Methods and compositions for preparing genetically engineered cells

Abstract

Methods for preparing T cells for cell therapy, compositions produced by the methods, and methods for administering the cells to a subject are provided. In particular, the present disclosure relates to the preparation of engineered T cells, e.g., engineered T cells expressing genetically engineered receptors, e.g., engineered (recombinant) TCRs and genetically engineered antigen receptors such as chimeric antigen receptors (CARs), or other recombinant chimeric receptors. Features of the methods include producing more consistent and / or predictable T cell products and / or lower toxicity compared to other methods. The provided methods include incubating cells under stimulatory conditions to induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulatory composition, which can then result in preferential transduction of cells derived from naive-like T cells. Features of the methods may also include reduced costs, number of steps, and resource expenditure compared to other methods.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 543,359, filed August 9, 2017, entitled "METHODS AND COMPOSITIONS FOR PREPARING GENETICALLY ENGINEERED CELLS," the contents of which are incorporated by reference in their entirety.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application has been submitted with an electronic Sequence Listing, which is provided as a file entitled 735042010540SEQLIST.txt, created on July 11, 2018, and is 35,434 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to methods for preparing T cells for cell therapy, compositions produced by the methods, and methods for administering the cells to a subject. In particular, the disclosure relates to the preparation of engineered T cells, e.g., engineered T cells expressing genetically engineered receptors, e.g., engineered (recombinant) TCRs and engineered antigen receptors such as chimeric antigen receptors (CARs), or other recombinant chimeric receptors. Features of the methods include producing a more consistent and / or predictable T cell product and / or lower toxicity compared to other methods. The provided methods include incubating cells under stimulatory conditions to induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in a stimulatory composition, which can then result in preferential transduction of cells derived from naive-like T cells. Features of the methods may also include reduced costs, number of steps, and resource expenditure compared to other methods. [Background technology]

[0004] background A variety of methods are available for preparing cells for therapeutic use and for administering cells.For example, methods are available for preparing cells, including T cells, for manipulation and cell therapy, including methods that include depletion or enrichment of certain subpopulations.For example, improved methods are needed to reduce the toxicity associated with certain adoptive cell therapy administration, to improve manufacturing process, to enable improved administration, and / or to reduce costs or other resources.Methods, cells, compositions, kits and systems that meet such needs are provided. Summary of the Invention

[0005] overview Provided herein is a method for genetically engineering T cells, comprising: incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, wherein the stimulatory conditions include a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating a stimulated composition; and introducing a nucleic acid encoding an engineered recombinant receptor into the composition of stimulated T cells, wherein the introducing step is performed during at least a portion of the incubating step.

[0006] Provided herein are methods for genetically engineering T cells, comprising incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, wherein the stimulatory conditions include the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating a stimulated composition; and wherein incubating the input composition under stimulatory conditions is performed before, during, and / or after introducing a nucleic acid encoding an engineered recombinant receptor. In some embodiments, the incubating step is performed for at least 3 days. In some cases, the incubating step is performed for at least 4 days. In some embodiments, the incubating step is performed for at least 5 days. In some embodiments, the incubating step is performed for at least 6 days.

[0007] Provided herein are methods for stimulating T cells, comprising: (a) incubating an input composition containing a culture starting amount of naive-like T cells or a CD8+ T cell subset thereof under stimulatory conditions, thereby producing a stimulated composition; and (b) introducing a nucleic acid encoding an engineered recombinant receptor into the stimulated cell composition, thereby producing an output composition containing T cells expressing the engineered recombinant receptor. In some embodiments, the T cells contain naive-like T cells and non-naive-like T cells, and the stimulatory conditions preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulated composition. In some embodiments, the introducing step is performed during at least a portion of the incubating step or after the incubating step.

[0008] In some embodiments, the culture starting dose of naive-like T cells or CD8+ T cell subsets thereof is 0.1 x 10 8 ~5×10 8 pieces or approximately 0.1 x 10 8 ~5×10 8 pieces, 0.1×10 8 ~4×10 8 pieces or approximately 0.1 x 10 8 ~4×10 8 pieces, 0.1×10 8 ~2×10 8 pieces or approximately 0.1 x 10 8 ~2×10 8 pieces, 0.1×10 8 ~1×10 8 pieces or approximately 0.1 x 10 8 ~1×10 8 pieces, 1×10 8 ~5×10 8 pieces or approximately 1 x 10 8 ~5×10 8 pieces, 1×10 8 ~4×10 8 pieces or approximately 1 x 10 8 ~4×10 8 pieces, 1×10 8 ~2×10 8 pieces or approximately 1 x 10 8 ~2×10 8 pieces, 2×10 8 ~5×10 8 pieces or approximately 2 x 10 8 ~5×10 8 pieces, 2×10 8 ~4×10 8 pieces or approximately 2 x 10 8 ~4×10 8 naive-like T cells or CD8+ T cell subsets thereof. In some cases, the starting culture dose of naive-like T cells or CD8+ T cell subsets thereof is at least 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8naive-like T cells or CD8+ T cell subsets thereof, or at least about 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells or their CD8+ T cell subsets, or 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or about 0.5 x 10 8 pieces, approximately 0.75×10 8 pieces, about 1×10 8 pieces, approximately 1.5×10 8 pieces, approximately 2×10 8 pieces, or approximately 4 x 10 8 In some examples, the starting amount of naive-like T cells or CD8+ T cell subsets thereof is at least 2×10 8 naive-like T cells or CD8+ T cell subsets thereof, or at least about 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or about 2 x 10 8 naive-like T cells or their CD8+ T cell subsets.

[0009] Under stimulation conditions, 1 × 10 8 ~4×10 8 pieces or approximately 1 x 10 8 ~4×10 8Methods for stimulating T cells are provided, comprising incubating an input composition comprising T cells containing a culture-starting amount of naive-like T cells or a CD8+ T cell subset thereof, thereby producing a stimulated composition. In some aspects, the T cells contain naive-like T cells and non-naive-like T cells, and the stimulation conditions preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulated composition.

[0010] In some embodiments, the culture starting dose of naive-like T cells or CD8+ T cell subsets thereof is at least 2×10 8 naive-like T cells or CD8+ T cell subsets thereof, or at least about 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or about 2 x 10 8 naive-like T cells or a CD8+ T cell subset thereof. In some aspects, the starting amount of culture is an amount of naive-like CD8+ T cells.

[0011] In some of any such embodiments, the naive-like T cells or naive-like CD8+ T cells are surface positive for T cell activation markers selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and / or are surface negative for markers selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1; and / or have low expression of CD95; and / or are negative for intracellular expression of cytokines selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10. In some embodiments, the naive-like cells or naive-like CD8+ cells are surface positive for T cell activation markers selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and / or are surface negative for markers selected from the group consisting of CD45RO, CD56, KLRG1; and / or have low expression of CD95. In some embodiments, the naive-like T cells or naive-like CD8+ cells are CD45RA+, CD27+, CCR7+, CD62-, and / or CD45RO-.

[0012] In some of any such embodiments, the non-naive-like T cells are surface negative for a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and / or are surface positive for a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1, and perforin; and / or are positive for intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, IL-10; and / or have high expression of CD95. In some aspects, the non-naive-like T cells are CD45RA-, CD27-, CCR7-, CD62+, and / or CD45RO+.

[0013] In some embodiments, the cells of the input composition have not been, and are not, subjected to a selection step based on endogenous T cell surface markers that distinguish between naive-like T cells and non-naive-like T cells prior to incubation.

[0014] In some embodiments, the method further comprises introducing the engineered recombinant receptor into the stimulated cells, thereby producing an output composition comprising T cells that express the engineered recombinant receptor. In some cases, incubating the composition under stimulatory conditions occurs before, during, and / or after introducing the nucleic acid encoding the engineered recombinant receptor.

[0015] In some embodiments, the recombinant receptor can bind to a target antigen associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition. In some examples, the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. In some examples, the target antigen is a tumor antigen. In some embodiments, the target antigen is selected from the group consisting of αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), and hepatitis B surface antigen, antifolate receptor, cyclin, cyclin A2, CC motif. Chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), erb-B2, erb-B3, erb-B4, erbB dimer, epidermal growth factor receptor type III variant (EGFR vIII), folate binding protein (FBP), Fc receptor-like 5 (FCRL5;Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor α, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erbB2), Her3 (erb-B3), Her4 (erb-B4), erbB dimer, human high-molecular-weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, and human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22R-α), IL-13R-alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV) ), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), fetal tumor antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2; also known as dopachrome tautomerase, dopachrome delta-isomerase, or DCT), folate receptor-a, 8H9, biantigen, glycoprotein 100 (gp100), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGF-R2), estrogen receptor, progesterone receptor, Wilms' tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, and antigens associated with universal tags;

[0016] In some embodiments, the recombinant receptor is or contains a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR).

[0017] In some of any such embodiments, the recombinant receptor contains an extracellular domain containing an antigen-binding domain, optionally the antigen-binding domain specifically binds to a target antigen. In some cases, the antigen-binding domain is or contains an antibody, or an antibody fragment thereof, optionally a single-chain fragment. In some aspects, the fragment contains antibody variable regions linked by a flexible linker. In some examples, the fragment contains an scFv.

[0018] In some embodiments, the recombinant receptor further contains a spacer and / or hinge region. In some aspects, the recombinant receptor contains an intracellular signaling region. In some examples, the intracellular signaling region contains an intracellular signaling domain. In some aspects, the intracellular signaling domain is or contains a primary signaling domain, a signaling domain capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain is or contains the intracellular signaling domain of a CD3 chain, optionally a CD3-zeta (CD3ζ) chain, or a signaling portion thereof.

[0019] In some embodiments, the recombinant receptor further comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some aspects, the intracellular signaling region further comprises a costimulatory signaling region. In some embodiments, the costimulatory signaling region comprises the intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof. In some cases, the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. In some embodiments, the CAR comprises an antigen-specific scFv, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or comprises a CD3ζ signaling domain, and optionally further comprises a spacer between the transmembrane domain and the scFv; the CAR comprises, in order, an antigen-specific scFv, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is optionally a CD3ζ signaling domain; or the CAR comprises, in order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is optionally a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or comprises a CD3ζ signaling domain.

[0020] In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region.

[0021] In some of any such embodiments, the stimulatory conditions include incubation with a stimulatory reagent capable of activating T cells, CD4+ T cells, and / or CD8+ T cells; inducing a signal through the TCR complex; and / or inducing proliferation of T cells, CD4+ T cells, and / or CD8+ T cells. In some embodiments, the stimulatory conditions include incubation with a stimulatory reagent 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 costimulatory molecules. In some cases, the stimulatory reagent contains a primary agent that specifically binds to a member of the TCR complex, optionally specifically binding to CD3. In some embodiments, the primary agent is an antibody or antigen-binding fragment.

[0022] In some examples, the stimulatory agent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, optionally the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. In some embodiments, the primary agent is an antibody or antigen-binding fragment. In some embodiments, the primary agent and the secondary agent comprise antibodies, and optionally, one or more stimulatory agents comprise incubation with an anti-CD3 antibody and an anti-CD28 antibody.

[0023] In some embodiments, the primary and / or secondary objects are present on the surface of a solid support. In some cases, the solid support is or contains a bead. In some embodiments, the bead contains a diameter greater than or 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 examples, the bead contains a diameter of 4.5 μm or about 4.5 μm. In some aspects, the bead contains a diameter that is the same size as or approximately the same size as a lymphocyte or antigen-presenting cell.

[0024] In some embodiments, the beads are inert. In some cases, the beads are or contain a polystyrene surface and, optionally, a magnetic or superparamagnetic core.

[0025] In some embodiments, the stimulation conditions are 1:1 to 10:1 or about 1:1 to 10:1, 1:1 to 8:1 or about 1:1 to 8:1, 1:1 to 6:1 or about 1:1 to 6:1, 1:1 to 4:1 or about 1:1 to 4:1, 1:1 to 3:1 or about 1:1 to 3:1, 2:1 to 4:1 or about 2:1 to 4:1, 2:1 to 3:1 or about 2:1 to 3:1, 1:1 to 2:1 or about 1:1 to 2:1, 4:1 to 10:1 or about 4:1 to 10:1, 4:1 to 8:1 or about 4:1 to 8:1. This method includes incubating cells at a bead-to-cell ratio of about 4:1 to 6:1, 4:1 to 6:1, 6:1 to 10:1, or about 6:1 to 10:1, 6:1 to 8:1, or about 6:1 to 8:1, 8:1 to 10:1, or about 8:1 to 10:1, 1:1 to 1:10, or about 1:1 to 1:8, 1:1 to 1:6, or about 1:1 to 1:4, 1:2 to 1:3, or about 1:2 to 1:3. In some examples, the bead-to-cell ratio is from 3:1 or about 3:1. In some embodiments, the bead-to-cell ratio is from 1:1 or about 1:1.

[0026] Provided herein is a method for genetically engineering T cells, comprising: incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, the stimulatory conditions including a stimulatory reagent comprising an anti-CD3 antibody and a secondary agent that is an anti-CD28 antibody attached to beads, wherein the ratio of beads to cells during the incubating step is at or about 1:1 to 4:1; and introducing a nucleic acid encoding an engineered recombinant receptor into the composition of stimulated T cells, wherein the introducing step is performed during at least a portion of the incubating step.

[0027] Provided herein is a method for genetically engineering T cells, comprising incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, wherein the stimulatory conditions include a stimulatory reagent comprising an anti-CD3 antibody and a secondary agent that is an anti-CD28 antibody attached to beads, wherein the ratio of beads to cells during the incubating step is at or about 1:1 to 4:1; and wherein the step of incubating the input composition under stimulatory conditions is performed before, during, and / or after the step of introducing a nucleic acid encoding an engineered recombinant receptor.

[0028] In some of any such embodiments, the T cells are derived from a biological sample, optionally from a human subject. In some cases, the biological sample is or contains 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.

[0029] In some embodiments, the T cells contain CD4+ and / or CD8+ cells. In some embodiments, the T cells comprise CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is 2:1 or about 2:1 to 1:5 or about 1:5. In some cases, the ratio of CD4+ cells to CD8+ cells is 1:1, 1:2, 2:1, 1:3, or 3:1, or about 1:1, about 1:2, about 2:1, about 1:3, or about 3:1. In some embodiments, the naive-like T cells comprise naive-like CD4+ T cells and / or naive-like CD8+ T cells.

[0030] In some of any such embodiments, the naive-like T cells are polyclonal. In some cases, the clonality of the naive-like T cells is determined by clonal sequencing, optionally next-generation sequencing, or spectratyping.

[0031] In some embodiments, the presence, amount, number, or percentage of naive-like T cells is detected by flow cytometry.

[0032] In some of any such embodiments, the stimulatory conditions do not include N-acetylcysteine ​​(NAC). In some embodiments, the stimulatory conditions do not contain IL-15 and / or IL-7. In some embodiments, the stimulatory conditions result in or induce death or non-naive-like T cells or subpopulations thereof. In some aspects, the stimulatory conditions result in activation-induced cell death (AICD) of non-naive-like T cells or subpopulations thereof.

[0033] In some embodiments, the method further comprises adding DNase during incubation and / or to the stimulated composition.

[0034] In some embodiments, incubation is carried out for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, or for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. In some embodiments, the percentage of cells derived from naive-like T cells in the stimulated composition is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, or more than about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, or more than about 100-fold compared to the percentage of naive-like cells in the input composition. In some embodiments, the ratio of cells derived from naive-like T cells compared to cells derived from non-naive-like T cells in the stimulated composition is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, or more than about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, or about 100-fold, compared to the ratio of naive-like T cells compared to non-naive-like T cells in the input composition.

[0035] In some cases, the stimulated composition contains more than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of cells derived from naive-like T cells of the input composition. In some embodiments, the stimulated composition contains less than 10% of cells derived from non-naive-like T cells. In some examples, the stimulated composition contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of cells derived from non-naive T cells.

[0036] In some embodiments, a greater percentage of naive-like T cells among the cells in the input composition are induced to proliferate and / or be activated compared to non-naive-like T cells. In some aspects, a greater percentage of naive-like T cells in the input composition are dividing compared to the percentage of non-naive-like T cells in the input composition 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the incubation. In some cases, the stimulatory conditions can induce a greater percentage of proliferation of cells of the human naive-like T cell population compared to human non-naive-like T cells 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of incubation under the conditions.

[0037] In some of any such embodiments, the non-naive-like T cells are effector T (T EFF ) cells, memory T cells, central memory T cells (T CM ), Effector Memory T(T EM ) cells, and combinations thereof; or the non-naive-like T cells are selected from the group consisting of effector T (T EFF ) cells and / or memory T cells, wherein the memory T cells are selected from the group consisting of central memory T cells (T CM ) and / or Effector Memory T(T EM ) cells.

[0038] In some embodiments, the percentage of naive-like T cells in the input composition is less than the percentage of engineered cells in the stimulated composition that are derived from naive-like T cells in the input composition. In some embodiments, a greater percentage of the introduced cells are naive-like T cells in the input composition or are derived from the expansion of naive-like T cells in the input composition compared to non-naive-like T cells in the input composition.

[0039] In some of any such embodiments, the introduction is by transduction. In some embodiments, the nucleic acid comprises a viral vector. In some cases, the viral vector is a retroviral vector. In some aspects, the viral vector is a lentiviral vector or a gammaretroviral vector. In some embodiments, the introduction is by transposition of a transposon containing the nucleic acid molecule. In some embodiments, the ratio of naive-like T cells compared to non-naive-like T cells in the stimulated composition is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, or 100-fold compared to the ratio of naive-like T cells compared to non-naive-like T cells in the input composition. In some embodiments, the stimulated composition is more polyclonal or multiclonal compared to the input composition. In some embodiments, the method is performed in vitro or ex vivo.

[0040] Provided are output compositions produced by any of the methods provided herein. Also provided are pharmaceutical compositions containing the described output compositions. In some embodiments, the pharmaceutical composition further contains a pharmaceutical carrier.

[0041] Methods of treatment are provided that include administering to a mammalian subject an output composition produced by any of the described methods or any of the described pharmaceutical compositions. In some embodiments, the cells are derived from the subject to which the cells are administered. [The present invention 1001] (a) incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, wherein the stimulatory conditions include a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating a stimulated composition; and (b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into the composition of stimulated T cells, wherein said introducing step is performed during at least a portion of said incubating step. 1. A method for genetically engineering T cells, comprising: [The present invention 1002] 1. A method for genetically engineering T cells, comprising incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, the stimulatory conditions include the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, thereby producing a stimulated composition; and the step of incubating the input composition under stimulatory conditions is performed before, during, and / or after the step of introducing the nucleic acid encoding the engineered recombinant receptor; method. [The present invention 1003] The method of claim 1001 or 1002, wherein said incubating step is carried out for at least 3 days. [The present invention 1004] The method of claim 1001 or 1002, wherein said incubating step is carried out for at least 4 days. [The present invention 1005] The method of claim 1001 or 1002, wherein said incubating step is carried out for at least 5 days. [The present invention 1006] The method of claim 1001 or 1002, wherein said incubating step is carried out for at least 6 days. [The present invention 1007] (a) incubating an input composition comprising a culture starting amount of T cells, including naive-like T cells or a CD8+ T cell subset thereof, under stimulatory conditions, thereby generating a stimulated composition, wherein the stimulatory conditions include the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating the stimulated composition; and (b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into the stimulated cell composition. 1. A method for stimulating T cells, comprising: [The present invention 1008] The method of claim 1007, wherein the T cells comprise naive-like T cells and non-naive-like T cells, and the stimulating conditions preferentially induce the expansion or proliferation of naive-like T cells compared to non-naive-like T cells in the stimulated composition. [The present invention 1009] The method of claim 1007 or claim 1008, wherein said introducing step is performed during at least a portion of said incubating step or after said incubating step. [The present invention 1010] The initial amount of cultured naive-like T cells or CD8+ T cell subsets thereof is 0.1 × 10 8 ~5×10 8 pieces or approximately 0.1 x 10 8 ~5×10 8 pieces, 0.1×10 8 ~4×10 8 pieces or approximately 0.1 x 10 8 ~4×10 8 pieces, 0.1×10 8 ~2×10 8 pieces or approximately 0.1 x 10 8 ~2×10 8 pieces, 0.1×10 8 ~1×10 8 pieces or approximately 0.1 x 10 8 ~1×10 8 pieces, 1×10 8 ~5×10 8 pieces or approximately 1 x 10 8 ~5×10 8 pieces, 1×10 8 ~4×10 8 pieces or approximately 1 x 10 8 ~4×10 8 pieces, 1×10 8 ~2×10 8 pieces or approximately 1 x 10 8 ~2×10 8 pieces, 2×10 8 ~5×10 8 pieces or approximately 2 x 10 8 ~5×10 8 pieces, 2×10 8 ~4×10 8 pieces or approximately 2 x 10 8 ~4×10 8 1009. The method of any of claims 1007 to 1009, wherein the cells are naive-like T cells or a CD8+ T cell subset thereof. [The present invention 1011] The starting amount of naive-like T cells or CD8+ T cell subsets thereof is at least 0.5×10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or at least about 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells or their CD8+ T cell subsets, or 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or about 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×108 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 10. The method of any one of claims 1007 to 1010, wherein the naive-like T cells are CD8+ T cell subsets thereof. [The present invention 1012] The starting amount of naive-like T cells or CD8+ T cell subsets thereof is at least 2×10 8 naive-like T cells or CD8+ T cell subsets thereof, or at least about 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or about 2 x 10 8 naive-like T cells or a CD8+ T cell subset thereof. [The present invention 1013] Under stimulation conditions, 1 × 10 8 ~4×10 8 pieces or approximately 1 x 10 8 ~4×10 8 incubating an input composition comprising T cells comprising a culture starting amount of naive-like T cells or a CD8+ T cell subset thereof, wherein the stimulatory conditions comprise the presence of a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, thereby generating a stimulated composition. A method for stimulating T cells, comprising: [The present invention 1014] The method of claim 1013, wherein the T cells comprise naive-like T cells and non-naive-like T cells, and the stimulatory conditions preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulated composition. [The present invention 1015] The starting amount of naive-like T cells or CD8+ T cell subsets thereof is at least 2×10 8 naive-like T cells or CD8+ T cell subsets thereof, or at least about 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or 2 x 10 8 naive-like T cells or CD8+ T cell subsets thereof, or about 2 x 10 8 The method of claim 1013 or 1014, wherein the naive-like T cells or CD8+ T cell subset thereof. [The present invention 1016] The method of any one of claims 1007 to 1015, wherein the initial amount of culture is the amount of naive-like CD8+ T cells. [The present invention 1017] The naive-like T cells or naive-like CD8+ T cells are is surface positive for a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and / or is surface negative for a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1; and / or Low expression of CD95; and / or negative for intracellular expression of cytokines selected from the group consisting of IL-2, IFN-γ, IL-4, and IL-10; Any of the methods 1001 to 1016 of the present invention. [The present invention 1018] The naive-like cells or naive-like CD8+ cells are is surface positive for a T cell activation marker selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and / or is surface negative for a marker selected from the group consisting of CD45RO, CD56, and KLRG1; and / or have low expression of CD95, Any of the methods 1001 to 1016 of the present invention. [The present invention 1019] The method of any of claims 1001 to 1018, wherein said naive-like T cells or naive-like CD8+ cells are CD45RA+, CD27+, CCR7+, and / or CD45RO-. [The present invention 1020] the non-naive-like T cells is surface negative for T cell activation markers selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and / or is surface positive for a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1, and perforin; and / or positive for intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, and IL-10; and / or High expression of CD95 Any of methods 1001 to 1007, 1008 to 1012, and 1014 to 1019 of the present invention. [The present invention 1021] Any of the methods of the present inventions 1001 to 1007, 1008 to 1012, and 1014 to 1020, wherein the non-naive-like T cells are CD45RA-, CD27-, CCR7-, and / or CD45RO+. [The present invention 1022] Any of the methods of claims 1001 to 1021, wherein the cells of said input composition have not or will not be subjected to a selection step based on an endogenous T cell surface marker that distinguishes between naive-like T cells and non-naive-like T cells prior to said incubation. [The present invention 1023] 10. The method of any of claims 1013 to 1022, further comprising the step of introducing a genetically engineered recombinant receptor into said stimulated cells, thereby producing an output composition comprising T cells expressing the genetically engineered recombinant receptor. [The present invention 1024] The method of claim 1023, wherein the step of incubating the composition under stimulatory conditions occurs before, during, and / or after the step of introducing a nucleic acid encoding an engineered recombinant receptor. [The present invention 1025] the recombinant receptor Target antigens associated with, specific for, and / or expressed on cells or tissues of a disease, disorder, or condition Any of the methods 1001 to 1012 and 1023 to 1024 of the present invention can be combined with the above. [The present invention 1026] 1026. The method of claim 1025, wherein said disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, or a tumor or cancer. [The present invention 1027] The method of claim 1025 or claim 1026, wherein the target antigen is a tumor antigen. [The present invention 1028] The target antigen is selected from the group consisting of αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), Her2 / neu (receptor tyrosine kinase erbB2), L1-CAM, B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), and hepatitis B surface antigen, antifolate receptor, cyclin, cyclin A2, and CC motif chemokine. ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), erb-B2, erb-B3, erb-B4, erbB dimer, epidermal growth factor receptor type III variant (EGFR vIII), folate binding protein (FBP), Fc receptor-like 5 (FCRL5;Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), folate-binding protein (FBP), folate receptor α, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glypican-3 (GPC3), G protein-coupled receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erbB2), Her3 (erb-B3), and Her4 (erb-B4). , erbB dimer, human high-molecular-weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22R-α), IL-13R-alpha 2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing 8-phosphorylated nucleotides Family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), fetal tumor antigen, preferentially expressed antigen in melanoma (PR) AME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2;Any of the methods of claims 1025 to 1027, wherein the antigen is selected from among dopachrome tautomerase, dopachrome delta-isomerase, or DCT (also known as folate receptor-a, 8H9, biantigen, glycoprotein 100 (gp100), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGF-R2), estrogen receptor, progesterone receptor, Wilms' tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, and antigens associated with a universal tag; [The present invention 1029] The method of any of claims 1001 to 1012 and 1023 to 1028, wherein said recombinant receptor is or comprises a functional non-TCR antigen receptor or TCR or an antigen-binding fragment thereof. [The present invention 1030] The method of any of claims 1001 to 1012 and 1023 to 1029, wherein the recombinant receptor is a chimeric antigen receptor (CAR). [The present invention 1031] Any of the methods of claims 1001 to 1012 and 1023 to 1030, wherein the recombinant receptor comprises an extracellular domain comprising an antigen-binding domain that specifically binds to a target antigen, and an intracellular signaling domain comprising an ITAM. [The present invention 1032] 1032. The method of claim 1031, wherein said antigen-binding domain is or comprises an antibody, or an antibody fragment thereof, optionally a single-chain fragment. [The present invention 1033] 1033. The method of claim 1031 or 1032, wherein said intracellular signalling domain is or comprises the intracellular signalling domain of a CD3 chain, optionally the CD3-zeta (CD3ζ) chain, or a signalling part thereof. [The present invention 1034] The method of any one of claims 1031 to 1033, wherein the intracellular signaling region further comprises a costimulatory signaling region. [This invention 1035] 1035. The method of claim 1034, wherein said costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. [The present invention 1036] 1036. The method of claim 1034 or claim 1035, wherein said costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. [This invention 1037] the CAR comprises an scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule that is or comprises 4-1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule that is or comprises a CD3ζ signaling domain, and optionally further comprises a spacer between the transmembrane domain and the scFv; the CAR comprises, in order, an scFv specific for an antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or includes a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3ζ signaling domain; or the CAR comprises, in order, an antigen-specific scFv, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or includes a CD3ζ signaling domain; Any of the methods 1030 to 1036 of the present invention. [The present invention 1038] The method of any of claims 1001 to 1037, wherein said stimulatory reagent comprises a primary agent that specifically binds to a member of the TCR complex. [This invention 1039] The method of any of claims 1001 to 1038, wherein said stimulating reagent comprises a primary reagent that specifically binds to CD3. [The present invention 1040] The method of claim 1038 or claim 1039, wherein said primary agent is an antibody or antigen-binding fragment. [This invention 1041] The method of claim 1038 or claim 1039, wherein the stimulatory agent further comprises a secondary agent that specifically binds to a T cell costimulatory molecule, and optionally the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. [The present invention 1042] 1042. The method of claim 1041, wherein said primary agent is an antibody or antigen-binding fragment. [This invention 1043] The method of any of claims 1001 to 1040, wherein said stimulating reagent comprises a primary agent which is an anti-CD3 antibody and a secondary agent which is an anti-CD28 antibody. [This invention 1044] The method of any of claims 1038 to 1043, wherein the primary and / or secondary substances are present on the surface of a solid support. [This invention 1045] 104. The method of claim 1044, wherein said solid support is or comprises a bead. [The present invention 1046] The method of claim 1045, wherein said beads comprise a diameter of 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. [This invention 1047] 1047. The method of claim 1045 or claim 1046, wherein said beads comprise a diameter of at or about 4.5 μm. [This invention 1048] 1048. The method of any of claims 1045 to 1047, wherein said beads comprise a diameter that is the same size as or approximately the same size as a lymphocyte or antigen presenting cell. [This invention 1049] 1049. The method of any one of claims 1045 to 1048, wherein the beads are inert. [The present invention 1050] 1049. The method of any one of claims 1045 to 1049, wherein said beads are or comprise a polystyrene surface. [This invention 1051] 1050. The method of any of claims 1045 to 1050, wherein the beads comprise a magnetic or superparamagnetic core. [This invention 1052] The stimulation conditions are 1:1 to 10:1 or about 1:1 to 10:1, 1:1 to 8:1 or about 1:1 to 8:1, 1:1 to 6:1 or about 1:1 to 6:1, 1:1 to 4:1 or about 1:1 to 4:1, 1:1 to 3:1 or about 1:1 to 3:1, 2:1 to 4:1 or about 2:1 to 4:1, 2:1 to 3:1 or about 2:1 to 3:1, 1:1 to 2:1 or about 1:1 to 2:1, 4:1 to 10:1 or about 4:1 to 10:1, 4:1 to 8:1 or about 4:1 to 8:1, 4:1 to 6:1 or about 4: 10. The method of any of claims 1045 to 1051, comprising incubating the cells at a ratio of beads to cells that is 1 to 6:1, 6:1 to 10:1 or about 6:1 to 10:1, 6:1 to 8:1 or about 6:1 to 8:1, 8:1 to 10:1 or about 8:1 to 10:1, 1:1 to 1:10 or about 1:1 to 1:10, 1:1 to 1:8 or about 1:1 to 1:8, 1:1 to 1:6 or about 1:1 to 1:6, 1:1 to 1:4 or about 1:1 to 1:4, 1:2 to 1:3 or about 1:2 to 1:3. [This invention 1053] (a) incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, the stimulatory conditions comprising a stimulatory reagent comprising an anti-CD3 antibody and a secondary agent that is an anti-CD28 antibody attached to beads, the ratio of beads to cells during the incubating step being at or about 1:1 to 4:1; and (b) introducing a nucleic acid encoding a genetically engineered recombinant receptor into the composition of stimulated T cells, wherein said introducing step is performed during at least a portion of said incubating step. 1. A method for genetically engineering T cells, comprising: [This invention 1054] 1. A method for genetically engineering T cells, comprising incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions for 2 to 6 days, the stimulating conditions include a stimulating reagent comprising an anti-CD3 antibody and a secondary agent that is an anti-CD28 antibody attached to beads, and the ratio of beads to cells during the incubating step is between or about 1:1 and 4:1; and The method, wherein the step of incubating the input composition under stimulatory conditions occurs before, during, and / or after the step of introducing a nucleic acid encoding an engineered recombinant receptor. [This invention 1055] 105. The method of any of claims 1052 to 1054, wherein the ratio of beads to cells is from or about 3:1. [The present invention 1056] 105. The method of any of claims 1052 to 1054, wherein the ratio of beads to cells is from 1:1 or from about 1:1. [This invention 1057] The method of any of claims 1001 to 1056, wherein said T cells are derived from a biological sample, and optionally, said biological sample is derived from a human subject. [This invention 1058] 1057. The method of claim 1057, wherein said 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 leukocyte apheresis product. [This invention 1059] The method of any of claims 1001 to 1058, wherein said T cells comprise CD4+ and / or CD8+ cells. [The present invention 1060] 1059. The method of any of claims 1001 to 1059, wherein said T cells comprise CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is 2:1, or about 2:1 to 1:5, or about 1:5. [The present invention 1061] The method of any of claims 1001 to 1060, wherein the T cells comprise CD4+ and CD8+ T cells, and the ratio of CD4+ cells to CD8+ cells is 1:1, 1:2, 2:1, 1:3, or 3:1, or is about 1:1, about 1:2, about 2:1, about 1:3, or about 3:1. [The present invention 1062] The method of any of claims 1001 to 1061, wherein the naive-like T cells comprise naive-like CD4+ T cells and / or naive-like CD8+ T cells. [The present invention 1063] The method of any of claims 1001 to 1062, wherein said naive-like T cells are polyclonal. [The present invention 1064] The method of any one of claims 1001 to 1063, wherein the stimulating conditions do not include N-acetylcysteine ​​(NAC). [This invention 1065] The method of any of claims 1001 to 1063, wherein said stimulating conditions do not include IL-15 and / or IL-7. [The present invention 1066] The method of any of claims 1001 to 1065, wherein said stimulatory conditions result in or induce death or non-naive-like T cells or a subpopulation thereof. [This invention 1067] The method of any of claims 1001 to 1066, wherein said stimulatory conditions result in activation-induced cell death (AICD) of non-naive-like T cells or a subpopulation thereof. [The present invention 1068] The method of any of claims 1001 to 1067, further comprising the step of adding DNase during said incubation and / or to said stimulated composition. [The present invention 1069] Any of the methods of claims 1007 to 1065, wherein the incubation is carried out for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, or for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 days. [The present invention 1070] Any of the methods of inventions 1001 to 1069, wherein the percentage of cells derived from naive-like T cells in the stimulated composition is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, or more than about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, or about 100-fold, compared to the percentage of naive-like cells in the input composition. [This invention 1071] Any of the methods of inventions 1001 to 1070, wherein the ratio of cells derived from naive-like T cells compared to cells derived from non-naive-like T cells in the stimulated composition is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, or more than about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, or about 100-fold, compared to the ratio of naive-like T cells compared to non-naive-like T cells in the input composition. [This invention 1072] Any of the methods of claims 1001 to 1071, wherein the stimulated composition comprises more than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% cells derived from naive-like T cells of the input composition. [This invention 1073] The method of any of claims 1001 to 1072, wherein said stimulated composition comprises less than 10% cells derived from non-naive-like T cells. [This invention 1074] Any of the methods of claims 1001 to 1073, wherein the stimulated composition comprises less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% cells derived from non-naive T cells. [This invention 1075] Any of the methods of claims 1001 to 1074, wherein a greater percentage of naive-like T cells among the cells in said input composition are induced to proliferate and / or be activated compared to non-naive-like T cells. [This invention 1076] Any of the methods of claims 1001 to 1075, wherein a greater percentage of T cells that were naive-like in the input composition have divided on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after the start of the incubation compared to the percentage of T cells that were non-naive-like in the input composition. [This invention 1077] Any of the methods of claims 1001 to 1076, wherein the stimulatory conditions are capable of inducing proliferation of a greater percentage of cells of the human naive-like T cell population compared to human non-naive-like T cells on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after initiation of incubation under said conditions. [This invention 1078] (i) Non-naive-like T cells evolve into effector T (T EFF ) cells, memory T cells, central memory T cells (T CM ), Effector Memory T(T EM ) cells, and combinations thereof, or (ii) the non-naive-like T cells are selected from the group consisting of effector T (T EFF ) cells and / or memory T cells, wherein the memory T cells are central memory T cells (T CM ) and / or Effector Memory T(T EM 1078. The method of any one of claims 1001 to 1077, optionally comprising a . [This invention 1079] Any of the methods of claims 1001 to 1078, wherein the percentage of naive-like T cells in said input composition is less than the percentage of engineered cells in said stimulated composition that are derived from naive-like T cells in said input composition. [The present invention 1080] Any of the methods of claims 1001 to 1012 and 1023 to 1079, wherein a greater percentage of the cells into which the nucleic acid has been introduced are or are derived from the expansion of naive-like T cells in the input composition compared to non-naive-like T cells in the input composition. [This invention 1081] The method of any of claims 1001 to 1012 and 1023 to 1080, wherein said introducing is by transduction with a viral vector comprising a nucleic acid encoding the recombinant receptor. [This invention 1082] 1081. The method of claim 1081, wherein said viral vector is a retroviral vector. [This invention 1083] 1083. The method of claim 1081 or 1082, wherein said viral vector is a lentiviral vector or a gammaretroviral vector. [This invention 1084] Any of the methods of inventions 1001 to 1083, wherein the ratio of naive-like T cells compared to non-naive-like T cells in the stimulated composition is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, or 100-fold, or more than about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, or about 100-fold, compared to the ratio of naive-like T cells compared to non-naive-like T cells in the input composition. [This invention 1085] The method of any of claims 1001 to 1084, wherein said stimulated composition is more polyclonal or multiclonal compared to said input composition. [The present invention 1086] Any of the methods of claims 1001 to 1085, carried out in vitro or ex vivo. [This invention 1087] An output composition produced by any of the methods of inventions 1001 to 1086. [This invention 1088] A pharmaceutical composition comprising the output composition of the present invention 1087. [This invention 1089] The pharmaceutical composition of the present invention 1088 further comprising a pharmaceutical carrier. [The present invention 1090] A method of treatment comprising administering to a mammalian subject an output composition produced by any of the methods of inventions 1001 to 1006, or a pharmaceutical composition of invention 1088 or invention 1089. [This invention 1091] The method of claim 1090, wherein the cells are derived from the subject to which the cells are administered. DETAILED DESCRIPTION OF THE INVENTION

[0042] Detailed Description Provided herein are methods for incubating (e.g., stimulating) cells in the process of preparing cells for adoptive cell therapy, and compositions and cells produced by the methods.In some embodiments, the cells comprise T cells, which in some aspects express engineered antigen receptors.In some embodiments, the engineered antigen receptors comprise engineered or recombinant T cell receptors (TCRs) and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).

[0043] In some embodiments, methods for incubating (e.g., stimulating) T cells are provided. Available methods use anti-CD3 and anti-CD28 for T cell stimulation. However, currently available methods do not focus on obtaining a specific target population of cells through the incubation process and generating a more desirable population of T cells and their beneficial outcomes. Available methods also do not result in the elimination of undesirable and specific T cell populations from the input T cell population in the incubation process, nor their beneficial outcomes. Furthermore, available methods are not designed to eliminate subpopulations from the T cell population during the incubation process, which are used to generate output compositions containing T cells expressing engineered recombinant receptors. Available methods also do not account for benefits including generating a more consistent and / or predictable population of T cells, which is beneficial for genetic engineering, medication, and / or clinical response.

[0044] In some aspects, provided embodiments provide methods for generating compositions of increased numbers of more uniform and / or predictable T cells compared to other stimulation methods, and in some aspects further provide for the reduction or elimination of undesirable T cell populations, e.g., cells whose presence may result in such heterogeneity in the final product that it is difficult to predict potency, efficacy, and safety. In some embodiments, provided methods address issues related to the generation of engineered T cells where a majority or proportion of such engineered cells are derived from non-naive-like T cells. In some aspects, issues related to lack of persistence, attrition, and / or toxicity may be relevant to adoptive T cell therapies comprising compositions containing engineered cells derived from non-naive-like T cells and / or where the percentage or number of engineered cells derived from non-naive-like T cells is above a certain threshold. In some embodiments, methods resulting in engineered T cell compositions enriched for cells derived from naive-like cells can exhibit characteristics related to the percentage of healthy cells and / or an overall increase in cells in the composition that exhibit increased persistence compared to other methods that are not as enriched for such naive-like cells.

[0045] Thus, some methods for preparing engineered T cells for adoptive therapy provided herein use conditions that result in the preferential expansion or proliferation, or genetic engineering, of cells derived from naive-like T cells (or derived from CD27+, CD45RA+, CCR7+, CD62L+, or CD45RO- T cells) compared to cells derived from non-naive-like T cells (or derived from CD27-, CD45RA-, CCR7-, CD62L+, or CD45RO+ T cells). In some embodiments, the method includes incubating T cells in an input composition containing naive-like T cells under stimulatory conditions that preferentially produce a stimulated composition containing cells derived from the naive-like T cells of the input composition. In some aspects, the T cell population contains a mixture of both naive-like and non-naive-like T cells. In some embodiments, the stimulatory conditions preferentially induce a response in non-naive-like T cells compared to naive-like T cells. In some cases, the response involves preferential activation of non-naive-like T cells, which in some embodiments can lead to cell death.

[0046] In some embodiments, the method includes incubating cells under stimulatory conditions, including incubation with a stimulatory 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 costimulatory molecules. In some cases, the primary agent specifically binds to CD3, and / or the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS. For example, in some embodiments, the primary agent is or comprises anti-CD3, and the secondary agent is or comprises anti-CD28. In some cases, the primary agent and the secondary agent comprise antibodies and / or are present on the surface of a solid support. In some examples, the solid support is a bead.

[0047] In some embodiments, the stimulatory conditions include incubating the cells of the input composition with such stimulatory reagents, e.g., anti-CD3 / anti-CD28 beads, at a ratio of beads to cells of 1:1 to 10:1 or about 1:1 to 10:1, 1:1 to 8:1 or about 1:1 to 8:1, 1:1 to 6:1 or about 1:1 to 6:1, 1:1 to 4:1 or about 1:1 to 4:1, 1:1 to 3:1 or about 1:1 to 3:1, 4:1 to 10:1 or about 4:1 to 10:1. , 4:1 to 8:1 or about 4:1 to 8:1, 4:1 to 6:1 or about 4:1 to 6:1, 6:1 to 10:1 or about 6:1 to 10:1, 6:1 to 8:1 or about 6:1 to 8:1, 8:1 to 10:1 or about 8:1 to 10:1, 1:1 to 1:10 or about 1:1 to 1:10, 1:1 to 1:8 or about 1:1 to 1:8, 1:1 to 1:6 or about 1:1 to 1:6, 1:1 to 1:4 or about 1:1 to 1:4, 1:2 to 1:3 or about 1:2 to 1:3. In some embodiments, the bead to cell ratio is from 3:1 or about 3:1. In some cases, the bead to cell ratio is from 1:3 or about 1:3.

[0048] The method generally further comprises a step for genetically engineering the composition of stimulated T cells. Genetic engineering may be performed or initiated on the stimulated composition following or at any time after the start of the initial incubation, and / or simultaneously with the incubation. In some embodiments, cells are incubated with a nucleic acid encoding such an engineered molecule, so that the nucleic acid is introduced and the engineered molecule is expressed in the cells in the composition, thereby producing the output composition. Among the engineered molecules are proteins, for example, engineered antigen receptors, including chimeric antigen receptors (CARs) and other recombinant receptors, such as chimeric receptors with a ligand-binding extracellular portion and an intracellular signaling portion.

[0049] Also provided are culture initiation compositions, stimulated compositions, and output compositions used in or produced by the described methods. Also provided are methods comprising administering such compositions and cells to subjects in need thereof, including cancer patients. Additionally, provided are kits comprising compositions and / or cells produced by any of the methods described herein.

[0050] The methods can be advantageous and can produce a more desirable product. In some embodiments, the provided methods allow for a more consistent manufacturing process. In some embodiments, the provided methods produce cells that result in more uniform transduction and / or expansion in later steps of cell manipulation. In some embodiments, more uniform transduction and / or expansion can result in a more predictable T cell product throughout the manufacturing process. In some cases, the T cell product can be more consistently dosed for administration to a subject. Such characteristics may, in some situations, reduce or prevent potential toxicity and / or associated outcomes and symptoms in a subject following adoptive cell therapy.

[0051] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein conflicts or otherwise contradicts a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0052] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [Brief explanation of the drawings]

[0053] [Figure 1A] Figures 1A-1D illustrate results from CAR+ T cell compositions generated from expansion and non-expansion processes involving bead-based stimulation reagent (beads), bead-based stimulation reagent and incubation in basal medium (beads-basal medium), or oligomeric stimulation reagent (oligomers). Figure 1A illustrates the percentage of CD4+ and CD8+ T cells positive for both CCR7 and CD27. [Figure 1B] Figures 1A-1D illustrate results from CAR+ T cell compositions generated from expansion and non-expansion processes involving bead-based stimulation reagent (beads), bead-based stimulation reagent and incubation in basal medium (beads-basal medium), or oligomeric stimulation reagent (oligomers). Figure 1B illustrates the percentage of CCR7+CD27+ cells relative to CD4+CAR+ T cells. [Figure 1C] Figures 1A-1D illustrate results from CAR+ T cell compositions generated from expansion and non-expansion processes involving bead-based stimulation reagent (beads), bead-based stimulation reagent and incubation in basal medium (beads-basal medium), or oligomeric stimulation reagent (oligomers). Figure 1C illustrates the percentage of CCR7+CD27+ cells relative to CD8+ CAR+ T cells. [Figure 1D] 1A-1D illustrate results from CAR+ T cell compositions generated from expansion and non-expansion processes involving bead-based stimulation reagent (beads), bead-based stimulation reagent and incubation in basal medium (beads-basal medium), or oligomeric stimulation reagent (oligomers). Figure 1D shows the percentage of CCR7+CD27+ cells generated from a representative donor from the expansion process on various days during the manufacturing process, including day 1 activation (d1 AMAT), day 2 transduction (d2 XMAT), and various times after initiation of culture (d4 INOC+2, d6 INOC+4, d7 INOC+5). [Figure 2A]Figures 2A-2D show Kaplan-Meier survival curves (Figure 2A for progression-free survival) for subjects receiving CAR+ T cell compositions divided into groups receiving compositions containing a percentage of CCR7+CD27+ CAR+ T cells among CD4+ CAR+ T cells that is above or below a certain threshold level. [Figure 2B] Figures 2A-2D show Kaplan-Meier survival curves (Figure 2B for progression-free survival) for subjects receiving CAR+ T cell compositions divided into groups receiving compositions containing a percentage of CCR7+CD27+ CAR+ T cells among CD8+ CAR+ T cells that is above or below a certain threshold level. [Figure 2C] Figures 2A-2D show Kaplan-Meier survival curves (vs. duration of response in Figure 2C) for subjects receiving CAR+ T cell compositions divided into groups receiving compositions containing a percentage of CCR7+CD27+ CAR+ T cells among CD4+ CAR+ T cells that was above or below a certain threshold level. [Figure 2D] Figures 2A-2D show Kaplan-Meier survival curves (vs. duration of response in Figure 2D) for subjects receiving CAR+ T cell compositions divided into groups receiving compositions containing a percentage of CCR7+CD27+ CAR+ T cells among CD8+ CAR+ T cells that was above or below a certain threshold level. [Figure 3] Figure 3 shows the T cell clonality (Shannon index applied) of the isolated CD4+ and CD8+ T cell composition (CMAT) before manipulation and of the CD4+ and CD8+ therapeutic CAR+ T cell composition after manipulation.

[0054] I. Methods for Incubating (e.g., Stimulating) T Cells Provided herein are methods for incubating (e.g., stimulating) cells in the process of preparing cells for adoptive cell therapy, as well as compositions and cells produced by the methods. In some embodiments, the cells typically comprise T cells, which in some embodiments express engineered antigen receptors, such as engineered or recombinant T cell receptors (TCRs), and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).

[0055] In some embodiments, a method for incubating (e.g., stimulating) T cells is provided, comprising incubating an input composition containing a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions, wherein the input composition contains a culture starting amount of naive-like T cells or a CD8+ T cell subset thereof, thereby generating a stimulated composition. In some aspects, the stimulatory conditions preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulated composition. In some embodiments, the method further comprises introducing a nucleic acid encoding an engineered recombinant receptor into the stimulated cell composition, thereby generating an output composition comprising T cells expressing the engineered recombinant receptor. In some aspects, the introducing step is performed during at least a portion of the incubating step or after the incubating step. In some embodiments, the cells of the input composition have not been or are not subjected to a selection step based on endogenous T cell surface markers that distinguish between naive-like T cells and non-naive-like T cells prior to incubation.

[0056] In some embodiments of the provided methods, the step of introducing a nucleic acid encoding an engineered recombinant receptor is performed before, during, and / or after the step of introducing a nucleic acid encoding an engineered recombinant receptor. In some embodiments, the method includes incubating the composition under stimulatory conditions before the step of introducing a nucleic acid encoding an engineered recombinant receptor. In some cases, the method includes incubating the composition under stimulatory conditions during the step of introducing a nucleic acid encoding an engineered recombinant receptor. In some embodiments, the method includes incubating the composition under stimulatory conditions after the step of introducing a nucleic acid encoding an engineered recombinant receptor.

[0057] In certain embodiments, the stimulatory conditions include a surface, such as a magnetic bead, having attached thereto one or more agents that bind to cell surface moieties. In one embodiment, the surface has at least an anti-CD3 antibody attached thereto. In another embodiment, the surface has an anti-CD3 and / or anti-CD28 antibody attached thereto. In some embodiments, at least a substantial portion of at least one population of T cells in the input composition is deleted approximately after the incubating step. In one embodiment, the cell-to-bead ratio in the stimulatory conditions is about 50:1 to about 5:1. In certain embodiments, the ratio is about 100:1 to about 1:1. In some embodiments, the ratio is about 1:1 to 1:50. In certain embodiments, the ratio is at least about 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In one particular embodiment, the ratio is about 3:1. In one particular embodiment, the ratio is about 1:3.

[0058] In some embodiments of the methods provided herein, the culture conditions preferentially induce proliferation, stimulation, and / or activation of non-naive-like T cells relative to naive-like T cells. In some embodiments, the incubation (e.g., stimulation) method produces a desired output composition comprised of a desired number of cells derived from the naive-like T cells of the input composition. In some embodiments using the methods of stimulating T cells described herein, a greater percentage of the cells in the input composition are induced to proliferate and / or expand compared to the non-naive-like T cells. In some aspects, the stimulated composition resulting from the methods of stimulating described herein contains less than 10% cells derived from non-naive-like T cells. In some examples, the stimulated composition contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% cells derived from non-naive T cells. In some embodiments, a greater percentage of naive-like T cells in the input composition have divided on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after the start of the incubation compared to the percentage of non-naive-like T cells in the input composition. In some cases, the stimulatory conditions of the method for stimulating T cells induce cell death in a specific subpopulation of cells. In certain instances, the stimulatory conditions of the method induce activation of non-naive-like T cells, thereby inducing activation-induced cell death (AICD).

[0059] In one aspect, the method preferentially activates non-naive-like T cells of the input composition, thereby inducing cell death of the non-naive-like T cells of the input composition, thereby eliminating T cells derived from the non-naive-like cells of the input composition from the stimulated composition. Concurrently, remaining desired cells, e.g., cells derived from the naive-like T cells of the input composition, are stimulated to activate, survive, and expand, resulting in a population of activated cells from which at least a substantial portion of unwanted T cell subpopulations have been eliminated. Furthermore, the incubation (e.g., stimulation) conditions provided by the methods described herein restore polyclonality to the population of T cells with respect to expressed TCR genes, as indicated by spectratyping or other methods of quantifying clonality. In some embodiments, the characteristics of cells in the output composition derived from the naive-like T cells of the input composition are indicated by the expression of specific markers.

[0060] A. Input Composition In the provided methods for incubating (e.g., stimulating) T cells, the method includes incubating an input composition comprising a population of T cells under stimulatory conditions. The input composition, in some aspects, comprises a population of T cells, e.g., including naive-like T cells and non-naive-like T cells. In some embodiments, the population of T cells comprises CD4+ and / or CD8+ cells. In some embodiments, the input composition comprises a culture starting amount of cells. In some cases, the culture starting amount of cells is based on the amount of naive-like T cells or their CD8+ T cell subsets in the input composition. In some aspects, specific subsets of T cells, such as naive-like T cells and / or non-naive-like T cells, can be identified using specific markers or characteristics. In some embodiments, expression of cell surface markers is used to evaluate and / or identify T cell subsets. In some aspects, the clonality of the input composition can be characterized.

[0061] In some aspects, a composition of cells to be incubated and / or manipulated, such as an input composition, has not been and is not subjected to pre-selection of naive-like T cells. In some embodiments, the method does not include positive or negative selection or enrichment of naive-like cells in the input composition. In some aspects, a composition of stimulated and / or manipulated cells, such as an input composition and / or stimulated composition, has not and is not subjected to such selection prior to incubation (e.g., stimulation). In some embodiments, the cells have not and / or are not subjected to a selection step based on the level or presence of T cell surface markers that distinguish naive-like T cells from non-naive-like T cells, such as CD27, CD28, CD45RA, CD45RO, CD56, CD62L, CD95, KLRG1, or CCR7, prior to incubation. For example, in some aspects, the cells in the input composition have not been subjected to selection based on expression or surface expression of such markers prior to incubation under stimulatory conditions. In some aspects, the cells in the stimulated composition are not subjected to selection based on expression or surface expression of such markers prior to genetic manipulation, e.g., incubation with a nucleic acid. In some aspects, the step of incubating the input composition is performed without selection based on surface expression of a marker to enrich for naive-like T cells.

[0062] In some embodiments, the method involves fewer selection steps compared to other methods, e.g., does not involve the selection of a subset of T cells, and is therefore simpler than multi-step selection methods, with the advantage of saving costs and / or resources. In some embodiments, the method does not involve enrichment based on the expression of markers characteristic of memory T cells or subsets thereof and / or naive-like T cells. In some aspects, the composition of cells to be incubated (e.g., stimulated), such as the input composition and / or the stimulated composition, has not been and is not subjected to such selection.

[0063] In some examples, the methods do not include positive selection based on markers that are characteristic of non-naive-like T cells or that distinguish particular subpopulations of T cells, such as CD62L, CCR7, CD27, CD28, CD56, CD3, CD122, CD95, CD25, IL7-Rα, and / or CD127. In some examples, the methods do not include positive selection or enrichment based on expression of CD62L, CCR7, CD27, CD28, CD56, CD3, CD122, CD95, CD25, IL7-Rα, and / or CD127. In some embodiments, the methods do not use samples or compositions that have been enriched or positively selected based on such markers or to enrich for particular subtypes.

[0064] In some embodiments, the method does not include an affinity-based selection step designed to separate or distinguish between naive-like T cells and non-naive-like T cells. In some examples, the method does not include positive selection based on markers that are characteristic of or distinguish between naive-like or non-naive-like cells, such as CD27, CD28, CD45RO, CD45RA, CD56, CCR7, CD95, KLRG1, and / or CD62L. In some examples, the method does not include positive selection or enrichment based on the expression of such markers. In some embodiments, the method does not use a sample or composition that has been enriched or positively selected based on such markers or to enrich for such subtypes.

[0065] cell In some embodiments, the methods provided herein include one or more steps for preparing a cellular input composition containing a culture starting amount of naive-like T cells, e.g., in connection with a method including one or more steps of stimulating, expanding, proliferating, and / or genetically manipulating, e.g., transducing, cells. The cells are generally eukaryotic cells, such as mammalian cells, and typically human cells. The input composition can be made or generated by various methods, including isolating or selecting cells from a biological sample. In some embodiments, the input composition contains CD4+ cells and / or CD8+ cells from a biological sample, e.g., obtained or derived from one or more isolation, selection, or enrichment steps. In some specific examples, the ratio of CD4+ cells to CD8+ cells in the input culture is 1:1, 1:2, 2:1, 1:3, or 3:1, or is about 1:1, about 1:2, about 2:1, about 1:3, or about 3:1. In some embodiments, the input composition containing isolated CD4+ and / or CD8+ T cells contains a mixture of naive-like and non-naive-like T cells.

[0066] In some embodiments, the cells of the input composition are cells of innate or adaptive immunity, including those derived from blood, bone marrow, lymph, or lymphoid organs, and from a sample containing immune system cells, e.g., lymphocytes, typically T cells, and myeloid or lymphoid cells. The cells are typically primary cells, e.g., isolated directly from the subject and / or isolated and frozen from the subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, defined by, for example, function; activation state; maturity; differentiation, expansion, recirculation, localization, and / or persistence potential; antigen specificity; antigen receptor type; presence in specific organs or compartments; marker or cytokine secretion profile; and / or degree of differentiation. The cells can be allogeneic and / or autologous to the subject being treated. Some methods include pre-made methods. In some embodiments, the methods include isolating cells from a subject, preparing, treating, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient, before or after cryopreservation.

[0067] Among the subtypes and subpopulations of T cells and / or CD4+ T cells and / or CD8+ T cells are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (T SCM ), Central Memory T(T CM ), Effector Memory T(T EM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells. In some embodiments, the cells are regulatory T cells (Tregs). In some embodiments, the cells further comprise recombinant FOXP3 or a variant thereof.

[0068] In some embodiments, the preparation of engineered cells comprises one or more culturing and / or preparation steps. Cells for manipulation can be isolated from a sample, such as a biological sample, for example, obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated has a disease or condition, or is in need of cell therapy, or is a subject to whom cell therapy is administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, for example, adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated.

[0069] Thus, in some embodiments, the cells are primary cells, for example, primary human cells. Samples include tissues, body fluids, and other samples directly collected from subjects, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with viral vectors), washing, and / or incubation. Biological samples can be samples directly obtained from biological sources or samples that are processed. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, including processed samples derived therefrom.

[0070] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0071] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from a heterologous source, e.g., a mouse, a rat, a non-human primate, or a pig.

[0072] In some embodiments, cell isolation comprises one or more preparation steps and / or cell separation steps that are not based on affinity.In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, concentrate desired components, or lyse or remove cells that are sensitive to specific reagents.In some examples, cells are separated based on one or more characteristics, such as density, adhesive properties, size, sensitivity and / or resistance to specific components.

[0073] In some instances, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and / or platelets, and in some aspects, cells other than red blood cells and platelets.

[0074] In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and place the cells in a buffer or medium appropriate for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution does not contain calcium and / or magnesium, and / or many or all divalent cations. In some aspects, the washing step is accomplished in a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are washed after washing, for example, by filtration with Ca. ++ / Mg ++ In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.

[0075] In some embodiments, the method comprises a density-based cell separation method, eg, preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0076] In some embodiments, the isolation method involves separating different cell types based on the expression or presence of one or more specific molecules in cells, such as surface markers, for example, surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation may be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some aspects, the isolation involves the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such marker, and then generally followed by a washing step and separation of cells that bind to the antibody or binding partner from cells that do not bind to the antibody or binding partner.

[0077] Such separation steps can be based on positive selection, in which cells bound to the reagent are retained for further use, and / or negative selection, in which cells that are not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, so that separation is best performed based on markers expressed by cells other than the desired population.

[0078] Separation does not necessarily result in the enrichment or removal of 100% of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0079] In some cases, multiple separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as subsequent positive or negative selection.In some cases, for example, by incubating cells with multiple antibodies or binding partners that are each specific to the markers targeted for negative selection, a single separation step can simultaneously deplete cells that express multiple markers.Similarly, by incubating cells with multiple antibodies or binding partners that are expressed on various cell types, multiple cell types can simultaneously be positively selected.

[0080] For example, in some aspects, specific subpopulations of T cells, e.g., cells that are positive for or express high levels of one or more surface markers, e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD56 + , CD45RA + , CD95 hi , and / or CD45RO + T cells are isolated by positive or negative selection techniques.

[0081] For example, CD3 + , CD28 + T cells can be positively selected using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0082] In some embodiments, isolation is performed by enriching for a particular cell population by positive selection, or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is performed by isolating cells from a marker expressed on the cells to be positively or negatively selected, respectively. +) or expressed at relatively high levels (markers 高 ) This is achieved by incubating with one or more antibodies or other binding agents that specifically bind to one or more surface markers.

[0083] In some embodiments, T cells are separated from the PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, e.g., CD14. + or CD8 + The selection process involves CD4 + Helper T cells and CD8 + It is used to isolate cytotoxic T cells. + and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high degree on one or more naive, memory, and / or effector T cell subpopulations.

[0084] In some embodiments, CD8 + The cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM Enrichment of T cells is performed to increase efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects are particularly robust in such subpopulations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, T CM enriched CD8 + T cells and CD4 + Combining with T cells further enhances efficacy.

[0085] In embodiments, the memory T cells are CD8 + CD62L on peripheral blood lymphocytes + and CD62L - PBMCs are expressed in both CD62L and CD8 subsets, for example, using anti-CD8 and anti-CD62L antibodies. - CD8 + and / or CD62L + CD8 + Fractions can be concentrated or depleted.

[0086] In some embodiments, central memory T (T CM ) Cell enrichment is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells that express or high express CD45RA and / or granzyme B. In some aspects, T CM Cell-enriched CD8 + Isolation of the population is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, central memory T (T CM Enrichment of CD8 cells is performed starting from a negative fraction of cells selected based on CD4 expression, which is subjected to negative selection based on expression of CD14 and CD45RA, and positive selection based on CD62L. In some aspects, such selections are performed simultaneously, and in other aspects, sequentially in any order. In some aspects, CD8 + The same selection step based on CD4 expression used in the preparation of the cell population or subpopulation may also be used to select CD4 expression-based populations or subpopulations, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more further positive or negative selection steps. + It can also be used to generate cell populations or subpopulations.

[0087] In certain instances, a sample of PBMCs or other white blood cell sample is subjected to CD4 + The cells are subjected to selection, where both the negative and positive fractions are retained, and the negative fraction is then subjected to negative selection based on expression of CD14 and CD45RA or ROR1, and positive selection based on markers characteristic of central memory T cells, such as CD62L or CCR7, where positive and negative selection are performed in either order.

[0088] CD4 + T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations bearing cell surface antigens. CD4 + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - , CD45RA + , CD62L - , CD4 + In some embodiments, the T cells are central memory CD4 + The cells are CD62L + and CD45RO + In some embodiments, the effector CD4 + The cells are CD62L - and CD45RO - is.

[0089] In one example, negative selection leads to CD4 +To enrich cells, monoclonal antibody cocktails typically contain antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for cell separation for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: SA Brooks and U. Schumacher (Copyright) Humana Press Inc., Totowa, NJ).

[0090] In some aspects, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, e.g., a paramagnetic bead (e.g., Dynalbeads or MACS beads, etc.). The magnetically responsive material, e.g., particle, is generally attached, directly or indirectly, to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on the cell, cells, or population of cells that is desired to be separated, e.g., negatively or positively selected.

[0091] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. In some aspects, magnetically responsive materials used in magnetic separation methods can be used. Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342 B, which are incorporated herein by reference. Other examples include colloidal-sized particles, such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al.

[0092] Incubation is generally carried out under conditions in which the antibody or binding partner attached to the magnetic particle or bead, or a molecule such as a secondary antibody or other reagent that specifically binds to such an antibody or binding partner, specifically binds to a cell surface molecule if present on cells in the sample.

[0093] In some aspects, the sample is placed in a magnetic field, and cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed in the same selection step, where the positive and negative fractions are retained and further processed or subjected to additional separation steps.

[0094] In certain embodiments, magnetically responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, magnetic particles are attached to cells via coating with a primary antibody specific for one or more markers. In certain embodiments, cells, rather than beads, are labeled with a primary antibody or binding partner, and then magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

[0095] In some embodiments, the magnetically responsive particles remain attached to the cells that are then incubated, cultured, and / or manipulated; in some aspects, the particles remain attached to the cells for administration to patients. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells can include, for example, the use of competitive unlabeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[0096] In some embodiments, affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The magnetic-activated cell sorting (MACS) system allows for high-purity selection of cells to which magnetized particles are attached. In certain embodiments, MACS operates in a mode in which non-target and target species are sequentially eluted after application of an external magnetic field. That is, cells attached to magnetized particles are held in place while unattached species are eluted. Then, after this initial elution step is complete, species that were trapped in the magnetic field and prevented from elution are released in some way so that they can be eluted and recovered. In certain aspects, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0097] In certain embodiments, isolation or separation is carried out using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method of the present invention.In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user handling, and / or contamination.In one example, the system is the system described in International PCT Publication No. WO2009 / 072003 or US 20110003380 A1.

[0098] In some embodiments, the system or device performs one or more, e.g., all, of the isolation, processing, manipulation, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer program connected to the system or device, which allows a user to program, control, evaluate the outcome of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0099] In some aspects, the separation and / or other steps are performed using, for example, the CliniMACS system (Miltenyi Biotec) for automated cell separation at clinical scale in a closed and sterile system. Components can include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some aspects, the integrated computer controls all components of the instrument and commands the system to perform repetitive procedures in a standardized sequence. In some aspects, the magnetic separation unit includes a movable permanent magnet and a holder for the selected column. The peristaltic pump controls the flow rate of the entire tubing set and, together with the pinch valves, ensures a controlled flow of buffer through the system and continuous suspension of the cells.

[0100] In some aspects, the CliniMACS system uses antibody-coupled magnetizable particles supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is then connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing including a pre-column and a separation column and is intended for single use only. After the separation program begins, the system automatically applies the cell sample to the separation column. Labeled cells are retained in the column while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population for use in the methods described herein is unlabeled and not retained in the column. In some embodiments, the cell population for use in the methods described herein is labeled and retained in the column. In some embodiments, the cell population for use in the methods described herein is eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0101] In certain embodiments, the separation and / or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system includes a cell processing unit that allows for automated cell washing and fractionation by centrifugation. The CliniMACS Prodigy system can also include an on-board camera and image recognition software that determines the optimal cell fractionation endpoint by identifying the macroscopic layers of the source cell product. For example, peripheral blood can be automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system can also include an integrated cell culture chamber that accomplishes cell culture protocols, such as cell differentiation and expansion, antigen loading, and long-term cell culture. An input port can allow for the sterile removal and replenishment of culture media, and cells can be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0102] In some embodiments, the cell populations described herein are collected and enriched (or depleted) through flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) through preparative-scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) through the use of a microelectromechanical system (MEMS) chip combined with a FACS-based detection system (see, for example, WO 2010 / 033140, Cho et al. (2010) Lab Chip 10:1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In either case, cells can be labeled with multiple markers, which allows for the isolation of clearly defined T cell subsets with high purity.

[0103] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection.For example, separation can be based on binding to fluorescently labeled antibodies.In some examples, cell separation based on the binding of antibodies or other binding partners specific to one or more cell surface markers is carried out in a fluid flow, for example, by fluorescence activated cell sorting (FACS), including preparative scale (FACS), and / or by a microelectromechanical system (MEMS) chip combined with a flow cytometry detection system.Such a method allows for positive and negative selection based on multiple markers simultaneously.

[0104] In some embodiments, the preparation method includes a step of freezing the cells, e.g., for cryopreservation, either before or after isolation, incubation, and / or manipulation. In some embodiments, the freezing and subsequent thawing step removes granulocytes and, to some extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters may be used in some aspects. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium, which is then diluted 1:1 with medium to achieve final DMSO and HSA concentrations of 10% and 4%, respectively. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0105] a. Naive-like T cells In some embodiments, the method includes incubating an input composition containing naive-like T cells or a certain threshold amount of naive-like T cells. One aspect of the method of the present invention provides an input composition that has been evaluated for cell subpopulations based on the expression of various markers, such as CD27, CD28, CD56, CD62L, CD95, KLRG1, CD45RA or CD45RO, cytokines (e.g., IL-2, IFN-γ, IL-4, IL-10), cytokine receptors (e.g., CD25), perforin, adhesion molecules (e.g., VLA-1, VLA-2, VLA-4, LPAM-1, LFA-1), and / or homing molecules (e.g., L-selectin), prior to the incubation step (e.g., stimulating step). In some embodiments, various characteristics of naive-like T cells can be utilized to identify naive-like T cells. In some embodiments, the expression of specific markers of naive-like T cells can be evaluated. For example, in some cases, naive-like T cells are surface positive for markers including T cell activation markers selected from the group consisting of CD27, CD28, CD45RA, CD62L, and CCR7. In some aspects, naive-like T cells are surface negative for CD56 and / or CD45RO. In some aspects, naive-like T cells are surface negative for CD45RO and cell surface positive for CD27, CD45RA, and CCR7. In some cases, naive-like T cells are negative for intracellular expression of cytokines such as IL-2, IFN-γ, IL-4, and / or IL-10. In some further examples, naive-like T cells are negative for expression of the markers CD25 and / or perforin. In some cases, naive-like T cells are negative for expression of CD95 lo is.

[0106] To evaluate the expression of markers of naive-like T cells, the method in some embodiments includes detecting markers by performing an in vitro assay. In some examples, the in vitro assay is an immunoassay, an aptamer-based assay, a histological or cytological assay, or an mRNA expression level assay. In some cases, the in vitro assay used can be an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunophenotyping, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay, or avidity assay. In some embodiments, the expression of markers of naive-like T cells is determined by RNA-seq.

[0107] The naive-like T cells of the input composition can also be evaluated by the clonality of the T cells. In some embodiments, evaluating the clonality of a population of T cells is evaluating the clonal diversity of the population of T cells. In some embodiments, the naive-like T cells are polyclonal or multiclonal. The polyclonality of the input composition of T cells is measured by the breadth of the population's response to a given antigen. In some aspects, the input composition can be evaluated by measuring the number of different epitopes recognized by antigen-specific cells. This can be performed using standard techniques for generating and cloning antigen-specific T cells in vitro. In some embodiments, the naive-like T cells are polyclonal (or multiclonal), without a single clonal population predominating in the population of naive-like T cells.

[0108] For example, in reference to a population of T cells in an input composition, in some aspects, the characteristic of polyclonality refers to a population of T cells with multiple and broad antigen specificities. In some embodiments, polyclonality relates to a population of T cells that exhibits high diversity in their TCR repertoire. In some cases, the diversity of the TCR repertoire is due in some respects to V(D)J recombination events triggered by selection events against self- and foreign antigens. In some embodiments, a population of diverse or polyclonal T cells is a population of T cells in which analysis indicates the presence of many diverse or different TCR transcripts or products present in the population. In some embodiments, a population of T cells that exhibits high or relatively high clonality is a population of T cells in which the TCR repertoire is less diverse. In some embodiments, T cells are oligoclonal when analysis indicates the presence of several, e.g., two or three, TCR transcripts or products in the population of T cells. In some embodiments, T cells are monoclonal when analysis indicates the presence of a single TCR transcript or product in the population of T cells.

[0109] In some cases, the clonality of the cells in input composition, such as naive-like T cells, is determined by clonal sequencing, optionally by next-generation sequencing or spectratyping.In some aspects, to evaluate TCR repertoire, including the sequence encoding complementarity-determining region 3 (CDR3), next-generation sequencing can be used with genomic DNA or cDNA from T cells.In some embodiments, whole transcriptome sequencing by RNA-seq can be used.In some embodiments, single-cell sequencing can be used.

[0110] In some embodiments, polyclonality can be assessed or determined by spectratyping (measurement of TCR Vβ, Vα, Vγ, or Vδ chain hypervariable region repertoires). A population of T cells is considered polyclonal when the Vβ spectratyping profile for a given TCR Vβ, Vα, Vγ, or Vδ family has multiple peaks, typically five or more major peaks, with a Gaussian distribution in most cases. Polyclonality can also be defined by generating and characterizing antigen-specific clones against an antigen of interest.

[0111] In some embodiments, the method for assessing clonality can include various features of the methods described in International Publication Nos. WO2012 / 048341, WO2014 / 144495, WO2017 / 053902, WO2016044227, WO2016176322, and WO2012048340, each of which is incorporated by reference in its entirety. In some embodiments, such methods can be used to obtain sequence information about a target polynucleotide of interest in a cell, such as a TCR. The target gene can be obtained from genomic DNA or mRNA of cells from a sample or a population of cells. The sample or population of cells can include immune cells. For example, for a target TCR molecule, the gene encoding the TCR chain can be obtained from genomic DNA or mRNA of immune cells or T cells. In some embodiments, the starting material is RNA from T cells composed of a gene encoding the TCR chain.

[0112] In some embodiments, the Shannon index is applied to clonality as a threshold to filter clones ("Shannon-adjusted clonality"). See Chaara et al. (2018) Front Immunol 9:1038.

[0113] In some embodiments, the provided methods promote or result in an increase in polyclonality of a population of T cells or subsets thereof derived from an input composition. In some embodiments, the provided methods promote or result in an increase in diversity of a population of T cells or subsets thereof derived from an input composition. In some embodiments, the T cells or CD4 or CD8 subsets thereof of the incubated or stimulated composition exhibit reduced or decreased clonality compared to the T cells or CD4 or CD8 subsets thereof in the input composition before performing the method. In some embodiments, the degree of clonality is reduced by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more, or by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more.

[0114] In one aspect of the provided methods, the population of T cells in the input composition are activated or stimulated to induce apoptosis, as described below in the section entitled "Cell Incubation," thereby eliminating a subpopulation from the population of cells. Concurrently, the remaining desired cells, e.g., naive-like cells, e.g., cells derived from the naive-like T cells of the input composition, are activated and stimulated to expand, thereby resulting in a population of activated cells from which at least a substantial portion of the unwanted T cell subpopulation (e.g., non-naive-like T cells) has been eliminated. As previously mentioned, stimulation / activation as described herein may be performed on the cells remaining after direct exposure of the population of cells to the pro-apoptotic composition. Furthermore, the subsequent stimulation and activation provided by the present invention restores polyclonality to the population of T cells with respect to expressed TCR genes, as shown by spectratyping or sequencing methods.

[0115] Culture starting amount In aspects of the provided methods, the input composition comprises a culture starting amount for preferential activation or expansion of naive-like T cells. In some cases, the culture starting amount comprises, is determined for, or is based on the number of naive-like T cells or a CD8+ T cell subset thereof in the composition. Thus, in aspects of the provided methods, incubation (e.g., stimulation) is performed regardless of the total number or percentage of non-naive T cells, as long as a threshold or culture starting amount of naive-like T cells is present in the input composition.

[0116] In some embodiments of the methods provided herein for incubating (e.g., stimulating) cells, the culture starting dose of naive-like T cells or CD8+ T cell subsets thereof is 0.1 x 10 8 ~5×10 8 pieces or approximately 0.1 x 10 8 ~5×10 8 pieces, 0.1×10 8 ~4×10 8 pieces or approximately 0.1 x 10 8 ~4×10 8 pieces, 0.1×10 8 ~2×10 8 pieces or approximately 0.1 x 10 8 ~2×10 8 pieces, 0.1×10 8 ~1×10 8 pieces or approximately 0.1 x 10 8 ~1×10 8 pieces, 1×10 8 ~5×10 8 pieces or approximately 1 x 10 8 ~5×10 8 pieces, 1×10 8 ~4×10 8 pieces or approximately 1 x 10 8 ~4×10 8 pieces, 1×10 8 ~2×10 8 pieces or approximately 1 x 10 8 ~2×10 8 pieces, 2×10 8 ~5×10 8 pieces or approximately 2 x 10 8 ~5×108 pieces, 2×10 8 ~4×10 8 pieces or approximately 2 x 10 8 ~4×10 8 In some cases, the starting culture dose of naive-like T cells or CD8+ T cell subsets thereof is at least 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells, or at least about 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells or 0.5 x 10 8 pieces, 0.75×10 8 pieces, 1×10 8 pieces, 1.5×10 8 pieces, 2×10 8 pieces, or 4 x 10 8 naive-like T cells, or approximately 0.5 x 10 8 pieces, approximately 0.75×10 8 pieces, about 1×10 8 pieces, approximately 1.5×10 8 pieces, approximately 2×10 8 pieces, or approximately 4 x 10 8 In some examples, the starting culture dose of naive-like T cells or CD8+ T cell subsets thereof is at least 2×10 8 cells, or at least about 2 x 10 8 cells or 2 x 10 8 cells, or approximately 2 x 10 8 Each cell is an individual cell.

[0117] In some embodiments of the methods for incubating provided herein under stimulatory conditions described herein, the input composition comprising a population of T cells comprising naive-like T cells and non-naive-like T cells comprises 1 x 10 8 ~4×10 8 pieces or approximately 1 x 10 8 ~4×10 8 The starting culture amount of naive-like T cells or CD8+ T cell subsets thereof comprises at least 2×10 naive-like T cells or CD8+ T cell subsets thereof. In some embodiments, the method results in a stimulated composition, whereby the stimulation conditions preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulated composition. In some aspects, the starting culture amount of naive-like T cells or CD8+ T cell subsets thereof comprises at least 2×10 naive-like T cells or CD8+ T cell subsets thereof. 8 cells, or at least about 2 x 10 8 cells or 2 x 10 8 cells, or approximately 2 x 10 8 In some embodiments, the amount of naive-like T cells and non-naive-like T cells in the input composition is approximately the same. In some cases, the culture starting amount is the amount of naive CD8+ T cells. In some cases, the culture starting amount does not take into account the amount of non-naive-like T cells in the input composition. In some aspects, the culture starting amount is determined based on the number of target non-naive-like T cells in the stimulated composition.

[0118] b. Non-naive-like T cells In some embodiments, the method comprises incubating an input composition comprising a population of T cells comprising naive-like T cells and non-naive-like T cells. In some embodiments, the non-naive-like T cells are effector T (T EFF ) cells, memory T cells, central memory T cells (T CM ), Effector Memory T(T EM) cells, and combinations thereof. One aspect of the methods of the present invention provides an input composition that has been evaluated for a population of cells expressing various markers, such as CD27, CD28, CD56, CD62L, CD95, KLRG1, CD45RA or CD45RO, cytokines (e.g., IL-2, IFN-γ, IL-4, IL-10), cytokine receptors (e.g., CD25), perforin, adhesion molecules (e.g., VLA-1, VLA-2, VLA-4, LPAM-1, LFA-1), and / or homing molecules (e.g., L-selectin), prior to the incubating step (e.g., stimulating step). To identify non-naive-like T cells, in some embodiments, various characteristics of non-naive-like T cells can be utilized. In some embodiments, the expression of specific markers of non-naive-like T cells can be evaluated. For example, in some cases, non-naive-like T cells are surface negative for markers including T cell activation markers such as CD27, CD28, CD45RA, and CCR7; and in some cases, non-naive-like T cells are surface positive for markers including CD62L. In some aspects, non-naive-like T cells are surface positive for CD56 and / or CD45RO. In some aspects, non-naive-like T cells are surface positive for CD45RO and cell surface negative for CD27, CD45RA, and CCR7. In some cases, non-naive-like T cells are positive for intracellular expression of cytokines such as IL-2, IFN-γ, IL-4, and / or IL-10. In some further examples, non-naive-like T cells are positive for expression of the markers CD25 and / or perforin. In some cases, non-naive-like T cells are positive for expression of CD95 hi is.

[0119] To evaluate the expression of markers of non-naive-like T cells, the method in some embodiments includes detecting markers by performing an in vitro assay. In some examples, the in vitro assay is an immunoassay, an aptamer-based assay, a histological or cytological assay, or an mRNA expression level assay. In some cases, the in vitro assay used can be an enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, flow cytometry assay, surface plasmon resonance (SPR), chemiluminescence assay, lateral flow immunoassay, inhibition assay, or avidity assay. In some embodiments, the expression of markers of non-naive-like T cells is determined by RNA-seq.

[0120] The non-naive-like T cells of the input composition can also be evaluated by the clonality of T cells. In some embodiments, the non-naive-like T cells are monoclonal. The clonality of the input composition of T cells is measured by the breadth of the population's response to a given antigen. In some embodiments, monoclonality refers to a population of T cells with low diversity. In some aspects, the input composition can be evaluated by measuring the number of different epitopes recognized by antigen-specific cells. This can be performed using standard techniques for generating and cloning antigen-specific T cells in vitro. In some embodiments, the non-naive-like T cells exhibit a predominance of a single TCR gene rearrangement pattern. The clonality of cells in the input composition, such as non-naive-like T cells, is determined in some instances by clonal sequencing, optionally next-generation sequencing, or spectratyping.

[0121] In some embodiments, assessing the clonality of a population of T cells is assessing the clonal diversity of a population of T cells. In some embodiments, a monoclonal population of T cells refers to a population of T cells that exhibits low diversity. For example, in relation to the population of T cells in the input composition, monoclonality refers to a population of T cells with a single specificity as defined by spectratyping (measurement of TCR Vβ, Vα, Vγ, or Vδ chain hypervariable region repertoire). A population of T cells is considered monoclonal (or monospecific) when the Vβ, Vα, Vγ, and / or Vδ spectratyping profile for a given TCR Vβ, Vα, Vγ, and / or Vδ family has a single dominant peak. Spectratyping distinguishes rearranged variable genes of a specific size, not sequence. It is understood, therefore, that a single peak may represent a population of T cells expressing any one of a limited number of rearranged TCR variable genes (Vβ, Vα, Vγ, or Vδ) that contain any one of the four possible nucleotides (adenine (a), guanine (g), cytosine (c), or thymine (t)) or combinations of the four nucleotides in the junction region. In certain embodiments, it may be desirable to clone and sequence a particular band to determine the sequence of the rearranged variable region present in the band corresponding to a particular length.

[0122] In some embodiments, the method for assessing clonality can include various features of the methods described in International Publication Nos. WO2012 / 048341, WO2014 / 144495, WO2017 / 053902, WO2016044227, WO2016176322, and WO2012048340, each of which is incorporated by reference in its entirety. In some embodiments, such methods can be used to obtain sequence information about a target polynucleotide of interest in a cell, such as a TCR. The target gene can be obtained from genomic DNA or mRNA of cells from a sample or a population of cells. The sample or population of cells can include immune cells. For example, for a target TCR molecule, the gene encoding the TCR chain can be obtained from genomic DNA or mRNA of immune cells or T cells. In some embodiments, the starting material is RNA from T cells composed of genes encoding the TCR chain. In some embodiments, the Shannon index is applied to clonality as a threshold for filtering clones ("Shannon-adjusted clonality"). See Chaara et al. (2018) Front Immunol 9:1038.

[0123] Thus, some methods for incubating (e.g., stimulating) T cells in preparation of engineered T cells for adoptive therapy use conditions that preferentially induce the expansion and proliferation of cells derived from naive-like T cells (or derived from CD45RA+ or CD45RO- T cells) relative to cells derived from non-naive-like T cells (or derived from CD45RA- or CD45RO+ T cells). In some embodiments, naive-like T cells are CD45RA+, CD45RO-, CD27+, and CCR7+. In some embodiments, non-naive-like T cells are CD45RA-, CD45RO+, CD27-, and CCR7-. In some embodiments, the method includes incubating T cells in a culture start composition under stimulatory conditions that preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells, thereby generating a stimulated composition. In using the provided methods, evaluation of the input composition using the above-described markers and characteristics may be utilized as part of the method.

[0124] B. Cell Incubation In some embodiments, the provided methods include growing, incubating, culturing, and / or genetically manipulating cells in an input composition, e.g., an input composition containing a culture starting amount of naive-like T cells. For example, in some embodiments, methods are provided for incubating and / or manipulating a culture starting amount of a provided input composition containing a threshold number of naive-like T cells as described. The incubation and / or manipulation can be performed in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other vessel for culturing or growing cells.

[0125] In some embodiments, cells are incubated and / or cultured before or in association with genetic manipulation, for example, according to any of the methods described in Section II. The incubation step can include culturing, cultivating, stimulating, activating, and / or expanding. In some embodiments, the composition or cells are incubated under stimulating conditions or in the presence of a stimulating substance. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime cells for genetic manipulation, such as for the introduction of recombinant receptors, e.g., CARs.

[0126] The conditions can include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0127] In some embodiments, the stimulatory conditions or stimulatory substances include one or more agents, e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, such as antibodies specific for the TCR, e.g., anti-CD3. In some embodiments, the stimulatory conditions include one or more agents, e.g., ligands, e.g., anti-CD28, that can stimulate a costimulatory receptor. In some embodiments, such agents and / or ligands may be bound to a solid support, such as beads, and / or one or more cytokines. Optionally, the expansion method may further include adding an anti-CD3 antibody and / or an anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml).

[0128] The provided methods generally involve the presence, design, and / or use of stimulatory conditions that preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells, and / or that preferentially do not induce the expansion or proliferation of non-naive-like T cells relative to naive-like T cells. In some aspects, the conditions include an agent that induces an activation signal such that only non-naive-like T cells present in the composition are activated in a manner that induces cell death. Such preferential conditions are typically used at a step prior to the introduction of an engineered molecule, such as a nucleic acid encoding an engineered antigen receptor.

[0129] Conditions include, but are not limited to, those designed to induce proliferation, expansion, activation, and / or survival of cells in a population. In some embodiments, the conditions induce a stimulatory signal, e.g., an activation signal, sufficient to activate and / or induce proliferation or division in non-naive-like T cells or a subset thereof. Naive-like T cells generally require a minimum signal via TCR / CD3 to reach an activation threshold, e.g., to become fully activated and enter the cell cycle. This minimum signal is generally higher than the signal required to induce activation / cell cycle entry in non-naive-like T cells and / or certain subsets thereof. Non-naive-like T cells generally require a much lower level of TCR / CD3 binding to be activated and enter the cell cycle. In some aspects, a stronger signal can cause or increase the level of activation-induced cell death in non-naive-like cells or certain populations thereof.

[0130] Thus, in some embodiments, when a composition comprises naive-like T cells and non-naive-like T cells, and conditions are used that induce an activation signal that is below the activation threshold required for naive-like T cell activation, primarily the non-naive-like T cells are activated and become susceptible to stimulation conditions that can result in cell death. For example, under certain stimulation conditions, cell death may be due to activation-induced cell death.

[0131] In some aspects, the stimulatory conditions are such that activation-induced cell death in non-naive-like cells is induced compared to other conditions, such as standard conditions. Thus, the present invention provides methods for eliminating at least a substantial portion of any unwanted T cell subpopulation, e.g., non-naive-like T cells of an input composition. For purposes of the provided methods, a substantial portion refers to an unwanted cell subpopulation, e.g., at least 70% of the non-naive-like T cells of the input composition. In certain embodiments, a substantial portion refers to 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and higher, of the unwanted cell subpopulation, e.g., non-naive-like T cells of the input composition. For example, cell elimination resulting from cell death of non-naive-like T cells can be measured using any number of techniques, including, but not limited to, flow cytometry analysis using various antibodies and / or peptide-MHC tetramers, and functional assays such as proliferation and chromium release assays.

[0132] In some embodiments, enzymes are added to the input composition to remove cellular components remaining from the elimination of cells. For example, deoxyribonuclease (DNase) or recombinant human deoxyribonuclease I (Pulmozyme®) is added to the input composition in some exemplary embodiments. In some aspects, the stimulation condition includes deoxyribonuclease (DNase) or recombinant human deoxyribonuclease I (Pulmozyme®).

[0133] In some cases, the stimulatory conditions do not include culture components supplemented to protect specific subsets of T cells, such as non-naive-like T cells. Thus, in some cases, removal of components from the stimulatory culture may contribute to the elimination of non-naive-like T cells. In some aspects, the stimulatory conditions are implemented, or additionally implemented, by excluding or reducing the concentration of culture reagents known or likely to reduce AICD and / or that promote the survival of older cells, such as non-naive cells. In some cases, the stimulatory conditions do not include N-acetylcysteine ​​or include a reduced amount or concentration of N-acetylcysteine. In some cases, the stimulatory conditions do not include recombinant IL-7 and / or recombinant IL-15 or include a reduced amount or concentration of recombinant IL-7 or IL-15. In some embodiments, a culture additive (e.g., a toxin attached to CD45RO) that additionally assists or promotes the elimination of non-naive cells may be included.

[0134] In certain embodiments, the stimulation and / or expansion time can be 2-15 days, 2-12 days, 2-10 days, 2-8 days, 2-6 days, 2-4 days, 4-12 days, 4-10 days, 4-8 days, 4-6 days, 6-12 days, 6-10 days, 6-8 days, 8-12 days, 8-10 days, or 10-12 days. In some embodiments, the cells are incubated for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or longer than 14 days. In one embodiment of the provided method, the mixture may be cultured for 30 minutes to several hours (about 3 hours), up to about 14 days, or any integer value of hours or minutes therebetween. In another embodiment, the mixture may be cultured for 21 days. In one embodiment, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for at least 2-3 days or at least about 2-3 days. In another embodiment, the beads and T cells are cultured together for at least 2 days or at least about 2 days, or for at least 48 hours or at least about 48 hours. In some aspects, multiple cycles of stimulation may also be desirable, such that the culture time of the T cells can be 60 days or longer.

[0135] Incubation and / or manipulation may be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other container for culturing or growing cells. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory substance. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in a population, mimic antigen exposure, and / or prime cells for genetic manipulation, such as for the introduction of a recombinant antigen receptor.

[0136] The provided methods generally involve incubation (e.g., of a T cell-containing input composition) under stimulatory conditions. In the context of stimulating T cells, the stimulatory conditions generally include a primary agent capable of binding to, ligating, cross-linking, and / or inducing activation via the intracellular signaling domain of the TCR complex or a member thereof, e.g., CD3. In some embodiments, the stimulatory conditions include a primary agent that specifically binds to a member of the TCR complex and a secondary agent that specifically binds to a T cell costimulatory molecule. In some embodiments, the primary agent specifically binds to CD3, and / or the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.

[0137] In some embodiments, the stimulatory conditions include immobilized anti-CD3 and anti-CD28 antibodies, soluble anti-CD3 antibodies, derivatives of such antibodies, and / or other ligands that bind the TCR / CD3 complex on T cells. In some aspects, the primary agent activates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include binding partners, e.g., antigen-binding antibody fragments, natural ligands and / or antibodies, including those specific for TCR components such as CD3. Exemplary anti-CD3 antibodies are BC3, OKT3, and G19-4.

[0138] In some embodiments, the stimulatory conditions further include incubation with a secondary agent, e.g., an agent capable of inducing a T cell costimulatory signal in the T cell, e.g., a signal that, in combination with a primary signal (e.g., TCR / CD3 ligation), leads to T cell proliferation and activation. Exemplary secondary agents are those that specifically bind to, ligate, crosslink, and / or induce intracellular signaling events through T cell costimulatory or accessory molecules, such as CD28, CD137 (4-1-BB), OX40, ICOS, CD40, LFA-1, DAP10, and / or CD54. Agents include antibodies, natural ligands, and other binding partners, including fragments thereof. In some embodiments, the secondary agent is an anti-CD28 antibody that binds to CD28, e.g., including an antigen-binding antibody fragment. In some aspects, it is an anti-CD28 antibody. Exemplary anti-CD28 antibodies are B-T3 and XR-CD28.

[0139] In some embodiments, a binding partner or agent that specifically binds to a particular molecule, e.g., a T cell stimulatory or costimulatory molecule, comprises an antibody specific for such a molecule, including an antigen-binding antibody fragment. In some aspects, the antibody, e.g., an anti-CD3 antibody and / or an anti-CD28 antibody, is present at a concentration of at least 0.5 ng / mL or at least about 0.5 ng / mL. In some aspects, the agent comprises one or more other binding partners for such a molecule, such as a natural binding partner. The agent may also comprise natural ligands and complexes, including molecules and / or complexes on antigen-presenting cells, and / or superantigens (e.g., Staphylococcus enterotoxin A (SEA), Staphylococcus enterotoxin B (SEB), toxic shock syndrome toxin 1 (TSST-1), endotoxin). Other exemplary agents are those that mimic signaling through primary or costimulatory T cell signaling molecules, such as PKC activators, phorbol myristate acetate (PMA), phytohemagglutinin (PHA), and / or calcium ionophores, such as ionomycin, mitogens, including lipopolysaccharide (LPS), T cell mitogens, and cytokines. In some aspects, the conditions include incubation with one or more stimulatory cytokines or other factors, such as IL-2 and / or IL-15. In some aspects, the concentration of the cytokine is at least about 10 units / mL.

[0140] In some embodiments, the primary agent and the secondary (co-stimulatory) agent are bound to a solid surface or support, such as a particle, e.g., a bead. In some embodiments, the cells can be incubated and / or contacted with a stimulatory agent capable of activating and / or expanding T cells. In some aspects, the primary agent and the secondary agent are coupled to a solid surface, such as a particle, e.g., a bead. In certain embodiments, the stimulatory agent comprises a particle, e.g., a bead, conjugated or bound to one or more agents, e.g., biomolecules, capable of activating and / or expanding 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., composed of a material suitable for biological use. In some embodiments, the beads are non-toxic to cultured cells, e.g., cultured T cells. In some embodiments, the beads can be any particle to which an agent can be attached in a manner that allows interaction between the agent and the cell.

[0141] In some embodiments, the stimulator comprises one or more agents capable of activating and / or expanding cells, e.g., T cells, that are bound to or otherwise attached to the beads, e.g., the surface of the beads. In certain embodiments, the beads are non-cellular particles. In certain embodiments, the beads may comprise colloidal particles, microspheres, nanoparticles, magnetic beads, etc. In some embodiments, the beads are agarose beads. In certain embodiments, the beads are sepharose beads.

[0142] In certain embodiments, the stimulant comprises beads that are monodisperse. In certain embodiments, beads that are monodisperse comprise a size distribution that has a diameter standard deviation of less than 5% from one another.

[0143] In some embodiments, the beads contain one or more agents, e.g., agents coupled, conjugated, or linked (directly or indirectly) to the surface of the beads. In some embodiments, agents contemplated 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 that have 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 certain embodiments, the desired target is CD3. In certain embodiments, the desired target is a costimulatory molecule, e.g., CD28. The one or more agents may be directly or indirectly attached to the beads by various methods. Attachment may be covalent, non-covalent, electrostatic, or hydrophobic, and may be achieved by various attachment means, including, for example, chemical, mechanical, or enzymatic means. In some embodiments, a biomolecule (e.g., a biotinylated anti-CD3 antibody) may be indirectly attached to a bead via another biomolecule (e.g., an anti-biotin antibody) that is directly attached to the bead.

[0144] In some embodiments, one or more of the agents attached to the beads are antibodies. Antibodies can include polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules, as well as antibody fragments (Fab, F(ab')2, and Fv). In some embodiments, the stimulating reagent is an antibody fragment (including an antigen-binding fragment), such as a Fab, Fab'-SH, Fv, scFv, or (Fab')2 fragment. It will be understood that constant regions of any isotype, including IgG, IgM, IgA, IgD, and IgE constant regions, can be used for the antibodies contemplated herein, and that such constant regions can be obtained from any human or animal species (e.g., murine species). In some embodiments, the agent is an antibody that binds to and / or recognizes one or more components of the T cell receptor. In certain embodiments, the agent is an anti-CD3 antibody. In certain embodiments, the agent is an antibody that binds to and / or recognizes a co-receptor. In some embodiments, the stimulatory reagent comprises an anti-CD28 antibody.

[0145] In some embodiments, the beads have a diameter 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 greater than about 1500 μm. In some embodiments, the beads have a diameter of 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 beads have a diameter of about 3 μm to about 5 μm. In some embodiments, the beads have a diameter of at least about or at least 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 certain embodiments, the beads have a diameter of 4.5 μm or about 4.5 μm. In certain embodiments, the beads have a diameter of 2.8 μm or about 2.8 μm.

[0146] In some embodiments, the beads have a density of 0.001 g / cm 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 / cm 3 greater than 0.6 g / cm, greater than 0.7 g / cm 3 Greater than 0.8 g / cm 3 Greater than 0.9 g / cm 3 greater than 1 g / cm 3 Greater than 1.1 g / cm 3 Greater than 1.2 g / cm 3 Greater than 1.3 g / cm 3Greater than 1.4 g / cm 3 Greater than 1.5 g / cm 3 Greater than 2 g / cm 3 Greater than 3 g / cm 3 Greater than 4 g / cm 3 or greater than 5g / cm 3 In some embodiments, the beads have a density of about 0.001 g / cm 3 ~about 100 g / cm 3 , about 0.01 g / cm 3 ~about 50 g / cm 3 , about 0.1 g / cm 3 ~about 10 g / cm 3 , about 0.1 g / cm 3 ~about .5 g / cm 3 , about 0.5 g / cm 3 ~about 1 g / cm 3 , about 0.5 g / cm 3 ~Approx. 1.5 g / cm 3 , about 1 g / cm 3 ~Approx. 1.5 g / cm 3 , about 1 g / cm 3 ~about 2 g / cm 3 , or about 1 g / cm 3 ~about 5 g / cm 3 In some embodiments, the beads have a density of about 0.5 g / cm 3 , about 0.6 g / cm 3 , about 0.7 g / cm 3 , about 0.8 g / cm 3 , about 0.9 g / cm 3 , about 1.0 g / cm 3 , about 1.1 g / cm 3 , about 1.2 g / cm 3 , about 1.3 g / cm 3 , about 1.4 g / cm 3 , about 1.5 g / cm 3 , about 1.6 g / cm 3 , about 1.7 g / cm 3 , about 1.8 g / cm 3 , about 1.9 g / cm 3 , or approximately 2.0 g / cm 3In certain embodiments, the beads have a density of about 1.6 g / cm 3 In certain embodiments, the beads or particles have a density of about 1.5 g / cm 3 In certain embodiments, the particles have a density of about 1.3 g / cm 3 It has a density of

[0147] In certain embodiments, the plurality of beads has a uniform density, hi certain embodiments, the uniform density comprises a density standard deviation of less than 10%, less than 5%, or less than 1% of the average bead density.

[0148] In some embodiments, the beads have a m of about 0.001 per gram of particles. 2 (m 2 / g) ~ approx. 1,000 m 2 / g, approx. 0.010 m 2 / g~approx. 100 m 2 / g, approx. 0.1 m 2 / g ~ approx. 10 m 2 / g, approx. 0.1 m 2 / g ~ approx. 1 m 2 / g, approx. 1 m 2 / g ~ approx. 10 m 2 / g, approx. 10 m 2 / g~approx. 100 m 2 / g, approx. 0.5 m 2 / g ~ approx. 20 m 2 / g, approx. 0.5 m 2 / g ~ approx. 5 m 2 / g, or approximately 1 m 2 / g ~ approx. 4 m 2 In some embodiments, the particles or beads have a surface area of ​​about 1 m 2 / g ~ approx. 4 m 2 / g of surface area.

[0149] In some embodiments, the beads contain at least one substance on or near the bead surface that can be coupled, linked, or conjugated to an agent. In some embodiments, the beads are surface-functionalized, i.e., contain functional groups that can form covalent bonds with binding molecules, such as polynucleotides or polypeptides. In certain embodiments, the beads contain surface-exposed carboxyl, amino, hydroxyl, tosyl, epoxy, and / or chloromethyl groups. In certain embodiments, the beads contain surface-exposed agarose and / or sepharose. In certain embodiments, the bead surface contains an attached stimulating reagent that can bind to or attach to the binding molecule. In certain embodiments, the biomolecule is a polypeptide. In some embodiments, the beads contain surface-exposed protein A, protein G, or biotin.

[0150] In some embodiments, the beads respond to 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 certain embodiments, the beads do not exhibit any magnetic properties unless exposed to a magnetic field.

[0151] In certain embodiments, the beads comprise a magnetic core, a paramagnetic core, or a superparamagnetic core. In some embodiments, the magnetic core contains a metal. In some embodiments, the metal can be, but is not limited to, iron, nickel, copper, cobalt, gadolinium, manganese, tantalum, zinc, zirconium, or any combination thereof. In certain 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 an alloy (e.g., CoTaZn). In some embodiments, the magnetic core comprises one or more of ferrite, a metal, an 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 (FeO), maghemite (γFeO), or greigite (FeS). In some embodiments, the inner core comprises iron oxide (e.g., FeO).

[0152] In certain embodiments, the beads contain a magnetic, paramagnetic, and / or superparamagnetic core covered by a surface-functionalized coat or coating. In some embodiments, the coat can contain materials including, but not limited to, polymers, polysaccharides, silica, fatty acids, proteins, carbon, agarose, sepharose, or combinations thereof. In some embodiments, the polymer can be polyethylene glycol, polylactic-co-glycolic acid, polyglutaraldehyde, polyurethane, polystyrene, or polyvinyl alcohol. In certain embodiments, the outer coat or coating comprises polystyrene. In certain embodiments, the outer coating is surface-functionalized.

[0153] In some embodiments, the stimulation reagent comprises beads containing a metal oxide core (e.g., an iron oxide core) and a coat, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and the coat comprises at least one polysaccharide (e.g., aminodextran), at least one polymer (e.g., polyurethane), and silica. In some embodiments, the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In certain embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulation reagent comprises an anti-CD3 antibody, an anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulation reagent comprises an anti-biotin antibody. In some embodiments, the beads have a diameter of about 3 μm to about 10 μm. In some embodiments, the beads have a diameter of about 3 μm to about 5 μm. In certain embodiments, the beads have a diameter of about 3.5 μm.

[0154] In some embodiments, the stimulatory reagent comprises one or more agents attached to beads comprising a metal oxide core (e.g., an iron oxide inner core) and a coat (e.g., a protective coat), wherein the coat comprises polystyrene. In certain embodiments, the beads are monodisperse superparamagnetic beads comprising a superparamagnetic iron core, e.g., a core comprising magnetite (Fe3O4) and / or maghemite (γFe2O3)c, and a polystyrene coat or coating. In some embodiments, the beads are non-porous. In some embodiments, the beads contain a functionalized surface to which one or more agents are attached. In certain embodiments, the one or more agents are covalently bound to the beads at the surface. In some embodiments, the one or more agents comprise an antibody or antigen-binding fragment thereof. In some embodiments, the one or more agents comprise an anti-CD3 antibody and an anti-CD28 antibody. In certain embodiments, the beads have a weight of about 1.5 g / cm 3 density, and approximately 1 m 2 / g~4 m 2 / g. In certain embodiments, the beads have a diameter of about 4.5 μm and a surface area of ​​about 1.5 g / cm 3 In some embodiments, the beads are monodisperse superparamagnetic beads having an average diameter of about 2.8 μm and a density of about 1.3 g / cm 3 The beads are monodisperse superparamagnetic beads with a density of 0.015 μm.

[0155] The exposure time to the particles may be varied to achieve isolation of various T cell populations. For example, in one preferred embodiment, T cells are isolated by incubation with 3x28 beads, such as DYNABEADS® M-450 or Dynabeads® CD3 / CD28 CTS™, for a period sufficient for positive selection of the desired T cells. In one embodiment, the period is approximately 30 minutes. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours or longer. In one preferred embodiment, the incubation period is 24 hours. For isolation of T cells from cancer patients, the use of a longer incubation time, such as 24 hours, can increase cell yield.

[0156] When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" configuration) or to separate surfaces (i.e., in "trans" configuration). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to the cell surface, and the agent providing the primary activation signal is in solution or coupled to a surface. In a preferred embodiment, the two agents are immobilized on beads, either on the same bead, i.e., in "cis," or on separate beads, i.e., in "trans." As an example, the agent providing the primary activation signal is an anti-CD3 antibody, and the agent providing the costimulatory signal is an anti-CD28 antibody; both agents are co-immobilized on the same bead in equivalent molecular amounts. In one embodiment, a 1:1 ratio of each antibody bound to beads is used for CD4+ T cell expansion and T cell proliferation. In certain aspects of the invention, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In one particular embodiment, an increase of about 0.5 to about 3-fold is observed compared to the expansion observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibodies than anti-CD3 antibodies are bound to the particles, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the invention, the ratio of anti-CD28 antibodies to anti-CD3 antibodies bound to beads is greater than 2:1. In one particular embodiment, a CD3:CD28 ratio of 1:200 is used for antibodies bound to beads. In one particular embodiment, a CD3:CD28 ratio of 1:150 is used for antibodies bound to beads. In one particular embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used.In another embodiment, a 1:45 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:40 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:35 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:25 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:20 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:15 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:5 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:4 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound to beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to beads is used.

[0157] In some embodiments, active substance, for example, antibody, is added to culture or composition in soluble form.In some embodiments, one active substance is included in soluble form, and another active substance is included by coupling on solid support.In some aspects, when two or more active substances are coupled to solid support, the two active substances are coupled to the same support or particle, for example, by incubation with anti-CD3 / anti-CD28 beads, which are beads that contain antibodies that recognize CD3 and CD28.In other aspects, two or more active substances are coupled to different supports, such as different beads.For example, in some aspects, anti-CD3 beads and anti-CD28 beads are added to culture separately.

[0158] In some embodiments, the culture conditions include artificial antigen-presenting cells.For example, in some aspects, the surface is an artificial antigen-presenting cell loaded with one or more agents that can strengthen the signal through TCR complex, such as anti-CD3 antibody and / or anti-CD28 antibody.Exemplary antigen-presenting cells are genetically engineered cells, such as myeloid cells (e.g., K562 or U937) that are engineered to express Fc receptors, for example, CD32 intermediate affinity Fc receptors or CD64 high affinity Fc receptors.To deliver signals in culture, such cells can be loaded with anti-CD3 antibody and / or anti-CD28 antibody that are recognized by Fc receptors, and incubated with T cells. Exemplary artificial APCs, as well as ratios and methods of their use in culture conditions, are described, for example, in Suhoski et al., Molecular Therapy (2007) 155, 981-988; Thomas et al., Clin Immunol (2002) 105(3): 259-72; Kim et al., Nature Biotechnology 22, 403-410 (2004). In some embodiments, the culture conditions include antigen-presenting cells, such as PBMCs, loaded with an antigen, e.g., an antigen recognized by a TCR complex or other receptor on the cells to be transduced, e.g., T cells specific for a particular tumor antigen.

[0159] In some embodiments, preferential expansion or proliferation of naive-like T cells over non-naive-like T cells is achieved by using stimulatory conditions designed to induce a signal of a particular strength, such as, for example, a stimulatory or activation signal strength greater than a certain level to induce cell death of non-naive-like T cells. In some aspects, a stronger signal induces cell death of non-naive-like T cells of the input composition, whereas a weaker signal does not induce cell death and / or maintains the survival of non-naive-like T cells compared to a stronger signal. Thus, in some embodiments, a stronger signal preferentially activates or causes cell death of non-naive-like T cells.

[0160] In some aspects, preferential expansion or proliferation of naive-like T cells over non-naive-like T cells is achieved by a specific ratio of beads to cells. In some embodiments, any stimulatory condition that biases expansion or survival or naive-like T cells over non-naive-like T cells can be used. In some embodiments, the stimulatory condition includes, for example, incubation in the presence of a bead reagent containing a primary and / or secondary signal for T cell activation, such as an anti-CD3 / anti-CD28 bead reagent, provided in an amount that favors the proliferation and / or survival of naive-like cells and / or preferentially induces activation-induced cell death (AICD) of non-naive-like cells. In some cases, such bead reagents are incubated with cells at a bead-to-cell ratio of 1:1 or higher, which in some aspects can lead to greater AICD and an increased survival percentage of naive-like cells. See, e.g., Kalamazz et al., J. Immunother. (2004) 27(5):405-418, and U.S. Pat. Nos. 7,977,095 and 9,528,088. In one embodiment, the ratio is about 50:1 to about 5:1. In certain embodiments, the ratio is about 100:1 to about 1:1. In one embodiment, the ratio is at least about 45:1. In certain embodiments, the ratio is at least about 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some aspects, a bead-to-cell ratio of less than 5:1 or less than 3:1 is used. In one particular embodiment, the ratio is about 3:1.

[0161] In one embodiment of the provided method, the bead:cell ratio can be tailored to obtain a desired T cell phenotype. In one particular embodiment, the bead:cell ratio can be varied to selectively expand or delete a subset of T cells. In one embodiment, the particular bead:cell ratio used selectively induces cell death in non-naive-like T cells or cells derived from non-naive-like T cells of the input composition. In a further embodiment, the particular bead:cell ratio used selectively expands naive-like T cells or cells derived from naive-like T cells. In some embodiments, a particular ratio can be used as long as the desired expansion or deletion of a subset of T cells occurs. Thus, the compositions and methods described herein can be used to expand or delete specific populations of T cells for use in any of the various immunotherapy settings described herein.

[0162] Also provided are stimulation conditions suitable for incubating (e.g., stimulating) T cells. Suitable conditions for T cell culture include an appropriate medium (e.g., OpTmizer™ (Gibco), or minimal essential medium, or RPMI medium 1640, or X-vivo 15 (BioWhittaker)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), serum replacement products, or interleukin-2 (IL-2), insulin, or any other additives for cell growth. Media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, either serum-free or supplemented with an appropriate amount of serum (or plasma) or serum replacement or a defined set of hormones, and / or cytokines in amounts sufficient for T cell proliferation and expansion, with added amino acids and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures, not in the culture of cells that will be infused into the subject. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0163] In another embodiment, the time of exposure to a stimulator, such as anti-CD3 / anti-CD28 (i.e., 3x28)-coated beads, may be modified or tailored to achieve the desired T cell phenotype. Because expansion of helper T cells (TH) can improve or restore overall immune responsiveness, a larger population of TH cells, typically CD4+, as opposed to CD8+ cytotoxic or regulatory T cells, may be desirable. While many specific immune responses are mediated by CD8+ antigen-specific T cells, which can directly lyse or kill target cells, most immune responses require the help of CD4+ T cells that express important immune regulatory molecules, such as GM-CSF, CD40L, and IL-2. When CD4-mediated help is preferred, methods for protecting or enhancing the CD4:CD8 ratio, such as those described herein, can be significantly beneficial. Increasing the number of CD4+ T cells can increase the amount of CD40L expressed by cells introduced into the patient, potentially improving target cell visibility (improving APC function). Similar effects can be seen by increasing the number of infused cells expressing GM-CSF or IL-2, all of which are primarily expressed by CD4+ T cells. Similarly, utilizing a population of regulatory T cells, which can be generated and expanded using the methods described herein, may be desirable in certain applications (e.g., Autoimmun Rev. 2002 August; 1(4):190-7; Curr Opin Immunol. 2002 December; 14(6):771-8). Alternatively, in situations where less CD4 help is required and increased numbers of CD8+ T cells are desired, the XCELLERATE™ approach described herein can also be utilized, for example, by preselecting for CD8+ cells prior to stimulation and / or culture. Such situations may exist when increased levels of IFN-γ or increased cytolysis of target cells is preferred. The time and type of exposure to stimulators may also be modified, for example, to expand T cells with a desired TCR repertoire expressing a desired Vβ family gene.

[0164] Other conditions among the stimulatory conditions, in some embodiments, also include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0165] In some embodiments, cells or compositions are evaluated and / or adjusted during the incubation process. For example, evaluation and / or adjustment can be any time after the start of incubation or culture, for example, during incubation. Evaluation can include taking one or more measurements of the composition or the container containing cells, for example, evaluating cells for proliferation rate, degree of survival, phenotype, for example, expression of one or more surface or intracellular markers, for example, protein or polynucleotide, and / or evaluating the composition or container for temperature, medium components, oxygen or carbon dioxide content, and / or the presence or absence or amount or relative amount of one or more factors, agents, components, and / or cell types, including subtypes. Evaluation can also include evaluation of indicators or predictors of toxicity outcomes, for example, using in vitro or ex vivo as described herein.

[0166] In some aspects, the assessment is performed in an automated manner, e.g., using a device as described herein, and / or is pre-set to be performed at a specific time point during incubation. In some aspects, the outcome of the assessment indicates that an adjustment should be made.

[0167] Adjustments can include adjusting any cell culture factor or parameter, such as temperature, the length of time for which the incubation or step is performed (incubation duration), supplementation, the addition and / or removal of one or more components in the composition being incubated, such as medium or buffer or components thereof, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, or cells or cell types or populations of cells. In some aspects, the removal or addition of various components or other adjustments is performed in an automated manner, for example, using a device or system as described herein. In some embodiments, the system is programmed so that adjustments are automatically initiated based on certain readings from interim assessments. For example, in some cases, the system or device is programmed to perform one or more assessments at specific times; in such cases, the system or device can be further programmed so that a specific outcome of such assessment, such as a specific ratio of one cell type to another, triggers a specific adjustment, such as the addition of one or more of the cell types.

[0168] In some aspects, adjustments are made in one or more devices or systems as described herein by adding or removing components in a manner that does not disrupt the enclosed environment containing the cells and compositions, for example, by input and / or removal valves designed to add or remove components while maintaining sterility. Various adjustments of stimulation conditions that favor responses and / or outcomes in specific cell types are disclosed, for example, in U.S. Patent No. 8,617,884 and U.S. Patent Application Publication No. 20030235908 A1.

[0169] In some embodiments, the cells are incubated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, or about 14 days, or for 1, 2, 3, 4 or more weeks or about 1, about 2, about 3, about 4 or more weeks, either in total or prior to manipulation. In some examples, incubation is carried out for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, or for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 days.

[0170] In some aspects, the incubation is carried out according to techniques such as those described in U.S. Pat. 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.

[0171] In some embodiments, the stimulatory conditions include adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., so that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to the addition of the population of T cells.

[0172] In some embodiments, the stimulatory conditions include a temperature suitable for the proliferation of human T lymphocytes, e.g., at least about 25°C, generally at least about 30°C, and generally at or about 37°C. Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some aspects, the LCL feeder cells are provided in any suitable amount, such as at a ratio of LCL feeder cells to primary T lymphocytes of at least about 10:1.

[0173] In some embodiments, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones can be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0174] Incubation and / or manipulation may be performed in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other vessel for culturing or growing cells.

[0175] Also provided are culture starting compositions, e.g., those containing T cells, e.g., human primary T cells, and stimulatory conditions, e.g., various agents at concentrations / ratios designed for preferential activation or expansion of non-naive T cells, used in the methods.

[0176] In some embodiments, when cells are engineered, for example to introduce an engineered antigen receptor, incubation in the presence of one or more stimulatory agents continues throughout the engineering step.

[0177] Bead-to-cell ratios ranging from 1:500 to 500:1 and any integer value therebetween may be used to stimulate T cells or other target cells. In some cases, the bead-to-cell ratio may depend on the particle size relative to the target cells. For example, small beads can only bind a few cells, while larger beads can bind many. In certain embodiments, the cell-to-particle ratio ranges from 1:100 to 100:1 and any integer value therebetween, and in further embodiments, the ratio includes 1:50 to 50:1 and any integer value therebetween. In other embodiments, cell-to-particle ratios ranging from 1:9 to 9:1 and any integer value therebetween may also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 coupled particles to T cells resulting in T cell stimulation can vary as described herein, but certain preferred values ​​include at least 1:150, 1:125, 1:100, 1:75, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2.5, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 beads per T cell. In one particular embodiment, the preferred ratio of beads to cells is 3:1. In some cases, the ratio of beads to cells is 1:3.

[0178] In further embodiments, the particle-to-cell ratio can vary depending on the day of stimulation. For example, in one embodiment, the particle-to-cell ratio is 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at a final ratio of 1:1 to 1:10 (based on the cell number on the day of addition). In another embodiment, the particle-to-cell ratio is at least about 1:2.5 on the first day, and additional particles are added to the cells at about 1:10, 1:25, 1:50, or 1:100 on the fifth day, 1:10, 1:25, 1:50, or 1:100 on the seventh day, and 1:10, 1:25, 1:50, or 1:100 on the ninth day. In one particular embodiment, the particle-to-cell ratio is 1:1 on the first day of stimulation, and is adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added daily or every other day to a final ratio of 1:1 on day 1 and 1:5 on days 3 and 5 of stimulation. In another embodiment, the particle to cell ratio is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation. In another embodiment, particles are added daily or every other day to a final ratio of 1:1 on day 1 and 1:10 on days 3 and 5 of stimulation. In some aspects, various other ratios may be suitable for use in the present invention. In particular, the ratio will vary depending on particle size and cell size and cell type.

[0179] One aspect of the present invention relates to the observation that using different bead (e.g., anti-CD3 / anti-CD28 bead reagent) to cell ratios can result in different outcomes in terms of expanding antigen-specific T cells. In particular, the bead to cell ratio can be varied to selectively expand or delete antigen-specific or antigen-experienced (e.g., memory, effector, or activated) T cells, as opposed to other cell types such as naive or naive-like T cells. In one embodiment, the specific bead to cell ratio used selectively deletes antigen-specific T cells. Specifically, a bead to cell ratio of 1:1 or greater, and / or a high bead to cell ratio, such as at least or about 3:1, 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and higher, can induce the deletion of antigen-specific T cells. Without being bound by theory, it is believed that antigen-specific T cells are sensitized to further stimulation. Thus, in some embodiments, the strength of the activation signal delivered to T cells via a reagent or composition can affect the expansion and / or death of one or more specific T cell subsets. In some aspects, selective expansion of memory T cells (antigen-specific T cells) can occur with signals via TCRs and / or co-receptors that are weaker than others, whereas in some embodiments, selective deletion of memory T cells and / or other antigen-experienced T cells can occur after incubation with a reagent that delivers a relatively stronger signal while sparing naive cells. Among other factors, the amount of CD3 / TCR (and CD28) receptors bound by the ligand can determine or contribute to the determination of signal strength in some aspects. Thus, stimulation at a high bead-to-cell ratio in some situations can provide a high concentration of stimulating antibody (i.e., a "strong" signal), resulting in overstimulation of antigen-specific T cells, causing their death by either apoptosis or other mechanisms.In this regard, the bead compositions described herein may function as pro-apoptotic compositions in some situations and / or with certain compositions. In some aspects, the signal should not be so strong that it also kills naive-like T cells, e.g., by activation-induced cell death. In some embodiments, the ratio of stimulatory bead reagents, e.g., anti-CD3 / anti-CD28 bead reagents, is less than 10:1.

[0180] Further in this regard, in certain embodiments, such reagents or compositions (e.g., surfaces having attached reagents that stimulate cell surface moieties, such as the bead compositions described herein) used as pro-apoptotic conditions, for example, for a particular cell type, are used to expand the population of remaining cells for use in any of the various immunotherapy settings described herein. In further embodiments, the particular bead-to-cell ratio used selectively expands naive-like T cells. The particular ratio may be adjusted to produce the desired expansion or deletion of specific T cells. Thus, the compositions and methods described herein can be used to expand or delete specific populations of T cells in a population of input compositions for use in any of the various immunotherapy settings described herein.

[0181] Further in this regard, in certain embodiments, for example, for a particular cell type, the same reagent or substance or composition (e.g., a surface having attached thereto a reagent that stimulates cell surface moieties, such as the bead compositions described herein) used as a pro-apoptotic composition is used to expand the population of remaining cells for use in any of the various immunotherapy settings described herein. Using a lower bead-to-cell ratio provides a stimulatory signal to antigen-specific T cells that does not overstimulate but induces rapid proliferation of these cells. In further embodiments, the particular bead-to-cell ratio used selectively expands antigen-specific T cells. In some aspects, any ratio can be used as long as the desired expansion or deletion occurs. Thus, the compositions and methods described herein can be used to expand or delete specific populations of T cells for use in any of the various immunotherapy settings described herein.

[0182] Using certain methodologies, it may be advantageous to maintain long-term stimulation of a population of T cells after initial activation and stimulation by separating T cells from stimulation after a period of about 2 to about 14 days. The rate of T cell proliferation is monitored periodically (e.g., daily) by examining the size of the T cells, for example, with a Coulter Counter, or by measuring their volume. In this regard, resting T cells have a mean diameter of about 6.8 microns; upon initial activation and stimulation, in the presence of a stimulatory ligand, the T cell mean diameter increases to more than 12 microns by day 4 and begins to decrease by about day 6. When the mean T cell diameter decreases to approximately 8 microns, the T cells may be reactivated and restimulated to induce further T cell proliferation. Alternatively, the rate of T cell proliferation and the time to T cell restimulation can be monitored by assaying for the presence of cell surface molecules, such as CD154, CD54, CD25, CD137, and CD134, that are induced on activated T cells.

[0183] To induce long-term stimulation of populations of CD4+ and / or CD8+ T cells, it may be necessary to reactivate and restimulate T cells several times with stimulators, such as anti-CD3 and anti-CD28 antibodies (e.g., B-T3, XR-CD28 (Diaclone, Besancon, France)), to generate populations of CD4+ or CD8+ cells that are expanded in number by about 10 to about 1,000-fold from the original T cell population. For example, in one embodiment of the present invention, T cells are stimulated as described two or three times. In a further embodiment, T cells are stimulated as described four or five times. Using the methodology of the present invention, it is possible to achieve T cell numbers that are about 100 to about 100,000-fold higher than pre-stimulation, with increased polyclonality. Furthermore, T cells expanded by the methods of the present invention secrete substantial levels of cytokines (e.g., IL-2, IFN-γ, IL-4, GM-CSF, and TNF-α) into the culture supernatant. For example, compared with stimulation with IL-2, CD4+ T cells expanded by the use of anti-CD3 and anti-CD28 costimulation secrete high levels of GM-CSF and TNF-α into the culture medium. These cytokines can be purified from the culture supernatant, or the supernatant can be used directly to maintain the cells in culture. Similarly, T cells expanded by the methods of the present invention can be administered together with the culture supernatant and cytokines to support cell growth in vivo.

[0184] In one embodiment, T cell stimulation is performed with, for example, anti-CD3 and anti-CD28 antibodies co-immobilized on beads (3x28 beads) for a period sufficient for the cells to return to a quiescent state (low or no proliferation) (approximately 8-14 days after initial stimulation). The stimulation signal is then removed from the cells, and the cells are washed and infused back into the patient. Cells at the end of the stimulation phase are rendered "super-inducible" by the methods of the present invention, as demonstrated by their ability to respond to antigen and their ability to exhibit a memory-like phenotype, as evidenced by the examples. Thus, upon in vivo restimulation after infusion, either exogenously or with antigen, activated T cells exhibit a robust response characterized by unique phenotypic characteristics, such as sustained CD154 expression and increased cytokine production.

[0185] In a further embodiment of the present invention, cells, such as T cells, are mixed with agent-coated or conjugated beads, the beads and cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, the agent-coated or conjugated beads and cells are not separated prior to culture, but are cultured together. In a further embodiment, the beads and cells are first concentrated by applying force, resulting in cell surface moiety ligation, thereby inducing polarization of cell stimulation and / or activation signals.

[0186] As an example, if T cells are the target cell population, cell surface moieties can be ligated by contacting prepared T cells with paramagnetic beads (3 x 28 beads) bearing anti-CD3 and anti-CD28 antibodies. In one embodiment, cells (e.g., 10-10 T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a 1:1 ratio) are combined in a buffer, preferably PBS (free of divalent cations such as calcium and magnesium). In some aspects, any cell concentration may be used. For example, target cells may be very rare in a sample, constituting only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the target cells of interest. Therefore, any cell number is within the scope of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum cell-particle contact. For example, in one embodiment, a concentration of about 2 billion cells / mL is used. In another embodiment, more than 100 million cells / mL is used. In a further embodiment, a cell concentration of 10 million cells / mL, 15 million cells / mL, 20 million cells / mL, 25 million cells / mL, 30 million cells / mL, 35 million cells / mL, 40 million cells / mL, 45 million cells / mL, or 50 million cells / mL is used. In yet another embodiment, a cell concentration of 75 million cells / mL, 80 million cells / mL, 85 million cells / mL, 90 million cells / mL, 95 million cells / mL, or 100 million cells / mL is used. In a further embodiment, a concentration of 125 million cells / mL or 150 million cells / mL can be used. Using a high concentration can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, populations of such cells that may have therapeutic value and would be desirable to obtain.For example, using a high concentration of cells allows for more efficient selection of CD8+ T cells, which normally have weaker CD28 expression.

[0187] In related embodiments, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of T cells and particles, the interaction between the particles and cells is minimized. This allows for the selection of a large number of cells expressing the desired antigen bound to the particles. For example, CD4+ T cells express higher levels of CD28 than CD8+ T cells at diluted concentrations, and are more efficiently captured and stimulated. In one embodiment, the concentration of cells used is about 5 x 10 6 In other embodiments, the concentration used is about 1 x 10 5 / mL to 1×106 / mL, and any integer value therebetween.

[0188] The buffer in which the cells are suspended may be any suitable for the particular cell type. When using a particular cell type, the buffer may contain other components necessary to maintain cell integrity during the process, such as 1-5% serum. In another embodiment, the cells and beads may be combined in cell culture medium. The cells and beads may be mixed for a period ranging from 1 minute to several hours, for example, by rotation, stirring, or any other means of mixing. The container of beads and cells is then concentrated by force, for example, by placing it in a magnetic field. The medium and unbound cells are removed, and the cells attached to the beads or other surfaces are washed, for example, by pumping via a peristaltic pump, and then resuspended in a medium appropriate for cell culture.

[0189] In one embodiment of the present invention, the mixture may be cultured for 30 minutes to several hours (approximately 3 hours), up to approximately 14 days, or any integer value of hours or minutes therebetween. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the present invention, the beads and T cells are cultured together for approximately 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. As noted above, several cycles of stimulation may also be desirable, allowing the T cell culture time to be 60 days or longer. Suitable conditions for T cell culture include an appropriate medium (e.g., minimal essential medium, or RPMI medium 1640, or X-vivo 15 (BioWhittaker)), which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), or interleukin-2 (IL-2), insulin, or any other additives for cell growth. Culture media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in amounts sufficient for T cell proliferation and expansion, with added amino acids and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0190] In one embodiment of the present invention, the bead:cell ratio can be tailored to obtain a desired T cell phenotype. In one particular embodiment, the bead:cell ratio can be varied to selectively expand or delete antigen-specific (memory) T cells. In one embodiment, the particular bead:cell ratio used selectively deletes antigen-specific T cells. In a further embodiment, the particular bead:cell ratio used selectively expands antigen-specific T cells. In some aspects, any ratio can be used as long as the desired expansion or deletion of antigen-specific T cells occurs. Thus, the compositions and methods described herein can be used to expand specific populations of T cells or to delete specific populations of T cells for use in any of the various immunotherapy settings described herein.

[0191] In another embodiment, the time of exposure to a stimulator, such as anti-CD3 / anti-CD28 (i.e., 3x28)-coated beads, may be modified or tailored to obtain a desired T cell phenotype. Alternatively, a desired population of T cells can be selected using any number of selection techniques prior to stimulation. Because expansion of helper T cells (TH) can improve or restore overall immune responsiveness, a larger population of TH cells, typically CD4+, may be desirable, as opposed to CD8+ cytotoxic T cells or regulatory T cells. While many specific immune responses are mediated by CD8+ antigen-specific T cells, which can directly lyse or kill target cells, most immune responses require the help of CD4+ T cells that express important immune regulatory molecules, such as GM-CSF, CD40L, and IL-2. When CD4-mediated help is preferred, methods for protecting or enhancing the CD4:CD8 ratio, such as those described herein, may be significantly beneficial. Increasing the number of CD4+ T cells can increase the amount of CD40L expressed by cells introduced into the patient, potentially improving target cell visibility (improving APC function). A similar effect can be seen by increasing the number of infused cells expressing GM-CSF or IL-2, all of which are primarily expressed by CD4+ T cells. Similarly, utilizing a population of regulatory T cells, which can be generated and expanded using the methods described herein, may be desirable in certain applications (e.g., Autoimmun Rev. 2002 August; 1(4):190-7; Curr Opin Immunol. 2002 December; 14(6):771-8). Alternatively, in situations where less CD4 help is required and increased numbers of CD8+ T cells are desired, the XCELLERATE™ approach described herein can also be utilized, for example, by preselecting for CD8+ cells prior to stimulation and / or culture. Such situations may exist when increased levels of IFN-γ or increased cytolysis of target cells is preferred.For example, the time and type of exposure to stimuli may also be modified to expand T cells expressing desired Vβ family genes and having a desired TCR repertoire.

[0192] The exposure time to particles may be varied to achieve isolation of various T cell populations. For example, in one preferred embodiment, T cells are isolated by incubation with 3x28 beads, such as DYNABEADS® M-450, for a period sufficient for positive selection of the desired T cells. In one embodiment, the period is approximately 30 minutes. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours or longer. In one preferred embodiment, the incubation period is 24 hours or approximately 24 hours. For isolation of T cells from cancer patients, the use of a longer incubation time, such as at least 24 hours or at least approximately 24 hours, can increase cell yield.

[0193] In certain embodiments, the total stimulation and / or augmentation time can be 2 to 15 days, 2 to 12 days, 2 to 12 days, 2 to 8 days, 2 to 6 days, 2 to 4 days, 4 to 12 days, 4 to 10 days, 4 to 8 days, 4 to 6 days, 6 to 12 days, 6 to 10 days, 6 to 8 days, 8 to 12 days, 8 to 10 days, or 10 to 12 days, all ranges inclusive. In some embodiments, the cells are incubated, and / or incubated with a stimulatory reagent (e.g., a particle as described herein) for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or for longer than 14 days. When T cell stimulation is performed for a shorter period of time, the population of T cells may not increase in number, may not increase dramatically in number, may decrease in number, or may remain the same in number, but the population will provide more robust and healthy activated T cells that can continue to expand in vivo and / or more closely resemble the natural effector T cell pool. For example, when T cell stimulation is performed for a shorter period of time, the population of T cells may contain a greater percentage and / or proportion of naive or naive-like T cells, or engineered T cells, compared to a parallel process in which T cell stimulation is performed for a longer period of time. In some embodiments, a shorter period of time may be characterized by a total incubation time, in some embodiments with the stimulator, of less than or less than about 6 days, less than or less than about 5 days, less than or less than about 4 days, less than or less than about 3 days, or less than or less than about 2 days. Because the availability of T cell help is often a rate-limiting factor in antibody responses to protein antigens, the ability to selectively expand or infuse into a subject a CD4+-enriched population of T cells is extremely beneficial.A further benefit of such enriched populations is readily apparent in that activated helper T cells, which recognize antigens presented by B lymphocytes, deliver two types of stimuli: physical contact and cytokine production, which result in B cell proliferation and differentiation.

[0194] In some cases, the stimulatory conditions do not include culture components or agents that are supplemented to protect specific subsets of T cells, such as non-naive-like T cells. Thus, in some cases, removing a component or agent from the stimulatory culture can contribute to the elimination of non-naive-like T cells. In some embodiments, the stimulatory conditions do not include agents, such as N-acetylcysteine, that can be used to regulate and / or fine-tune the strength of TCR / CD3 signaling. In some aspects, such agents are removed or reduced in amounts / ratios that increase the strength of the activation signal. In some aspects, the stimulatory conditions are implemented, or additionally implemented, by excluding or reducing the concentration of culture reagents that are known or likely to reduce AICD and / or promote the survival of older cells, such as non-naive cells. In some cases, the stimulatory conditions do not include N-acetylcysteine ​​or include N-acetylcysteine ​​in reduced amounts or concentrations. In some cases, the stimulatory conditions do not include recombinant IL-7 and / or recombinant IL-15, or include reduced amounts or concentrations of recombinant IL-7 or IL-15. In some embodiments, culture additives (e.g., toxins attached to CD45RO) that additionally aid or facilitate the removal of non-naive cells may be included.

[0195] C. Stimulated Composition Also provided are stimulated compositions produced by the described incubation (e.g., stimulation) methods. In some embodiments, the cells of the stimulated composition are further manipulated after incubation with the input composition, for example, to introduce a genetically engineered antigen receptor. In some embodiments, the method further comprises incubating in the presence of one or more stimulatory substances during the genetic engineering stage.

[0196] Cells of the stimulated composition that have not undergone apoptosis using the incubation (e.g., stimulation) methods described herein can increase the polyclonality of the remaining T cell population, as measured by the breadth of the population's response to a given antigen. Restoration or increase in polyclonality can be measured, for example, by determining the breadth of the response to a particular antigen of interest by measuring the number of different epitopes recognized by the antigen-specific cells. This can be performed using standard techniques for generating and cloning antigen-specific T cells in vitro.

[0197] In some embodiments of the methods provided herein, the culture conditions preferentially induce the expansion, proliferation, and / or survival of non-naive-like T cells relative to naive-like T cells. Preferential expansion, proliferation, and / or survival of a first cell type or population compared to a second cell type or population means that the relative expansion, proliferation, and / or survival is greater for the first type or population than for the second type or population. This can include scenarios in which a greater percentage of the first cell type or population expands, proliferates, and / or survives, and / or the extent to which cells expand, proliferate, and / or survive (e.g., the overall or average extent among the populations or types of cells) is greater for the first population or type than for the second population or type.

[0198] In some embodiments, preferential expansion, proliferation, and / or survival is expressed by comparing the percentage of naive-like cells in the input composition with the percentage of engineered cells in the stimulated composition that are derived from naive-like cells in the original input composition. In some instances, the percentage of engineered cells in the stimulated composition that are derived from naive-like cells in the original input composition is generally greater than the former under the stimulation conditions described herein. For example, in one embodiment, the percentage of cells in the stimulated composition that are derived from naive-like cells in the original input composition is greater than the percentage of naive cells in the culture initiation composition. In one aspect, the input composition (or the T cells therein) contains 70% or about 70% naive-like T cells and 30% non-naive-like T cells, whereas more than 50%, e.g., at least 70%, of the engineered cells in the resulting stimulated composition are derived from naive-like cells in the input composition. In some cases, using other conditions, e.g., stimulation with an agent that induces a strong signal through the TCR complex, will result in fewer than 50%, e.g., at or about 5-10%, of cells in the stimulated composition being of non-naive-like origin. In some embodiments, the stimulation conditions induce a strong signal that induces activation-induced cell death in the non-naive-like T cells of the input composition.

[0199] In some embodiments, the percentage of naive-like T cells or cells derived from naive-like T cells of the input composition in the stimulated composition is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, or more than about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, or about 100-fold compared to the input composition. In some cases, the ratio of cells derived from naive-like T cells in the input composition compared to cells derived from non-naive-like T cells in the input composition, or the ratio of naive-like T cells compared to non-naive-like T cells in the stimulated composition, is increased by more than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold relative to the ratio of naive-like T cells compared to non-naive-like T cells in the input composition. In some embodiments, the stimulated composition comprises greater than 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% cells derived from the naive-like T cells of the input composition.

[0200] In some embodiments using the methods for stimulating T cells described herein, a greater percentage of naive-like T cells in the input composition are induced to proliferate and / or be activated compared to non-naive-like T cells. In some aspects, the stimulated composition resulting from the methods for stimulating described herein contains less than 10% cells derived from non-naive-like T cells. In some cases, the stimulated composition contains less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% cells derived from non-naive T cells. In some embodiments, a greater percentage of naive-like T cells in the input composition have divided compared to the percentage of non-naive-like T cells in the input composition 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the start of the incubation. In some cases, the stimulating conditions induce cell death. In certain instances, the stimulatory conditions of the method induce activation of non-naive-like T cells, thereby inducing activation-induced cell death (AICD).

[0201] In some embodiments, the methods include stimulatory conditions that can induce proliferation of a greater percentage of cells of the naive-like T cell population compared to non-naive-like T cells on days 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after initiation of incubation under the conditions.

[0202] In some embodiments, the stimulation conditions result in a stimulated composition, where the stimulation conditions preferentially induce the expansion of naive-like T cells, such that a target amount of T cells in the stimulated composition is derived from naive-like T cells in the input composition. In some embodiments, the stimulated composition is further adjusted to achieve a preferred CD4:CD8 ratio of T cells. In some cases, specific cell populations can be removed from the stimulated composition to achieve a preferred CD4:CD8 ratio.

[0203] In some embodiments, for example, the stimulatory conditions under which the input composition is incubated preferentially induce the expansion, proliferation, and / or survival of naive-like T cells or a subset thereof compared to non-naive-like T cells or a subset thereof. In some aspects, the stimulatory conditions strongly activate the non-naive-like T cells of the input composition, thereby activating cell death, particularly in the non-naive-like T cells of the input composition. In some aspects, this preferentially favors the survival of naive-like T cells. In some aspects, such stimulatory conditions result in reduced levels of toxicity and / or toxicity-related outcomes or symptoms after the cells and compositions produced by the method are administered to a subject.

[0204] Whether a particular response is induced or preferentially induced in one population over another can be measured by various methods. For example, whether cells are induced to enter the cell cycle can be measured by flow cytometry-based methods, including the use of dyes and other agents, such as CFSE and other intercalating agents to evaluate cell division and subsequent flow cytometry evaluation, the evaluation of the incorporation of tritium (H3)-labeled thymidine and similar agents, and / or cell count. Comparisons can be made by evaluating various pure test populations, for example, by separately comparing naive T cell populations and non-naive T cell populations under specific conditions. Activation can be measured, for example, by the secretion of various cytokines and / or the upregulation or expression of various activation markers, including CD25, CD69, and / or cell size (e.g., forward scatter as measured by flow cytometry). Viability and / or apoptosis can be assessed by a variety of methods, including flow cytometry, uptake of various dyes including propidium iodide, and staining with agents such as Annexin V and similar agents.

[0205] In some embodiments, the percentage of naive-like T cells in the input composition is less than the percentage of T cells in the stimulated composition that are derived from the naive-like T cells in the input composition.In some embodiments, the method provided herein produces a greater percentage of transfected cells that are naive-like T cells in the input composition or derived from the proliferation thereof compared to the non-naive-like T cells in the input composition.Various adjustments of stimulation conditions that favor the response and / or outcome of specific cell types are disclosed, for example, in U.S. Patent No. 8,617,884 and U.S. Patent Application Publication No. 20030235908 A1.

[0206] In some embodiments, the stimulated composition contains cells that express or are derived from cells that express specific markers of naive-like T cells. For example, the stimulated composition produced by the provided methods is derived from a population of cells that are surface-positive for a T cell activation marker selected from the group consisting of CD27, CD28, CD45RA, and CCR7. In some cases, the stimulated composition produced by the provided methods is derived from a population of cells that are surface-negative for a T cell activation marker such as CD62L. In some aspects, the cells of the stimulated composition are derived from cells that are surface-negative or surface-negative for CD56 and / or CD45RO. In some specific embodiments, the stimulated composition produced by the provided methods is derived from a population of cells that are CD27+, CD45RA+, CD45RO-, and CCR7+. In some cases, the cells of the stimulated composition are derived from cells that are negative or negative for intracellular expression of cytokines such as IL-2, IFN-γ, IL-4, and / or IL-10. In some further examples, the cells of the stimulated composition are negative or derived from cells that are negative for expression of the markers CD25 and / or perforin. In some cases, the cells of the stimulated composition are negative for expression of the markers CD95 lo or CD95 loIt originates from cells that are

[0207] In some cases, the stimulated composition contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% cells derived from T cells that are surface positive for T cell activation markers such as CD27, CD28, CD45RA, and CCR7, and surface negative for CD62L. In some embodiments, the stimulated composition contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% cells derived from T cells that are surface negative for CD56 and / or CD45RO. In some aspects, the stimulated composition contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% cells derived from T cells that are surface negative for CD45RO and cell surface positive for CD27, CD45RA, and CCR7. In some instances, the stimulated composition contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% cells derived from T cells that are negative for intracellular expression of cytokines such as IL-2, IFN-γ, IL-4, and IL-10. In some aspects, the stimulated composition contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% cells derived from T cells that are negative for expression of the markers CD25 and / or perforin. In some instances, the stimulated composition contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% cells derived from T cells that are negative for expression of the markers CD25 and / or perforin. lo The present invention contains cells derived from T cells that are

[0208] In certain embodiments, the stimulated composition is more polyclonal or multiclonal compared to the input composition. In some embodiments, the stimulated composition is more diverse compared to the input composition. In some embodiments, this increase in polyclonality includes a shift in the T cell population from monoclonal to oligoclonal or polyclonal, as measured by the Vβ, Vα, Vγ, or Vδ spectrotype profile of at least one Vβ, Vα, Vγ, or Vδ family gene. Stimulation and activation of the remaining cells that survive, expand, or proliferate using the provided methods can increase the polyclonality of the remaining T cells of the stimulated composition, as measured by the breadth of the population's response to a given antigen. Restoration or increase in polyclonality of the stimulated composition can be measured by determining the breadth of the response to a particular antigen of interest, for example, by measuring the number of different epitopes recognized by the antigen-specific cells. This can be performed using standard techniques for generating and cloning antigen-specific T cells in vitro.

[0209] II. Methods for Genetically Manipulating Cells In some embodiments, the method further comprises introducing an engineered recombinant receptor, e.g., a chimeric receptor such as a chimeric antigen receptor (CAR), into the composition of stimulated T cells, generating an output composition comprising T cells expressing the engineered recombinant receptor. In some cases, incubating the input composition under stimulatory conditions occurs before, during, and / or after introducing a nucleic acid encoding the engineered recombinant receptor. In some examples, the introduction is by transduction. In some embodiments, the provided method produces a stimulated composition that can be homogeneously transduced. In some aspects, the nucleic acid contains a viral vector. In some cases, the viral vector is a retroviral vector. In some examples, the viral vector is a lentiviral vector or a gammaretroviral vector. In some embodiments, the method comprising introducing is performed in vitro or ex vivo.

[0210] Thus, the methods provided herein include one or more steps for preparing cells for genetic manipulation. In certain embodiments, the one or more steps include isolating cells from a biological sample, stimulating an input composition of cells, and preparing a composition of cells to be genetically manipulated. Also provided are compositions containing and / or enriched in a population of such cells, e.g., cells expressing a recombinant receptor, e.g., a chimeric receptor, in which the cells account for at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent of the total cells in the composition, or a specific type of cell, such as T cells, CD8+ cells, or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, e.g., adoptive cell therapy. Also provided are methods for manipulating, producing, or generating such cells, methods for administering cells and compositions to a subject, e.g., a patient, and methods for detecting, selecting, isolating, or separating such cells. Thus, genetically engineered cells expressing a recombinant receptor, e.g., a CAR, are provided.

[0211] In some embodiments, the cells contain one or more nucleic acids introduced through genetic engineering, thereby expressing recombinant or engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or sample obtained from the cell, e.g., obtained from another organism or cell that is not normally found in the cell being engineered and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, and is not found in nature, including, for example, a chimeric combination of nucleic acids encoding various domains from multiple different cell types.

[0212] A. Genetic manipulation 1. Recombinant antigen receptor In some embodiments, engineered cells, e.g., T cells, are provided that express a CAR with specificity for a particular antigen (or marker or ligand), e.g., an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0213] In certain embodiments, the recombinant receptor, e.g., the chimeric receptor, contains an intracellular signaling region, which includes a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), such as a cytoplasmic (intracellular) region that can induce a primary activation signal in a T cell, e.g., a cytoplasmic signaling domain of a T cell receptor (TCR) component (e.g., the cytoplasmic signaling domain of the ζ chain of the CD3 zeta (CD3ζ) chain, or a functional variant or signaling portion thereof), and / or includes an immunoreceptor tyrosine-based activation motif (ITAM).

[0214] In some embodiments, the chimeric receptor further comprises an extracellular ligand-binding domain that specifically binds to a ligand (e.g., antigen) antigen. In some embodiments, the chimeric receptor is a CAR that comprises an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand, such as an antigen, is a protein expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which is recognized on the cell surface in association with a major histocompatibility complex (MHC) molecule, like a TCR.

[0215] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells are described in, for example, International Patent Application Publication Nos. WO2000 / 14257, WO2013 / 126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, 20130149337, U.S. Patent Nos. 6, 4 51,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP 2537416; and / or Sadelain et al., Cancer Discov. 2013 April;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr.Opin.Immunol., 2012 October;24(5):633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some aspects, the antigen receptor includes the CAR described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668 A1.Examples of CARs include those disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Pat. No. 7,446,190, U.S. Pat. No. 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Pat. No. 7,446,190, and U.S. Pat. No. 8,389,282.

[0216] In some embodiments, CARs are constructed with specificity for a particular antigen (or marker or ligand) intended to induce a dampening response, such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen expressed on a normal or non-diseased cell type. Thus, a CAR typically comprises, in its extracellular portion, one or more antigen-binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, a CAR comprises a variable heavy chain (VH) of a monoclonal antibody (mAb). H ) and variable light chain (V L ), and contain one or more antigen-binding portions of an antibody molecule, such as single-chain antibody fragments (scFv) derived from

[0217] In some embodiments, the antibody or its antigen-binding portion is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among antigen receptors, there are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, CARs containing antibodies or antigen-binding fragments that exhibit TCR-like specificity for peptide-MHC complexes can also be called TCR-like CARs. In some embodiments, the extracellular antigen-binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked to one or more intracellular signaling components via a linker and / or transmembrane domain in some aspects. In some embodiments, such molecules can typically mimic or approximate the signaling through natural antigen receptors, such as TCRs, and optionally the signaling through such receptors in combination with costimulatory receptors.

[0218] In some embodiments, the recombinant receptor, for example, a chimeric receptor (e.g., CAR), comprises a ligand-binding domain that binds to, for example, specifically binds to, an antigen (or ligand). Some of the antigens targeted by chimeric receptors are expressed in association with the disease, condition, or cell type targeted through adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including blood cancers, immune system cancers, for example, B, T, and myeloid leukemias, lymphomas, and lymphomas, such as multiple myeloma, leukemias, and / or myelomas.

[0219] In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or overexpressed on disease or condition cells, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0220] In some embodiments, the CAR contains an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an antigen, e.g., an intact antigen, expressed on the surface of a cell.

[0221] In certain embodiments, the antigen is αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9; also known as CAIX or G250), cancer-testis antigen, cancer / testis antigen 1B (CTAG; also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epidermal growth factor receptor type III mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like 5 (FCRL5;Fc receptor homolog 5 or also known as FCRH5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor α, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erbB2), Her3 (erb-B 3), Her4 (erb-B4), erbB dimer, human high-molecular-weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor α (IL-22Rα), IL-13 receptor α2 (IL-13Rα2), kinase insert domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat Melan-containing 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligand, melan-A (MART-1), neural cell adhesion molecule (NCAM), fetal tumor antigen, melanoma preferentially expressed antigen ( PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein G (TPBG; also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1; also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2;The receptor may be a dopachrome tautomerase, dopachrome delta-isomerase, or DCT (also known as vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms' tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or a biotinylated molecule, and / or a molecule expressed by HIV, HCV, HBV, or other pathogens. In some embodiments, the antigen targeted by the receptor includes an antigen associated with a B-cell malignancy, such as any of a number of known B-cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b, or CD30.

[0222] In some embodiments, the antigen is or comprises a pathogen-specific or pathogen-expressed antigen, hi some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and / or a parasitic antigen.

[0223] In some embodiments, the antigen or antigen-binding domain is CD19. In some embodiments, the scFv comprises VH and VL derived from an antibody or antibody fragment specific to CD19. In some embodiments, the antibody or antibody fragment that binds to CD19 is a mouse-derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, such as that described in US Patent Application Publication No. 2016 / 0152723.

[0224] In some embodiments, the scFv is derived from FMC63. FMC63 generally refers to a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al. (1987). Leucocyte typing III. 302). The FMC63 antibody comprises CDRH1 and H2 as set forth in SEQ ID NO:38, 39, and CDRH3 as set forth in SEQ ID NO:40 or 54, respectively, and CDRL1 as set forth in SEQ ID NO:35, and CDR L2 36 or 55, and CDR L3 sequence 37 or 34. The FMC63 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO:41. H ), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO:42 L In some embodiments, the scFv comprises a variable light chain containing the CDRL1 sequence of SEQ ID NO:35, the CDRL2 sequence of SEQ ID NO:36, and the CDRL3 sequence of SEQ ID NO:37, and / or a variable heavy chain containing the CDRH1 sequence of SEQ ID NO:38, the CDRH2 sequence of SEQ ID NO:39, and the CDRH3 sequence of SEQ ID NO:40. In some embodiments, the scFv comprises the variable heavy chain region of FMC63 shown in SEQ ID NO:41 and the variable light chain region of FMC63 shown in SEQ ID NO:42. In some embodiments, the variable heavy chain and variable light chain are connected by a linker. In some embodiments, the linker is shown in SEQ ID NO:56. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V HIn some embodiments, the svFc is encoded by the sequence of nucleotides set forth in SEQ ID NO:57, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:57. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:43, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43.

[0225] In some embodiments, the scFv is derived from SJ25C1. SJ25C1 is a murine monoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, NR, et al. (1987). Leucocyte typing III. 302). The SJ25C1 antibody comprises CDRH1, H2, and H3 sequences shown in SEQ ID NOs: 47-49, respectively, and CDRL1, L2, and L3 sequences shown in SEQ ID NOs: 44-46, respectively. The SJ25C1 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 50. H ), and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO:51. LIn some embodiments, the scFv comprises a variable light chain containing the CDRL1 sequence of SEQ ID NO:44, the CDRL2 sequence of SEQ ID NO:45, and the CDRL3 sequence of SEQ ID NO:46, and / or a variable heavy chain containing the CDRH1 sequence of SEQ ID NO:47, the CDRH2 sequence of SEQ ID NO:48, and the CDRH3 sequence of SEQ ID NO:49. In some embodiments, the scFv comprises the variable heavy chain region of SJ25C1 shown in SEQ ID NO:50 and the variable light chain region of SJ25C1 shown in SEQ ID NO:51. In some embodiments, the variable heavy chain and variable light chain are connected by a linker. In some embodiments, the linker is shown in SEQ ID NO:52. In some embodiments, the scFv comprises, in order, V H , linker, and V L In some embodiments, the scFv comprises, in order, V L , linker, and V H In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:53, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53.

[0226] In some embodiments, the antigen or antigen-binding domain is BCMA. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for BCMA. In some embodiments, the antibody or antibody fragment that binds to BCMA is or contains a VH and a VL derived from an antibody or antibody fragment set forth in International Patent Application Publication Nos. WO 2016 / 090327 and WO 2016 / 090320.

[0227] In some embodiments, the antigen or antigen-binding domain is GPRC5D. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for GPRC5D. In some embodiments, the antibody or antibody fragment that binds to GPRC5D is or contains a VH and a VL derived from the antibody or antibody fragment set forth in International Patent Application Publication Nos. WO 2016 / 090329 and WO 2016 / 090312.

[0228] In some embodiments, CAR contains a TCR-like antibody, for example, an antibody or antigen-binding fragment (e.g., scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen that is presented on the cell surface as an MHC-peptide complex. In some embodiments, the antibody or its antigen-binding portion that recognizes an MHC-peptide complex can be expressed on a cell as part of a recombinant receptor, for example, an antigen receptor. Among antigen receptors, there are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, CARs that contain an antibody or antigen-binding fragment that exhibits TCR-like specificity for peptide-MHC complexes can also be called TCR-like CARs.

[0229] Reference to a "major histocompatibility complex" (MHC) refers, in some cases, to a protein, generally a glycoprotein, containing a polymorphic peptide-binding site or binding groove capable of forming a complex with a peptide antigen of a polypeptide, including a peptide antigen processed by cellular machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as a complex with a peptide, i.e., an MHC-peptide complex, for presentation of the antigen in a conformation recognizable by an antigen receptor on a T cell, such as a TCR or TCR-like antibody. Generally, MHC class I molecules are heterodimers, in some cases having a transmembrane α chain with three α domains and non-covalently bound β2-microglobulin. MHC class II molecules are generally composed of two transmembrane glycoproteins, α and β, both of which typically span the membrane. An MHC molecule can comprise an effective portion of an MHC that contains one or more antigen-binding sites for binding peptides and the sequences necessary for recognition by an appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides originating from the cytosol to the cell surface, where the MHC-peptide complexes are generally expressed by CD8 + In some embodiments, MHC class II molecules deliver peptides originating from the vesicular system to the cell surface, where they are typically recognized by CD4+ T cells. + They are recognized by T cells. Generally, MHC molecules are encoded by a group of linked gene loci collectively called H-2 in mice and human leukocyte antigens (HLA) in humans. Therefore, typically, human MHC can also be called human leukocyte antigens (HLA).

[0230] The terms "MHC-peptide complex" or "peptide-MHC complex" or variations thereof refer to a complex or association of a peptide antigen with an MHC molecule, e.g., generally through non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of a cell. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, e.g., a TCR, a TCR-like CAR, or an antigen-binding portion thereof.

[0231] In some embodiments, peptides, such as peptide antigens or epitopes of polypeptides, can associate with MHC molecules, e.g., for recognition by antigen receptors. Generally, peptides are derived from or based on fragments of longer biological molecules, such as polypeptides or proteins. In some embodiments, peptides are typically about 8 to about 24 amino acids in length. In some embodiments, peptides have a length of 9 to 22 amino acids or about 9 to 22 amino acids for recognition in an MHC class II complex. In some embodiments, peptides have a length of 8 to 13 amino acids or about 8 to 13 amino acids for recognition in an MHC class I complex. In some embodiments, upon recognition of the peptide in association with an MHC molecule, such as an MHC-peptide complex, the antigen receptor, e.g., a TCR or TCR-like CAR, generates or triggers an activation signal to a T cell, inducing a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response, or other response.

[0232] In some embodiments, TCR-like antibodies or antigen-binding portions can be generated (see, e.g., U.S. Patent Application Publication Nos. 2002 / 0150914; 2003 / 0223994; 2004 / 0191260; 2006 / 0034850; 2007 / 00992530; 20090226474; 20090304679; and International PCT Publication No. WO 03 / 068201).

[0233] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of an antigen that can bind to MHC, such as a tumor antigen, e.g., a universal tumor antigen, a myeloma antigen, or other antigens described below. In some embodiments, an effective amount of the immunogen is then administered to the host to elicit an immune response, wherein the immunogen retains its three-dimensional form for a period of time sufficient to elicit an immune response to the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine whether the desired antibody that recognizes the three-dimensional presentation of the peptide in the binding groove of the MHC molecule has been produced. In some embodiments, the produced antibody can be evaluated to confirm that the antibody can distinguish the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and a complex with an unrelated peptide. The desired antibody can then be isolated.

[0234] In some embodiments, antibodies or antigen-binding portions thereof that specifically bind to MHC-peptide complexes can be generated by using antibody library display methods, such as phage antibody libraries. In some embodiments, for example, phage display libraries of mutant Fab, scFv, or other antibody types can be generated, in which library members are mutated at one or more residues in one or more CDRs. See, e.g., U.S. Patent Application Publication Nos. 20020150914, 2014 / 0294841; and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.

[0235] The term "antibody" is used herein in the broadest sense and includes intact antibodies as well as fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and fragments containing variable heavy chains (V) capable of specifically binding to an antigen.H (scFv) fragments, and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0236] In some embodiments, antigen-binding proteins, antibodies, and antigen-binding fragments thereof specifically recognize the antigen of a full-length antibody. In some embodiments, the heavy and light chains of the antibody can be full-length or can be antigen-binding portions (Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly selected from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is selected from, e.g., kappa or lambda, particularly kappa.

[0237] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain (V); H ) domains, scFv and single domain V Hand multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0238] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L A V domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen can be identified by combining V domains derived from the antibody that binds the antigen. H or V L domains, and each complementary V L or V H Libraries of domains may be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0239] Single domain antibody is an antibody fragment that comprises all or part of the heavy chain variable domain of antibody, or all or part of the light chain variable domain.In certain embodiments, single domain antibody is a human single domain antibody.In some embodiments, CAR comprises an antibody heavy chain domain that specifically binds to antigen, such as cancer marker or cell surface antigen of targeted cell or disease, such as tumor cell or cancer cell, for example, any of the target antigens described herein or known in the art.

[0240] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment containing a non-naturally occurring configuration, such as one having two or more antibody regions or chains linked by a synthetic linker, e.g., a peptide linker, and / or a configuration that cannot be produced by enzymatic digestion of a naturally occurring intact antibody. In some embodiments, the antibody fragment is an scFv.

[0241] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0242] Thus, in some embodiments, the chimeric antigen receptor, including the TCR-like CAR, comprises an extracellular portion containing an antibody or antibody fragment. In some embodiments, the antibody or fragment comprises an scFv. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM).

[0243] In some embodiments, the recombinant receptor, e.g., CAR, e.g., the antibody portion thereof, further comprises a spacer, which comprises a hinge region, e.g., an IgG4 hinge region, and / or a C H 1 / C LThe recombinant receptor may be or comprise at least a portion of an immunoglobulin constant region or a variant or modified version thereof, such as an Fc region. In some embodiments, the recombinant receptor further comprises a spacer and / or hinge region. In some embodiments, the constant region or portion is that of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen recognition component, e.g., scFv, and the transmembrane domain. The spacer can be of a length that provides increased cellular responsiveness after antigen binding compared to the absence of the spacer. In some examples, the spacer is 12 or about 12 amino acids in length, or is 12 amino acids or less in length. Exemplary spacers include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and including any integer between any of the endpoints of the recited range. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to CH2 and CH3 domains, or an IgG4 hinge linked to a CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or International Patent Application Publication No. WO2014031687. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 1 and is encoded by the sequence set forth in SEQ ID NO: 2. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 3. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 4.

[0244] In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:5. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:1, 3, 4, and 5. In some embodiments, the spacer has the sequence set forth in SEQ ID NOs:23-31. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs:27-31, 58, 59.

[0245] The antigen recognition domain is generally linked to one or more intracellular signaling components, for example, in the case of CAR, the signaling components mimic the activation through an antigen receptor complex, such as a TCR complex, and / or the signal through another cell surface receptor.Thus, in some embodiments, the antigen binding component (e.g., antibody) is linked to one or more transmembrane domains and intracellular signaling domains.In some embodiments, the transmembrane domain is fused to the extracellular domain.In one embodiment, the transmembrane domain that naturally associates with one of the domains in the receptor, such as CAR, is used.In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid the binding of such domain to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0246] In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprising at least the transmembrane region of) the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, synthetic transmembrane domains primarily comprise hydrophobic residues such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is via a linker, spacer, and / or transmembrane domain.

[0247] Some intracellular signaling regions mimic or approximate signaling through natural antigen receptors, signaling through such receptors in combination with costimulatory receptors, and / or signaling through costimulatory receptors alone. In some embodiments, a short oligopeptide or polypeptide linker, e.g., a linker 2-10 amino acids in length, such as one containing glycine and serine, e.g., a glycine-serine doublet, is present to form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0248] Receptors, e.g., CARs, generally comprise at least one or more intracellular signaling components. In some embodiments, receptors comprise intracellular components of the TCR complex, such as the TCR CD3 chain, e.g., the CD3ζ chain, which mediates T cell activation and cytotoxicity. Thus, in some aspects, ROR1-binding antibodies are linked to one or more cell signaling modules. In some embodiments, the cell signaling modules comprise a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, receptors, e.g., CARs, further comprise portions of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, CARs comprise chimeric molecules between CD3 zeta (CD3ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0249] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of immune cells, such as T cells engineered to express the CAR. For example, in some situations, the CAR induces T cell function, such as cytolytic activity or T helper activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling region of an antigen receptor component or costimulatory molecule is used in place of the intact immunostimulatory chain, for example, when it transmits an effector function signal. In some embodiments, the intracellular signaling region, for example, comprising one or more intracellular domains, comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects also comprises that of a co-receptor that acts cooperatively with such receptors in a natural context to initiate signal transduction after antigen receptor binding, and / or any derivative or variant of such molecules, and / or any synthetic sequence with the same functional capability.

[0250] In relation to natural TCR, complete activation generally requires not only TCR-mediated signal transduction but also costimulatory signal.Therefore, in some embodiments, CAR also comprises the component for generating secondary signal or costimulatory signal to promote complete activation.In other embodiments, CAR does not comprise the component for generating costimulatory signal.In some aspects, additional CAR is expressed in the same cell and provides the component for generating secondary signal or costimulatory signal.

[0251] T cell activation, in some aspects, is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, a CAR comprises one or both of such signaling components.

[0252] In some aspects, CAR comprises a primary cytoplasmic signaling sequence that controls the primary activation of TCR complex.The primary cytoplasmic signaling sequence that acts in a stimulatory manner can contain a signaling motif known as immunoreceptor tyrosine-based activation motif or ITAM.Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from TCR or CD3ζ, FcRγ or FcRβ.In some embodiments, the cytoplasmic signaling molecule in CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0253] In some embodiments, the CAR comprises the signaling region and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR comprises both the signaling region and the costimulatory component.

[0254] In some embodiments, the signaling region is contained within one CAR, while the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises an activating or stimulatory CAR and a costimulatory CAR, both expressed on the same cell (see WO2014 / 055668).

[0255] In certain embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3ζ) intracellular domain. In some embodiments, the intracellular signaling region comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3ζ intracellular domain.

[0256] In some embodiments, the CAR includes one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3ζ, CD28, and 4-1BB.

[0257] In some cases, CARs are referred to as first-generation, second-generation, and / or third-generation CARs. In some aspects, first-generation CARs only provide CD3 chain-inducing signals upon antigen binding; in some aspects, second-generation CARs provide such signals and costimulatory signals, for example, contain intracellular signaling domains derived from costimulatory receptors such as CD28 or CD137; in some aspects, third-generation CARs contain multiple costimulatory domains from different costimulatory receptors.

[0258] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment is an scFv or single domain V H In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3 zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain disposed between the extracellular domain and the intracellular signaling region.

[0259] In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane domain can be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any of those described herein. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule, for example, between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0260] In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of CD28 or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains the transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains the signaling portion of 4-1BB or a functional variant thereof and the signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer containing a portion of an Ig molecule, such as a human Ig molecule, e.g., an Ig hinge, e.g., an IgG4 hinge, e.g., a hinge-only spacer.

[0261] In some embodiments, the transmembrane domain of the receptor, e.g., CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27 amino acid transmembrane domain of human CD28 (Accession No. P10747.1), or is a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8; in some embodiments, the transmembrane domain-containing portion of the recombinant receptor is the transmembrane domain of SEQ ID NO:8. The amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0262] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.

[0263] In some embodiments, the intracellular signaling region comprises the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, e.g., the 41 amino acid domain thereof and / or such a domain having an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, the intracellular signaling domain can comprise the amino acid sequence set forth in SEQ ID NO:10 or 11, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10 or 11. In some embodiments, the intracellular region comprises the intracellular costimulatory signaling domain of 4-1BB or a functional variant or portion thereof, e.g., the 42 amino acid cytoplasmic domain of human 4-1BB (Accession No. Q07011.1) or a functional variant or portion thereof, e.g., the amino acid sequence set forth in SEQ ID NO:12, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12.

[0264] In some embodiments, the intracellular signaling region comprises a human CD3 chain, optionally the CD3 zeta stimulatory signaling domain or a functional variant thereof, e.g., the 112 AA cytoplasmic domain of human CD3 zeta isoform 3 (Accession No. P20963.2), or the CD3 zeta signaling domain described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling region comprises the amino acid sequence set forth in SEQ ID NO: 13, 14, or 15, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14, or 15.

[0265] In some aspects, the spacer contains only the hinge region of an IgG, e.g., an IgG4 or IgG1 hinge only, e.g., the hinge only spacer shown in SEQ ID NO: 1. In other embodiments, the spacer comprises C H 2 and / or C H In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the C3 domain. In some embodiments, the spacer is a C4 domain, as shown in SEQ ID NO:3. H 2 and C H In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to the 3 domains. In some embodiments, the spacer is a C hinge, as shown in SEQ ID NO:4. H It is an Ig hinge, such as an IgG4 hinge, linked to only 3 domains. In some embodiments, the spacer is or includes a glycine-serine rich sequence or other flexible linker, such as a known flexible linker.

[0266] 2. Chimeric Autoantibody Receptor (CAAR) In some embodiments, the recombinant receptors expressed by the engineered cells used in connection with the provided methods, uses, articles of manufacture, and compositions include chimeric autoantibody receptors (CAARs). In some embodiments, the CAARs are specific to autoantibodies. In some embodiments, cells expressing CAARs, such as T cells engineered to express CAARs, can be used to specifically bind to and kill autoantibody-expressing cells, but not to specifically bind to and kill normal antibody-expressing cells. In some embodiments, CAAR-expressing cells can be used to treat autoimmune diseases associated with the expression of autoantigens, such as autoimmune diseases. In some embodiments, CAAR-expressing cells can target B cells that ultimately produce autoantibodies and display autoantibodies on their cell surface, marking these B cells as disease-specific targets for therapeutic intervention. In some embodiments, CAAR-expressing cells can be used to efficiently target and kill pathogenic B cells in autoimmune diseases by targeting disease-causing B cells with antigen-specific chimeric autoantibody receptors. In some embodiments, the recombinant receptor is a CAAR, such as any described in U.S. Patent Application Publication No. 2017 / 0051035.

[0267] In some embodiments, the CAAR comprises an autoantibody binding domain, a transmembrane domain, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain capable of inducing a primary activation signal in T cells, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling region comprises a secondary or costimulatory signaling region (secondary intracellular signaling region).

[0268] In some embodiments, the autoantibody binding domain comprises an autoantigen or a fragment thereof. The selection of the autoantigen can depend on the type of autoantibody to be targeted. For example, the autoantigen may be selected so that it recognizes an autoantibody on a target cell, such as a B cell, associated with a particular disease state, e.g., an autoimmune disease, such as an autoantibody-mediated autoimmune disease. In some embodiments, the autoimmune disease comprises pemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1 (Dsg1) and Dsg3.

[0269] 3. Multi-targeting In some embodiments, the cells used in connection with the provided methods, uses, articles of manufacture, and compositions include cells that use a multi-targeting strategy. In some embodiments, the cells express a multi-chain chimeric antigen receptor (CAR) or express two or more engineered receptors on the cell, each recognizing the same different antigen, and typically each containing a different intracellular signaling component. Such multi-targeting strategies are described, for example, in International Patent Application Publication No. WO 2014055668 A1 (describes, e.g., the combination of an activating CAR and a costimulatory CAR that target off-targets, e.g., two different antigens that are present individually on normal cells but together only on cells of the disease or condition to be treated), and Fedorov et al., Sci. Transl. Medicine, 5(215)(2013) (describes cells expressing an activating CAR and an inhibitory CAR, e.g., the activating CAR binds to one antigen expressed on both normal or non-diseased cells and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on normal cells or cells that are not desired to be treated).

[0270] For example, in some embodiments, the cells generally comprise a receptor expressing a first engineered antigen receptor (e.g., a CAR or TCR) that can induce an activation or stimulatory signal to the cell upon specific binding to an antigen, e.g., the first antigen, recognized by the first receptor. In some embodiments, the cells further comprise a second engineered antigen receptor (e.g., a CAR or TCR), e.g., a chimeric costimulatory receptor, that can induce a costimulatory signal to the immune cell upon specific binding to a second antigen, generally, recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

[0271] In some embodiments, the first and / or second engineered antigen receptor (e.g., CAR or TCR) can induce an activation signal to a cell. In some embodiments, the receptor comprises an intracellular signaling component containing an ITAM or ITAM-like motif. In some embodiments, the activation induced by the first receptor comprises signal transduction or changes in protein expression in the cell, resulting in the initiation of an immune response, such as ITAM phosphorylation and / or the initiation of an ITAM-mediated signaling cascade, the formation of an immunological synapse and / or the clustering of molecules (e.g., CD4 or CD8) near the bound receptor, the activation of one or more transcription factors, such as NF-κB and / or AP-1, and / or the induction of gene expression of factors such as cytokines, proliferation, and / or survival.

[0272] In some embodiments, the first and / or second receptor comprises the intracellular signaling domain or region of a costimulatory receptor such as CD28, CD137 (4-1BB), OX40, and / or ICOS. In some embodiments, the first receptor and the second receptor comprise the intracellular signaling domain of different costimulatory receptors. In one embodiment, the first receptor contains a CD28 costimulatory signaling region and the second receptor contains a 4-1BB costimulatory signaling region, or vice versa.

[0273] In some embodiments, the first and / or second receptor comprises both an intracellular signaling domain containing an ITAM or ITAM-like motif and an intracellular signaling domain of a costimulatory receptor.

[0274] In some embodiments, the first receptor contains an intracellular signaling domain containing an ITAM or ITAM-like motif, and the second receptor contains an intracellular signaling domain of a costimulatory receptor. The costimulatory signal, combined with an activation signal induced in the same cell, results in a robust and sustained immune response, such as increased gene expression, secretion of cytokines and other factors, and T cell-mediated effector functions such as cell killing.

[0275] In some embodiments, the ligation of either the first receptor alone or the second receptor alone does not induce a robust immune response.In some aspects, when only one receptor is ligated, cells become tolerant or unresponsive to antigens, or are inhibited, and / or are not induced to proliferate, secrete factors, or perform effector functions.However, in some such embodiments, when multiple receptors are ligated, such as when the cells that express the first and second antigens meet, a desired response is achieved, such as complete immune activation or stimulation, as indicated by the secretion of one or more cytokines, proliferation, persistence, and / or the performance of immune effector functions, such as the cytotoxic killing of target cells.

[0276] In some embodiments, the two receptors induce activation signal and inhibitory signal to cell, respectively, so that the binding of one receptor to its antigen activates cell or induces response, while the binding of the second inhibitory receptor to its antigen induces the signal that suppresses or dampens its response.An example is the combination of activating CAR and inhibitory CAR or iCAR.For example, the strategy can be used in which activating CAR binds to the antigen that is expressed in disease or condition but also expressed on normal cells, and inhibitory receptor binds to the antigen that is expressed on normal cells but not expressed on disease or condition cells.

[0277] In some embodiments, multi-targeting strategies are used when the antigen associated with a particular disease or condition is expressed on non-diseased cells and / or on the engineered cells themselves, either transiently (for example, upon stimulation associated with genetic engineering) or permanently.In such cases, by requiring the ligation of two separate and individually specific antigen receptors, specificity, selectivity and / or efficacy can be improved.

[0278] In some embodiments, multiple antigens, for example, the first and second antigens, are expressed in targeted cells, tissues, or diseases or conditions, such as cancer cells.In some aspects, the cells, tissues, diseases, or conditions are multiple myeloma or multiple myeloma cells.In some embodiments, one or more of the multiple antigens are generally also expressed in cells that are not desired to be targeted by cell therapy, such as normal or non-diseased cells or tissues, and / or in the engineered cells themselves.In such embodiments, specificity and / or efficacy are achieved by requiring the ligation of multiple receptors to achieve cellular response.

[0279] 4. T cell receptor In some embodiments, engineered cells, e.g., T cells, are provided that express a T cell receptor (TCR) or antigen-binding portion thereof that recognizes a peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, a virus, or an autoimmune protein.

[0280] In some embodiments, a "T cell receptor" or "TCR" is a molecule that contains variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRα and TCRβ, respectively), or an antigen-binding portion thereof, and can specifically bind to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. Typically, TCRs that exist in the αβ and γδ forms are generally structurally similar, although the T cells that express them may have distinct anatomical locations or functions. TCRs can be found on the surface of cells or in a soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0281] Unless otherwise specified, the term "TCR" should be understood to encompass complete TCRs and antigen-binding portions or fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including αβ or γδ forms of TCR. In some embodiments, the TCR is less than full-length, but is an antigen-binding portion that binds to a specific peptide bound to an MHC molecule, e.g., an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR can contain only a portion of the structural domain of a full-length or intact TCR, but can still bind to a peptide epitope, such as an MHC-peptide complex, to which the complete TCR binds. In some cases, the antigen-binding portion contains sufficient variable domains of the TCR, e.g., the variable α and β chains of the TCR, to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of the TCR contain the complementarity-determining regions involved in recognizing peptides, MHC, and / or MHC-peptide complexes.

[0282] In some embodiments, the variable domain of a TCR contains hypervariable loops or complementarity-determining regions (CDRs), which are generally the main contributors to antigen recognition and binding capacity and specificity. In some embodiments, the CDRs of a TCR or a combination thereof form all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs in the variable region of a TCR chain are generally separated by framework regions (FRs), which generally show lower variability between TCR molecules compared to CDRs (see, for example, Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; also see Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity, or is the most important of the three CDRs on a given TCR variable region for antigen recognition and / or interaction with the processed peptide portion of a peptide-MHC complex. In some circumstances, CDR1 of the α chain can interact with the N-terminal portion of a particular antigen peptide. In some circumstances, CDR1 of the β chain can interact with the C-terminal portion of a peptide. In some circumstances, CDR2 is the primary CDR that most strongly contributes to or is responsible for interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the β chain can contain an additional hypervariable region (CDR4 or HVR4) that is generally involved in superantigen binding and not involved in antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

[0283] In some embodiments, the TCR can also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with the invariant protein of the CD3 complex, which is involved in mediating signal transduction.

[0284] In some embodiments, a TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (e.g., an α chain or a β chain) comprises two immunoglobulin-like domains, such as a variable domain (e.g., Vα or Vβ; typically amino acids 1-116 according to the Kabat numbering system, Kabat et al., "Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain adjacent to the cell membrane (e.g., an α chain constant domain or Cα, typically positions 117-259 of the chain according to the Kabat numbering system, or a β chain constant domain or C βThe constant domains of the TCR may contain a short connecting sequence in which cysteine ​​residues form disulfide bonds, thereby linking the two chains of the TCR. In some embodiments, the TCR may have additional cysteine ​​residues in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.

[0285] In some embodiments, the TCR chain contains a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules, such as CD3 and its subunits. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contain one or more immunoreceptor tyrosine-based activation motifs or ITAMs that are involved in the signaling ability of the TCR complex.

[0286] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β, or γ and δ), linked, for example, by one or more disulfide bonds.

[0287] In some embodiments, TCRs can be generated from known TCR sequences, such as those of the Vα and β chains, for which substantially full-length coding sequences are readily available. Methods can be used to obtain full-length TCR sequences, including V chain sequences, from cellular sources. In some embodiments, nucleic acids encoding TCRs can be obtained from a variety of sources, for example, by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids within or isolated from a given cell or cells, or by synthesis of publicly available TCR DNA sequences.

[0288] In some embodiments, the TCR is obtained from a biological source, e.g., from a cell, e.g., from a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, the T cell can be obtained from an in vivo isolated cell. In some embodiments, the TCR is a thymically selected TCR. In some embodiments, the TCR is a neoepitope-restricted TCR. In some embodiments, the T cell can be a cultured T cell hybridoma or clone. In some embodiments, the TCR or antigen-binding portion thereof or antigen-binding fragment thereof can be synthetically generated from knowledge of the sequence of the TCR.

[0289] In some embodiments, TCRs are generated from TCRs identified or selected from screening a library of candidate TCRs against a target polypeptide antigen or its target T cell epitope. TCR libraries can be generated by amplifying Vα and Vβ repertoires from T cells isolated from a subject, including cells present in PBMCs, spleen, or other lymphoid organs. In some cases, T cells can be amplified from tumor-infiltrating lymphocytes (TILs). In some embodiments, TCR libraries can be generated from CD4+ or CD8+ cells. In some embodiments, TCRs can be amplified from a T cell source of a normal, healthy subject, i.e., a normal TCR library. In some embodiments, TCRs can be amplified from a T cell source of a diseased subject, i.e., a diseased TCR library. In some embodiments, degenerate primers are used to amplify the Vα and Vβ gene repertoire in a sample, such as T cells obtained from a human, for example, by RT-PCR. In some embodiments, scTv libraries can be assembled from naive Vα and Vβ libraries, where the amplification products are cloned or assembled so that they are separated by a linker. Depending on the subject and cell source, the library can be HLA allele-specific. Alternatively, in some embodiments, TCR libraries can be generated by mutagenesis or diversification of parent or framework TCR molecules. In some aspects, TCRs are subjected to directed evolution, such as by mutagenesis of the α or β chain. In some aspects, specific residues within the CDRs of the TCR are altered. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells can be selected, such as by screening to evaluate CTL activity against peptides. In some aspects, for example, TCRs present on antigen-specific T cells can be selected by avidity, such as a specific affinity or avidity for an antigen.

[0290] In some embodiments, the TCR or its antigen-binding portion is modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as higher affinity for specific MHC-peptide complexes. In some embodiments, directed evolution is achieved by display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display approach involves engineering or modifying a known, parental, or reference TCR. For example, in some cases, a wild-type TCR can be used as a template to create mutagenized TCRs in which one or more residues in the CDRs are mutated, and mutants with desired altered properties, such as higher affinity for a desired target antigen, are selected.

[0291] In some embodiments, the peptide of the target polypeptide for use in the creation or generation of the TCR of interest is known or can be easily identified. In some embodiments, the peptide suitable for use in the generation of TCR or antigen-binding portion can be determined based on the presence of HLA-restricted motifs in the target polypeptide of interest, such as the target polypeptide described below. In some embodiments, the peptide is identified using available computer prediction models. In some embodiments, for predicting MHC class I binding site, such models include but are not limited to ProPred1 (Singh and Raghava (2001) Bioinformatics 17(12):1236-1237) and SYFPEITHI (Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1): 75-93 2007). In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasians and is therefore a suitable choice of MHC antigen for use in preparing TCRs or other MHC-peptide binding molecules.

[0292] HLA-A0201 binding motif and proteasome and immunoproteasome cleavage site can be used by computer prediction model.For predicting MHC class I binding site, such model includes but is not limited to ProPred1 (Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17 (12): 1236-1237 2001, more detailed description) and SYFPEITHI (referring to Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology, vol. 409 (1): 75-93 2007).

[0293] In some embodiments, the TCR or its antigen-binding portion may be a recombinantly produced natural protein or a variant thereof, which has one or more altered characteristics, such as binding characteristics. In some embodiments, the TCR may be derived from one of various animal species, such as human, mouse, rat, or other mammal. The TCR may be cell-bound or soluble. In some embodiments, for the purposes of the provided methods, the TCR is cell-bound and expressed on the surface of a cell.

[0294] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). In some embodiments, the dTCR or scTCR has a structure described in WO 03 / 020763, WO 04 / 033685, WO2011 / 044186.

[0295] In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some embodiments, the TCR contains a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR can form a TCR complex with CD3. In some embodiments, either a TCR, including a dTCR or a scTCR, can be linked to a signaling domain to generate an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell.

[0296] In some embodiments, the dTCR contains a first polypeptide in which a sequence corresponding to a TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR α chain constant region extracellular sequence, and a second polypeptide in which a sequence corresponding to a TCR β chain variable region sequence is fused to the N-terminus of a sequence corresponding to a TCR β chain constant region extracellular sequence, and the first and second polypeptides are linked by a disulfide bond. In some embodiments, the bond can correspond to the natural interchain disulfide bond present in naturally occurring dimeric αβ TCRs. In some embodiments, the interchain disulfide bond does not exist in naturally occurring TCRs. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequence of the dTCR polypeptide pair. In some cases, both natural and non-natural disulfide bonds may be desirable. In some embodiments, the TCR contains a transmembrane sequence for membrane anchoring.

[0297] In some embodiments, the dTCR contains a TCR alpha chain containing a variable alpha domain, a constant alpha domain, and a first dimerization motif attached to the C-terminus of the constant alpha domain, and a TCR beta chain containing a variable beta domain, a constant beta domain, and a first dimerization motif attached to the C-terminus of the constant beta domain, wherein the first and second dimerization motifs readily interact to form a covalent bond between an amino acid in the first dimerization motif and an amino acid in the second dimerization motif, linking the TCR alpha chain and the TCR beta chain together.

[0298] In some embodiments, TCR is scTCR.Typically, scTCR can be produced by methods known to those skilled in the art.See, for example, Soo Hoo, WF et al. PNAS (USA) 89, 4759 (1992);Wulfing, C. and Pluckthun, A., J. Mol. Biol. 242, 655 (1994);Kurucz, I. et al. PNAS (USA) 90, 3830 (1993);International Publication No. PCT No. WO 96 / 13593, WO 96 / 18105, WO99 / 60120, WO99 / 18129, WO 03 / 020763, WO2011 / 044186;And Schlueter, CJ et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, the scTCR contains a non-native disulfide interchain bond introduced to facilitate TCR chain association (see, e.g., International Publication No. WO 03 / 020763). In some embodiments, the scTCR is a non-disulfide truncated TCR in which a heterologous leucine zipper fused to its C-terminus facilitates chain association (see, e.g., International Publication No. WO 99 / 60120). In some embodiments, the scTCR contains a TCR alpha variable domain covalently linked to a TCR beta variable domain via a peptide linker (see, e.g., International Publication No. WO 99 / 18129).

[0299] In some embodiments, the scTCR contains a first segment composed of an amino acid sequence corresponding to a TCR alpha chain variable region, a second segment composed of an amino acid sequence corresponding to a TCR beta chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to a TCR beta chain constant domain extracellular sequence, and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0300] In some embodiments, the scTCR contains a first segment composed of an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant domain sequence, and a second segment composed of a beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant sequence and transmembrane sequence sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0301] In some embodiments, the scTCR contains a first segment composed of a TCR beta chain variable region sequence fused to the N-terminus of a beta chain extracellular constant domain sequence, and a second segment composed of an alpha chain variable region sequence fused to the N-terminus of an alpha chain extracellular constant sequence and transmembrane sequence sequence, and optionally a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[0302] In some embodiments, the linker of the scTCR linking the first and second TCR segments can be any linker capable of forming a single polypeptide chain while retaining the binding specificity of the TCR. In some embodiments, the linker sequence can have the formula, for example, -P-AA-P-, where P is proline and AA represents an amino acid sequence, where the amino acids are glycine and serine. In some embodiments, the first and second segments are paired such that their variable region sequences are oriented for such binding. Thus, in some cases, the linker has sufficient length to bridge the distance between the C-terminus of the first segment and the N-terminus of the second segment, or vice versa, but is not so long as to block or reduce binding of the scTCR to the target ligand. In some embodiments, the linker can contain 10 to 45 amino acids or about 10 to 45 amino acids, e.g., 10 to 30 amino acids or 26 to 41 amino acid residues, e.g., 29, 30, 31, or 32 amino acids. In some embodiments, the linker has the formula TIFF0007824728000001.tif4128, where P is proline, G is glycine, and S is serine (SEQ ID NO:23). In some embodiments, the linker has the sequence I have TIFF0007824728000002.tif4128.

[0303] In some embodiments, the scTCR contains a covalent disulfide bond linking residues of the immunoglobulin region of the constant domain of the α chain to residues of the immunoglobulin region of the constant domain of the β chain. In some embodiments, there are no interchain disulfide bonds in native TCRs. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. In some cases, both natural and non-natural disulfide bonds may be desirable.

[0304] In some embodiments of dTCR or scTCR containing introduced interchain disulfide bond, there is no natural disulfide bond.In some embodiments, one or more of the natural cysteines that form natural interchain disulfide bonds are replaced with another residue, such as serine or alanine.In some embodiments, the introduced disulfide bond can be formed by mutating non-cysteine ​​residues on the first and second segments to cysteine.Exemplary non-natural disulfide bonds of TCR are described in International Publication No. PCT WO2006 / 000830.

[0305] In some embodiments, the TCR or antigen-binding fragment thereof exhibits an affinity for the target antigen with an equilibrium binding constant of 10 to 10 M or about 10 to 10 M, and all individual values ​​and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or a ligand.

[0306] In some embodiments, one or more nucleic acids encoding TCRs, such as α and β chains, can be amplified by PCR, cloning, or other suitable means and cloned into one or more suitable expression vectors. The expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification, or for expression, or both, such as plasmids and viruses.

[0307] In some embodiments, the vector can be a pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.) vector. In some cases, bacteriophage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. In some embodiments, plant expression vectors can be used, including pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some embodiments, viral vectors such as retroviral vectors are used.

[0308] In some embodiments, recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, the vector can contain regulatory sequences, such as transcription and translation initiation and termination codons, that are specific to the type of host into which the vector will be introduced (e.g., bacteria, fungi, plants, or animals), as appropriate, taking into account whether the vector is DNA-based or RNA-based. In some embodiments, the vector can contain a non-native promoter operably linked to the nucleotide sequence encoding the TCR or antigen-binding portion (or other MHC-peptide binding molecule). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long terminal repeat of murine stem cell virus. Other known promoters are also contemplated.

[0309] In some embodiments, to generate a vector encoding a TCR, the α chain and β chain are PCR amplified from the total cDNA isolated from the T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α chain and β chain are cloned into the same vector. In some embodiments, the α chain and β chain are cloned into different vectors. In some embodiments, the generated α chain and β chain are incorporated into a retroviral vector, for example, a lentiviral vector.

[0310] B. Nucleic Acids and Vectors Also provided are one or more polynucleotides (e.g., nucleic acid molecules) encoding the recombinant receptor, vectors for genetically engineering cells to express the receptor, and methods for producing engineered cells. In some aspects, the recombinant receptor is or contains a chimeric antigen receptor (CAR). In some aspects, the recombinant receptor is or contains a T cell receptor (TCR), for example, a transgenic TCR.

[0311] In some cases, the nucleic acid sequence encoding a recombinant receptor, such as a chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. Non-limiting illustrative examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide shown in SEQ ID NO:26 and encoded by the nucleotide sequence shown in SEQ ID NO:25, or the CD8 alpha signal peptide shown in SEQ ID NO:18.

[0312] In certain cases where a nucleic acid molecule encodes two or more different polypeptide chains, each of the polypeptide chains can be encoded by a separate nucleic acid molecule, e.g., two separate nucleic acids are provided, each of which can be individually transferred or introduced into a cell for expression in the cell.

[0313] In some embodiments, such as those in which a polynucleotide contains a first and a second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains can be operably linked to promoters, which can be the same or different. In some embodiments, the nucleic acid molecule can contain promoters that drive the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules can be multicistronic (bicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). In some embodiments, the transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), allowing co-expression of gene products by messages from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of RNA in a single open reading frame (ORF) containing two or three genes separated from each other by sequences encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into individual proteins either during translation (in the case of 2A) or post-translation. In some cases, peptides such as T2A can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skipping), resulting in a separation between the end of the 2A sequence and the next downstream peptide (see, e.g., de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). A variety of 2A elements are known.Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO:22), equine rhinitis A virus (E2A, e.g., SEQ ID NO:21), Thosea asigna virus (T2A, e.g., SEQ ID NO:6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:19 or 20), as described in U.S. Patent Application Publication No. 20070116690.

[0314] In some embodiments, exogenous marker genes may be utilized in some cases for engineered cell therapy, allowing for cell detection or selection, and in some cases, may also promote cell suicide. Exemplary surrogate markers may include truncated forms of cell surface polypeptides, for example, truncated forms that are non-functional and do not or cannot transduce signals or signals normally transduced by the full-length form of the cell surface polypeptide, and / or do not or cannot internalize. Exemplary truncated cell surface polypeptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequences set forth in SEQ ID NO: 7 or 16), or prostate-specific membrane antigen (PSMA) or modified forms thereof. tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells engineered with tEGFR constructs and the encoded exogenous protein, and / or to eliminate or isolate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g., surrogate marker, comprises all or a portion (e.g., a truncated form) of CD34, NGFR, CD19, or a truncated CD19, e.g., a truncated non-human CD19, or epidermal growth factor receptor (e.g., tEGFR).

[0315] In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), e.g., super-fold GFP (sfGFP), red fluorescent protein (RFP), e.g., tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, such as species variants, monomeric variants, and codon-optimized and / or highly sensitive variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as luciferase, the lacZ gene from Escherichia coli (E. coli), alkaline phosphatase, secreted placental alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0316] In some embodiments, the marker is a selectable marker. In some embodiments, the selectable marker is or comprises a polypeptide that confers resistance to an exogenous agent or drug. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the selectable marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selectable marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, a geneticin resistance gene, or a zeocin resistance gene, or modified versions thereof.

[0317] In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, e.g., a cleavable linker sequence, e.g., a polynucleotide encoding T2A. For example, the marker, and optionally the linker sequence, can be any of those disclosed in PCT Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR), optionally linked to a linker sequence, e.g., a T2A cleavable linker sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include the amino acid sequence set forth in SEQ ID NO: 7 or 16, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. In some instances, a truncated epidermal growth factor receptor (EGFRt), such as that shown in SEQ ID NO: 7 or 16, can be co-expressed with a transgene of interest (CAR or TCR) in transduced cells (see, e.g., U.S. Patent No. 8,802,374). The EGFRt can contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells engineered with the EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and / or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434.

[0318] Also provided is a vector or construct containing such nucleic acid and / or polynucleotide.In some embodiments, the vector or construct contains one or more promoters that are operably linked to the nucleic acid encoding the recombinant receptor, so as to drive its expression.In some embodiments, the promoter is operably linked to one or more than one nucleic acid molecule or polynucleotide.Therefore, also provided is a vector, such as the vector that contains any of the polynucleotides provided herein.

[0319] In some cases, vector is a viral vector such as retroviral vector, for example, lentiviral vector or gammavirus vector.Also provided is a composition that contains such vector or a combination of vectors.In some embodiments, a set or combination of vectors is used together for cell manipulation.In some embodiments, the first and second vectors in the set are introduced into cell simultaneously or sequentially in any order for manipulation.

[0320] In some embodiments, the vector includes a viral vector, e.g., a retrovirus or lentivirus, a non-viral vector, or a vector derived from a transposon, e.g., the Sleeping Beauty transposon system, simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV), a lentiviral vector, or a retroviral vector, e.g., a gamma retroviral vector, Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or a retroviral vector derived from an adeno-associated virus (AAV).

[0321] C. Vectors and Methods for Genetic Engineering Various methods for introducing genetically engineered components, such as recombinant receptors, for example, CAR or TCR, are well known.Exemplary methods include those for transferring nucleic acids encoding receptors, including those via viruses, such as retroviruses or lentiviruses, transduction, transposons, and electroporation.In some embodiments, surface glycan expression is evaluated in the cell composition that is collected before, during, or immediately after the genetic engineering process.In some embodiments, surface glycan expression is evaluated and compared between the cell compositions at corresponding stages of the process of introducing genetically engineered components.In some embodiments, the composition is engineered or has been engineered to express the same recombinant receptor, but by different methods of introducing genetic material.

[0322] In some embodiments, gene transfer is achieved by first stimulating the cells, such as by combining the cells with a stimulatory agent that induces a response such as proliferation, survival, and / or activation, as measured by the expression of cytokines or activation markers, followed by transduction of the activated cells and expansion in culture to numbers sufficient for clinical application.

[0323] In some embodiments, recombinant nucleic acids are introduced into cells using recombinant infectious viral particles, such as vectors derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are introduced into T cells using recombinant lentiviral or retroviral vectors, such as gamma-retroviral vectors (see, for example, Koste et al. Gene Therapy doi: 10.1038 / gt.2014.25 (2014); Carlens et al. Exp Hematol., 28(10): 1137-46 (2000); Alonso-Cam...

Claims

1. (a) incubating an input composition comprising a population of T cells, including naive-like T cells and non-naive-like T cells, under stimulatory conditions comprising IL-2, IL-7, and IL-15 for 2 to 6 days; (i) the cells of said input composition have not been or are not subjected to a selection step based on an endogenous T cell surface marker that distinguishes between naive-like T cells and non-naive-like T cells prior to said incubation; (ii) the stimulatory conditions include a stimulatory reagent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex and / or one or more intracellular signaling domains of one or more costimulatory molecules, thereby producing a stimulated composition, wherein the stimulatory reagent includes a first agent that is an anti-CD3 antibody and a second agent that is an anti-CD28 antibody; and (iii) at least 2 × 10 naive-like T cells; 8 The culture contains a starting amount of naive-like T cells, and the naive-like T cells are CD27+ and CCR7+. process; and (b) introducing a nucleic acid encoding an engineered recombinant chimeric antigen receptor (CAR) into the population of T cells, wherein the introducing step is performed during at least a portion of the incubating step such that the nucleic acid is introduced and the engineered recombinant chimeric antigen receptor (CAR) is expressed in the population of T cells.

1. A method for genetically engineering T cells, comprising:

2. 10. The method of claim 1, wherein the incubating step is carried out for at least 3 days, at least 4 days, or at least 5 days.

3. 3. The method of claim 1 or 2, wherein the stimulatory conditions preferentially induce the expansion or proliferation of naive-like T cells relative to non-naive-like T cells in the stimulated composition.

4. the naive-like T cells Surface positivity for the T-cell activation marker CD28; and / or is surface negative for a marker selected from the group consisting of CD25, CD56, CD62L, KLRG1; and / or Low expression of CD95; and / or negative for intracellular expression of cytokines selected from the group consisting of IL-2, IFN-γ, IL-4, and IL-10; The method according to any one of claims 1 to 3.

5. the non-naive-like T cells is surface negative for T cell activation markers selected from the group consisting of CD45RA, CD27, CD28, and CCR7; and / or is surface positive for a marker selected from the group consisting of CD25, CD45RO, CD56, CD62L, KLRG1, and perforin; and / or positive for intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-γ, IL-4, and IL-10; and / or High expression of CD95 The method according to any one of claims 1 to 4.

6. 6. The method of any one of claims 1 to 5, wherein the CAR comprises an extracellular domain comprising an antigen-binding domain that specifically binds to an antigen, and an intracellular signaling domain that is or comprises the intracellular signaling domain of the CD3-zeta (CD3ζ) chain.

7. 7. The method of claim 6, wherein the intracellular signaling domain further comprises a costimulatory signaling region, and the costimulatory signaling region comprises 4-1-BB, CD28, or ICOS, or a signaling portion thereof.

8. The method of any one of claims 1 to 7, wherein the primary agent and / or secondary agent is present on the surface of a bead.

9. 9. The method of claim 8, wherein the beads comprise a diameter of greater than 3.5 μm but not greater than 9 μm, or not greater than 8 μm, or not greater than 7 μm, or not greater than 6 μm, or not greater than 5 μm.

10. 10. The method of claim 8 or 9, wherein the beads comprise a diameter of 4.5 μm.

11. 11. The method of any one of claims 8 to 10, wherein the stimulation conditions comprise incubating cells at a bead to cell ratio of 1:1 to 10:

1.

12. 12. The method of any one of claims 8 to 11, wherein the stimulatory conditions comprise a stimulatory reagent comprising an anti-CD3 antibody and a secondary agent that is an anti-CD28 antibody attached to beads, and wherein the ratio of beads to cells during the incubating step is between 1:1 and 4:

1.

13. 13. The method of any one of claims 1 to 12, wherein the T cells are derived from a biological sample, which 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.

14. 14. The method of claim 13, wherein the biological sample is from a human subject.

15. The method of any one of claims 1 to 14, wherein the T cells comprise CD4+ and / or CD8+ cells.

16. 16. The method of any one of claims 1 to 15, wherein the T cells comprise CD4+ and CD8+ T cells, and the ratio of CD4+ to CD8+ T cells is between 2:1 and 1:

5.

17. 17. The method of any one of claims 1 to 16, wherein the T cells comprise CD4+ and CD8+ T cells, and the ratio of CD4+ cells to CD8+ cells is 1:1, 1:2, 2:1, or 1:

3.

18. 18. The method of any one of claims 1 to 17, wherein the stimulatory conditions do not include N-acetylcysteine ​​(NAC).

19. 19. The method of any one of claims 1 to 18, wherein the stimulatory conditions result in activation-induced cell death (AICD) of non-naive-like T cells or a subpopulation thereof.

20. the percentage of cells derived from naive-like T cells in the stimulated composition is increased by more than 1.5-fold compared to the percentage of naive-like cells in the input composition; the ratio of cells derived from naive-like T cells compared to cells derived from non-naive-like T cells in the stimulated composition is increased by more than 1.5-fold compared to the ratio of naive-like T cells compared to non-naive-like T cells in the input composition; the ratio of naive-like T cells to non-naive-like T cells in the stimulated composition is increased by more than 1.5-fold compared to the ratio of naive-like T cells to non-naive-like T cells in the input composition; the stimulated composition comprises greater than 75% cells derived from the naive-like T cells of the input composition; the stimulated composition comprises less than 10% cells derived from non-naive-like T cells; and / or the stimulated composition comprises less than 9% cells derived from non-naive T cells; 20. The method according to any one of claims 1 to 19.

21. 21. The method of any one of claims 1 to 20, wherein the stimulated composition is more polyclonal or multiclonal compared to the input composition.

22. The method of any one of claims 1 to 21, which is carried out in vitro or ex vivo.

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