The culturing step is carried out for 2 to 10 days including both of the values, and until the output composition contains at least a threshold number of T cells or viable T cells, where the threshold number of T cells or viable T cells is at least four times the number of T cells or viable T cells of the manipulated cell composition before culturing the manipulated composition.

2. The method according to claim 1, wherein the input composition contains more than 70% CD3+ primary human T cells.

3. The input composition is 200 × 10 6 Cell ~300×10 6 The method according to claim 1 or 2, comprising cells.

4. The method according to any one of claims 1 to 3, wherein the incubation step is carried out in the presence of recombinant IL-2, recombinant IL-15, and recombinant IL-7.

5. The method according to any one of claims 1 to 4, wherein in the incubation step, the concentration of recombinant IL-2 is 10 IU / mL to 200 IU / mL.

6. The method according to any one of claims 1 to 5, wherein the concentration of recombinant IL-15 in the incubation step is 1 IU / mL to 25 IU / mL.

7. The method according to any one of claims 4 to 6, wherein in the incubation step, the concentration of recombinant IL-7 is 100 IU / mL to 1000 IU / mL.

8. The method according to any one of claims 1 to 7, wherein the input composition is a first input composition enriched with CD8+ primary human T cells, and the method further comprises the following: (a) A step of separately incubating a second input composition enriched with CD4+ primary human T cells; here, These CD8+ primary human T cells were isolated from the same biological sample as the CD4+ primary human T cells, and The incubation step of the second input composition is carried out in the presence of (i) an anti-CD3 antibody or its CD3-binding fragment, and an anti-CD28 antibody or its CD28-binding fragment, and (ii) one or more cytokines, thereby generating the second stimulated composition; (b) a step of introducing a recombinant receptor, which is a CAR or TCR, into the cells of the second stimulated composition, thereby generating a second engineered composition containing engineered T cells; and (c) A step of culturing the second manipulated composition under conditions that promote the expansion of the manipulated T cells, thereby producing a second output composition containing the manipulated T cells.

9. The method according to claim 8, wherein the recombinant receptor introduced into the second stimulated composition is the same recombinant receptor introduced into the first stimulated composition.

10. The method according to any one of claims 1 to 9, wherein the anti-CD3 antibody or its CD3-binding fragment, and / or the anti-CD28 antibody or its CD28-binding fragment, are present on the surface of the beads.

11. The method according to claim 10, wherein the bead-to-cell ratio is less than 3:

1.

12. The method according to any one of claims 1 to 11, wherein the concentration of N-acetylcysteine ​​(NAC) is 0.2 mg / mL to 2.0 mg / mL.

13. The method according to any one of claims 1 to 12, wherein the introduction step is performed by a viral vector containing a polynucleotide encoding the recombinant receptor.

14. The aforementioned viral vector (a) Retroviral vectors; (b) lentiviral vector; or (c) Gamma retrovirus vector The method according to claim 13.

15. The method according to any one of claims 1 to 14, wherein the introduction step is carried out in the presence of a transduction adjuvant.

16. The method according to claim 15, wherein the transduction adjuvant is a transduction adjuvant derived from protamine sulfate and / or fibronectin, or comprises the same.

17. The method according to any one of claims 1 to 16, wherein the incubation step is carried out in the presence of recombinant IL-2, recombinant IL-15, and recombinant IL-7.

18. The method according to any one of claims 1 to 17, wherein the concentration of recombinant IL-2 in the culture step is 50 IU / mL to 500 IU / mL.

19. The method according to any one of claims 1 to 18, wherein the concentration of recombinant IL-15 in the culture step is 5 IU / mL to 50 IU / mL.

20. The method according to any one of claims 17 to 19, wherein the concentration of recombinant IL-7 in the culture step is 500 IU / mL to 2000 IU / mL.

21. The method according to any one of claims 1 to 20, comprising the step of removing the anti-CD3 antibody or its CD3-binding fragment, and the anti-CD28 antibody or its CD28-binding fragment from the manipulated composition before the culturing step.

22. The method according to claim 21, wherein the anti-CD3 antibody or its CD3-binding fragment, and the anti-CD28 antibody or its CD28-binding fragment are removed within 7 days from the start of the incubation step.

23. The method according to any one of claims 1 to 22, wherein the culturing step is carried out until the output composition contains at least a threshold number of viable T cells.

24. The method according to claim 23, wherein the culturing step is continued for at least one day after a threshold number of viable T cells has been reached.

25. The method according to claim 23 or claim 24, wherein the threshold number of viable T cells is at least five times the number of viable T cells in the manipulated cell composition before culturing the manipulated composition.

26. The method according to any one of claims 1 to 25, wherein the culturing step is performed until at least 9 days after the start of the incubation step.

27. The method according to any one of claims 1 to 26, wherein the culture step is carried out for 2 to 8 days including both end values, until the output composition contains a threshold number of T cells or a threshold number of viable T cells, wherein the threshold number of T cells or the threshold number of viable T cells are at least four times the number of manipulated cells or viable T cells of the manipulated composition before culture.

28. The threshold number of T cells or viable T cells is 50 × 10 6 Cells or at least 50 × 10 6 The method according to any one of claims 24 to 27, wherein the cell is a cell.

29. The method according to any one of claims 1 to 28, further comprising the step of collecting cells of the output composition after the culturing step.

30. The method according to claim 29, wherein the time between the start of the incubation step of the output composition and the collection of cells is 7 to 15 days.

31. The method according to claim 29 or 30, further comprising the step of formulating collected cells into an output composition for cryopreservation and / or administration to a subject.

32. The method according to claim 31, wherein the cells collected from the output composition are formulated in the presence of pharmaceutically acceptable excipients and / or cryoprotectants.

33. The method according to any one of claims 1 to 32, wherein the input composition comprises primary T cells obtained from a human subject having cancer.

34. The method according to claim 33, wherein the recombinant receptor is capable of binding to a target antigen that is associated with cancer cells or tissues, specific to cancer cells or tissues, and / or expressed on cancer cells or tissues.

35. The method according to any one of claims 1 to 34, wherein the recombinant receptor is a CAR.

36. The method according to any one of claims 1 to 35, wherein the recombinant receptor is an anti-CD19 CAR.

37. The predetermined concentration is at least 0.6 × 10 6 The method according to any one of claims 1 to 36, wherein the amount is live cells / mL.

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