Methods for producing therapeutic compositions of engineered cells

Abstract

The present disclosure provides methods for genetically engineered T cells, such as CD4+T cells, for use in cell therapy. In some aspects, the method provided includes one or more steps for the following: incubating the cells under stimulating conditions, introducing recombinant polypeptides into the cells by transduction or transfection, and cultivating the cells under conditions that promote proliferation and / or amplification. In some aspects, the incubation and / or cultivation are carried out in the presence of recombinant IL-2. In some aspects, the method provided is an effective and reliable means for producing genetically engineered T cells with a high success rate.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to the following applications: U.S. Provisional Application No. 62 / 580,409, filed November 1, 2017, entitled "Procedure for Generating Therapeutic Compositions of Engineered Cells"; U.S. Provisional Application No. 62 / 596,771, filed December 8, 2017, entitled "Procedure for Generating Therapeutic Compositions of Engineered Cells"; and U.S. Provisional Application No. 62 / 721,603, filed August 22, 2018, entitled "Procedure for Generating Therapeutic Compositions of Engineered Cells," the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] By referencing and incorporating into the sequence list

[0004] This application is submitted together with an electronic sequence list. The sequence list is provided as a file titled 735042013240SeqList.txt, created on October 31, 2018, and is 35,994 bytes in size. Information from the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure provides methods for genetically engineered T cells, such as CD4+ T cells and / or CD8+ T cells, for use in cell therapy. In some aspects, the provided methods include one or more steps of: incubating the cells under stimulating conditions, introducing a recombinant polypeptide into the cells by transduction or transfection, and culturing the cells under conditions that promote proliferation and / or expansion. In some aspects, the incubation and / or the culture is performed in the presence of recombinant IL-2. In some aspects, the provided methods are an efficient and reliable means of generating genetically engineered T cells with a high success rate. Background Technology

[0006] Various cell therapy approaches can be used to treat diseases and conditions. Cell therapy approaches include those involving the genetic engineering of immune cells (such as T cells) with recombinant receptors (such as chimeric antigen receptors). There is a need for improved methods for manufacturing and / or engineering such cell therapies, including providing more efficient methods and / or improved cell composition products. Methods, kits, and articles that meet these needs are provided. Summary of the Invention

[0007] In some aspects, a method for producing compositions for engineered cells is provided, the method comprising: (a) incubating an input composition containing T cells enriched for CD4+ primary human T cells under stimulating conditions, the stimulating conditions including the presence of: (i) a stimulating agent capable of activating one or more intracellular signal transduction domains of one or more components of the TCR complex and / or one or more intracellular signal transduction domains of one or more co-stimulatory molecules and (ii) one or more cytokines, wherein at least one cytokine is or includes 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.

[0008] In some embodiments of the methods provided herein, the input composition comprises greater than or greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD4+ primary human T cells; and / or the input composition consists substantially of CD4+ primary human T cells. In some embodiments, the concentration of recombinant IL-2 is from 10 IU / mL to 200 IU / mL or from about 10 to about 200 IU / mL. In some embodiments of the methods provided herein, the one or more cytokines further comprise IL-7 and / or IL-15, optionally wherein the concentration of IL-7 is from 100 IU / mL to 1000 IU / mL or from about 100 IU / mL to about 1000 IU / mL and / or the concentration of IL-15 is from 1 IU / mL to 50 IU / mL or from about 1 IU / mL to about 50 IU / mL. In some embodiments of the methods provided herein, the incubation is carried out in the presence of one or more antioxidants.

[0009] In some aspects, a method for producing a composition for engineered cells is provided, the method comprising: (a) incubating an input composition containing T cells enriched for one or both of CD4+ and CD8+ primary human T cells to produce a stimulated composition, wherein the incubation is performed under: (1) one or more stimulating conditions including the presence of: (i) a stimulating agent capable of activating one or more intracellular signal transduction domains of one or more components of the TCR complex and / or one or more intracellular signal transduction domains of one or more co-stimulatory molecules and (ii) one or more cytokines; and / or (2) in the presence of one or more antioxidants; and (b) introducing a recombinant receptor into the stimulated composition to produce an engineered composition containing engineered T cells.

[0010] In some embodiments of the methods provided herein, the input composition comprises greater than or greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD4+ and / or CD8+ primary human T cells; and / or the input composition consists substantially of CD4+ and / or CD8+ primary human T cells. In some embodiments of the methods provided herein, the 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 from 10 to 200 IU / mL or from about 10 to about 200 IU / mL; the concentration of recombinant IL-7 is from 100 IU / mL to 1000 IU / mL or from about 100 IU / mL to about 1000 IU / mL; and / or the concentration of recombinant IL-15 is from 1 IU / mL to 25 IU / mL or from about 1 IU / mL to about 25 IU / mL.

[0011] In some embodiments of the methods provided herein, the input composition comprises greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD4+ primary human T cells; and / or the input composition consists substantially of CD4+ primary human T cells. In some embodiments, the one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15. In some embodiments, the input composition comprises greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD8+ primary human T cells; and / or the input composition consists substantially of CD8+ primary human T cells. In some embodiments of the methods provided herein, the one or more cytokines are selected from recombinant IL-2 and recombinant IL-15.

[0012] In some embodiments of the methods provided herein, the stimulating agent comprises a primary agent that specifically binds to members 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 co-stimulatory molecule, optionally wherein said co-stimulatory 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, optionally wherein said stimulating agent comprises incubation with anti-CD3 antibody and 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.

[0013] 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 greater than about 3.5 μm, but not greater than about 9 μm, or not greater than about 8 μm, or not greater than about 7 μm, or not greater than about 6 μm, or not greater than about 5 μm. In some embodiments, the beads have a diameter of or about 4.5 μm. In some embodiments, the beads are inert. In some embodiments, the beads are 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 3:1 or less than about 3:1. In some embodiments, the bead-to-cell ratio is from 2:1 to 0.5:1 or from about 2:1 to about 0.5:1. In some embodiments, the bead-to-cell ratio is or about 1:1.

[0014] In some embodiments of the methods provided herein, the one or more antioxidants include sulfur-containing antioxidants. In some embodiments, the one or more antioxidants include glutathione precursors. In some embodiments, the one or more antioxidants include N-acetylcysteine ​​(NAC), optionally wherein the concentration of said NAC is from 0.2 mg / mL to 2.0 mg / mL or from about 0.2 mg / mL to about 2.0 mg / mL.

[0015] In some embodiments of the methods provided herein, the introduction comprises transducing the stimulated cells of the composition with a viral vector containing a polynucleotide encoding the 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 performed in the presence of a transduction adjuvant. In some embodiments, the transduction adjuvant is or includes protamine sulfate, optionally from 1 μg / ml to 50 μg / ml or from about 1 μg / ml to about 50 μg / ml; a transduction adjuvant derived from fibronectin; and / or RetroNectin. In other embodiments of the methods provided herein, the introduction comprises transfecting the stimulated cells of the composition with a vector containing a polynucleotide encoding the recombinant receptor. In some embodiments, the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a Piggybac transposon.

[0016] In some embodiments of the methods provided herein, the methods further include culturing the engineered composition under conditions that promote the proliferation or expansion of the engineered cells, thereby producing an output composition containing the engineered T cells. In some embodiments, the culturing is performed in the presence of one or more cytokines, wherein at least one cytokine is or includes recombinant human IL-2. In some embodiments, the stimulating agent is removed from the engineered composition prior to the culturing.

[0017] In some embodiments, the stimulant is removed within 7 days or less after the start of the incubation. In some embodiments, the stimulant is removed from 3 to 6 days, or from about 3 to about 6 days, after the start of the incubation. In some embodiments, the stimulant is removed 4 days or about 4 days after the start of the incubation. In some embodiments, removing the beads includes exposing the cells of the engineered composition to a magnetic field.

[0018] In other aspects, methods are provided for producing compositions of engineered cells, the methods comprising culturing an engineered cell composition containing CD4+ primary human T cells, including cells engineered with a recombinant receptor, in the presence of one or more cytokines, wherein at least one cytokine is or includes recombinant human IL-2, wherein the method results in the proliferation or expansion of the cells in the composition to produce an output composition containing engineered CD4+ cells. In some embodiments, the proliferation or expansion results in an increase, about or at least 2-fold, 3-fold, 4-fold, 5-fold, or greater than 5-fold, in the number of CD4+ T cells engineered with the recombinant receptor.

[0019] In some embodiments of any of the methods provided herein, the engineered cell composition comprises greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD4+ primary human T cells or CD4+ recombinant receptor-expressing cells; and / or the engineered cell composition consists substantially of CD4+ primary human T cells. In some embodiments of the methods provided herein, the concentration of recombinant IL-2 is from 50 IU / mL to 500 IU / mL or from about 50 IU / mL to about 500 IU / mL. In some embodiments of any of the methods provided herein, the one or more cytokines further comprise IL-7 and / or IL-15, optionally wherein the concentration of IL-7 is from 500 IU / mL to 2000 IU / mL or from about 500 IU / mL to about 2000 IU / mL and / or the concentration of IL-15 is from 5 IU / mL to 50 IU / mL or from about 5 IU / mL to about 50 IU / mL.

[0020] In some embodiments of any of the methods provided herein, the engineered cell composition is produced by a method comprising: (a) incubating an input composition containing primary T cells enriched for CD4+ primary human T cells under stimulating conditions, the stimulating conditions comprising the presence of: (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 cytokines, wherein at least one cytokine is or includes 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.

[0021] In some embodiments of any of the methods provided herein, the input composition comprises greater than or greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD4+ primary human T cells; and / or the input composition consists substantially of CD4+ primary human T cells. In some embodiments of any of the methods provided herein, the 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 performed in the presence of a surfactant. In some embodiments, at least a portion of the culture is performed under continuous mixing and / or perfusion conditions.

[0022] In another aspect, a method is provided for producing a composition of engineered cells, the method comprising: culturing an engineered cell composition containing one or both of 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 performed in the presence of a surfactant and / or at least a portion of the culturing is performed in the presence of continuous mixing and / or perfusion; wherein the method results in the proliferation or expansion of cells in the composition to produce an output composition containing engineered CD4+ and / or CD8+ T cells.

[0023] In some embodiments of any of the above methods, the engineered cell composition comprises more than or greater than about 70%, more than or greater than about 75%, more than or greater than about 80%, more than or greater than about 85%, more than or greater than about 90%, more than or greater than about 95%, or more than or greater than 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 engineered cell composition is substantially composed of CD4+ and / or CD8+ primary human T cells.

[0024] In some implementations of any of the methods provided herein, the proliferation or amplification results in an increase, approximately or at least 2-fold, 3-fold, 4-fold, 5-fold or greater than 5-fold, in the number of CD4+ and / or CD8+ T cells engineered with recombinant receptors.

[0025] In some embodiments of any of the methods provided herein, the 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 from 50 IU / mL to 500 IU / mL or from about 50 IU / mL to about 500 IU / mL; the concentration of recombinant IL-7 is from 500 IU / mL to 2000 IU / mL or from about 500 IU / mL to about 2000 IU / mL; and / or the concentration of recombinant IL-15 is from 5 IU / mL to 50 IU / mL or from about 5 IU / mL to about 50 IU / mL.

[0026] In some embodiments of any of the methods provided herein, the engineered cell composition comprises more than about 70%, more than or more than about 75%, more than or more than about 80%, more than or more than about 85%, more than or more than about 90%, more than or more than about 95%, or more than or more than about 98% CD4+ primary human T cells or CD4+ and recombinant receptor-expressing primary human T cells; and / or the engineered cell composition consists substantially of CD4+ primary human T cells.

[0027] In some embodiments of any of the methods provided herein, the proliferation or expansion results in an increase, approximately or at least 2-fold, 3-fold, 4-fold, 5-fold, or greater than 5-fold, in the number of CD8+ T cells engineered with the recombinant receptor. In some embodiments, the one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15.

[0028] In some embodiments of any of the methods provided herein, the engineered cell composition comprises more than about 70%, more than or more than about 75%, more than or more than about 80%, more than or more than about 85%, more than or more than about 90%, more than or more than about 95%, or more than or more than about 98% CD8+ primary human T cells or CD8+ and recombinant receptor-expressing primary human T cells; and / or the engineered cell composition consists substantially of CD8+ primary human T cells.

[0029] In some embodiments of any of the methods provided herein, the 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 poloxamer, optionally wherein the concentration of said poloxamer is from 0.5 μL / mL to 5 μL / mL or from about 0.5 μL / mL to about 5 μL / mL. In some embodiments, said poloxamer is poloxamer 188.

[0030] In some embodiments of any of the methods provided herein, the engineered cell composition is produced by a method comprising: (a) incubating an input composition containing primary T cells enriched for one or both of CD4+ and CD8+ primary human T cells under stimulating conditions, the stimulating conditions comprising the presence of: (i) a stimulating agent capable of activating one or more intracellular signal transduction domains of one or more components of the TCR complex and / or one or more intracellular signal transduction domains of one or more co-stimulatory molecules and (ii) 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.

[0031] In some embodiments of any of the methods provided herein, the input composition comprises greater than or greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD4+ and / or CD8+ primary human T cells; and / or the input composition consists substantially of CD4+ and / or CD8+ primary human T cells. In some embodiments, the one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and / or recombinant IL-15.

[0032] In some embodiments of any of the methods provided herein, the input composition comprises greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD4+ primary human T cells; and / or the input composition consists substantially of CD4+ primary human T cells. In some embodiments, the one or more cytokines are selected from recombinant IL-2, recombinant IL-7, and recombinant IL-15.

[0033] In some embodiments of any of the methods provided herein, the input composition comprises greater than about 70%, greater than or greater than about 75%, greater than or greater than about 80%, greater than or greater than about 85%, greater than or greater than about 90%, greater than or greater than about 95%, or greater than or greater than about 98% CD8+ primary human T cells; and / or the input composition consists substantially of CD8+ primary human T cells. In some embodiments, the one or more cytokines are selected from recombinant IL-2 and recombinant IL-15.

[0034] In some embodiments of any of the methods provided herein, the stimulating agent comprises a primary agent that specifically binds to members 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 co-stimulatory molecule, optionally wherein said co-stimulatory molecule is selected from CD28, CD137(4-1-BB), OX40, or ICOS.

[0035] In some embodiments, the primary and / or secondary agents comprise antibodies, optionally wherein the stimulating agent comprises incubation with anti-CD3 antibodies and anti-CD28 antibodies or antigen-binding fragments thereof. 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 greater than about 3.5 μm, but not greater than about 9 μm, or not greater than about 8 μm, or not greater than about 7 μm, or not greater than about 6 μm, or not greater than about 5 μm. In some embodiments, the beads comprise a diameter of or greater than about 4.5 μm. In some embodiments, the beads are inert. In some embodiments, the beads are 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 less than about 3:1. In some embodiments, the bead-to-cell ratio is from 2:1 to 0.5:1 or from about 2:1 to about 0.5:1. In some implementations, the ratio of beads to cells is 1:1 or approximately 1:1.

[0036] In some embodiments of any of the methods provided herein, the incubation is carried out in the presence of one or more antioxidants. In some embodiments, the one or more antioxidants include sulfur-containing antioxidants. In some embodiments, the one or more antioxidants include glutathione precursors. In some embodiments, the one or more antioxidants include N-acetylcysteine ​​(NAC), optionally wherein the concentration of said NAC is from 0.2 mg / mL to 2.0 mg / mL or from about 0.2 mg / mL to about 2.0 mg / mL.

[0037] In some embodiments of any of the methods provided herein, the introduction comprises transducing the stimulated cells of the composition with a viral vector containing a polynucleotide encoding the 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 any of the methods provided herein, the introduction is performed in the presence of a transduction adjuvant. In some embodiments, the transduction adjuvant is or includes protamine sulfate, optionally from 1 μg / ml to 50 μg / ml or from about 1 μg / ml to about 50 μg / ml; a transduction adjuvant derived from fibronectin; and / or RetroNectin. In some embodiments of any of the methods provided herein, the introduction comprises transfecting the stimulated cells of the composition with a vector containing a polynucleotide encoding the recombinant receptor. In some embodiments, the vector is a transposon, optionally a Sleeping Beauty (SB) transposon or a Piggybac transposon.

[0038] In some embodiments of any of the methods provided herein, the engineered cell composition does not contain a stimulating agent and / or the stimulating agent has been substantially removed from the composition prior to the culture, the stimulating agent containing an agent capable of activating one or more intracellular signal transduction domains of one or more components of the TCR complex and / or one or more intracellular signal transduction domains of one or more co-stimulatory molecules.

[0039] In some embodiments of any of the methods provided herein, the culture is performed at least until the output composition contains a threshold number of T cells. In some embodiments, the culture continues for at least one day after the threshold number of T cells is reached. In some embodiments, the threshold number is at least 2, 3, 4, 5, or more times greater than the amount of the engineered cell composition prior to the culture. In some embodiments of any of the methods provided herein, the culture is performed for 2 to 10 days and includes an end value, and / or the culture is performed for at least 10 days. In some embodiments of any of the methods provided herein, after the culture, the cells of the output composition are collected. In some embodiments, the time between the start of the incubation and the collection of the cells of the output composition is from 7 to 15 days or from about 7 to about 15 days. In some embodiments, the time between the start of the incubation and the collection of the cells of the output composition is from 9 to 13 days or from about 9 to about 13 days. In some embodiments, the time between the start of the incubation and the collection of the cells of the output composition is from 8 to 13 days or from about 8 to about 13 days.

[0040] In some embodiments of any of the methods provided herein, the method further includes optionally formulating the cells of the output composition in the presence of a pharmaceutically acceptable excipient for cryopreservation and / or administration to a subject. In some embodiments, the cells of the output composition are formulated in the presence of a cryoprotectant. In some embodiments, the cryoprotectant comprises DMSO. In some embodiments, the cells of the output composition are formulated in a container, optionally in a vial or bag.

[0041] In some embodiments of any of the methods provided herein, the method further includes isolating CD4+ and / or CD8+ T cells from a biological sample prior to the incubation. In some embodiments, the isolation includes selecting cells based on the surface expression of CD4 and / or CD8, optionally by positive or negative selection. In some embodiments, the isolation includes performing immunoaffinity-based selection. In some embodiments, the biological sample includes primary T cells obtained from a subject. In some embodiments, the subject is a human subject. In some embodiments, the biological sample is or includes whole blood samples, erythrocyte sedimentation rate (ESR) amber layer samples, peripheral blood mononuclear cell (PBMC) samples, ungraded T cell samples, lymphocyte samples, leukocyte samples, apheresis products, or leukoablation products.

[0042] In some embodiments of any of the methods provided herein, the recombinant receptor is capable of binding to a target antigen that is associated with, specific to, and / or expressed on cells or tissues of the disease, disorder, or condition. In some embodiments, the disease, disorder, or 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 antigen is selected from 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, dual antigen, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, liver glycoside 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-13Ra2), kinase insertion domain receptor (kdr), κ light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-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), oncoemulsification antigen, melanoma preferred expression antigen (PRAME), PSCA, progesterone receptor, survival protein, ROR1, TAG72, tEGFR, VEGF receptor, VEGF-R2, Wilms tumor 1 (WT-1), pathogen-specific antigens, and antigens associated with universal labels.

[0043] In some embodiments of any of the methods provided herein, the recombinant receptor is or comprises a functional non-TCR antigen receptor or a TCR or an antigen-binding fragment thereof. In some embodiments of any of the methods provided herein, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments of any of the methods provided herein, the recombinant receptor is an anti-CD19 CAR. In some embodiments, the chimeric antigen receptor comprises an extracellular domain containing an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, the antibody fragment optionally being a single-chain fragment. In some embodiments, the fragment comprises an antibody variable region linked by a flexible linker. In some embodiments, the fragment comprises scFv.

[0044] In some embodiments of any of the methods provided herein, the chimeric antigen receptor further comprises a spacer and / or a 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 includes a primary signaling domain, a signaling domain capable of inducing primary activation signals in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immune receptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or includes an intracellular signaling domain of the CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0045] In some embodiments of any of the methods provided herein, the chimeric antigen receptor further includes a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some embodiments, the intracellular signaling region further includes a co-stimulatory signaling region. In some embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of a T cell co-stimulatory molecule or a signaling portion thereof. In some embodiments, the co-stimulatory signaling region includes an intracellular signaling domain of CD28, 4-1BB, or ICOS or a signaling portion thereof. In some embodiments, the co-stimulatory signaling region is located between the transmembrane domain and the intracellular signaling region.

[0046] In some embodiments of any of the methods provided herein, the output composition containing the threshold number or greater of cells is produced between repetitions of the method greater than or greater than about 85%, greater than or greater than about 90%, or greater than or greater than about 95%. In some embodiments of any of the methods described herein, the method is performed in less than 21 days (including end values).

[0047] In other embodiments, compositions are provided containing engineered cells produced by the methods described in any of the embodiments herein. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions comprise a cryoprotectant, optionally DMSO.

[0048] In another embodiment, an article is provided comprising any of the compositions described herein, and instructions for administering the output composition to a subject. In some embodiments of the article, the subject suffers from a disease or condition, optionally wherein the recombinant receptor specifically recognizes or specifically binds to an antigen associated with or expressed or present on cells of the disease or condition. In some embodiments, the output composition is a composition engineered for CD4+ T cells. In some embodiments, the output composition is an engineered composition for CD8+ T cells.

[0049] In some aspects, articles of manufacture are provided comprising a composition of engineered CD4+ T cells produced by any of the methods described herein, a composition of engineered CD8+ T cells produced by any of the methods described herein, and instructions for administering the engineered CD4+ T cells and the engineered CD8+ T cells to a subject. In some embodiments, the instructions specify that the CD4+ T cells and CD8+ T cells be administered to the subject separately. In other embodiments, the instructions specify that the CD4+ T cells and the CD8+ T cells be administered to the subject at a desired ratio.

[0050] In some implementations of any of the methods described herein, the method is performed in less than 21 days (including end values). Attached Figure Description

[0051] Figure 1 The diagram illustrates the total cell counts of CD4+ (solid symbols) and CD8+ (hollow symbols) cell compositions obtained from the same leukapheresis sample, measured at different time points during cell stimulation, transduction, and amplification, in alternative (triangle) and exemplary (circle) procedures for generating anti-CD19 chimeric antigen receptor (CAR) expressing cells as described in Example 1. The horizontal dashed line indicates the threshold cell count required to meet the harvest criteria.

[0052] Figures 2A-2D Depicting the situation from donor 1 of Experiment 1 ( Figure 2A Experiment 1 Donor 2 ( Figure 2B ) or Experiment 2 donor 3 ( Figure 2C CD4+ cells from donor 3 in experiment 2, or from donor 3 in experiment 2. Figure 2DIn CD8+ cells, viable cell counts (VCC; x 10⁻¹⁰) were assessed using either differential DHM continuous monitoring (“continuous”, line) or manual sampling (“manual”, point). 6 Cell count / mL and cell viability (%). The top figure depicts the measurements for each, and the bottom figure depicts the linear regression analysis and R0. 2 And slope, to be used to compare continuous monitoring and manual sampling.

[0053] Figure 3 The study describes the improvement in viable cell counts (VCCs) assessed by continuous differential DHM monitoring during automated amplification compared to manual amplification. 6 Cell count / mL and cell viability (%). Detailed Implementation

[0054] This document provides methods for generating or producing compositions of engineered cells (such as engineered CD4+ and / or CD8+ T cells) expressing recombinant receptors. In certain embodiments, the methods are used in conjunction with processes including: incubating cells under stimulating conditions; genetically engineering cells, for example, by introducing a polynucleotide encoding a recombinant receptor; and / or culturing the engineered cells under conditions that promote cell proliferation and / or expansion. In some embodiments, the cells are cell compositions enriched for CD4+ T cells (hereinafter also referred to as compositions enriched for CD4+ T cells). In some embodiments, the cells are cell compositions enriched for CD8+ T cells (hereinafter also referred to as compositions enriched for CD8+ T cells). In some embodiments, the methods are performed to generate or produce two or separate compositions of engineered T cells, each engineered from cells derived from the same biological sample (e.g., from the same subject) with the same recombinant receptor, such as by separately incubating, engineering, and culturing separate compositions of CD4+ T cells and CD8+ T cells.

[0055] Different methods can be used to generate genetically engineered T cell populations, including generating engineered T cells expressing chimeric antigen receptors. However, in some embodiments, some of these methods may require a longer or relatively longer time to generate engineered cells. In some embodiments, the time required to generate engineered T cells from samples obtained from different subjects may vary with some existing methods. For example, in some embodiments, the same method may take 5, 6, 7 days or more longer to generate engineered cells for one subject than for another. In some embodiments, the ability of some methods to successfully generate therapy-suitable engineered cells across different subjects may vary. In certain embodiments, the variability and / or lack of predictability of some methods may pose problems for clinicians, such as difficulty in determining whether cell therapy can be generated for a given subject, or difficulty in planning or coordinating cell therapy administration, for example, when the timing of cell therapy availability is not yet known.

[0056] The provided implementations address one or more of these problems. In certain implementations, the provided methods generate engineered T cells suitable for therapies, such as autologous cell therapies, in a short or relatively short time frame compared to some existing methods. Furthermore, in some implementations, the provided methods produce a more consistent and less variable process in terms of the time required to generate engineered cells from samples collected from different subjects. In certain implementations, the provided methods are capable of successfully generating engineered T cells suitable for cell therapy from a high proportion of subjects. Therefore, in some implementations, the methods described herein provide a relatively rapid and efficient means of generating engineered T cells for therapy. These features may allow for the treatment of more potential subjects with T-cell therapies such as autologous T-cell therapies and may alleviate some of the difficulties associated with planning or coordinating cell therapy for subjects.

[0057] In some implementations, compared to other products, the provided method shortens the amplification duration and / or enables product generation within a narrower duration window across a wider range of starting samples (including those that might otherwise not have reached the harvest threshold), and in some respects reduces failures due to poor cell amplification.

[0058] In some embodiments, the provided method is used in conjunction with a process for generating engineered CD4+ T cells expressing recombinant receptors, such as chimeric antigen receptors. In specific embodiments, CD4+ T cells are incubated and / or cultured in the presence of recombinant IL-2. Typically, alternative processes for generating engineered CD4+ T cells do not involve or require the addition of recombinant IL-2 along with the CD4+ T cells, as in some embodiments, cultured CD4+ T cells are generally understood to produce and / or secrete IL-2. However, in the absence of a desire to be bound by theory, some embodiments consider that CD4+ T cells derived from certain patients (such as diseased subjects and / or subjects whose T cells contain one or more characteristics associated with unhealthy cells) do not produce or secrete sufficient amounts of IL-2. In some embodiments, such CD4+ T cells will not grow, proliferate, and / or expand without supplementation with recombinant IL-2. Therefore, in some implementations, by including recombinant IL-2 in the process of culturing and engineering CD4+ T cells, the provided method expands the pool of subjects who can provide engineered CD4+ T cells, and thereby expands the pool of subjects who can be treated with autologous cell therapies containing engineered CD4+ T cells.

[0059] In some embodiments, cells are incubated under stimulating conditions using a stimulating agent (e.g., beads conjugated with anti-CD3 and anti-CD28 antibodies) prior to genetic engineering (e.g., transduction or transfection). In some embodiments, the stimulating agent is isolated or removed from the cells before the start of the culture step and at least 7 days after the start of incubation (e.g., at or around 4 or 5 days). Specific embodiments contemplate that when the stimulating agent is removed or isolated from the cells at an earlier point in time during the process, the cells have improved survival rates and will undergo more robust proliferation and / or expansion during the culture step compared to cells cultured in an alternative process where the stimulating agent is not isolated or removed, or at a later point in time during the process. In such embodiments, the cultured cells achieve target or threshold cell counts, densities, and / or expansion more quickly compared to cells cultured in an alternative process. Therefore, in some implementations, removing or separating the stimulating agent from the cells within 7 days or earlier from the start of incubation allows the process of generating or producing engineered cells to be completed in a shorter amount of time than alternative processes.

[0060] In some embodiments, the methods provided herein are used in conjunction with a process for generating genetically engineered T cells over a short period of time. In some embodiments, the short duration of the process can increase the rate, occurrence, and / or probability of generating an engineered T-cell composition that can be administered to a subject for cell therapy. In some embodiments, the manufacturing protocol for the therapeutic cell composition may require the generation and release of the cell composition for infusion within a specified timeframe, for example, to validate and / or determine its suitability for administration to a subject. In some aspects, the short duration of the provided process may be expected to reduce or eliminate process failures due to the cell composition not expanding within the required timeframe.

[0061] In certain embodiments, the methods provided herein are used in conjunction with culturing engineered cells under conditions that promote proliferation and / or expansion. In some embodiments, the culturing is carried out in a setting that allows for constant mixing and / or perfusion of the cultured cells, such as in or connected to a bioreactor. In some embodiments, at least a portion of the culturing is performed under constant mixing and slow, constant perfusion to replace used culture medium with fresh medium. In some embodiments, cells are initially cultured under static conditions, e.g., without perfusion or mixing, and then cultured under constant mixing and perfusion when the cultured cells reach a predetermined cell count or density, and / or once the cells have been initially cultured for a predetermined amount of time. In some such embodiments, the cultured cells achieve the target or threshold cell count, density, and / or expansion more quickly than cells cultured in alternative processes. Therefore, in some embodiments, culturing under constant mixing and / or perfusion allows the generation or production of engineered cells to be completed in a shorter amount of time compared to alternative processes of culturing cells under static conditions.

[0062] In certain embodiments, the provided methods are used in conjunction with processes for efficiently producing or generating engineered cells suitable for use in cell therapy. In some embodiments, the timing, conditions, and reagents used for each step of the process improve the efficiency of each subsequent step and / or the entire process. For example, in some embodiments, cells may be incubated with reagents (e.g., stimulating agents or transduction adjuvants) at concentrations high enough to achieve the desired effect (e.g., stimulating cells or improving transduction efficiency), but at concentrations low enough to avoid slowing growth or reducing survival in subsequent treatment steps. Furthermore, in some embodiments, the steps of the process are timed to begin or end at specific points in time to improve the efficiency of subsequent process steps and / or the entire process. For example, in some embodiments, the steps of incubation and engineering (e.g., transducing or transfecting cells) are completed earlier in the process than in alternative methods, which in some embodiments improves survival and / or health status, and / or the rate of cell proliferation and expansion during subsequent culture steps. Thus, in one aspect, the specific timing, conditions, and reagents of each step affect the cells beyond the individual steps and, in some embodiments, affect the performance of the entire process.

[0063] In some embodiments, the method is used in conjunction with a process that can generate or produce genetically engineered cells suitable for cell therapy in a manner that is faster and more efficient than alternative processes. In some embodiments, the method provided herein has a high success rate in generating or producing engineered cell compositions from a broader population of subjects compared to what might be achieved by alternative processes. In some embodiments, engineered cells generated or produced by the provided method can have superior health, viability, activation, and higher recombinant receptor expression compared to cells generated by alternative methods. Therefore, in some respects, the speed and efficiency with which the provided method produces engineered cells for cell therapy allows for easier planning and coordination of cell therapy treatments, such as autologous therapies, for a broader population of subjects compared to what might be achieved by some alternative methods.

[0064] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or expressions used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the commonly understood meaning in the art.

[0065] For all purposes, all publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety as if each individual publication were individually incorporated by reference. Where the definitions described herein are contrary to or otherwise inconsistent with those described in patents, applications, published applications, and other publications incorporated herein by reference, the definitions described herein shall prevail.

[0066] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0067] I. Methods for producing engineered cells

[0068] This document provides methods for producing output compositions of engineered cells, such as engineered CD4+ T cells and / or engineered CD8+ T cells, said engineered cells expressing recombinant proteins, such as recombinant receptors like T cell receptors (TCRs) or chimeric antigen receptors (CARs). In some embodiments, the methods provided herein are used in conjunction with the manufacture, generation, or production of cell therapies and may be combined with additional processing steps, such as isolation, separation, selection, activation or stimulation, transduction, washing, suspension, dilution, concentration, and / or formulation of cells. In some embodiments, methods for generating or producing engineered cells, such as engineered CD4+ T cells and / or engineered CD8+ T cells, include one or more of the following: isolating cells from a subject, preparing cells, processing cells, incubating cells under stimulating conditions, and / or engineering (e.g., transducing) cells. In some embodiments, the method includes processing steps performed in the following order: firstly, isolating, such as selecting or separating, input cells, e.g., primary cells, from a biological sample; incubating the input cells under stimulating conditions; engineering them with vector particles, such as viral vector particles, to introduce recombinant polynucleotides into the cells, e.g., by transduction or transfection; culturing the engineered cells (e.g., transduced cells), such as to amplify the cells; and collecting, harvesting all or part of the cells, and / or filling containers with them 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 engineered cells is suitable for use in therapies, such as autologous cell therapy.

[0069] In certain embodiments, the provided method is used in conjunction with an output composition that produces cells expressing the recombinant receptor from an initial or input cell composition. 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 as enriched T cell composition, enriched CD4+ T cell composition, and enriched CD8+ T cell composition, respectively). In some embodiments, the provided method is used in conjunction with one or more of the following: activating or stimulating the cell composition enriched for T cells; genetically engineering the enriched T cell composition, for example, by introducing a polynucleotide encoding a recombinant protein through transduction or transfection; and / or culturing the engineered composition of enriched T cells, for example, under conditions that promote proliferation and / or expansion. In some embodiments, the method may also be used in conjunction with an input composition that isolates or selects cells from a biological sample (such as a biological sample obtained, collected, and / or acquired from a subject) to produce enriched T cells. In certain embodiments, the provided method can be used in conjunction with compositions for harvesting, collecting, and / or formulating enriched T cells after cells have been incubated, activated, stimulated, engineered, transduced, transfected, and / or cultured.

[0070] In some embodiments, the provided methods are used in combination 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 embodiments, the enriched CD4+ T cell composition contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD4+ T cells. In a particular embodiment, the enriched CD4+ T cell composition contains up to 100% CD4+ T cells or contains about 100% CD4+ T cells. In some embodiments, the T-cell enriched composition comprises or contains 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 contains no CD8+ T cells, and / or contains no or substantially no CD8+ T cells. In some embodiments, the population consists primarily of CD4+ T cells.

[0071] In some embodiments, the provided method is used in conjunction with two or more separate output compositions for generating enriched T cells. In some embodiments, the provided method is performed separately on two or more separate compositions for enriched T cells (e.g., one or more separate compositions for enriched CD4+ T cells and one or more separate compositions for enriched CD8+ T cells). In some embodiments, the method may be used in conjunction with: separately activating and / or stimulating two or more compositions for enriched T cells; separately engineering two or more compositions for enriched T cells; and / or separately culturing two or more compositions for enriched T cells. In some embodiments, the method may also be used in conjunction with separate input compositions, such as separate compositions for enriched CD4+ T cells and CD8+ T cells, for isolating or selecting different cells from a biological sample to generate enriched T cells. In a particular embodiment, the provided method may be used in conjunction with separate compositions for harvesting, collecting, and / or formulating enriched T cells after T cells have been incubated, activated, stimulated, engineered, transduced, transfected, and / or cultured.

[0072] In a particular embodiment, the provided method can be used in combination with: incubating at least one single composition of enriched CD4+ T cells and at least one single composition of enriched CD8+ T cells, respectively; activating and / or stimulating at least one single composition of enriched CD4+ T cells and at least one single composition of enriched CD8+ T cells, respectively, after said incubation; engineering, transducing and / or transfecting at least one single composition of enriched CD4+ T cells and at least one single composition of enriched CD8+ T cells, respectively, after said activation and / or stimulation; culturing at least one single composition of enriched CD4+ T cells and at least one single composition of enriched CD8+ T cells, respectively, after said engineering, transducing and / or transfecting; harvesting and / or collecting at least one single composition of enriched CD4+ T cells and at least one single composition of enriched CD8+ T cells, respectively, after said culturing; and formulating at least one single composition of enriched CD4+ T cells and at least one single composition of enriched CD8+ T cells, respectively, after said harvesting and / or collecting. At least one single composition of T cells; and / or administer the formulated compositions to subjects in need, respectively.

[0073] In some embodiments, the two or more individual compositions of enriched T cells include a composition of enriched CD4+ T cells. In some embodiments, the two or more individual compositions include CD8+ T cells. In some embodiments, the two or more individual compositions include a composition of enriched CD4+ T cells and a composition of enriched CD8+ T cells. In a particular embodiment, the individual compositions of enriched CD4+ T cells and CD8+ T cells are derived from the same biological sample, such as the same biological sample obtained, collected, and / or acquired from a single subject, for example, initially isolated, selected, and / or enriched from it. In some embodiments, the same biological sample is first subjected to selection by CD4+ T cells, wherein negative and positive fractions are preserved, and the negative fraction is further subjected to selection by CD8+ T cells. In another embodiment, the same biological sample is first subjected to selection by CD8+ T cells, wherein negative and positive fractions are preserved, and the negative fraction is further subjected to selection by CD4+ T cells.

[0074] In some embodiments, the T-cell enriched composition is a CD8+ T-cell enriched composition. In some embodiments, the CD8+ T-cell enriched composition contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD8+ T cells, or contains or contains about 100% CD8+ T cells. In some embodiments, the CD8+ T-cell enriched composition contains or contains 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 contains no CD4+ T cells, and / or contains no or substantially no CD4+ T cells. In some embodiments, the cell population consists primarily of CD8+ T cells.

[0075] In some embodiments, recombinant IL-2 is used to incubate, activate, stimulate, engineer, transduce, transfect, and / or cultivate compositions enriched with CD4+ T cells and / or engineered compositions enriched with CD4+ T cells in the presence of recombinant IL-2. In certain embodiments, the method is used in conjunction with an input composition that incubates CD4+ T cells enriched with recombinant IL-2 under stimulating conditions and / or in the presence of recombinant IL-2. In some embodiments, the method is used in conjunction with engineered compositions that cultivate CD4+ T cells enriched with recombinant IL-2 under conditions promoting proliferation and / or expansion.

[0076] In some embodiments, incubating the enriched T cells under stimulating conditions is or includes incubating the cells with a stimulating agent, such as that described in Section IB-1. In some embodiments, the stimulating agent is removed or isolated from the cells prior to the culture step. In some embodiments, the stimulating agent is removed or isolated from the cells after genetic engineering, such as transfection or transduction. In some embodiments, the stimulating agent is removed from the cells within a set time period from the start of incubation with the stimulating agent, for example, within 7 days or less from the start of the incubation. In a particular embodiment, incubation under stimulating conditions is carried out in the presence of one or more antioxidants, such as sulfur-containing antioxidants and / or glutathione precursors.

[0077] In certain embodiments, the T-cell enriched composition, such as a T-cell enriched stimulated composition, is engineered in the presence of a polycation to improve transfection or transduction efficiency. In some embodiments, the polycation is present in low and / or relatively low amounts and / or concentrations.

[0078] In some embodiments, at least a portion of the culture step is performed under constant mixing and / or perfusion conditions, for example, in a closed system using a bioreactor. In some embodiments, the mixing and / or perfusion incorporates stabilization and / or gradual replacement of used or old cell culture media or solutions with fresh culture media or solutions. In particular embodiments, cells are cultured in the presence of surfactants and / or agents that reduce or prevent cell shearing (such as shearing during constant mixing and / or perfusion).

[0079] In some embodiments, the provided methods are performed such that one, more, or all of the steps in the preparation of cells for clinical use (e.g., in adoptive cell therapy) are performed without exposing the cells to non-sterile conditions. In some embodiments of this method, cell isolation, separation or selection, transduction, washing, optional activation or stimulation, and formulation are all performed within a closed system. In some embodiments, one or more steps are performed outside the closed system or device. In some such embodiments, the cell-enriched composition is transferred out of the closed system or device under sterile conditions, such as by aseptic transfer to a separate closed system.

[0080] In certain embodiments, the enriched T-cell composition may be collected, formulated for cryoprotection, cryopreservation, and / or storage at below 0°C, below -20°C, or below -70°C or -80°C, or below -70°C or -80°C, before, during, or after any stage or step of the process for generating an output composition of enriched T cells expressing the recombinant receptor. In some embodiments, cells may be stored for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or less than 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or more than 8 weeks. After storage, the enriched T-cell composition may be thawed, and processing may be resumed from the same point in the process. In some embodiments, the enriched T-cell input composition is cryopreserved and stored, followed by further processing, such as incubation under stimulating conditions. In a particular implementation, the cultured and / or formulated composition of enriched T cells is cryogenically frozen and stored, and then administered to a subject, for example, as an autologous cell therapy.

[0081] In some embodiments, individual cell compositions enriched with T cells are combined into a single composition. For example, in some embodiments, compositions enriched with CD4+ T cells and compositions enriched with CD8+ T cells are combined into a single composition enriched with both CD4+ and CD8+ T cells. In some embodiments, the individual compositions are derived from the same biological sample, such as the same biological sample obtained, collected, and / or acquired from a single subject, from which they were initially isolated, selected, and / or enriched. In some embodiments, the individual compositions are processed separately for one or more steps or stages of the process for generating the output composition, such as a process combined with the provided method. In some embodiments, the individual compositions may be combined into a single composition before, during, or after any step or stage of the process for generating the output composition. Thus, in some embodiments, individual input, stimulation, engineering, culture, formulation, and / or harvesting compositions of enriched T cells from the same biological sample are combined into a single composition, and in some embodiments, this is further processed as a single composition. In some embodiments, individual output compositions of enriched cells are combined into a single output composition before the cells are administered to the subject.

[0082] In some embodiments, at any stage or step of the process, a portion of the cells may be sampled or collected, for example, cells may be removed from the composition enriched with T cells while the composition is held in a closed system, such as during isolation, incubation, engineering, cultivation, and / or formulation. In some embodiments, markers, characteristics, or properties of such cells may be analyzed, including but not limited to viability, apoptosis, activation, stimulation, growth, and / or consumption. In some embodiments, cells are sampled or collected via an automated process while the composition enriched with T cells is held in a closed system. In some embodiments, analysis of the sampled or collected cells is automated. In a particular embodiment, the analysis is performed under aseptic conditions in a closed system.

[0083] In some embodiments, cells or cell compositions produced and / or processed by the provided methods can be compared with cells or cell compositions processed or produced by exemplary and / or alternative processes. In some embodiments, alternative and / or exemplary processes may differ in one or more specific aspects, but in other aspects contain similar or identical features, aspects, steps, stages, reagents, and / or conditions of embodiments or aspects of the provided methods being compared. For example, when the provided methods are used in conjunction with incubating cells in the presence of a reagent, such cells can be compared with cells not incubated with said reagent in exemplary and / or alternative processes. In some embodiments, unless otherwise stated, the provided methods and exemplary and / or alternative processes are similar and / or identical in other respects, such as having similar or identical steps of isolation, selection, enrichment, activation, stimulation, engineering, transfection, transduction, culture, and / or formulation. In some embodiments, unless otherwise stated, the provided methods and alternative processes isolate, select, and / or enrich cells from the same or similar types of biological samples, and / or process cells of the same cell type and / or input cells.

[0084] Cells and compositions (including pharmaceutical compositions and formulations) prepared by the method are also provided, as well as kits, systems, and apparatus for performing the method. Methods for using cells and compositions prepared by the method (including therapeutic methods, such as methods for adoptive cell therapy) and pharmaceutical compositions for administration to subjects are also provided.

[0085] A. Sample and Cell Preparation

[0086] In certain embodiments, the provided method is used in conjunction with one or more input compositions that isolate, select, and / or enrich cells from biological samples to generate enriched cells, such as T cells. In some embodiments, the provided method includes isolating cells or compositions thereof from biological samples, such as those obtained from or derived from a subject, such as a subject suffering from a specific disease or condition or requiring or to be given cell therapy. In some aspects, the subject is a human being, such as a subject who is a patient requiring a specific therapeutic intervention (e.g., adoptive cell therapy, where cells are isolated, processed, and / or engineered for use in the therapeutic intervention). Thus, in some embodiments, the cells are primary cells, such as primary human cells. The samples include tissues, fluids, and other samples taken directly from the subject. The biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, bodily fluids (e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived from them.

[0087] In some respects, the sample is blood or a blood-derived sample, or is derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, bowel-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived therefrom. In the case of cell therapy (e.g., adoptive cell therapy), the sample includes samples from autologous and allogeneic sources.

[0088] In some instances, cells are obtained from the subject's circulating blood via apheresis or leukoablation. In some aspects, the sample contains lymphocytes (including T cells, monocytes, granulocytes, and B cells), other nucleated leukocytes, erythrocytes, and / or platelets, and in some aspects, cells other than erythrocytes and platelets.

[0089] In some embodiments, blood cells collected from the subject are washed, for example to remove plasma fractions, and the cells are placed in 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 wash solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is performed in a semi-automatic "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, Baxter), according to the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF), according to the manufacturer's instructions. In some embodiments, the cells are resuspended after washing in a variety of biocompatible buffers (e.g., calcium-free buffers). ++ / Mg ++ In some embodiments, components of the blood cell sample are removed and the cells are directly resuspended in the culture medium.

[0090] In some embodiments, the preparation method includes the steps of isolating, selecting and / or enriching, and / or incubating for transduction and engineering, before or after incubation, and / or freezing (e.g., cryopreserving) the cells after culturing and / or harvesting the engineered cells. In some embodiments, the freezing and subsequent thawing steps remove granulocytes from the cell population and, to some extent, monocytes. In some embodiments, for example, after a washing step to remove plasma and platelets, the cells are suspended in a freezing solution. In some aspects, any of a variety of known freezing solutions and parameters can be used. In some embodiments, the cells are frozen in a medium and / or solution, such as cryopreservation or cryopreservation, the medium and / or solution having a final concentration of or about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO. In a particular embodiment, the cells are frozen in a medium and / or solution, such as cryopreservation or cryopreservation, the medium and / or solution having a final concentration of HSA of about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25%, or between 0.1% and -5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2%. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with the medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then typically frozen to -80°C or about -80°C at a rate of about 1° / min and stored in the gas phase of a liquid nitrogen tank.

[0091] In some embodiments, the isolation of the cells or population includes one or more preparative and / or non-affinity-based cell isolation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to a specific reagent. In some examples, cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity to a specific component, and / or resistance. In some embodiments, the method includes density-based cell isolation methods, such as preparing leukocytes from peripheral blood by lysing erythrocytes and centrifuging via Percoll or Ficoll gradient.

[0092] In some embodiments, at least a portion of the selection step includes incubating cells with a selection reagent. For example, incubation with one or more selection reagents as part of a selection method can be performed using one or more selection reagents to select one or more different cell types based on the expression or presence of one or more specific molecules in or on cells, such as surface markers (e.g., surface proteins), intracellular markers, or nucleic acids. In some embodiments, any known method can be used that employs one or more selection reagents for separation based on such markers. In some embodiments, one or more selection reagents result in separation based on affinity or the administration of immunoaffinity. For example, in some aspects, selection includes incubation with one or more reagents for separating cells and cell populations based on the cellular expression or expression level of one or more markers (typically cell surface markers), such as by incubation with antibodies or binding couplers specifically bound to such markers, followed typically by a washing step and separation of cells bound to the antibody or binding coupler from those not bound to the antibody or binding coupler.

[0093] In some aspects of such processes, a volume of cells is mixed with a desired amount of affinity-based selection reagent. Immune affinity-based selection can be performed using any system or method that induces a favorable energy interaction between the separated cells and labeled molecules (e.g., antibodies or other binding partners) that specifically bind to the cells on a solid surface (e.g., particles). In some embodiments, the method uses particles such as beads, for example magnetic beads, coated with a selection agent (e.g., antibody) that is specific to the cell label. The particles (e.g., beads) can be incubated or mixed with the cells in a container (e.g., tube or bag) while agitated or mixed, wherein the ratio of cell density to particles (e.g., beads) is constant to help promote an energy-favorable interaction. In other cases, the method includes selecting cells, wherein all or part of the selection is performed within the internal cavity of a centrifuge chamber, for example, under centrifugal rotation. In some embodiments, incubation of cells with the selection reagent (e.g., an immunoaffinity-based selection reagent) is performed in a centrifuge chamber. In some embodiments, the separation or detachment is performed using the system, apparatus, or device described in International Patent Application Publication No. WO 2009 / 072003 or US 20110003380 A1. In one example, the system is as described in International Publication No. WO 2016 / 073602.

[0094] In some embodiments, by performing such a selection step or part thereof within the cavity of a centrifuge chamber (e.g., incubating with antibody-coated particles (e.g., magnetic beads)), the user is able to control certain parameters, such as the volume of various solutions, the addition of solutions during treatment, and their timing, which can provide several advantages compared to other available methods. For example, the ability to reduce the volume of liquid in the cavity during incubation can increase the concentration of the particles used in the selection (e.g., bead reagents), and thereby increase the chemical potential of the solution without affecting the total number of cells in the cavity. This, in turn, can enhance the pairwise interaction between the cells being treated and the particles used for selection. In some embodiments, for example, when associated with systems, circuitry, and controls as described herein, performing the incubation step in a chamber allows the user to agitate the solution at one or more desired times during incubation, which can also improve the interaction.

[0095] In some embodiments, at least a portion of the selection step is performed in a centrifuge chamber, including incubating the cells with a selection reagent. In some aspects of such methods, a volume of cells is mixed with a desired amount of affinity-based selection reagent, said amount being significantly less than that typically used when performing a similar selection in a tube or container to select the same number and / or volume of cells according to the manufacturer's instructions. In some embodiments, the amount of one or more selection reagents used is no more than 5%, 10%, 15%, 20%, 25%, 50%, 60%, 70%, or 80% of the amount of the same one or more selection reagents used for selecting cells in a tube or container-based incubation, according to the manufacturer's instructions.

[0096] In some embodiments, for cell selection, such as selection based on immunoaffinity, cells are incubated in a chamber cavity in a composition that also contains a selection buffer with a selection reagent, such as an antibody, specifically binding to a surface-labeled molecule, like an antibody, on cells that are desired to be enriched and / or depleted (but not on other cells in the composition). This molecule is optionally coupled to a scaffold, such as a polymer, or a surface, such as beads, like magnetic beads, such as magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8. In some embodiments, as described, the selection reagent is added to the cells in the chamber cavity in an amount significantly smaller than, or less than, the amount required to achieve substantially the same or similar selection efficiency for the same number or volume of cells when selected in an oscillating or rotating tube (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of said amount). In some embodiments, incubation is performed with the addition of a selection buffer to the cells and the selection reagent to achieve a target incubation volume, such as 10 mL to 200 mL, including at least or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL of reagent. In some embodiments, the selection buffer and the selection reagent are premixed before being added to the cells. In some embodiments, the selection buffer and the selection reagent are added to the cells separately. In some embodiments, selection incubation is performed under periodic, mildly mixed conditions, which can help promote energy-favorable interactions, thereby allowing for the use of less total selection reagent while achieving high selection efficiency.

[0097] In some implementations, the total incubation time with the selected reagent is from 5 minutes to 6 hours or from about 5 minutes to about 6 hours, such as 30 minutes to 3 hours, for example at least or about 30 minutes, 60 minutes, 120 minutes or 180 minutes.

[0098] In some embodiments, incubation is typically carried out under mixed conditions, such as in the presence of rotation, usually at a relatively low force or speed, such as a speed lower than that used to precipitate cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at a certain RCF at the sample or wall of a chamber or other container, said RCF being from 80 g to 100 g or from about 80 g to about 100 g (e.g., or about or at least 80 g, 85 g, 90 g, 95 g or 100 g). In some embodiments, rotation is performed using repeated intervals of such low-speed rotation followed by a pause, such as rotation and / or pause for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds, for example, rotation for about 1 or 2 seconds, followed by a pause for about 5, 6, 7 or 8 seconds.

[0099] In some embodiments, the process is carried out within a completely closed system integral with the chamber. In some embodiments, the process (and in some respects one or more additional steps, such as a pre-washing step for washing samples containing cells, such as agarose samples) is automated, such that cells, reagents, and other components are drawn into and expelled from the chamber at appropriate times and centrifuged, so that the washing and binding steps are completed in a single closed system using an automated procedure.

[0100] In some embodiments, after incubating and / or mixing cells with one and / or more selection reagents, the incubated cells are separated to select cells based on the presence or absence of one or more specific reagents. In some embodiments, separation is performed in the same closed system in which cells are incubated together with the selection reagent. In some embodiments, after incubation with the selection reagent, the incubated cells (including cells in which the selection reagent has been bound) are transferred to a system for separating cells based on immunoaffinity. In some embodiments, the system for immunoaffinity-based separation is or contains a magnetic separation column.

[0101] Such separation steps can be based on positive selection (where cells that have bound to the reagent (e.g., antibody or binding coupler) are retained for further use) and / or negative selection (where cells that have not yet bound to the reagent (e.g., antibody or binding coupler) are retained). In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when antibodies specifically identifying cell types in a heterogeneous population are unavailable, making it preferable to perform separation based on markers expressed by cells that are not from the desired population.

[0102] In some embodiments, the process steps further include negative and / or positive selection of the incubated cells, such as using a system or device capable of affinity-based selection. In some embodiments, separation is performed by enriching a specific cell population via positive selection or depleting a specific cell population via negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers expressed (labeled +) or expressed at relatively high levels (labeled high) on the cells of the positive or negative selection, respectively. Multiple rounds of the same selection steps, such as positive or negative selection steps, may be performed. In some embodiments, fractions of positive or negative selection undergo the selection process, such as by repeating the positive or negative selection steps. In some embodiments, the selection is repeated two, three, four, five, six, seven, eight, nine, or more than nine times. In some embodiments, the same selection is performed up to five times. In some embodiments, the same selection step is performed three times.

[0103] Isolation does not need to result in 100% enrichment or removal of a specific cell population or cells expressing a specific marker. For example, positive selection or enrichment for a specific type of cell (such as those expressing a marker) means increasing the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion for a specific type of cell (such as those expressing a marker) means reducing the number or percentage of such cells, but does not need to result in the complete removal of all such cells.

[0104] In some examples, multiple rounds of separation steps are performed, where fractions of positive or negative selection from one step undergo another separation step, such as subsequent positive or negative selection. In some examples, a single separation step can simultaneously deplete cells expressing multiple markers, such as by incubating cells with multiple antibodies or binding partners (each antibody or binding partner is specific to the marker targeted by negative selection). 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 embodiments, one or more separation steps are repeated and / or performed more than once. In some embodiments, fractions of positive or negative selection resulting from a separation step undergo the same separation step, such as 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 negative selection fraction. In some 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 implementations, 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 implementations, one or more selection steps are repeated three times.

[0105] For example, in some aspects, specific subsets of T cells, such as cells that are positive for or express high levels of one or more surface markers, are isolated using positive or negative selection techniques, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells. In some embodiments, such cells are selected by incubating with one or more antibodies or binding conjugates that specifically bind to such markers. In some embodiments, the antibodies or binding conjugates may be directly or indirectly conjugated to a solid support or matrix used to achieve selection, such as magnetic beads or paramagnetic beads. For example, CD3+, CD28+ T cells can be selected using CD3 / CD28 conjugated magnetic beads (e.g., M-450CD3 / CD28 T cell expander and / or Positive selection was performed on the pearl.

[0106] In some implementations, T cells are isolated from PBMC samples through negative selection of markers (such as CD14) expressed on non-T cells (such as B cells, monocytes, or other leukocytes). In some aspects, a CD4+ or CD8+ selection step is 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 of markers expressed or expressed at relatively high levels on one or more naive, memory, and / or effector T cell subsets.

[0107] In some implementations, CD8+ T cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the corresponding subsets. In some implementations, central memory T (TCM) cells are enriched to increase efficacy, such as to improve long-term survival, expansion, and / or transplantation after administration, which is particularly robust in some respects in such subsets. See Terakura et al., (2012) Blood. 1:72–82; Wang et al. (2012) J Immunother. 35(9):689-701. In some implementations, TCM-rich CD8+ T cells are combined with CD4+ T cells to further enhance efficacy.

[0108] In the implementation scheme, memory T cells are present in two subsets of CD8+ peripheral blood lymphocytes: CD62L+ and CD62L-. PBMCs can be enriched or depleted for the CD62L-CD8+ and / or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

[0109] In some implementations, the enrichment of central memory T (TCM) cells is based on the 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, the isolation of a CD8+ population rich in 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, the enrichment of central memory T (TCM) cells begins with a negative cell fraction selected based on CD4 expression, which is then negatively selected based on the expression of CD14 and CD45RA and positively selected based on CD62L.

[0110] In some aspects, such selection is performed simultaneously, and in others, sequentially in any order. In some aspects, the same CD4 expression-based selection step used to prepare a CD8+ T cell population or subset is also used to generate a CD4+ T cell population or subset, such that positive and negative fractions from CD4-based isolation are retained and used in subsequent steps of the method, optionally after one or more other 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 selection of the CD8+ T cell population are performed sequentially in any order. In some embodiments, the methods for selecting cells may include those described in published U.S. Application No. US20170037369. In some embodiments, the selected CD4+ T cell population and the selected CD8+ T cell population may be combined after selection. 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, such as according to the provided method, selected CD4+ T cell populations and selected CD8+ T cell populations are treated separately, thereby enriching the selected CD4+ T cell population with CD4+ T cells and incubating it with a stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads), transducing it with a viral vector encoding a recombinant protein (e.g., CAR), and culturing it under T cell amplification conditions; and enriching the selected CD8+ T cell population with CD8+ T cells and incubating it with a stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads), transducing it with a viral vector encoding a recombinant protein (e.g., CAR) (such as the same recombinant protein from the same donor used for engineered CD4+ T cells), and culturing it under T cell amplification conditions.

[0111] In certain embodiments, biological samples, such as PBMCs or other leukocyte samples, undergo selection for CD4+ T cells, where negative and positive fractions are retained. In some embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, biological samples undergo selection for CD8+ T cells, where negative and positive fractions are retained. In some embodiments, CD4+ T cells are selected from the negative fraction.

[0112] In a specific example, PBMC samples or other leukocyte samples were subjected to selection by CD4+ T cells, in which negative and positive fractions were preserved. The negative fractions were then subjected to negative selection based on the expression of CD14 and CD45RA or CD19, and positive selection based on markers specific to central memory T cells (such as CD62L or CCR7), wherein the positive and negative selections were performed in any order.

[0113] CD4+ T helper cells can be classified into naive, central memory, and effector cells by identifying cell populations possessing cell surface antigens. CD4+ lymphocytes can be obtained using 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-.

[0114] In one example, to enrich CD4+ T cells through negative selection, monoclonal antibody mixtures typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding couplers are bound to a solid support or matrix (such as magnetic or paramagnetic beads) to allow cell separation for positive and / or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (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 S. Brooks and U. Schumacher). Humana Press Inc., Totowa, NJ.

[0115] In some respects, the incubation sample or composition of cells to be isolated is mixed with small magnetizable or magnetically responsive materials containing selective reagents (such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., like Dynalbeads or...). The materials are incubated together with the beads. Magnetic responsive materials (e.g., particles) are typically attached directly or indirectly to binding partners (e.g., antibodies) that specifically bind to molecules (e.g., surface markers) present on a single cell, multiple cells, or cell population that need to be separated (e.g., for negative or positive selection).

[0116] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material that binds to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials used in magnetic separation methods are known, such as those described in Molday, U.S. Patent No. 4,452,773, and European Patent Specification EP 452342B, which are hereby incorporated by reference. Colloidal-sized particles, such as those described in Owen, U.S. Patent No. 4,795,698; and Liberti et al., U.S. Patent No. 5,200,084, may also be used.

[0117] Incubation is typically carried out under conditions in which antibodies or binding couplers, or molecules (such as secondary antibodies or other reagents) that specifically bind to such antibodies or binding couplers attached to magnetic particles or beads, specifically bind to cell surface molecules (if present on the cells within the sample).

[0118] In some embodiments, the magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In some embodiments, the magnetic particles are attached to cells by coating with a primary antibody specific to one or more labels. In some embodiments, the cells, instead of 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 embodiments, the streptavidin-coated magnetic particles are used in conjunction with a biotinylated primary or secondary antibody.

[0119] In some aspects, separation is achieved in a procedure in which the sample is placed in a magnetic field, and those cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted by the magnet are retained; for negative selection, cells not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, wherein positive and negative fractions are retained for further processing or subjected to further separation steps.

[0120] In some embodiments, affinity-based selection is performed via magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetically activated cell sorting (MACS), such as the CliniMACS system, is capable of selecting cells with high purity attached to magnetized particles. In some embodiments, the MACS operates in a manner where non-target species and target species are eluted sequentially after an external magnetic field is applied. That is, while eluting unattached species, cells attached to the magnetized particles are held in place. Then, after this first elution step is completed, the species trapped in the magnetic field and prevented from elution are released in a way that allows them to be eluted and recovered. In some embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0121] In some embodiments, the magnetically responsive particles remain attached to cells that are subsequently incubated, cultured, and / or engineered; 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 competitive unlabeled antibodies, magnetizable particles, or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0122] In some embodiments, the isolation and / or selection yields one or more input compositions enriched with T cells, such as CD3+ T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, two or more separate 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, acquired, and / or obtained from the same subject.

[0123] In some embodiments, the one or more input compositions are or comprise a composition enriched with T cells, the composition 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 about 100% CD3+ T cells. In a particular embodiment, the input composition enriched with T cells consists substantially of CD3+ T cells.

[0124] In some embodiments, the one or more input compositions are or comprise compositions enriched with CD4+ T cells, the 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 about 100% CD4+ T cells. In some 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 contains no CD8+ T cells, and / or contains no or substantially no CD8+ T cells. In some embodiments, the T cell enriched composition consists substantially of CD4+ T cells.

[0125] In some embodiments, the one or more compositions are or comprise CD8+ T cell compositions, wherein the compositions are or contain 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 are or are about 100% CD8+ T cells. In some embodiments, the CD8+ T cell 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% CD4+ T cells, and / or contains no CD4+ T cells, and / or contains no or substantially no CD4+ T cells. In some embodiments, the T cell-enriched composition consists substantially of CD8+ T cells.

[0126] In some embodiments, one or more input compositions of the enriched T cells are frozen, e.g., cryopreserved and / or cryopreserved, after isolation, selection, and / or enrichment. In some embodiments, the one or more input compositions are frozen, e.g., cryopreserved and / or cryopreserved, before any step of incubation, activation, stimulation, engineering, transduction, transfection, culture, expansion, harvesting, and / or formulation of the cells. In a particular embodiment, the one or more cryopreserved input compositions are stored, for example, at or at about -80°C for 12 hours to 7 days, 24 hours to 120 hours, or 2 days to 5 days. In a particular embodiment, the one or more cryopreserved input compositions are stored at or at about -80°C for less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the one or more cryopreserved input compositions are stored at or at about -80°C for 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, or about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0127] B. Cell activation and stimulation

[0128] In some embodiments, the provided method is used in conjunction with incubating cells under stimulating conditions. In some embodiments, the stimulating conditions include conditions that activate or stimulate, and / or are capable of activating or stimulating signals in cells (e.g., CD4+ T cells or CD8+ T cells), such as signals generated from TCRs and / or co-receptors. In some embodiments, the stimulating conditions include one or more steps of culturing, cultivating, incubating, activating, or proliferating cells with a stimulating agent (e.g., an agent that activates or stimulates, and / or is capable of activating or stimulating signals in cells) and / or in the presence of said stimulating agent. In some embodiments, the stimulating agent stimulates and / or activates TCRs and / or co-receptors. In a particular embodiment, the stimulating agent is the agent described in section IB-1.

[0129] In some embodiments, one or more compositions of enriched T cells are incubated under stimulating conditions prior to transfection and / or transduction of genetically engineered cells, such as cells using the techniques provided in Chapter IC. In a particular embodiment, the one or more compositions of enriched T cells are incubated under stimulating conditions after they have been isolated, selected, enriched, or obtained from a biological sample. In a particular embodiment, the one or more compositions are input compositions. In a particular embodiment, the one or more input compositions have been previously cryogenically frozen and stored, and thawed prior to incubation.

[0130] In some embodiments, the one or more compositions of enriched T cells are or comprise two separate compositions of enriched T cells, such as separate input compositions. In a particular embodiment, two separate compositions of enriched T cells, for example, two separate compositions of enriched T cells selected, isolated, and / or enriched from the same biological sample, are incubated separately under stimulation conditions. In some embodiments, the two separate compositions comprise a composition of enriched CD4+ T cells. In a particular embodiment, the two separate compositions comprise a composition of enriched CD8+ T cells. In some embodiments, two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are incubated separately under stimulation conditions.

[0131] In some embodiments, a single composition enriched with T cells is incubated under stimulating conditions. In some embodiments, the single composition is a composition enriched with CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells that have been combined from individual compositions prior to incubation.

[0132] In some embodiments, the composition of CD4+ T cells incubated under stimulation conditions 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%, at least 99.5%, at least 99.9%, or about 100% CD4+ T cells. In some embodiments, the composition of CD4+ T cells incubated under stimulation conditions comprises 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 contains no CD8+ T cells, and / or contains no or substantially no CD8+ T cells.

[0133] In some embodiments, the composition of CD8+ T cells incubated under stimulation conditions 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%, at least 99.5%, at least 99.9%, or about 100% CD8+ T cells. In some embodiments, the composition of CD8+ T cells incubated under stimulation conditions comprises 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 contains no CD4+ T cells, and / or contains no or substantially no CD4+ T cells.

[0134] 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 embodiments, separate stimulated compositions of enriched CD4+ and CD8+ T cells are combined into a single composition after incubation has been performed and / or completed. In some embodiments, as provided by the method, separate stimulated compositions of stimulated CD4+ T cells and stimulated CD8+ T cells are treated separately after incubation has been performed and / or completed, thereby transducing a population of stimulated CD4+ T cells (e.g., incubated with stimulating anti-CD3 / anti-CD28 magnetic bead stimulating reagent) with a viral vector encoding a recombinant protein (e.g., CAR) and culturing it under T cell expansion conditions, and transducing a population of stimulated CD8+ T cells (e.g., incubated with stimulating anti-CD3 / anti-CD28 magnetic bead stimulating reagent) with a viral vector encoding a recombinant protein (e.g., CAR) (e.g., the same recombinant protein from the same donor used for engineered CD4+ T cells) with a viral vector and culturing it under T cell expansion conditions.

[0135] In some embodiments, the incubation under stimulating conditions may include culturing, nurturing, stimulating, activating, or propagating, including incubation in the presence of stimulating conditions designed, for example, to induce cell proliferation, expansion, activation, and / or survival in a population, to mimic antigen exposure, and / or to induce cell use for genetic engineering, such as for the introduction of recombinant antigen receptors. In certain embodiments, the stimulating conditions may include one or more of the following: specific culture media, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulating factors (e.g., cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells)).

[0136] In some respects, stimulation and / or incubation under stimulation conditions are carried out according to a variety of techniques, such as those described in the following documents: U.S. Patent No. 6,040,177 to Riddell et al.; 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.

[0137] In some implementations, cells (e.g., T cells), cell compositions, and / or cell populations, such as CD4, are expanded through methods such as... +And CD8+ T cells or combinations thereof, populations or subsets: Add feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the culture starting composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells per T lymphocyte in the initial population to be expanded); and incubate the culture (e.g., for a duration sufficient to expand the number of said T cells). In some aspects, the non-dividing feeder cells may comprise γ-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with γ rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, feeder cells are added to the culture medium prior to the addition of the T cell population.

[0138] In some embodiments, stimulation conditions include temperatures suitable for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically or about 37 degrees Celsius. In some embodiments, a temperature transition is achieved during culture, such as from 37 degrees Celsius to 35 degrees Celsius. Optionally, incubation may further include the addition of non-dividing EBV-transformed lymphoid stem cells (LCLs) as feeder cells. LCLs can be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some aspects, LCL feeder cells are provided in any suitable amount, such as an LCL feeder cell to naïve T lymphocyte ratio of at least about 10:1.

[0139] In the implementation plan, antigen-specific CD4 cells can be obtained by stimulating naive or antigen-specific T lymphocytes with the antigen. + and CD8 + A population. 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. Naïve T cells can also be used.

[0140] In certain embodiments, the stimulating conditions include incubating, culturing, and / or cultivating cells with a stimulating agent. In certain embodiments, the stimulating agent is the agent described in section IB-1. In some embodiments, the stimulating agent contains or includes beads. An exemplary stimulating agent is or includes anti-CD3 / anti-CD28 magnetic beads. In some embodiments, the initiation and / or commencement of incubation, culturing, and / or cultivation of cells under stimulating conditions occurs when cells are contacted with and / or incubated with the stimulating agent. In certain embodiments, cells are incubated before, during, and / or after genetically engineered cells (e.g., as introduced into cells via transfection or transduction).

[0141] In some embodiments, the composition enriched T cells is incubated with a stimulating agent and / or beads (e.g., anti-CD3 / anti-CD28 magnetic beads) at a ratio of 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1 to cells. In a particular embodiment, the ratio of the stimulating agent and / or beads to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, or between 1.1:1 and 0.9:1. In a particular embodiment, the ratio of the stimulating agent to cells is about 1:1 or 1:1.

[0142] In certain embodiments, incubating the cells at a ratio of less than 3:1 or less than 3 beads per cell (e.g., a 1:1 ratio) of the stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads) reduces the amount of cell death (e.g., activation-induced death) that occurs during incubation. In some embodiments, the cells are incubated together with the stimulating agent (e.g., 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 the cells at a ratio of less than 3:1 or less than 3 beads per cell (e.g., a 1:1 ratio) reduces the amount of cell death (e.g., activation-induced death) that occurs during incubation.

[0143] In a particular embodiment, the composition enriched with T cells is incubated with the stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads) at a stimulating agent to cell ratio of less than 3:1 (e.g., a 1:1 ratio), 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 example, being viable and / or not undergoing necrosis, programmed cell death, or apoptosis, during 1, 2, 3, 4, 5, 6, 7, or more days after the completion of incubation. In a particular embodiment, the composition enriched with T cells is incubated with the stimulating agent at a stimulating agent to cell ratio of less than 3:1 and / or a bead to cell ratio of less than 1:1, and during incubation, 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 the cells undergo activation-induced cell death.

[0144] In some embodiments, the composition enriched with T cells is incubated with the stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads) at a bead-to-cell ratio of less than 3:1 (e.g., a 1:1 ratio), and the cells of the composition have a survival rate of 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 compared to cells that have undergone exemplary and / or alternative processes (where the composition enriched with T cells is incubated with the stimulating agent at a ratio of 3:1 or greater).

[0145] In some embodiments, the composition of the enriched T cells incubated with the stimulating agent comprises 1.0 x 10⁻⁶ T cells. 5 cells / mL to 1.0 x 10⁻⁶ 8 1.0 x 10⁻⁶ cells / mL or from approximately 1.0 x 10⁻ 5 cells / mL to approximately 1.0 x 10⁻⁶ 8 Cells / mL, such as at least or about 1.0 x 10⁻⁶. 5 cells / mL, 5 x 10 5 cells / mL, 1 x 10 6 cells / mL, 5 x 10 6 cells / mL, 1 x 10 7 cells / mL, 5 x 10 7 1 cell / mL or 1 x 10 8 Cells / mL. In some embodiments, the composition of enriched T cells incubated with the stimulating agent contains about 0.5 x 10⁻⁶ cells / mL. 6 cells / mL, 1x10 6 cells / mL, 1.5 x 10 6 cells / mL, 2 x 10 6 cells / mL, 2.5 x 10 6 cells / mL, 3 x 10 6 1 cell / mL, 3.5 x 10 6 cells / mL, 4 x 10 6 4.5 x 10 cells / mL 6 cells / mL, 5 x 10 6 5.5 x 10 cells / mL 6 cells / mL, 6 x 10 6 1 cell / mL, 6.5 x 10 61 cell / mL, 7 x 10 6 1 cell / mL, 7.5 x 10 6 1 cell / mL, 8 x 10 6 8.5 x 10 cells / mL 6 cells / mL, 9 x 10 6 1 cell / mL, 9.5 x 10 6 1 cell / mL or 10 x 10 6 Cells / mL, such as approximately 2.4 x 10⁻⁶ cells / mL 6 Cells / mL.

[0146] In some embodiments, the composition enriched with T cells is incubated with the stimulating agent at a temperature ranging from about 25°C to about 38°C, such as from about 30°C to about 37°C, for example, at or above about 37°C ± 2°C. In some embodiments, the composition enriched with T cells is incubated with the stimulating agent at a CO2 level ranging from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example, at or above about 5% ± 0.5%. In some embodiments, the composition enriched with T cells is incubated with the stimulating agent at a temperature of or above about 37°C and / or at a CO2 level of or above about 5%.

[0147] In certain embodiments, the stimulation conditions include incubating, culturing, and / or cultivating a composition enriched with T cells in the presence of one or more cytokines and / or in the presence of one or more cytokines. In certain embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In some embodiments, the one or more cytokines bind to and / or are capable of binding to receptors expressed by T cells and / or endogenous to T cells. In certain embodiments, the one or more cytokines are or include members of the 4-α-helical bundle cytokine family. In some embodiments, members of the 4-α-helical bundle cytokine family 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, the one or more cytokines are or include IL-15. In a particular embodiment, the one or more cytokines are or include IL-7. In a particular embodiment, the one or more cytokines are or include IL-2. In some embodiments, the stimulation conditions comprise incubating a composition of enriched T cells (e.g., enriched CD4+ T cells or enriched CD8+ T cells) in the presence of the described stimulation reagent (anti-CD3 / anti-CD28 magnetic beads) and in the presence of one or more recombinant cytokines.

[0148] In certain embodiments, the composition enriched with CD4+ T cells is incubated with IL-2, such as recombinant IL-2. Where it is not desired to be bound by theory, certain embodiments consider that CD4+ T cells obtained from some subjects do not produce or insufficiently produce an amount of IL-2 that allows for growth, division, and expansion throughout the process of producing the composition used to generate export cells (e.g., engineered cells suitable for use in cell therapy). In some embodiments, incubating the composition enriched with CD4+ T cells under stimulation conditions in the presence of recombinant IL-2 increases the probability or likelihood that the CD4+ T cells of the composition will continue to survive, grow, expand, and / or be activated during the incubation step and throughout the process. In some embodiments, compared to alternative and / or exemplary methods of incubating the composition enriched with CD4+ T cells in the presence of recombinant IL-2, incubating the composition enriched with CD4+ T cells in the presence of recombinant IL-2 will generate enriched CD4+ T cells (e.g., engineered CD4+ T cells suitable for use in cell therapy) from the composition enriched with CD4+ T cells. The probability and / or likelihood of the output composition of T cells increases 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%, 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.

[0149] In some embodiments, the amount or concentration of one or more cytokines is measured and / or quantified using 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 a unit of measurement for the potency of a biological agent by comparison with an international reference standard having specific gravity and specific strength (e.g., WHO First International Standard for Human IL-2, 86 / 504). International Units are the only recognized and standardized method for reporting units of bioactivity, which are published and derived from international collaborative research. In certain embodiments, the IU of a composition, sample, or source of cytokines can be obtained through product comparison testing with similar WHO standard products. For example, in some embodiments, a composition, sample, or source of human recombinant IL-2, IL-7, or IL-15 is compared with WHO standard IL-2 products (NIBSC code: 86 / 500), WHO standard IL-17 products (NIBSC code: 90 / 530), and WHO standard IL-15 products (NIBSC code: 95 / 554), respectively.

[0150] In some implementations, the bioactivity, expressed in IU / mg, is equivalent to (ED) in ng / ml. 50 ) -1 x10 6 In a specific implementation, the ED of recombinant human IL-2 or IL-15 50 This is equivalent to the concentration required for half-maximal stimulation (XTT cleavage) of cell proliferation using CTLL-2 cells. In some embodiments, the ED of recombinant human IL-7... 50 This is equivalent to the half-maximal stimulation required for the proliferation of PHA-activated human peripheral blood lymphocytes. Details relating to the determination and calculation of IU of 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; details relating to the determination and calculation of IU of IL-15 are discussed in Soman et al., Journal of Immunological Methods (2009), 348(1-2):83-94; all of which are hereby incorporated by reference in their entirety.

[0151] In certain embodiments, the composition enriched with CD8+ T cells is incubated under stimulating conditions in the presence of IL-2 and / or IL-15. In some embodiments, the composition enriched with CD4+ T cells is incubated under stimulating conditions in the presence of IL-2, IL-7, and / or IL-15. In some embodiments, the IL-2, IL-7, and / or IL-15 are recombinant. In some embodiments, the IL-2, IL-7, and / or IL-15 are human. In certain embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and / or IL-15. In some aspects, incubation of the enriched T cell composition further includes the presence of a stimulating agent, such as anti-CD3 / anti-CD28 magnetic beads.

[0152] In some embodiments, the cells are incubated with cytokines, such as recombinant human cytokines, at concentrations between 1 IU / ml and 1,000 IU / ml, between 10 IU / ml and 50 IU / ml, between 50 IU / ml and 100 IU / ml, between 100 IU / ml and 200 IU / ml, between 100 IU / ml and 500 IU / ml, between 250 IU / ml and 500 IU / ml, or between 500 IU / ml and 1,000 IU / ml.

[0153] In some embodiments, the composition enriched with T cells is incubated with IL-2, such as recombinant human IL-2, at a concentration between 1 IU / ml and 200 IU / ml, between 10 IU / ml and 200 IU / ml, between 10 IU / ml and 100 IU / ml, between 50 IU / ml and 150 IU / ml, between 80 IU / ml and 120 IU / ml, between 60 IU / ml and 90 IU / ml, or between 70 IU / ml and 90 IU / ml. In certain embodiments, the composition enriched with T cells is incubated with recombinant IL-2 at a concentration of 50 IU / ml, 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml, 75 IU / ml, 80 IU / ml, 85 IU / ml, 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 composition enriched with T cells is incubated in the presence of recombinant IL-2 at a concentration of 85 IU / ml or higher. In some embodiments, the composition incubated with recombinant IL-2 enriches a population of T cells, such as CD4+ T cells and / or CD8+ T cells. In some embodiments, the T cell population is a CD4+ T cell population. In some embodiments, the T-cell enriched composition is a CD8+ T-cell enriched composition. In a particular embodiment, the T-cell enriched composition enriches CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the T-cell enriched composition is a CD4+ T-cell enriched composition. In a particular embodiment, the T-cell enriched composition enriches CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition. In some embodiments, the CD4+ T-cell enriched composition incubated with recombinant IL-2 may also be incubated with recombinant IL-7 and / or recombinant IL-15 as described. In some embodiments, the CD8+ T-cell enriched composition incubated with recombinant IL-2 may also be incubated with recombinant IL-15 as described.

[0154] In some embodiments, the composition enriched with T cells is incubated with recombinant IL-7, such as human recombinant IL-7, at a concentration between 100 IU / ml and 2,000 IU / ml, between 500 IU / ml and 1,000 IU / ml, between 100 IU / ml and 500 IU / ml, between 500 IU / ml and 750 IU / ml, between 750 IU / ml and 1,000 IU / ml, or between 550 IU / ml and 650 IU / ml. In a particular embodiment, the composition enriched T cells is incubated with recombinant IL-7 at a concentration of 50 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml, 300 IU / ml, 350 IU / ml, 400 IU / ml, 450 IU / ml, 500 IU / ml, 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, or 1,000 IU / ml. In a particular embodiment, the composition enriched T cells is incubated in the presence of recombinant IL-7 at a concentration of 600 IU / ml or higher. In some embodiments, the composition incubated with recombinant IL-7 enriches a population of T cells, such as CD4+ T cells. In some embodiments, the CD4+ T cell enriched composition incubated with recombinant IL-7 may also be incubated with recombinant IL-2 and / or recombinant IL-15 as described. In a particular embodiment, the T cell enriched composition enriches CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected from or depleted from the composition. In some embodiments, the CD8+ T cell enriched composition is not incubated with recombinant IL-7.

[0155] In some embodiments, the composition enriched with T cells is incubated with recombinant IL-15, such as human recombinant IL-15, at a concentration between 0.1 IU / ml and 100 IU / ml, between 1 IU / ml and 100 IU / ml, between 1 IU / ml and 50 IU / ml, between 5 IU / ml and 25 IU / ml, between 25 IU / ml and 50 IU / ml, between 5 IU / ml and 15 IU / ml, or between 10 IU / ml and 100 IU / ml. In certain embodiments, the composition enriched with T cells is incubated with recombinant IL-15 at a concentration of 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 6 IU / ml, 7 IU / ml, 8 IU / ml, 9 IU / ml, 10 IU / ml, 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 composition enriched with T cells is incubated in or in about 10 IU / ml of recombinant IL-15. In some embodiments, the composition incubated with recombinant IL-15 enriches a population of T cells, such as CD4+ T cells and / or CD8+ T cells. In some embodiments, the T cell population is a CD4+ T cell population. In some embodiments, the T cell-enriched composition is a CD8+ T cell-enriched composition. In a particular embodiment, the T cell-enriched composition enriches CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the T cell-enriched composition is a CD4+ T cell-enriched composition. In a particular embodiment, the T cell-enriched composition enriches CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition. In some embodiments, the CD4+ T cell-enriched composition incubated with recombinant IL-15 may also be incubated with recombinant IL-7 and / or recombinant IL-2 as described. In some embodiments, the CD8+ T cell-enriched composition incubated with recombinant IL-15 may also be incubated with recombinant IL-2 as described.

[0156] In a particular embodiment, the cells (such as enriched CD4+ T cells and / or enriched CD8+ T cells) are incubated with the stimulating agent in the presence of one or more antioxidants. In some embodiments, the antioxidant includes, but is not limited to, one or more antioxidants including tocopherol, tocotrienol, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, α-tocoquinone, Trolox (6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-dicarboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, β-carotene, selenium, ubiquinone, luetin, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine ​​(NAC), citric acid, L-carnitine, BHT, thioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine and cysstathionine, and / or glycine-glycine-histidine. In some aspects, incubation of the enriched T cell composition (such as enriched CD4+ T cells and / or enriched CD8+ T cells) with an antioxidant also includes the presence of a stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads) and one or more recombinant cytokines (as described).

[0157] In some embodiments, the one or more antioxidants are or include sulfur-containing antioxidants. In some embodiments, sulfur-containing antioxidants may include thiol antioxidants and / or, for example, antioxidants exhibiting one or more sulfur moieties within a ring structure. In some embodiments, the sulfur-containing antioxidants may include, for example, N-acetylcysteine ​​(NAC) and 2,3-dimercaptopropanol (DMP), L-2-oxo-4-thiazolidinyl carboxylate (OTC), and lipoic acid. In a particular embodiment, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified into a derived glutathione in one or more steps within the cell. In a particular embodiment, the glutathione precursor may include, but is not limited to, N-acetylcysteine ​​(NAC), L-2-oxothiazolidinyl-4-carboxylic acid (procysteine), lipoic acid, S-allylcysteine, or methylmethionine sulfonium chloride.

[0158] In some embodiments, incubating the cells under stimulating conditions (such as enriched CD4+ T cells and / or enriched CD8+ T cells) includes incubating the cells in the presence of one or more antioxidants. In specific embodiments, the cells are stimulated with the stimulating agent in the presence of one or more antioxidants. In some embodiments, the cells are incubated in the presence of one or more antioxidants at concentrations between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 μg / ml, between 100 ng / ml and 10 μg / ml, between 1 μg / ml and 100 μg / ml, between 10 μg / ml and 1 mg / ml, between 100 μg / ml and 1 mg / ml, between 1500 μg / ml and 2 mg / ml, between 500 μg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml. In some embodiments, the cells are incubated in the presence of one or more of the antioxidants at or above 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, or 500 mg / ml. In some embodiments, the one or more antioxidants are or include sulfur-containing antioxidants. In a particular embodiment, the one or more antioxidants are or include glutathione precursors.

[0159] In some embodiments, the one or more antioxidants are or include N-acetylcysteine ​​(NAC). In some embodiments, incubating the cells under stimulating conditions (such as enriched CD4+ T cells and / or enriched CD8+ T cells) includes incubating the cells in the presence of NAC. In a particular embodiment, the cells are stimulated with the stimulating agent in the presence of NAC. In some embodiments, the cells are incubated in the presence of NAC between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 μg / ml, between 100 ng / ml and 10 μg / ml, between 1 μg / ml and 100 μg / ml, between 10 μg / ml and 1 mg / ml, between 100 μg / ml and 1 mg / ml, between 1-500 μg / ml and 2 mg / ml, between 500 μg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml. In some embodiments, the cells are incubated in the presence of NAC at or above 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, or 500 mg / ml. In some embodiments, the cells are incubated with or above 0.8 mg / ml.

[0160] In certain embodiments, the composition of enriched T cells (such as enriched CD4+ T cells and / or enriched CD8+ T cells) incubated in the presence of one or more antioxidants, such as NAC, reduces activation in cells compared to cells incubated in the absence of an antioxidant in alternative and / or exemplary processes. In some embodiments, the reduced activation is measured by the expression of one or more activation markers in the cells. In some embodiments, activation markers include, but are not limited to, increased intracellular complexity (e.g., as determined by measuring side scattering (SSC), increased cell size (e.g., as determined by measuring cell diameter and / or forward scattering (FSC), increased CD27 expression, and / or decreased CD25 expression. In some embodiments, the cells of the composition have negative, reduced, or low expression and / or levels of activation markers when examined during or after incubation, engineering, transduction, transfection, amplification, or formulation, or during or after any stage of the process performed after incubation. In some embodiments, after the process is completed, the cells of the composition have negative, reduced, or low expression and / or levels of activating markers. In a particular embodiment, the cells of the output composition have negative, reduced, or low expression and / or levels of activating markers.

[0161] In some embodiments, flow cytometry is used to determine the relative size of cells. In specific embodiments, FSC and SSC parameters are used to analyze cells and distinguish them from one another based on size and internal complexity. In specific embodiments, particles or beads of known size can be measured as a standard for determining the actual size of cells. In some embodiments, flow cytometry is used in combination with staining agents such as labeled antibodies to measure or quantify the expression of surface proteins (such as activation markers, such as CD25 or CD27).

[0162] In some embodiments, the 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, such as NAC, and the cell diameter is reduced by at least 0.25 μm, 0.5 μm, 0.75 μm, 1.0 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 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 an antioxidant. In a particular embodiment, the composition enriched with T cells is incubated in the presence of one or more antioxidants, such as NAC, and the cell size 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%, as measured by FSC, compared to cells incubated in alternative and / or exemplary processes where incubation was not performed in the presence of an antioxidant.

[0163] In some embodiments, the 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, such as 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 no incubation is performed in the presence of an antioxidant.

[0164] In a particular embodiment, the 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, such as NAC, and the CD27 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%, as measured by flow cytometry, compared to cells incubated in alternative and / or exemplary processes in which incubation is not performed in the presence of an antioxidant.

[0165] In some embodiments, the 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, such as NAC, and the CD25 expression 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 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 compared to cells incubated in alternative and / or exemplary processes in which no incubation is performed in the presence of an antioxidant, for example, by flow cytometry.

[0166] In certain embodiments, incubating the 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, for example, during the incubation or culture steps or phases described in Section ID. In some embodiments, the cell-enriched composition achieves 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or greater than 10-fold expansion within 14, 12, 10, 9, 8, 7, 6, 5, 4, or 3 days after the start of culture. In some embodiments, the composition of enriched T cells is incubated in the presence of one or more antioxidants, and the cells of the composition experience a rapid expansion rate of 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 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold during culture, compared to cultured cells in alternative and / or exemplary processes in which incubation is not carried out in the presence of antioxidants.

[0167] In certain embodiments, incubating the 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, reduces the amount of cell death (e.g., due to apoptosis). In some embodiments, incubating the composition of enriched T cells in the presence of one or more antioxidants, such as NAC, and after 1, 2, 3, 4, 5, 6, 7 days or more following the completion of incubation, or for at least 1, 2, 3, 4, 5, 6, 7 days or more, results in 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 surviving, for example, without undergoing apoptosis. In some embodiments, the composition is incubated in the presence of one or more antioxidants, such as NAC, and the cells of the composition have a survival rate of 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 compared to cells undergoing exemplary and / or alternative processes in which cells are not incubated in the presence of one or more antioxidants.

[0168] In a particular embodiment, the 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, such as NAC, and caspase expression, such as 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 no incubation in the presence of an antioxidant.

[0169] In some embodiments, the composition or cells (e.g., enriched CD4+ T cells and / or enriched CD8+ T cells) are incubated under the stimulation conditions or in the presence of the stimulating agents described above. Such conditions include those designed to induce cell proliferation, expansion, activation, and / or survival in a population, mimic antigen exposure, and / or initiate cell use for genetic engineering (e.g., for the introduction of recombinant antigen receptors). Exemplary stimulating agents (e.g., anti-CD3 / anti-CD28 magnetic beads) are described below. Incubation with the stimulating agents can also be performed in the presence of one or more stimulating cytokines, such as one or more of 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, the composition enriched CD4+ T cells is incubated under stimulation conditions with a stimulating agent (i.e., recombinant IL-2, recombinant IL-7, recombinant IL-15) and NAC in the amounts described above. In some embodiments, the composition enriched with CD8+ T cells is incubated under stimulation conditions with a stimulator (i.e., recombinant IL-2, recombinant IL-15) and NAC in the amounts described.

[0170] In some implementations, the conditions for stimulation and / or activation may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulating factors (such as cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors and any other agents designed to activate cells)).

[0171] In some respects, incubation is carried out using a variety of techniques, such as those described in the following documents: U.S. Patent No. 6,040,177 to Riddell et al.; 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.

[0172] In some embodiments, at least a portion of the incubation in the presence of one or more stimulating conditions or stimulants is carried out within the internal cavity of a centrifuge chamber, for example, under centrifugal rotation, as described in International Publication No. WO 2016 / 073602. In some embodiments, at least a portion of the incubation carried out in the centrifuge chamber includes mixing with one or more reagents to induce stimulation and / or activation. In some embodiments, cells (e.g., selected cells) are mixed with stimulating conditions or stimulants in the centrifuge chamber. In some aspects of such processes, a volume of cells is mixed with a quantity of one or more stimulating conditions or stimulants, said quantity being much smaller than that typically used when performing similar stimulation in cell culture plates or other systems.

[0173] In some embodiments, a stimulant is added to the cells in the chamber cavity in an amount that achieves approximately the same or similar selection efficiency as, for example, when selected in a tube or bag under periodic oscillation or rotation without mixing in the centrifugation chamber, typically using or significantly reducing the amount of stimulant required (e.g., no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the amount). In some embodiments, incubation is performed with the addition of an incubation buffer to the cells and the stimulant to achieve a target incubation volume of, for example, about 10 mL to about 200 mL, or about 20 mL to about 125 mL, such as at least or about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 105 mL, 110 mL, 115 mL, 120 mL, 125 mL, 130 mL, 135 mL, 140 mL, 145 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, or 200 mL of reagent. In some embodiments, the incubation buffer and the stimulant are premixed before the cells are added. In some embodiments, the incubation buffer and the stimulant are added to the cells separately. In some implementations, stimulation incubation is performed under periodic, mildly mixed conditions, which may help promote energy-friendly interactions and thus allow for the use of less overall stimulant while achieving cell stimulation and activation.

[0174] In some embodiments, incubation is typically carried out under mixed conditions, such as in the presence of rotation, usually at a relatively low force or speed, such as a speed lower than that used to precipitate cells, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at a certain RCF at the sample or wall of a chamber or other container, said RCF being from 80 g to 100 g or from about 80 g to about 100 g (e.g., or about or at least 80 g, 85 g, 90 g, 95 g or 100 g). In some embodiments, rotation is performed using repeated intervals of such low-speed rotation followed by a pause, such as rotation and / or pause for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds, for example, rotation for about 1 or 2 seconds, followed by a pause for about 5, 6, 7 or 8 seconds.

[0175] In some embodiments, the total duration of incubation with the stimulant is, for example, between about 1 hour and 96 hours, between 1 hour and 72 hours, between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, between 18 hours and 30 hours, or between 12 hours and 24 hours, such as at least or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, further incubation is performed for the following durations: between or about 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours, including the end values.

[0176] In some embodiments, the cells are cultured, cultivated, and / or incubated under stimulating conditions before and / or during steps for introducing polynucleotides, such as those encoding recombinant receptors, into cells, for example, by transduction and / or transfection (as described in section IC). In some embodiments, the cells are cultured, cultivated, and / or incubated under stimulating conditions for the following durations prior to genetic engineering: between 30 minutes and 2 hours, between 1 hour and 8 hours, between 1 hour and 6 hours, between 6 hours and 12 hours, between 12 hours and 18 hours, between 16 hours and 24 hours, between 12 hours and 36 hours, between 24 hours and 48 hours, between 24 hours and 72 hours, between 42 hours and 54 hours, between 60 hours and 120 hours, between 96 hours and 120 hours, between 90 hours, and between 1 day and 7 days, between 3 days and 8 days, between 1 day and 3 days, between 4 days and 6 days, or between 4 days and 5 days. In some implementations, the cells are incubated for 2 days or about 2 days prior to engineering.

[0177] In some embodiments, the cells are incubated with and / or in the presence of a stimulating agent before and / or during the genetic engineering of the cells. In some embodiments, the cells are incubated with and / or in the presence of a stimulating agent for the following durations: between 12 and 36 hours, between 24 and 48 hours, between 24 and 72 hours, between 42 and 54 hours, between 60 and 120 hours, between 96 and 120 hours, between 90 hours, between 2 and 7 days, between 3 and 8 days, between 1 and 8 days, between 4 and 6 days, or between 4 and 5 days. In certain embodiments, the cells are cultured, cultivated, and / or incubated under stimulating conditions for the following durations before and / or during the genetic engineering of the cells: less than 10 days, 9 days, 8 days, 7 days, 6 days, or 5 days, 4 days, or for the following durations: less than 168 hours, 162 hours, 156 hours, 144 hours, 138 hours, 132 hours, 120 hours, 114 hours, 108 hours, 102 hours, or 96 hours. In certain embodiments, the cells are incubated with and / or in the presence of a stimulating agent for 4 days, 5 days, 6 days, or 7 days, or about 4 days, 5 days, 6 days, or 7 days. In some embodiments, the cells are incubated with and / or in the presence of a stimulating agent for 4 days or about 4 days. In certain embodiments, the cells are incubated with and / or in the presence of a stimulating agent for 5 days or about 5 days. In some embodiments, the cells are incubated with and / or in the presence of a stimulating agent for less than 7 days.

[0178] In some embodiments, incubating the cells under stimulating conditions includes incubating the cells with the stimulating agent described in Section IB-1. In some embodiments, the stimulating agent contains or includes beads, such as paramagnetic beads, and the cells are incubated with the stimulating agent at a ratio less than 3:1 (beads:cells), such as 1:1. In certain embodiments, the cells are incubated with the stimulating agent in the presence of one or more cytokines and / or one or more antioxidants. In some embodiments, a composition enriched with CD4+ T cells is incubated with the stimulating agent at a 1:1 (beads:cells) ratio in the presence of recombinant IL-2, IL-7, IL-15, and NAC. In some embodiments, a composition enriched with CD8+ T cells is incubated with the stimulating agent at 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 isolated from the cells within 6, 5, or 4 days from the start of incubation, such as from the time the stimulating agent is added to or comes into contact with the cells.

[0179] 1. Stimulants

[0180] In some embodiments, incubating the composition of enriched cells under stimulating conditions is or includes incubating and / or contacting the composition of enriched cells with a stimulating agent capable of activating and / or expanding T cells. In some embodiments, the stimulating agent is capable of stimulating and / or activating one or more signals in the cells. In some embodiments, the one or more signals are receptor-mediated. In a particular embodiment, the one or more signals are changes in the level or amount of signal transduction and / or second messengers (e.g., cAMP and / or intracellular calcium), changes in the amount, cell localization, conformation, phosphorylation, ubiquitination, and / or truncation of one or more cellular proteins, and / or changes in or related to cell activity (e.g., transcription, translation, protein degradation, cell morphology, activation state, and / or cell division). In a particular embodiment, the stimulating agent activates and / or is capable of activating one or more intracellular signal transduction domains of one or more components of the TCR complex and / or one or more intracellular signal transduction domains of one or more co-stimulatory molecules.

[0181] In some embodiments, the stimulating agent contains particles (e.g., beads) conjugated or linked to one or more agents (e.g., biomolecules) capable of activating and / or amplifying cells (e.g., T cells). In some embodiments, the one or more agents are bound to the beads. In some embodiments, the beads are biocompatible, i.e., made of materials suitable for biological use. In some embodiments, the beads are non-toxic to the cultured cells (e.g., cultured T cells). In some embodiments, the beads can be any particle capable of attaching the agent in a manner that allows interaction between the agent and the cells.

[0182] In some embodiments, the stimulating agent contains one or more agents capable of activating and / or amplifying cells (e.g., T cells), said one or more agents binding to or otherwise attaching to the beads, such as binding to or attaching to the surface of the beads. In some 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 embodiments, the beads are agarose gel beads.

[0183] In certain embodiments, the stimulating agent contains monodisperse beads. In some embodiments, the monodisperse beads comprise a size dispersion in which the standard deviation of their diameters is less than 5%.

[0184] In some embodiments, the beads contain one or more agents, such as agents coupled, conjugated, or linked (directly or indirectly) to the surface of the beads. In some embodiments, agents such as those considered herein may include, but are not limited to, RNA, DNA, proteins (e.g., enzymes), antigens, polyclonal antibodies, monoclonal antibodies, antibody fragments, carbohydrates, lipid lectins, or any other biomolecules having an 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 embodiments, the desired target is CD3. In some embodiments, the desired target is a T-cell co-stimulatory molecule, such as CD28, CD137 (4-1-BB), OX40, or ICOS. The one or more agents can be attached directly or indirectly to the beads by various methods known and available in the art. The attachment can be covalent, non-covalent, electrostatic, or hydrophobic, and can be achieved by various attachment means, including, for example, chemical, mechanical, or enzymatic means. In some implementations, a biomolecule (e.g., a biotinylated anti-CD3 antibody) can be indirectly attached to the bead via another biomolecule (e.g., an anti-biotinylated antibody) that is directly attached to the bead.

[0185] In some embodiments, the stimulating agent contains beads and one or more agents that interact directly with macromolecules on the cell surface. In some embodiments, the beads (e.g., paramagnetic beads) interact with the cells via one or more agents (e.g., antibodies) that are specific to one or more macromolecules on the cells (e.g., one or more cell surface proteins). In some embodiments, the beads (e.g., paramagnetic beads) are labeled with a first agent described herein (such as a primary antibody (e.g., an anti-biotinylated antibody) or other biomolecule), and then a second agent (such as a secondary antibody (e.g., a biotinylated anti-CD3 antibody) or other second biomolecule (e.g., streptavidin)) is added, whereby the secondary antibody or other second biomolecule specifically binds to such a primary antibody or other biomolecule on the particle.

[0186] In some embodiments, the stimulating agent contains one or more agents (e.g., antibodies) that attach to beads (e.g., paramagnetic beads) and specifically bind to one or more of the following macromolecules on cells (e.g., T cells): 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 macromolecules include LA (LFA-1), CD62L (L-selectin), CD29 / CD49d (VLA-4), Notch ligands (e.g., delta-like 1 / 4, serrated 1 / 2, etc.), CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, and CXCR3, or fragments thereof, including the corresponding ligands or fragments thereof. In some embodiments, the agent (e.g., antibody) attached to the beads (e.g., paramagnetic beads) specifically binds to one or more of the following macromolecules on cells (e.g., T cells): CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA, and / or CD45RO.

[0187] In some embodiments, one or more agents attached to the bead are antibodies. The antibodies may include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having the Fc region of an immunoglobulin), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, biantibodies, 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), such as Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragments. It should be understood that any isotype constant region can be used for the antibodies considered herein, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species (e.g., rodent 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 a particular embodiment, the agent is an anti-CD3 antibody. In some embodiments, the agent is an antibody that binds to and / or recognizes a co-receptor. In some embodiments, the stimulating agent comprises an anti-CD28 antibody. In some embodiments, the diameter of the beads is greater than about 0.001 μm, greater than about 0.01 μm, greater than about 0.1 μm, greater than about 1.0 μm, greater than about 10 μm, greater than about 50 μm, greater than about 100 μm, or greater than about 1000 μm but not exceeding about 1500 μm. In some embodiments, the diameter of the beads is about 1.0 μm to about 500 μm, about 1.0 μm to about 150 μm, about 1.0 μm to about 30 μm, about 1.0 μm to about 10 μm, about 1.0 μm to about 5.0 μm, about 2.0 μm to about 5.0 μm, or about 3.0 μm to about 5.0 μm. In some embodiments, the diameter of the beads is about 3 μm to about 5 μm. In some embodiments, the diameter of the beads is at least or at least about or about 0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm, 8.5 μm, 9.0 μm, 9.5 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, or 20 μm. In some embodiments, the diameter of the beads is or is about 4.5 μm. In some embodiments, the diameter of the beads is or is about 2.8 μm.

[0188] In some implementations, the density of the beads is greater than 0.001 g / cm³. 3 Greater than 0.01 g / cm 3 Greater than 0.05 g / cm 3 Greater than 0.1 g / cm 3 Greater than 0.5 g / cm3 Greater than 0.6 g / cm 3 Greater than 0.7 g / cm 3 Greater than 0.8 g / cm 3 Greater than 0.9 g / cm 3 Greater than 1g / cm 3 Greater than 1.1 g / cm 3 Greater than 1.2 g / cm 3 Greater than 1.3 g / cm 3 Greater than 1.4 g / cm 3 Greater than 1.5 g / cm 3 Greater than 2g / cm 3 Greater than 3g / cm 3 Greater than 4g / cm 3 or greater than 5g / cm 3 In some implementations, the density of the beads is approximately 0.001 g / cm³. 3 With approximately 100g / cm 3 Approximately 0.01 g / cm³ 3 With approximately 50g / cm 3 Approximately 0.1 g / cm³ 3 With approximately 10g / cm 3 Approximately 0.1 g / cm³ 3 With approximately 0.5g / cm 3 Approximately 0.5 g / cm 3 With approximately 1g / cm 3 Approximately 0.5 g / cm 3 With approximately 1.5 g / cm 3 Approximately 1g / cm 3 With approximately 1.5 g / cm 3 Approximately 1g / cm 3 With approximately 2g / cm 3 or about 1g / cm 3 With approximately 5g / cm 3 Between. In some implementations, the density of the beads is approximately 0.5 g / cm³. 3 Approximately 0.5 g / cm 3 Approximately 0.6 g / cm³ 3 Approximately 0.7 g / cm³ 3 Approximately 0.8 g / cm³ 3 Approximately 0.9 g / cm³ 3 Approximately 1.0 g / cm³ 3 Approximately 1.1 g / cm³ 3 Approximately 1.2 g / cm³ 3 Approximately 1.3 g / cm³ 3 Approximately 1.4 g / cm³ 3 Approximately 1.5g / cm 3Approximately 1.6 g / cm³ 3 Approximately 1.7 g / cm³ 3 Approximately 1.8 g / cm³ 3 Approximately 1.9 g / cm³ 3 or approximately 2.0 g / cm³ 3 In some embodiments, the density of the beads is approximately 1.6 g / cm³. 3 In a particular embodiment, the density of the beads or particles is approximately 1.5 g / cm³. 3 In some embodiments, the particle density is approximately 1.3 g / cm³. 3 .

[0189] In some embodiments, the multiple beads have a uniform density. In some embodiments, uniform density includes a density standard deviation of less than 10%, less than 5%, or less than 1% of the average bead density.

[0190] In some implementations, the surface area of ​​the beads is approximately 0.001 m². 2 / per gram of particles (m 2 / g) to about 1,000m 2 / g, approximately 0.010m 2 / g to approximately 100m 2 / g, approximately 0.1m 2 / g to approximately 10m 2 / g, approximately 0.1m 2 / g to approximately 1m 2 / g, approximately 1m 2 / g to approximately 10m 2 / g, approximately 10m 2 / g to approximately 100m 2 / g, approximately 0.5m 2 / g to approximately 20m 2 / g, approximately 0.5m 2 / g to approximately 5m 2 / g or approximately 1m 2 / g to approximately 4m 2 Between / g. In some embodiments, the surface area of ​​the particles or beads is approximately 1m². 2 / g to approximately 4m 2 / g.

[0191] In some embodiments, the beads contain at least one material on or near their surface that can be coupled, linked, or conjugated with a pharmaceutical agent. In some embodiments, the beads are surface-functionalized, i.e., containing functional groups capable of forming covalent bonds with binding molecules (e.g., polynucleotides or polypeptides). In specific embodiments, the beads contain surface-exposed carboxyl, amino, hydroxyl, toluenesulfonyl, epoxy, and / or chloromethyl groups. In specific embodiments, the beads contain surface-exposed agarose and / or agarose gel. In some embodiments, the bead surface contains an attached stimulating agent that can bind or attach to binding molecules. In specific embodiments, the biomolecule is a polypeptide. In some embodiments, the beads contain surface-exposed protein A, protein G, or biotin.

[0192] In some embodiments, the beads react in a magnetic field. In some embodiments, the beads are magnetic beads. In some embodiments, the magnetic beads are paramagnetic. In certain embodiments, the magnetic beads are superparamagnetic. In some embodiments, the beads do not exhibit any magnetic properties unless they are exposed to a magnetic field.

[0193] In certain embodiments, the bead comprises a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains 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 embodiments, the magnetic core comprises a metal oxide (e.g., iron oxide), a ferrite (e.g., manganese ferrite, cobalt ferrite, nickel ferrite, etc.), hematite, and a metal alloy (e.g., CoTaZn). In some embodiments, the magnetic core comprises one or more of a ferrite, a metal, a metal alloy, iron oxide, or chromium dioxide. In some embodiments, the magnetic core comprises elemental iron or a compound thereof. In some embodiments, the magnetic core comprises one or more of magnetite (Fe3O4), maghematite (γFe2O3), or pyrite (Fe3S4). In some embodiments, the core comprises iron oxide (e.g., Fe3O4).

[0194] In some embodiments, the beads contain magnetic, paramagnetic, and / or superparamagnetic cores covered by a surface-functionalized coating (coat or coating). In some embodiments, the coating may contain materials that include, but are not limited to, polymers, polysaccharides, silica, fatty acids, proteins, carbon, agarose, agarose gel, or combinations thereof. In some embodiments, the polymer may be polyethylene glycol, poly(lactic-co-glycolic acid), polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In some embodiments, the outer coating (coat or coating) comprises polystyrene. In a particular embodiment, the outer coating is surface-functionalized.

[0195] In some embodiments, the stimulating agent comprises beads containing a metal oxide core (e.g., an iron oxide core) and a coating, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coating comprises at least one polysaccharide (e.g., aminoglucan), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In some embodiments, the one or more agents comprise an antibody or an antigen-binding fragment thereof. In specific embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulating agent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulating agent comprises an anti-biotin antibody. In some embodiments, the diameter of the beads is from about 3 μm to about 10 μm. In some embodiments, the diameter of the beads is from about 3 μm to about 5 μm. In some embodiments, the diameter of the beads is about 3.5 μm.

[0196] In some embodiments, the stimulating agent comprises one or more agents attached to beads comprising a metal oxide core (e.g., an iron oxide core) and a coating (e.g., a protective coating), wherein the coating comprises polystyrene. In some embodiments, the beads are monodisperse paramagnetic (e.g., superparamagnetic) beads comprising a paramagnetic (e.g., superparamagnetic) iron core (e.g., a core comprising magnetite (Fe3O4) and / or maghemite (γFe2O3)) and a polystyrene coating. In some embodiments, the beads are non-porous. In some embodiments, the beads contain functionalized surfaces attached to the one or more agents. In some embodiments, the one or more agents are covalently bound to the beads on the surface. In some embodiments, the one or more agents comprise antibodies or antigen-binding fragments thereof. In some embodiments, the one or more agents comprise anti-CD3 antibodies and anti-CD28 antibodies. In some embodiments, the stimulating agent is or comprises anti-CD3 / anti-CD28 magnetic beads. In some embodiments, the one or more agents comprise anti-CD3 antibodies and / or anti-CD28 antibodies, as well as antibodies or antigen fragments thereof capable of binding to labeled antibodies (e.g., biotinylated antibodies, such as labeled anti-CD3 or anti-CD28 antibodies). In some embodiments, the beads have a concentration of approximately 1.5 g / cm³. 3 The density and approximately 1m 2 / g to approximately 4m 2 / g surface area. In a particular embodiment; the beads are monodisperse superparamagnetic beads having a diameter of about 4.5 μm and a surface area of ​​about 1.5 g / cm². 3 The density. In some embodiments, the beads are monodisperse superparamagnetic beads with an average diameter of about 2.8 μm and a density of about 1.3 g / cm³. 3 The density.

[0197] In some embodiments, the composition enriched with T cells is incubated with a stimulating agent at a bead-to-cell ratio of 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In a particular embodiment, the bead-to-cell ratio is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, or between 1.1:1 and 0.9:1. In a particular embodiment, the stimulating agent-to-cell ratio is about 1:1 or 1:1.

[0198] 2. Removal of stimulants from cells

[0199] In some embodiments, the stimulating agent (e.g., anti-CD3 / anti-CD28 magnetic beads) is removed and / or isolated from the cells. Where it is not desired to be bound by theory, specific embodiments consider that, in some cases, the binding and / or association between the stimulating agent and the cells may decrease over time during incubation. In some embodiments, one or more agents may be added to reduce the binding and / or association between the stimulating agent and the cells. In specific embodiments, changes in cell culture conditions (e.g., culture medium temperature or pH) may reduce the binding and / or association between the stimulating agent and the cells. Therefore, in some embodiments, the stimulating agent may be removed separately from the incubation, cell culture system, and / or solution, e.g., the cells are not removed from the incubation, cell culture system, and / or solution.

[0200] Methods for removing stimulating agents (e.g., as or containing particles such as bead particles or magnetizable particles) from cells are known. In some embodiments, the use of competitive antibodies (e.g., unlabeled antibodies) can be employed, which, for example, bind to a primary antibody of the stimulating agent and alter the affinity of said primary antibody for antigens on its cells, thereby allowing for gentle detachment. In some cases, after detachment, the competitive antibody may remain associated with the particles (e.g., bead particles), while unreacted antibodies are washed away or can be washed away, and the cells are free of detaching, selecting, enriching, and / or activating antibodies. An example of such an agent is DETACaBEAD (Friedl et al. 1995; Entschladen et al. 1997). In some embodiments, particles (e.g., bead particles) can be removed in the presence of a cleavable adapter (e.g., a DNA adapter), thereby conjugating the particle-bound antibody to the adapter (e.g., CELLection, Dynal). In some cases, the adapter region provides a cleavable site to remove particles (e.g., bead particles) from the cells after detachment, for example, by adding a DNase or other release buffer. In some embodiments, other enzymatic methods may also be used to release particles (e.g., bead particles) from cells. In some embodiments, the particles (e.g., bead particles or magnetizable particles) are biodegradable.

[0201] In some embodiments, the stimulant is magnetic, paramagnetic, and / or superparamagnetic, and / or contains magnetic beads, paramagnetic beads, and / or superparamagnetic beads, and the stimulant can be removed from the cells by exposing the cells to a magnetic field. Examples of suitable devices containing magnets for generating a magnetic field include DynaMag CTS (Thermo Fisher), Takara, and EasySep magnets (Stem Cell Technologies).

[0202] In certain embodiments, the stimulating agent is removed or isolated from the cells before completing the provided method, for example, before harvesting, collecting, and / or formulating engineered cells produced by the methods provided herein. In some embodiments, the stimulating agent is removed and / or isolated from the cells before engineering (e.g., transducing or transfecting) the cells. In certain embodiments, the stimulating agent is removed and / or isolated from the cells after the cell engineering step. In some embodiments, the stimulating agent is removed before culturing the cells, for example, before culturing engineered, such as transfected or transduced, cells under conditions that promote proliferation and / or expansion.

[0203] In some embodiments, the stimulating agent is isolated and / or removed from the cells after a certain time period. In a particular embodiment, the time period is the amount of time from the start and / or the initial incubation under the stimulating conditions. In a particular embodiment, incubation is considered to begin when the cells come into contact with the stimulating agent and / or the culture medium or solution containing the stimulating agent. In a particular embodiment, the stimulating agent is removed or isolated from the cells within 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, or about 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the start or initial incubation. In a particular embodiment, the stimulating agent is removed and / or isolated from the cells after 9, 8, 7, 6, 5, 4, 3, or 2 days, or about 9, 8, 7, 6, 5, 4, 3, or 2 days after the start or initial incubation. In some embodiments, the stimulant is removed and / or isolated from the cells at 168, 162, 156, 144, 138, 132, 120, 114, 108, 102, or 96 hours, or approximately 168, 162, 156, 144, 138, 132, 120, 114, 108, 102, or 96 hours after the start of incubation. In certain embodiments, the stimulant is removed and / or isolated from the cells 5 days or approximately 5 days after the start of incubation. In some embodiments, the stimulant is removed and / or isolated from the cells 4 days or approximately 4 days after the start of incubation.

[0204] C. Engineered cells

[0205] In some embodiments, the provided methods involve administering cells expressing recombinant antigen receptors to a subject suffering from a disease or condition. Various methods for introducing genetically engineered components such as recombinant receptors (e.g., CARs or TCRs) are well known and can be used in conjunction with the provided methods and compositions. Exemplary methods include those for transferring nucleic acids encoding receptors, including via viruses (e.g., retroviruses or lentiviruses), transduction, transposons, and electroporation.

[0206] Cells expressing the receptor and administered via the provided methods include engineered cells. Genetic engineering typically involves introducing nucleic acids encoding recombinant or engineered components, such as through retroviral transduction, transfection, or transformation, into a composition containing the cells.

[0207] In some embodiments, the methods provided herein are used in conjunction with one or more compositions of engineered enriched T cells. In some embodiments, engineering is or includes the introduction of a polynucleotide, such as a recombinant polynucleotide encoding a recombinant protein. In a particular embodiment, the recombinant protein is a recombinant receptor, such as any receptor described in Section II. The introduction of a nucleic acid molecule encoding a recombinant protein (such as a recombinant receptor) into cells can be performed using any of a number of known vectors. Such vectors include viral and nonviral systems, including lentivirus and gamma retrovirus systems, and transposon-based systems, such as the PiggyBac or Sleeping Beauty gene transfer systems. Exemplary methods include those for transferring nucleic acids encoding receptors, including via viruses (e.g., retroviruses or lentiviruses), transduction, transposons, and electroporation. In some embodiments, engineering produces one or more engineered compositions of enriched T cells.

[0208] In some embodiments, one or more compositions of enriched T cells are engineered, for example, by transduction or transfection, before culturing cells under conditions that promote proliferation and / or expansion, as provided in Section ID. In a particular embodiment, one or more compositions of enriched T cells are engineered after the compositions have been stimulated, activated, and / or incubated under stimulating conditions (as described in the methods provided in Section IB). In a particular embodiment, the compositions are stimulated compositions. In a particular embodiment, the stimulated compositions have been previously cryopreserved and stored, and thawed prior to engineering.

[0209] In some embodiments, the one or more compositions of stimulated T cells are or comprise two separate stimulated compositions of enriched T cells. In a particular embodiment, two separate compositions of enriched T cells, for example, two separate compositions of enriched T cells selected, isolated, and / or enriched from the same biological sample, are engineered separately. In some embodiments, the two separate compositions comprise a composition of enriched CD4+ T cells. In a particular embodiment, the two separate compositions comprise a composition of enriched CD8+ T cells. In some embodiments, two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are genetically engineered separately, such as after incubation under the stimulation conditions described above. In some embodiments, a single composition of enriched T cells is genetically engineered. In some 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 that has been combined from the separate compositions prior to genetic engineering.

[0210] In some embodiments, the engineered, e.g., transduced or transfected enriched CD4+ T cell (e.g., stimulated 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%, at least 99.5%, at least 99.9%, or about 100% CD4+ T cells. In some embodiments, the engineered enriched CD4+ T cell (e.g., stimulated CD4+ T cell) composition comprises 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 contains no CD8+ T cells, and / or contains no or substantially no CD8+ T cells.

[0211] In some embodiments, the engineered, e.g., transduced or transfected enriched CD8+ T cell (e.g., stimulated CD8+ 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%, at least 99.5%, at least 99.9%, or about 100% CD8+ T cells. In some embodiments, the engineered enriched CD8+ T cell (e.g., stimulated CD8+ T cell) composition comprises 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 contains no CD4+ T cells, and / or contains no or substantially no CD4+ T cells.

[0212] In some embodiments, separate compositions enriched with CD4+ and CD8+ T cells are combined into a single composition and genetically engineered, for example, through transduction or transfection. In some embodiments, separately engineered compositions enriched with CD4+ and CD8+ T cells are combined into a single composition after genetic engineering has been performed and / or completed. In a particular embodiment, separate compositions enriched with CD4+ and CD8+ T cells (such as separate compositions stimulating DD4+ and CD8+ T cells) are engineered separately and treated separately after genetic engineering has been performed and / or completed for T cell culture and / or expansion.

[0213] In some embodiments, the introduction of polynucleotides, such as recombinant polynucleotides encoding recombinant proteins, is performed by contacting enriched CD4+ or CD8+ T cells (e.g., stimulated CD4+ or CD8+ T cells) with viral particles containing said polynucleotides. In some embodiments, contact can be achieved by centrifugation, such as spinoculation (e.g., centrifugal inoculation). In some embodiments, the composition containing cells, viral particles, and reagents can be rotated, typically at a relatively low force or speed, such as a speed lower than that used for cell precipitation, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some implementations, rotation is performed with a force (e.g., relative centrifugal force) ranging from 100g to 3200g or from about 100g to about 3200g (e.g., about or at least about 100g, 200g, 300g, 400g, 500g, 1000g, 1500g, 2000g, 2500g, 3000g, or 3200g), such as about 693g, measured, for example, at the inner or outer wall of the chamber or cavity. The term "relative centrifugal force" or RCF is generally understood as the effective force exerted on an object or substance (e.g., a cell, sample, or granule and / or a point in the rotating chamber or other container) at a specific point in space, relative to the Earth's gravity, such as compared to the axis of rotation. This value can be determined using well-known formulas that take into account gravity, rotational speed, and the radius of rotation (the distance between the axis of rotation and the object, substance, or particle from which the RCF is measured). In some embodiments, at least a portion of the contact, incubation, and / or engineering of the cells (e.g., cells from a stimulated composition enriched with CD4+ T cells or CD8+ T cells) with the virus is performed under rotation between about 100g and 3200g, 1000g and 2000g, 1000g and 3200g, 500g and 1000g, 400g and 1200g, 600g and 800g, 600g and 700g, or 500g and 700g. In some embodiments, the rotation is performed between 600g and 700g, for example at or around 693g.

[0214] In some embodiments, at least a portion of the engineering, transduction, and / or transfection is performed under rotation, such as rotational inoculation and / or centrifugation. In some embodiments, rotation is performed for at least 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 1 form, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or at least 7 days. In some embodiments, rotation is performed for about 60 minutes. In some embodiments, rotation is performed for about 30 minutes. In some embodiments, rotation is performed at between 600g and 700g, for example at or at about 693g for about 30 minutes.

[0215] In some implementations, the number of live cells to be engineered, transduced, and / or transfected ranges from approximately 5 x 10⁻⁶. 6 One cell to approximately 100 x 10⁻⁶ 7 Cells, such as from approximately 10 x 10 6 One cell to approximately 100 x 10⁻⁶ 6 Cells, from approximately 100 x 10 6 One cell to approximately 200 x 10⁻⁶ 6 Cells, from approximately 200 x 10 6 One cell to approximately 300 x 10⁻⁶ 6 Cells, from approximately 300 x 10 6 One cell to approximately 400 x 10⁻⁶ 6 Cells, from approximately 400 x 10 6 One cell to approximately 500 x 10⁻⁶ 6 One cell or from approximately 500 x 10 6 One cell to approximately 100 x 10⁻⁶ 7 The number of live cells to be engineered, transduced, and / or transfected is approximately or less than approximately 300 x 10⁻⁶ cells. In specific examples, the number of live cells to be engineered, transduced, and / or transfected is approximately 300 x 10⁻⁶ cells. 6 Each cell.

[0216] In some embodiments, at least a portion of the engineering, transduction, and / or transfection is performed in volumes (e.g., rotational seeding volumes) ranging from about 5 mL to about 100 mL, such as from about 10 mL to about 50 mL, from about 15 mL to about 45 mL, from about 20 mL to about 40 mL, from about 25 mL to about 35 mL, or about 30 mL. In some embodiments, the volume of the cell clumps after rotational seeding ranges from about 1 mL to about 25 mL, such as from about 5 mL to about 20 mL, from about 5 mL to about 15 mL, from about 5 mL to about 10 mL, or about 10 mL.

[0217] In some embodiments, gene transfer is accomplished by first stimulating cells, such as by combining them with a stimulant that induces a response (e.g., proliferation, survival, and / or activation, as measured by the expression of cytokines or activation markers), then transducing the activated cells, and expanding them in culture to a quantity sufficient for clinical application. In some embodiments, gene transfer is accomplished by first incubating cells under stimulating conditions, such as by any of the methods described in Section IB.

[0218] In some embodiments, the method for genetic engineering is carried out by contacting one or more cells of the composition with a nucleic acid molecule encoding a recombinant protein (e.g., a recombinant receptor). In some embodiments, the contact can be achieved by centrifugation, such as rotary inoculation (e.g., centrifugal inoculation). Such methods include any of those described in International Publication No. WO 2016 / 073602. Exemplary centrifuge chambers include those manufactured and sold by Biosafe SA, including those for... and 2. Systems including A-200 / F and A-200 centrifuge chambers and various kits for such systems. Exemplary chambers, systems, and processing instruments and cabinets are described in, for example, the following documents: U.S. Patent No. 6,123,655, U.S. Patent No. 6,733,433 and published U.S. Patent Application Publication No. US 2008 / 0171951, and published International Patent Application Publication No. WO 00 / 38762, the contents of which are incorporated herein by reference in their entirety. Exemplary kits for such systems include, but are not limited to, single-use kits sold by BioSafe SA under the product names CS-430.1, CS-490.1, CS-600.1, or CS-900.2.

[0219] In some embodiments, the system includes other instruments and / or is associated with other instruments, including instruments for operating, automating, controlling, and / or monitoring transduction steps and one or more other processing steps performed in the system (e.g., one or more processing steps that may be performed using or in combination with a centrifuge system as described herein or in International Publication No. WO 2016 / 073602). In some embodiments, such instruments are housed in a cabinet. In some embodiments, the instruments include a cabinet comprising a housing containing control circuitry, a centrifuge, a shroud, a motor, a pump, sensors, a display, and a user interface. Exemplary devices are described in U.S. Patent Nos. 6,123,655, 6,733,433, and US 2008 / 0171951.

[0220] In some embodiments, the system comprises a series of containers, such as bags, tubing, stopcocks, clamps, connectors, and centrifuge chambers. In some embodiments, the containers (e.g., bags) include one or more containers (e.g., bags) containing cells and viral vector particles to be transduced in the same container or in separate containers (e.g., the same bag or separate bag). In some embodiments, the system further includes one or more containers (e.g., bags) containing media, such as diluents and / or washing solutions, which are drawn into chambers and / or other components during the method to dilute, resuspend, and / or wash components and / or compositions. The containers may be connected to one or more locations within the system, for example, corresponding to inlet lines, diluent lines, washing lines, waste lines, and / or outlet lines.

[0221] In some embodiments, the chamber is associated with a centrifuge capable of rotating the chamber, for example, about its axis of rotation. Rotation can occur before, during, and / or after incubation for cell transduction, and / or in one or more other processing steps. Thus, in some embodiments, one or more of the various processing steps are performed under rotation (e.g., under a specific force). The chamber is typically capable of rotating vertically or substantially vertically such that the chamber is positioned vertically during centrifugation, with the sidewalls and axis vertical or substantially vertical, and one or more end walls horizontal or substantially horizontal.

[0222] In some embodiments, the cell-containing composition and the viral vector particle-containing composition, along with optional air, may be combined or mixed before the composition is provided to the cavity. In some embodiments, the cell-containing composition and the viral vector particle-containing composition, along with optional air, are provided separately to the cavity and combined and mixed therein. In some embodiments, the cell-containing composition, the viral vector particle-containing composition, and optional air may be provided to the inner cavity in any order. In any of these embodiments, the cell-containing and viral vector particle-containing compositions are input compositions that have been combined or mixed together, whether the input compositions are combined and / or mixed inside or outside the centrifuge chamber, and / or whether the cells and viral vector particles are provided to the centrifuge chamber together or separately (e.g., simultaneously or sequentially).

[0223] In some embodiments, the transduction method involves the intake of a volume of gas (e.g., air) prior to incubation of the cells and viral vector particles (e.g., rotation). In other embodiments, the transduction method involves the intake of a volume of gas (e.g., air) during incubation (e.g., rotation) of the cells and viral vector particles.

[0224] In some embodiments, the liquid volume and optionally the air volume of the cell or viral vector particles constituting the transduction composition can be a predetermined volume. This volume can be programmed into the system and / or controlled by circuitry associated with the system.

[0225] In some embodiments, the intake of the transduction composition and optional gas (such as air) is manually, semi-automatically, and / or automatically controlled until the desired or predetermined volume has been introduced into the internal cavity of the chamber. In some embodiments, sensors associated with the system can detect the inflow and outflow of liquids and / or gases into the centrifuge chamber, such as by their color, flow rate, and / or density, and can communicate with associated circuitry to stop or continue intake as needed until such a desired or predetermined volume has been achieved. In some aspects, sensors programmed or capable only of detecting liquids in the system and not gases (e.g., air) can be made to allow gas (such as air) to be introduced into the system without stopping intake. In some such embodiments, an opaque tube can be placed in the tubing near the sensor when gas (such as air) intake is required. In some embodiments, the intake of gas (such as air) can be manually controlled.

[0226] In aspects of the provided method, the internal cavity of the centrifuge chamber is subjected to high-speed rotation. In some embodiments, rotation is performed before, simultaneously, subsequently, or intermittently before the intake of the liquid input composition and optionally air. In some embodiments, rotation is performed after the intake of the liquid input composition and optionally air. In some embodiments, rotation is performed by centrifugation at a relative centrifugal force at the inner surface of the internal cavity sidewalls and / or the cell surface layer at about or at least about 800 g, 1000 g, 1100 g, 1500 g, 1600 g, 1800 g, 2000 g, 2200 g, 2500 g, 3000 g, 3500 g, or 4000 g. In some embodiments, rotation is achieved by centrifugation with a force greater than or greater than about 1100g, such as greater than or greater than about 1200g, greater than or greater than about 1400g, greater than or greater than about 1600g, greater than or greater than about 1800g, greater than or greater than about 2000g, greater than or greater than about 2400g, greater than or greater than about 2800g, greater than or greater than about 3000g, or greater than or greater than about 3200g. In some embodiments, rotation is achieved by centrifugation with a force of less than or greater than about 1600g.

[0227] In some embodiments, the transduction method includes rotating or centrifuging the transduction composition and optional air in a centrifuge chamber for more than or greater than about 5 minutes, such as more than or greater than about 10 minutes, more than or greater than about 15 minutes, more than or greater than about 20 minutes, more than or greater than about 30 minutes, more than or greater than about 45 minutes, more than or greater than about 60 minutes, more than or greater than about 90 minutes, or more than or greater than about 120 minutes. In some embodiments, rotating or centrifuging the transduction composition and optional air in a centrifuge chamber for more than 5 minutes, but not exceeding 60 minutes, 45 minutes, 30 minutes, or 15 minutes. In a particular embodiment, transduction includes rotating or centrifuging for 60 minutes or about 60 minutes.

[0228] In some embodiments, the transduction method includes rotating or centrifuging the transduction composition and optionally air in a centrifuge chamber for the following durations: between about 10 and 60 minutes, between 15 and 60 minutes, between 15 and 45 minutes, between 30 and 60 minutes, or between 45 and 60 minutes, each containing an end value, and the rotation or centrifugation is performed at a force of at least or greater than or about 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g, 2000 g, 2200 g, 2400 g, 2800 g, 3200 g, or 3600 g at the inner surface of the internal cavity sidewall and / or the cell surface. In a particular embodiment, the transduction method includes rotating or centrifuging the transduction composition (e.g., cells and viral vector particles) at 1600 g or about 1600 g for 60 minutes or about 60 minutes.

[0229] In some embodiments, gas (e.g., air) in the chamber cavity is vented from the chamber. In some embodiments, gas (e.g., air) is vented to a container operatively connected to the centrifuge chamber as part of a closed system. In some embodiments, the container is a free or empty container. In some embodiments, air (e.g., gas) in the chamber cavity is vented through a filter operatively connected to the internal cavity of the centrifuge chamber via a sterile line. In some embodiments, air is vented using a manual, semi-automatic, or automatic process. In some embodiments, air is vented from the chamber before, simultaneously, intermittently, or after the expulsion of an output composition containing incubated cells and viral vector particles (e.g., cells that have been initiated for transduction or have been transduced with a viral vector) from the chamber cavity.

[0230] In some embodiments, transduction and / or other incubation are performed as a continuous or semi-continuous process or as part of a continuous or semi-continuous process. In some embodiments, the continuous process involves continuously taking up cell and viral vector particles, such as transduction compositions (as a pre-existing single composition or by continuously aspirating portions of them into the same vessel, such as a cavity, and thereby mixing), and / or continuously expelling or discharging liquids and optionally gases (such as air) from the vessel during at least a portion of the incubation, such as during a portion of centrifugation. In some embodiments, continuous take-up and continuous expulsion are at least partially simultaneous. In some embodiments, continuous take-up is performed during a portion of the incubation, such as during a portion of centrifugation, and continuous expulsion is performed during a separate portion of the incubation. The two may be performed alternately. Therefore, continuous take-up and expulsion during incubation can allow for the processing (e.g., transduction) of a larger total sample volume.

[0231] In some embodiments, incubation is part of a continuous process, the method including, during at least a portion of the incubation, continuously ingesting the transduction composition into the cavity during chamber rotation, and during the portion of the incubation, continuously expelling liquid and optionally discharging gas (e.g., air) from the cavity through at least one opening during chamber rotation.

[0232] In some embodiments, semi-continuous incubation is performed by alternating between the intake of a composition into a cavity, incubation, expulsion of liquid from the cavity, and optionally expulsion of gas (e.g., air) from the cavity, such as into an output container, and subsequent intake of a subsequent (e.g., second, third, etc.) composition containing more cells and other reagents (e.g., viral vector particles) for treatment, and repeating the process. For example, in some embodiments, incubation is part of a semi-continuous process, the method comprising, before incubation, intake of a transduction composition into the cavity through the at least one opening, and after incubation, expulsion of fluid from the cavity; intake of another transduction composition containing cells and viral vector particles into the inner cavity; and incubation of another transduction composition in the inner cavity under certain conditions, thereby transducing the cells in the other transduction composition by the vector. The process can be repeated several times. In this respect, semi-continuous or continuous methods can allow for the production of even larger volumes and / or numbers of cells.

[0233] In some implementations, a portion of the transduction incubation is carried out in a centrifuge chamber under conditions including rotation or centrifugation.

[0234] In some embodiments, the method includes incubation, wherein another portion of the incubation of the cells and viral vector particles is performed without rotation or centrifugation, said other portion generally following said at least one portion of the incubation, including rotation or centrifugation of the chamber. In some embodiments, the incubation of the cells and viral vector particles is performed without rotation or centrifugation for at least 1 hour, 6 hours, 12 hours, 24 hours, 32 hours, 48 ​​hours, 60 hours, 72 hours, 90 hours, 96 hours, 3 days, 4 days, 5 days, or more than 5 days. In some embodiments, the incubation will be 72 hours or about 72 hours.

[0235] In some such embodiments, further incubation is performed under specific conditions to allow the viral vector to integrate into the host genome of one or more cells. A skilled technician can expertly assess or determine whether incubation has resulted in the integration of the viral vector particles into the host genome, and thus empirically determine the conditions for further incubation. In some embodiments, the integration of the viral vector into the host genome can be assessed by measuring the expression level of recombinant proteins (such as heterologous proteins) encoded by the nucleic acids of the viral vector particles contained in the genome after incubation. Various well-known methods for assessing the expression levels of recombinant molecules can be used, for example, in the case of cell surface proteins, detection can be performed by affinity-based methods (e.g., immunoaffinity-based methods), such as flow cytometry. In some examples, expression is measured by detecting transduction markers and / or reporter constructs. In some embodiments, the nucleic acids encoding truncated surface proteins are included within the vector and used as markers for their expression and / or enhancement.

[0236] In some embodiments, the composition containing cells, a carrier (e.g., viral particles), and reagents may be rotated, typically at a relatively low force or speed, such as a speed lower than that used for cell precipitation, such as from 600 rpm to 1700 rpm or from about 600 rpm to about 1700 rpm (e.g., about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some embodiments, rotation is performed with a force (e.g., relative centrifugal force) from 100 g to 3200 g or from about 100 g to about 3200 g (e.g., about or at least about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, or 3200 g), said force being measured, for example, at the inner or outer wall of the chamber or cavity. The term "relative centrifugal force" or RCF is generally understood as the effective force exerted on an object or substance (e.g., a cell, sample, or granule and / or a point in a rotating chamber or other container) at a specific point in space, relative to the Earth's gravity, such as compared to the axis of rotation. This value can be determined using well-known formulas that take into account gravity, the speed of rotation, and the radius of rotation (the distance between the axis of rotation and the object, substance, or particle on which the RCF is measured).

[0237] In some implementations, during at least a portion of genetic engineering (e.g., transduction) and / or after genetic engineering, cells are transferred to a bioreactor bag assembly for culturing the genetically engineered cells, such as for cell culture or expansion, as described above.

[0238] In some embodiments, a composition engineered, for example, to transduce or transfect enriched T cells, is engineered in the presence of a transduction adjuvant. In some embodiments, a composition engineered for enriched T cells is engineered in the presence of one or more polycations. In some embodiments, a composition of enriched T cells is transduced, for example, by incubating with viral vector particles, in the presence of one or more transduction adjuvants. In certain embodiments, a composition of enriched T cells is transfected, for example, by incubating with a non-viral vector, in the presence of one or more transduction adjuvants. In some embodiments, the presence of one or more transduction adjuvants increases gene delivery efficiency, such as by increasing the amount, fraction, and / or percentage of engineered (e.g., transduced or transfected) cells in the composition. In some embodiments, the presence of one or more transduction adjuvants increases transfection efficiency. In some embodiments, the presence of one or more transduction adjuvants increases transduction efficiency. 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 the cells engineered in the presence of a polycation contain or express a recombinant polynucleotide. In some embodiments, compared to alternative and / or exemplary methods of engineering cells in the absence of a transduction adjuvant, 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 of the cells in the presence of a polycation are engineered to contain or express a recombinant transduction adjuvant.

[0239] In some embodiments, compositions of enriched cells are engineered in the presence of transduction adjuvants at 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 10 μg / ml. In some embodiments, transduction adjuvants suitable for the provided methods include, but are not limited to, polycations, fibronectin or fragments or variants derived from fibronectin, RetroNectin, and combinations thereof.

[0240] In some embodiments, the cells are engineered in the presence of cytokines, such as recombinant human cytokines, at concentrations between 1 IU / ml and 1,000 IU / ml, between 10 IU / ml and 50 IU / ml, between 50 IU / ml and 100 IU / ml, between 100 IU / ml and 200 IU / ml, between 100 IU / ml and 500 IU / ml, between 250 IU / ml and 500 IU / ml, or between 500 IU / ml and 1,000 IU / ml.

[0241] In some embodiments, compositions engineered to enrich T cells are used in the presence of IL-2, such as recombinant human IL-2, wherein the concentration of IL-2 is between 1 IU / ml and 200 IU / ml, between 10 IU / ml and 100 IU / ml, between 50 IU / ml and 150 IU / ml, between 80 IU / ml and 120 IU / ml, between 60 IU / ml and 90 IU / ml, or between 70 IU / ml and 90 IU / ml. In certain embodiments, a composition for enriching T cells is engineered in the presence of recombinant IL-2 at a concentration of 50 IU / ml, 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml, 75 IU / ml, 80 IU / ml, 85 IU / ml, 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 composition for enriching T cells is engineered in the presence of 85 IU / ml or approximately. In some embodiments, the T cell population is a CD4+ T cell population. In certain embodiments, the composition for enriching T cells enriches CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected from or depleted from the composition. In a particular embodiment, the T-cell enriched composition is a CD8+ T-cell enriched composition. In a particular embodiment, the T-cell enriched composition enriches CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition.

[0242] In some embodiments, a composition engineered to enrich T cells is used in the presence of recombinant IL-7, such as human recombinant IL-7, wherein the concentration of the recombinant IL-7 is between 100 IU / ml and 2,000 IU / ml, between 500 IU / ml and 1,000 IU / ml, between 100 IU / ml and 500 IU / ml, between 500 IU / ml and 750 IU / ml, between 750 IU / ml and 1,000 IU / ml, or between 550 IU / ml and 650 IU / ml. In a particular embodiment, the composition of enriched T cells is engineered in the presence of IL-7 at a concentration of 50 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml, 300 IU / ml, 350 IU / ml, 400 IU / ml, 450 IU / ml, 500 IU / ml, 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, or 1,000 IU / ml. In a particular embodiment, the composition of enriched T cells is engineered in the presence of IL-7 at a concentration of approximately 600 IU / ml. In some embodiments, the composition engineered in the presence of recombinant IL-7 enriches a population of T cells, such as CD4+ T cells. In a particular embodiment, the T-cell enriched composition enriches CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected from or depleted from the composition.

[0243] In some embodiments, compositions of T-cell enriched cells are engineered in the presence of recombinant IL-15, such as human recombinant IL-15, wherein the concentration of the recombinant IL-15 is between 0.1 IU / ml and 100 IU / ml, between 1 IU / ml and 50 IU / ml, between 5 IU / ml and 25 IU / ml, between 25 IU / ml and 50 IU / ml, between 5 IU / ml and 15 IU / ml, or between 10 IU / ml and 100 IU / ml. In certain embodiments, the composition enriched with T cells is engineered in the presence of IL-15 at a concentration of 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 6 IU / ml, 7 IU / ml, 8 IU / ml, 9 IU / ml, 10 IU / ml, 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 composition enriched with T cells is engineered in or in about 10 IU / ml of IL-15. In some embodiments, the composition enriched with T cells is incubated in or in about 10 IU / ml of recombinant IL-15. In some embodiments, the composition engineered in the presence of recombinant IL-15 enriches a population of T cells, such as CD4+ T cells and / or CD8+ T cells. In some embodiments, the T cell-enriched composition is a CD8+ T cell-enriched composition. In a particular embodiment, the T cell-enriched composition enriches CD8+ T cells, wherein CD4+ T cells are not enriched and / or wherein CD4+ T cells are negatively selected or depleted from the composition. In some embodiments, the T cell-enriched composition is a CD4+ T cell-enriched composition. In a particular embodiment, the T cell-enriched composition enriches CD4+ T cells, wherein CD8+ T cells are not enriched and / or wherein CD8+ T cells are negatively selected or depleted from the composition.

[0244] In certain embodiments, a composition engineered with enriched CD8+ T cells is used in the presence of IL-2 and / or IL-15. In some embodiments, a composition engineered with enriched CD4+ T cells is used in the presence of IL-2, IL-7, and / or IL-15. In some embodiments, the IL-2, IL-7, and / or IL-15 are recombinant. In some embodiments, the IL-2, IL-7, and / or IL-15 are human. In certain embodiments, the one or more cytokines are or include recombinant human IL-2, IL-7, and / or IL-15.

[0245] In certain embodiments, the cells are engineered in the presence of one or more antioxidants. In some embodiments, the antioxidants include, but are not limited to, one or more antioxidants including tocopherol, tocotrienol, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, α-tocoquinone, Trolox (6-hydroxy-2,5,7,8-tetramethylbenzodihydropyran-2-dicarboxylic acid), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), flavonoids, isoflavones, lycopene, β-carotene, selenium, ubiquinone, syphilitic acid, S-adenosylmethionine, glutathione, taurine, N-acetylcysteine ​​(NAC), citric acid, L-carnitine, BHT, thioglycerol, ascorbic acid, propyl gallate, methionine, cysteine, homocysteine, glutathione, cystamine and cystathionine, and / or glycine-glycine-histidine.

[0246] In some embodiments, the one or more antioxidants are or include sulfur-containing antioxidants. In some embodiments, sulfur-containing antioxidants may include thiol antioxidants and / or, for example, antioxidants exhibiting one or more sulfur moieties within a ring structure. In some embodiments, the sulfur-containing antioxidants may include, for example, N-acetylcysteine ​​(NAC) and 2,3-dimercaptopropanol (DMP), L-2-oxo-4-thiazolidinyl carboxylate (OTC), and lipoic acid. In a particular embodiment, the sulfur-containing antioxidant is a glutathione precursor. In some embodiments, the glutathione precursor is a molecule that can be modified into a derived glutathione in one or more steps within the cell. In a particular embodiment, the glutathione precursor may include, but is not limited to, N-acetylcysteine ​​(NAC), L-2-oxothiazolidinyl-4-carboxylic acid (procysteine), lipoic acid, S-allylcysteine, or methylmethionine sulfonium chloride.

[0247] In some embodiments, the cells are engineered in the presence of one or more antioxidants. In some embodiments, the cells are engineered in the presence of one or more antioxidants at concentrations between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 μg / ml, between 100 ng / ml and 10 μg / ml, between 1 μg / ml and 100 μg / ml, between 10 μg / ml and 1 mg / ml, between 100 μg / ml and 1 mg / ml, between 1500 μg / ml and 2 mg / ml, between 500 μg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml. In some embodiments, the cells are engineered in the presence of one or more of the antioxidants at or above 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, or 500 mg / ml. In some embodiments, the one or more antioxidants are or include sulfur-containing antioxidants. In a particular embodiment, the one or more antioxidants are or include glutathione precursors.

[0248] In some embodiments, the cells are engineered in the presence of NAC. In some embodiments, the cells are engineered in the presence of NAC between 1 ng / ml and 100 ng / ml, between 10 ng / ml and 1 μg / ml, between 100 ng / ml and 10 μg / ml, between 1 μg / ml and 100 μg / ml, between 10 μg / ml and 1 mg / ml, between 100 μg / ml and 1 mg / ml, between 1,500 μg / ml and 2 mg / ml, between 500 μg / ml and 5 mg / ml, between 1 mg / ml and 10 mg / ml, or between 1 mg / ml and 100 mg / ml. In some embodiments, the cells are engineered in the presence of NAC at or above 1 ng / ml, 10 ng / ml, 100 ng / ml, 1 μg / ml, 10 μg / ml, 100 μg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 200 mg / ml, 300 mg / ml, 400 mg / ml, or 500 mg / ml. In some embodiments, the cells are engineered with or above 0.8 mg / ml.

[0249] In some embodiments, compositions of enriched T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), are engineered in the presence of one or more polycations. In some embodiments, compositions of enriched T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), are transduced in the presence of one or more polycations, for example, by incubating enriched T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), with a non-viral vector. In certain embodiments, compositions of enriched T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), are transfected in the presence of one or more polycations. In some embodiments, the presence of one or more polycations increases the efficiency of gene delivery, such as by increasing the amount, fraction, and / or percentage of engineered (e.g., transduced or transfected) cells in the composition. In some embodiments, the presence of one or more polycations increases the efficiency of transfection. In some embodiments, the presence of one or more polycations increases 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 the cells engineered in the presence of a polycation contain or express recombinant polynucleotides. In some embodiments, compared to alternative and / or exemplary methods of engineering cells in the absence of a transduction adjuvant, 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 of the cells in the presence of a polycation are engineered to contain or express recombinant polynucleotides.

[0250] In some embodiments, for example, the cell-enriched composition, such as a composition enriched with CD4+ T cells or CD8+ T cells (e.g., those enriched with CD4+ T cells or those enriched with CD8+ T cells), is engineered in the presence of low concentrations or amounts of polycations, relative to exemplary and / or alternative methods of engineered cells in the presence of polycations. In some embodiments, the cell-enriched composition, such as those enriched with CD4+ T cells (e.g., those enriched with CD8+ T cells or those enriched with CD4+ T cells), is engineered in the presence of 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 amounts or concentrations of polycations used in exemplary and / or alternative methods of cell engineered cells. In some embodiments, the enriched cells, such as the composition of stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), are engineered in the presence of the polycation at 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 10 μg / ml. In a particular embodiment, the enriched cells, such as those stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), are engineered in the presence of the polycation at or above 1 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, or 50 μg / ml.

[0251] In a particular embodiment, the composition engineered with the 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 (e.g., due to necrosis, programmed cell death, or apoptosis). In some embodiments, the enriched T cells, such as the composition of stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), are engineered in the presence of low amounts of polycations (e.g., less than 100 μg / ml, 50 μg / ml, or 10 μg / ml). After the engineering step is completed, 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 example, without experiencing necrosis, programmed cell death, or apoptosis, during or after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days. In some embodiments, compared with alternative and / or exemplary methods of engineering cells in the presence of higher amounts or concentrations of polycations (e.g., more than 50 μg / ml, 100 μg / ml, 500 μg / ml, or 1,000 μg / ml), the composition is engineered in the presence of low concentrations or amounts of polycations, and the cells of the composition have a survival rate of 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 compared with cells undergoing the exemplary and / or alternative processes.

[0252] In some embodiments, the polycation is positively charged. In some embodiments, the polycation reduces the repulsion between the cell and the carrier (e.g., a viral or non-viral carrier) and mediates the contact and / or binding of the carrier to the cell surface. In some embodiments, the polycation is a polyglucan, DEAE-glucan, protamine sulfate, poly-L-lysine, or a cationic liposome.

[0253] 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 engineered in the presence of protamine sulfate at concentrations of 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 10 μg / ml. In specific embodiments, the concentrations are approximately 1 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, 55 μg / ml, 60 μg / ml, 75 μg / ml, 80 μg / ml, 85 μg / ml, or 90 μg / ml. The composition engineered with enriched cells, such as stimulated T cells (e.g., stimulated CD4+ T cells or stimulated CD8+ T cells), in the presence of protamine sulfate at concentrations of 1, 95 μg / ml, 100 μg / ml, 105 μg / ml, 110 μg / ml, 115 μg / ml, 120 μg / ml, 125 μg / ml, 130 μg / ml, 135 μg / ml, 140 μg / ml, 145 μg / ml, or 150 μg / ml.

[0254] In some embodiments, the engineered composition enriched with CD4+ T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells), comprises 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 about 100% CD4+ T cells. In some embodiments, the engineered composition enriched with CD4+ T cells, such as stimulated T cells (e.g., stimulated CD4+ T cells), comprises 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 contains no CD8+ T cells, and / or contains no or substantially no CD8+ T cells.

[0255] In some embodiments, the engineered composition of enriched CD8+ T cells, such as stimulated T cells (e.g., stimulated CD8+ T cells), 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%, at least 99.5%, at least 99.9%, or about 100% CD8+ T cells. In some embodiments, the engineered composition of enriched CD8+ T cells, such as stimulated T cells (e.g., stimulated CD8+ T cells), comprises 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 contains no CD4+ T cells, and / or contains no or substantially no CD4+ T cells.

[0256] In some embodiments, engineering the cells includes culturing, contacting, or incubating them with a vector, such as a viral or non-viral vector. In some embodiments, the engineering includes culturing, contacting, and / or incubating the cells with the vector for about or at least 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 54 hours, 60 hours, 72 hours, 84 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more than 7 days. In a particular embodiment, the engineering includes culturing, contacting, and / or incubating the cells with the vector for about 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or 84 hours, or for about 2 days, 3 days, 4 days, or 5 days. In some embodiments, the engineering step is performed for about 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or 84 hours. In some implementations, the engineering process is carried out for about 60 hours or about 84 hours, or for about 72 hours, or for about 2 days.

[0257] In some embodiments, the engineering is performed at a temperature ranging from about 25°C to about 38°C, such as from about 30°C to about 37°C, from about 36°C to about 38°C, or at or above about 37°C ± 2°C. In some embodiments, the T-cell enriched composition is engineered at a CO2 level ranging from about 2.5% to about 7.5%, such as from about 4% to about 6%, for example at or above about 5% ± 0.5%. In some embodiments, the T-cell enriched composition is engineered at a temperature of or above about 37°C and / or at a CO2 level of or above about 5%.

[0258] In some embodiments, the cells are cultured after steps for genetic engineering, such as transducing or transfecting cells (e.g., CD4+ and / or CD8+ T cells) to contain one or more polynucleotides encoding recombinant receptors. In some embodiments, culture may include culturing, incubation, stimulation, activation, amplification, and / or proliferation. In some such embodiments, further culture is performed under conditions designed to integrate the viral vector into the host genome of one or more cells. Incubation and / or engineering may be performed in culture vessels, such as units, chambers, wells, columns, tubes, tube sets, valves, vials, culture dishes, bags, or other containers used for culturing or cultured cells. In some embodiments, the composition or cells are incubated in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce cell proliferation, amplification, activation, and / or survival in a population, simulate antigen exposure, and / or induce cell use for genetic engineering (e.g., for introducing recombinant antigen receptors).

[0259] In some embodiments, further incubation is carried out at a temperature above room temperature, for example, above or above about 25°C, typically above or above about 32°C, 35°C, or 37°C. In some embodiments, further incubation is carried out at a temperature of or above about 37°C ± 2°C, for example, at or above about 37°C.

[0260] In some embodiments, further incubation is carried out under conditions for stimulating and / or activating cells, said conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulating factors (e.g., cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors and any other agents designed to activate cells)).

[0261] In some embodiments, the stimulating condition or agent includes one or more agents (e.g., stimulating and / or adjuvant agents) capable of activating intracellular signaling domains of the TCR complex, such as ligands. In some aspects, the agent initiates or activates the TCR / CD3 intracellular signaling cascade in T cells, for example, an agent suitable for delivering a primary signal to, for example, initiate the activation of ITAM-induced signals (e.g., those specific to TCR components), and / or agents that promote co-stimulatory signals (e.g., co-stimulatory signals specific to T cell co-stimulatory receptors), such as anti-CD3, anti-CD28, or anti-41-BB (e.g., which optionally bind to a solid support such as beads) and / or one or more cytokines. The stimulant includes anti-CD3 / anti-CD28 beads (e.g., M-450CD3 / CD28T cell expander and / or (beads). Optionally, the amplification method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium. In some embodiments, the stimulant includes IL-2 and / or IL-15, for example, the IL-2 concentration is at least about 10 units / mL.

[0262] In some embodiments, the stimulating condition or stimulant includes one or more agents (e.g., ligands) capable of activating intracellular signaling domains of the TCR complex. In some aspects, the agent initiates or activates the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include, for example, antibodies that bind to a solid support (such as beads), such as those specific to TCR components and / or co-stimulatory receptors (e.g., anti-CD3, anti-CD28); and / or one or more cytokines. Optionally, the amplification method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies (e.g., at a concentration of at least about 0.5 ng / mL) to the culture medium. In some embodiments, the stimulant includes IL-2 and / or IL-15, for example, at an IL-2 concentration 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.

[0263] The conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, and agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulating factors (such as cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors and any other agents designed to activate cells)).

[0264] In some respects, incubation is carried out using a variety of techniques, such as those described in the following documents: U.S. Patent No. 6,040,177 to Riddell et al.; 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.

[0265] In some embodiments, further incubation is performed in the same container or apparatus where the contact took place. In some embodiments, further incubation is performed without rotation or centrifugation, typically following at least a portion of an incubation performed under rotation (e.g., in combination with centrifugation or rotational inoculation). In some embodiments, further incubation is performed outside the stationary phase, such as outside the chromatographic matrix, for example, in solution.

[0266] In some implementations, further incubation is carried out in a different container or device than the one in which the contact was made, for example by transferring (e.g., automatically transferring) the cell composition to a different container or device after contact with the virus particles and reagents.

[0267] In some implementations, further culturing or incubation, for example to promote in vitro amplification, may be carried out for more than or greater than about 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some implementations, further culturing or incubation may be carried out for no more than 6 days, no more than 5 days, no more than 4 days, no more than 3 days, no more than 2 days, or no more than 24 hours.

[0268] In some embodiments, the total duration of incubation with the stimulant is, for example, between about 1 hour and 96 hours, between 1 hour and 72 hours, between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours, such as at least or about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, further incubation is performed for the following durations: between or about 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours, including the end values.

[0269] In some implementations, the methods provided herein do not include further culture or incubation, for example, do not include an in vitro amplification step, or include a significantly shorter in vitro amplification step.

[0270] In some embodiments, the stimulating agent is removed and / or isolated from the cells prior to engineering. In certain embodiments, the stimulating agent is removed and / or isolated from the cells after engineering. In some embodiments, the stimulating agent is removed and / or isolated from the cells after engineering and before culturing engineered cells under conditions that promote proliferation and / or expansion. In some embodiments, the stimulating agent is the stimulating agent described in section IB-1. In certain embodiments, the stimulating agent is removed and / or isolated from the cells as described in section IB-2.

[0271] 1. Carrier and Method

[0272] Also 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 the provided methods for generating said engineered cells. In some embodiments, the vector contains nucleic acid encoding the recombinant receptor. In specific 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, for example a lentiviral vector or a gamma retroviral vector.

[0273] In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) contains a signal sequence encoding a signal peptide. Non-limiting exemplary examples of signal peptides include, for example, the GMCSFRα chain signal peptide shown in SEQ ID NO:61 and encoded by the nucleotide sequence shown in SEQ ID NO:60, the CD8α signal peptide shown in SEQ ID NO:59, or the CD33 signal peptide shown in SEQ ID NO:58.

[0274] In some implementations, the vector includes viral vectors (e.g., retroviruses or lentiviruses), nonviral vectors, or transposons, such as the Sleeping Beauty transposon system; vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV); lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors; and retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen lesion-forming virus (SFFV), or adeno-associated virus (AAV).

[0275] In some embodiments, the viral vector or non-viral DNA contains nucleic acid encoding a heterologous recombinant protein. In some embodiments, the heterologous recombinant molecule is or includes a recombinant receptor (e.g., an antigen receptor), an SB transposon (e.g., for gene silencing), a capsid-encapsulated transposon, a homologous double-stranded nucleic acid (e.g., for genomic recombination), or a reporter gene (e.g., a fluorescent protein such as GFP) or a luciferase.

[0276] a. Viral vector particles

[0277] In some implementations, recombinant nucleic acids are transferred to cells using recombinant infectious viral particles (e.g., vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV)). In some implementations, recombinant nucleic acids are transferred to T cells using recombinant lentiviral vectors or retroviral vectors (e.g., 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 Nov 29(11):550-557).

[0278] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focal formation virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. Retroviruses are generally amphiphilic, meaning they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Numerous illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740, 6,207,453, 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).

[0279] Methods of lentiviral transduction are known. Exemplary methods are described in, for example, the following literature: 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.

[0280] In some embodiments, the viral vector particle contains a genome derived from a retroviral genome-based vector (e.g., a vector derived from a lentiviral genome). In some aspects of the provided viral vector, a heterologous nucleic acid encoding a recombinant receptor (e.g., an antigen receptor, such as a CAR) is contained and / or located between the 5'LTR and 3'LTR sequences of the vector genome.

[0281] In some embodiments, the viral vector genome is a lentiviral genome, such as the HIV-1 genome or the SIV genome. For example, lentiviral vectors have been generated by repeatedly attenuating virulence genes, for example, by deleting genes env, vif, vpu, and nef, making the vector safer for therapeutic purposes. Lentiviral vectors are known. See Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry essential sequences for incorporating foreign nucleic acids, for selection, and for transferring said nucleic acids into host cells. Known lentiviruses can be readily obtained from depository institutions or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Virginia 20110-2209) or isolated from known sources using common techniques.

[0282] Non-limiting 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 infection anemia virus (E1AV). For example, lentiviral vectors have been generated by repeatedly attenuating HIV virulence genes, for example, by deleting genes env, vif, vpr, vpu, and nef, making the vectors safer for therapeutic purposes. Lentiviral vectors are known in the art (see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based and are configured to carry essential sequences for incorporating foreign nucleic acids, for selection, and for transferring said nucleic acids into host cells. Known lentiviruses can be readily obtained from depository institutions or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Virginia 20110-2209) or isolated from known sources using common techniques.

[0283] In some implementations, the viral genome vector may contain sequences of the 5' and 3' LTRs of a retrovirus (e.g., lentivirus). In some aspects, the viral genome construct may contain sequences of the 5' and 3' LTRs from a lentivirus, and specifically may contain the R and U5 sequences of the 5' LTR from the lentivirus and an inactivated or self-inactivated 3' LTR from the lentivirus. The LTR sequence can be an LTR sequence 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.

[0284] In some embodiments, the nucleic acid of the viral vector (e.g., the HIV viral vector) lacks an additional transcription unit. 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 the nucleic acid used to generate 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 typically have deletions of enhancer and promoter sequences in the 3' long terminal repeat (LTR), which are copied into the 5'LTR during vector integration. In some embodiments, sufficient sequences, including removal of the TATA box, can be eliminated to eliminate the transcriptional activity of the LTR. This prevents the generation 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-κB sites. Due to the self-inactivation of the 3'LTR, the provirus generated after entry and reverse transcription contains an inactivated 5'LTR. This can improve safety by reducing the risk of mobilizing the vector genome and the effect of the LTR on nearby cellular promoters. The self-inactivating 3'LTR can be constructed using 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.

[0285] Optionally, the U3 sequence from the lentivirus 5'LTR can be replaced by a promoter sequence (e.g., a heterologous promoter sequence) in the viral construct. This can increase the titer of virus recovered from the packaging cell line. Enhancer sequences may also be included. Any enhancer / promoter combination that increases viral RNA genome expression in the packaging cell line can be used. In one example, a CMV enhancer / promoter sequence (US Patent Nos. 5,385,839 and 5,168,062) is used.

[0286] In some embodiments, the risk of insertional mutagenesis can be minimized by constructing the retroviral vector genome (e.g., a lentiviral vector genome) as integration-deficient. Various approaches can be used to generate non-integrating vector genomes. In some embodiments, one or more mutations can be engineered into the integrase component of the pol gene, such that it encodes a protein with an inactive integrase. In some embodiments, the vector genome itself can be modified to prevent integration, for example by mutating or deleting one or both attachment sites, or by deleting or modifying the proximal polypurine segment (PPT) of the 3'LTR to render it non-functional. In some embodiments, non-genetic methods can be used; these methods include pharmacological agents that inhibit one or more functions of the integrase. These methods are not mutually exclusive; that is, more than one of the methods can be used at a time. For example, neither the integrase nor the attachment site may be functional, or neither the integrase nor the PPT site may be functional, or neither the attachment site nor the PPT site may be functional, or both may be non-functional. Such methods and viral vector genomes are known and available (see Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al. J Virol 69:2729, 1995; Brown et al. J Virol 73:9011 (1999); WO 2009 / 076524; McWilliams et al., J Virol 77:11150, 2003; Powell and Levin J Virol 70:5288, 1996).

[0287] In some embodiments, the vector contains sequences for replication in host cells (e.g., prokaryotic host cells). In some embodiments, the nucleic acid of the viral vector contains one or more origins of replication for replication in prokaryotic cells (e.g., bacterial cells). In some embodiments, vectors including prokaryotic origins of replication may also contain genes whose expression confers detectable or selectable markers, such as antibiotic resistance.

[0288] Viral vector genomes are typically constructed in the form of plasmids that can be transfected into packaging or production cell lines. Retroviral particles containing an RNA copy of the viral vector genome can be generated using any of a variety of known methods. In some embodiments, at least two components are involved in the preparation of a virus-based gene delivery system: first, a packaging plasmid, including structural proteins and enzymes necessary for the generation of viral vector particles; and second, the viral vector itself, i.e., the genetic material to be transferred. Biosafety precautions may be incorporated when designing one or both of these components.

[0289] In some embodiments, the packaging plasmid may contain all retroviral (e.g., HIV-1) proteins except for the envelope proteins (Naldini et al., 1998). In other embodiments, the viral vector may lack additional viral genes (e.g., those related to virulence, such as vpr, vif, vpu, and nef) and / or Tat (the major transactivator of HIV). In some embodiments, the lentiviral vector (e.g., HIV-based lentiviral vectors) contains only the three genes of the parent virus: gag, pol, and rev, which reduces or eliminates the possibility of reconstructing the wild-type virus through recombination.

[0290] In some embodiments, a viral vector genome is introduced into a packaging cell line containing all the components required to package viral genomic RNA transcribed from the viral vector genome into viral particles. Alternatively, in addition to one or more target sequences (e.g., recombinant nucleic acids), the viral vector genome may contain one or more genes encoding viral components. However, in some aspects, to prevent genome replication in target cells, endogenous viral genes required for replication are removed and provided separately in the packaging cell line.

[0291] In some embodiments, packaging cell lines are transfected with one or more plasmid vectors containing the components required to generate the particles. In some embodiments, packaging cell lines are transfected with plasmids containing the viral vector genome (including the LTR, cis-acting packaging sequence, and target sequence, i.e., nucleic acid encoding an antigen receptor (e.g., CAR)); and one or more helper plasmids encoding viral enzymes and / or structural components (e.g., Gag, pol, and / or rev). In some embodiments, various genetic components for generating retroviral vector particles are isolated using multiple vectors. In some such embodiments, providing packaging cells with separate vectors reduces the likelihood of recombination events that could otherwise generate replicating viruses. In some embodiments, a single plasmid vector containing all retroviral components can be used.

[0292] In some embodiments, retroviral vector particles (e.g., lentiviral vector particles) are pseudotyped to increase transduction efficiency in host cells. For example, in some embodiments, retroviral vector particles (e.g., lentiviral vector particles) are pseudotyped with VSV-G glycoprotein, which provides a broad cellular host range, thereby expanding the cell types that can be transduced. In some embodiments, packaging cell lines are transfected with plasmids or polynucleotides encoding non-natural envelope glycoproteins to include, for example, heterophilic, polyphilic, or biphilic envelopes, such as Sindbis virus envelopes, GALV, or VSV-G.

[0293] In some embodiments, the packaging cell line provides the necessary components, including viral regulatory proteins and structural proteins, for packaging viral genomic RNA into lentiviral vector particles. In some embodiments, the packaging cell line can be any cell line capable of expressing lentiviral proteins and producing functional lentiviral vector particles. In some aspects, suitable packaging cell lines include 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10), and Cf2Th (ATCC CRL 1430) cells.

[0294] In some embodiments, the packaging cell line stably expresses one or more viral proteins. For example, in some aspects, packaging cell lines containing gag, pol, rev, and / or other structural genes but lacking LTRs and packaging components can be constructed. In some embodiments, the packaging cell line can be transiently transfected with nucleic acid molecules encoding one or more viral proteins, as well as a viral vector genome containing nucleic acid molecules encoding heterologous proteins and / or nucleic acids encoding envelope glycoproteins.

[0295] In some embodiments, viral vectors and packaging plasmids and / or helper plasmids are introduced into packaging cell lines via transfection or infection. The packaging cell lines produce viral vector particles containing the viral vector genome. Methods used for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-glucan, and lipid transfection methods, electroporation, and microinjection.

[0296] When introducing recombinant plasmids and retroviral LTRs and packaging sequences into specialized cell lines (e.g., via calcium phosphate precipitation), the packaging sequences can allow the packaging of the recombinant plasmid's RNA transcripts into viral particles, which may then be secreted into the culture medium. In some embodiments, the culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. For example, in some aspects, after co-transfecting the packaging plasmid and transfer vector into the packaging cell line, the viral vector particles are recovered from the culture medium and titrated by a person skilled in the art using standard methods.

[0297] In some embodiments, retroviral vectors, such as lentiviral vectors, can be generated in packaging cell lines (such as the exemplary HEK 293T cell line) by introducing plasmids to allow the production of lentiviral particles. In some embodiments, the packaging cells are transfected and / or contain polynucleotides encoding gag and pol, as well as polynucleotides encoding recombinant receptors (e.g., antigen receptors, such as CAR). In some embodiments, the packaging cell lines are optionally and / or additionally transfected with and / or contain said polynucleotides encoding rev proteins. In some embodiments, the packaging cell lines are optionally and / or additionally transfected with and / or contain said polynucleotides encoding non-natural envelope glycoproteins (e.g., VSV-G). In some such embodiments, approximately two days after transfection of cells (e.g., HEK 293T cells), the cell supernatant contains the recombinant lentiviral vector, which can be recovered and titrated.

[0298] The recovered and / or generated retroviral vector particles can be used to transduce target cells as described above. Once inside the target cell, the viral RNA is reverse transcribed, enters the cell nucleus, and stably integrates into the host genome. Expression of recombinant proteins (e.g., antigen receptors, such as CARs) can be detected one or two days after viral RNA integration.

[0299] In some embodiments, the provided method relates to transducing cells by contacting (e.g., incubating) a cell composition containing multiple cells with viral particles. In some embodiments, the cells to be transfected or transduced are or contain primary cells obtained from a subject, such as cells enriched and / or selected from the subject.

[0300] In some embodiments, the concentration of cells to be transduced in the composition is from 1.0 x 10⁻⁶. 5 cells / mL to 1.0 x 10⁻⁶ 8 1.0 x 10⁻⁶ cells / mL or from approximately 1.0 x 10⁻⁶ cells / mL 5 cells / mL to approximately 1.0 x 10⁻⁶ 8 Cells / mL, such as at least or about 1.0 x 10⁻⁶ cells / mL, if water is stopped or at least about 1.0 x 10⁻⁶. 5 cells / mL, 5 x 10 5 cells / mL, 1 x 10 6 cells / mL, 5 x 10 6 cells / mL, 1 x 10 7 cells / mL, 5 x 10 7 1 cell / mL or 1 x 10 8 Cells / mL.

[0301] In some embodiments, the viral particles are provided at a ratio (IU / cell) of viral vector particle copies or infectious units (IU) to the total number of cells to be transduced. For example, in some embodiments, the viral particles are present during exposure at approximately or at least 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU of viral vector particles per cell.

[0302] In some implementations, the titer of the viral vector particles is at or around 1 x 10⁻⁶. 6 IU / mL and 1x 10 8 Between IU / mL, for example at or around 5 x 10⁻⁶. 6 IU / mL and 5x 10 7 Between IU / mL, for example at least 6 x 10⁻⁶ 6 IU / mL, 7 x 10 6 IU / mL, 8 x 10 6 IU / mL, 9 x 10 6 IU / mL, 1x 10 7 IU / mL, 2 x 10 7 IU / mL, 3 x 10 7 IU / mL, 4 x 10 7 IU / mL or 5x10 7 IU / mL.

[0303] In some implementations, transduction can be achieved at a multiplicity of infection (MOI) of less than 100, for example typically less than 60, 50, 40, 30, 20, 10, 5 or smaller.

[0304] In some embodiments, the method involves contacting or incubating cells with viral particles. In some embodiments, the contact is carried out for 30 minutes to 72 hours, such as 30 minutes to 48 hours, 30 minutes to 24 hours, or 1 hour to 24 hours, for example, at least or about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, or longer.

[0305] In some embodiments, the contact is carried out in solution. In some embodiments, the cells and virus particles are contacted in volumes ranging from 0.5 mL to 500 mL, or from about 0.5 mL to about 500 mL, such as from or from about 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5 mL to 500 mL, 5 mL to 200 mL, 5 mL to 100 mL, 5 mL to 50 mL, 5 mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL, or 200 mL to 500 mL.

[0306] In some implementations, the input cells are treated, incubated, or contacted with particles containing binding molecules that bind to or recognize recombinant receptors encoded by viral DNA.

[0307] In some implementations, incubating cells with viral vector particles results in or produces an output composition containing cells transduced with viral vector particles.

[0308] b. Non-viral vector

[0309] In some embodiments, the recombinant nucleic acid is transferred to T cells via electroporation (see, for example, Chicaybam et al., (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al., (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is transferred to T cells via transposition (see, for example, Manuri et al., (2010) Hum Gene Ther 21(4):427-437; Sharma et al., (2013) Molec Ther NuclAcids 2,e74; and Huang et al., (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0310] Other methods and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0311] In some implementations, recombinant nucleic acids are transferred to T cells via transposons. Transposons (transposable elements) are mobile segments of DNA that can move from one locus within the genome to another. These elements move via a conserved “cut-and-paste” mechanism: transposases catalyze the excision of the transposon from its original location and facilitate its reintegration at other locations in the genome. If the transposase is provided by another transposase gene, elements lacking the transposase can be mobilized. Therefore, transposons can be used to incorporate foreign DNA into the host genome without the use of viral transduction systems. Examples of transposons suitable for use with mammalian cells (e.g., human primary leukocytes) include, but are not limited to, SleepingBeauty and Piggybac.

[0312] Transposon-based transfection is a two-component system consisting of a transposon and a transposon. In some embodiments, the system comprises a transposon engineered to contain foreign DNA (also referred to herein as cargo DNA) (e.g., a gene encoding a recombinant receptor), flanked by inverted / homing repeat (IR / DR) sequences recognized by the accompanying transposon. In some embodiments, the nonviral plasmid encodes a transposon under promoter control. Transfection of the plasmid into a host cell results in transient expression of the transposon, thereby allowing it to be expressed at a sufficient level in the initial post-transfection period to integrate the transposon into the genomic DNA. In some embodiments, the transposon itself does not integrate into the genomic DNA, and therefore transposon expression decreases over time. In some embodiments, transposase expression is performed by the host cell at a level sufficient to integrate the corresponding transposon for the following durations: less than about 4 hours, less than about 8 hours, less than about 12 hours, less than about 24 hours, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, less than about 7 days, less than about 2 weeks, less than about 3 weeks, less than about 4 weeks, less than about 1 week, or less than about 8 weeks. In some embodiments, the cargo DNA introduced into the host genome is subsequently not removed from the host genome, at least because the host does not express endogenous transposases capable of excising the cargo DNA.

[0313] Sleeping Beauty (SB) is a synthetic member of the Tc / 1-Sailor superfamily of transposons, reconstructed from a dormant element present in the salmon genome. SB transposon-based transfection is a two-component system consisting of a transposon containing an inverted / directed repeat (IR / DR) sequence that leads to precise integration into a TA dinucleotide. The transposon is engineered with a target expression cassette flanked by IR / DR sequences. The SB transposon binds to a specific binding site located on the Sleeping Beauty transposon's IR. The SB transposon mediates the integration of the transposon, a mobile element encoding a cargo sequence flanked by inverted terminal repeat sequences with catalytic enzyme (SB) binding sites. Stable expression is achieved when the SB inserts the gene sequence into the vertebrate chromosome at the TA target dinucleotide via a cut-and-paste mechanism. This system has been used to engineer various vertebrate cell types, including human primary peripheral blood leukocytes. In some embodiments, cells are contacted with, incubated with, and / or treated with SB transposons containing a cargo gene (e.g., a gene encoding a recombinant receptor or CAR) flanked by an SBIR sequence. In a particular embodiment, cells to be transfected are contacted with, incubated with, and / or treated with a plasmid containing an SB transposon containing a cargo gene (e.g., a gene encoding a CAR) flanked by an SBIR sequence. In some embodiments, the plasmid also contains a gene encoding an SB transposase without an SBIR sequence flanking it.

[0314] PiggyBac (PB) is another transposon system that can be used to integrate cargo DNA into the genomic DNA of a host (e.g., human). PB transposases recognize PB transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon and efficiently remove the contents from the original site and efficiently integrate the contents into the TTAA chromosomal site. The PB transposon system enables the mobilization of a target gene between the two ITRs in a PB vector into the target genome. The PB system has been used to engineer various vertebrate cell types, including primary human cells. In some embodiments, cells to be transfected are contacted with, incubated with, and / or treated with PB transposons containing a cargo gene (e.g., a gene encoding a CAR) flanked by PB IR sequences. In certain embodiments, cells to be transfected are contacted with a plasmid containing a PB transposon, incubated with a plasmid containing a PB transposon, and / or treated with a plasmid containing a PB transposon that contains a cargo gene (e.g., a gene encoding a CAR) flanked by a PB IR sequence. In some embodiments, the plasmid also contains a gene encoding an SB transposase without a PB IR sequence flanking it.

[0315] In some embodiments, various elements of transposons / transposases used in the subject approach, such as one or more SB or PB vectors, can be generated using standard methods of restriction enzyme digestion, ligation, and molecular cloning. One approach for constructing the subject vector includes the following steps. First, a purified nucleic acid fragment containing the desired component nucleotide sequence as well as an external sequence is digested with a restriction endonuclease from an initial source (e.g., a vector containing a transposase gene). The fragment containing the desired nucleotide sequence is then separated from unwanted fragments of different sizes using conventional separation methods (e.g., by agarose gel electrophoresis). The desired fragment is excised from the gel and ligated together in an appropriate conformation to produce a circular nucleic acid or plasmid containing the desired sequence (e.g., a sequence corresponding to the various elements of the subject vector as described above). The circular molecule thus constructed is then amplified in a prokaryotic host (e.g., *E. coli*), if desired. The procedures for digestion, plasmid construction, cell transformation, and plasmid generation involved in these steps are well known to those skilled in the art, and the enzymes required for restriction enzyme digestion and ligation are commercially available. (See, for example, R. Wu, Ed., Methods in Enzymology, Vol. 68, Academic Press, New York (1979); T. Maniatis, EFFritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1982); Catalogue Nos. 1982-83, New England Biolabs, Inc.; Catalogue Nos. 1982-83, Bethesda Research Laboratories, Inc. Examples of how to construct the vectors used in the subject method are provided in the experimental section below. The preparation of the representative Sleeping Beauty transposon system is also disclosed in WO 98 / 40510 and WO 99 / 25817.)

[0316] In some embodiments, transposon transduction is performed using a plasmid containing a transposase gene and a plasmid containing a transposon, the transposon containing a cargo DNA sequence flanked by inverted / homing repeat (IR / DR) sequences recognized by the transposase. In some embodiments, the cargo DNA sequence encodes a heterologous protein, such as a recombinant T-cell receptor or CAR. In some embodiments, the plasmid contains both a transposase and a transposon. In some embodiments, the transposase is under the control of a pervasive promoter or any promoter suitable for driving the expression of the transposase in target cells. Pervasive promoters include, but are not limited to, EF1a, CMB, SV40, PGK1, Ubc, human β-actin, CAG, TRE, UAS, Ac5, CaMKIIa, and U6. In some embodiments, the cargo DNA contains a selection cassette that allows selection of cells that stably integrate the cargo DNA into the genomic DNA. Suitable selection boxes include, but are not limited to, those encoding the following selection boxes: kanamycin resistance gene, spectinomycin resistance gene, streptomycin resistance gene, ampicillin resistance gene, carbenicillin resistance gene, hygromycin resistance gene, bleomycin resistance gene, erythromycin resistance gene, and polymyxin B resistance gene.

[0317] In some implementations, the components for transposon transduction (e.g., plasmids containing SB transposase and SB transposons) are introduced into the target cells. Any convenient protocol can be used, depending on the location of the target cells, to introduce the system components into the target cells in vitro or in vivo. For example, when the target cells are isolated cells, the system can be introduced directly into the cells, for example, using standard transformation techniques under cell culture conditions that allow for target cell viability. Such techniques include, but are not limited to, viral infection, transformation, conjugation, protoplast fusion, electroporation, particle gun techniques, calcium phosphate precipitation, direct microinjection, viral vector delivery, etc. The choice of method generally depends on the type of cells to be transformed and the environment in which the transformation occurs (i.e., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0318] In some embodiments, an SB transposon and SB transposase source are introduced into target cells of a multicellular organism (e.g., a mammal or a human) under conditions sufficient to cleave nucleic acids flanked by inverted repeat sequences from a vector carrying the transposon, and the cleaved nucleic acids are subsequently integrated into the genome of the target cell. Some embodiments also include the step of ensuring the presence of the necessary transposase activity and the introduced transposon in the target cell. Depending on the structure of the transposon vector itself, i.e., whether the vector includes a region encoding a product with transposase activity, the method may further include introducing a second vector encoding the necessary transposase activity into the target cell.

[0319] In some implementations, the amount of vector nucleic acid containing the transposon and the amount of vector nucleic acid encoding the transposase introduced into the cell are sufficient to provide the necessary excision of the transposon nucleic acid and insertion into the target cell genome. Thus, the amount of vector nucleic acid introduced should provide sufficient transposase activity and a sufficient copy number of the desired nucleic acid to be inserted into the target cell. The amount of vector nucleic acid introduced into the target cell varies depending on the efficiency of the specific introduction protocol employed (e.g., the specific in vitro administration protocol).

[0320] Once the vector DNA, combined with the necessary transposase, enters the target cell, the vector nucleic acid region flanked by inverted repeat sequences (i.e., the vector nucleic acid located between inverted repeat sequences recognized by the Sleeping Beauty transposase) is cleaved from the vector via the provided transposase and inserted into the genome of the target cell. Thus, after the vector DNA is introduced into the target cell, a transposase-mediated excision of the exogenous nucleic acid carried by the vector is subsequently performed and inserted into the genome of the target cell. In certain embodiments, the vector integrates into at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, or at least 20% of the genome of cells transfected with SB transposons and / or SB transposase. In some embodiments, the integration of the nucleic acid into the target cell genome is stable; that is, the vector nucleic acid remains present in the target cell genome for more than a transient period and is passed on to the offspring of the target cell in a portion of the chromosomal genetic material.

[0321] In some embodiments, transposons are used to integrate nucleic acids (i.e., polynucleotides) of various sizes into the target cell genome. In some embodiments, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 0.1 kb to 200 kb, from about 0.5 kb to 100 kb, from about 1.0 kb to about 8.0 kb, from about 1.0 kb to about 200 kb, from about 1.0 kb to about 10 kb, from about 10 kb to about 50 kb, from about 50 kb to about 100 kb, or from about 100 kb to about 200 kb. In some embodiments, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 1.0 kb to about 8.0 kb. In some embodiments, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 1.0 kb to about 200 kb. In a particular embodiment, the size of the DNA inserted into the target cell genome using a subject-matter approach ranges from about 1.0 kb to about 8.0 kb.

[0322] D. Cell culture and / or expansion

[0323] In some embodiments, the provided method includes one or more steps for culturing cells (e.g., culturing cells under conditions that promote proliferation and / or expansion). In some embodiments, cells are cultured under conditions that promote proliferation and / or expansion following a genetic engineering step (e.g., introducing a recombinant polypeptide into cells via transduction or transfection). In a particular embodiment, cells are cultured after incubation under stimulating conditions and transduction or transfection with a recombinant polynucleotide (e.g., a polynucleotide encoding a recombinant receptor). In some embodiments, the culture produces one or more culture compositions enriched with T cells.

[0324] In some embodiments, one or more compositions of enriched T cells (including stimulated and transduced T cells) are cultured prior to cell preparation, for example, under conditions that promote proliferation and / or expansion (such as separate compositions of CD4+ and CD8+ T cells). In some aspects, the culture method (e.g., for promoting proliferation and / or expansion) includes methods provided herein as in section IF. In a particular embodiment, the one or more compositions of enriched T cells are cultured after they have been engineered (e.g., transduced or transfected). In a particular embodiment, the one or more compositions are engineered compositions. In a particular embodiment, the one or more engineered compositions have been previously cryogenically frozen and stored, and thawed prior to culture.

[0325] In some embodiments, the engineered T cell composition is or comprises two separate compositions of enriched T cells. In a particular embodiment, two separate compositions of enriched T cells incorporating a recombinant receptor (e.g., CAR) are cultured separately under conditions promoting cell proliferation and / or expansion, wherein the separate compositions are, for example, two separate compositions of enriched T cells selected, isolated, and / or enriched from the same biological sample. In some embodiments, the conditions are stimulation conditions. In some embodiments, the two separate compositions comprise a composition of enriched CD4+ T cells (e.g., engineered CD4+ T cells incorporating a nucleic acid encoding a recombinant receptor and / or expressing a recombinant receptor). In a particular embodiment, the two separate compositions comprise a composition of enriched CD8+ T cells (e.g., engineered CD8+ T cells incorporating a nucleic acid encoding a recombinant receptor and / or expressing a recombinant receptor). In some embodiments, two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells (e.g., engineered CD4+ T cells and engineered CD8+ T cells) are cultured separately, for example, under conditions promoting proliferation and / or expansion. In some embodiments, a single composition enriched with T cells is cultured. In some embodiments, the single composition is a composition enriched with CD4+ T cells. In some embodiments, the single composition is a composition of enriched CD4+ and CD8+ T cells that have been combined from individual compositions prior to culture.

[0326] In some embodiments, the composition comprising enriched CD4+ T cells (such as engineered CD4+ T cells) cultured under conditions promoting proliferation and / or expansion 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%, at least 99.5%, at least 99.9%, or about 100% CD4+ T cells. In some embodiments, the composition comprises at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or about 100% CD4+ T cells expressing recombinant receptors and / or transduced or transfected with recombinant polynucleotides encoding recombinant receptors. In some embodiments, the cultured composition enriched with 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% of CD8+ T cells, and / or contains no CD8+ T cells, and / or contains no or substantially no CD8+ T cells.

[0327] In some embodiments, a composition comprising enriched CD8+ T cells (such as engineered CD8+ T cells) cultured under conditions promoting proliferation and / or expansion 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%, at least 99.5%, at least 99.9%, or about 100% CD8+ T cells. In a particular embodiment, the composition comprises at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or about 100% CD8+ T cells expressing a recombinant receptor and / or transduced or transfected with a recombinant polynucleotide encoding the recombinant receptor. In some embodiments, the composition of CD8+ T cells incubated under stimulation 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% of CD4+ T cells, and / or contains no CD4+ T cells, and / or contains no or substantially no CD4+ T cells.

[0328] In some embodiments, separate compositions of enriched CD4+ and CD8+ T cells (such as separate compositions of engineered CD4+ and engineered CD8+ T cells) are combined into a single composition and cultured, for example, under conditions that promote proliferation and / or expansion. In some embodiments, compositions of separately cultured enriched CD4+ and CD8+ T cells are combined into a single composition after culture has been performed and / or completed. In a particular embodiment, separate compositions of enriched CD4+ and CD8+ T cells (such as separate compositions of engineered CD4+ and engineered CD8+ T cells) are cultured separately, for example, under conditions that promote proliferation and / or expansion.

[0329] In some embodiments, the cells, such as the engineered cells, are cultured in a volume of culture medium, said volume being about or at least 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1,000 mL, 1,200 mL, 1,400 mL, 1,600 mL, 1,800 mL, 2,000 mL, 2,200 mL, or 2,400 mL. In some embodiments, cells are cultured at an initial volume and then adjusted to different volumes. In certain embodiments, the volume is adjusted after the culture period. In certain embodiments, the volume is increased from said initial volume during the culture period. In some embodiments, the volume is increased during the culture period when the cells achieve a certain density. In some embodiments, said initial volume is or is about 500 mL.

[0330] In a particular embodiment, during cultivation, the volume is increased from the initial volume when the cells achieve a certain density or concentration. In a particular embodiment, the volume is increased when the cells achieve a density and / or concentration of: [value missing], approximately [value missing], or at least 0.1 x 10⁻⁶. 6 cells / ml, 0.2 x 10 6 cells / ml, 0.4 x 10 6 cells / ml, 0.6 x 10 6 cells / ml, 0.8 x 10 6 cells / ml, 1 x 10 6 1.2 x 10 cells / ml 6 1.4 x 10 cells / ml 6 1.6 x 10 cells / ml 6 1.8 x 10 cells / ml 6 1 cell / ml, 2.0 x 10 6 1 cell / ml, 2.5 x 10 6 1 cell / ml, 3.0 x 10 6 1 cell / ml, 3.5 x 10 6 4.0 x 10 cells / ml 6 4.5 x 10 cells / ml 6 5.0 x 10 cells / ml 6 1 cell / ml, 6 x 10 6 1 cell / ml, 8 x 10 6 1 cell / ml or 10 x 10 6 Cells / ml. In some implementations, the cell count is at least or about 0.6 x 10⁻⁶. 6The volume is increased from the initial volume when the cell density and / or concentration reaches 100 cells / ml. In some embodiments, the density and / or concentration is the density and / or concentration of live cells in the culture. In a particular embodiment, the volume is increased when the cells achieve a density and / or concentration of: 1, about, or at least 0.1 x 10⁻⁶ cells / ml. 6 live cells / ml, 0.2 x 10 6 live cells / ml, 0.4 x 10 6 live cells / ml, 0.6 x 10 6 live cells / ml, 0.8 x 10 6 live cells / ml, 1 x 10 6 1.2 x 10 live cells / ml 6 1.4 x 10 live cells / ml 6 1.6 x 10 live cells / ml 6 1.8 x 10 live cells / ml 6 live cells / ml, 2.0 x 10 6 live cells / ml, 2.5 x 10 6 3.0 x 10 live cells / ml 6 3.5 x 10 live cells / ml 6 live cells / ml, 4.0 x 10 6 4.5 x 10 live cells / ml 6 5.0 x 10 live cells / ml 6 live cells / ml, 6 x 10 6 live cells / ml, 8 x 10 6 1 live cells / ml or 10 x 10 6 1 live cells / ml. In some embodiments, the live cell count is achieved as at least or about 0.6 x 10⁻⁶. 6 When the density and / or concentration of cells / ml is reached, the volume is increased from the initial volume. In some embodiments, the density and / or concentration of cells or live cells can be determined or monitored during culture, such as by using methods as described, including optical methods, including digital holographic microscopy (DHM) or differential digital holographic microscopy (DDHM).

[0331] In some embodiments, the cells achieve a certain density and / or concentration, and the volume is increased by about or at least 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1,000 mL, 1,200 mL, 1,400 mL, 1,600 mL, 1,800 mL, 2,000 mL, 2,200 mL, or 2,400 mL. In some embodiments, the volume is increased by 500 mL. In a particular embodiment, the volume is increased to about or at least 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1,000 mL, 1,200 mL, 1,400 mL, 1,600 mL, 1,800 mL, 2,000 mL, 2,200 mL, or 2,400 mL. In some embodiments, the volume is increased to 1,000 mL. In some embodiments, the volume is increased at a rate of 5 mL, 10 mL, 20 mL, 25 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 75 mL, 80 mL, 9 mL, or 100 mL every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the rate is 50 mL every 8 minutes or approximately.

[0332] In some embodiments, compositions of enriched T cells (such as engineered T cells) are cultured under conditions that promote proliferation and / or expansion. In some embodiments, such conditions may be designed to induce proliferation, expansion, activation, and / or survival of cells in a population. In particular embodiments, the stimulating conditions may include one or more of the following: a specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents (e.g., nutrients, amino acids, antibiotics, ions, and / or stimulating factors (e.g., cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors, and any other agents designed to promote cell growth, division, and / or expansion)).

[0333] In some embodiments, incubation is performed under conditions typically including temperatures suitable for the growth of primary immune cells (such as human T lymphocytes), for example at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically about 37 degrees Celsius. In some embodiments, the composition enriched with T cells is incubated at temperatures between 25°C and 38°C, such as between 30°C and 37°C, for example, about 37°C ± 2°C. In some embodiments, incubation is carried out for a period of time until the culture (e.g., incubation or expansion) produces the desired or threshold density, concentration, number, or dose of cells. In some embodiments, incubation is carried out for a period of time until the culture (e.g., incubation or expansion) produces the desired or threshold density, concentration, number, or dose of live cells. In some embodiments, incubation is carried out for a duration greater than or equal to about 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or longer. In some implementations, the density, concentration, and / or number or dose of cells can be determined or monitored during cultivation, such as by using methods as described, including optical methods, including digital holographic microscopy (DHM) or differential digital holographic microscopy (DDHM).

[0334] In some embodiments, the stimulating agent is removed and / or isolated from the cells prior to culture. In some embodiments, the stimulating agent is removed and / or isolated from the cells after engineering and before cultured engineered cells, for example, under conditions that promote proliferation and / or expansion. In some embodiments, the stimulating agent is the stimulating agent described herein, for example, in section IB-1. In certain embodiments, the stimulating agent is removed and / or isolated from the cells as described herein, for example, in section IB-2.

[0335] In certain embodiments, compositions of enriched T cells, such as engineered T cells (e.g., a single composition of engineered CD4+ T cells and engineered CD8+ T cells), are cultured in the presence of one or more cytokines. In some embodiments, the one or more cytokines are recombinant cytokines. In certain embodiments, the one or more cytokines are human recombinant cytokines. In some embodiments, the one or more cytokines bind to and / or are capable of binding to receptors expressed by T cells and / or endogenous to T cells. In certain embodiments, the one or more cytokines are or include members of the 4-α-helical bundle cytokine family. In some embodiments, members of the 4-α-helical bundle cytokine family 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, the one or more cytokines are or include IL-15. In a particular embodiment, the one or more cytokines are or include IL-7. In a particular embodiment, the one or more cytokines are or include recombinant IL-2.

[0336] In certain embodiments, the composition enriched with CD4+ T cells (such as engineered CD4+ T cells) is cultured with recombinant IL-2. In some embodiments, culturing the composition enriched with CD4+ T cells (such as engineered CD4+ T cells) in the presence of recombinant IL-2 increases the probability or likelihood that the CD4+ T cells of the composition will continue to survive, grow, expand, and / or be activated during the culture step and throughout the process. In some embodiments, compared to alternative and / or exemplary methods of culturing the enriched CD4+ T cells in the presence of recombinant IL-2 without culturing the composition, the composition of culturing the enriched CD4+ T cells (such as engineered CD4+ T cells) in the presence of recombinant IL-2 increases the probability and / or likelihood of producing an output composition of enriched CD4+ T cells (e.g., engineered CD4+ T cells suitable for cell therapy) from the composition of the enriched CD4+ T cells 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%, 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%, or at least 200% CD4+.

[0337] In some embodiments, the cells (such as a single composition of engineered CD4+ T cells and engineered CD8+ T cells) are cultured with cytokines, such as recombinant human cytokines, in concentrations between 1 IU / ml and 2,000 IU / ml, between 10 IU / ml and 100 IU / ml, between 50 IU / ml and 500 IU / ml, between 100 IU / ml and 200 IU / ml, between 500 IU / ml and 1400 IU / ml, between 250 IU / ml and 500 IU / ml, or between 500 IU / ml and 2,500 IU / ml.

[0338] In some embodiments, a composition of enriched T cells (such as a single composition of engineered CD4+ T cells and engineered CD8+ T cells) is cultured with recombinant IL-2, such as human recombinant IL-2, at a concentration between 2 IU / ml and 500 IU / ml, between 10 IU / ml and 250 IU / ml, between 100 IU / ml and 500 IU / ml, or between 100 IU / ml and 400 IU / ml. In a particular embodiment, the composition of enriched T cells is cultured with IL-2 at a concentration of or about 50 IU / ml, 75 IU / ml, 100 IU / ml, 125 IU / ml, 150 IU / ml, 175 IU / ml, 200 IU / ml, 225 IU / ml, 250 IU / ml, 300 IU / ml, or 400 IU / ml. In some embodiments, the enriched T cell composition is cultured with recombinant IL-2 at a concentration of 200 IU / ml. In some embodiments, the enriched T cell composition is a composition enriched with CD4+ T cells, such as a composition of engineered CD4+ T cells. In a particular embodiment, the enriched T cell composition is a composition enriched with CD8+ T cells, such as a composition of engineered CD8+ T cells.

[0339] In some embodiments, compositions of enriched T cells (such as a single composition of engineered CD4+ T cells and CD8+ T cells) are cultured with IL-7, such as recombinant human IL-7, in concentrations between 10 IU / ml and 5,000 IU / ml, between 500 IU / ml and 2,000 IU / ml, between 600 IU / ml and 1,500 IU / ml, between 500 IU / ml and 2,500 IU / ml, between 750 IU / ml and 1,500 IU / ml, or between 1,000 IU / ml and 2,000 IU / ml. In certain embodiments, the composition of enriched T cells is cultured with IL-7 at a concentration of 100 IU / ml, 200 IU / ml, 300 IU / ml, 400 IU / ml, 500 IU / ml, 600 IU / ml, 700 IU / ml, 800 IU / ml, 900 IU / ml, 1000 IU / ml, 1200 IU / ml, 1400 IU / ml, or 1600 IU / ml. In some embodiments, the cells are cultured in the presence of recombinant IL-7 at a concentration of 1200 IU / ml or higher. In some embodiments, the composition of enriched T cells is a composition of enriched CD4+ T cells (such as engineered CD4+ T cells).

[0340] In some embodiments, compositions of enriched T cells (such as a single composition of engineered CD4+ T cells and CD8+ T cells) are cultured with IL-15, such as recombinant human IL-15, wherein the concentration of said IL-15 is between 0.1 IU / ml and 200 IU / ml, between 1 IU / ml and 50 IU / ml, between 5 IU / ml and 25 IU / ml, between 25 IU / ml and 50 IU / ml, between 5 IU / ml and 15 IU / ml, or between 10 IU / ml and 00 IU / ml. In a particular embodiment, the composition of enriched T cells is cultured with IL-15 at a concentration of 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 6 IU / ml, 7 IU / ml, 8 IU / ml, 9 IU / ml, 10 IU / ml, 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, 50 IU / ml, 100 IU / ml, or 200 IU / ml. In a particular embodiment, the composition of enriched T cells is cultured with recombinant IL-15 at a concentration of 20 IU / ml. In some embodiments, the composition of enriched T cells is a composition of enriched CD4+ T cells (such as engineered CD4+ T cells). In a particular embodiment, the composition enriched with T cells is a composition enriched with CD8+ T cells (such as engineered CD8+ T cells).

[0341] In certain embodiments, a composition is used to culture enriched CD8+ T cells (such as engineered CD8+ T cells) in the presence of IL-2 and / or IL-15 (as described in the amounts). In some embodiments, a composition is used to culture enriched CD4+ T cells (such as engineered CD4+ T cells) in the presence of IL-2, IL-7, and / or IL-15 (as described in the amounts). In some embodiments, the IL-2, IL-7, and / or IL-15 are recombinant. In some embodiments, the IL-2, IL-7, and / or IL-15 are human. In certain embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and / or IL-15.

[0342] In certain embodiments, the culture is performed in a closed system. In some embodiments, the culture is performed under aseptic conditions in a closed system. In certain embodiments, the culture is performed in a closed system using one or more steps of the provided system. In some embodiments, the composition enriched with T cells is removed from the closed system and placed in and / or connected to a bioreactor for culture. Examples of bioreactors suitable for the culture include, but are not limited to, the GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20|50, Finesse SmartRocker bioreactor system, and Pall XRS bioreactor system. In some embodiments, the bioreactor is used for perfusion and / or mixing of cells during at least a portion of the culture step.

[0343] In some embodiments, cells cultured simultaneously under closed, ligated, and / or controlled bioreactor conditions experience faster expansion during culture compared to cells cultured without a bioreactor, such as cells cultured under static conditions like no mixing, shaking, movement, and / or perfusion. In some embodiments, cells cultured simultaneously under closed, ligated, and / or controlled bioreactor conditions reach or achieve threshold expansion, cell count, and / or density within 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 60 hours, 48 ​​hours, 36 hours, 24 hours, or 12 hours. In some embodiments, compared with cells cultured in exemplary and / or alternative processes in which cells are cultured simultaneously under the control of a bioreactor without being cultured in a closed, connected and / or controlled manner, cells cultured simultaneously under the control of a bioreactor reach or achieve 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, or at least 5-fold thresholds for cell expansion, cell count, and / or density.

[0344] In some embodiments, the mixing is or includes rocking and / or motion. In some cases, the bioreactor may undergo motion or rocking, which can increase oxygen transfer in some respects. Motion of the bioreactor can include, but is not limited to, rotation along a horizontal axis, rotation along a vertical axis, rocking motion along a tilted (or inclined) horizontal axis of the bioreactor, or any combination thereof. In some embodiments, at least a portion of the incubation is carried out with rocking. The rocking speed and rocking angle can be adjusted to achieve the desired agitation. In some embodiments, the rocking angle is 20°, 19°, 18°, 17°, 16°, 15°, 14°, 13°, 12°, 11°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, or 1°. In some embodiments, the rocking angle is between 6 and 16°. In other embodiments, the rocking angle is between 7 and 16°. In other embodiments, the rocking angle is between 8 and 12°. In some embodiments, the swing rate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 rpm. In some embodiments, the swing rate is between 4 rpm and 12 rpm, for example, between 4 rpm and 6 rpm and includes extreme values.

[0345] In some embodiments, the bioreactor is maintained at a temperature equal to or close to 37°C and a CO2 level equal to or close to 5%, and has a stable airflow rate of approximately or at least 0.01 L / min, 0.05 L / min, 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 1.0 L / min, 1.5 L / min, or 2.0 L / min or greater than 2.0 L / min. In some embodiments, at least a portion of the cultivation is carried out under perfusion, such as at a rate of 290 ml / day, 580 ml / day, and / or 1160 ml / day, for example, depending on the timing associated with the initiation of cultivation and / or the density of the cultured cells. In some embodiments, at least a portion of the cell culture expansion is carried out under rocking motion with an angle between 5° and 10° (e.g., 6°) and a constant rocking speed, such as between 5 RPM and 15 RPM, e.g., 6 RPM or 10 RPM.

[0346] In some embodiments, at least a portion of the cultivation step is performed under constant perfusion (e.g., perfusion at a slow, stable rate). In some embodiments, perfusion is or includes the outflow of liquid, such as used culture medium, and the inflow of fresh culture medium. In some embodiments, the perfusion replaces the used culture medium with fresh culture medium. In some embodiments, at least a portion of the cultivation is performed under perfusion having a stable rate of: or at least 100 ml / day, 200 ml / day, 250 ml / day, 275 ml / day, 290 ml / day, 300 ml / day, 350 ml / day, 400 ml / day, 450 ml / day, 500 ml / day, 550 ml / day, 575 ml / day, 580 ml / day, 600 ml / day, etc. ml / day, 650ml / day, 700ml / day, 750ml / day, 800ml / day, 850ml / day, 900ml / day, 950ml / day, 1000ml / day, 1100ml / day, 1160ml / day, 1200ml / day, 1400ml / day, 1600ml / day, 1800ml / day, 2000ml / day, 2200ml / day, or 2400ml / day.

[0347] In certain embodiments, culture is initiated under no-perfusion conditions, and perfusion is initiated after a set and / or predetermined time period, such as at the start of or initial culture, for about or at least 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or more than 72 hours. In certain embodiments, perfusion is initiated when the cell density or concentration reaches a set or predetermined density or concentration. In some embodiments, perfusion is initiated when the cultured cells reach a density or concentration of about or at least 0.1 x 10⁻⁶. 6 cells / ml, 0.2 x 10 6 cells / ml, 0.4 x 10 6 cells / ml, 0.6 x 10 6 cells / ml, 0.8 x 10 6 cells / ml, 1 x 10 6 1.2 x 10 cells / ml 6 1.4 x 10 cells / ml 6 1.6 x 10 cells / ml 6 1.8 x 10 cells / ml 6 1 cell / ml, 2.0 x 10 6 1 cell / ml, 2.5 x 10 6 1 cell / ml, 3.0 x 10 6 1 cell / ml, 3.5 x 10 6 4.0 x 10 cells / ml6 4.5 x 10 cells / ml 6 5.0 x 10 cells / ml 6 1 cell / ml, 6 x 10 6 1 cell / ml, 8 x 10 6 1 cell / ml or 10 x 10 6 Cells / ml. In a particular embodiment, perfusion is initiated when the density or concentration of live cells reaches a set or predetermined density or concentration. In some embodiments, perfusion is initiated when the cultured live cells reach a density or concentration of: [missing information - likely a specific density or concentration], approximately 0.1 x 10⁻⁶ cells / ml. 6 live cells / ml, 0.2 x 10 6 live cells / ml, 0.4 x 10 6 live cells / ml, 0.6 x 10 6 live cells / ml, 0.8 x 10 6 live cells / ml, 1 x 10 6 1.2 x 10 live cells / ml 6 1.4 x 10 live cells / ml 6 1.6 x 10 live cells / ml 6 1.8 x 10 live cells / ml 6 live cells / ml, 2.0 x 10 6 live cells / ml, 2.5 x 10 6 3.0 x 10 live cells / ml 6 3.5 x 10 live cells / ml 6 live cells / ml, 4.0 x 10 6 4.5 x 10 live cells / ml 6 5.0 x 10 live cells / ml 6 live cells / ml, 6 x 10 6 live cells / ml, 8 x 10 6 1 live cells / ml or 10 x 10 6 live cells / ml.

[0348] In certain embodiments, perfusion is performed at different rates during culture. For example, in some embodiments, the perfusion rate depends on the density and / or concentration of the cultured cells. In some embodiments, the perfusion rate is increased when the cells reach a set or predetermined density or concentration. The perfusion rate can be varied during culture, for example, from a stable perfusion rate to an increased stable perfusion rate, changing once, twice, three times, four times, five times, more than five times, more than ten times, more than 15 times, more than 20 times, more than 25 times, more than 50 times, or more than 100 times. In some embodiments, the stable perfusion rate is increased when the cells reach a set or predetermined cell density or concentration of: [value missing], [value missing], or approximately or at least 0.6 x 10⁻⁶. 6 cells / ml, 0.8 x 10 6 cells / ml, 1 x 10 6 1.2 x 10 cells / ml 6 1.4 x 10 cells / ml 6 1.6 x 10 cells / ml 6 1.8 x 10 cells / ml 6 1 cell / ml, 2.0 x 10 6 1 cell / ml, 2.5 x 10 6 1 cell / ml, 3.0 x 10 6 1 cell / ml, 3.5 x 10 6 4.0 x 10 cells / ml 6 4.5 x 10 cells / ml 6 5.0 x 10 cells / ml 6 1 cell / ml, 6 x 10 6 1 cell / ml, 8 x 10 6 1 cell / ml or 10 x 10 6 Cells / ml. In some embodiments, a stable perfusion rate is increased when the cell density or concentration reaches the following set or predetermined values: [value missing], [value missing], or approximately or at least 0.6 x 10⁻⁶ cells / ml. 6 live cells / ml, 0.8 x 10 6 live cells / ml, 1 x 10 6 1.2 x 10 live cells / ml 6 1.4 x 10 live cells / ml 6 1.6 x 10 live cells / ml 6 1.8 x 10 live cells / ml 6 live cells / ml, 2.0 x 10 6 live cells / ml, 2.5 x 10 6 3.0 x 10 live cells / ml 63.5 x 10 live cells / ml 6 live cells / ml, 4.0 x 10 6 4.5 x 10 live cells / ml 6 5.0 x 10 live cells / ml 6 live cells / ml, 6 x 10 6 live cells / ml, 8 x 10 6 1 live cells / ml or 10 x 10 6 Cells / ml. In some embodiments, the density and / or concentration of cells or live cells during culture (e.g., under perfusion) can be determined or monitored, such as by using methods as described, including optical methods, including digital holographic microscopy (DHM) or differential digital holographic microscopy (DDHM).

[0349] In some embodiments, culturing begins under non-perfusion conditions, and perfusion is initiated when the cell density or concentration reaches a set or predetermined density or concentration. In some embodiments, when the cell density or concentration reaches a set or predetermined density or concentration, perfusion is initiated at rates of: approximately or at least 100 ml / day, 200 ml / day, 250 ml / day, 275 ml / day, 290 ml / day, 300 ml / day, 350 ml / day, 400 ml / day, 450 ml / day, 500 ml / day, 550 ml / day, 575 ml / day, and 580 ml / day. 600ml / day, 650ml / day, 700ml / day, 750ml / day, 800ml / day, 850ml / day, 900ml / day, 950ml / day, 1000ml / day, 1100ml / day, 1160ml / day, 1200ml / day, 1400ml / day, 1600ml / day, 1800ml / day, 2000ml / day, 2200ml / day, or 2400ml / day. In some embodiments, perfusion is initiated when the cultured cells or cultured live cells reach the following density or concentration: [missing information - likely a specific concentration], approximately or at least 0.1 x 10 [missing information - likely a specific concentration]. 6 cells / ml, 0.2 x 10 6 cells / ml, 0.4 x 10 6 cells / ml, 0.6 x 10 6 cells / ml, 0.8 x 10 6 cells / ml, 1x10 6 1.2 x 10 cells / ml 6 1.4 x 10 cells / ml 6 1.6 x 10 cells / ml 6 1.8 x 10 cells / ml 6 1 cell / ml, 2.0 x 106 1 cell / ml, 2.5 x 10 6 1 cell / ml, 3.0 x 10 6 1 cell / ml, 3.5 x 10 6 4.0 x 10 cells / ml 6 1 cell / ml, 4.5 x 10 6 5.0 x 10 cells / ml 6 1 cell / ml, 6 x 10 6 1 cell / ml, 8 x 10 6 1 cell / ml or 10 x 10 6 Cells / ml

[0350] In some embodiments, at least a portion of the culture is perfused at a certain rate, and when the cell density or concentration reaches a set or predetermined density or concentration, the perfusion rate is increased to the following rates: 100 ml / day, 200 ml / day, 250 ml / day, 275 ml / day, 290 ml / day, 300 ml / day, 350 ml / day, 400 ml / day, 450 ml / day, 500 ml / day, 550 ml / day, 575 ml / day. ml / day, 580ml / day, 600ml / day, 650ml / day, 700ml / day, 750ml / day, 800ml / day, 850ml / day, 900ml / day, 950ml / day, 1000ml / day, 1100ml / day, 1160ml / day, 1200ml / day, 1400ml / day, 1600ml / day, 1800ml / day, 2000ml / day, 2200ml / day, or 2400ml / day. In some embodiments, perfusion is initiated when the cultured cells or cultured live cells reach the following density or concentration: [missing value], [missing value], approximately or at least 0.1 x 10 [missing value]. 6 cells / ml, 0.2 x 10 6 cells / ml, 0.4 x 10 6 cells / ml, 0.6 x 10 6 cells / ml, 0.8 x 10 6 cells / ml, 1 x 10 6 1.2 x 10 cells / ml 6 1.4 x 10 cells / ml 6 1.6 x 10 cells / ml 6 1.8 x 10 cells / ml 6 1 cell / ml, 2.0 x 10 6 1 cell / ml, 2.5 x 10 6 1 cell / ml, 3.0 x 10 61 cell / ml, 3.5 x 10 6 4.0 x 10 cells / ml 6 1 cell / ml, 4.5 x 10 6 5.0 x 10 cells / ml 6 1 cell / ml, 6 x 10 6 1 cell / ml, 8 x 10 6 1 cell / ml or 10 x 10 6 Cells / ml. In some embodiments, perfusion is performed when cells are cultured in volumes of approximately 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1000 mL. In some embodiments, the volume is 1000 mL.

[0351] In some embodiments, culture is initiated under conditions of no perfusion or perfusion at a certain rate, and the cell density or concentration reaches approximately 0.61 x 10⁻⁶. 6 When the concentration is 1000 cells / ml, the perfusion rate is increased to about 290 ml / day. In some embodiments, when cells are cultured in a volume of 1000 mL or more, the perfusion rate is increased to about 290 ml / day. 6 At a concentration of [number] cells / ml, cells are perfused at a rate of [number], [number], or at least 290 ml / day. In some embodiments, when the cell density or concentration reaches [number], [number], or at least 0.81 x 10⁻⁶ cells / ml, the cells are perfused at a rate of [number], [number], or at least 0.81 x 10⁻⁶ cells / ml. 6 At a concentration of [number] cells / ml, the perfusion rate is increased to approximately 580 ml / day. In some embodiments, when the cell density or concentration reaches approximately 1.01 x 10⁻⁶ cells / ml, the perfusion rate is increased to [number], [number], or at least 580 ml / day. 6 At a concentration of [number] cells / ml, the perfusion rate is increased to approximately 1160 ml / day. In some embodiments, when the cell density or concentration reaches approximately 1.2 x 10⁻⁶ cells / ml, the perfusion rate is increased to [number], [number], or at least 1160 ml / day. 6 When the concentration is 1 cell / ml, the perfusion rate will be increased to approximately 1160 ml / day.

[0352] In aspects of the provided embodiments, the perfusion rate is determined by assessing cell density and / or concentration or assessing the density and / or concentration of live cells during culture, including the timing of initiating or increasing perfusion as described herein and above. In some embodiments, cell density and / or concentration can be determined using methods as described, including optical methods, including digital holographic microscopy (DHM) or differential digital holographic microscopy (DDHM).

[0353] In some embodiments, the composition of enriched cells, such as engineered T cells (e.g., engineered CD4+ T cells or engineered CD8+ T cells), is cultured in the presence of a surfactant. In a particular embodiment, culturing the cells of the composition reduces the amount of shear stress that may occur during culture, such as due to mixing, rocking, movement, and / or perfusion. In a particular embodiment, the composition of enriched T cells, such as engineered T cells (e.g., engineered CD4+ T cells or engineered CD8+ T cells), is cultured with a surfactant, 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 example, being viable and / or not undergoing necrosis, programmed cell death, or apoptosis, during or after 1, 2, 3, 4, 5, 6, 7, or more days following completion of culture. In a particular embodiment, the composition of enriched T cells, such as engineered T cells (e.g., engineered CD4+ T cells or engineered CD8+ T cells), is cultured in the presence of a surfactant, 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 the cells do not experience cell death such as that caused by shear or shear-induced stress, such as programmed cell death, apoptosis, and / or necrosis.

[0354] In a particular embodiment, a composition of enriched T cells, such as engineered T cells (e.g., engineered CD4+ T cells or engineered CD8+ T cells), is cultured in the presence of surfactant in amounts between 0.1 μl / ml and 10.0 μl / ml, between 0.2 μl / ml and 2.5 μl / ml, between 0.5 μl / ml and 5 μl / ml, between 1 μl / ml and 3 μl / ml, or between 2 μl / ml and 4 μl / ml. In some embodiments, the composition of enriched T cells, such as engineered T cells (e.g., engineered CD4+ T cells or engineered CD8+ T cells), is cultured in the presence of surfactant in amounts of: 0.1 μl / ml, 0.2 μl / ml, 0.4 μl / ml, 0.6 μl / ml, 0.8 μl / ml, 1 μl / ml, 1.5 μl / ml, 2.0 μl / ml, 2.5 μl / ml, 5.0 μl / ml, 10 μl / ml, 25 μl / ml, or 50 μl / ml. In some embodiments, the composition of enriched T cells is cultured in the presence of surfactant in the presence of 0.1 μl / ml or about 2 μl / ml.

[0355] In some embodiments, the surfactant i...

Claims

1. A method for producing a composition of engineered cells, the method comprising: (a) incubating a first input composition comprising T cells enriched for CD8+ primary human T cells, thereby producing a first stimulated composition, and incubating a second input composition comprising T cells enriched for CD4+ primary human T cells, thereby producing a second stimulated composition, wherein the T cells enriched for CD8+ primary human T cells and the T cells enriched for CD4+ primary human T cells are isolated from the same biological sample, and the incubation of the first input composition and the second input composition are each performed under stimulating conditions, the stimulating conditions comprising the presence of: (i) an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof; (ii) an antioxidant, wherein the antioxidant comprises N-acetylcysteine ​​(NAC), and (iii) recombinant IL-2 and recombinant IL-15, wherein the concentration of recombinant IL-2 is 10 IU / mL to 200 IU / mL, and the concentration of recombinant IL-15 is 1 IU / mL to 25 IU / mL; wherein the incubation of the second input composition is also performed in the presence of recombinant IL-7, wherein the concentration of the recombinant IL-7 is 100 IU / mL to 1000 IU / mL; (b) introducing a first chimeric antigen receptor CAR into the cells of the first stimulated composition, thereby generating a first engineered composition comprising engineered T cells, and introducing a second CAR into the cells of the second stimulated composition, thereby generating a second engineered composition comprising engineered T cells, wherein the first CAR and the second CAR are the same; and (c) culturing the first engineered composition under conditions that promote expansion of the first engineered T cells, thereby producing a first output composition comprising engineered CD8+ T cells, and culturing the second engineered composition under conditions that promote expansion of the second engineered T cells, thereby producing a second output composition comprising engineered CD4+ T cells, wherein: (i) the incubation of the first engineered composition and the second engineered composition is carried out in the presence of: (1) recombinant IL-2 and recombinant IL-15, wherein the concentration of recombinant IL-2 is 50 IU / mL to 500 IU / mL, and the concentration of recombinant IL-15 is 5 IU / mL to 50 IU / mL; and (2) a surfactant, wherein the surfactant comprises a poloxamer; (ii) wherein the incubation of the second input composition is also performed in the presence of recombinant IL-7, wherein the concentration of the recombinant IL-7 is 500 IU / mL to 2000 IU / mL; (iii) at least a portion of the growing of the first engineered composition and the second engineered composition is performed using perfusion; (iv) culturing the first engineered composition and the second engineered composition for 2 days to 8 days, inclusive, wherein the culturing is performed at least until the output composition comprises 4 times more viable T cells than the composition prior to culturing.

2. The method of claim 1, wherein the first input composition comprises 200 × 10 6 Cells up to 300 × 10 6 cell.

3. The method of claim 1, wherein the incubation of the first output composition is performed in the presence of recombinant IL-7.

4. The method of claim 1, wherein the incubation of the first output composition is performed in the presence of recombinant IL-7, and for the incubation: The concentration of recombinant IL-7 ranged from 100 IU / mL to 1000 IU / mL.

5. The method of claim 1, wherein the first input composition comprises greater than 70% CD8+ primary human T cells.

6. The method of claim 1, wherein the first input composition comprises greater than 85% CD8+ primary human T cells.

7. The method of claim 1, wherein the first input composition consists of CD8+ primary human T cells.

8. The method of claim 1, wherein the second input composition comprises greater than 70% CD4+ primary human T cells.

9. The method of claim 1, wherein the second input composition comprises greater than 85% CD4+ primary human T cells.

10. The method of claim 1, wherein the second input composition consists of CD4+ primary human T cells.

11. The method of claim 1, wherein the second input composition comprises 200 × 10 6 Cells up to 300 × 10 6 cell.

12. The method of any one of claims 1-11, wherein the anti-CD3 antibody or antigen-binding fragment thereof and the anti-CD28 antibody or antigen-binding fragment thereof are present on the surface of a solid support.

13. The method of claim 12, wherein the solid support is or comprises beads.

14. The method of claim 13, wherein the diameter of the beads is greater than 3.5 μm but not greater than 9 μm.

15. The method of claim 13, wherein the beads are inert.

16. The method of claim 13, wherein the beads are or comprise a polystyrene surface.

17. The method of claim 13, wherein the beads are magnetic or superparamagnetic.

18. The method of claim 13, wherein the ratio of beads to cells is less than 3:

1.

19. The method of claim 13, wherein the ratio of beads to cells is from 2:1 to 0.5:

1.

20. The method of claim 13, wherein the ratio of beads to cells is 1:

1.

21. The method of any one of claims 1-11, wherein the concentration of NAC is from 0.2 mg / mL to 2.0 mg / mL.

22. The method of any one of claims 1-11, wherein the introducing comprises transducing the cells of the first stimulated composition and / or the second stimulated composition with a viral vector comprising a polynucleotide encoding the first CAR and / or the second CAR.

23. The method of claim 22, wherein the viral vector is a retroviral vector.

24. The method of claim 22, wherein the viral vector is a lentiviral vector or a gammaretroviral vector.

25. The method of claim 22, wherein the introduction of the first stimulated composition and / or the second stimulated composition is performed in the presence of a transduction adjuvant.

26. The method of claim 25, wherein the transduction adjuvant is or comprises protamine sulfate.

27. The method of claim 25, wherein the transduction adjuvant is or comprises a transduction adjuvant derived from fibronectin.

28. The method of claim 25, wherein the transduction adjuvant is or comprises RetroNectin.

29. The method of claim 25, wherein the transduction adjuvant is or comprises from 1 µg / ml to 50 µg / ml of protamine sulfate.

30. The method of any one of claims 1-11, wherein the introducing comprises transfecting the cells of the first stimulated composition and / or the second stimulated composition with a vector comprising a polynucleotide encoding the first CAR and / or the second CAR.

31. The method of claim 30, wherein the vector is a transposon.

32. The method of claim 30, wherein the vector is a Sleeping Beauty (SB) transposon or a Piggybac transposon.

33. The method of any one of claims 1-11, wherein the poloxamer is present at a concentration of from 0.5 μL / mL to 5 μL / mL.

34. The method of any one of claims 1-11, wherein at least a portion of the incubation of the first engineered composition and / or the second engineered composition is performed with continuous mixing and / or perfusion.

35. The method of any one of claims 1-11, wherein the method comprises removing the anti-CD3 antibody or antigen-binding fragment thereof and the anti-CD28 antibody or antigen-binding fragment thereof from the first engineered composition and / or the second engineered composition prior to the incubation.

36. The method of claim 35, wherein the anti-CD3 antibody or antigen-binding fragment thereof and the anti-CD28 antibody or antigen-binding fragment thereof are removed from the first engineered composition and / or the second engineered composition within 7 days or less after initiating the incubation.

37. The method of claim 35, wherein the anti-CD3 antibody or antigen-binding fragment thereof and the anti-CD28 antibody or antigen-binding fragment thereof are removed from the first engineered composition and / or the second engineered composition from 3 to 6 days after initiating the incubation.

38. The method of claim 35, wherein the anti-CD3 antibody or antigen-binding fragment thereof and the anti-CD28 antibody or antigen-binding fragment thereof are removed from the first engineered composition and / or the second engineered composition 4 days after initiating the incubation.

39. The method of claim 35, wherein the removing comprises exposing cells of the first engineered composition and / or the second engineered composition to a magnetic field.

40. The method of any one of claims 1-11, wherein the incubation of the first engineered composition is performed in the presence of recombinant IL-7.

41. The method of any one of claims 1-11, wherein the cultivation of the first engineered composition is performed in the presence of recombinant IL-7, and for the cultivation of the engineered composition: The concentration of recombinant IL-7 ranged from 500 IU / mL to 2000 IU / ml.

42. The method of any one of claims 1-11, wherein the threshold number of T cells or live T cells is at least 5 times greater than the number of T cells or live T cells of the first engineered cell composition and / or the second engineered cell composition prior to the culturing.

43. The method of any one of claims 1-11, wherein the threshold number of T cells or viable T cells of the first output composition and / or the second output composition is or is at least 50×10 6 cells.

44. The method of any one of claims 1-11, wherein after said cultivating, said method comprises collecting cells of said first output composition and / or said second output composition.

45. The method of claim 43, wherein after said incubating, said method comprises collecting cells of said first output composition and / or said second output composition.

46. ​​The method of claim 44, wherein the amount of time between initiation of the incubation and harvesting of cells of the first output composition and / or the second output composition is from 7 days to 15 days.

47. The method of claim 44, wherein the amount of time between the initiation of the incubation and the collection of the cells of the first output composition and / or the second output composition is from 9 days to 13 days.

48. The method of claim 44, wherein the amount of time between initiation of the incubation and collection of cells of the first output composition and / or the second output composition is from 8 days to 13 days.

49. The method of claim 44, further comprising formulating the cells of the first output composition and / or the second output composition for cryopreservation and / or administration to a subject.

50. The method of claim 49, wherein the cells of the first output composition and / or the second output composition are formulated in the presence of a pharmaceutically acceptable excipient and / or a cryoprotectant.

51. The method of claim 50, wherein the cryoprotectant comprises DMSO.

52. The method of claim 49, wherein the cells of the first output composition and / or the second output composition are formulated in a container.

53. The method of claim 49, wherein the cells of the first output composition and / or the second output composition are formulated in a vial or a bag.

54. The method of any one of claims 1-11, further comprising isolating the CD4+ T cells and / or the CD8+ T cells from a biological sample prior to the incubating.

55. The method of claim 54, wherein the separating comprises selecting cells based on surface expression of CD4 and / or CD8.

56. The method of claim 54, wherein said separating comprises performing an immunoaffinity-based selection.

57. The method of claim 55, wherein the biological sample is or comprises 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 leukapheresis product.

58. The method of any one of claims 1-11, wherein the biological sample is obtained from a human subject suffering from a disease, disorder or condition.

59. The method of claim 58, wherein the first CAR and / or the second CAR is capable of binding to a target antigen that is associated with, is specific to, and / or is expressed on cells or tissues of the disease, disorder, or condition.

60. The method of claim 59, wherein the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

61. The method of claim 59, wherein the target antigen is a tumor antigen.

62. The method of any one of claims 1-11, wherein the first CAR and / or the second CAR is capable of binding to a target antigen that is associated with a disease, disorder, or condition, is specific to a disease, disorder, or condition, and / or is expressed on cells or tissues of a disease, disorder, or condition.

63. The method of claim 62, wherein the disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer.

64. The method of claim 62, wherein the target antigen is a tumor antigen.

65. The method of claim 62, wherein the target antigen is selected from 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, dual antigen, 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 insert domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MART-1, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, NCAM, NKG2D, NKG2D ligand, NY-ESO-1, O-acetylated GD2 (OGD2), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), PSCA, progesterone receptor, survivin, ROR1, TAG72, tEGFR, VEGF receptor, VEGF-R2, Wilms tumor 1 (WT-1), pathogen-specific antigens, and antigens associated with universal tags.

66. The method of any one of claims 1-11, wherein the first CAR and / or the second CAR is an anti-CD19 CAR.

67. The method of any one of claims 1-11, wherein the first CAR and / or the second CAR comprises an extracellular domain comprising an antigen binding domain.

68. The method of claim 67, wherein the antigen binding domain is or comprises an antibody or an antigen binding fragment thereof.

69. The method of claim 67, wherein the antigen binding domain is or comprises a single chain fragment.

70. The method of claim 67, wherein the antigen binding domain is or comprises an antibody variable region connected by a flexible linker.

71. The method of claim 67, wherein the antigen binding domain is or comprises a scFv.

72. The method of claim 67, wherein the chimeric antigen receptor further comprises a spacer and / or a hinge region.

73. The method of claim 67, wherein the chimeric antigen receptor comprises an intracellular signaling domain.

74. The method of claim 73, wherein the intracellular signaling region comprises a primary signaling domain.

75. The method of claim 73, wherein the intracellular signaling region comprises a signaling domain capable of inducing a primary activation signal in a T cell.

76. The method of claim 73, wherein the intracellular signaling region comprises a signaling domain of a T cell receptor (TCR) component.

77. The method of claim 73, wherein the intracellular signaling region comprises a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM).

78. The method of claim 73, wherein the intracellular signaling region comprises an intracellular signaling domain of a CD3 chain or a signaling portion thereof.

79. The method of claim 73, wherein the intracellular signaling region comprises the intracellular signaling domain of the CD3-zeta (CD3ζ) chain or a signaling portion thereof.

80. The method of claim 73, wherein the chimeric antigen receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

81. The method of claim 74, wherein the chimeric antigen receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

82. The method of claim 81, wherein the intracellular signaling region further comprises a co-stimulatory signaling region.

83. The method of claim 82, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of a T cell co-stimulatory molecule or a signaling portion thereof.

84. The method of claim 82, wherein the co-stimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof.

85. The method of claim 82, wherein the co-stimulatory signaling region is located between the transmembrane domain and the primary signaling domain.

86. The method of any one of claims 1-11, wherein the method is performed for less than 21 days, inclusive.

87. The method of any one of claims 1-11, wherein the first output composition and / or the second output composition is ready for administration to the subject within less than 21 days and a 95% confidence interval including the end values.

88. The method of any one of claims 1-11, wherein cell viability, concentration, density, number, or a combination thereof, of the cells is monitored during at least a portion of the cultivation of the first engineered composition and / or second engineered composition.

89. The method of claim 88, wherein the monitoring of the first engineered composition and / or the second engineered composition is performed by optical methods.

90. The method of claim 88, wherein the monitoring of the first engineered composition and / or the second engineered composition is performed by bright field microscopy, fluorescence microscopy, differential interference contrast microscopy, phase contrast microscopy, digital holographic microscopy (DHM), differential digital holographic microscopy (DDHM), or a combination thereof.

91. The method of claim 88, wherein the monitoring of the first engineered composition and / or the second engineered composition is performed by differential digital holographic microscopy (DDHM).

92. The method of claim 88, wherein the monitoring is performed intermittently or continuously during at least a portion of the cultivation of the first engineered composition and / or the second engineered composition.

93. The method of claim 88, wherein the monitoring is performed at least every 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, or 26 hours during the incubation of the first engineered composition and / or the second engineered composition.

94. The method of claim 88, wherein the monitoring is performed at least every 1 hour, 6 hours, 12 hours, 18 hours, 24 hours, or 26 hours during the incubation of the first engineered composition and / or the second engineered composition.

95. The method of claim 88, wherein the monitoring and cultivation of the first engineered composition and / or the second engineered composition is performed in a closed system.

96. A composition comprising engineered cells produced by the method of any one of claims 1-95.

97. The composition of claim 96, further comprising a pharmaceutically acceptable carrier.

98. The composition of claim 96 or 97 comprising a cryoprotectant.

99. The composition of claim 98, wherein the cryoprotectant is DMSO.

100. An article of manufacture comprising an output composition according to any one of claims 96-99; and instructions for administering the output composition to a subject.

101. The article of claim 100, wherein the subject suffers from a disease or disorder.

102. The product of claim 101, wherein the first CAR and / or the second CAR specifically recognizes or specifically binds to an antigen associated with the disease or condition, expressed by the disease or condition, or present in cells of the disease or condition.

103. The article of any one of claims 100-102, wherein the output composition is a composition of engineered CD4+ T cells.

104. The article of any one of claims 100-102, wherein the output composition is a composition of engineered CD8+ T cells.

105. An article of manufacture comprising a composition of engineered CD4+ T cells produced by the method of any one of claims 1-95, a composition of engineered CD8+ T cells produced by the method of any one of claims 1-95; and instructions for administering the engineered CD4+ T cells and the engineered CD8+ T cells to a subject.

106. The article of manufacture of claim 105, wherein the instructions provide for separate administration of the CD4+ T cells and CD8+ T cells to the subject.

107. The article of manufacture of claim 105 or 106, wherein the instructions provide for administering the CD4+ T cells and the CD8+ T cells to the subject in a desired ratio.

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